**Meet the editor**

Dr Mehnaz Atiq is a Paediatric Cardiologist at the Aga Khan University hospital, Karachi, Pakistan. This is a tertiary care hospital with a reputed cardiology service, adult and congenital. The center provides state of art care to sick cardiac patients. These include General and Interventional cardiology for children and adults, nuclear studies, electrophysiology, cardiac magnetic reso-

nance and computerized tomographic angiography for adults and children with heart disease. Dr Mehnaz Atiq did her undergraduate medical school from India and then migrated to Pakistan. She trained in Pediatric Cardiology locally and in Gothenburg, Sweden. Thereafter she pursued training in Interventional cardiology in the Hospital for Sick children, Toronto, Canada. She was the first to start fetal cardiology service in the country. Dr Mehnaz Atiq is presently a Professor in Pediatric Cardiology and runs a fellowship training program.

Contents

**Preface IX** 

Chapter 4 **Pathogenesis of Renovascular** 

Chapter 5 **Cardiovascular Disease in Inflammatory** 

Chapter 6 **Cardiovascular Risk Factors in Elderly** 

Chapter 7 **Erectile Dysfunction Complicating** 

Chapter 9 **The Polycystic Ovary Syndrome Status –** 

Arun Kumar

Chapter 8 **Vascular Dysfunction in** 

Mikiya Nakastuka

Aizuri Murad and Anne-Marie Tobin

Chapter 1 **Lipoprotein (a) and Cardiovascular Risk 1**  José Antonio Díaz Peromingo

Chapter 2 **Remnant Lipoproteins are a Stronger Risk Factor** 

Katsuyuki Nakajima and Masaki Q. Fujita

**for Cardiovascular Events than LDL-C – From** 

Chapter 3 **Cardiovascular Risk Factors and Liver Transplantation 37**  Anna Rossetto, Umberto Baccarani and Vittorio Bresadola

> **Hypertension: Challenges and Controversies 49**  Blake Fechtel, Stella Hartono and Joseph P. Grande

**Disorders – Psoriasis and Psoriatic Arthritis 67** 

**Cardiovascular Risk Factors and Disease 99**  Irekpita Eshiobo, Emeka Kesieme and Taofik Salami

**Women with Recurrent Pregnancy Loss 123** 

**A Risk Factor for Future Cardiovascular Disease 151**  Ioana Ilie, Razvan Ilie, Lucian Mocan, Carmen Georgescu, Ileana Duncea, Teodora Mocan, Steliana Ghibu and Cornel Iancu

**Normolipidaemic Acute Myocardial Infarct Patients 83** 

**the Studies of Autopsies in Sudden Cardiac Death Cases 15** 

### Contents

#### **Preface XIII**


Ioana Ilie, Razvan Ilie, Lucian Mocan, Carmen Georgescu, Ileana Duncea, Teodora Mocan, Steliana Ghibu and Cornel Iancu X Contents



Contents VII

Chapter 21 **Health Related Quality of Life in Coronary Patients 399** 

**Affect: Relation to Cardiovascular Disease 415** Marco A.A. Torquato Jr., Bruno P.F. de Souza,

María Dueñas, Alejandro Salazar, Begoña Ojeda and Inmaculada Failde

Chapter 22 **Anger, Hostility and Other Forms of Negative** 

Dan V. Iosifescu and Renerio Fraguas

Chapter 23 **"Recognizing Hunger" – A Training to Abate** 

Mario Ciampolini

Chapter 24 **Effects of Dietary Fiber Intake**

Mercedes Sotos Prieto

**Insulin Resistance, Associated Subclinical Inflammation and Cardiovascular Risks 437**

**on Cardiovascular Risk Factors 459**  Sara Arranz, Alex Medina-Remón,

Chapter 25 **Mediterranean Diet and Gene-Mediterranean**

Rosa M. Lamuela-Raventós and Ramón Estruch

**Diet Interactions in Determining Intermediate Cardiovascular Disease Phenotypes 489**


VI Contents

Chapter 10 **Premature Atherosclerosis**

Chapter 11 **Dysmetabolic Syndrome 219** 

**Long After Kawasaki Disease 201**  Nobutaka Noto and Tomoo Okada

Elvira Craiu, Lucia Cojocaru, Andrei Rusali,

Changshun Xie, Yali Liu and Chunming Ma

Chapter 14 **Adolescent Obesity Predicts Cardiovascular Risk 275** Jarosław Derejczyk, Barbara Kłapcińska, Ewa Sadowska-Krępa, Olga Stępień-Wyrobiec,

Chapter 15 **Peculiarities of Coronary Artery Disease in Athletes 291**

**Bathing: Is There a Cardiovascular Risk? 309**

**Risk Assessment in Apparently Healthy Bosnian Men and Women: Cross-Sectional Study 341**

**the Anthropometric Measure of Cardiovascular Risk 319** 

**Cardiovascular Risk Factors Among Adult Population 359** 

**Among Multiple Risk Factors Causing Morning Onset of Cardiac Events due to Circadian Variations 383** 

Elżbieta Kimsa and Katarzyna Kempa

Halna du Fretay Xavier, Akoudad Hafid, Hamadou Ouceyni and Benhamer Hakim

Takeshi Otsuki and Yasuko Okuda

Edita Stokić, Biljana Srdić, Vladimir Brtka

Chapter 18 **The Use of Reynolds Risk Score in Cardiovascular** 

Chapter 19 **The Assessment of Prevalence of Hypertension as**

Chapter 20 **Theoretical Identification of Behavioral Risk Factors** 

Fumiko Furukawa and Tatsuya Morimoto

Chapter 16 **Blood Pressure Regulation During** 

Chapter 17 **Sagittal Abdominal Diameter as** 

Asija Začiragić

Aida Pilav

and Dragana Tomić-Naglić

**and Metabolic Syndrome in Han Young Adults – AST/ALT Ratio and Metabolic Syndrome 247**

Chapter 13 **On the Mechanism of Action of Prolylcarboxypeptidase 255** B. Shariat-Madar, M. Taherian and Z. Shariat-Madar

Razvan Maxim and Irinel Parepa

Chapter 12 **The Relationship Between AST/ALT Ratio**

Qiang Lu, Xiaoli Liu, Shuhua Liu,


Preface

disease which may prove fatal or disabling.

research throws light on interventions and their results.

committed to cardiovascular risk prevention.

Among the non-communicable diseases, cardiovascular disorders are the leading cause of morbidity and mortality in both the developed and the developing countries. The spectrum of risk factors is wide and their understanding is imperative to prevent the first and recurrent episodes of myocardial infarction, stroke or peripheral vascular

There is ample evidence from longitudinal studies to prove that cardiovascular diseases are preventable. Individuals with low levels of risk factors generally have a healthy lifestyle. Genetic factors have to be kept in mind when risk stratification is done for cardiovascular diseases. Despite our knowledge of risk factors, huge differences exist in the prevalence between populations within the same region, between men and women and in the racial and ethnic subgroups. Much of this variability is explained on the basis of behavioral and cultural differences rather than genetic or clinical reason. Moreover, risk factors are frequently redefined as newer

This book has tried to present an update on risk factors incorporating new research which has thrown more light on the existing knowledge. It has also tried to highlight regional diversity addressing such issues. It will hopefully be resourceful to the cardiologists, general practitioners, family physicians, researchers, graduate students

**Dr. Mehnaz Atiq**

Karachi, Pakistan

Division of Cardiac Services Aga Khan University Hospital

### Preface

Among the non-communicable diseases, cardiovascular disorders are the leading cause of morbidity and mortality in both the developed and the developing countries. The spectrum of risk factors is wide and their understanding is imperative to prevent the first and recurrent episodes of myocardial infarction, stroke or peripheral vascular disease which may prove fatal or disabling.

There is ample evidence from longitudinal studies to prove that cardiovascular diseases are preventable. Individuals with low levels of risk factors generally have a healthy lifestyle. Genetic factors have to be kept in mind when risk stratification is done for cardiovascular diseases. Despite our knowledge of risk factors, huge differences exist in the prevalence between populations within the same region, between men and women and in the racial and ethnic subgroups. Much of this variability is explained on the basis of behavioral and cultural differences rather than genetic or clinical reason. Moreover, risk factors are frequently redefined as newer research throws light on interventions and their results.

This book has tried to present an update on risk factors incorporating new research which has thrown more light on the existing knowledge. It has also tried to highlight regional diversity addressing such issues. It will hopefully be resourceful to the cardiologists, general practitioners, family physicians, researchers, graduate students committed to cardiovascular risk prevention.

> **Dr. Mehnaz Atiq** Division of Cardiac Services Aga Khan University Hospital Karachi, Pakistan

**1** 

*Spain* 

José Antonio Díaz Peromingo

**Lipoprotein (a) and Cardiovascular Risk** 

First epidemiological studies of Lp(a) and CHD were reported at the end of the last century (1-3) but the investigation of this lipoprotein as a potential cardiovascular risk factor has been hampered by the lack of consistent approaches to its measurement for decades. Lp(a) laboratory standardization emerged in 2000 (4) and was accepted by the World Health Organization in 2004 (5). Another challenge associated to its measurement is the fact that population differences can also contribute to variation in Lp(a) serum concentration (6). Since Lp(a) characterization, evidences favoring its association with cardiovascular risk have been reported. At the same time, studies against this association have also been published leading to some confusion regarding to the possible role of Lp(a) in cardiovascular disease. The last years have clarified somewhat this issue and evidences of Lp(a) as an independent cardiovascular risk factor have been proposed (7-13). Several key points such as its homology with plasminogen, differences among the apo(a) isoforms, genetic considerations as well as special circumstances such as the relationship of Lp(a) and atrial fibrillation, dialysis, alcohol consumption and blood coagulation have been investigated. In this chapter, Lp(a) metabolism, epidemiological and genetic considerations, association with coronary heart disease and stroke, special situations as well as controversies and current treatment

Lipoprotein (a), Lp (a), is a low density lipoprotein (LDL)-like particle synthesized in the liver by hepatocytes and then secreted into plasma. It was first described by Berg in 1963 (14). It consists of an apolipoprotein B100 (apoB100) molecule that is linked covalently by a disulfide bond to a large glycoprotein known as apolipoprotein (a), [apo(a)] (15). Lp(a) metabolic route is shown in figure 1. Its molecular weight ranges from 200 kDa to more than 800 kDa (16). The apo(a) gene (*LPA*) is a major determinant of the plasma concentration of Lp(a), including variations in the kringle region-coding repeats, with accounts for the size polymorphism of apo(a) leading to different apo(a) sizes (17). This fact is very important because small size isofoms seem to be associated to worse cardiovascular profile. Apo(a) chain contains 5 cysteine-rich domains known as kringles, and especially Kringle IV (KIV) is very similar to plasminogen (18,19). This particle is not only located in the plasma but also has been shown to enter the arterial intima of humans and has an increased affinity by the

**1. Introduction** 

options are related.

**2. Lipoprotein (a) metabolism** 

*Short Stay Medical Unit, Department of Internal Medicine, Hospital Clínico Universitario, Santiago de Compostela,* 

### **Lipoprotein (a) and Cardiovascular Risk**

#### José Antonio Díaz Peromingo

*Short Stay Medical Unit, Department of Internal Medicine, Hospital Clínico Universitario, Santiago de Compostela, Spain* 

#### **1. Introduction**

First epidemiological studies of Lp(a) and CHD were reported at the end of the last century (1-3) but the investigation of this lipoprotein as a potential cardiovascular risk factor has been hampered by the lack of consistent approaches to its measurement for decades. Lp(a) laboratory standardization emerged in 2000 (4) and was accepted by the World Health Organization in 2004 (5). Another challenge associated to its measurement is the fact that population differences can also contribute to variation in Lp(a) serum concentration (6). Since Lp(a) characterization, evidences favoring its association with cardiovascular risk have been reported. At the same time, studies against this association have also been published leading to some confusion regarding to the possible role of Lp(a) in cardiovascular disease. The last years have clarified somewhat this issue and evidences of Lp(a) as an independent cardiovascular risk factor have been proposed (7-13). Several key points such as its homology with plasminogen, differences among the apo(a) isoforms, genetic considerations as well as special circumstances such as the relationship of Lp(a) and atrial fibrillation, dialysis, alcohol consumption and blood coagulation have been investigated. In this chapter, Lp(a) metabolism, epidemiological and genetic considerations, association with coronary heart disease and stroke, special situations as well as controversies and current treatment options are related.

#### **2. Lipoprotein (a) metabolism**

Lipoprotein (a), Lp (a), is a low density lipoprotein (LDL)-like particle synthesized in the liver by hepatocytes and then secreted into plasma. It was first described by Berg in 1963 (14). It consists of an apolipoprotein B100 (apoB100) molecule that is linked covalently by a disulfide bond to a large glycoprotein known as apolipoprotein (a), [apo(a)] (15). Lp(a) metabolic route is shown in figure 1. Its molecular weight ranges from 200 kDa to more than 800 kDa (16). The apo(a) gene (*LPA*) is a major determinant of the plasma concentration of Lp(a), including variations in the kringle region-coding repeats, with accounts for the size polymorphism of apo(a) leading to different apo(a) sizes (17). This fact is very important because small size isofoms seem to be associated to worse cardiovascular profile. Apo(a) chain contains 5 cysteine-rich domains known as kringles, and especially Kringle IV (KIV) is very similar to plasminogen (18,19). This particle is not only located in the plasma but also has been shown to enter the arterial intima of humans and has an increased affinity by the

Lipoprotein (a) and Cardiovascular Risk 3

*Repeats (No.) Molecular weight (kDa)*

Respecting to apo(a) isofoms, it has been suggested a most important pathogenic role of Lp(a) particles with smaller apo(a) isoforms (18,31). This is probably due to several factors. First, an increased capacity to bind oxidized phospholipids, second, the ability to localize in blood vessel walls, and eventually related to its thrombogenic effect by increasing inhibition of plasmin activity. Apo(a) size heterogeneity is related to a copy number variation in the protein domain kringle IV type 2 (KIV2) (32) (Table 1). This copy number variation (5-50 identically repeated copies) confers heterogeneity in the molecular mass of apo(a) ranging between 200 and 800 kDa. Ethnical differences in the frequency distribution of apo(a) KIV repeated alleles have been reported (33,34). In all ethnic groups, Caucasians, Asians and African-Americans, higher levels of circulating Lp(a) concentrations tend to be associated with smaller apo(a) isoforms (35,36). This finding could explain partially the association of higher Lp(a) levels and cardiovascular disease. People with smaller apo(a) isoforms have an approximately 2-fold higher risk of coronary artery disease and ischemic stroke than those with larger apo(a) isoforms. Furthermore, isoforms with less KIV repetitions (isoforms F, B, S1 and S2) have the

greater analogy with plasminogen being associated with higher coronary risk (37,38).

Lp(a) concentration showing fewer number of KIV2 repeats (41,46), (Table 1).

Apo(a) gen (6q2.6-q2.7) (39,40) have different kringle domains that show a high degree of

Genetic variants associated with Lp(a) level have been associated with coronary disease (42). More specifically, the apo(a) gen is the major determinant of variation in some populations like African-Americans modulating the plasmatic concentration of Lp(a) (43). It has been reported that apo(a) gene accounts for greater than 90% of the variation of plasmatic Lp(a) concentrations (28). Apo(a) gen polymorphisoms as well certain gene cluster associated to *LPA* have been shown to modulate Lp(a) concentrations leading to an increase in the risk for coronary artery disease (44). The genetic basis for apo(a) isoform variation is a segment existing in multiple repeats (KIV2 polymorphism) located in the *LPA* gene (41). Variations in nucleotide polymorphisms in *LPA* may be an important contributor to the observed Lp(a) between-population variance and increase Lp(a) level in some populations (45-47). Once again, ethnical differences have been reported in people of European continental ancestry where apo(a) isoform polymorphism contributes between 40% and 70% of the variation of

>25 >700

Table 1. Relation between KIV2 repeats and apo(a) isoforms size

homology to the kringle domains IV and V of plasminogen (41).

**4. Genetic considerations** 

20-25 500-650

13-20 400-500

5-12 <400

extracellular matrix (20). This issue confers a greater opportunity to Lp(a) oxidation (21) and interaction of Lp(a) with macrophages (22,23). Recently, it has been suggested that Lp(a) could be a preferential carrier of oxidized phospholipids in human plasma (24). These oxidized Lp(a) have a greater atherosclerotic effect as compared to native Lp(a) and this action may be increased by hyperglucemia (25). Different Lp(a) subtypes have been proposed regarding to apo(a) isoforms and these apo(a) isoforms predict the risk for CHD independently of the ethnic group (26). These isoforms are classified in order to their different size (16). Table 1 shows classification of these isoforms and its relation with KIV repeats.

Fig. 1. Metabolic route of Lp(a).

#### **3. Epidemiological aspects**

Plasma levels of Lp(a) show great diversity regarding to different ethnical groups but a plasmatic concentration greater than 30 mg/dl is currently considered an independent cardiovascular risk factor (27). In this sense, African-Americans have higher Lp(a) concentrations than Caucasians. These levels may also be very different even in individuals carrying apo(a) of the same size polymorphism. It has been suggested the possibility of the presence of additional factors affecting this ethnical differences or the existence of high risk-Lp(a) or low risk-Lp(a) (28,29). By the other hand, not all ethnic groups show the same relation with Lp(a). In American-Indians, Lp(a) level has been reported to be low and non independently associated with cardiovascular disease (30).

extracellular matrix (20). This issue confers a greater opportunity to Lp(a) oxidation (21) and interaction of Lp(a) with macrophages (22,23). Recently, it has been suggested that Lp(a) could be a preferential carrier of oxidized phospholipids in human plasma (24). These oxidized Lp(a) have a greater atherosclerotic effect as compared to native Lp(a) and this action may be increased by hyperglucemia (25). Different Lp(a) subtypes have been proposed regarding to apo(a) isoforms and these apo(a) isoforms predict the risk for CHD independently of the ethnic group (26). These isoforms are classified in order to their different size (16). Table 1 shows classification of these isoforms and its relation with KIV

Plasma levels of Lp(a) show great diversity regarding to different ethnical groups but a plasmatic concentration greater than 30 mg/dl is currently considered an independent cardiovascular risk factor (27). In this sense, African-Americans have higher Lp(a) concentrations than Caucasians. These levels may also be very different even in individuals carrying apo(a) of the same size polymorphism. It has been suggested the possibility of the presence of additional factors affecting this ethnical differences or the existence of high risk-Lp(a) or low risk-Lp(a) (28,29). By the other hand, not all ethnic groups show the same relation with Lp(a). In American-Indians, Lp(a) level has been reported to be low and non

Apo(a)

B100 Lp(a)

Oxidation Receptor?

Cell

repeats.

Macrophage

Fig. 1. Metabolic route of Lp(a).

**3. Epidemiological aspects** 

LIVER

B100

LDL

B100

Lp(a) oxidated

independently associated with cardiovascular disease (30).


Table 1. Relation between KIV2 repeats and apo(a) isoforms size

Respecting to apo(a) isofoms, it has been suggested a most important pathogenic role of Lp(a) particles with smaller apo(a) isoforms (18,31). This is probably due to several factors. First, an increased capacity to bind oxidized phospholipids, second, the ability to localize in blood vessel walls, and eventually related to its thrombogenic effect by increasing inhibition of plasmin activity. Apo(a) size heterogeneity is related to a copy number variation in the protein domain kringle IV type 2 (KIV2) (32) (Table 1). This copy number variation (5-50 identically repeated copies) confers heterogeneity in the molecular mass of apo(a) ranging between 200 and 800 kDa. Ethnical differences in the frequency distribution of apo(a) KIV repeated alleles have been reported (33,34). In all ethnic groups, Caucasians, Asians and African-Americans, higher levels of circulating Lp(a) concentrations tend to be associated with smaller apo(a) isoforms (35,36). This finding could explain partially the association of higher Lp(a) levels and cardiovascular disease. People with smaller apo(a) isoforms have an approximately 2-fold higher risk of coronary artery disease and ischemic stroke than those with larger apo(a) isoforms. Furthermore, isoforms with less KIV repetitions (isoforms F, B, S1 and S2) have the greater analogy with plasminogen being associated with higher coronary risk (37,38).

#### **4. Genetic considerations**

Apo(a) gen (6q2.6-q2.7) (39,40) have different kringle domains that show a high degree of homology to the kringle domains IV and V of plasminogen (41).

Genetic variants associated with Lp(a) level have been associated with coronary disease (42). More specifically, the apo(a) gen is the major determinant of variation in some populations like African-Americans modulating the plasmatic concentration of Lp(a) (43). It has been reported that apo(a) gene accounts for greater than 90% of the variation of plasmatic Lp(a) concentrations (28). Apo(a) gen polymorphisoms as well certain gene cluster associated to *LPA* have been shown to modulate Lp(a) concentrations leading to an increase in the risk for coronary artery disease (44). The genetic basis for apo(a) isoform variation is a segment existing in multiple repeats (KIV2 polymorphism) located in the *LPA* gene (41). Variations in nucleotide polymorphisms in *LPA* may be an important contributor to the observed Lp(a) between-population variance and increase Lp(a) level in some populations (45-47). Once again, ethnical differences have been reported in people of European continental ancestry where apo(a) isoform polymorphism contributes between 40% and 70% of the variation of Lp(a) concentration showing fewer number of KIV2 repeats (41,46), (Table 1).

Lipoprotein (a) and Cardiovascular Risk 5





outcomes in dialysis patients (75).

valvular atrial fibrillation (78).

contributing to the increase in atherosclerotic risk (83).

Lp(a) to inhibit elastolysis in the vessels wall (90).

#### **5. Evidences favoring association with cardiovascular disease**


#### **6. Special situations**

There are some common medical conditions that may be influenced by the level of Lp(a). Conversely, serum Lp(a) levels can be modified by the existence of some medical disorders. These medical conditions are summarized as follows:



statistical relationship between higher level of Lp(a) and thrombotic stroke (67).

There are some common medical conditions that may be influenced by the level of Lp(a). Conversely, serum Lp(a) levels can be modified by the existence of some medical disorders.

**5. Evidences favoring association with cardiovascular disease** 

59) contributing to some degree of controversy.

These medical conditions are summarized as follows:

**6. Special situations** 


Lipoprotein (a) and Cardiovascular Risk 7

vascular disease independent from Lp(a) concentration is not completely clear at present.

The effect of the change in Lp(a) level and its relation with inflammation as well as its

It has been suggested that Lp(a) is associated with CHD only at very high concentrations but this affirmation remains somewhat controversial making very important to identify possible

Lp(a) results from the association of apo(a) and LDL particles. Since first studies linking Lp(a) and cardiovascular disease, an important amount of clinical and laboratory evidences have supported the fact that Lp(a) is and independent cardiovascular risk factor, especially

Many ethnical differences and variations in apo(a) size have been reported. Moreover, small apo(a) size isoforms have been related with an increased cardiovascular risk. Its relation with the number of KIV repeats determines genetically variation in apo(a) size. Several studies including methanalysis have related higher levels of Lp(a) with CHD and stroke.

It seems also that Lp(a) is elevated in patients under dialysis, and possibly in those with atrial fibrillation increasing the cardiovascular risk of these patients, normally already high. An interesting link between laboratory and clinical effects of Lp(a) is its action modulating the fibrinolytic system because of the great homology between Lp(a) and plasminogen.

The association between higher levels of Lp(a) and alcohol intake remains more

Current treatment options are not very useful except for niacin and plasma apheresis but

Finally, large prospective studies are needed focusing on Lp(a)-associated small apo(a) isoforms and cardiovascular disease, and also in order to ensure treatment approaches.

[1] Schriewer H, Assmann G, Sandkamp M, Schulte H. The relationship of lipoprotein (a)

[2] Schernthaner G, Kostner GM, Dieplinger H, Prager R, Mühlhauser I. Apolipoproteins (A-

[3] Murai A, Miyahara T, Fujimoto N, Matsuda M, Kameyama M. Lp(a) lipoprotein as a risk

diabetes mellitus. Atherosclerosis. 1983;49:277-293.

(Lp(a)) to risk factors of coronary heart disease: initial results of the prospective epidemiological study on company employees in Westfalia. J Clin Chem Clin

I, A-II, B), Lp(a) lipoprotein and lecithin: cholesterol acyltransferase activity in

factor for coronary heart disease and cerebral infarction. Atherosclerosis.

both therapies are not easy to use because of toxicity, tolerability and availability.

Moreover, many studies have used different cut-offs to define smaller apo(a) size.

influence on endothelial function are unknown at present.

in younger people with premature cardiovascular disease.

**9. Conclusions** 

controversial at present.

**10. References** 

Biochem. 1984;22:591-596.

1986;59:199-204.

ethnical differences as well as an adequate cut-off level we can rely on.

#### **7. Treatment**

Treatment possibilities are scarce at present when the aim is to reduce Lp(a) plasma concentration. Only niacin, in a dose dependent fashion, and certain inhibitors of cholesteryl ester transfer protein have shown limited effect ranging between 20%-40% lowering from baseline levels (91,92). Other drugs such as acetylsalicylic acid and L-carnitine can decrease mildly elevated Lp(a) concentrations (91,93,94). Contradictory findings have been reported with statins (95-98). Promising molecules like mipomersen, an antisense oligonucleotide directed to human apoB100 have been shown to reduce Lp(a) concentrations by 70% in transgenic mice (99). Similar molecules such as eprotirone, tibolone and proprotein convertase subtilisin/kexin type 9 (PCSK-9) inhibitors can also decrease Lp(a) concentrations being currently under development (91,100-102). Nevertheless, the most dramatic change in Lp(a) concentrations can be achieved with regular lipid apheresis (103,104). Table 2 shows the efficacy of different treatment options in reducing Lp(a) plasmatic level.


Table 2. Effect of different pharmacological therapies on Lp(a) serum concentration.

#### **8. Controversies**

The risk associated to Lp(a) concentration is only about one-quarter of that seen with LDL cholesterol so any clinical implication of this moderate association currently appeared limited. The role of specific Lp(a) subtypes could help to clarify the vascular risk. Particularly, smaller apo(a) isoforms could act associated with other factors such as smalldense LDL and oxidized LDL particles in the vessel wall increasing inflammation and accelerating atherosclerotic disease. This fact needs for more investigation.

Studies reporting association of apo(a) isoforms size variations with the risk of vascular disease have reported divergent relative risks, involve wide confidence intervals and the number of individuals included has been small. If smaller apo(a) isoforms are relevant to vascular disease independent from Lp(a) concentration is not completely clear at present. Moreover, many studies have used different cut-offs to define smaller apo(a) size.

The effect of the change in Lp(a) level and its relation with inflammation as well as its influence on endothelial function are unknown at present.

It has been suggested that Lp(a) is associated with CHD only at very high concentrations but this affirmation remains somewhat controversial making very important to identify possible ethnical differences as well as an adequate cut-off level we can rely on.

#### **9. Conclusions**

6 Recent Advances in Cardiovascular Risk Factors

Treatment possibilities are scarce at present when the aim is to reduce Lp(a) plasma concentration. Only niacin, in a dose dependent fashion, and certain inhibitors of cholesteryl ester transfer protein have shown limited effect ranging between 20%-40% lowering from baseline levels (91,92). Other drugs such as acetylsalicylic acid and L-carnitine can decrease mildly elevated Lp(a) concentrations (91,93,94). Contradictory findings have been reported with statins (95-98). Promising molecules like mipomersen, an antisense oligonucleotide directed to human apoB100 have been shown to reduce Lp(a) concentrations by 70% in transgenic mice (99). Similar molecules such as eprotirone, tibolone and proprotein convertase subtilisin/kexin type 9 (PCSK-9) inhibitors can also decrease Lp(a) concentrations being currently under development (91,100-102). Nevertheless, the most dramatic change in Lp(a) concentrations can be achieved with regular lipid apheresis (103,104). Table 2 shows the efficacy of different treatment options in reducing Lp(a)

> *Change in Lp(a) concentration (%)*

Apheresis 40-60

accelerating atherosclerotic disease. This fact needs for more investigation.

Table 2. Effect of different pharmacological therapies on Lp(a) serum concentration.

The risk associated to Lp(a) concentration is only about one-quarter of that seen with LDL cholesterol so any clinical implication of this moderate association currently appeared limited. The role of specific Lp(a) subtypes could help to clarify the vascular risk. Particularly, smaller apo(a) isoforms could act associated with other factors such as smalldense LDL and oxidized LDL particles in the vessel wall increasing inflammation and

Studies reporting association of apo(a) isoforms size variations with the risk of vascular disease have reported divergent relative risks, involve wide confidence intervals and the number of individuals included has been small. If smaller apo(a) isoforms are relevant to

Estrogen substitutive 15-40

Neomicine 25

Nicotinic acid 35

Statins 5

Resins 0

Diet and exercise 0

Fibrates 5-10

therapy

**8. Controversies** 

*Treatment*

**7. Treatment** 

plasmatic level.

Lp(a) results from the association of apo(a) and LDL particles. Since first studies linking Lp(a) and cardiovascular disease, an important amount of clinical and laboratory evidences have supported the fact that Lp(a) is and independent cardiovascular risk factor, especially in younger people with premature cardiovascular disease.

Many ethnical differences and variations in apo(a) size have been reported. Moreover, small apo(a) size isoforms have been related with an increased cardiovascular risk. Its relation with the number of KIV repeats determines genetically variation in apo(a) size. Several studies including methanalysis have related higher levels of Lp(a) with CHD and stroke.

It seems also that Lp(a) is elevated in patients under dialysis, and possibly in those with atrial fibrillation increasing the cardiovascular risk of these patients, normally already high.

An interesting link between laboratory and clinical effects of Lp(a) is its action modulating the fibrinolytic system because of the great homology between Lp(a) and plasminogen.

The association between higher levels of Lp(a) and alcohol intake remains more controversial at present.

Current treatment options are not very useful except for niacin and plasma apheresis but both therapies are not easy to use because of toxicity, tolerability and availability.

Finally, large prospective studies are needed focusing on Lp(a)-associated small apo(a) isoforms and cardiovascular disease, and also in order to ensure treatment approaches.

#### **10. References**


Lipoprotein (a) and Cardiovascular Risk 9

[19] Kraft HG, Lingenhel A, Pang RW, Delport R, Trommsdorff M, Vermaak H, Janus ED,

[20] Williams KJ, Flees GM, Petrie KA, Snyder ML, Brocia RW, Swenson TL. Mechanisms by

[22] Zioncheck TF, Powell LM, Rice GC, Eaton DL, Lawn RM. Interaction of recombinant

[23] Tsimikas S, Witztum JL. The role of oxidized phospholipids in mediating lipoprotein(a)

[24] Bergmark C, Dewan A, Orsoni A, Merki E, Miller ER, Shin MJ, Binder CJ, Hörkkö S,

[25] Kotani K, Yamada S, Uurtuya S, Yamada T, Taniguchi N, Sakurabayashi I. The

[26] Sandholzer C, Saha N, Kark JD, Rees A, Jaross W, Dieplinger H, Hoppichler F,

[28] Boerwinkle E, Leffert CC, Lin J, Lackner C, Chiesa G, Hobbs HH. Apolipoprotein(a)

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**2** 

*Japan* 

**Remnant Lipoproteins are a Stronger** 

**Risk Factor for Cardiovascular Events** 

**Autopsies in Sudden Cardiac Death Cases** 

*1Graduate School of Health Sciences, Gunma University, Maebashi, Gunma,* 

Plasma LDL-C level is the most well established risk factor for coronary heart disease (CHD) (1). Accordingly, the numerous studies have shown that the LDL-C lowering drugs, statins significantly reduced plasma LDL-C together with approximately 30% reduction in cardiovascular events (2). Therefore, it has been generally believed that the cardiovascular events are directly associated with the elevated LDL-C or its modified oxidized LDL (3). In this manuscript, we have reviewed the patho-physiological role of LDL-C and remnant lipoproteins at cardiovascular events in Japanese sudden cardiac death (SCD) cases (Table 1), especially in SCD cases with nearly normal coronary arteries (coronary atherosclerosis grade (-) and (±), namely Pokkuri Death Syndrome (PDS). As the formation and physiological role of LDL in liver and plasma has been well established, those of remnant lipoproteins (RLP) have also been established recently as a risk for CHD (4-6). As shown in Figure 1, TG-rich lipoprotein (TRL) remnants are formed in the circulation when apoB-48 containing chylomicrons (CM) of intestinal origin or apoB-100 containing VLDL of hepatic origin are converted by lipoprotein lipase (and to a lesser extent by hepatic lipase) into smaller and denser particles of LDL. Compared with their nascent precursors, TRL remnants are depleted of triglycerides, phospholipids, apoA-I and apoA-IV in the case of CM and are enriched in cholesteryl esters, apoCs and apoE (6). They can thus be identified, separated, or quantified in plasma on the basis of their density, charge, size, specific lipid components, apolipoprotein composition and apolipoprotein immunospecificity. This should mean that we have now two identified cardiovascular risk factors, LDL and RLP (CM remnants and VLDL remnants) (Figure 1), in SCD and PDS cases and attempted to

Recent evidences have suggested that elevated plasma levels of remnant lipoprotein – cholesterol (RLP-C) and reduced lipoprotein lipase (LPL) activity relate to the promotion of coronary artery events associated with spasm (7-9), which has been often observed as a

understand the differences in their contributions to CHD.

**1. Introduction** 

**than LDL-C – From the Studies of** 

Katsuyuki Nakajima1,2 and Masaki Q. Fujita2

*2Department of Legal Medicine (Forensic Medicine), Keio University School of Medicine, Shinjuku-ku, Tokyo,* 


### **Remnant Lipoproteins are a Stronger Risk Factor for Cardiovascular Events than LDL-C – From the Studies of Autopsies in Sudden Cardiac Death Cases**

Katsuyuki Nakajima1,2 and Masaki Q. Fujita2

*1Graduate School of Health Sciences, Gunma University, Maebashi, Gunma, 2Department of Legal Medicine (Forensic Medicine), Keio University School of Medicine, Shinjuku-ku, Tokyo, Japan* 

#### **1. Introduction**

14 Recent Advances in Cardiovascular Risk Factors

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Opin Lipidol. 2010;21:499-506.

2002;164:305-311.

Plasma LDL-C level is the most well established risk factor for coronary heart disease (CHD) (1). Accordingly, the numerous studies have shown that the LDL-C lowering drugs, statins significantly reduced plasma LDL-C together with approximately 30% reduction in cardiovascular events (2). Therefore, it has been generally believed that the cardiovascular events are directly associated with the elevated LDL-C or its modified oxidized LDL (3). In this manuscript, we have reviewed the patho-physiological role of LDL-C and remnant lipoproteins at cardiovascular events in Japanese sudden cardiac death (SCD) cases (Table 1), especially in SCD cases with nearly normal coronary arteries (coronary atherosclerosis grade (-) and (±), namely Pokkuri Death Syndrome (PDS). As the formation and physiological role of LDL in liver and plasma has been well established, those of remnant lipoproteins (RLP) have also been established recently as a risk for CHD (4-6). As shown in Figure 1, TG-rich lipoprotein (TRL) remnants are formed in the circulation when apoB-48 containing chylomicrons (CM) of intestinal origin or apoB-100 containing VLDL of hepatic origin are converted by lipoprotein lipase (and to a lesser extent by hepatic lipase) into smaller and denser particles of LDL. Compared with their nascent precursors, TRL remnants are depleted of triglycerides, phospholipids, apoA-I and apoA-IV in the case of CM and are enriched in cholesteryl esters, apoCs and apoE (6). They can thus be identified, separated, or quantified in plasma on the basis of their density, charge, size, specific lipid components, apolipoprotein composition and apolipoprotein immunospecificity. This should mean that we have now two identified cardiovascular risk factors, LDL and RLP (CM remnants and VLDL remnants) (Figure 1), in SCD and PDS cases and attempted to understand the differences in their contributions to CHD.

Recent evidences have suggested that elevated plasma levels of remnant lipoprotein – cholesterol (RLP-C) and reduced lipoprotein lipase (LPL) activity relate to the promotion of coronary artery events associated with spasm (7-9), which has been often observed as a

Remnant Lipoproteins are a Stronger Risk Factor for Cardiovascular

Events than LDL-C – From the Studies of Autopsies in Sudden Cardiac Death Cases 17

Fig. 1. Metabolic map of lipoproteins. After fat intake, the intestine secretes chylomicrons (CM), the triglycerides of which are lipolyzed by lipoprotein lipase (LPL). The LPL reaction

endothelial-bound lipoprotein lipase and VLDL remnant particles are formed. The final TRL remnant composition is modulated by the cholesterol ester transfer protein (CETP) reaction with HDL, hepatic lipase (HL), and the exchange of soluble apolipoproteins such as C-I, C-II, C-III and E. The great majority of the remnants are removed from plasma by receptormediated processes and the principal receptors are the LDL receptor and the LDL-receptorrelated protein (LRP) in liver. It is probable that the CM remnants use both of these routes,

Control (n = 76) SCD(n = 165)

(mg/dl) 177 134-209 211 175-243 < 0.0001

(mg/dl) 116 81-157 148 100-230 < 0.001 VLDL-C (mg/dl) 20.3 6.0-35.6 27.0 16.1-47.6 < 0.001 LDL-C (mg/dl) 92 67-132 134 99-167 < 0.0001 HDL-C (mg/dl) 42 31-60 41 33-53 NS RLP-C (mg/dl) 9.1 5.4-13.8 16.4 10.0-26.9 < 0.001 RLP-TG (mg/dl) 49 33-78 81 51-132 < 0.001 Table 2(a). Plasma lipid and lipoprotein levels in total cases of sudden cardiac death (SCD)

median 25-75% tile median 25-75% tile P valuea

constitutes the initial process in the formation of triglyceride-rich lipoprotein (TRL) remnants (CM remnants and VLDL remnants) . The VLDL secretion process is partly regulated by the rate of FFA influx to the liver. VLDL triglycerides are lipolyzed by

whereas the VLDL remnants are more likely to use only the LDL receptor.

Total cholesterol

and control death.

Triglyceride

major risk of sudden cardiac death (10). Likewise, we previously reported a significant association between sudden cardiac death and plasma levels of RLP-C and RLP-triglyceride (RLP-TG), especially in cases of Pokkuri death (11). Pokkuri death in Japanese refers the cases who "die suddenly and unexpectedly", and had not taken any medications prior to death. PDS has been categorized as one type of SCD syndrome, but not having coronary atherosclerosis and without cardiac hyperplasia. Most of such cases have been observed in Asian young males, and as yet, no report of PDS is seen in Caucasians.


SCD cases without atherosclerosis (Non-athero; n=48) are categorized as PDS in this manuscript.

Table 1. Demography data of sudden cardiac death and control death cases with and without coronary artery atherosclerosis

Both SCD with coronary atherosclerosis and PDS without coronary atherosclerosis showed abnormally high plasma RLP-C and RLP-TG level, namely postprandial remnant hyperlipoproteinemia in postmortem plasma (11-14) (Table 2). RLP isolated from postmortem plasma by an immunoaffinity gel separation method (15) showed atherogenic and inflammatory effects (16, 17) similar to the RLP isolated from plasma of living subjects (6). In particular, Shimokawa and colleagues (18) reported that RLP isolated from plasma of SCD cases induced strong spasm in in-vivo setting by up-regulating the Rho-kinase pathway in healthy porcine coronary arteries, which might mimic the etiological phenomenon of PDS. But LDL (or Ox-LDL) did not enhance the formation of coronary vascular lesions in regions where coronary spasm could be induced in the same experimental model (19).

Further, Takeichi et al. (11-13) suggested RLP as one of the major risk factors in SCD and PDS. Although LDL-C levels were also elevated in parallel with the majority of SCD cases who have severe coronary atherosclerosis, the role of LDL-C in fatal clinical events was not fully understood in these cases. Therefore, the relationship between plasma levels of RLP-C, RLP-TG, LDL-C and the incidence of cardiovascular events has been studied in SCD cases with and without coronary atherosclerosis as well as in control death cases (Table2). In particular, we were interested in PDS cases among SCD cases as a disease model of coronary artery events which were neither associated with the severity of coronary artery atherosclerosis nor plaque ruptures (20).

major risk of sudden cardiac death (10). Likewise, we previously reported a significant association between sudden cardiac death and plasma levels of RLP-C and RLP-triglyceride (RLP-TG), especially in cases of Pokkuri death (11). Pokkuri death in Japanese refers the cases who "die suddenly and unexpectedly", and had not taken any medications prior to death. PDS has been categorized as one type of SCD syndrome, but not having coronary atherosclerosis and without cardiac hyperplasia. Most of such cases have been observed in

> Control (n=76) SCD (n=165) mean±SD mean±SD

> > (n=48)

Athero

(n=117) P value

(n=27) P value Non-athero

Age in years 42.7±16.5 51.3±14.5 < 0.01 37.5±13.1 54.5±10.5 < 0.0001 Male/Female 41/8 24/3 NS 43/5 90/27 NS Heart weight (g) 358±86 387±80 NS 356±82 414±91 < 0.001 Body weight (kg) 61.7±11.7 62.9±9.5 NS 65.2±11.9 63.9±12.7 NS Body height (cm) 165±8.2 164±9.1 NS 168±9.6 164±8.5 NS BMI 22.6±4.8 23.5±3.3 NS 22.9±3.5 23.8±3.7 NS

(h) 8.7±2.9 8.6±3.5 NS 8.9±3.3 8.2±3.4 NS

SCD cases without atherosclerosis (Non-athero; n=48) are categorized as PDS in this manuscript. Table 1. Demography data of sudden cardiac death and control death cases with and

Both SCD with coronary atherosclerosis and PDS without coronary atherosclerosis showed abnormally high plasma RLP-C and RLP-TG level, namely postprandial remnant hyperlipoproteinemia in postmortem plasma (11-14) (Table 2). RLP isolated from postmortem plasma by an immunoaffinity gel separation method (15) showed atherogenic and inflammatory effects (16, 17) similar to the RLP isolated from plasma of living subjects (6). In particular, Shimokawa and colleagues (18) reported that RLP isolated from plasma of SCD cases induced strong spasm in in-vivo setting by up-regulating the Rho-kinase pathway in healthy porcine coronary arteries, which might mimic the etiological phenomenon of PDS. But LDL (or Ox-LDL) did not enhance the formation of coronary vascular lesions in regions where coronary spasm could be induced in the same

Further, Takeichi et al. (11-13) suggested RLP as one of the major risk factors in SCD and PDS. Although LDL-C levels were also elevated in parallel with the majority of SCD cases who have severe coronary atherosclerosis, the role of LDL-C in fatal clinical events was not fully understood in these cases. Therefore, the relationship between plasma levels of RLP-C, RLP-TG, LDL-C and the incidence of cardiovascular events has been studied in SCD cases with and without coronary atherosclerosis as well as in control death cases (Table2). In particular, we were interested in PDS cases among SCD cases as a disease model of coronary artery events which were neither associated with the severity of coronary artery

Asian young males, and as yet, no report of PDS is seen in Caucasians.

Athero

Non-athero (n=49)

Postmortem period

experimental model (19).

without coronary artery atherosclerosis

atherosclerosis nor plaque ruptures (20).

Fig. 1. Metabolic map of lipoproteins. After fat intake, the intestine secretes chylomicrons (CM), the triglycerides of which are lipolyzed by lipoprotein lipase (LPL). The LPL reaction constitutes the initial process in the formation of triglyceride-rich lipoprotein (TRL) remnants (CM remnants and VLDL remnants) . The VLDL secretion process is partly regulated by the rate of FFA influx to the liver. VLDL triglycerides are lipolyzed by endothelial-bound lipoprotein lipase and VLDL remnant particles are formed. The final TRL remnant composition is modulated by the cholesterol ester transfer protein (CETP) reaction with HDL, hepatic lipase (HL), and the exchange of soluble apolipoproteins such as C-I, C-II, C-III and E. The great majority of the remnants are removed from plasma by receptormediated processes and the principal receptors are the LDL receptor and the LDL-receptorrelated protein (LRP) in liver. It is probable that the CM remnants use both of these routes, whereas the VLDL remnants are more likely to use only the LDL receptor.


Table 2(a). Plasma lipid and lipoprotein levels in total cases of sudden cardiac death (SCD) and control death.

Remnant Lipoproteins are a Stronger Risk Factor for Cardiovascular

Sensitivity (%)

Cut-off (mg/dl)

(1) Total (SCD : Control)

 (2) Non-athero (Pokkuri: Control)

 (3) Athero (SCD : Control)

control (n=27).

cardiac death

**and cardiovascular events** 

Events than LDL-C – From the Studies of Autopsies in Sudden Cardiac Death Cases 19

RLP-C 12.8 67.9 68.2 84.4 45.7 2.12 RLP-TG 53.3 72.9 60.8 82.5 46.9 1.86 LDL-C 92.5 79.4 52.7 81.0 50.2 1.68 Total Cholesterol 181 69.3 49.3 77.6 38.7 1.37 Triglycerides 117 66.3 49.3 76.8 36.6 1.31

RLP-C 10.1 70.2 77.5 75.4 72.7 3.13 RLP-TG 67 66.7 75.6 72.8 62.9 2.73 LDL-C 106 58.3 61.7 59.9 60.2 1.52 Total Cholesterol 165 63.8 51.1 56.1 59.0 1.30 Triglycerides 113 53.3 50.0 51.1 52.2 1.07

RLP-C 13.4 65.3 69.2 90.2 31.5 2.12 RLP-TG 79.4 52.5 72.4 89.2 26.0 1.90 LDL-C 102 79.7 57.1 89.0 39.4 1.86 Total Cholesterol 217 55.2 78.6 91.8 28.8 2.58 Triglycerides 131 64.9 64.3 88.7 29.7 1.82 Number of cases are as follows: (1) total SCD; SCD (n=165) and Control (n=76) : (2) Non-athero; Pokkuri (Non-athero SCD) (n=48) and Non-athero control (n=49): (3) Athero; Athero SCD (n=117) and Athero-

Table 3. Cut-off value of RLP-C and RLP-TG from ROC analysis in predicting sudden

**2. Role of plasma LDL and remnant lipoproteins at coronary atherosclerosis** 

Based on our autopsy studies, more than two thirds of SCD cases were found to be associated with postprandial remnant hyperlipoproteinemia [11-15]. If severe spasm of the coronary artery is to be a crucial event prior to cardiac death in PDS, we may say that the vasospasm is not very likely to occur in coronary arteries with severe coronary artery atherosclerotic lesions due to reduced elasticity and increased stiffness or hardness of the vascular wall. Caucasians experience more severe coronary atherosclerosis than Japanese or other Southeastern Asians. Accordingly, this might be one explanation why PDS is uncommon among Caucasians. In view of this background, PDS could be an interesting disease case to study coronary heart disease (CHD), which is independent of severity of coronary atherosclerosis and plaque ruptures in spite of remnant hyperlipoproteinemia. Significantly younger age of PDS cases compared to the other SCD cases may be one of the reasons why PDS cases were not associated with severe coronary atherosclerosis. The

PPV Positive predictive value

NPV Negative predictive value

Likelihood ratio

Specificity (%)


Table 2(b). Plasma lipid and lipoprotein levels in cases of Pokkuri death and control death (without coronary artery atherosclerosis).


Table 2(c). Plasma lipid and lipoprotein levels in cases of sudden cardiac death (SCD) and control death( with coronary artery atherosclerosis).

We have established the cut-off values and the likelihood ratios of total cholesterol (TC), TG, RLP-C, RLP-TG and LDL-C in plasma of SCD and control death cases with and without coronary atherosclerosis after adjusting for the postmortem conditions (Table 3) (21). The cut-off values and likelihood ratios of these major plasma lipids and lipoproteins were calculated by using an ROC analysis model for predicting the risk of fatal clinical events with and without coronary atherosclerosis. In approximately two-third of sudden cardiac death cases, we found postprandial remnant hyperlipoproteinemia, especially rich in very low density lipoprotein (VLDL) remnants (14) and significantly elevated LDL-C. Both lipoproteins were significantly elevated in SCD, but LDL-C in PDS cases was within the normal range, together with significantly elevated remnant lipoproteins in fatal clinical events. Therefore, we found that PDS cases were a good disease model to distinguish the role of LDL and RLP as cardiovascular risk factors.

(mg/dl) 165 121-205 182 149-221 < 0.01

(mg/dl) 114 75-129 120 96-216 < 0.005 VLDL-C (mg/dl) 20.3 5.1-35.2 27.0 16.2-43.7 NS LDL-C (mg/dl) 89 67-130 118 83-140 < 0.001 HDL-C (mg/dl) 39 30-59 41 34-56 NS RLP-C (mg/dl) 7.1 5.1-10.1 14.3 9.5-26.4 < 0.001 RLP-TG (mg/dl) 48 33-67 78 50-117 < 0.001 Table 2(b). Plasma lipid and lipoprotein levels in cases of Pokkuri death and control death

Total cholesterol

(without coronary artery atherosclerosis).

control death( with coronary artery atherosclerosis).

role of LDL and RLP as cardiovascular risk factors.

Triglyceride

Total cholesterol

Control (n = 49) Pokkuri (n = 48)

Control (n = 27) SCD(n = 117)

(mg/dl) 203 171-216 219 185-248 < 0.05 Triglyceride (mg/dl) 120 82-188 154 106-232 < 0.05 VLDL-C (mg/dl) 21.5 7.0-39.0 26.9 15.8-49.4 NS LDL-C (mg/dl) 95 69-136 148 103-179 < 0.01 HDL-C (mg/dl) 45 34-63 41 33-51 NS RLP-C (mg/dl) 12.5 10.1-18.5 17.2 11.4-46.8 < 0.05 RLP-TG (mg/dl) 55 38-84 83 51-142 < 0.05 Table 2(c). Plasma lipid and lipoprotein levels in cases of sudden cardiac death (SCD) and

We have established the cut-off values and the likelihood ratios of total cholesterol (TC), TG, RLP-C, RLP-TG and LDL-C in plasma of SCD and control death cases with and without coronary atherosclerosis after adjusting for the postmortem conditions (Table 3) (21). The cut-off values and likelihood ratios of these major plasma lipids and lipoproteins were calculated by using an ROC analysis model for predicting the risk of fatal clinical events with and without coronary atherosclerosis. In approximately two-third of sudden cardiac death cases, we found postprandial remnant hyperlipoproteinemia, especially rich in very low density lipoprotein (VLDL) remnants (14) and significantly elevated LDL-C. Both lipoproteins were significantly elevated in SCD, but LDL-C in PDS cases was within the normal range, together with significantly elevated remnant lipoproteins in fatal clinical events. Therefore, we found that PDS cases were a good disease model to distinguish the

median 25-75% tile median 25-75% tile P valuea

median 25-75% tile median 25-75% tile P valuea


Number of cases are as follows: (1) total SCD; SCD (n=165) and Control (n=76) : (2) Non-athero; Pokkuri (Non-athero SCD) (n=48) and Non-athero control (n=49): (3) Athero; Athero SCD (n=117) and Atherocontrol (n=27).

Table 3. Cut-off value of RLP-C and RLP-TG from ROC analysis in predicting sudden cardiac death

#### **2. Role of plasma LDL and remnant lipoproteins at coronary atherosclerosis and cardiovascular events**

Based on our autopsy studies, more than two thirds of SCD cases were found to be associated with postprandial remnant hyperlipoproteinemia [11-15]. If severe spasm of the coronary artery is to be a crucial event prior to cardiac death in PDS, we may say that the vasospasm is not very likely to occur in coronary arteries with severe coronary artery atherosclerotic lesions due to reduced elasticity and increased stiffness or hardness of the vascular wall. Caucasians experience more severe coronary atherosclerosis than Japanese or other Southeastern Asians. Accordingly, this might be one explanation why PDS is uncommon among Caucasians. In view of this background, PDS could be an interesting disease case to study coronary heart disease (CHD), which is independent of severity of coronary atherosclerosis and plaque ruptures in spite of remnant hyperlipoproteinemia. Significantly younger age of PDS cases compared to the other SCD cases may be one of the reasons why PDS cases were not associated with severe coronary atherosclerosis. The

Remnant Lipoproteins are a Stronger Risk Factor for Cardiovascular

because of it low concentration in plasma.

**death** 

Events than LDL-C – From the Studies of Autopsies in Sudden Cardiac Death Cases 21

be an appropriate diagnostic marker for predicting cardiovascular events but not the severity of coronary atherosclerosis, whereas RLP-C could be a marker for predicting both cardiovascular events and the severity of coronary atherosclerosis. LDL-C could be a marker for predicting the severity of coronary atherosclerosis, but not cardiovascular events. Elevated Oxidized LDL seems to be associated with the presence of vulnerable plaque at blood vessels (6), not a causative factor for the formation or initiation of atherosclerosis

**3. Postprandial remnant hyperlipoproteinemia as a risk for sudden cardiac** 

Several clinical studies have shown that elevated plasma TG levels greatly increase the risk of sudden cardiac death. Results from the Paris Prospective Study (26) and The Apolipoprotein Related Mortality Risk Study (AMORIS) in Sweden (27) demonstrated that increased TG was a strong risk factor for fatal myocardial infarction. However, plasma TG levels can alter very easily within a short time. Therefore it has been difficult to identify the clinical events of elevated TG in the long term prospective studies until recently (28-30).

If the lipid and lipoprotein levels in postmortem plasma correctly reflected the antemortem levels, these data could probably provide the same values with the results obtained from the prospective studies, which require long-term observation for evaluation. The plasma levels of lipids and lipoproteins in sudden death cases may reflect the feature at the moment of fatal clinical events followed by certain inevitable postmortem alterations, but still may reflect the physiological conditions when the cardiac events had occurred. Therefore, we analyzed postmortem plasma under well-controlled conditions to clarify the cause of sudden cardiac death. Plasma RLP-C and RLP-TG levels vary greatly within a short time as the TG levels, compared with other stable plasma markers such as HDL-C and LDL-C. Hence, we believe that the cross-sectional study of RLPs at the moment of sudden death is a superior analytical method than a prospective study of RLP (31) to identify potential risks of CHD. During the investigations of sudden death cases, we found that the postmortem alterations of lipids and lipoproteins in plasma were unexpectedly slight (21) compared with proteins or other bio-markers. Moreover, these plasma lipoprotein levels were very

similar to those determined in living patients from the studies in our laboratory.

plasma of SCD cases compared with the control death cases.

Remnant lipoproteins are known to increase postprandially as chylomicron (CM) remnants, but very low density lipoprotein (VLDL) remnants also increase at the same time. The remarkable close correlation between the increment in the concentration of TG-rich lipoprotein (TRL) apoB-48 (CM) and apoB-100 (VLDL) after a fat meal indicates that reduced efficiency of CM particle clearance is closely coupled to the accumulation of VLDL particles as proposed by Karpe et al (32). Delayed clearance of CM particles, as evidently occurs in many hypertriglyceridemic states, may thus contribute to the elevation of apoB-100 in TRL. More than two thirds of the SCD including PDS case observed in this study showed stomach full, indicating the strong association with postprandial remnant hyperlipoproteinemia. Significant remnant hyperlipoproteinemia was observed in the

The postprandial increase of apoB-48-carrying CM and CM remnants after fat load is known to correlate well with the increase of RLP-C and RLP-TG (33). These data suggested the

prevalence of severe coronary atherosclerosis is known to be strongly associated with age (Table1). We found that plasma lipid (TC, TG) and lipoprotein (LDL-C, RLP-C, and RLP-TG) levels were significantly elevated in these sudden cardiac death cases as compared with those in control death cases when coronary atherosclerosis was pathologically graded above (1+), reflecting the clinical feature of severe coronary atherosclerosis (11-13). Most of the coronary arteries in PDS cases were pathologically graded as () and (±), indicating no coronary atherosclerosis [11]. Plasma LDL-C in SCD cases was shown to be correlated with the severity of coronary artery atherosclerosis [13]. This is in line with the perception (albeit by implication) that LDL-C plays a major role in the progression of coronary atherosclerosis in CHD patients. We found that the incidence of elevated plasma LDL-C was significantly greater in SCD cases with coronary atherosclerosis compared with than in controls and PDS cases. However, plasma LDL-C levels were all within normal range in PDS cases (22). Hence, LDL-C did not seem to play a significant role at cardiovascular events in PDS, despite being elevated within normal range , rather the data strongly indicated an association between plasma LDL-C and the progression of coronary atherosclerosis in SCD cases.

Elevated plasma remnant lipoproteins (RLP) levels were the most striking observation in PDS (RLP-C likelihood ratio; 3.13, RLP-TG; 2.73, LDL-C; 1.52, TC; 1.30, TG; 1.07) for predicting sudden cardiac death (Table 3). Despite the high plasma concentration of RLPs in PDS cases, the progression of atherosclerosis at coronary arteries was not observed. It might be valid to say that increased plasma RLPs may initiate the vascular endothelial damage and this is followed by an influx of large amounts of LDL into the vascular wall. Then it follows to form an advanced atherosclerotic lesion with macrophages and smooth muscle cells as Nakajima et al reviewed previously (6, 23). PDS cases may be in the early stage of atherosclerosis, which can lead cardiovascular events under certain conditions such as with severe stress without strong morphological changes. Therefore, the authors proposed that the occurrence of cardiovascular events at coronary arteries and the severity of atherosclerotic lesions in CHD should be considered as separate factors. Therefore, the intervention should be targeted to suppress the cardiovascular events more aggressively than to slow down the progression of atherosclerosis. Takeichi and Fujita did not observe frequent plaque ruptures in coronary arteries at autopsy in Japanese SCD cases [24].

The literature on atherosclerosis has long been dominated by data in Caucasian patients who in most cases had severe atherosclerosis at the time of fatal clinical events. Hence, fatal clinical events have been believed to occur in relation to the severity of atherosclerosis in coronary arteries. In contrast, fatal clinical events of PDS cases had occurred in the absence of coronary atherosclerosis or plaque rupture. Plasma LDL-C levels were also within normal range associated with no coronary atherosclerosis in PDS cases. This again puts more weight on RLP as the causative factor of cardiovascular events. Interestingly, we found that RLP-TG (TG concentration in remnant lipoproteins) was not an indicator for predicting the presence or progression of coronary atherosclerosis even in SCD [22]; however, it was significantly associated with fatal clinical events in SCD including PDS (Table 2). The bioactive components co-localized with triglycerides in RLP such as oxidized phospholipids or their metabolites [25] may enhance the formation of coronary vascular lesions and may induce severe spasm in coronary arteries. These results also suggested that triglycerides in RLP were not associated with the progression of atherosclerotic plaques, but cholesterol in RLP was strongly associated with the severity of atherosclerosis [13, 22]. Therefore RLP-TG could

prevalence of severe coronary atherosclerosis is known to be strongly associated with age (Table1). We found that plasma lipid (TC, TG) and lipoprotein (LDL-C, RLP-C, and RLP-TG) levels were significantly elevated in these sudden cardiac death cases as compared with those in control death cases when coronary atherosclerosis was pathologically graded above (1+), reflecting the clinical feature of severe coronary atherosclerosis (11-13). Most of the coronary arteries in PDS cases were pathologically graded as () and (±), indicating no coronary atherosclerosis [11]. Plasma LDL-C in SCD cases was shown to be correlated with the severity of coronary artery atherosclerosis [13]. This is in line with the perception (albeit by implication) that LDL-C plays a major role in the progression of coronary atherosclerosis in CHD patients. We found that the incidence of elevated plasma LDL-C was significantly greater in SCD cases with coronary atherosclerosis compared with than in controls and PDS cases. However, plasma LDL-C levels were all within normal range in PDS cases (22). Hence, LDL-C did not seem to play a significant role at cardiovascular events in PDS, despite being elevated within normal range , rather the data strongly indicated an association between plasma LDL-C and the progression of coronary atherosclerosis in SCD

Elevated plasma remnant lipoproteins (RLP) levels were the most striking observation in PDS (RLP-C likelihood ratio; 3.13, RLP-TG; 2.73, LDL-C; 1.52, TC; 1.30, TG; 1.07) for predicting sudden cardiac death (Table 3). Despite the high plasma concentration of RLPs in PDS cases, the progression of atherosclerosis at coronary arteries was not observed. It might be valid to say that increased plasma RLPs may initiate the vascular endothelial damage and this is followed by an influx of large amounts of LDL into the vascular wall. Then it follows to form an advanced atherosclerotic lesion with macrophages and smooth muscle cells as Nakajima et al reviewed previously (6, 23). PDS cases may be in the early stage of atherosclerosis, which can lead cardiovascular events under certain conditions such as with severe stress without strong morphological changes. Therefore, the authors proposed that the occurrence of cardiovascular events at coronary arteries and the severity of atherosclerotic lesions in CHD should be considered as separate factors. Therefore, the intervention should be targeted to suppress the cardiovascular events more aggressively than to slow down the progression of atherosclerosis. Takeichi and Fujita did not observe

frequent plaque ruptures in coronary arteries at autopsy in Japanese SCD cases [24].

The literature on atherosclerosis has long been dominated by data in Caucasian patients who in most cases had severe atherosclerosis at the time of fatal clinical events. Hence, fatal clinical events have been believed to occur in relation to the severity of atherosclerosis in coronary arteries. In contrast, fatal clinical events of PDS cases had occurred in the absence of coronary atherosclerosis or plaque rupture. Plasma LDL-C levels were also within normal range associated with no coronary atherosclerosis in PDS cases. This again puts more weight on RLP as the causative factor of cardiovascular events. Interestingly, we found that RLP-TG (TG concentration in remnant lipoproteins) was not an indicator for predicting the presence or progression of coronary atherosclerosis even in SCD [22]; however, it was significantly associated with fatal clinical events in SCD including PDS (Table 2). The bioactive components co-localized with triglycerides in RLP such as oxidized phospholipids or their metabolites [25] may enhance the formation of coronary vascular lesions and may induce severe spasm in coronary arteries. These results also suggested that triglycerides in RLP were not associated with the progression of atherosclerotic plaques, but cholesterol in RLP was strongly associated with the severity of atherosclerosis [13, 22]. Therefore RLP-TG could

cases.

be an appropriate diagnostic marker for predicting cardiovascular events but not the severity of coronary atherosclerosis, whereas RLP-C could be a marker for predicting both cardiovascular events and the severity of coronary atherosclerosis. LDL-C could be a marker for predicting the severity of coronary atherosclerosis, but not cardiovascular events. Elevated Oxidized LDL seems to be associated with the presence of vulnerable plaque at blood vessels (6), not a causative factor for the formation or initiation of atherosclerosis because of it low concentration in plasma.

#### **3. Postprandial remnant hyperlipoproteinemia as a risk for sudden cardiac death**

Several clinical studies have shown that elevated plasma TG levels greatly increase the risk of sudden cardiac death. Results from the Paris Prospective Study (26) and The Apolipoprotein Related Mortality Risk Study (AMORIS) in Sweden (27) demonstrated that increased TG was a strong risk factor for fatal myocardial infarction. However, plasma TG levels can alter very easily within a short time. Therefore it has been difficult to identify the clinical events of elevated TG in the long term prospective studies until recently (28-30).

If the lipid and lipoprotein levels in postmortem plasma correctly reflected the antemortem levels, these data could probably provide the same values with the results obtained from the prospective studies, which require long-term observation for evaluation. The plasma levels of lipids and lipoproteins in sudden death cases may reflect the feature at the moment of fatal clinical events followed by certain inevitable postmortem alterations, but still may reflect the physiological conditions when the cardiac events had occurred. Therefore, we analyzed postmortem plasma under well-controlled conditions to clarify the cause of sudden cardiac death. Plasma RLP-C and RLP-TG levels vary greatly within a short time as the TG levels, compared with other stable plasma markers such as HDL-C and LDL-C. Hence, we believe that the cross-sectional study of RLPs at the moment of sudden death is a superior analytical method than a prospective study of RLP (31) to identify potential risks of CHD. During the investigations of sudden death cases, we found that the postmortem alterations of lipids and lipoproteins in plasma were unexpectedly slight (21) compared with proteins or other bio-markers. Moreover, these plasma lipoprotein levels were very similar to those determined in living patients from the studies in our laboratory.

Remnant lipoproteins are known to increase postprandially as chylomicron (CM) remnants, but very low density lipoprotein (VLDL) remnants also increase at the same time. The remarkable close correlation between the increment in the concentration of TG-rich lipoprotein (TRL) apoB-48 (CM) and apoB-100 (VLDL) after a fat meal indicates that reduced efficiency of CM particle clearance is closely coupled to the accumulation of VLDL particles as proposed by Karpe et al (32). Delayed clearance of CM particles, as evidently occurs in many hypertriglyceridemic states, may thus contribute to the elevation of apoB-100 in TRL. More than two thirds of the SCD including PDS case observed in this study showed stomach full, indicating the strong association with postprandial remnant hyperlipoproteinemia. Significant remnant hyperlipoproteinemia was observed in the plasma of SCD cases compared with the control death cases.

The postprandial increase of apoB-48-carrying CM and CM remnants after fat load is known to correlate well with the increase of RLP-C and RLP-TG (33). These data suggested the

Remnant Lipoproteins are a Stronger Risk Factor for Cardiovascular

daily food intake as remnant lipoproteins.

precursor of sdLDL (59).

plasma levels of remnant lipoproteins as well as LDL-C.

HepG2, in which NK-104 enabled to induce LDL receptor.

the removal of RLP and LDL simultaneously.

Events than LDL-C – From the Studies of Autopsies in Sudden Cardiac Death Cases 23

lipoproteins are the atherogenic lipoproteins which appear and increase postprandially into plasma at the initial step of lipoprotein metabolism after food intake and then change to further metabolized lipoproteins, such as LDL particles (Figure 1). The postprandial state with increased remnant lipoproteins in plasma continues almost whole day except in the early morning (6), while not the case in LDL. Therefore, if postprandial lipoproteins are atherogenic risks as Zilversmit proposed (53), those should be the primary therapeutic target to prevent cardiovascular disease. Increased LDL is not directly associated with the

Possible mechanisms suggested for abnormal accumulation of lipoproteins postprandially in plasma are defective clearance in liver via receptor-mediated pathways and/or increased competition for high-affinity processes because of increased numbers of intestinally and hepatically derived particles in the postprandial state [32]. HMG-CoA reductase inhibitors decrease cellular cholesterol synthesis and consequently reduce the hepatic production of very-low-density lipoproteins (VLDL) and increase the expression of LDL-receptors in liver [54]. These properties of statins suggest that they are potential agents for regulating the

Atorvastatin is an HMG-CoA reductase inhibitor found to be effective in lowering fasting LDL-C and triglyceride levels [55]. Favorable effects of atorvastatin on postprandial lipoprotein metabolism have been reported in healthy normolipidemic human subjects [56- 58]. Recently, atorvastatin and rozuvastatin are reported to decrease small dense LDL-C significantly, which is highly correlated with remnant lipoproteins in plasma, possibly as a

We investigated whether RLP bound to LDL receptor more efficiently than LDL itself via apoE-ligand which is rich in RLP (6). RLP competed more efficiently with ,-VLDL than LDL in LDL receptor transfected cells (to be published in Clin Chim Acta 2012 by Takahashi et al). These results suggested that RLP which is mainly apoE-rich VLDL more efficiently binds and internalizes into LDL receptor transfected cells than LDL. Similar results were observed in VLDL receptor transfected cells, although VLDL receptor is not present in liver (60). Takahashi et al found that pitabastatin (NK-104) induced VLDL receptor in skeletal muscle cells with significantly higher concentration (more than 10 folds) compared to

In FH of a LDL receptor deficiency, statins have a dual mechanism of action involving an increase in the catabolism of LDL via up-regulation of LDL-receptors and a decrease in the hepatic secretion of apolipoprotein (apo) B-100. The net effect is a decrease in the concentration of apoB-containing lipoproteins. As CM remnants are also apo E-rich and mainly cleared via the LDL-receptor [61, 62], an increase in receptor activity and reduced competition from apoB-100-containing lipoproteins was hypothesized to increase the removal rate of remnant lipoproteins from circulation. A recent study investigating the effects of high-dose, long-term statin treatment on the metabolism of postprandial lipoproteins in heterozygous FH, reported a decrease in the fasting and postprandial RLP-C as well as LDL-C [63]. Statins can induce LDL receptor in heterozygous FH which enhance

However, it has not been known which, RLP or LDL, is removed earlier or primarily from plasma by increased LDL receptor with statin treatment. Takahashi et al suggested the

possibility that increased RLP in the postprandial state may be mainly composed of CM remnants. However, unexpectedly, we found no significant differences of apoB-48 levels in plasma or RLP apoB-48, but found significant differences of RLP aoB-100 levels between SCD and control death cases (14). As previously reported by Schneeman et al. (34), postprandial responses (after fat load) of apoB-48 and apoB-100 were highly correlated with those of TRL triglycerides. Although the increase in apoB-48 represented a 3.5-fold difference in concentration as compared with a 1.6-fold increase in apoB-100, apoB-100 accounted for about 80% of the increase in lipoprotein particles in TRL. Our results on plasma evaluation in SCD cases were very similar to the results reported by Schneeman et al (34). RLP apoB-100 levels were significantly elevated in SCD cases in the postprandial state (when RLP-C and RLP-TG were significantly elevated), however, plasma apoB-48 or RLP apoB-48 was not significantly elevated (14). These results strongly suggested that the major subset of RLP associated with fatal clinical events was apoB-100 carrying particles, but not apoB-48 particles.

The absolute amount of apoB-100 in RLP is much greater than that of apoB-48 in RLP. Hence, VLDL remnants, endogenous lipoprotein remnants, generated in the liver, may be more closely associated with the risk of sudden cardiac death than exogenous CM remnants, irrespective of the severity of coronary atherosclerosis. Furthermore, we often found SCD cases that had consumed alcohol on a full stomach. It is known that alcohol increases fatty acids in the liver and enhances VLDL production, and inhibit LPL activity (35). Alcohol intake with a fatty meal is known to greatly enhance TG increase in the postprandial state. The intake of alcohol together with a fatty meal may easily enhance the production of apoB-100 carrying VLDL in the liver, and increase VLDL remnants by inhibiting LPL activity and increase the potential risk of coronary artery in SCD cases.

#### **4. Comparative reactivity of LDL and remnant lipoproteins to LDL receptor in liver**

Clinical trials have shown that improvements in plasma LDL-C levels are associated with retardation of atherosclerosis and reduction in coronary artery morbidity and mortality [2, 36]. The major mechanism of statin therapeutic effect has been recognized as the increase of LDL receptor in liver to remove an elevated LDL-C in plasma. However, remnant lipoproteins have been also implicated in progression of atherosclerosis [37-40], with elevated remnant lipoprotein levels shown to independently predict clinical events in coronary artery disease (CAD) patients (4]. The direct comparison of reactivity between remnant lipoproteins and LDL to LDL receptor in liver has not been studied enough until recently.

Major target for remnant lipoprotein research has been focused on postprandial dyslipidemia. Postprandial dyslipidemia has been found to be associated with endothelial dysfunction [42, 43] an early indicator of atherogenesis. Previous studies have shown that normolipidemic patients with coronary disease have elevated postprandial levels of triglyceride-rich lipoproteins (TRLs) and their remnants compared with healthy control subjects [44-49]. Elevated remnant lipoprotein levels have also been associated with coronary endothelial dysfunction [50], with remnants shown to stimulate expression of proatherothrombotic molecules in endothelial cells [51]. Therefore, the prevention and treatment of atherosclerosis merits pharmacotherapy targeted at regulating postprandial dyslipidemia, namely remnant lipoproteins beyond LDL-C [52]. Postprandial remnant

possibility that increased RLP in the postprandial state may be mainly composed of CM remnants. However, unexpectedly, we found no significant differences of apoB-48 levels in plasma or RLP apoB-48, but found significant differences of RLP aoB-100 levels between SCD and control death cases (14). As previously reported by Schneeman et al. (34), postprandial responses (after fat load) of apoB-48 and apoB-100 were highly correlated with those of TRL triglycerides. Although the increase in apoB-48 represented a 3.5-fold difference in concentration as compared with a 1.6-fold increase in apoB-100, apoB-100 accounted for about 80% of the increase in lipoprotein particles in TRL. Our results on plasma evaluation in SCD cases were very similar to the results reported by Schneeman et al (34). RLP apoB-100 levels were significantly elevated in SCD cases in the postprandial state (when RLP-C and RLP-TG were significantly elevated), however, plasma apoB-48 or RLP apoB-48 was not significantly elevated (14). These results strongly suggested that the major subset of RLP associated with fatal clinical events was apoB-100 carrying particles, but not

The absolute amount of apoB-100 in RLP is much greater than that of apoB-48 in RLP. Hence, VLDL remnants, endogenous lipoprotein remnants, generated in the liver, may be more closely associated with the risk of sudden cardiac death than exogenous CM remnants, irrespective of the severity of coronary atherosclerosis. Furthermore, we often found SCD cases that had consumed alcohol on a full stomach. It is known that alcohol increases fatty acids in the liver and enhances VLDL production, and inhibit LPL activity (35). Alcohol intake with a fatty meal is known to greatly enhance TG increase in the postprandial state. The intake of alcohol together with a fatty meal may easily enhance the production of apoB-100 carrying VLDL in the liver, and increase VLDL remnants by inhibiting LPL activity and

**4. Comparative reactivity of LDL and remnant lipoproteins to LDL receptor in** 

Clinical trials have shown that improvements in plasma LDL-C levels are associated with retardation of atherosclerosis and reduction in coronary artery morbidity and mortality [2, 36]. The major mechanism of statin therapeutic effect has been recognized as the increase of LDL receptor in liver to remove an elevated LDL-C in plasma. However, remnant lipoproteins have been also implicated in progression of atherosclerosis [37-40], with elevated remnant lipoprotein levels shown to independently predict clinical events in coronary artery disease (CAD) patients (4]. The direct comparison of reactivity between remnant lipoproteins and LDL

Major target for remnant lipoprotein research has been focused on postprandial dyslipidemia. Postprandial dyslipidemia has been found to be associated with endothelial dysfunction [42, 43] an early indicator of atherogenesis. Previous studies have shown that normolipidemic patients with coronary disease have elevated postprandial levels of triglyceride-rich lipoproteins (TRLs) and their remnants compared with healthy control subjects [44-49]. Elevated remnant lipoprotein levels have also been associated with coronary endothelial dysfunction [50], with remnants shown to stimulate expression of proatherothrombotic molecules in endothelial cells [51]. Therefore, the prevention and treatment of atherosclerosis merits pharmacotherapy targeted at regulating postprandial dyslipidemia, namely remnant lipoproteins beyond LDL-C [52]. Postprandial remnant

increase the potential risk of coronary artery in SCD cases.

to LDL receptor in liver has not been studied enough until recently.

apoB-48 particles.

**liver** 

lipoproteins are the atherogenic lipoproteins which appear and increase postprandially into plasma at the initial step of lipoprotein metabolism after food intake and then change to further metabolized lipoproteins, such as LDL particles (Figure 1). The postprandial state with increased remnant lipoproteins in plasma continues almost whole day except in the early morning (6), while not the case in LDL. Therefore, if postprandial lipoproteins are atherogenic risks as Zilversmit proposed (53), those should be the primary therapeutic target to prevent cardiovascular disease. Increased LDL is not directly associated with the daily food intake as remnant lipoproteins.

Possible mechanisms suggested for abnormal accumulation of lipoproteins postprandially in plasma are defective clearance in liver via receptor-mediated pathways and/or increased competition for high-affinity processes because of increased numbers of intestinally and hepatically derived particles in the postprandial state [32]. HMG-CoA reductase inhibitors decrease cellular cholesterol synthesis and consequently reduce the hepatic production of very-low-density lipoproteins (VLDL) and increase the expression of LDL-receptors in liver [54]. These properties of statins suggest that they are potential agents for regulating the plasma levels of remnant lipoproteins as well as LDL-C.

Atorvastatin is an HMG-CoA reductase inhibitor found to be effective in lowering fasting LDL-C and triglyceride levels [55]. Favorable effects of atorvastatin on postprandial lipoprotein metabolism have been reported in healthy normolipidemic human subjects [56- 58]. Recently, atorvastatin and rozuvastatin are reported to decrease small dense LDL-C significantly, which is highly correlated with remnant lipoproteins in plasma, possibly as a precursor of sdLDL (59).

We investigated whether RLP bound to LDL receptor more efficiently than LDL itself via apoE-ligand which is rich in RLP (6). RLP competed more efficiently with ,-VLDL than LDL in LDL receptor transfected cells (to be published in Clin Chim Acta 2012 by Takahashi et al). These results suggested that RLP which is mainly apoE-rich VLDL more efficiently binds and internalizes into LDL receptor transfected cells than LDL. Similar results were observed in VLDL receptor transfected cells, although VLDL receptor is not present in liver (60). Takahashi et al found that pitabastatin (NK-104) induced VLDL receptor in skeletal muscle cells with significantly higher concentration (more than 10 folds) compared to HepG2, in which NK-104 enabled to induce LDL receptor.

In FH of a LDL receptor deficiency, statins have a dual mechanism of action involving an increase in the catabolism of LDL via up-regulation of LDL-receptors and a decrease in the hepatic secretion of apolipoprotein (apo) B-100. The net effect is a decrease in the concentration of apoB-containing lipoproteins. As CM remnants are also apo E-rich and mainly cleared via the LDL-receptor [61, 62], an increase in receptor activity and reduced competition from apoB-100-containing lipoproteins was hypothesized to increase the removal rate of remnant lipoproteins from circulation. A recent study investigating the effects of high-dose, long-term statin treatment on the metabolism of postprandial lipoproteins in heterozygous FH, reported a decrease in the fasting and postprandial RLP-C as well as LDL-C [63]. Statins can induce LDL receptor in heterozygous FH which enhance the removal of RLP and LDL simultaneously.

However, it has not been known which, RLP or LDL, is removed earlier or primarily from plasma by increased LDL receptor with statin treatment. Takahashi et al suggested the

Remnant Lipoproteins are a Stronger Risk Factor for Cardiovascular

plaques.

Events than LDL-C – From the Studies of Autopsies in Sudden Cardiac Death Cases 25

Fig. 2. Effect of RLP and Ox-LDL on athegenesis. The endothelial cell dysfunction is initiated by RLP in plasma followed by the induction of LOX-1 receptor and the associated pathway of various cytokines and enzymes. Ox-LDL promotes the progression of atherosclerosis in subendothelial space after a large efflux of LDL from plasma and form atherosclerotic

end of a 4 week treatment with oral administration of alpha-tocopherol acetate (300 IU/day). Alpha-tocopherol improved the impairment of endothelium-dependent vasodilation in patients with high RLP-C, but not in patients with low RLP-C. Similarly, RLP and their extracted lipids impaired endothelium-dependent vasorelaxation (EDR) of isolated rabbit aorta at the same concentration of serum RLP-C as found in patients with coronary artery disease (70). In contrast, non-RLP in the VLDL fraction had no effect on EDR. This in vitro study further showed that co-incubation of N-acetylcysteine and reduced glutathione (GSH), antioxidants, that were added to incubation mixture in isolated rabbit aorta containing RLP, almost completely reversed the impaired EDR, suggesting that reactive oxygen species contained in RLP or those generated by RLP played a significant role in the impairment of EDR. Further, Doi et al (51) showed that RLP isolated from patients undergoing treatment with alpha-tocopherol lost their inhibitory action on vasorelaxation of isolated rabbit aorta in response to Ach, whereas RLP from patients receiving placebo had inhibitory action on vasorelaxation. These results suggested that high RLP-C level being oxidized in plasma increased the oxidative stress and contribute to endothelial vasomotor dysfunction in patients with high plasma concentration of RLP-C. Ohara et al (17) reported that remnant lipoproteins isolated from SCD cases suppressed nitric oxide (NO) synthetase activity and attenuate endothelium-dependent vasorelaxation.

possibility that remnant lipoproteins are removed more primarily from plasma by statins and prevent cardiovascular disease, while LDL are more likely reduced as a consequence of reduction of the precursor lipoproteins (to be published in Clin Chim Acta 2012 by Takahashi et al).

Moreover, VLDL receptor which does not affect the removal of remnant lipoproteins in liver may affect on rhabdomyolysis in skeletal muscle cells, in case when VLDL receptors are significantly induced by statins in those cells. When plasma concentration of statins increased abnormally high, VLDL receptor could be induced in the skeletal muscle cells. Then, RLP binds and internalizes into skeletal muscle cells with significantly increased concentration and may cause the rhabdomyolysis in skeletal muscle cells by the cytotoxic effect of remnant lipoproteins (6, 64, 65).

The direct comparison between LDL and RLP has shown that RLP with its apoE-rich ligand has superior binding and internalization reactivity to LDL receptor than LDL in liver, which is a similar reactivity with VLDL receptor. These results suggest that RLP may be more primarily and efficiently metabolized in liver than LDL through increased LDL receptor when treated with statins.

#### **5. Possible molecular mechanism of remnant lipoproteins associated with coronary artery vasospasm**

Followings are the hypothesis of molecular mechanism on the initiation and progression of atherosclerosis associated with fatal cardiovascular events we have proposed from our studies on sudden cardiac death during last two decades (Figure 2).

Elevated plasma RLP first cause the initiation of vascular dysfunction at endothelial cell and smooth muscle cells through LOX-1 receptor and activate Rho-kinase pathway in vascular smooth muscle cells to induce coronary artery spasm as vascular smooth muscle hyperconstriction. However, LDL has no such biological properties to initiate the vascular dysfunction. Although Ox-LDL, derived from LDL modified, has very similar biological properties with remnant lipoproteins, the plasma concentration of Ox-LDL is significantly low and can not influence to the following phenomenon like remnant lipoproteins shown by in vitro studies (6).

#### **5.1 Remnant lipoproteins and impaired endothelialium-dependent vasorelaxation**

Endothelial activation or dysfunction is known to be an early event in the development of atherosclerosis which is not necessarily associated with strong morphological changes. Kugiyama et al (66) and Inoue et al (67) first found that plasma RLP-C levels, but not LDL-C levels, showed significant and independent correlation with impaired endothelial function reflected as impaired endothelium-dependent vasomotor function (vasorelaxation) in large and resistance coronary arteries in humans. These observations indicated the possibility that high plasma concentration of remnant lipoproteins impair endothelial cell function in human coronary arteries.

Flow-mediated vasodilation (FMD) of the brachial artery during reactive hyperemia has been used as a noninvasive method to assess endothelial function. Kugiyama et al (68) and Funada et al (69) examined FMD by high resolution ultrasound technique before and at the

possibility that remnant lipoproteins are removed more primarily from plasma by statins and prevent cardiovascular disease, while LDL are more likely reduced as a consequence of reduction of the precursor lipoproteins (to be published in Clin Chim Acta 2012 by

Moreover, VLDL receptor which does not affect the removal of remnant lipoproteins in liver may affect on rhabdomyolysis in skeletal muscle cells, in case when VLDL receptors are significantly induced by statins in those cells. When plasma concentration of statins increased abnormally high, VLDL receptor could be induced in the skeletal muscle cells. Then, RLP binds and internalizes into skeletal muscle cells with significantly increased concentration and may cause the rhabdomyolysis in skeletal muscle cells by the cytotoxic

The direct comparison between LDL and RLP has shown that RLP with its apoE-rich ligand has superior binding and internalization reactivity to LDL receptor than LDL in liver, which is a similar reactivity with VLDL receptor. These results suggest that RLP may be more primarily and efficiently metabolized in liver than LDL through increased LDL receptor

**5. Possible molecular mechanism of remnant lipoproteins associated with** 

studies on sudden cardiac death during last two decades (Figure 2).

Followings are the hypothesis of molecular mechanism on the initiation and progression of atherosclerosis associated with fatal cardiovascular events we have proposed from our

Elevated plasma RLP first cause the initiation of vascular dysfunction at endothelial cell and smooth muscle cells through LOX-1 receptor and activate Rho-kinase pathway in vascular smooth muscle cells to induce coronary artery spasm as vascular smooth muscle hyperconstriction. However, LDL has no such biological properties to initiate the vascular dysfunction. Although Ox-LDL, derived from LDL modified, has very similar biological properties with remnant lipoproteins, the plasma concentration of Ox-LDL is significantly low and can not influence to the following phenomenon like remnant lipoproteins shown by

**5.1 Remnant lipoproteins and impaired endothelialium-dependent vasorelaxation** 

Endothelial activation or dysfunction is known to be an early event in the development of atherosclerosis which is not necessarily associated with strong morphological changes. Kugiyama et al (66) and Inoue et al (67) first found that plasma RLP-C levels, but not LDL-C levels, showed significant and independent correlation with impaired endothelial function reflected as impaired endothelium-dependent vasomotor function (vasorelaxation) in large and resistance coronary arteries in humans. These observations indicated the possibility that high plasma concentration of remnant lipoproteins impair endothelial cell function in

Flow-mediated vasodilation (FMD) of the brachial artery during reactive hyperemia has been used as a noninvasive method to assess endothelial function. Kugiyama et al (68) and Funada et al (69) examined FMD by high resolution ultrasound technique before and at the

Takahashi et al).

effect of remnant lipoproteins (6, 64, 65).

when treated with statins.

in vitro studies (6).

human coronary arteries.

**coronary artery vasospasm** 

Fig. 2. Effect of RLP and Ox-LDL on athegenesis. The endothelial cell dysfunction is initiated by RLP in plasma followed by the induction of LOX-1 receptor and the associated pathway of various cytokines and enzymes. Ox-LDL promotes the progression of atherosclerosis in subendothelial space after a large efflux of LDL from plasma and form atherosclerotic plaques.

end of a 4 week treatment with oral administration of alpha-tocopherol acetate (300 IU/day). Alpha-tocopherol improved the impairment of endothelium-dependent vasodilation in patients with high RLP-C, but not in patients with low RLP-C. Similarly, RLP and their extracted lipids impaired endothelium-dependent vasorelaxation (EDR) of isolated rabbit aorta at the same concentration of serum RLP-C as found in patients with coronary artery disease (70). In contrast, non-RLP in the VLDL fraction had no effect on EDR. This in vitro study further showed that co-incubation of N-acetylcysteine and reduced glutathione (GSH), antioxidants, that were added to incubation mixture in isolated rabbit aorta containing RLP, almost completely reversed the impaired EDR, suggesting that reactive oxygen species contained in RLP or those generated by RLP played a significant role in the impairment of EDR. Further, Doi et al (51) showed that RLP isolated from patients undergoing treatment with alpha-tocopherol lost their inhibitory action on vasorelaxation of isolated rabbit aorta in response to Ach, whereas RLP from patients receiving placebo had inhibitory action on vasorelaxation. These results suggested that high RLP-C level being oxidized in plasma increased the oxidative stress and contribute to endothelial vasomotor dysfunction in patients with high plasma concentration of RLP-C. Ohara et al (17) reported that remnant lipoproteins isolated from SCD cases suppressed nitric oxide (NO) synthetase activity and attenuate endothelium-dependent vasorelaxation.

Remnant Lipoproteins are a Stronger Risk Factor for Cardiovascular

**A** 

**B** 

Events than LDL-C – From the Studies of Autopsies in Sudden Cardiac Death Cases 27

Fig. 3. RLPs, but not nascent VLDL (n-VLDL), induce LOX-1 expression in BVSMCs. (A ) After BVSMCs were treated with the indicated concentrations of RLPs for 16 h, total cell lysates were subjected to immunoblotting for LOX-1. TNF-+ served as a positive control. (B) After treatment with 25/g/ml of RLPs for the indicated time periods, total cellular RNA was subjected to Northern blot analyses. Bands for 28S and 18S ribosomal RNAs were visualized by ethidium bromide staining to control the amount of RNA loaded. (\*) *p* < 0.001

vs. 0g/ml of RLPs, (#) *p* < 0.05 vs. 0 h incubation (cited from Ref 83).

Probucol is known to inhibit the oxidative modification of LDL (71), lowering serum cholesterol levels. Ox-LDL has been shown to impair endothelium-dependent vasorelaxation and antioxidants, including probucol, suppressing the impaired EDR (72) as RLP described above.

#### **5.2 Both Ox-LDL and remnant lipoproteins activate LOX-1 receptor in endothelial cells**

A scavenger receptor independent pathway for acetyl LDL and oxidized LDL in cultured endothelial cells, has long been known; however, it has been difficult to isolate. Recently, Sawamura and his colleagues (73-76) discovered and characterized lectin-like oxidized LDL receptor-1 (LOX-1) as a vascular endothelial receptor for Ox-LDL. Endothelial dysfunction or activation invoked by oxidatively modified LDL has been implicated in the pathogenesis of atherosclerosis by enhanced intimal thickening and lipid deposition in the arteries. Ox-LDL and its lipid constituents, mainly composed of oxidized products of phospholipids such as lysophosphatidylcholine, impair endothelial production of NO, and induce the endothelial expression of leukocyte adhesion molecules and smooth muscle growth factors, which can contribute to atherogenesis via LOX-1 receptor. Vascular endothelial cells in culture and in vivo internalize and degrade Ox-LDL through a putative receptor-mediated pathway that does not involve macrophage scavenger receptor. The treatment of HUVECs with RLP increased LOX-1 expression in a dose dependent manner (Figure 3) and was completely inhibited by LOX-1- antisense, but not by LOX-1-sence. Monoclonal antibody to LOX-1 reported by Shin et al (77) and antisence LOX-1 oligodeoxynucleotide reported by Park et al (78) significantly reduced RLP-mediated production of superoxide (NADPH oxidase dependent), TNF-alpha, and interleukin-beta, NF-.B activation, DNA fragmentation (cell death: apoptosis). Further Shin et al (77) have emphasized the importance of RLP in increasing the expression of LOX-1 receptor protein in NADPH oxidase dependent superoxide production; the expression of adhesion molecules such as ICAM-1, VCAM -1 and MCP-1 stimulated by RLP is dependent on the activation of LOX-1 receptors. These findings strongly suggest that LOX-1 may play the role of a receptor of RLP as well as Ox-LDL in endothelial cells. Endothelial cell injury caused by RLP via LOX-1 receptor activation evidently can initiate atherosclerosis. Cilostazol, a platelet aggregation inhibitor and vasodilator (79), is known to reduce plasma RLP-C levels in patients with peripheral artery disease (80) and has showed significant protective effect against RLP-induced endothelial dysfunction by suppressing these variables both in-vitro and in-vivo with its antioxidative activity (81).

#### **5.3 Remnant lipoproteins activate LOX-1 receptor in smooth muscle cell**

Coronary vasospasm has been considered to occur at vascular smooth muscle cells (VSMCs) and the migration of VSMCs from media to intima and subsequent proliferation play key roles in atherogenesis. A previous report has demonstrated that RLPs induce VSMC proliferation [82]; however, receptors for RLPs in VSMCs have not yet been well characterized until recently reported by Aramaki et al (83), although LRP in the liver, apoB-48-R in macrophages, and VLDL receptor in heart, skeletal muscle, adipose tissue, brain and macrophages [84, 85] have been shown to act as a receptor for RLPs. LOX-1 expression is dynamically inducible by various proatherogenic stimuli, including tumor necrosis factor- +(TNF-+), heparin-binding epidermal growth factor-like growth factor (HB-EGF), and Ox-

Probucol is known to inhibit the oxidative modification of LDL (71), lowering serum cholesterol levels. Ox-LDL has been shown to impair endothelium-dependent vasorelaxation and antioxidants, including probucol, suppressing the impaired EDR (72) as

**5.2 Both Ox-LDL and remnant lipoproteins activate LOX-1 receptor in endothelial cells**  A scavenger receptor independent pathway for acetyl LDL and oxidized LDL in cultured endothelial cells, has long been known; however, it has been difficult to isolate. Recently, Sawamura and his colleagues (73-76) discovered and characterized lectin-like oxidized LDL receptor-1 (LOX-1) as a vascular endothelial receptor for Ox-LDL. Endothelial dysfunction or activation invoked by oxidatively modified LDL has been implicated in the pathogenesis of atherosclerosis by enhanced intimal thickening and lipid deposition in the arteries. Ox-LDL and its lipid constituents, mainly composed of oxidized products of phospholipids such as lysophosphatidylcholine, impair endothelial production of NO, and induce the endothelial expression of leukocyte adhesion molecules and smooth muscle growth factors, which can contribute to atherogenesis via LOX-1 receptor. Vascular endothelial cells in culture and in vivo internalize and degrade Ox-LDL through a putative receptor-mediated pathway that does not involve macrophage scavenger receptor. The treatment of HUVECs with RLP increased LOX-1 expression in a dose dependent manner (Figure 3) and was completely inhibited by LOX-1- antisense, but not by LOX-1-sence. Monoclonal antibody to LOX-1 reported by Shin et al (77) and antisence LOX-1 oligodeoxynucleotide reported by Park et al (78) significantly reduced RLP-mediated production of superoxide (NADPH oxidase dependent), TNF-alpha, and interleukin-beta, NF-.B activation, DNA fragmentation (cell death: apoptosis). Further Shin et al (77) have emphasized the importance of RLP in increasing the expression of LOX-1 receptor protein in NADPH oxidase dependent superoxide production; the expression of adhesion molecules such as ICAM-1, VCAM -1 and MCP-1 stimulated by RLP is dependent on the activation of LOX-1 receptors. These findings strongly suggest that LOX-1 may play the role of a receptor of RLP as well as Ox-LDL in endothelial cells. Endothelial cell injury caused by RLP via LOX-1 receptor activation evidently can initiate atherosclerosis. Cilostazol, a platelet aggregation inhibitor and vasodilator (79), is known to reduce plasma RLP-C levels in patients with peripheral artery disease (80) and has showed significant protective effect against RLP-induced endothelial dysfunction by suppressing these variables both in-vitro and in-vivo with its antioxidative

**5.3 Remnant lipoproteins activate LOX-1 receptor in smooth muscle cell** 

Coronary vasospasm has been considered to occur at vascular smooth muscle cells (VSMCs) and the migration of VSMCs from media to intima and subsequent proliferation play key roles in atherogenesis. A previous report has demonstrated that RLPs induce VSMC proliferation [82]; however, receptors for RLPs in VSMCs have not yet been well characterized until recently reported by Aramaki et al (83), although LRP in the liver, apoB-48-R in macrophages, and VLDL receptor in heart, skeletal muscle, adipose tissue, brain and macrophages [84, 85] have been shown to act as a receptor for RLPs. LOX-1 expression is dynamically inducible by various proatherogenic stimuli, including tumor necrosis factor- +(TNF-+), heparin-binding epidermal growth factor-like growth factor (HB-EGF), and Ox-

RLP described above.

activity (81).

Fig. 3. RLPs, but not nascent VLDL (n-VLDL), induce LOX-1 expression in BVSMCs. (A ) After BVSMCs were treated with the indicated concentrations of RLPs for 16 h, total cell lysates were subjected to immunoblotting for LOX-1. TNF-+ served as a positive control. (B) After treatment with 25/g/ml of RLPs for the indicated time periods, total cellular RNA was subjected to Northern blot analyses. Bands for 28S and 18S ribosomal RNAs were visualized by ethidium bromide staining to control the amount of RNA loaded. (\*) *p* < 0.001 vs. 0g/ml of RLPs, (#) *p* < 0.05 vs. 0 h incubation (cited from Ref 83).

Remnant Lipoproteins are a Stronger Risk Factor for Cardiovascular

spasm in pigs.

Events than LDL-C – From the Studies of Autopsies in Sudden Cardiac Death Cases 29

equivalent concentration of plasma RLP) isolated from the plasma of SCD cases. After 1 week, intracoronary serotonin caused hyperconstriction in the segment treated with RLP but not in the non-RLP VLDL treated segment (Figure 4). Likewise, RLP treated with hydroxyfasudil, a selective Rho-kinase inhibitor, dose dependently inhibited the coronary

Fig. 4. RLP ( RLP in VLDL fraction; RLP-VLDL) from patients with SCD markedly enhance

intracoronary serotonin (B). Black arrows indicate RLP site at coronary artery; white arrows, non-RLP site. RLP induced significant hyperconstriction at treated coronary site after 1 week, while hydroxyfasudil completely inhibited serotonin (5HT)-induced coronary hyperconstriction at RLP site (cited from Ref 18). These results were explained by the induction of Rho-kinase + and Rho-kinase ,, of which mRNA expression was enhanced by

It has been recently reported that sphingosine 1-phosphate (S1P) and sphingosylphosphorylcholine, present in serum lipoproteins, behave as a lipid mediator and cause vasoconstriction through upregulation of Rho/Rho-kinase pathway (89). The possible role of S1P and sphingosylphosphorylcholine in the RLP fraction remains to be elucidated. These results suggested the importance of intervention to suppress the cardiovascular events more aggressively by such as inhibiting Rho-kinase activation than to slow down the

Sudden and unexplained cardiac death has been known for many years in Southeast Asian countries, including Japan. These deaths were named differently in each country such as Pokkuri Death Syndrome in Japan, "Lai Tai" in Thailand, "Bangungut " in the Philippines, "Dream Disease" in Hawaii, and "Sudden Unexpected Nocturnal Death Syndrome" among South Asian immigrants in the USA. However, the clinical and pathological features of these

coronary vasospastic activity in pigs. Coronary angiograms before (A) and after

the treatment with RLP but not that with non-RLP.

progression of atherosclerosis.

**6. Conclusion remarks** 

LDL. Furthermore, LOX-1 is highly expressed by macrophages and VSMCs accumulate in the intima of advanced atherosclerotic lesions, as well as endothelial cells covering early atherosclerotic lesions in vivo, indicating that LOX-1 appears to play important roles at various stages of atherogenesis.. Aramaki et al (83) recently provided direct evidence, by cDNA and short interference RNAs (siRNAs) transfection, that LOX-1 acts as a receptor for RLP (Figure 1) and whereby induce VSMC migration, depending upon HB-EGF shedding and the downstream signal transduction cascades. The direct evidences that LOX-1 serves as a receptor for RLPs in vascular smooth muscle cells (VSMCs) were shown by use of two cell lines which stably express human or bovine LOX-1 and siRNA directed to LOX-1. In addition, involvement of metalloproteinase activation, HB-EGF shedding, EGFR transactivation, and activation of ERK, p38 MAPK and PI3K were also observed in RLP induced migration of BVSMCs. Competition studies in cells stably expressing LOX-1 indicated binding site(s) on the LOX-1 molecule for RLPs and oxidized LDL appear to be identical or overlapped, suggesting the C-terminal cysteine-rich C-type lectin-like domain was shown to be the responsible binding site(s) for RLPs [86]. These studies suggested the importance of LOX-1 in RLP-induced atherogenesis, as well as that induced by oxidized LDL. RLPs induced cell migration and LOX-1 expression by RLP-LOX-1 interactions, thus making a positive-feed back loop to further enhance the RLP-induced vascular dysfunction, as already showed in oxidized LDL-induced vascular dysfunction. In accordance with a previous report [77, 78], RLP-induced LOX-1 expression and cell migration depend upon HB-EGF shedding and subsequent EGFR transactivation demonstrated. Furthermore, the involvement of ERK, p38 MAPK and Akt as signal transducrion cascades located downstream to the EGFR transactivation were shown. JNK was not activated by RLPs or not involved in RLP-induced LOX-1 expression or cell migration (84).

These results suggested that RLP induced LOX-1 expression and enhance the activation of smooth muscle cells.

#### **5.4 Remnant lipoproteins activate Rho-kinase in smooth muscle cells and induce vasospasm**

Coronary vasospasm has been postulated to play an important role in SCD, although a direct demonstration for the hypothesis is still lacking. Shimokawa and his colleagues demonstrated the close relation between RLP and coronary vasospasm that is mediated by upregulated Rhokinase pathway (18). The expression and the activity of Rho-kinase are enhanced at the inflammatory coronary lesions in the porcine model with interleukin-1 (19, 88).

RLP isolated from the plasma of SCD cases exert a potent upregulating effect on Rho-kinase in hcVSMC (18). In organ chamber experiments, serotonin caused hyperconstriction of vascular smooth muscle cells (VSMC) from RLP-treated segment, which was significantly inhibited by hydoxyfasudil (a selective Rho-kinase inhibitor). In cultured human coronary VSMC, the treatment with RLP significantly enhanced the expression and the activity of Rho-kinase. These results indicated that RLP isolated from the plasma of sudden cardiac death cases upregulated Rho-kinase in coronary VSMC (promoted inflammation) and markedly enhanced coronary vasospasmic activity.

Further, Oi et al (18) performed in vivo study on the formation of coronary vascular lesion by RLP, using healthy pigs in which they treated pig coronary arteries with RLP (an equivalent concentration of plasma RLP) isolated from the plasma of SCD cases. After 1 week, intracoronary serotonin caused hyperconstriction in the segment treated with RLP but not in the non-RLP VLDL treated segment (Figure 4). Likewise, RLP treated with hydroxyfasudil, a selective Rho-kinase inhibitor, dose dependently inhibited the coronary spasm in pigs.

Fig. 4. RLP ( RLP in VLDL fraction; RLP-VLDL) from patients with SCD markedly enhance coronary vasospastic activity in pigs. Coronary angiograms before (A) and after intracoronary serotonin (B). Black arrows indicate RLP site at coronary artery; white arrows, non-RLP site. RLP induced significant hyperconstriction at treated coronary site after 1 week, while hydroxyfasudil completely inhibited serotonin (5HT)-induced coronary hyperconstriction at RLP site (cited from Ref 18). These results were explained by the induction of Rho-kinase + and Rho-kinase ,, of which mRNA expression was enhanced by the treatment with RLP but not that with non-RLP.

It has been recently reported that sphingosine 1-phosphate (S1P) and sphingosylphosphorylcholine, present in serum lipoproteins, behave as a lipid mediator and cause vasoconstriction through upregulation of Rho/Rho-kinase pathway (89). The possible role of S1P and sphingosylphosphorylcholine in the RLP fraction remains to be elucidated.

These results suggested the importance of intervention to suppress the cardiovascular events more aggressively by such as inhibiting Rho-kinase activation than to slow down the progression of atherosclerosis.

#### **6. Conclusion remarks**

28 Recent Advances in Cardiovascular Risk Factors

LDL. Furthermore, LOX-1 is highly expressed by macrophages and VSMCs accumulate in the intima of advanced atherosclerotic lesions, as well as endothelial cells covering early atherosclerotic lesions in vivo, indicating that LOX-1 appears to play important roles at various stages of atherogenesis.. Aramaki et al (83) recently provided direct evidence, by cDNA and short interference RNAs (siRNAs) transfection, that LOX-1 acts as a receptor for RLP (Figure 1) and whereby induce VSMC migration, depending upon HB-EGF shedding and the downstream signal transduction cascades. The direct evidences that LOX-1 serves as a receptor for RLPs in vascular smooth muscle cells (VSMCs) were shown by use of two cell lines which stably express human or bovine LOX-1 and siRNA directed to LOX-1. In addition, involvement of metalloproteinase activation, HB-EGF shedding, EGFR transactivation, and activation of ERK, p38 MAPK and PI3K were also observed in RLP induced migration of BVSMCs. Competition studies in cells stably expressing LOX-1 indicated binding site(s) on the LOX-1 molecule for RLPs and oxidized LDL appear to be identical or overlapped, suggesting the C-terminal cysteine-rich C-type lectin-like domain was shown to be the responsible binding site(s) for RLPs [86]. These studies suggested the importance of LOX-1 in RLP-induced atherogenesis, as well as that induced by oxidized LDL. RLPs induced cell migration and LOX-1 expression by RLP-LOX-1 interactions, thus making a positive-feed back loop to further enhance the RLP-induced vascular dysfunction, as already showed in oxidized LDL-induced vascular dysfunction. In accordance with a previous report [77, 78], RLP-induced LOX-1 expression and cell migration depend upon HB-EGF shedding and subsequent EGFR transactivation demonstrated. Furthermore, the involvement of ERK, p38 MAPK and Akt as signal transducrion cascades located downstream to the EGFR transactivation were shown. JNK was not activated by RLPs or not

involved in RLP-induced LOX-1 expression or cell migration (84).

smooth muscle cells.

**vasospasm** 

These results suggested that RLP induced LOX-1 expression and enhance the activation of

Coronary vasospasm has been postulated to play an important role in SCD, although a direct demonstration for the hypothesis is still lacking. Shimokawa and his colleagues demonstrated the close relation between RLP and coronary vasospasm that is mediated by upregulated Rhokinase pathway (18). The expression and the activity of Rho-kinase are enhanced at the

RLP isolated from the plasma of SCD cases exert a potent upregulating effect on Rho-kinase in hcVSMC (18). In organ chamber experiments, serotonin caused hyperconstriction of vascular smooth muscle cells (VSMC) from RLP-treated segment, which was significantly inhibited by hydoxyfasudil (a selective Rho-kinase inhibitor). In cultured human coronary VSMC, the treatment with RLP significantly enhanced the expression and the activity of Rho-kinase. These results indicated that RLP isolated from the plasma of sudden cardiac death cases upregulated Rho-kinase in coronary VSMC (promoted inflammation) and

Further, Oi et al (18) performed in vivo study on the formation of coronary vascular lesion by RLP, using healthy pigs in which they treated pig coronary arteries with RLP (an

**5.4 Remnant lipoproteins activate Rho-kinase in smooth muscle cells and induce** 

inflammatory coronary lesions in the porcine model with interleukin-1 (19, 88).

markedly enhanced coronary vasospasmic activity.

Sudden and unexplained cardiac death has been known for many years in Southeast Asian countries, including Japan. These deaths were named differently in each country such as Pokkuri Death Syndrome in Japan, "Lai Tai" in Thailand, "Bangungut " in the Philippines, "Dream Disease" in Hawaii, and "Sudden Unexpected Nocturnal Death Syndrome" among South Asian immigrants in the USA. However, the clinical and pathological features of these

Remnant Lipoproteins are a Stronger Risk Factor for Cardiovascular

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mediated formation of coronary vascular lesions in pigs in vivo. Arterioscler

surrogate markers of antemortem level in cases of sudden coronary death. *Clin* 

Particles or LDL-C as Major Pathologic Factors in Sudden Cardiac Death Cases.

sudden death cases are surprisingly similar with no coronary atherosclerosis and mainly occur among young males during sleep in the midnight, together with an excessive food and alcohol intake.

We have proposed a hypothesis that could explain a possible cause of PDS based on the postprandial increase of remnant lipoproteins in plasma and narrowed circumferences of coronary arteries in PDS cases. The elevated plasma RLP initiates the vascular dysfunction at endothelial cells in narrowed coronary arteries as an early event in the development of atherosclerosis and induces severe coronary spasm under stress or genetic disorder, possibly for example, through activating LOX-1 receptor and Rho-kinase pathway, at smooth muscle cells to cause cardiac arrest. LDL or low concentration of Ox-LDL could not explain these phenomena as RLP. Taken together, we have proposed that the severity of coronary atherosclerosis and the occurrence of cardiovascular events in CHD cases could be considered as separate factors, judging from the physiological role of LDL and RLP in plasma. Therefore, the intervention should be more targeted to suppress the plasma remnant lipoproteins to prevent cardiovascular events more aggressively rather than to slow down the progression of atherosclerosis by LDL.

#### **7. Acknowledgment**

This work is supported in part by Grant-in-Aid from Japan Society for the Promotion of Science (JSPS), No. 20406020. The authors deeply thank Dr. Richard Havel, University of California San Francisco and Dr. Ernest Schaefer, Tufts University for their long term collaboration on remnant lipoprotein research. Also we greatly thank Dr. Sanae Takeichi, Director of Takeichi Medical Research Laboratory (former professor of Tokai University School of Medicine, Department of Forensic Medicine) for her extensive research collaboration on sudden cardiac death.

#### **8. References**


sudden death cases are surprisingly similar with no coronary atherosclerosis and mainly occur among young males during sleep in the midnight, together with an excessive food

We have proposed a hypothesis that could explain a possible cause of PDS based on the postprandial increase of remnant lipoproteins in plasma and narrowed circumferences of coronary arteries in PDS cases. The elevated plasma RLP initiates the vascular dysfunction at endothelial cells in narrowed coronary arteries as an early event in the development of atherosclerosis and induces severe coronary spasm under stress or genetic disorder, possibly for example, through activating LOX-1 receptor and Rho-kinase pathway, at smooth muscle cells to cause cardiac arrest. LDL or low concentration of Ox-LDL could not explain these phenomena as RLP. Taken together, we have proposed that the severity of coronary atherosclerosis and the occurrence of cardiovascular events in CHD cases could be considered as separate factors, judging from the physiological role of LDL and RLP in plasma. Therefore, the intervention should be more targeted to suppress the plasma remnant lipoproteins to prevent cardiovascular events more aggressively rather than to slow down the progression of

This work is supported in part by Grant-in-Aid from Japan Society for the Promotion of Science (JSPS), No. 20406020. The authors deeply thank Dr. Richard Havel, University of California San Francisco and Dr. Ernest Schaefer, Tufts University for their long term collaboration on remnant lipoprotein research. Also we greatly thank Dr. Sanae Takeichi, Director of Takeichi Medical Research Laboratory (former professor of Tokai University School of Medicine, Department of Forensic Medicine) for her extensive research

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**3** 

*Italy* 

*University of Udine,* 

**Cardiovascular Risk Factors** 

Anna Rossetto, Umberto Baccarani and Vittorio Bresadola

In the last decades, the survival of liver transplanted patients and grafts have had a great improvement due to many factors. Careful preoperative evaluation in transplant recipients, experience and a multidisciplinary approach have, without any doubt, a major role in the selection of candidates and in the diagnosis and treatment of preoperative complications. Moreover, with the introduction of new generation immunosuppressive drugs and careful pharmacological monitoring, both the episodes of acute rejection and toxic effects have been

In consequence, since the graft and the patient post-transplant survival have been improved,

Besides the risk correlated with graft rejection, the transplanted population has an increased risk of developing many malignancies. A number of hematologic diseases, skin cancer, gastrointestinal tumors seems to recognize a possible trigger factor in immunosuppressive drugs and the patient's immunological status but also in serological status and viral infections quite common in immunosuppressed patients. As for frequency, after these two groups of complications, cardiovascular diseases are the third cause of death in the

Eligibility to liver transplant, once based on Child Pugh system, has been regulated, since a few years, by MELD score, an index of survival probability of the end stage liver disease (2). This score is obtained on three variables, INR, creatinine and bilirubine. Due to the well known inadequate number of available grafts, if compared to patients requiring liver transplantation and to the risk of mortality while in the waiting list, this score supplies a priority system, based on the severity of the disease, for the organ allocation. The cut off value normally considered for eligibility to liver transplantation is a MELD score 15. Since

10{0.957Ln(Scr)+0.378Ln(Tbil)+1.12 Ln (INR) +0.643} eligibility to transplantation undergoes the parameter of severity of the disease, selecting the population of patients with generally worse clinical conditions. Moreover, the median age of the patients waiting for transplantation seems to be higher, essentially due to the improvement in the antiviral therapies and the better medical treatment in the hepatological

the evaluation of the MELD score is obtained with this mathematical formula:

the transplanted population has started showing long term medical complications.

**1. Introduction** 

minimized (1)

transplanted population (1)

**and Liver Transplantation** 


### **Cardiovascular Risk Factors and Liver Transplantation**

Anna Rossetto, Umberto Baccarani and Vittorio Bresadola *University of Udine, Italy* 

#### **1. Introduction**

36 Recent Advances in Cardiovascular Risk Factors

[87] Shimokawa H, Ito A, Fukumoto Y, et al. Chronic treatment with interleukin-1 beta

[88] Tosaka M, Okajima F, Hashiba Y,et al. Sphingosine 1-phosphate contracts canine basilar

of platelet-derived growth factor. J Clin Invest. 1996; 97:769–776.

*Stroke.* 2001; 32: 2913–19.

induces coronary intimal lesions and vasospastic responses in pigs in vivo. The role

arteries in vitro and in vivo: possible role in pathogenesis of cerebral vasospasm.

In the last decades, the survival of liver transplanted patients and grafts have had a great improvement due to many factors. Careful preoperative evaluation in transplant recipients, experience and a multidisciplinary approach have, without any doubt, a major role in the selection of candidates and in the diagnosis and treatment of preoperative complications. Moreover, with the introduction of new generation immunosuppressive drugs and careful pharmacological monitoring, both the episodes of acute rejection and toxic effects have been minimized (1)

In consequence, since the graft and the patient post-transplant survival have been improved, the transplanted population has started showing long term medical complications.

Besides the risk correlated with graft rejection, the transplanted population has an increased risk of developing many malignancies. A number of hematologic diseases, skin cancer, gastrointestinal tumors seems to recognize a possible trigger factor in immunosuppressive drugs and the patient's immunological status but also in serological status and viral infections quite common in immunosuppressed patients. As for frequency, after these two groups of complications, cardiovascular diseases are the third cause of death in the transplanted population (1)

Eligibility to liver transplant, once based on Child Pugh system, has been regulated, since a few years, by MELD score, an index of survival probability of the end stage liver disease (2). This score is obtained on three variables, INR, creatinine and bilirubine. Due to the well known inadequate number of available grafts, if compared to patients requiring liver transplantation and to the risk of mortality while in the waiting list, this score supplies a priority system, based on the severity of the disease, for the organ allocation. The cut off value normally considered for eligibility to liver transplantation is a MELD score 15. Since the evaluation of the MELD score is obtained with this mathematical formula:

#### 10{0.957Ln(Scr)+0.378Ln(Tbil)+1.12 Ln (INR) +0.643}

eligibility to transplantation undergoes the parameter of severity of the disease, selecting the population of patients with generally worse clinical conditions. Moreover, the median age of the patients waiting for transplantation seems to be higher, essentially due to the improvement in the antiviral therapies and the better medical treatment in the hepatological

Cardiovascular Risk Factors and Liver Transplantation 39

the initial over activity with hyperinsulinemia, there is an impairment of islet beta cells; on the other hand, the liver glycogenogenesis and glycogenolysis pathways for the regulation of carbohydrate metabolism can be impaired due to the hepatic disease itself (10, 11, 12, 13,

Renal dysfunction is not uncommon (microalbuminuria, hepato-renal syndrome), and also pulmonary heart disease has to be considered such as Hepatopulmonary Syndrome where a mismatch between ventilation and perfusion is involved with hypoxemia due to an excess

Portopulmonary Hypertension, is a condition where pulmonary arterial hypertension is associated with liver disease or portal hypertension due to many factors such as hyperdinamic circulation, release of mediators from the congested bowel because of splancnic overload, vasoconstriction and remodeling of the lung vascular endothelium.(16) The American Association for the Study of Liver Diseases (2) recommends a rigorous pre-

CAD= coronary artery disease; CTA= computed tomography angiogram; ECG= electrocardiogram; H=

portopulmonary hypertension; Pulm.= pulmonary; QTc= corrected QT interval; RV= right ventricular;

Fig. 1. Suggested strategy for pre-operative cardiac assessment of liver transplant candidates

Patients who undergo liver transplantation have an around doubled risk of developing cardiovascular disease if compared to non transplanted population. To investigate the risk of such complications, multiple risk factors have to be added up. On one hand, there are all the risk factors of the general population such as advanced age, male gender, smoking history, high body mass index, pre-transplant diabetes mellitus, marital status. On the other hand, some peculiar risk related to the etiology of the underlying liver cirrhosis have to be

heart failure; LV= left ventricular; LVOTO= left ventricular outflow tract obstruction; POPH=

of perfusion because of abnormal intrapulmonary vascular dilation. (5, 9, 11, 16)

14, 15, 16, 17).

operative Assessment

TTE= transthoracic

**3. Post transplant cardiovascular risk** 

patient; for these reasons, the impairment of the liver function resulting in an eligible score can be delayed, with a longer conservative treatment and an older age of the patients who undergo liver transplantation.

Like in general population, the risk of developing cardiovascular disease increases with age, but liver disease by itself is often related to cardiovascular disease or higher cardiovascular risk factors (2, 3). Therefore, an accurate cardiovascular risk assessment of these patients, besides the whole pretransplant evaluation, is essential, both for the increased risk of cardiovascular diseases after liver transplant and for the cardiovascular risk assessment of the pre-transplant condition.

#### **2. Pretransplant cardiovascular risk**

Advanced hepatic liver disease is responsible for many changes in the physiology and biochemistry of the cardiovascular system, affecting contractility, heart rate, conduction and repolarization (4, 5, 6).

Cirrhotic cardiomyopathy is a pathological condition characterized by an increased cardiac output, impaired ventricular response to stress, decreased beta-agonist transduction, increased circulating inflammatory mediators with cardio depressant effect, alteration in repolarization, low systemic vascular resistance and bradycardia, altered function of muscarinic function and beta adrenergic stimulatory system, heart cell membrane abnormalities due to altered membrane fluidity and a modified calcium concentration, overproduction of nitric oxide, cardio depressant effects of an increased level of carbon monoxide (7).

Both the systolic and diastolic functions seem to be damages: the former, even if in cirrhotic cardiomyopathy the cardiac output is high, can be revealed by stress test, and can be caused by a reduced cardiovascular reactivity during exercise; the latter determined by fibrosis, myocardial hypertrophy and sub endothelial edema results in the impairment of compliance and relaxation. (4, 5, 6)

Abnormalities at the ECG, such electromechanical dissociation, which results in a prolongation of the QT interval , and chronotropic incompetence given by an impaired response to beta stimulation, are very common in cirrhosis and seem to be mainly related to portal hypertension and portosystemic shunting, but also to alterations of the heart rate with a central hypovolemia, loss of renal excretion of water and sodium and an altered baroreflex sensitivity which contribute to impair the cardiovascular system.(4, 5, 6, 8)

These conditions can lead to a higher risk of torsade de pointes, rhythm disturbances but also to the inability to develop physiologic tachycardia when required.

Some liver diseases seem to show a correlation also on the coronary blood flow. Diffuse but undetectable coronary atherosclerosis, reduced coronary micro vascular bed and impaired endothelium function are reported in NAFLD (9, 10).

Pre-transplant diabetes is another cardiovascular risk factor, and, in liver diseases the possibility to find an insulin resistance can be related to the pathogenesis of liver disease ( non alcoholic fatty liver disease), where the deregulation of fat metabolism causes an overproduction of very low density proteins involved in the metabolic syndrome, and, after

patient; for these reasons, the impairment of the liver function resulting in an eligible score can be delayed, with a longer conservative treatment and an older age of the patients who

Like in general population, the risk of developing cardiovascular disease increases with age, but liver disease by itself is often related to cardiovascular disease or higher cardiovascular risk factors (2, 3). Therefore, an accurate cardiovascular risk assessment of these patients, besides the whole pretransplant evaluation, is essential, both for the increased risk of cardiovascular diseases after liver transplant and for the cardiovascular risk assessment of

Advanced hepatic liver disease is responsible for many changes in the physiology and biochemistry of the cardiovascular system, affecting contractility, heart rate, conduction and

Cirrhotic cardiomyopathy is a pathological condition characterized by an increased cardiac output, impaired ventricular response to stress, decreased beta-agonist transduction, increased circulating inflammatory mediators with cardio depressant effect, alteration in repolarization, low systemic vascular resistance and bradycardia, altered function of muscarinic function and beta adrenergic stimulatory system, heart cell membrane abnormalities due to altered membrane fluidity and a modified calcium concentration, overproduction of nitric oxide, cardio depressant effects of an increased level of carbon

Both the systolic and diastolic functions seem to be damages: the former, even if in cirrhotic cardiomyopathy the cardiac output is high, can be revealed by stress test, and can be caused by a reduced cardiovascular reactivity during exercise; the latter determined by fibrosis, myocardial hypertrophy and sub endothelial edema results in the impairment of

Abnormalities at the ECG, such electromechanical dissociation, which results in a prolongation of the QT interval , and chronotropic incompetence given by an impaired response to beta stimulation, are very common in cirrhosis and seem to be mainly related to portal hypertension and portosystemic shunting, but also to alterations of the heart rate with a central hypovolemia, loss of renal excretion of water and sodium and an altered baroreflex sensitivity which contribute to impair the cardiovascular system.(4, 5, 6, 8)

These conditions can lead to a higher risk of torsade de pointes, rhythm disturbances but

Some liver diseases seem to show a correlation also on the coronary blood flow. Diffuse but undetectable coronary atherosclerosis, reduced coronary micro vascular bed and impaired

Pre-transplant diabetes is another cardiovascular risk factor, and, in liver diseases the possibility to find an insulin resistance can be related to the pathogenesis of liver disease ( non alcoholic fatty liver disease), where the deregulation of fat metabolism causes an overproduction of very low density proteins involved in the metabolic syndrome, and, after

also to the inability to develop physiologic tachycardia when required.

endothelium function are reported in NAFLD (9, 10).

undergo liver transplantation.

the pre-transplant condition.

repolarization (4, 5, 6).

monoxide (7).

compliance and relaxation. (4, 5, 6)

**2. Pretransplant cardiovascular risk** 

the initial over activity with hyperinsulinemia, there is an impairment of islet beta cells; on the other hand, the liver glycogenogenesis and glycogenolysis pathways for the regulation of carbohydrate metabolism can be impaired due to the hepatic disease itself (10, 11, 12, 13, 14, 15, 16, 17).

Renal dysfunction is not uncommon (microalbuminuria, hepato-renal syndrome), and also pulmonary heart disease has to be considered such as Hepatopulmonary Syndrome where a mismatch between ventilation and perfusion is involved with hypoxemia due to an excess of perfusion because of abnormal intrapulmonary vascular dilation. (5, 9, 11, 16)

Portopulmonary Hypertension, is a condition where pulmonary arterial hypertension is associated with liver disease or portal hypertension due to many factors such as hyperdinamic circulation, release of mediators from the congested bowel because of splancnic overload, vasoconstriction and remodeling of the lung vascular endothelium.(16)

The American Association for the Study of Liver Diseases (2) recommends a rigorous preoperative Assessment

CAD= coronary artery disease; CTA= computed tomography angiogram; ECG= electrocardiogram; H= heart failure; LV= left ventricular; LVOTO= left ventricular outflow tract obstruction; POPH= portopulmonary hypertension; Pulm.= pulmonary; QTc= corrected QT interval; RV= right ventricular; TTE= transthoracic

Fig. 1. Suggested strategy for pre-operative cardiac assessment of liver transplant candidates

#### **3. Post transplant cardiovascular risk**

Patients who undergo liver transplantation have an around doubled risk of developing cardiovascular disease if compared to non transplanted population. To investigate the risk of such complications, multiple risk factors have to be added up. On one hand, there are all the risk factors of the general population such as advanced age, male gender, smoking history, high body mass index, pre-transplant diabetes mellitus, marital status. On the other hand, some peculiar risk related to the etiology of the underlying liver cirrhosis have to be

Cardiovascular Risk Factors and Liver Transplantation 41

by the common onset of rejection and graft loss, when the immunosuppressive strategies were poor and crude such as the whole-body x-radiation at the very beginning (1960s) and azathioprine, prendisolone and anti-lymphocytes antibodies about ten years later. (27)

The experience in liver transplant shows that liver transplant recipients develop a lower rate of rejection if compared to other organs (heart, kidney) possibly because a form of microchimerism due to a large number of donor's cells within the allograft. Also the production by the liver of soluble donor MHC class 1 molecules has been mentioned to explain this resistance to rejection in liver transplantation. The size and regenerative

Nevertheless, although the liver is less prone to rejection then other organs, the immunosuppressive regimen remains mandatory after liver transplantation. The selection of immunosuppressive drugs is not universal but has to be done considering the pretransplant history and the medical conditions, considering for example pretransplant poor renal function, diabetes or hepatocellular carcinoma. Therefore a careful balance between pros

At present, calcineurin inhibitors are the most large used immunosuppressive drugs for liver transplantation, with well known side effects such as neurotoxicity, nephrotoxicity, hypertension, increased risk of death due to cardiovascular risk factors, gingival

 Cyclosporine, the oldest one, is derived from the fungus Tolypocladium inflatum and is a polypeptide of 11 amino acids. Tacrolimus, the most recent one, is derived from

Cyclosporine inhibits interleukin 2 gene transcription, binding cyclophilin, inhibiting the calcium/calmodulin phosphatase dependent calcineurin complex. It causes a dephosphorilation of activated T cell which is important for the transcription of cytokines for the activation of T cells, while Tacrolimus, from the same group of immunosuppressive drugs and with a similar mechanism of action, inhibits calcineurin binding to another

These drugs seem to have complex activities besides immunosuppression, causing endothelial dysfunction and more specifically a decrease in the production of the vasodilator nitric oxide (NO) by endothelial NO synthase (eNOS), affecting the vasodilator function by negatively altering endothelial intracellular Ca2+ and eNOS phosphorylation. Ca2+ concentration is altered by intracellular Ca2+ leak and decreased agonist-induced intracellular Ca2+ release which negatively affects eNOS phosphorylation, NO production, and endothelium-dependent dilatation. The precise mechanisms leading to hyperlipidemia are not completely known. There are contributing factors such as corticosteroids use and obesity. Anyway cyclosporine is related to an increase of VLDL and LDL while Tacrolimus seems to be characterized by VLDL increase alone. The mechanisms which underlie these side effects seem to be the increase in free intracellular cholesterol levels due to an impaired cholesterol esterification, but also the activation of the transcription factor responsible for the expression of lipid related genes. Oxidative processes may also underlie the

properties of liver can also play a role.(27, 28)

and cons is essential.

atherosclerotic effect.

hyperplasia, hirsutism and diabetes.

Streptomyces tsukabaensis and is a macrolide compound.

specific immunophilin, FK binding protein 12. (27, 28, 29)

considered, since liver disease caused by criptogenetic, alcohol and hepatitis C seems to be related to an increased cardiovascular risk. Moreover, after transplantation, a new physiological condition arises from the hemodynamic, biochemical and drug related standpoint. The vasodilatation existing in the cirrhotic patient has been solved and a systemic vasoconstriction is quite often present in the transplanted patient. Many factors seem to be involved in such systemic resistance modification but a definite pathogenesis is still under debate. The renin-angiotensin system seems to be involved but also the effect of endothelin, which seems to be higher then normal after LT, with its vasoconstrictor effect and which could determine the arterial pressure increase as well as the arterial stiffness which are clearly related to high pressure. Anyway a high arterial blood pressure before transplant seems to be a factor contributing the post transplant development of arterial hypertension (1, 18, 19)

The metabolic syndrome is apparently another risk factor, quite common in transplanted patient. In liver cirrhosis the patient generally suffers from a hypermetabolic condition and insulin resistance is very common. The normalization of the metabolic status, and the change induced in the metabolism of lipoproteins, in absence or combined with insulin resistance or diabetes due to the pancreas cells function impairment, can explain some effects of the post-transplant status while the underlying liver disease itself (HCV infection and relapse) seems to be related to diabetes affecting the insulin pathway and directly affecting the pancreatic beta cells (20, 21, 22, 23)

A major role in the cardiovascular risk factors lies in the irreplaceable use of immunosuppressive drugs (1, 18, 24)

Immunosuppressive drugs are related to arterial hypertension, and especially calcineurin inhibitors, have turned out to affect the vascular bed causing endothelial dysfunction. Besides their direct effect on the vascular endothelium, they also affect the vascular smooth muscle cells and have effects on the release and production of nitric oxide and endotheline. The renal function is impaired because of the nephrotoxic effects of many of these drugs that cause an impairment of the glomerular filtration rate and side effects on arterial pressure. Immunosuppressive drugs have also a pancreatic beta cells toxicity leading to a decrease in the production and secretion of insulin and an increased risk of diabetes (18). This condition by itself impairs the micro vascular bed and renal function. These drugs, especially M-tor inhibitors, affect the lipid metabolism, with the development of high levels of cholesterol and triglyceride concentration requiring lipid-lowering drugs. This side effect seems to be due to a decreased drug related bile acid synthesis but also to the agonist effects of some immunosuppressive drugs with low density lipoprotein cholesterol receptors, with a higher quantity of circulating low density lipoprotein. These side effects cause a further impairing in the vascular beds status, with effects of stiffness on the vascular walls. Also the steroids, frequently used in combination with immunosuppressive drugs or as treatment of acute rejection, have their well known effects on the vascular system, causing vasoconstriction, arterial hypertension, truncal fat deposition, and on the glucydic metabolism, causing a decreased insulin production, and impairing the peripheral glucose utilization, resulting in an insulin resistance or diabetes (1, 18, 25, 26).

#### **4. Immunosuppression and cardiovascular risk factors**

Transplantation soared in the 1980s thanks to the introduction of a new immunosuppressive drug: Cyclosporine. Before that, survival after liver transplantation was strongly impaired

considered, since liver disease caused by criptogenetic, alcohol and hepatitis C seems to be related to an increased cardiovascular risk. Moreover, after transplantation, a new physiological condition arises from the hemodynamic, biochemical and drug related standpoint. The vasodilatation existing in the cirrhotic patient has been solved and a systemic vasoconstriction is quite often present in the transplanted patient. Many factors seem to be involved in such systemic resistance modification but a definite pathogenesis is still under debate. The renin-angiotensin system seems to be involved but also the effect of endothelin, which seems to be higher then normal after LT, with its vasoconstrictor effect and which could determine the arterial pressure increase as well as the arterial stiffness which are clearly related to high pressure. Anyway a high arterial blood pressure before transplant seems to be a factor

The metabolic syndrome is apparently another risk factor, quite common in transplanted patient. In liver cirrhosis the patient generally suffers from a hypermetabolic condition and insulin resistance is very common. The normalization of the metabolic status, and the change induced in the metabolism of lipoproteins, in absence or combined with insulin resistance or diabetes due to the pancreas cells function impairment, can explain some effects of the post-transplant status while the underlying liver disease itself (HCV infection and relapse) seems to be related to diabetes affecting the insulin pathway and directly

A major role in the cardiovascular risk factors lies in the irreplaceable use of

Immunosuppressive drugs are related to arterial hypertension, and especially calcineurin inhibitors, have turned out to affect the vascular bed causing endothelial dysfunction. Besides their direct effect on the vascular endothelium, they also affect the vascular smooth muscle cells and have effects on the release and production of nitric oxide and endotheline. The renal function is impaired because of the nephrotoxic effects of many of these drugs that cause an impairment of the glomerular filtration rate and side effects on arterial pressure. Immunosuppressive drugs have also a pancreatic beta cells toxicity leading to a decrease in the production and secretion of insulin and an increased risk of diabetes (18). This condition by itself impairs the micro vascular bed and renal function. These drugs, especially M-tor inhibitors, affect the lipid metabolism, with the development of high levels of cholesterol and triglyceride concentration requiring lipid-lowering drugs. This side effect seems to be due to a decreased drug related bile acid synthesis but also to the agonist effects of some immunosuppressive drugs with low density lipoprotein cholesterol receptors, with a higher quantity of circulating low density lipoprotein. These side effects cause a further impairing in the vascular beds status, with effects of stiffness on the vascular walls. Also the steroids, frequently used in combination with immunosuppressive drugs or as treatment of acute rejection, have their well known effects on the vascular system, causing vasoconstriction, arterial hypertension, truncal fat deposition, and on the glucydic metabolism, causing a decreased insulin production, and impairing the peripheral glucose utilization, resulting in

Transplantation soared in the 1980s thanks to the introduction of a new immunosuppressive drug: Cyclosporine. Before that, survival after liver transplantation was strongly impaired

contributing the post transplant development of arterial hypertension (1, 18, 19)

affecting the pancreatic beta cells (20, 21, 22, 23)

an insulin resistance or diabetes (1, 18, 25, 26).

**4. Immunosuppression and cardiovascular risk factors** 

immunosuppressive drugs (1, 18, 24)

by the common onset of rejection and graft loss, when the immunosuppressive strategies were poor and crude such as the whole-body x-radiation at the very beginning (1960s) and azathioprine, prendisolone and anti-lymphocytes antibodies about ten years later. (27)

The experience in liver transplant shows that liver transplant recipients develop a lower rate of rejection if compared to other organs (heart, kidney) possibly because a form of microchimerism due to a large number of donor's cells within the allograft. Also the production by the liver of soluble donor MHC class 1 molecules has been mentioned to explain this resistance to rejection in liver transplantation. The size and regenerative properties of liver can also play a role.(27, 28)

Nevertheless, although the liver is less prone to rejection then other organs, the immunosuppressive regimen remains mandatory after liver transplantation. The selection of immunosuppressive drugs is not universal but has to be done considering the pretransplant history and the medical conditions, considering for example pretransplant poor renal function, diabetes or hepatocellular carcinoma. Therefore a careful balance between pros and cons is essential.

At present, calcineurin inhibitors are the most large used immunosuppressive drugs for liver transplantation, with well known side effects such as neurotoxicity, nephrotoxicity, hypertension, increased risk of death due to cardiovascular risk factors, gingival hyperplasia, hirsutism and diabetes.

 Cyclosporine, the oldest one, is derived from the fungus Tolypocladium inflatum and is a polypeptide of 11 amino acids. Tacrolimus, the most recent one, is derived from Streptomyces tsukabaensis and is a macrolide compound.

Cyclosporine inhibits interleukin 2 gene transcription, binding cyclophilin, inhibiting the calcium/calmodulin phosphatase dependent calcineurin complex. It causes a dephosphorilation of activated T cell which is important for the transcription of cytokines for the activation of T cells, while Tacrolimus, from the same group of immunosuppressive drugs and with a similar mechanism of action, inhibits calcineurin binding to another specific immunophilin, FK binding protein 12. (27, 28, 29)

These drugs seem to have complex activities besides immunosuppression, causing endothelial dysfunction and more specifically a decrease in the production of the vasodilator nitric oxide (NO) by endothelial NO synthase (eNOS), affecting the vasodilator function by negatively altering endothelial intracellular Ca2+ and eNOS phosphorylation. Ca2+ concentration is altered by intracellular Ca2+ leak and decreased agonist-induced intracellular Ca2+ release which negatively affects eNOS phosphorylation, NO production, and endothelium-dependent dilatation. The precise mechanisms leading to hyperlipidemia are not completely known. There are contributing factors such as corticosteroids use and obesity. Anyway cyclosporine is related to an increase of VLDL and LDL while Tacrolimus seems to be characterized by VLDL increase alone. The mechanisms which underlie these side effects seem to be the increase in free intracellular cholesterol levels due to an impaired cholesterol esterification, but also the activation of the transcription factor responsible for the expression of lipid related genes. Oxidative processes may also underlie the atherosclerotic effect.

Cardiovascular Risk Factors and Liver Transplantation 43

ethanolamine N-methyltransferase and altered availability of Alfa1-adrenergic receptors in vascular smooth muscles, leading to an increased vascular reactivity, pressure responsiveness and hypertension. The vasoconstriction and hypertension induced by steroids are also mediated through enhanced synthesis and action of vasoactive substances and their receptors, including neuro peptide Y (NPY), arginine vasopressin (AVP) and atrial

Another interesting issue is the role of the renin-angiotensin system activation in the development of steroids induced hypertension. Steroids act directly at the liver site,

On the other hand, steroids negatively affect various vasodilatory systems causing nitric oxide (NO) deficiency through a range of a negative influences on the NO biosynthetic pathways involving alteration in the activity and expression of NO synthase, decreased availability of tetrahydrobiopterin (BH4) and decreased NO precursor L-arginine. Moreover steroids affect the production of other vasodilatatory substances as prostacyclin,

Steroids induce insulin resistance in skeletal muscle by directly interfering with the insulin signaling cascade. The same effect is produced also in hepatic cells and thus endogenous

Steroids appear to have a direct causal effect relationship with cardiovascular disease depending on dose, duration, cumulative dose of exposure and route of administration. The increased risk is mediated through the induction of several risk factors for cardiovascular

Since the late 1990s other immunosuppressive drugs have been introduced into the world of transplantation: the group of m-Tor inhibitors (Sirolimus and Everolimus) (27, 28, 29).

Sirolimus is derived from actinomycete Streptomyces hygroscopicus. It has a homologous structure if compared to Cyclosporine and Tacrolimus and it also binds to FK bindig proteins family (FKBP-12) which binds to mammalian targets of rapamycin (MTOR) which has a kinase activity. Sirolimus plays its action on the signal transduction pathway, blocking the IL-2 and IL-5 induction of B and T cell proliferation. It inhibits the normal cell proliferation in response to IL-2. Everolimus, is a macrolide derived from Sirolimus, with a similar mechanism of action, inhibiting the activation of immunophyllin FKBP-12. Unlike

These drugs are particularly interesting and under debate. Based on their anti proliferative effects, they also seem to have anti-neoplastic effects based on the fact that they inhibit angiogenesis, inhibit cancer cells survival and also cancer stem cells survival (27, 28, 29).

They seem to have less nephrotoxic effects than calcineurin-inhibitors provided there is no pre-existing renal disease, basing their action on m-Tor contrasting the renal fibrosis caused by TGF-beta. There's anyway a risk of proteinuria or of renal damage amplification in an

They show interesting properties in promoting tolerance but Sirolimus is considered an unsafe drug for the 1st month after transplantation for a higher risk of developing hepatic artery thrombosis and for a slower and more difficult wounds healing. Mouth ulcers and leg

Sirolimus it has pharmacokinetics properties which make it easier to handle.

natriuretic peptide (ANP).

prostaglandin E2 and kallikrein.

disease.

glucose production is increased. (28, 29, 37)

already compromised renal function.

enhancing the synthesis of angiotensinogen. (29, 37)

The nephrotoxic effects are well known, and they can by themselves be related to the development of arterial hypertension. The mechanism, beside a possible involvement of the tubular epithelium, seems to be more strongly related to vascular alteration of the afferent arterioles, with consequent ions alteration and hyperkaliemia. (28, 29, 30)

The arteriolar vasoconstriction and renal ischemia are related to an imbalance between the vasoactive messengers ( endothelin-1 and tromboxane A2), the dysregulation of nitric-oxide formation and the renin-angiotensin system. Besides these vascular effects altering the renal control of arterial pressure, the nephrotoxic direct effects should also be mentioned, with tubular cell apoptosis and necrosis, but also the effects on the cell cycle compromising the proliferation capacity and accelerating cell aging possibly mediated by an oxidative stress caused by reactive oxygen species (ROS) and lipid peroxides. This effect has been demonstrated as an abnormality in permeability of the mitochondria causing an isometric vacuolization resulting in the presence of giant mitochondria. Thus, the endoplasmic reticulum undergoes enlargement and the proteic syntesis is affected. Cyclosporine can act as an endoplasmic reticulum stress inducer causing epithelial phenotypic changes leading to nephrotoxicity (30, 31, 32, 33, 34).

Endoplasmic reticulum stress seems to play a major role in many diseases such as atherosclerosis, Alzheimer, diabetes and inflammatory bowel diseases, leading the unfolded protein response to an adaptative response. (35, 36)

The above mentioned stress is involved in post transplant diabetes, promoting insulin resistance and cells death. Insulin secerning cells undergo proteins synthesis stress and are very sensitive to any status causing accumulation of anomalous proteins. The pathophisiology of pancreatic beta cells damage during treatment with Tacrolimus and Cyclosporine is still matter of debate, but it seems related to the endoplasmic reticulum stress which modify cells vitality, since in animal models treated with calcineurin inhibitors, nuclear inclusions, dilatation in cistern of the granulous endoplasmic reticulum with degranulation and degeneration of pancreatic beta islets have been shown (35, 36)

Steroids are often used in immunosuppressive therapeutic schemes in the perioperative period, both intraoperatively, and after transplantation for a period ranging from 3 to 6 months for liver transplantation. Moreover, at the possible onset of acute rejection, bolus of steroids are administered. The steroid immunosuppressive function derives from the inactivation of the response of lymphocytes and macrophages by inhibiting the production of cytokines, but also suppressing antigens and stimulating the migration of T cells to the lymphoid tissue.(30)

A large body of evidence supports the theory that steroids induce an imbalance between vasoconstriction and vasodilatation, favoring vasoconstriction, resulting in arterial hypertension.

The increased vasoconstriction is mediated by several mechanisms. In large part vasoconstriction is mediated by the increased endothelin-1 synthesis and secretion, increased erythropoietin levels by the increased level of cytosolic calcium, increased sympathetic activity that is mediated by the increase of Beta1-adrenergic receptor expression and increased synthesis of catecholamines, by the increased expression of various enzymes involved in the catecholamines biosynthesis including tyrosine hydroxilase and phenyl

The nephrotoxic effects are well known, and they can by themselves be related to the development of arterial hypertension. The mechanism, beside a possible involvement of the tubular epithelium, seems to be more strongly related to vascular alteration of the afferent

The arteriolar vasoconstriction and renal ischemia are related to an imbalance between the vasoactive messengers ( endothelin-1 and tromboxane A2), the dysregulation of nitric-oxide formation and the renin-angiotensin system. Besides these vascular effects altering the renal control of arterial pressure, the nephrotoxic direct effects should also be mentioned, with tubular cell apoptosis and necrosis, but also the effects on the cell cycle compromising the proliferation capacity and accelerating cell aging possibly mediated by an oxidative stress caused by reactive oxygen species (ROS) and lipid peroxides. This effect has been demonstrated as an abnormality in permeability of the mitochondria causing an isometric vacuolization resulting in the presence of giant mitochondria. Thus, the endoplasmic reticulum undergoes enlargement and the proteic syntesis is affected. Cyclosporine can act as an endoplasmic reticulum stress inducer causing epithelial phenotypic changes leading to

Endoplasmic reticulum stress seems to play a major role in many diseases such as atherosclerosis, Alzheimer, diabetes and inflammatory bowel diseases, leading the unfolded

The above mentioned stress is involved in post transplant diabetes, promoting insulin resistance and cells death. Insulin secerning cells undergo proteins synthesis stress and are very sensitive to any status causing accumulation of anomalous proteins. The pathophisiology of pancreatic beta cells damage during treatment with Tacrolimus and Cyclosporine is still matter of debate, but it seems related to the endoplasmic reticulum stress which modify cells vitality, since in animal models treated with calcineurin inhibitors, nuclear inclusions, dilatation in cistern of the granulous endoplasmic reticulum with

Steroids are often used in immunosuppressive therapeutic schemes in the perioperative period, both intraoperatively, and after transplantation for a period ranging from 3 to 6 months for liver transplantation. Moreover, at the possible onset of acute rejection, bolus of steroids are administered. The steroid immunosuppressive function derives from the inactivation of the response of lymphocytes and macrophages by inhibiting the production of cytokines, but also suppressing antigens and stimulating the migration of T cells to the

A large body of evidence supports the theory that steroids induce an imbalance between vasoconstriction and vasodilatation, favoring vasoconstriction, resulting in arterial

The increased vasoconstriction is mediated by several mechanisms. In large part vasoconstriction is mediated by the increased endothelin-1 synthesis and secretion, increased erythropoietin levels by the increased level of cytosolic calcium, increased sympathetic activity that is mediated by the increase of Beta1-adrenergic receptor expression and increased synthesis of catecholamines, by the increased expression of various enzymes involved in the catecholamines biosynthesis including tyrosine hydroxilase and phenyl

degranulation and degeneration of pancreatic beta islets have been shown (35, 36)

arterioles, with consequent ions alteration and hyperkaliemia. (28, 29, 30)

nephrotoxicity (30, 31, 32, 33, 34).

lymphoid tissue.(30)

hypertension.

protein response to an adaptative response. (35, 36)

ethanolamine N-methyltransferase and altered availability of Alfa1-adrenergic receptors in vascular smooth muscles, leading to an increased vascular reactivity, pressure responsiveness and hypertension. The vasoconstriction and hypertension induced by steroids are also mediated through enhanced synthesis and action of vasoactive substances and their receptors, including neuro peptide Y (NPY), arginine vasopressin (AVP) and atrial natriuretic peptide (ANP).

Another interesting issue is the role of the renin-angiotensin system activation in the development of steroids induced hypertension. Steroids act directly at the liver site, enhancing the synthesis of angiotensinogen. (29, 37)

On the other hand, steroids negatively affect various vasodilatory systems causing nitric oxide (NO) deficiency through a range of a negative influences on the NO biosynthetic pathways involving alteration in the activity and expression of NO synthase, decreased availability of tetrahydrobiopterin (BH4) and decreased NO precursor L-arginine. Moreover steroids affect the production of other vasodilatatory substances as prostacyclin, prostaglandin E2 and kallikrein.

Steroids induce insulin resistance in skeletal muscle by directly interfering with the insulin signaling cascade. The same effect is produced also in hepatic cells and thus endogenous glucose production is increased. (28, 29, 37)

Steroids appear to have a direct causal effect relationship with cardiovascular disease depending on dose, duration, cumulative dose of exposure and route of administration. The increased risk is mediated through the induction of several risk factors for cardiovascular disease.

Since the late 1990s other immunosuppressive drugs have been introduced into the world of transplantation: the group of m-Tor inhibitors (Sirolimus and Everolimus) (27, 28, 29).

Sirolimus is derived from actinomycete Streptomyces hygroscopicus. It has a homologous structure if compared to Cyclosporine and Tacrolimus and it also binds to FK bindig proteins family (FKBP-12) which binds to mammalian targets of rapamycin (MTOR) which has a kinase activity. Sirolimus plays its action on the signal transduction pathway, blocking the IL-2 and IL-5 induction of B and T cell proliferation. It inhibits the normal cell proliferation in response to IL-2. Everolimus, is a macrolide derived from Sirolimus, with a similar mechanism of action, inhibiting the activation of immunophyllin FKBP-12. Unlike Sirolimus it has pharmacokinetics properties which make it easier to handle.

These drugs are particularly interesting and under debate. Based on their anti proliferative effects, they also seem to have anti-neoplastic effects based on the fact that they inhibit angiogenesis, inhibit cancer cells survival and also cancer stem cells survival (27, 28, 29).

They seem to have less nephrotoxic effects than calcineurin-inhibitors provided there is no pre-existing renal disease, basing their action on m-Tor contrasting the renal fibrosis caused by TGF-beta. There's anyway a risk of proteinuria or of renal damage amplification in an already compromised renal function.

They show interesting properties in promoting tolerance but Sirolimus is considered an unsafe drug for the 1st month after transplantation for a higher risk of developing hepatic artery thrombosis and for a slower and more difficult wounds healing. Mouth ulcers and leg

Cardiovascular Risk Factors and Liver Transplantation 45

receptor

presenting cell;

Interferes with Signal 1

cells and monocytes

dislipidemia, gingival hyperplasia, hirsutism.

Dislipidemia, anemia, leucopenia, thrombocytopenia, HAT, wound dehiscence, aphtous ulcers, arthralgia,

Interferes with Signal 1, 2, 3 Inhibits lymphocytes trafficking

thymocytes

Inhibit Signal 3 trasduction via IL-2 receptor

Selective lysis of immature cortical

IMMUNOSUPPRESSIVE AGENT MECHANISM OF ACTION

MAMMALIAN TARGET OF RAPAMYCIN

IMMUNOSUPPRESSIVE AGENT SIDE EFFECTS

MAMMALIAN TARGET OF RAPAMYCIN INHIBITORS

INHIBITORS

CALINEURINE INHIBITORS Inhibit signal 2 trasduction via T cell

MYCOPHENOLIC ACID Inhibit purine and DNA synthesis

CORTICOSTEROIDS Inhibit cytokine transcription by antigen

AntiCD3 monoclonal antibodies Depletion and receptor modulation in T cell

Antithymocyte globuline Depletion and receptor modulation in T cells

Anti –CD52 monoclonal antibodies Cause depletion of thymocytes, T cells, B

CALINEURINE INHIBITORS Hypertension, Renal toxicity, neurotoxicity, diabetes,

proteinuria

MYCOPHENOLIC ACID Anorexia, abdominal pain, gastritis, diarrhea, neutropenia

Anti IL-2 alpha chain receptor antibodies Inhibit T cell proliferation to IL-2

edema are common. It's strongly related to high risk of developing of hyperlipidemia which seems to be caused by an increased hepatic secretion of VLDL, increased hepatic synthesis of apoB100 and a reduction in the hepatic catabolism of LDL. The action of lipoprotein lipase is decreased and the expression of apoCIII and lipase in adipose tissue are increased. Anyway these drugs have effects on macrophages and antiproliferative effects which could protect from the cardiovascular risk of atherosclerosis (27, 28, 29, 38, 39).

Antimetabolites such as mycophenolate mofetil and mycophenolate sodium have immunosuppressive properties which were recognized in the 1990s although these drugs are older. Their action derive from the blockage of inosine-51-monophosphate deydrogenase (IMPDH) resulting in the selective lymphocyte proliferation blockage. They have probably less cardiovascular side effects, mainly gastrointestinal disorders, bone marrow depression, some infections. Their exclusive use is hardly ever considered due to their insufficient protection from acute rejection if not combined to m-Tor or CNI. Yet their use anyway often allows a lower CNI or m-Tor dosage with side effects sparing properties. (27, 28, 29, 40)

Both polyclonal and monoclonal antibodies [ anti- thymocites globilin (ATG), antilymphocyte globulin (ALG), monoclonal antibodies (OKT3, Campath, Basiliximab)] are also largely used in combination with CNI or m-Tor delayed introduction as induction therapies, minimizing the side effects of CNI and m-Tor inhibitors.

Other immunosuppressive drugs are currently undergoing trials such as Belatacept, which inhibits T cell activation binding CD80 and CD86, and looks promising, with less renal toxicity, and Efalizumab which inhibits T cell-APC stabilization and blocks lymphocyte adhesion to endothelial cells, with good results as for immunosuppressive properties but with an increased risk of onset of post-transplant lymphoproliferative disorders (27, 28, 29).

#### **5. Risk of new onset of diabetes (36)**

edema are common. It's strongly related to high risk of developing of hyperlipidemia which seems to be caused by an increased hepatic secretion of VLDL, increased hepatic synthesis of apoB100 and a reduction in the hepatic catabolism of LDL. The action of lipoprotein lipase is decreased and the expression of apoCIII and lipase in adipose tissue are increased. Anyway these drugs have effects on macrophages and antiproliferative effects which could

Antimetabolites such as mycophenolate mofetil and mycophenolate sodium have immunosuppressive properties which were recognized in the 1990s although these drugs are older. Their action derive from the blockage of inosine-51-monophosphate deydrogenase (IMPDH) resulting in the selective lymphocyte proliferation blockage. They have probably less cardiovascular side effects, mainly gastrointestinal disorders, bone marrow depression, some infections. Their exclusive use is hardly ever considered due to their insufficient protection from acute rejection if not combined to m-Tor or CNI. Yet their use anyway often allows a lower CNI or m-Tor dosage with side effects sparing properties. (27, 28, 29, 40)

Both polyclonal and monoclonal antibodies [ anti- thymocites globilin (ATG), antilymphocyte globulin (ALG), monoclonal antibodies (OKT3, Campath, Basiliximab)] are also largely used in combination with CNI or m-Tor delayed introduction as induction

Other immunosuppressive drugs are currently undergoing trials such as Belatacept, which inhibits T cell activation binding CD80 and CD86, and looks promising, with less renal toxicity, and Efalizumab which inhibits T cell-APC stabilization and blocks lymphocyte adhesion to endothelial cells, with good results as for immunosuppressive properties but with an increased risk of onset of post-transplant lymphoproliferative disorders (27, 28, 29).

NON-MODIFIABLE POTENTIALLY MODIFIABLE MODIFIABLE

• HCV • CMV • Pre-tx IFG/IGT • Proteinuria • HypoMg

IGT: impaired glucose tolerance, IFG: impaired fasting glucose, Anti CD25 mAb, Anti CD25 monoclonal antibody, CMV:cytomegalovirus, HCV: hepatitis C, HypoMg: hypomagnesemia, Pre-

Individualization of Immunosuppressive

therapy • Tacrolimus • Cyclosporine • Corticosteroid • mTOR inhibitors • Anti CD25 mAB?

protect from the cardiovascular risk of atherosclerosis (27, 28, 29, 38, 39).

therapies, minimizing the side effects of CNI and m-Tor inhibitors.

**5. Risk of new onset of diabetes (36)** 

• African American, Hispanic • Age > 40–45 yrs • Recipient male gender • Family history of DM • HLA A30, B27, B42 • HLA mismatches

LEGEND:

Tx: pre-transplant


#### IMMUNOSUPPRESSIVE AGENT SIDE EFFECTS


Cardiovascular Risk Factors and Liver Transplantation 47

[5] Lee RF, Van Tassel,Glenn TK, Lee SS. Cardiac dysfunction in cirrhosis. Best Pract Res

[6] Møller S, Henriksen JH. Cirrhotic cardiomyopathy. J Hepatol. 2010 Jul;53(1):179-90. Epub

[7] Rockey DC.Vascular mediators in the injured liver. Hepatology. 2003 Jan;37(1):4-12.

[8] Yilmaz Y, Kurt R, Yonal O, Polat N, Celikel CA, Gurdal A, Oflaz H, Ozdogan O,

[9] Mandell MS, Lindenfeld J, Tsou MY, Zimmerman M. Cardiac evaluation of liver transplant candidates.World J Gastroenterol. 2008 Jun 14;14(22):3445-51. Review. [10] Fon Tacer K, Rozman D.Nonalcoholic Fatty liver disease: focus on lipoprotein and lipid

[11] Garcia-Compean D, Jaquez-Quintana JO, Gonzalez-Gonzalez JA, Maldonado-Garza

and management. World J Gastroenterol. 2009 Jan 21;15(3):280-8. Review. [12] Parekh S, Anania FA. Abnormal lipid and glucose metabolism in obesity: implications for

[13] Alexander S. Petrides, Leif C. Groop, Caroline A. Riely, and Ralph A. DeFronzo. Effect

[14] Krok KL, Milwalla F, Maheshwari A, Rankin R, Thuluvath PJ.Insulin resistance and

[15] García-Compean D, Jaquez-Quintana JO, Maldonado-Garza H.Hepatogenous diabetes. Current views of an ancient problem. Ann Hepatol. 2009 Jan-Mar;8(1):13-20. [16] Hoeper MM, Krowka MJ, Strassburg CP. Portopulmonary hypertension and hepatopulmonary syndrome. Lancet. 2004 May 1;363(9419):1461-8. Review. [17] Laish I, Braun M, Mor E, Sulkes J, Harif Y, Ben Ari Z. Metabolic syndrome in liver

[18] Tepperman E, Ramzy D, Prodger J, Sheshgiri R, Badiwala M, Ross H, Raoa V. Surgical

[19] Neal DA, Brown MJ, Wilkinson IB, Alexander GJ. Mechanisms of hypertension after

[20] Watt KD, Charlton MR.Metabolic syndrome and liver transplantation: a review and

[21] Sorice GP, Muscogiuri G, Mezza T, Prioletta A, Giaccari A. Metabolic syndrome in

[22] Pagadala M, Dasarathy S, Eghtesad B, McCullough AJ. Posttransplant metabolic

events. Liver Transpl. 2011 Jan;17(1):15-22. doi: 10.1002/lt.22198.

liver transplantation. Transplantation. 2005 Apr 27;79(8):935-40.

deregulation J Lipids. 2011;2011:783976. Epub 2011 Jul 2.

Clin. Invest. Volume 88, August 1991, 561-570

cirrhosis.Liver Transpl. 2009 Sep;15(9):1036-42.

Imeryuz N, Kalayci C, Avsar E. Coronary flow reserve is impaired in patients with nonalcoholic fatty liver disease: association with liver fibrosis. Atherosclerosis. 2010

H.Liver cirrhosis and diabetes: risk factors, pathophysiology, clinical implications

nonalcoholic fatty liver disease. Gastroenterology. 2007 May;132(6):2191-207. Review.

on physiologic hyperinsulinemia on glucose and lipid metabolism in cirrhosis. J.

microalbuminuria are associated with microvascular disease in patients with

transplant recipients: prevalence, risk factors, and association with cardiovascular

biology for the clinician: vascular effects of immunosuppression. Can J Surg. 2010

guide to management. J Hepatol. 2010 Jul;53(1):199-206. Epub 2010 Mar 31. Review.

transplant patients: an academic or a health burden? Transplant Proc. 2011 Jan-

syndrome: an epidemic waiting to happen. Liver Transpl. 2009 Dec;15(12):1662-70.

Clin Gastroenterol. 2007;21(1):125-40.

Jul;211(1):182-6. Epub 2010 Feb 7.

2010 Mar 31. Review.

Review.

Feb;53(1):57-63.

Feb;43(1):313-7.

Review.


#### **6. Conclusions**

Besides the cardiovascular risk factors related to age, family disease and life habits, the transplant candidate has a particular hemodynamic, biochemical, cardiac and systemic condition depending on the hepatic disease. After transplantation, these paraphysiologic modifications suddenly change, with the onset of a new systemic condition, the appearance of organ damages not evident before, a new risk factors profile related to the new situation and to unavoidable life-saving drugs treatment.

For these reasons the pre-transplant cardiovascular evaluation and the post-transplant accurate monitoring followed by a careful choice of the immunosuppressive therapeutic regimen, drug level monitoring, educational efforts to ameliorate life style and risk factors are mandatory for a satisfactory outcome.

#### **7. References**


aseptic necrosis of femoral head, diabetes,

hyperlipidemia, risk of infections, difficult wound

CORTICOSTEROIDS Hypertension, pancreatitis, peptic ulcer, osteoporosis,

healing

Antithymocyte globuline Leucopenia, thrombocytopenia

Anti IL-2 alpha chain receptor

and to unavoidable life-saving drugs treatment.

are mandatory for a satisfactory outcome.

Anti –CD52 monoclonal

antibodies

antibodies

**6. Conclusions** 

**7. References** 

AntiCD3 monoclonal antibodies Fever, hypotension, headache, aseptic meningitis,

dyspnea, vomiting, diarrhea

Risk of acute rejection

Risk of acute rejection

Besides the cardiovascular risk factors related to age, family disease and life habits, the transplant candidate has a particular hemodynamic, biochemical, cardiac and systemic condition depending on the hepatic disease. After transplantation, these paraphysiologic modifications suddenly change, with the onset of a new systemic condition, the appearance of organ damages not evident before, a new risk factors profile related to the new situation

For these reasons the pre-transplant cardiovascular evaluation and the post-transplant accurate monitoring followed by a careful choice of the immunosuppressive therapeutic regimen, drug level monitoring, educational efforts to ameliorate life style and risk factors

[1] Rossetto A, Bitetto D, Bresadola V, Lorenzin D, Baccarani U, De Anna D, Bresadola F,

[2] Raval Z, Harinstein ME, Skaro AI, Erdogan A, DeWolf AM, Shah SJ, Fix OK, Kay N,

[4] Zardi EM, Abbate A, Zardi DM, Dobrina A, Margiotta D, Van Tassell BW, Afeltra A,

liver transplant candidate. J Am Coll Cardiol. 2011 Jul 12;58(3):223-31. [3] Aberg F, Jula A, Höckerstedt K, Isoniemi H. Cardiovascular risk profile of patients with

Review. Erratum in: J Am Coll Cardiol. 2010 Sep 14;56(12):1000.

transplantation. Transplant Proc. 2010 Sep;42(7):2576-8.

population. Transplantation. 2010 Jan 15;89(1):61-8.

Adani GL. Cardiovascular risk factors and immunosuppressive regimen after liver

Abecassis MI, Gheorghiade M, Flaherty JD. Cardiovascular risk assessment of the

acute liver failure after liver transplantation when compared with the general

Sanyal AJ. Cirrhotic cardiomyopathy. J Am Coll Cardiol. 2010 Aug 10;56(7):539-49.


**4** 

*Mayo Clinic,* 

*USA* 

**Pathogenesis of Renovascular Hypertension:** 

With an aging population, renovascular hypertension has become a major public health problem (Safian & Textor, 2001). Although various forms of fibromuscular disease of the renal arteries and/or traumatic disruption of renal vessels are the most common cause of RVH among the younger individuals, atherosclerotic renal artery disease (ARAD) is the most common lesion producing hypertension by far (Garovic & Textor, 2005). ARAD is present in over 6.8% of individuals over 65 years of age and is found in up to 49.1% of patients with coronary artery disease or aortoiliac disease (Iglesias et al. 2000; Valabhji et al. 2000; Hansen et al. 2002; Rihal et al. 2002; Weber-Mzell et al. 2002; Textor 2003). Although many patients with asymptomatic renovascular disease do not develop progressive renal dysfunction, overall morbidity and mortality is significantly increased (Chabova et al. 2000; Textor 2002; Textor 2003; Textor 2003; Foley et al. 2005; Foley et al. 2005). On the other hand, some studies suggest that from 10% to 40% of elderly hypertensive patients with newly discovered end stage renal disease and no identifiable parenchymal renal disease have significant RAS (Textor and Wilcox 2001). As in other forms of renal disease, the severity of interstitial fibrosis, tubular atrophy, interstitial inflammation, and glomerular sclerosis are important predictors of renal outcome (Wright et al. 2001). It has been postulated that this acquired tubulointerstitial injury may contribute to at least some forms of essential hypertension (Raghow 1994). Mechanisms underlying vascular and renal dysfunction in RAS have not been well delineated, despite intense study (Textor 2004). This information is

essential for the development of therapies – surgical or medical – to treat RAS.

The hallmark of RVH arising from unilateral RAS is atrophy of the stenotic kidney and compensatory hyperplasia/hypertrophy of the contralateral kidney. Although this compensatory hypertrophy serves an adaptive function, this process may render the contralateral kidney more susceptible to other injuries (due to diabetes, glomerulonephritis, etc.) (Wenzel et al. 2002). Although the corresponding histologic, hemodynamic, and tubular alterations in the stenotic and contralateral kidneys have been superficially described in experimental animals, mechanisms underlying these alterations and the identification of markers that predict response to therapy have not been well defined. In particular, the stage in evolution of RAS at which the atrophic changes in the stenotic kidney preclude recovery of renal function after revascularization is not known. This lack of basic mechanistic knowledge is underscored by the variable response of RAS to surgical revascularization; significant

**1. Introduction** 

**Challenges and Controversies** 

Blake Fechtel, Stella Hartono and Joseph P. Grande


### **Pathogenesis of Renovascular Hypertension: Challenges and Controversies**

Blake Fechtel, Stella Hartono and Joseph P. Grande *Mayo Clinic, USA* 

#### **1. Introduction**

48 Recent Advances in Cardiovascular Risk Factors

[23] Ruiz-Rebollo ML, Sánchez-Antolín G, García-Pajares F, Fernández-Orcajo P, González-

[24] Pérez MJ, García DM, Taybi BJ, Daga JA, Rey JM, Grande RG, Lombardo JD, López JM.

[25] Farge D, Julien J.Effects of transplantation on the renin angiotensin system (RAS). J

[26] Bahirwani R, Reddy KR.Outcomes after liver transplantation: chronic kidney disease.

[27] Geissler E.K., Schlitt H.J. Immunosuppression for liver transplantation. Gut 2009;

[28] Pillai A. A., Levitsky J. Overview of immunosuppression in liver transplantation. Worl

[29] Mukherjee S., Mukherjee U. A comprehensive revies of immunosuppression used for

[30] Kockx M., Jessup W., Kritharides L. Cyclosporin A and atherosclerosis- Cellular pathways in atherogenesis. Pharmacology & Therapeutics 128 (2010) 106-118 [31] Halloran P.F. Mechanism of action of the calcineurin inhibitors. Transplant Proc. 2001;

[32] J. R. Chapman. Chronic Calcineurin Inhibitor Nephrotoxicity—Lest We Forget

[33] F. Lamoureux, E. Mestre, M. Essig, F.L. Sauvage, P. Marquet, L.N. Gastinel.

[34] Nicolas Pallet, Sophie Fougeray,Philippe Beaune,Christophe Legendre,Eric Thervet,

[35] Nicolas Pallet, Nicolas Bouvier, Philippe Beaune, Christophe Legendre, Dany

transplantation d'organe solide MEDECINE/SCIENCES 2010 ; 26 : 397-403 [36] Phuong-Thu T Pham, Phuong-Mai T Pham, Son V Pham, Phuong-Anh T Pham,

[37] Gabardi S., Pharm D., Baroletti S., Pharm D. Everolimus: A proliferation signal inhibitor

[38] Webster A. C., Lee V.W.S., Chapman J. R., Craig J.C. Target of rapamycin inhibitors

[39] Allison A. C., Eugui E.M. Mycophenolate mofetil and its mechanism of action.

Solid Organ Transplantation. Transplantation 2009;88: 605–613

Quantitative proteomic analysis of cyclosporine-induced toxicity in a human kidney cell line and comparison with tacrolimus. Journal of Proteomics (2011)

and Dany Anglicheau. Endoplasmic Reticulum Stress: An Unrecognized Actor in

Anglicheau, Éric Thervet. Implication du stress du reticulum endoplasmique en

Phuong-Chi T Pham. New onset diabetes after transplantation (NODAT): an overview Diabetes, Metabolic Syndrome and Obesity: Targets and Therapy

with clinical applications in organ transplantation, oncology and cardiology.

(Sirolimus and Everolimus) for primary immunosuppression of kidney transplant recipients: a systematic review and meta-analysis of randomized trials.

Transplant Proc. 2010 Mar;42(2):663-5.

Transplant Proc. 2011 Apr;43(3):739-41.

Liver Transpl. 2009 Nov;15 Suppl 2:S70-4.

58:452-463

33: 3067-3069

article in press

Hum Hypertens. 1998 Dec;12(12):827-32. Review.

J Gastroentol 2009 September 14; 15(34): 4225-4233.

American Journal of Transplantation 2011; 11: 693–697

liver transplantation. Journal of transplantation

Dovepress DOI: 10.2147/DMSO.S19027

Phramacotherapy 2010; 30(10): 1044-1056

Transplantation 2006; 9 (15): 1234-1248

Immunopharmacology 2000; 47:85-118

Sagrado M, Cítores-Pascual MA, Velicia-Llames R, Caro-Patón A.Risk of development of the metabolic syndrome after orthotopic liver transplantation.

Cardiovascular risk factors after liver transplantation: analysis of related factors.

With an aging population, renovascular hypertension has become a major public health problem (Safian & Textor, 2001). Although various forms of fibromuscular disease of the renal arteries and/or traumatic disruption of renal vessels are the most common cause of RVH among the younger individuals, atherosclerotic renal artery disease (ARAD) is the most common lesion producing hypertension by far (Garovic & Textor, 2005). ARAD is present in over 6.8% of individuals over 65 years of age and is found in up to 49.1% of patients with coronary artery disease or aortoiliac disease (Iglesias et al. 2000; Valabhji et al. 2000; Hansen et al. 2002; Rihal et al. 2002; Weber-Mzell et al. 2002; Textor 2003). Although many patients with asymptomatic renovascular disease do not develop progressive renal dysfunction, overall morbidity and mortality is significantly increased (Chabova et al. 2000; Textor 2002; Textor 2003; Textor 2003; Foley et al. 2005; Foley et al. 2005). On the other hand, some studies suggest that from 10% to 40% of elderly hypertensive patients with newly discovered end stage renal disease and no identifiable parenchymal renal disease have significant RAS (Textor and Wilcox 2001). As in other forms of renal disease, the severity of interstitial fibrosis, tubular atrophy, interstitial inflammation, and glomerular sclerosis are important predictors of renal outcome (Wright et al. 2001). It has been postulated that this acquired tubulointerstitial injury may contribute to at least some forms of essential hypertension (Raghow 1994). Mechanisms underlying vascular and renal dysfunction in RAS have not been well delineated, despite intense study (Textor 2004). This information is essential for the development of therapies – surgical or medical – to treat RAS.

The hallmark of RVH arising from unilateral RAS is atrophy of the stenotic kidney and compensatory hyperplasia/hypertrophy of the contralateral kidney. Although this compensatory hypertrophy serves an adaptive function, this process may render the contralateral kidney more susceptible to other injuries (due to diabetes, glomerulonephritis, etc.) (Wenzel et al. 2002). Although the corresponding histologic, hemodynamic, and tubular alterations in the stenotic and contralateral kidneys have been superficially described in experimental animals, mechanisms underlying these alterations and the identification of markers that predict response to therapy have not been well defined. In particular, the stage in evolution of RAS at which the atrophic changes in the stenotic kidney preclude recovery of renal function after revascularization is not known. This lack of basic mechanistic knowledge is underscored by the variable response of RAS to surgical revascularization; significant

Pathogenesis of Renovascular Hypertension: Challenges and Controversies 51

The 2K1C model has been established in mice using a clip of 0.12 mm (Wiesel et al. 1997). Four weeks after clipping, these investigators reported that 2K1C hypertensive mice exhibited blood pressure approximately 20 mm Hg higher than their sham operated controls. We have recently defined the histopathologic alterations connected with renal artery stenosis in animal model (Figure 1) (Cheng et al., 2009) and human (Keddis et al., 2010). In a murine model of 2K1C of RVH, we found that both the clipped and the contralateral kidney underwent minimal histopathological alterations during the first two weeks following surgery. Subsequently, the clipped kidney underwent atrophy, with generalized tubular atrophy, interstitial fibrosis and focal mononuclear infiltrates, whereas the contralateral kidney underwent hypertrophy/hyperplasia with minimal histopathologic alterations (figure 2). We propose that the murine 2K1C model is a good model of renovascular disease in humans with moderate hypertension. These animal models have helped to elucidate and suggest which cytokines and pathways are involved in RVH. Of these, the renin-angiotensin-aldosterone system, long known to have effects on the hemodynamics of RAS, is becoming more interesting for the inflammatory effects it causes

Fig. 1. Gross picture of the stenotic and contralateral kidney of mice (A) after placement of

Fig. 2. Glomerular appearance of the (A) contralateral and (B) stenotic kidney with H&E

renal artery clip, the 2K1C model, compared to (B) sham procedure.

A **B** 

A B

as well.

staining.

improvement in blood pressure control and recovery of renal function is achieved in only about half of patients, with approximately one-quarter showing no significant changes, and up to one-quarter of patients developing progressive deterioration of renal function (Textor and Wilcox 2001; Textor 2003; Textor 2004). Furthermore, angiotensin-converting enzyme (ACE) inhibitors or angiotensin receptor blockers potentiate hypoperfusion of the stenotic kidney, but have been advocated to prevent deterioration of function in the contralateral kidney (Mann et al. 2001; Schoolwerth et al. 2001). The stage during development of RVH and circumstances in which this treatment should be initiated are not known. This lack of understanding of basic mechanisms underlying the development of human RVH has prompted the development of animal models to address this issue.

#### **1.1 Animal model of renovascular disease**

The classic "Goldblatt" 2K1C rat model of RAS has been extensively used to model human RVH (Goldblatt et al. 1934). In the stenotic kidney, reduced renal perfusion stimulates renin secretion through the renal baroreceptor system, leading to increased plasma levels of angiotensin II (A-II), provoking systemic hypertension (Martinez-Maldonado 1991). A-II may increase blood pressure directly or through elaboration of other vasoconstrictors (such as endothelin, thromboxanes, etc.); aldosterone promotes sodium and water retention and secondarily suppresses renin release. Over time, secondary structural damage occurs to the kidneys, vessels, and other end organs. In this chronic phase, the role of A-II in maintaining elevated blood pressure is not clear, as this phase no longer completely responds to ACE inhibitor therapy. In this chronic phase, the renal damage and endothelial dysfunction may be associated with near-normal renin and A-II levels (Okamura et al. 1986; Carretero and Scicli 1991). Indeed, lack of response to A-II inhibition in experimental animals with sustained RVH may predict lack of response to surgical intervention to remove the RAS (Pipinos et al. 1998).

In the 2K1C model, the weight of the stenotic kidney tends to be lower than that of normal or sham-treated controls, indicating that the kidney has undergone atrophy. The weight of the contralateral kidney is higher than that of normal controls, indicative of a hypertrophic/hyperplastic response. Histopathologic alterations in this model are variable, and probably depend upon the extent of blood pressure elevation. As originally described, the "Goldblatt" 2K1C is a model of accelerated, or "malignant" hypertension, with mean systolic blood pressures >200 mmHg (Goldblatt et al. 1934; Wilson and Byrom 1939; Wilson and Byrom 1940). Under these conditions, the contralateral kidney, despite low renin expression, develops interstitial fibrosis, tubular atrophy, interstitial inflammation, glomerulosclerosis, and hyalinosis (Mai et al. 1993; Sebekova et al. 1998; Kobayashi et al. 1999; Gauer et al. 2003). These chronic tubulointerstitial alterations are associated with increased TGF-β expression (Wenzel et al. 2003). Reported histopathologic alterations in the stenotic kidney are variable, and range from minimal alterations (Eng et al. 1994) to focal interstitial fibrosis and tubular atrophy without significant glomerulosclerosis (Wenzel et al. 2002).

In a rat 2K1C model that develops moderate hypertension (mean arterial pressure 158 mmHg), atrophy of the stenotic kidney and hypertrophy of the contralateral kidney is observed. The stenotic kidney shows increased staining for renin associated with interstitial fibrosis and tubular atrophy, with minimal alterations observed in the contralateral kidney (Richter et al. 2004). In this model, COX-2 inhibitors significantly reduce interstitial fibrosis in the stenotic kidney (Richter et al. 2004).

improvement in blood pressure control and recovery of renal function is achieved in only about half of patients, with approximately one-quarter showing no significant changes, and up to one-quarter of patients developing progressive deterioration of renal function (Textor and Wilcox 2001; Textor 2003; Textor 2004). Furthermore, angiotensin-converting enzyme (ACE) inhibitors or angiotensin receptor blockers potentiate hypoperfusion of the stenotic kidney, but have been advocated to prevent deterioration of function in the contralateral kidney (Mann et al. 2001; Schoolwerth et al. 2001). The stage during development of RVH and circumstances in which this treatment should be initiated are not known. This lack of understanding of basic mechanisms underlying the development of human RVH has prompted the development of

The classic "Goldblatt" 2K1C rat model of RAS has been extensively used to model human RVH (Goldblatt et al. 1934). In the stenotic kidney, reduced renal perfusion stimulates renin secretion through the renal baroreceptor system, leading to increased plasma levels of angiotensin II (A-II), provoking systemic hypertension (Martinez-Maldonado 1991). A-II may increase blood pressure directly or through elaboration of other vasoconstrictors (such as endothelin, thromboxanes, etc.); aldosterone promotes sodium and water retention and secondarily suppresses renin release. Over time, secondary structural damage occurs to the kidneys, vessels, and other end organs. In this chronic phase, the role of A-II in maintaining elevated blood pressure is not clear, as this phase no longer completely responds to ACE inhibitor therapy. In this chronic phase, the renal damage and endothelial dysfunction may be associated with near-normal renin and A-II levels (Okamura et al. 1986; Carretero and Scicli 1991). Indeed, lack of response to A-II inhibition in experimental animals with sustained RVH may predict lack of response to surgical intervention to remove the RAS (Pipinos et al. 1998). In the 2K1C model, the weight of the stenotic kidney tends to be lower than that of normal or sham-treated controls, indicating that the kidney has undergone atrophy. The weight of the contralateral kidney is higher than that of normal controls, indicative of a hypertrophic/hyperplastic response. Histopathologic alterations in this model are variable, and probably depend upon the extent of blood pressure elevation. As originally described, the "Goldblatt" 2K1C is a model of accelerated, or "malignant" hypertension, with mean systolic blood pressures >200 mmHg (Goldblatt et al. 1934; Wilson and Byrom 1939; Wilson and Byrom 1940). Under these conditions, the contralateral kidney, despite low renin expression, develops interstitial fibrosis, tubular atrophy, interstitial inflammation, glomerulosclerosis, and hyalinosis (Mai et al. 1993; Sebekova et al. 1998; Kobayashi et al. 1999; Gauer et al. 2003). These chronic tubulointerstitial alterations are associated with increased TGF-β expression (Wenzel et al. 2003). Reported histopathologic alterations in the stenotic kidney are variable, and range from minimal alterations (Eng et al. 1994) to focal interstitial fibrosis and tubular

animal models to address this issue.

**1.1 Animal model of renovascular disease** 

atrophy without significant glomerulosclerosis (Wenzel et al. 2002).

in the stenotic kidney (Richter et al. 2004).

In a rat 2K1C model that develops moderate hypertension (mean arterial pressure 158 mmHg), atrophy of the stenotic kidney and hypertrophy of the contralateral kidney is observed. The stenotic kidney shows increased staining for renin associated with interstitial fibrosis and tubular atrophy, with minimal alterations observed in the contralateral kidney (Richter et al. 2004). In this model, COX-2 inhibitors significantly reduce interstitial fibrosis The 2K1C model has been established in mice using a clip of 0.12 mm (Wiesel et al. 1997). Four weeks after clipping, these investigators reported that 2K1C hypertensive mice exhibited blood pressure approximately 20 mm Hg higher than their sham operated controls. We have recently defined the histopathologic alterations connected with renal artery stenosis in animal model (Figure 1) (Cheng et al., 2009) and human (Keddis et al., 2010). In a murine model of 2K1C of RVH, we found that both the clipped and the contralateral kidney underwent minimal histopathological alterations during the first two weeks following surgery. Subsequently, the clipped kidney underwent atrophy, with generalized tubular atrophy, interstitial fibrosis and focal mononuclear infiltrates, whereas the contralateral kidney underwent hypertrophy/hyperplasia with minimal histopathologic alterations (figure 2). We propose that the murine 2K1C model is a good model of renovascular disease in humans with moderate hypertension. These animal models have helped to elucidate and suggest which cytokines and pathways are involved in RVH. Of these, the renin-angiotensin-aldosterone system, long known to have effects on the hemodynamics of RAS, is becoming more interesting for the inflammatory effects it causes as well.

Fig. 1. Gross picture of the stenotic and contralateral kidney of mice (A) after placement of renal artery clip, the 2K1C model, compared to (B) sham procedure.

Fig. 2. Glomerular appearance of the (A) contralateral and (B) stenotic kidney with H&E staining.

Pathogenesis of Renovascular Hypertension: Challenges and Controversies 53

stenotic kidney caused by these anti-hypertensive agents. With already compromised blood flow, concern about increased damage to the ischemic kidney must be weighed with the benefits to the contralateral kidney (Jackson et al., 1986). With substanstial arterial obstruction, simply reducing perfusion pressure can reduce post-stenotic blood flow beyond that required for metabolic demands in the kidney. Early experimental studies in rats emphasized the potential for irreversible damage to the clipped kidney in animals treated with ACE inhibitors, resulting in "medical nephrectomy" (Jackson, 1990). However, it is important to note that this adverse reaction can develop with any forms of antihypertensive therapy (Textor et al., 1983). The risk factors for this adverse event include older age groups, pre-existing renal dysfunction, and episodes of acute illness leading to volume depletion (such as diarrhea or reduced intake during diuretic administration) (Speirs, et al., 1988).

With the increasing evidence of the involvement of TGF-, in RAS induced damage, the role of the RAAS in RAS, especially with respect to its induction of an inflammatory response, is being re-thought. There should be careful consideration and evaluation of the role of the immunologic and cytokine-associated effects of RAAS in the pathological process and initiation of RAS induced kidney damage. Such research may shed new light on whether the

Mechanisms underlying the differential response of the stenotic and contralateral kidney during the development and progression of RVH have not been adequately defined, despite numerous studies (Goldblatt et al., 1934; Martinez-Maldonado, 1991; Carretero, 1991). TGFβ is involved in a number of processes relevant to the development of RVH, including cell cycle regulation leading to hypertrophy and/or apoptosis, MAPK activation, inflammation,

It is well recognized that TGF-β plays a central role in fibrotic diseases (Cheng & Grande, 2002; Border et al., 1990; Border & Noble, 1994, 1997; Border & Ruoslahti, 1992). All aspects of fibrogenesis have been shown to be regulated by TGF-β, including the initial inflammatory phase in which infiltrating inflammatory cells and macrophages set the stage for the subsequent fibrotic phase in which activated fibroblasts and myofibroblasts contribute to the pathogenic accumulation of matrix (Cheng et al., 2005). In the past few years, receptors and signal transduction pathways mediating the effects of TGF-β on cells have been identified, enabling the identification of specific pathways involved in pathogenic events dependent on this cytokine. TGF-β signals through a set of transmembrane receptor serine/threonine kinases unique to the larger superfamily of TGF-β-related proteins. The active heteromeric receptor complex is formed by binding of ligand to a type II receptor, recruitment and activation of the type I receptor kinase, and phosphorylation of intracellular mediating target proteins (Massague, 1992, 1998; Attisano et al., 1994). Increased TGFβ receptor expression is observed in experimental glomerulonephritis (Shankland et al., 1996; Tamaki et al., 1994). In experimental renal disease associated with epithelial to mesenchymal transformation, TGF-β type 1 receptor expression is increased in tubular epithelial cells (Yang & Liu, 2002). Downstream mediators are the Smad family of proteins (Piek et al., 1999). Smad2 and 3 are phosphorylated directly by the type I receptor kinase, after which they partner with Smad4 and translocate to the nucleus where they act as transcriptional regulators of target genes, including those essential for apoptosis,

benefits of RAAS inhibition outweigh the costs in patients with RAS.

and extracellular matrix synthesis (Cheng & Grande, 2002).

**1.3 TGF-**β **and renovascular disease** 

#### **1.2 The Renin-Angiotensin System and renovascular disease**

Though its normal function is to preserve organ perfusion by regulating sodium and water balance, extracellular fluid volume and cardiac activity, the Renin-Angiotensin-Aldosterone System (RAAS) also plays an integral part in RVH. The RAAS has been found to be significantly activated in the presence of RAS. It is known that the increase in blood pressure caused by the RAAS has significant detrimental effects on the body, but it may also play a number of other roles in the development and progress of RVH.

Renin is made primarily by the juxtaglomerular cells in the Juxtaglomerular apparatus in response to 1) low pressure in the afferent renal artery, 2) sympathetic nervous system activity 3) A-II levels and 4) low sodium delivered to the macular densa in the distal convoluted tubule of the nephron. Other signals, such as potassium concentration, atrial natruretic peptide and endothelin also modulate renin synthesis. Renin enzymatically converts angiotensinogen, made in the liver, to angiotensin I. ACE, synthesized primarily in the lungs (though also in other tissues), then converts angiotensin I to A-II, a significantly biologically active molecule. A-II, in addition to its numerous tissue effects, induces the synthesis of aldosterone in the zona glomerulosa of the adrenal medulla, which then acts on mineralocorticoid receptors throughout the body, though when considering RAS, their most notable function is in the kidney (Laragh et al., 1992). This pathway is also located completely within other organs, including, the kidney. The proximal tubule, interstitium and medulla of the kidney have higher-than-systemic concentrations of A-II, because of local synthesis, which allows it to act in a paracrine function (Johnston et al., 1992). A-II and aldosterone can also be synthesized through an ACE-independent pathway, which is what allows for the return to normal A-II levels in the presence of ACE inhibition. This effect is also particularly prominent in the kidneys, where an estimated 40% of renally-synthesized A-II does not rely on ACE (Hollenberg, 1999).

A-II acts on 2 different receptors (angiotensin receptor type 1, or AT-1, and angiotensin receptor type 2, or AT-2) producing very different biological responses. Of these, AT-1 appears to play the most significant role in renal vascular disease (AT-2 is mostly known for its role in fetal organ development). Stimulation of the AT-1 receptor is best known for its systemic vasoconstrictive effects, and its vasoconstrictive effects on the efferent renal arteriole. The former causes general rises in systolic blood pressure, while the later decreases renal plasma flow, but increases glomerular filtration fraction. AT-1 stimulation also causes salt-retention through a number of mechanisms, increasing blood pressure even further (Dzau & Re, 1994; Liu & Cogan, 1989; Brewster & Perazella, 2004). A-II also mediates nonhemodynamic effects. A-II has the ability to promote fibrosis through a number of mechanisms, including induction of collagen synthesis, inhibition of collagen-cleaving proteases, stimulation of the secretion of platelet-derived growth factor, and, most interestingly, direct stimulation of TGF-, receptor type II(Luft, 2003; Wolf, 2000). AT-1 stimulation also has the ability to promote fibrosis by up-regulating expression and synthesis of NF-.B and thus TGF-,, as well as a number of other cytokines (Tsuzuki, 1996). Downstream, aldosterone, in addition to its hypertensive effects mediated through the mineralocorticoid receptor, also up-regulates the expression of TGF-, (Juknevicius et al., 2000). These studies have supported a widespread use of RAAS inhibitors (ACE-inhibitors, AT-1 inhibitors and aldosterone receptor inhibitors) to prevent renal disease progression. However, there is concern with the often-seen deleterious effects on renal function of the stenotic kidney caused by these anti-hypertensive agents. With already compromised blood flow, concern about increased damage to the ischemic kidney must be weighed with the benefits to the contralateral kidney (Jackson et al., 1986). With substanstial arterial obstruction, simply reducing perfusion pressure can reduce post-stenotic blood flow beyond that required for metabolic demands in the kidney. Early experimental studies in rats emphasized the potential for irreversible damage to the clipped kidney in animals treated with ACE inhibitors, resulting in "medical nephrectomy" (Jackson, 1990). However, it is important to note that this adverse reaction can develop with any forms of antihypertensive therapy (Textor et al., 1983). The risk factors for this adverse event include older age groups, pre-existing renal dysfunction, and episodes of acute illness leading to volume depletion (such as diarrhea or reduced intake during diuretic administration) (Speirs, et al., 1988).

With the increasing evidence of the involvement of TGF-, in RAS induced damage, the role of the RAAS in RAS, especially with respect to its induction of an inflammatory response, is being re-thought. There should be careful consideration and evaluation of the role of the immunologic and cytokine-associated effects of RAAS in the pathological process and initiation of RAS induced kidney damage. Such research may shed new light on whether the benefits of RAAS inhibition outweigh the costs in patients with RAS.

#### **1.3 TGF-**β **and renovascular disease**

52 Recent Advances in Cardiovascular Risk Factors

Though its normal function is to preserve organ perfusion by regulating sodium and water balance, extracellular fluid volume and cardiac activity, the Renin-Angiotensin-Aldosterone System (RAAS) also plays an integral part in RVH. The RAAS has been found to be significantly activated in the presence of RAS. It is known that the increase in blood pressure caused by the RAAS has significant detrimental effects on the body, but it may also play a

Renin is made primarily by the juxtaglomerular cells in the Juxtaglomerular apparatus in response to 1) low pressure in the afferent renal artery, 2) sympathetic nervous system activity 3) A-II levels and 4) low sodium delivered to the macular densa in the distal convoluted tubule of the nephron. Other signals, such as potassium concentration, atrial natruretic peptide and endothelin also modulate renin synthesis. Renin enzymatically converts angiotensinogen, made in the liver, to angiotensin I. ACE, synthesized primarily in the lungs (though also in other tissues), then converts angiotensin I to A-II, a significantly biologically active molecule. A-II, in addition to its numerous tissue effects, induces the synthesis of aldosterone in the zona glomerulosa of the adrenal medulla, which then acts on mineralocorticoid receptors throughout the body, though when considering RAS, their most notable function is in the kidney (Laragh et al., 1992). This pathway is also located completely within other organs, including, the kidney. The proximal tubule, interstitium and medulla of the kidney have higher-than-systemic concentrations of A-II, because of local synthesis, which allows it to act in a paracrine function (Johnston et al., 1992). A-II and aldosterone can also be synthesized through an ACE-independent pathway, which is what allows for the return to normal A-II levels in the presence of ACE inhibition. This effect is also particularly prominent in the kidneys, where an estimated 40% of renally-synthesized

A-II acts on 2 different receptors (angiotensin receptor type 1, or AT-1, and angiotensin receptor type 2, or AT-2) producing very different biological responses. Of these, AT-1 appears to play the most significant role in renal vascular disease (AT-2 is mostly known for its role in fetal organ development). Stimulation of the AT-1 receptor is best known for its systemic vasoconstrictive effects, and its vasoconstrictive effects on the efferent renal arteriole. The former causes general rises in systolic blood pressure, while the later decreases renal plasma flow, but increases glomerular filtration fraction. AT-1 stimulation also causes salt-retention through a number of mechanisms, increasing blood pressure even further (Dzau & Re, 1994; Liu & Cogan, 1989; Brewster & Perazella, 2004). A-II also mediates nonhemodynamic effects. A-II has the ability to promote fibrosis through a number of mechanisms, including induction of collagen synthesis, inhibition of collagen-cleaving proteases, stimulation of the secretion of platelet-derived growth factor, and, most interestingly, direct stimulation of TGF-, receptor type II(Luft, 2003; Wolf, 2000). AT-1 stimulation also has the ability to promote fibrosis by up-regulating expression and synthesis of NF-.B and thus TGF-,, as well as a number of other cytokines (Tsuzuki, 1996). Downstream, aldosterone, in addition to its hypertensive effects mediated through the mineralocorticoid receptor, also up-regulates the expression of TGF-, (Juknevicius et al., 2000). These studies have supported a widespread use of RAAS inhibitors (ACE-inhibitors, AT-1 inhibitors and aldosterone receptor inhibitors) to prevent renal disease progression. However, there is concern with the often-seen deleterious effects on renal function of the

**1.2 The Renin-Angiotensin System and renovascular disease** 

number of other roles in the development and progress of RVH.

A-II does not rely on ACE (Hollenberg, 1999).

Mechanisms underlying the differential response of the stenotic and contralateral kidney during the development and progression of RVH have not been adequately defined, despite numerous studies (Goldblatt et al., 1934; Martinez-Maldonado, 1991; Carretero, 1991). TGFβ is involved in a number of processes relevant to the development of RVH, including cell cycle regulation leading to hypertrophy and/or apoptosis, MAPK activation, inflammation, and extracellular matrix synthesis (Cheng & Grande, 2002).

It is well recognized that TGF-β plays a central role in fibrotic diseases (Cheng & Grande, 2002; Border et al., 1990; Border & Noble, 1994, 1997; Border & Ruoslahti, 1992). All aspects of fibrogenesis have been shown to be regulated by TGF-β, including the initial inflammatory phase in which infiltrating inflammatory cells and macrophages set the stage for the subsequent fibrotic phase in which activated fibroblasts and myofibroblasts contribute to the pathogenic accumulation of matrix (Cheng et al., 2005). In the past few years, receptors and signal transduction pathways mediating the effects of TGF-β on cells have been identified, enabling the identification of specific pathways involved in pathogenic events dependent on this cytokine. TGF-β signals through a set of transmembrane receptor serine/threonine kinases unique to the larger superfamily of TGF-β-related proteins. The active heteromeric receptor complex is formed by binding of ligand to a type II receptor, recruitment and activation of the type I receptor kinase, and phosphorylation of intracellular mediating target proteins (Massague, 1992, 1998; Attisano et al., 1994). Increased TGFβ receptor expression is observed in experimental glomerulonephritis (Shankland et al., 1996; Tamaki et al., 1994). In experimental renal disease associated with epithelial to mesenchymal transformation, TGF-β type 1 receptor expression is increased in tubular epithelial cells (Yang & Liu, 2002). Downstream mediators are the Smad family of proteins (Piek et al., 1999). Smad2 and 3 are phosphorylated directly by the type I receptor kinase, after which they partner with Smad4 and translocate to the nucleus where they act as transcriptional regulators of target genes, including those essential for apoptosis,

Pathogenesis of Renovascular Hypertension: Challenges and Controversies 55

experimental anti-glomerular basement membrane antibody mediated glomerulonephritis (Stambe et al. 2003). In a variety of other human renal diseases, p-ERK expression is observed in regions of tubulointerstitial damage, within α-SMA positive myofibroblasts (Masaki et al. 2004). In cultured cells, ERK is involved in epithelial to mesenchymal transformation (Li et al. 2004; Xie et al. 2004; Yang et al. 2004). We have previously identified ERK, p38, and JNK as essential intermediates for MC mitogenesis, and ERK and p38 as essential intermediates for TGF-β stimulated collagen IV mRNA expression and MCP-1 production (Cheng et al. 2002; Cheng et al. 2004). We have also shown that ERK is significantly upregulated in a rat model of salt sensitive hypertension (Diaz et al., 2008). Others have shown that p38 activation is necessary for TGF-β stimulation of fibronectin

The MAPK pathways are involved in regulation of cell cycle arrest and apoptosis, which is of direct relevance to the renal atrophy which occurs in the stenotic kidney of the 2K1C RAS model. Activation of ERK is necessary for TGF-β-mediated induction of p21 and cell cycle arrest (Hu et al. 1999). High glucose promotes hypertrophy of MC through ERK mediated phosphorylation of p27 (Wolf et al. 2003). Activation of p38 or JNK is frequently associated with cell cycle arrest or apoptosis (Cardone et al. 1997; Frasch et al. 1998). Induction of apoptosis in MC requires sustained activation of JNK (Guo et al. 1998). Apoptosis and other cellular responses may be directed by a balance between ERK and JNK activation (Xia et al.

Based on these considerations, there has been intense interest in developing low molecular weight pathway specific MAPK inhibitors as therapeutic agents to treat cancer and fibroproliferative inflammatory conditions (Duncia et al. 1998; Sebolt-Leopold et al. 1999; Clemons et al. 2002; Duan et al. 2004; Sebolt-Leopold and Herrera 2004; Jo et al. 2005; McDaid et al. 2005). These agents have been employed in experimental renovascular disease, with mixed results. The ERK inhibitor U0126 was effective in reducing acute renal injury in an experimental mesangial proliferative glomerulonephritis model (Bokemeyer et al. 2002). In human renal diseases associated with injury to podocytes, p38 is induced. The p38 inhibitor FR167653 prevents renal dysfunction and glomerulosclerosis in chronic adriamycin nephropathy (Koshikawa et al. 2005) and in experimental crescenteric glomerulonephritis (Wada et al. 2001). Similarly, the p38 inhibitor NPC31145 reduced acute inflammatory injury in an experimental anti-glomerular basement membrane glomerulonephritis model (Stambe et al. 2003). On the other hand, the p38 inhibitor FR167653 increased proteinuria in a passive Heymann nephritis model of podocyte injury (Aoudjit et al. 2003), suggesting that activation of p38 protects podocytes from complement mediated injury. Furthermore, the p38 inhibitor NPC31169 exacerbated renal damage in a

remnant kidney model due to in vivo induction of ERK (Ohashi et al. 2004).

RVH initiates activation of the renin–angiotensin system and structural remodeling, evidenced by fibrosis and vascular deterioration in the affected kidney. Although the renin– angiotensin system tends to resolve once a stable blood pressure (BP) is reached, it has been suggested that transient elevation of plasma A-II could precipitate macrophage infiltration, thereby initiating an inflammatory response within the kidney (Ozawa et al., 2007). This inflammatory cascade may well underlie the degenerative processes within the kidney as its

**1.5 The role of inflammation in renovascular disease** 

renal artery begins to narrow.

production (Suzuki et al. 2004).

1995).

inflammation, differentiation, and growth inhibition (Massague & Wotton, 2000; Derynck et al., 1998; Attisano et al., 2001). TGF-β plays a critical role in chronic inflammatory changes of the interstitium and extracellular matrix accumulation during fibrogenesis (Cheng & Grande, 2002; Grande et al., 1997, 2002). TGF-β initiates the transition of renal tubular epithelial cells to myofibroblasts, the cellular source for extracellular matrix deposition, leading to irreversible renal failure (Yang & Liu, 2001; Iwano et al., 2002; Li et al., 2002).

A predominant role of TGF-β1 in regulation of extracellular matrix deposition is highlighted in our published studies employing renal tubular epithelial cells derived from animals bearing a homozygous deletion of the TGF-β1 gene (Grande et al, 2002). Although the most direct means to test the hypothesis that T TGF-β1 plays a central role in the development of RVH would be to perform these studies in mice bearing homozygous deletion of the TGF-β1 gene, the phenotype of these animals precludes such studies. TGF-β1 KO animals have an extremely high rate of embryonic lethality, and the few surviving mice develop a systemic inflammatory syndrome, leading to their death within 2-4 weeks of age (Letterio et al, 1994; Martin et al., 1995; Kulkarni & Karlsson, 1993; Boivin et al., 1995). For this reason, more recent studies have employed mice with genetic manipulation of the Smad proteins to define potential mechanisms by which the TGF-β signaling pathway is involved in chronic tissue injury. Smad3 KO mice show accelerated healing of wounds, in association with decreased local inflammation (Ashcroft et al., 1999). Smad3-null mice have been used in several chronic injury models, including ureteric obstruction (Sato et al., 2003). In WT mice, unilateral ureteric obstruction (UUO) produces extensive interstitial fibrosis and tubular atrophy, with TGF-β1-driven epithelial to mesenchymal transformation of tubular epithelial cells, as evidenced by reduction in E-cadherin staining and de novo induction of α-smooth muscle actin (α-SMA) staining. This is associated with extensive influx of monocytes. In Smad3 KO animals subjected to UUO, there was a marked reduction in interstitial fibrosis, and epithelial to mesenchymal transformation, indicating that the Smad pathway is necessary for epithelial to mesenchymal transformation by TGF-β (Itoh et al., 2003; Yu et al., 2002).

#### **1.4 MAPK pathways and renovascular disease**

It is well recognized that cellular adaptive responses to environmental stimuli, including hypertrophy, hyperplasia, and atrophy associated with increased apoptotic activity, are transduced through the MAPK pathway(s) (Kyriakis 2000; Kyriakis and Avruch 2001). Cardiac hypertrophy in A-II dependent hypertension is associated with activation of p38, whereas ERK and JNK are preferentially stimulated in an A-II independent model of RVH (Pellieux et al. 2000). The development of hypertension is associated with persistent ERK activation in the aorta of Dahl salt-sensitive rats and stroke-prone spontaneously hypertensive rats (Kim et al. 1997; Hamaguchi et al. 2000). In human diabetic nephropathy, there is increased immunohistochemical staining for p-ERK in glomeruli which correlates with the severity of glomerular lesions and increased p-p38 staining which correlates with severity of tubulointerstitial lesions and number of CD68-positive macrophages (Adhikary et al. 2004; Toyoda et al. 2004; Sakai et al. 2005). Hypertension accelerates the development of diabetic nephropathy in a rat model of type 2 diabetes through induction of ERK and p38, as well as TGF-β (Imai et al. 2003). Similarly, the p38 and JNK pathways are activated in the early stages of experimental proliferative glomerulonephritis, whereas the ERK pathway is persistently activated (Bokemeyer et al. 1998). Both p38 and JNK are activated in

inflammation, differentiation, and growth inhibition (Massague & Wotton, 2000; Derynck et al., 1998; Attisano et al., 2001). TGF-β plays a critical role in chronic inflammatory changes of the interstitium and extracellular matrix accumulation during fibrogenesis (Cheng & Grande, 2002; Grande et al., 1997, 2002). TGF-β initiates the transition of renal tubular epithelial cells to myofibroblasts, the cellular source for extracellular matrix deposition, leading to irreversible renal failure (Yang & Liu, 2001; Iwano et al., 2002; Li et al., 2002).

A predominant role of TGF-β1 in regulation of extracellular matrix deposition is highlighted in our published studies employing renal tubular epithelial cells derived from animals bearing a homozygous deletion of the TGF-β1 gene (Grande et al, 2002). Although the most direct means to test the hypothesis that T TGF-β1 plays a central role in the development of RVH would be to perform these studies in mice bearing homozygous deletion of the TGF-β1 gene, the phenotype of these animals precludes such studies. TGF-β1 KO animals have an extremely high rate of embryonic lethality, and the few surviving mice develop a systemic inflammatory syndrome, leading to their death within 2-4 weeks of age (Letterio et al, 1994; Martin et al., 1995; Kulkarni & Karlsson, 1993; Boivin et al., 1995). For this reason, more recent studies have employed mice with genetic manipulation of the Smad proteins to define potential mechanisms by which the TGF-β signaling pathway is involved in chronic tissue injury. Smad3 KO mice show accelerated healing of wounds, in association with decreased local inflammation (Ashcroft et al., 1999). Smad3-null mice have been used in several chronic injury models, including ureteric obstruction (Sato et al., 2003). In WT mice, unilateral ureteric obstruction (UUO) produces extensive interstitial fibrosis and tubular atrophy, with TGF-β1-driven epithelial to mesenchymal transformation of tubular epithelial cells, as evidenced by reduction in E-cadherin staining and de novo induction of α-smooth muscle actin (α-SMA) staining. This is associated with extensive influx of monocytes. In Smad3 KO animals subjected to UUO, there was a marked reduction in interstitial fibrosis, and epithelial to mesenchymal transformation, indicating that the Smad pathway is necessary for

epithelial to mesenchymal transformation by TGF-β (Itoh et al., 2003; Yu et al., 2002).

It is well recognized that cellular adaptive responses to environmental stimuli, including hypertrophy, hyperplasia, and atrophy associated with increased apoptotic activity, are transduced through the MAPK pathway(s) (Kyriakis 2000; Kyriakis and Avruch 2001). Cardiac hypertrophy in A-II dependent hypertension is associated with activation of p38, whereas ERK and JNK are preferentially stimulated in an A-II independent model of RVH (Pellieux et al. 2000). The development of hypertension is associated with persistent ERK activation in the aorta of Dahl salt-sensitive rats and stroke-prone spontaneously hypertensive rats (Kim et al. 1997; Hamaguchi et al. 2000). In human diabetic nephropathy, there is increased immunohistochemical staining for p-ERK in glomeruli which correlates with the severity of glomerular lesions and increased p-p38 staining which correlates with severity of tubulointerstitial lesions and number of CD68-positive macrophages (Adhikary et al. 2004; Toyoda et al. 2004; Sakai et al. 2005). Hypertension accelerates the development of diabetic nephropathy in a rat model of type 2 diabetes through induction of ERK and p38, as well as TGF-β (Imai et al. 2003). Similarly, the p38 and JNK pathways are activated in the early stages of experimental proliferative glomerulonephritis, whereas the ERK pathway is persistently activated (Bokemeyer et al. 1998). Both p38 and JNK are activated in

**1.4 MAPK pathways and renovascular disease** 

experimental anti-glomerular basement membrane antibody mediated glomerulonephritis (Stambe et al. 2003). In a variety of other human renal diseases, p-ERK expression is observed in regions of tubulointerstitial damage, within α-SMA positive myofibroblasts (Masaki et al. 2004). In cultured cells, ERK is involved in epithelial to mesenchymal transformation (Li et al. 2004; Xie et al. 2004; Yang et al. 2004). We have previously identified ERK, p38, and JNK as essential intermediates for MC mitogenesis, and ERK and p38 as essential intermediates for TGF-β stimulated collagen IV mRNA expression and MCP-1 production (Cheng et al. 2002; Cheng et al. 2004). We have also shown that ERK is significantly upregulated in a rat model of salt sensitive hypertension (Diaz et al., 2008). Others have shown that p38 activation is necessary for TGF-β stimulation of fibronectin production (Suzuki et al. 2004).

The MAPK pathways are involved in regulation of cell cycle arrest and apoptosis, which is of direct relevance to the renal atrophy which occurs in the stenotic kidney of the 2K1C RAS model. Activation of ERK is necessary for TGF-β-mediated induction of p21 and cell cycle arrest (Hu et al. 1999). High glucose promotes hypertrophy of MC through ERK mediated phosphorylation of p27 (Wolf et al. 2003). Activation of p38 or JNK is frequently associated with cell cycle arrest or apoptosis (Cardone et al. 1997; Frasch et al. 1998). Induction of apoptosis in MC requires sustained activation of JNK (Guo et al. 1998). Apoptosis and other cellular responses may be directed by a balance between ERK and JNK activation (Xia et al. 1995).

Based on these considerations, there has been intense interest in developing low molecular weight pathway specific MAPK inhibitors as therapeutic agents to treat cancer and fibroproliferative inflammatory conditions (Duncia et al. 1998; Sebolt-Leopold et al. 1999; Clemons et al. 2002; Duan et al. 2004; Sebolt-Leopold and Herrera 2004; Jo et al. 2005; McDaid et al. 2005). These agents have been employed in experimental renovascular disease, with mixed results. The ERK inhibitor U0126 was effective in reducing acute renal injury in an experimental mesangial proliferative glomerulonephritis model (Bokemeyer et al. 2002). In human renal diseases associated with injury to podocytes, p38 is induced. The p38 inhibitor FR167653 prevents renal dysfunction and glomerulosclerosis in chronic adriamycin nephropathy (Koshikawa et al. 2005) and in experimental crescenteric glomerulonephritis (Wada et al. 2001). Similarly, the p38 inhibitor NPC31145 reduced acute inflammatory injury in an experimental anti-glomerular basement membrane glomerulonephritis model (Stambe et al. 2003). On the other hand, the p38 inhibitor FR167653 increased proteinuria in a passive Heymann nephritis model of podocyte injury (Aoudjit et al. 2003), suggesting that activation of p38 protects podocytes from complement mediated injury. Furthermore, the p38 inhibitor NPC31169 exacerbated renal damage in a remnant kidney model due to in vivo induction of ERK (Ohashi et al. 2004).

#### **1.5 The role of inflammation in renovascular disease**

RVH initiates activation of the renin–angiotensin system and structural remodeling, evidenced by fibrosis and vascular deterioration in the affected kidney. Although the renin– angiotensin system tends to resolve once a stable blood pressure (BP) is reached, it has been suggested that transient elevation of plasma A-II could precipitate macrophage infiltration, thereby initiating an inflammatory response within the kidney (Ozawa et al., 2007). This inflammatory cascade may well underlie the degenerative processes within the kidney as its renal artery begins to narrow.

Pathogenesis of Renovascular Hypertension: Challenges and Controversies 57

Fig. 3. MCP-1 staining in the (A) contralateral kidney and (B) stenotic kidney of C57BLKS

At a cellular level, it is apparent that the renal artery stenosis did elicit an inflammatory cascade in the kidney as evidenced by macrophage infiltration, the rise in MCP-1 and its receptor chemokine (C-C motif) receptor 2 (CCR2), NF.B, protein kinase C (PKC) and TGF- ,. Remarkably, we also saw transient increase in MCP-1 and TGF-, in the contralateral kidney which indicates some inflammatory process taking place despite lack of inflammatory cells and/or tissue damage. It is apparent that blockade of the MCP-1 receptor does offer renal protection and prevents the progressive fibrosis development in renovascular hypertension. Elucidating the underlying mechanisms of this protection will allow us to develop preventive measures and novel therapeutic interventions that could

Hypertension is one of the most common reasons for a visit to a physician. There are several key issues that need to be addressed during evaluation of a patient with hypertension: accurate blood pressure reading, determination of target organ damage due to hypertension, screening for other cardiovascular risk factor, stratification of cardiovascular disease, and assessment for the cause of hypertension (primary vs. secondary hypertension). Thorough assessment of the cause of hypertension is essential for determining the correct treatment approach, especially in children where atherosclerosis is not common. For children with hypertension, it is necessary to consider genetic diseases (i.e. coarctation of the aorta, primary aldosteronism) and auto-immune diseases (i.e. post-infectiousglomerulonephritis). When initiating treatment, it is important to maintain low systolic pressure as systolic pressure is a stronger predictor of cardiovascular event (Mancia et al. 2009; Cherubini et al. 2010). Maintenance of blood pressure goal should ideally be achieved

Optimal management of renovascular hypertension requires an understanding of the disease process and remains an important challenge for clinicians caring for patients with hypertension. Although the pathophysiology and the consequence to human health caused

mouse 4 weeks after placement of renal artery clip.

**A B** 

possibly be applied to other renal diseases.

within 6 to 9 months of therapeutic initiation.

**1.6 Therapeutic implication** 

**2. Conclusion** 

We performed PCR array studies of renal homogenates obtained from mice subjected to RAS or sham surgery. The results of our PCR Array studies (Table 1) implicate monocyte chemoattractant protein-1 (MCP-1) as a key chemokine in this inflammatory response. In addition to infiltrating inflammatory cells, tubular epithelial cells of the stenotic kidney of mice exposed to RAS express high level of MCP-1 (figure 3). Our findings are in accord with those of other investigators who have studied renovascular hypertension. High salt diet in DSS rats caused expression of NADPH oxidase and MCP-1 in the dilated renal tubules and resulting in interstitial inflammation and migration of mononuclear cells (Shigemoto et al., 2007). Increased MCP-1 levels also seem to stimulate TGF-, formation in glomerular cells despite the absence of infiltrating inflammatory cells (Wolf et al., 2002). Studies in the swine model of renovascular hypertension using bindarit, a selective MCP-1 blocker, show that inhibition of MCP-1 confers renal protective effects by blunting renal inflammation and reducing the level of collagen deposition, thereby preserving the kidney in chronic RAS (Zoja et al., 1998; Zhu et al., 2009). It was further indicated that MCP-1 contributes to functional and structural impairment in the RAS kidney, specifically in the tubulo-interstitial compartment.


Table 1. PCR array results of proinflammatory cytokine expression in stenotic kidneys of RAS mouse

Fig. 3. MCP-1 staining in the (A) contralateral kidney and (B) stenotic kidney of C57BLKS mouse 4 weeks after placement of renal artery clip.

At a cellular level, it is apparent that the renal artery stenosis did elicit an inflammatory cascade in the kidney as evidenced by macrophage infiltration, the rise in MCP-1 and its receptor chemokine (C-C motif) receptor 2 (CCR2), NF.B, protein kinase C (PKC) and TGF- ,. Remarkably, we also saw transient increase in MCP-1 and TGF-, in the contralateral kidney which indicates some inflammatory process taking place despite lack of inflammatory cells and/or tissue damage. It is apparent that blockade of the MCP-1 receptor does offer renal protection and prevents the progressive fibrosis development in renovascular hypertension. Elucidating the underlying mechanisms of this protection will allow us to develop preventive measures and novel therapeutic interventions that could possibly be applied to other renal diseases.

#### **1.6 Therapeutic implication**

56 Recent Advances in Cardiovascular Risk Factors

We performed PCR array studies of renal homogenates obtained from mice subjected to RAS or sham surgery. The results of our PCR Array studies (Table 1) implicate monocyte chemoattractant protein-1 (MCP-1) as a key chemokine in this inflammatory response. In addition to infiltrating inflammatory cells, tubular epithelial cells of the stenotic kidney of mice exposed to RAS express high level of MCP-1 (figure 3). Our findings are in accord with those of other investigators who have studied renovascular hypertension. High salt diet in DSS rats caused expression of NADPH oxidase and MCP-1 in the dilated renal tubules and resulting in interstitial inflammation and migration of mononuclear cells (Shigemoto et al., 2007). Increased MCP-1 levels also seem to stimulate TGF-, formation in glomerular cells despite the absence of infiltrating inflammatory cells (Wolf et al., 2002). Studies in the swine model of renovascular hypertension using bindarit, a selective MCP-1 blocker, show that inhibition of MCP-1 confers renal protective effects by blunting renal inflammation and reducing the level of collagen deposition, thereby preserving the kidney in chronic RAS (Zoja et al., 1998; Zhu et al., 2009). It was further indicated that MCP-1 contributes to functional and structural impairment in the

**Gene function Gene symbol Gene name Fold change**

Ccl3 MIP-1+ (macrophage inflammatory protein

Ccl4 MIP-1, (macrophage inflammatory protein

Ccl5 RANTES (regulated upon activation, normal

Ccl20 MIP-3+ (macrophage inflammatory protein

Cxcl3 MIP-2, (macrophage inflammatory protein

Cxcl5 AMCF-II (alveolar macrophage chemotactic

Table 1. PCR array results of proinflammatory cytokine expression in stenotic kidneys of

Cxcl9 Mig (monokine induced by --interferon) +5 IL1+ Interleukin-1+ +18 IL1, Interleukin-1, +11

Ccl22 MDC (macrophage derived chemokine) +41 Ccr2 Chemokine (C-C motif) receptor 2 +30 Ccr3 Chemokine (C-C motif) receptor 3 +35 Ccr4 Chemokine (C-C motif) receptor 4 +6 Cxcl2 MIP-2+ (macrophage inflammatory protein

T-cell expressed and secreted cytokine) Ccl7 MCP-3 (monocyte chemoattractant protein 3) +153 Ccl8 MCP-2 (monocyte chemoattractant protein 2) +149 Ccl12 MCP-5 (monocyte chemoattractant protein 5) +56

+22

+8

+105

+208

+7

+653

**Inflammation** Ccl2 MCP-1 (monocyte chemoattractant protein 2) +43

RAS kidney, specifically in the tubulo-interstitial compartment.

1+)

1,)

3+)

2+)

2,)

RAS mouse

factor)

Hypertension is one of the most common reasons for a visit to a physician. There are several key issues that need to be addressed during evaluation of a patient with hypertension: accurate blood pressure reading, determination of target organ damage due to hypertension, screening for other cardiovascular risk factor, stratification of cardiovascular disease, and assessment for the cause of hypertension (primary vs. secondary hypertension). Thorough assessment of the cause of hypertension is essential for determining the correct treatment approach, especially in children where atherosclerosis is not common. For children with hypertension, it is necessary to consider genetic diseases (i.e. coarctation of the aorta, primary aldosteronism) and auto-immune diseases (i.e. post-infectiousglomerulonephritis). When initiating treatment, it is important to maintain low systolic pressure as systolic pressure is a stronger predictor of cardiovascular event (Mancia et al. 2009; Cherubini et al. 2010). Maintenance of blood pressure goal should ideally be achieved within 6 to 9 months of therapeutic initiation.

#### **2. Conclusion**

Optimal management of renovascular hypertension requires an understanding of the disease process and remains an important challenge for clinicians caring for patients with hypertension. Although the pathophysiology and the consequence to human health caused

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Blood Pressure Research of the American Heart Association. Circulation 2001;104(16):1985-91.


Sebekova K, Dammrich J, Fierlbeck W, Krivosikova Z, Paczek L, Heidland A. Effect of

Sebolt-Leopold JS, Herrera R. Targeting the mitogen-activated protein kinase cascade to

Shankland SJ, Pippin J, Pichler RH, Gordon KL, Friedman S, Gold LI, et al. Differential

Shigemoto Fujii, Ling Zhang and Hiroaki Kosaka. Albuminuria, Expression of Nicotinamide

Speirs CJ, Dollery CT, Inman WHW, et al. Postmarketing surveillance of enalapril II:

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Stambe C, Atkins RC, Tesch GH, Kapoun AM, Hill PA, Schreiner GF, et al. Blockade of

Suzuki H, Uchida K, Nitta K, Nihei H. Role of mitogen-activated protein kinase in the

Tamaki K, Okuda S, Ando T, Iwamoto T, Nakayama M, Fujishima M. TGF-β1 in

Textor SC, Novick AC, Steinmuller DR, Streem SB. Renal failure limiting antihypertensive

Textor SC, Wilcox CS. Renal artery stenosis: a common, treatable cause of renal failure?

Textor SC. Ischemic nephropathy: where are we now? J Am Soc Nephrol 2004;15(8):1974-82. Textor SC. Managing renal arterial disease and hypertension. Curr Opin Cardiol

Textor SC. Progressive hypertension in a patient with "incidental" renal artery stenosis.

cultured renal interstitial fibroblasts. Clin Exp Nephrol 2004;8(3):188-95. Swartbol P, Thorvinger BO, Parsson H, et al. Renal Artery Stenosis in patients with

the rat model of Goldblatt hypertension. Am J Nephrol 1998;18(6):570-6. Sebolt-Leopold JS, Dudley DT, Herrera R, Van Becelaere K, Wiland A, Gowan RC, et al.

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Adenine Dinucleotide Phosphate Oxidase and Monocyte Chemoattractant Protein-1 in the Renal Tubules of Hypertensive Dahl Salt-Sensitive Rats. Hypertension

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therapy as an indication for renal revascularization. Arch Int Med. 1983;143:2208–


**5** 

*Ireland* 

**Cardiovascular Disease in Inflammatory** 

Aizuri Murad and Anne-Marie Tobin

**Disorders – Psoriasis and Psoriatic Arthritis** 

*Department of Dermatology, Adelaide and Meath Hospital and Trinity College Dublin,* 

Psoriasis, a papulosquamous skin disease, was originally thought to be a disorder primarily of epidermal keratinocytes, but is now recognised as one of the commonest immunemediated disorders. It is a chronic skin disorder that causes areas of thickened, inflamed, red skin, often covered with silvery scales. Worldwide psoriasis prevalence rates range from 0.6 percent to 4.8 percent. Children and adolescents can develop psoriasis, but it occurs primarily in adults. There seem to be two peaks in onset: one between ages 20 and 30 and another between 50 and 60. Women and men are equally affected. The immune system is involved and appears to be overactive in a way that causes inflammation. Specifically, there is excessive production of T-Helper 1 cytokines, particularly TNF+. These have many effects, including growth of extra blood vessels within the skin and increased turnover of the skin cells. Like most diseases, psoriasis is influenced by inherited characteristics. Up to 50% of people with psoriasis will know of another affected family member. Patients with a family history of psoriasis tend to develop psoriasis earlier in life than those without a family history. It is associated with comorbidities that include metabolic syndrome and increased cardiovascular risk. These conditions share etiologic features and health consequences that directly correlate with the severity of psoriatic disease. Up to thirty percent of patients with psoriasis develop psoriatic arthritis, this is an erosive, seronegative arthritis, which is associated with inflammation of tendon insertion points (enthesitis). An increased risk of cardiovascular disease was first noted in patients with rheumatoid arthritis and it became apparent that chronic inflammation was associated with increased risk of cardiovascular or

While heart disease remains a quiet killer, psoriasis is a visible disease whose impact on social interaction and quality of life usually prompt earlier physician consultation. Psoriasis patients are at increased risk of being obese and therefore are at greater risk than the general population to develop myocardial infarction, metabolic syndrome and other comorbidities. It has become evident that patients with psoriasis and psoriatic arthritis have an increased incidence of cardiovascular disease and also certain cardiovascular risk factors such as smoking, hypertension and metabolic syndrome compared to the normal population. They also have increased non-conventional risk factors such as raised levels of homocysteine and

**1. Introduction** 

cerebrovascular disease.

excessive alcohol consumption.


### **Cardiovascular Disease in Inflammatory Disorders – Psoriasis and Psoriatic Arthritis**

Aizuri Murad and Anne-Marie Tobin

*Department of Dermatology, Adelaide and Meath Hospital and Trinity College Dublin, Ireland* 

#### **1. Introduction**

66 Recent Advances in Cardiovascular Risk Factors

Yang J, Liu Y. Blockage of tubular epithelial to myofibroblast transition by hepatocyte

Yang J, Liu Y. Dissection of key events in tubular epithelial to myofibroblast transition and its implications in renal interstitial fibrosis. Am J Pathol 2001;159(4):1465-75. Yang M, Huang H, Li J, Li D, Wang H. Tyrosine phosphorylation of the LDL receptor-

Yu L, Hebert MC, Zhang YE. TGF-beta receptor-activated p38 MAP kinase mediates Smad-

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Zoja C, Corna D, Benedetti G, Morigi M, Donadelli R, Guglielmotti A, et al. Bindarit retards

independent TGF-beta responses. Embo J 2002;21(14):3749-59.

swine renovascular hypertension. J Hypertens 2009; 27:2063–2073

107.

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growth factor prevents renal interstitial fibrosis. J Am Soc Nephrol 2002;13(1):96-

related protein (LRP) and activation of the ERK pathway are required for connective tissue growth factor to potentiate myofibroblast differentiation. Faseb J

chemokine monocyte chemoattractant protein-1 contributes to renal dysfunction in

renal disease and prolongs survival in murine lupus autoimmune disease. Kidney

Psoriasis, a papulosquamous skin disease, was originally thought to be a disorder primarily of epidermal keratinocytes, but is now recognised as one of the commonest immunemediated disorders. It is a chronic skin disorder that causes areas of thickened, inflamed, red skin, often covered with silvery scales. Worldwide psoriasis prevalence rates range from 0.6 percent to 4.8 percent. Children and adolescents can develop psoriasis, but it occurs primarily in adults. There seem to be two peaks in onset: one between ages 20 and 30 and another between 50 and 60. Women and men are equally affected. The immune system is involved and appears to be overactive in a way that causes inflammation. Specifically, there is excessive production of T-Helper 1 cytokines, particularly TNF+. These have many effects, including growth of extra blood vessels within the skin and increased turnover of the skin cells. Like most diseases, psoriasis is influenced by inherited characteristics. Up to 50% of people with psoriasis will know of another affected family member. Patients with a family history of psoriasis tend to develop psoriasis earlier in life than those without a family history.

It is associated with comorbidities that include metabolic syndrome and increased cardiovascular risk. These conditions share etiologic features and health consequences that directly correlate with the severity of psoriatic disease. Up to thirty percent of patients with psoriasis develop psoriatic arthritis, this is an erosive, seronegative arthritis, which is associated with inflammation of tendon insertion points (enthesitis). An increased risk of cardiovascular disease was first noted in patients with rheumatoid arthritis and it became apparent that chronic inflammation was associated with increased risk of cardiovascular or cerebrovascular disease.

While heart disease remains a quiet killer, psoriasis is a visible disease whose impact on social interaction and quality of life usually prompt earlier physician consultation. Psoriasis patients are at increased risk of being obese and therefore are at greater risk than the general population to develop myocardial infarction, metabolic syndrome and other comorbidities. It has become evident that patients with psoriasis and psoriatic arthritis have an increased incidence of cardiovascular disease and also certain cardiovascular risk factors such as smoking, hypertension and metabolic syndrome compared to the normal population. They also have increased non-conventional risk factors such as raised levels of homocysteine and excessive alcohol consumption.

Cardiovascular Disease in Inflammatory Disorders – Psoriasis and Psoriatic Arthritis 69

endothelial dysfunction and increased insulin resistance in patients with psoriasis (Karadag et al., 2010). In a separate Turkish study, aortic elasticity in patients with psoriasis was found to be significantly lower than the control group. In psoriatic patients without cardiac involvement, aortic elasticity was decreased and this decrease was correlated with the duration and severity of the disease (Bicer et al., 2009). A study by El-Mongey and colleague showed an increase in carotid artery intima-media thickness in patients with chronic psoriasis suggesting that chronic psoriasis is associated with subclinical atherosclerosis with increased risk of cardiovascular disease (El-Mongy et al., 2010). Patients with psoriatic arthritis also had a higher prevalence of subclinical atherosclerosis as measured by intimamedia wall thickness (Eder et al., 2008; Kimhi et al., 2007) and endothelial dysfunction even without any overt cardiovascular disease (Gonzalez-Juanatey et al., 2007). More recently, subclinical left ventricular dysfunction has also been shown in psoriatic arthritis patients who had no established cardiovascular disease or risk factors (Shang et al., 2011). Risk factors for cardiovascular disease as well as other vascular diseases were shown by Kaye and colleagues to occur with higher incidence in patients with psoriasis than in the general population (Kaye et al., 2008). The study by Jamnitski and colleague showed the prevelance of cardiovascular diseases in psoriatic arthritis resembles that of rheumatoid arthritis

There are several theories to explain the association between psoriasis and increased cardiovascular risk. Both psoriasis and atherosclerosis involve Th-1 lymphocytes and cytokines and it is suggested that the excess inflammatory cells and mediators produced in psoriasis may contribute to the development of atherosclerotic plaques. A recent casecontrol study in 2011 on inpatients with psoriasis and dermatitis by Shapiro and colleagues supports previous reports of an association between psoriasis and CVD risk factors, suggesting that the inflammatory process in psoriasis, but not in dermatitis which is a T helper-2 mediated condition is involved in athersclerosis (Shapiro et al., 2011). Subsequently, Armstrong and colleagues have investigated the different inflammatory pathways which are common in psoriasis and atherosclerosis. They have found that psoriasis and atherosclerosis are diseases in which effector T lymphocytes such as Helper T cells type 1 (Th1) and 17 (Th17) play integral roles in disease pathogenesis and progression. Regulatory T cells (Treg) also exert clinically important anti-inflammatory effects that are pathologically altered in psoriasis and atherosclerosis. These shared pathways provide the basis for mechanisms that may explain the epidemiologic observation that patients with

In patients with psoriatic arthritis, it was shown recently that better control of inflammation with TNF-alpha blockers may inhibit the cascade that causes raised vascular risks in this cohort of patients. They were found to have thinner carotid intima-media thickness compared to those who were treated with DMARDs (Di Minno et al., 2011). With these findings, it is possible that control of chronic inflammation in these patients may have a significant role in preventing vasculopathy. Apart from this, screening and modification other cardiovascular risk factors in these patients should form part of their

Cardiovascular risk factors found with increased frequency in patients with psoriasis include conventional cardiovascular risk factors such as obesity, diabetes mellitus, hypertension, dyslipidaemia and smoking. Oxidative stress, endothelial cell dysfunction,

psoriasis have an increased risk of heart disease (Armstrong et al., 2011).

(Jamnitski et al., 2011).

assessment.

#### **2. Psoriasis, psoriatic arthritis and cardiovascular disease**

An association between psoriasis and cardiovascular risk was first described in 1978. Patients with psoriasis in an outpatient clinic had 2.2 times higher incidence of venous and arterial vascular disease in a clinic-based control study (McDonald & Calabresi, 1978). Gelfand and colleagues published a large cross -sectional study of the UK General Practice Database reporting a higher death rate from cardiovascular disease in those with severe psoriasis compared to the general population (Gelfand et al.,2006). The investigators controlled for diabetes, hyperlipidaemia, hypertension, body mass index, age, sex and smoking and found that psoriasis appeared to confer an independent risk for myocardial infarction. This risk is greater in younger patients (Gelfand et al.,2006). A second group utilising the same data found an increased incidence of risk factors for cardiovascular disease, as well as increased rates of myocardial infarction, angina, stroke and peripheral vascular disease (Henseler & Christophers, 1995). Patients were also found to have an increased risk of cardiovascular mortality that is independent of traditional cardiovascular risk factors (Mehta et al., 2010). More recently, a study has shown that psoriasis conferred an additional 6.2% absolute risk of 10 year major adverse cardiac events after adjusting for age, gender, diabetes, hypertension, tobacco use and hyperlipidaemia (Mehta et al., 2011).

A hospital based study from Sweden, Germany and Finland previously documented increased rates of risk factors such as hypertension, diabetes and obesity in patients with psoriasis. Poikolainen and Mallbris found that patients with severe psoriasis who required hospitalisation for treatment of their psoriasis had increased mortality from cardiovascular disease (Mallbris et al, 2004; Poikolainen et al., 1999). Patients managed as out-patients however did not have excess risk, suggesting that more severe disease was associated with a higher risk of cardiovascular disease. In a Danish nationwide cohort study, psoriasis was shown to be associated with increased risk of adverse cardiovascular events and all cause of mortality especially in young patients with severe disease (Ahlehoff et al., 2011). Furthermore, a separate Danish study showed that psoriasis significantly impaired prognosis in patients after myocardial infarction (Ahlehoff et al., 2011).

An observational study by Prodanovich and colleagues examined the cardiovascular risk factors in psoriasis and found psoriasis to be associated with atherosclerosis and this association applies to coronary artery, cerebrovascular and peripheral vascular diseases (Prodanovich et al., 2009). A cross sectional prevalence-based study from 2 American healthcare databases showed an increase prevalence of cardiovascular diseases and risk factors in patients with psoriasis compared with general population (Kimball et al., 2008). In Israel, Shapiro and colleagues showed a strong association between psoriasis, atherosclerosis, heart failure and diabetes (Shapiro et al., 2007). In addition to large clinical studies, several studies have documented subclinical cardiovascular disease.

A Chinese study showed that young patients with psoriasis have increased arterial stiffness compared with healthy controls. More importantly, CRP positively correlated with, and independently predicted, arterial stiffness. This suggests that systemic inflammation in patients with psoriasis is associated with premature atherosclerosis (Yiu et al., 2011). In another Asian study, Mazlan and colleagues also showed that there was a significant association between cardiovascular risk and intima-media thickness in psoriatic arthritis patients. However, it was not associated with disease activity, disease severity and DMARDS therapy (Mazlan et al., 2009). Karadag and colleagues demonstrated a significant

An association between psoriasis and cardiovascular risk was first described in 1978. Patients with psoriasis in an outpatient clinic had 2.2 times higher incidence of venous and arterial vascular disease in a clinic-based control study (McDonald & Calabresi, 1978). Gelfand and colleagues published a large cross -sectional study of the UK General Practice Database reporting a higher death rate from cardiovascular disease in those with severe psoriasis compared to the general population (Gelfand et al.,2006). The investigators controlled for diabetes, hyperlipidaemia, hypertension, body mass index, age, sex and smoking and found that psoriasis appeared to confer an independent risk for myocardial infarction. This risk is greater in younger patients (Gelfand et al.,2006). A second group utilising the same data found an increased incidence of risk factors for cardiovascular disease, as well as increased rates of myocardial infarction, angina, stroke and peripheral vascular disease (Henseler & Christophers, 1995). Patients were also found to have an increased risk of cardiovascular mortality that is independent of traditional cardiovascular risk factors (Mehta et al., 2010). More recently, a study has shown that psoriasis conferred an additional 6.2% absolute risk of 10 year major adverse cardiac events after adjusting for age, gender, diabetes, hypertension, tobacco use and hyperlipidaemia (Mehta et al., 2011).

A hospital based study from Sweden, Germany and Finland previously documented increased rates of risk factors such as hypertension, diabetes and obesity in patients with psoriasis. Poikolainen and Mallbris found that patients with severe psoriasis who required hospitalisation for treatment of their psoriasis had increased mortality from cardiovascular disease (Mallbris et al, 2004; Poikolainen et al., 1999). Patients managed as out-patients however did not have excess risk, suggesting that more severe disease was associated with a higher risk of cardiovascular disease. In a Danish nationwide cohort study, psoriasis was shown to be associated with increased risk of adverse cardiovascular events and all cause of mortality especially in young patients with severe disease (Ahlehoff et al., 2011). Furthermore, a separate Danish study showed that psoriasis significantly impaired

An observational study by Prodanovich and colleagues examined the cardiovascular risk factors in psoriasis and found psoriasis to be associated with atherosclerosis and this association applies to coronary artery, cerebrovascular and peripheral vascular diseases (Prodanovich et al., 2009). A cross sectional prevalence-based study from 2 American healthcare databases showed an increase prevalence of cardiovascular diseases and risk factors in patients with psoriasis compared with general population (Kimball et al., 2008). In Israel, Shapiro and colleagues showed a strong association between psoriasis, atherosclerosis, heart failure and diabetes (Shapiro et al., 2007). In addition to large clinical

A Chinese study showed that young patients with psoriasis have increased arterial stiffness compared with healthy controls. More importantly, CRP positively correlated with, and independently predicted, arterial stiffness. This suggests that systemic inflammation in patients with psoriasis is associated with premature atherosclerosis (Yiu et al., 2011). In another Asian study, Mazlan and colleagues also showed that there was a significant association between cardiovascular risk and intima-media thickness in psoriatic arthritis patients. However, it was not associated with disease activity, disease severity and DMARDS therapy (Mazlan et al., 2009). Karadag and colleagues demonstrated a significant

prognosis in patients after myocardial infarction (Ahlehoff et al., 2011).

studies, several studies have documented subclinical cardiovascular disease.

**2. Psoriasis, psoriatic arthritis and cardiovascular disease** 

endothelial dysfunction and increased insulin resistance in patients with psoriasis (Karadag et al., 2010). In a separate Turkish study, aortic elasticity in patients with psoriasis was found to be significantly lower than the control group. In psoriatic patients without cardiac involvement, aortic elasticity was decreased and this decrease was correlated with the duration and severity of the disease (Bicer et al., 2009). A study by El-Mongey and colleague showed an increase in carotid artery intima-media thickness in patients with chronic psoriasis suggesting that chronic psoriasis is associated with subclinical atherosclerosis with increased risk of cardiovascular disease (El-Mongy et al., 2010). Patients with psoriatic arthritis also had a higher prevalence of subclinical atherosclerosis as measured by intimamedia wall thickness (Eder et al., 2008; Kimhi et al., 2007) and endothelial dysfunction even without any overt cardiovascular disease (Gonzalez-Juanatey et al., 2007). More recently, subclinical left ventricular dysfunction has also been shown in psoriatic arthritis patients who had no established cardiovascular disease or risk factors (Shang et al., 2011). Risk factors for cardiovascular disease as well as other vascular diseases were shown by Kaye and colleagues to occur with higher incidence in patients with psoriasis than in the general population (Kaye et al., 2008). The study by Jamnitski and colleague showed the prevelance of cardiovascular diseases in psoriatic arthritis resembles that of rheumatoid arthritis (Jamnitski et al., 2011).

There are several theories to explain the association between psoriasis and increased cardiovascular risk. Both psoriasis and atherosclerosis involve Th-1 lymphocytes and cytokines and it is suggested that the excess inflammatory cells and mediators produced in psoriasis may contribute to the development of atherosclerotic plaques. A recent casecontrol study in 2011 on inpatients with psoriasis and dermatitis by Shapiro and colleagues supports previous reports of an association between psoriasis and CVD risk factors, suggesting that the inflammatory process in psoriasis, but not in dermatitis which is a T helper-2 mediated condition is involved in athersclerosis (Shapiro et al., 2011). Subsequently, Armstrong and colleagues have investigated the different inflammatory pathways which are common in psoriasis and atherosclerosis. They have found that psoriasis and atherosclerosis are diseases in which effector T lymphocytes such as Helper T cells type 1 (Th1) and 17 (Th17) play integral roles in disease pathogenesis and progression. Regulatory T cells (Treg) also exert clinically important anti-inflammatory effects that are pathologically altered in psoriasis and atherosclerosis. These shared pathways provide the basis for mechanisms that may explain the epidemiologic observation that patients with psoriasis have an increased risk of heart disease (Armstrong et al., 2011).

In patients with psoriatic arthritis, it was shown recently that better control of inflammation with TNF-alpha blockers may inhibit the cascade that causes raised vascular risks in this cohort of patients. They were found to have thinner carotid intima-media thickness compared to those who were treated with DMARDs (Di Minno et al., 2011). With these findings, it is possible that control of chronic inflammation in these patients may have a significant role in preventing vasculopathy. Apart from this, screening and modification other cardiovascular risk factors in these patients should form part of their assessment.

Cardiovascular risk factors found with increased frequency in patients with psoriasis include conventional cardiovascular risk factors such as obesity, diabetes mellitus, hypertension, dyslipidaemia and smoking. Oxidative stress, endothelial cell dysfunction,

Cardiovascular Disease in Inflammatory Disorders – Psoriasis and Psoriatic Arthritis 71

prevalence of hypertension in psoriasis based on a sample of Spanish population. The prevalence of hypertension was significantly higher in psoriasis patients than controls (Armesto et al., 2011). Armstrong and colleague showed that compared to hypertensive patients without psoriasis, psoriasis patients with hypertension were 5 times more likely to be on a monotherapy antihypertensive regimen, 9.5 times more likely to be on dual antihypertensive therapy, 16.5 times more likely to be on triple antihypertensive regimen, and 19.9 times more likely to be on quadruple therapy or centrally-acting agent (Armstrong

Several mechanisms including unhealthy lifestyle, activation of T Helper-1 lymphocytes and autoantibodies recognizing oxidized low-density lipoprotein may induce dyslipidaemia in psoriatic patients. Moreover, the levels of antibodies against oxidized low-density lipoprotein correlate with the disease activity. A large study on lipid profile at the onset of psoriasis showed significantly higher very low-density lipoprotein and high density lipoprotein fractions (Mallbris et al., 2006). This study was controlled for gender, blood pressure, BMI, physical activity, smoking and alcohol consumption. Later, a large crosssectional study using a population-based database by Dreiher and colleagues found that psoriatic patients had higher triglyceride and lower high-density lipoprotein cholesterol

In a separate study, children with psoriasis were found to have elevated total plasma cholesterol and HDL cholesterol and a decrease in the ratio of HDL to LDL cholesterol (Ferretti et al.,1993,1994). Dyslipidaemia observed in patients with psoriasis is compounded by increased oxidative stress and decreased anti-oxidant capacity. Autoantibodies recognizing oxidized LDL have been found in psoriasis, with their levels correlating with

More recently, it has been suggested that statin therapy may have beneficial effects by downregulating lymphocyte function-associated antigen-1, inhibiting leukocyte endothelial adhesion, extravasation and natural killer cell activity, all of which are key to the development of psoriasis lesions. They also reduce levels of proinflammatory cytokines such as tumour necrosis factor-alpha, interleukin 1 and 6, lowering C-reactive protein promote T (H) 1 cytokine receptors on T cell, leading to inhibition of activation of lymphocytes and

Overall, statin therapy for associated dyslipidaemia in patients with psoriasis has shown clinical improvement due to its immunomodulatory and anti-inflammatory effects

The association between psoriasis and hyperglycaemia was documented as early as 1967 (Lynch, 1967). Since then, numerous studies have confirmed the association between psoriasis, hyperglycaemia and relative insulin resistance (Fratino et al., 1979; Neimann et al., 2006; Pelfini et al., 1979; Reynoso-von Drateln et al., 2003; Sommer et al., 2006)**.** A crosssectional study by Ucak and colleagues found that psoriatic patients were more insulin resistant than healthy subjects and type II psoriatics(late onset) were more susceptible than

et al., 2011).

**3.3 Dyslipidaemia and psoriasis** 

levels compared to control (Dreiher et al., 2008).

infiltration into inflammatory sites.

**3.4 Diabetes mellitus and psoriasis** 

(Ghazizadeh et al., 2011).

disease activity as measured by PASI (Offidani et al., 1994).

abnormal platelet adhesion and hyperhomocysteinemia which may also increase cardiovascular risk have all been reported to occur with greater prevalence in psoriasis.

#### **3. Conventional risk factors in psoriasis**

#### **3.1 Smoking and psoriasis**

Smoking and psoriasis have been associated by several studies and from the disease perspective, it appeared to have an adverse effect with treatment response and patients in this cohort seem to have more severe disease. There is an increased rate of smoking in patients with psoriasis compared to controls (Christophers, 2001; Griffiths & Barker, 2007; McDonald & Calabresi, 1978; Stern et al., 2004; Veale & Fitzgerald, 2002). Poikolainen and colleagues found that smoking is a risk factor for psoriasis in women. Negative life events and smoking were more common among psoriasis patients than among controls (Poikolainen et al., 2004). Furthermore, the risk for psoriasis was higher in ex-smokers and current smokers compared to individuals who had never smoked (Mills et al., 1992; Naldi et al., 1999,2005; Poikolainen et al., 2004; Williams, 1994).

Behnam and colleagues found that women who are smokers have an up to 3.3-fold increased risk of developing plaque-type psoriasis. Men who are smokers do not exhibit such an increased risk, but studies have shown that smoking more than 10 cigarettes per day by men who are psoriasis patients may be associated with a more severe expression of disease in their extremities. In addition, smoking among both men and women who are psoriasis patients has been shown to reduce improvement rates (Behnam et al., 2005). Smoking also adversely affects the natural history of psoriasis in both genders especially in those who smoked more than 20 cigarettes a day. An Italian hospital-based cross-sectional study by Fortes and colleagues showed high intensity of smoking (>20 cigarettes daily) vs a lower level of consumption (< or =10 cigarettes daily) was associated with a more than 2 fold increased risk of clinically more severe. Separate analyses for men and women showed that the effect of cigarette-years was stronger for women (Fortes et al., 2005). Therefore, patients with psoriasis who smoke tend to have a less favourable outcome and disease that is more difficult to control.

Some researchers have found that heavier smokers have a greater risk of developing psoriasis and this only falls back to normal 20 years after quitting. There are good reasons for these patients to cease smoking to improve their psoriasis and more importantly, for their general health (Behnam et al., 2005; Fortes et al., 2005; Setty et al., 2007).

#### **3.2 Hypertension and psoriasis**

The association between elevated blood pressure and psoriasis was first described in 1977 and further studies showed an increased prevalence of essential hypertension in patients with psoriasis (Gisondi et al., 2007). In these patients, their hypertension is likely to be more severe and requires more medication to control it.

Enhanced activity of the renin-angiotensin system (Cohen et al., 2008) and increased levels of endothelin-1 released from vascular endothelium (Binazzi et al., 1975) were factors that contribute to the increased incidence of hypertension in patients with psoriasis. More recently, a hospital-based case-controlled study by Armesto and colleague evaluated the prevalence of hypertension in psoriasis based on a sample of Spanish population. The prevalence of hypertension was significantly higher in psoriasis patients than controls (Armesto et al., 2011). Armstrong and colleague showed that compared to hypertensive patients without psoriasis, psoriasis patients with hypertension were 5 times more likely to be on a monotherapy antihypertensive regimen, 9.5 times more likely to be on dual antihypertensive therapy, 16.5 times more likely to be on triple antihypertensive regimen, and 19.9 times more likely to be on quadruple therapy or centrally-acting agent (Armstrong et al., 2011).

#### **3.3 Dyslipidaemia and psoriasis**

70 Recent Advances in Cardiovascular Risk Factors

abnormal platelet adhesion and hyperhomocysteinemia which may also increase cardiovascular risk have all been reported to occur with greater prevalence in psoriasis.

Smoking and psoriasis have been associated by several studies and from the disease perspective, it appeared to have an adverse effect with treatment response and patients in this cohort seem to have more severe disease. There is an increased rate of smoking in patients with psoriasis compared to controls (Christophers, 2001; Griffiths & Barker, 2007; McDonald & Calabresi, 1978; Stern et al., 2004; Veale & Fitzgerald, 2002). Poikolainen and colleagues found that smoking is a risk factor for psoriasis in women. Negative life events and smoking were more common among psoriasis patients than among controls (Poikolainen et al., 2004). Furthermore, the risk for psoriasis was higher in ex-smokers and current smokers compared to individuals who had never smoked (Mills et al., 1992; Naldi et

Behnam and colleagues found that women who are smokers have an up to 3.3-fold increased risk of developing plaque-type psoriasis. Men who are smokers do not exhibit such an increased risk, but studies have shown that smoking more than 10 cigarettes per day by men who are psoriasis patients may be associated with a more severe expression of disease in their extremities. In addition, smoking among both men and women who are psoriasis patients has been shown to reduce improvement rates (Behnam et al., 2005). Smoking also adversely affects the natural history of psoriasis in both genders especially in those who smoked more than 20 cigarettes a day. An Italian hospital-based cross-sectional study by Fortes and colleagues showed high intensity of smoking (>20 cigarettes daily) vs a lower level of consumption (< or =10 cigarettes daily) was associated with a more than 2 fold increased risk of clinically more severe. Separate analyses for men and women showed that the effect of cigarette-years was stronger for women (Fortes et al., 2005). Therefore, patients with psoriasis who smoke tend to have a less favourable outcome and disease that

Some researchers have found that heavier smokers have a greater risk of developing psoriasis and this only falls back to normal 20 years after quitting. There are good reasons for these patients to cease smoking to improve their psoriasis and more importantly, for

The association between elevated blood pressure and psoriasis was first described in 1977 and further studies showed an increased prevalence of essential hypertension in patients with psoriasis (Gisondi et al., 2007). In these patients, their hypertension is likely to be more

Enhanced activity of the renin-angiotensin system (Cohen et al., 2008) and increased levels of endothelin-1 released from vascular endothelium (Binazzi et al., 1975) were factors that contribute to the increased incidence of hypertension in patients with psoriasis. More recently, a hospital-based case-controlled study by Armesto and colleague evaluated the

their general health (Behnam et al., 2005; Fortes et al., 2005; Setty et al., 2007).

**3. Conventional risk factors in psoriasis** 

al., 1999,2005; Poikolainen et al., 2004; Williams, 1994).

**3.1 Smoking and psoriasis** 

is more difficult to control.

**3.2 Hypertension and psoriasis** 

severe and requires more medication to control it.

Several mechanisms including unhealthy lifestyle, activation of T Helper-1 lymphocytes and autoantibodies recognizing oxidized low-density lipoprotein may induce dyslipidaemia in psoriatic patients. Moreover, the levels of antibodies against oxidized low-density lipoprotein correlate with the disease activity. A large study on lipid profile at the onset of psoriasis showed significantly higher very low-density lipoprotein and high density lipoprotein fractions (Mallbris et al., 2006). This study was controlled for gender, blood pressure, BMI, physical activity, smoking and alcohol consumption. Later, a large crosssectional study using a population-based database by Dreiher and colleagues found that psoriatic patients had higher triglyceride and lower high-density lipoprotein cholesterol levels compared to control (Dreiher et al., 2008).

In a separate study, children with psoriasis were found to have elevated total plasma cholesterol and HDL cholesterol and a decrease in the ratio of HDL to LDL cholesterol (Ferretti et al.,1993,1994). Dyslipidaemia observed in patients with psoriasis is compounded by increased oxidative stress and decreased anti-oxidant capacity. Autoantibodies recognizing oxidized LDL have been found in psoriasis, with their levels correlating with disease activity as measured by PASI (Offidani et al., 1994).

More recently, it has been suggested that statin therapy may have beneficial effects by downregulating lymphocyte function-associated antigen-1, inhibiting leukocyte endothelial adhesion, extravasation and natural killer cell activity, all of which are key to the development of psoriasis lesions. They also reduce levels of proinflammatory cytokines such as tumour necrosis factor-alpha, interleukin 1 and 6, lowering C-reactive protein promote T (H) 1 cytokine receptors on T cell, leading to inhibition of activation of lymphocytes and infiltration into inflammatory sites.

Overall, statin therapy for associated dyslipidaemia in patients with psoriasis has shown clinical improvement due to its immunomodulatory and anti-inflammatory effects (Ghazizadeh et al., 2011).

#### **3.4 Diabetes mellitus and psoriasis**

The association between psoriasis and hyperglycaemia was documented as early as 1967 (Lynch, 1967). Since then, numerous studies have confirmed the association between psoriasis, hyperglycaemia and relative insulin resistance (Fratino et al., 1979; Neimann et al., 2006; Pelfini et al., 1979; Reynoso-von Drateln et al., 2003; Sommer et al., 2006)**.** A crosssectional study by Ucak and colleagues found that psoriatic patients were more insulin resistant than healthy subjects and type II psoriatics(late onset) were more susceptible than

Cardiovascular Disease in Inflammatory Disorders – Psoriasis and Psoriatic Arthritis 73

 Metabolic syndrome has been reported to be strongly associated with psoriasis. A hospitalbased case-control study by Gisondi and colleagues showed a higher prevalence of metabolic syndrome in patients with psoriasis. These patients were found to be older and had longer disease duration compared with those without metabolic syndrome. However, there was no correlation between disease severity and prevalence of this syndrome (Gisondi et al., 2007). Another case-control study by Cohen and colleagues showed metabolic syndrome was more common in male psoriatic patients who are above 50 years of age (Cohen et al., 2008). Management of these patients should also target these metabolic conditions associated with psoriasis which are significant predictors of a cardiovascular

There is less research on cardiovascular disease and risk factors in psoriatic arthritis compared to psoriasis but it has been shown that patients with psoriatic arthritis have an increased prevalence of cardiovascular risk factors, type 2 diabetes, hyperlipidaemia and

A slightly different pattern of dyslipidaemia was found in these patients. They had higher HDL cholesterol and apolipoprotein A1 levels, lower total cholesterol and low density lipoprotein cholesterol levels and lower total cholesterol to HDL cholesterol ratio (Tam et al., 2008). Older studies have shown that psoriatic arthritis patients with synovitis had lower total cholesterol, low density lipoprotein (LDL) and high density lipoprotein (HDL)

Individual components of metabolic syndrome such as hypertension, obesity, insulin resistance and dyslipidaemia (Jones et al., 2000; Tam et al., 2008) have been reported in psoriatic arthritis patients but the full spectrum of metabolic syndrome has not been studied

Chronic inflammation is known to play a role in the development of atherosclerosis involving the innate immune system and T helper-1 lymphocytes (Hansson et al., 2002, 2006). This is similar to the pattern of immune mediated inflammation seen in psoriasis and with chronic low grade circulating inflammatory cells and cytokines which invoke endothelial inflammation, could ultimately lead to plaque formation (Wakkee et al., 2007). The increase prevalence of obesity in psoriasis and psoriatic arthritis may also increase the

C - reactive protein is a moderate predictor for cardiovascular disease (Danesh et al., 2004) and a marker that correlates well with joint inflammation. However, a large Italian study showed that it is more valuable in severe joint disease (Cervini et al., 2005). In another study, raised plasma fibrinogen levels are shown to be associated with an increased risk of vascular events. This may be mediated by adverse effects of fibrinogen on plasma viscosity, coagulation, platelet activity, inflammation and atherogenesis (Dziedzic, 2008; Kakfika et al., 2007). It is also known to be elevated in psoriasis and psoriatic arthritis (Marongiu et al.,

**5. Non-conventional risk factors in psoriasis and psoriatic arthritis** 

event. Therefore, healthy diet and lifestyle should be emphasized.

**4. Conventional risk factors in psoriatic arthritis** 

(Lazarevic et al., 1992; Skoczynska et al., 2003).

burden of inflammation (Hammings et al., 2006).

hypertension.

in these patients.

**5.1 Inflammation** 

type I psoriatics(onset before 35 years of age) to develop impaired glucose tolerance (Ucak et al., 2006). Qureshi and colleagues found that women with psoriasis were 63% more likely to develop diabetes and around 17% more likely to develop hypertension, than women without psoriasis (Qureshi et al., 2009). Genetic analysis of two non-major histocompatibility complex (MHC) in patients with psoriasis found the strongest phenotypic marker for a loci mapping to chromosome 6p22. This marker maps to CDKAL1, a gene associated to type 2 diabetes, suggesting a possible role for pleiotropic susceptibility loci for both conditions (Wol et al., 2008).

Boehncke and colleagues found a significant correlation between the Psoriasis Area and Severity Index (PASI) score and insulin secretion. The PASI score was significantly correlated with serum resistin levels, a cytokine known to be increased in insulin resistance (Boehncke et al., 2007). Patients with psoriasis demonstrate hyperinsulinaemia which correlates with disease severity (Boehncke et al., 2007;Ucak et al., 2006) and increased levels of insulin results in excessive levels if insulin-like growth factors (IGF) which appear to have a role in epidermal hyperproliferation in psoriasis (Hodak et al., 1996; Wraight et al., 2000; Xu et al, 1996). Induction of interleukin-6 and vascular endothelial growth factor has been postulated as underpinning IGF's role in the development of psoriatic plaques (Kwon et al., 2000, 2004).

More recently, a cohort analysis by Solomon and colleagues on patients with psoriasis showed a reduced risk of diabetes mellitus (DM) with the use of a tumor necrosis factor + (TNF-+)inhibitor or hydroxychloroquine, but not with methotrexate, compared with other nonbiologic disease-modifying antirheumatic drugs (DMARDs). Evidence suggests a possible role for DMARDs and immunosuppression in DM prevention (*Solomon* et al., 2011) along with adopting a healthy lifestyle, regular physical activity and good nutrition.

#### **3.5 Obesity and metabolic syndrome in psoriasis.**

In a case control study, we found that patients with psoriasis had higher Body Mass Index (BMI) compared to controls (Tobin et al., 2011). Individuals with psoriasis were more likely than controls to be obese. An increased adiposity and weight gain were strong risk factors for the development of psoriasis. A case-controlled study by Chen and colleagues found high levels of leptin more often in females, the obese and those with high blood pressure, metabolic syndrome and psoriasis. Hyperleptinemia in psoriasis is associated with higher risk of developing metabolic syndrome, which may be defined as the concurrence of hypertension, dyslipidaemia, diabetes and central adiposity. This finding links the chronic inflammation of psoriasis with metabolic disturbances. The high circulating leptin levels in individuals with psoriasis may derive not only from fat tissue but also from inflammation (Chen et al., 2008). Body weight loss has been reported to significantly decrease leptin levels and improve insulin sensitivity and may reduce the likelihood of developing metabolic syndrome and adverse cardiovascular diseases. Psoriasis and obesity are linked through a common pathophysiological mechanism of chronic low grade inflammation. Not only is obesity associated with a higher incidence of psoriasis and greater severity, it also affects response to treatment. Weight loss could potentially become part of the general treatment of psoriasis, especially in patients with obesity.

Furthermore, when age, smoking and alcohol intake were all controlled for, there was a strong association between BMI and psoriasis (Setty et al., 2007).

 Metabolic syndrome has been reported to be strongly associated with psoriasis. A hospitalbased case-control study by Gisondi and colleagues showed a higher prevalence of metabolic syndrome in patients with psoriasis. These patients were found to be older and had longer disease duration compared with those without metabolic syndrome. However, there was no correlation between disease severity and prevalence of this syndrome (Gisondi et al., 2007). Another case-control study by Cohen and colleagues showed metabolic syndrome was more common in male psoriatic patients who are above 50 years of age (Cohen et al., 2008). Management of these patients should also target these metabolic conditions associated with psoriasis which are significant predictors of a cardiovascular event. Therefore, healthy diet and lifestyle should be emphasized.

#### **4. Conventional risk factors in psoriatic arthritis**

There is less research on cardiovascular disease and risk factors in psoriatic arthritis compared to psoriasis but it has been shown that patients with psoriatic arthritis have an increased prevalence of cardiovascular risk factors, type 2 diabetes, hyperlipidaemia and hypertension.

A slightly different pattern of dyslipidaemia was found in these patients. They had higher HDL cholesterol and apolipoprotein A1 levels, lower total cholesterol and low density lipoprotein cholesterol levels and lower total cholesterol to HDL cholesterol ratio (Tam et al., 2008). Older studies have shown that psoriatic arthritis patients with synovitis had lower total cholesterol, low density lipoprotein (LDL) and high density lipoprotein (HDL) (Lazarevic et al., 1992; Skoczynska et al., 2003).

Individual components of metabolic syndrome such as hypertension, obesity, insulin resistance and dyslipidaemia (Jones et al., 2000; Tam et al., 2008) have been reported in psoriatic arthritis patients but the full spectrum of metabolic syndrome has not been studied in these patients.

#### **5. Non-conventional risk factors in psoriasis and psoriatic arthritis**

#### **5.1 Inflammation**

72 Recent Advances in Cardiovascular Risk Factors

type I psoriatics(onset before 35 years of age) to develop impaired glucose tolerance (Ucak et al., 2006). Qureshi and colleagues found that women with psoriasis were 63% more likely to develop diabetes and around 17% more likely to develop hypertension, than women without psoriasis (Qureshi et al., 2009). Genetic analysis of two non-major histocompatibility complex (MHC) in patients with psoriasis found the strongest phenotypic marker for a loci mapping to chromosome 6p22. This marker maps to CDKAL1, a gene associated to type 2 diabetes, suggesting a possible role for pleiotropic susceptibility loci for both conditions

Boehncke and colleagues found a significant correlation between the Psoriasis Area and Severity Index (PASI) score and insulin secretion. The PASI score was significantly correlated with serum resistin levels, a cytokine known to be increased in insulin resistance (Boehncke et al., 2007). Patients with psoriasis demonstrate hyperinsulinaemia which correlates with disease severity (Boehncke et al., 2007;Ucak et al., 2006) and increased levels of insulin results in excessive levels if insulin-like growth factors (IGF) which appear to have a role in epidermal hyperproliferation in psoriasis (Hodak et al., 1996; Wraight et al., 2000; Xu et al, 1996). Induction of interleukin-6 and vascular endothelial growth factor has been postulated as underpinning IGF's role in the development of psoriatic plaques (Kwon et al., 2000, 2004).

More recently, a cohort analysis by Solomon and colleagues on patients with psoriasis showed a reduced risk of diabetes mellitus (DM) with the use of a tumor necrosis factor + (TNF-+)inhibitor or hydroxychloroquine, but not with methotrexate, compared with other nonbiologic disease-modifying antirheumatic drugs (DMARDs). Evidence suggests a possible role for DMARDs and immunosuppression in DM prevention (*Solomon* et al., 2011)

In a case control study, we found that patients with psoriasis had higher Body Mass Index (BMI) compared to controls (Tobin et al., 2011). Individuals with psoriasis were more likely than controls to be obese. An increased adiposity and weight gain were strong risk factors for the development of psoriasis. A case-controlled study by Chen and colleagues found high levels of leptin more often in females, the obese and those with high blood pressure, metabolic syndrome and psoriasis. Hyperleptinemia in psoriasis is associated with higher risk of developing metabolic syndrome, which may be defined as the concurrence of hypertension, dyslipidaemia, diabetes and central adiposity. This finding links the chronic inflammation of psoriasis with metabolic disturbances. The high circulating leptin levels in individuals with psoriasis may derive not only from fat tissue but also from inflammation (Chen et al., 2008). Body weight loss has been reported to significantly decrease leptin levels and improve insulin sensitivity and may reduce the likelihood of developing metabolic syndrome and adverse cardiovascular diseases. Psoriasis and obesity are linked through a common pathophysiological mechanism of chronic low grade inflammation. Not only is obesity associated with a higher incidence of psoriasis and greater severity, it also affects response to treatment. Weight loss could potentially become part of the general treatment of

Furthermore, when age, smoking and alcohol intake were all controlled for, there was a

along with adopting a healthy lifestyle, regular physical activity and good nutrition.

**3.5 Obesity and metabolic syndrome in psoriasis.** 

psoriasis, especially in patients with obesity.

strong association between BMI and psoriasis (Setty et al., 2007).

(Wol et al., 2008).

Chronic inflammation is known to play a role in the development of atherosclerosis involving the innate immune system and T helper-1 lymphocytes (Hansson et al., 2002, 2006). This is similar to the pattern of immune mediated inflammation seen in psoriasis and with chronic low grade circulating inflammatory cells and cytokines which invoke endothelial inflammation, could ultimately lead to plaque formation (Wakkee et al., 2007). The increase prevalence of obesity in psoriasis and psoriatic arthritis may also increase the burden of inflammation (Hammings et al., 2006).

C - reactive protein is a moderate predictor for cardiovascular disease (Danesh et al., 2004) and a marker that correlates well with joint inflammation. However, a large Italian study showed that it is more valuable in severe joint disease (Cervini et al., 2005). In another study, raised plasma fibrinogen levels are shown to be associated with an increased risk of vascular events. This may be mediated by adverse effects of fibrinogen on plasma viscosity, coagulation, platelet activity, inflammation and atherogenesis (Dziedzic, 2008; Kakfika et al., 2007). It is also known to be elevated in psoriasis and psoriatic arthritis (Marongiu et al.,

Cardiovascular Disease in Inflammatory Disorders – Psoriasis and Psoriatic Arthritis 75

with alcoholic liver disease (Tobin et al., 2009). Patients undergoing phototherapy for psoriasis also had an increased prevalence of excessive alcohol intake (Kirby et al., 2011). Treatment outcome is adversely affected in patients who consume excess alcohol (Gupta et

Overall, alcohol misuse is common in patients with moderate to severe psoriasis. Proper screening allows identification of these patients who would benefit from appropriate intervention. There are limited studies about alcohol consumption in patients with psoriatic

Sporadic heavy drinking (binge drinking) increases the risk of developing coronary heart disease, the most common form of heart disease. Men nearly double their chances of developing coronary heart disease by drinking more than eight units of alcohol a day. Women have a 1.3 times greater risk of developing coronary heart disease when they drink more than six units a day. Women who persistently drink more than three units of alcohol a day and men, who drink more than four, are more likely to suffer from the risk factors associated with cardiovascular disorders such as high blood pressure. Alcohol can increase levels of homocysteine. High homocysteine levels increase the risk of thrombosis. Long-term drinking and heavy alcohol consumption is linked with weakness of the heart muscle,

There is evidence to suggest that a regular pattern of drinking relatively small amounts of alcohol (one or two drinks a few times a week) reduces the risk of heart disease in men over the age of 40 and post-menopausal women. Therefore, excess amount of alcohol appear to

Much has been said about the effects of chronic inflammation on cardiovascular risk factors and cardiac events. Researchers have tried to investigate if systemic therapy may improve cardiac biomarkers in patients with severe psoriasis. A German study by Boehncke and colleagues investigated the effects of continuous systemic therapy on the cardiovascular risk of patients with severe psoriasis. There was a trend towards reduced serum levels of vascular endothelial growth factor (VEGF) and resistin, while the potentially cardioprotective adiponectin showed a trend toward increased serum levels under therapy. This was parallel to improvement in C-reactive protein, PASI and insulin responsiveness (Boehncke et al., 2011). The impact on the metabolic state was found to be better if the psoriatic inflammation was controlled for longer (Boehncke et al., 2011). However, Abuabara and colleagues investigated on the effect of systemic treatment on the incidence of myocardial infarction in a control group treated with UVB therapy that has limited systemic anti-inflammatory effects. The risk of developing myocardial infarction in patients with severe psoriasis receiving systemic therapy was not reduced compared to a group

The increased cardiovascular risks in patients with psoriasis and psoriatic arthritis may be due to higher prevalence of multiple risk factors in these patient cohorts. It is very unlikely

be harmful whereas small amounts seem to be cardioprotective.

undergoing phototherapy (Abuabara et al., 2011).

**7. Conclusion** 

**6. Does treatment of inflammation ameliorate cardiovascular risk?** 

al., 1993).

arthritis.

known as cardiomyopathy.

1994; Rocha – Pereira et al., 2004; Vanizor Kural et al., 2003). This would indicate chronic inflammation and a possible role in increasing cardiovascular risk.

#### **5.2 Endothelial dysfunction**

Patients with psoriasis were found to have increased calcification in their coronary arteries compared to controls. Ludwig and colleagues found a significantly increased prevalence and severity of coronary artery calcification in patients with psoriasis and is likely to be an independent risk factor (Ludwig et al., 2007). Chronic inflammation causes endothelial dysfunction which leads to formation of atherosclerotic plaques in association with raised plasma lipids. Increased levels of oxidized low density lipoprotein in psoriatic plaques have been reported compared to controls (Rocha – Pereira et al., 2004; Vanizor Kural et al., 2003). Oxidized low-density lipoprotein is thought to promote atherosclerosis through complex inflammatory and immunologic mechanisms that lead to lipid dysregulation and foam cell formation. Recent findings suggested that oxidized LDL forms complexes with beta2 glycoprotein I (beta2GPI) and/or C-reactive protein (CRP) in the intima of atherosclerotic lesions. Oxidative stress has a critical role in causing damage to endothelial cells (Matsuura et al., 2006).

#### **5.3 Fibronectin and platelets: Atherothrombotic markers**

Atherothrombosis results in myocardial infarction, stroke and peripheral vascular disease. Low levels of fibronectin have been suggested as a marker of atherothrombosis and this has been shown in patients with psoriasis with active disease and in remission (De Pita et al., 1996). Increased platelet aggregation has also been found in these patients compared to controls. Hayashi and colleagues found platelet aggregation was significantly increased in psoriatics compared with normal controls. An additive effect was observed when diabetes was associated with psoriasis, with platelet aggregation being further increased by ADP. The increased platelet aggregation with ADP and epinephrine was significantly reduced when the skin lesions had cleared (Hayashi et al., 1985).

#### **5.4 Homocysteine**

Homocysteine causes endothelial dysfunction and is an independent risk factor for development of cardiovascular disease (Graham et al., 1997). In a case controlled study we showed that psoriatic patients have a relative risk 7.1 times greater than controls of having significantly raised levels of homocysteine (Tobin et al., 2011). This was also found in 2 uncontrolled studies and one in the context of patients who are taking methotrexate (Vanizor Kural et al., 2003). Refsum and colleagues investigated the effect of low-dose methotrexate on plasma homocysteine in patients who had psoriasis. Psoriasis patients had significantly higher basal plasma homocysteine levels (Refsum et al., 1989). High levels of homocysteine have been documented in small number of patients with psoriatic arthritis (Segal et al., 2004).

#### **5.5 Increased alcohol consumption**

Excessive alcohol consumption has been widely documented in patient with psoriasis (Higgins, 2000) and some researchers believe that drinking large amounts of alcohol predisposes to psoriasis (Segal et al., 2004). There is a high prevalence of psoriasis in patients with alcoholic liver disease (Tobin et al., 2009). Patients undergoing phototherapy for psoriasis also had an increased prevalence of excessive alcohol intake (Kirby et al., 2011). Treatment outcome is adversely affected in patients who consume excess alcohol (Gupta et al., 1993).

Overall, alcohol misuse is common in patients with moderate to severe psoriasis. Proper screening allows identification of these patients who would benefit from appropriate intervention. There are limited studies about alcohol consumption in patients with psoriatic arthritis.

Sporadic heavy drinking (binge drinking) increases the risk of developing coronary heart disease, the most common form of heart disease. Men nearly double their chances of developing coronary heart disease by drinking more than eight units of alcohol a day. Women have a 1.3 times greater risk of developing coronary heart disease when they drink more than six units a day. Women who persistently drink more than three units of alcohol a day and men, who drink more than four, are more likely to suffer from the risk factors associated with cardiovascular disorders such as high blood pressure. Alcohol can increase levels of homocysteine. High homocysteine levels increase the risk of thrombosis. Long-term drinking and heavy alcohol consumption is linked with weakness of the heart muscle, known as cardiomyopathy.

There is evidence to suggest that a regular pattern of drinking relatively small amounts of alcohol (one or two drinks a few times a week) reduces the risk of heart disease in men over the age of 40 and post-menopausal women. Therefore, excess amount of alcohol appear to be harmful whereas small amounts seem to be cardioprotective.

#### **6. Does treatment of inflammation ameliorate cardiovascular risk?**

Much has been said about the effects of chronic inflammation on cardiovascular risk factors and cardiac events. Researchers have tried to investigate if systemic therapy may improve cardiac biomarkers in patients with severe psoriasis. A German study by Boehncke and colleagues investigated the effects of continuous systemic therapy on the cardiovascular risk of patients with severe psoriasis. There was a trend towards reduced serum levels of vascular endothelial growth factor (VEGF) and resistin, while the potentially cardioprotective adiponectin showed a trend toward increased serum levels under therapy. This was parallel to improvement in C-reactive protein, PASI and insulin responsiveness (Boehncke et al., 2011). The impact on the metabolic state was found to be better if the psoriatic inflammation was controlled for longer (Boehncke et al., 2011). However, Abuabara and colleagues investigated on the effect of systemic treatment on the incidence of myocardial infarction in a control group treated with UVB therapy that has limited systemic anti-inflammatory effects. The risk of developing myocardial infarction in patients with severe psoriasis receiving systemic therapy was not reduced compared to a group undergoing phototherapy (Abuabara et al., 2011).

#### **7. Conclusion**

74 Recent Advances in Cardiovascular Risk Factors

1994; Rocha – Pereira et al., 2004; Vanizor Kural et al., 2003). This would indicate chronic

Patients with psoriasis were found to have increased calcification in their coronary arteries compared to controls. Ludwig and colleagues found a significantly increased prevalence and severity of coronary artery calcification in patients with psoriasis and is likely to be an independent risk factor (Ludwig et al., 2007). Chronic inflammation causes endothelial dysfunction which leads to formation of atherosclerotic plaques in association with raised plasma lipids. Increased levels of oxidized low density lipoprotein in psoriatic plaques have been reported compared to controls (Rocha – Pereira et al., 2004; Vanizor Kural et al., 2003). Oxidized low-density lipoprotein is thought to promote atherosclerosis through complex inflammatory and immunologic mechanisms that lead to lipid dysregulation and foam cell formation. Recent findings suggested that oxidized LDL forms complexes with beta2 glycoprotein I (beta2GPI) and/or C-reactive protein (CRP) in the intima of atherosclerotic lesions. Oxidative stress has a critical role in causing damage to endothelial cells (Matsuura

Atherothrombosis results in myocardial infarction, stroke and peripheral vascular disease. Low levels of fibronectin have been suggested as a marker of atherothrombosis and this has been shown in patients with psoriasis with active disease and in remission (De Pita et al., 1996). Increased platelet aggregation has also been found in these patients compared to controls. Hayashi and colleagues found platelet aggregation was significantly increased in psoriatics compared with normal controls. An additive effect was observed when diabetes was associated with psoriasis, with platelet aggregation being further increased by ADP. The increased platelet aggregation with ADP and epinephrine was significantly reduced

Homocysteine causes endothelial dysfunction and is an independent risk factor for development of cardiovascular disease (Graham et al., 1997). In a case controlled study we showed that psoriatic patients have a relative risk 7.1 times greater than controls of having significantly raised levels of homocysteine (Tobin et al., 2011). This was also found in 2 uncontrolled studies and one in the context of patients who are taking methotrexate (Vanizor Kural et al., 2003). Refsum and colleagues investigated the effect of low-dose methotrexate on plasma homocysteine in patients who had psoriasis. Psoriasis patients had significantly higher basal plasma homocysteine levels (Refsum et al., 1989). High levels of homocysteine have been

Excessive alcohol consumption has been widely documented in patient with psoriasis (Higgins, 2000) and some researchers believe that drinking large amounts of alcohol predisposes to psoriasis (Segal et al., 2004). There is a high prevalence of psoriasis in patients

documented in small number of patients with psoriatic arthritis (Segal et al., 2004).

inflammation and a possible role in increasing cardiovascular risk.

**5.3 Fibronectin and platelets: Atherothrombotic markers** 

when the skin lesions had cleared (Hayashi et al., 1985).

**5.2 Endothelial dysfunction** 

et al., 2006).

**5.4 Homocysteine** 

**5.5 Increased alcohol consumption** 

The increased cardiovascular risks in patients with psoriasis and psoriatic arthritis may be due to higher prevalence of multiple risk factors in these patient cohorts. It is very unlikely

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61-7


**6** 

Arun Kumar

*Malaysia* 

**Cardiovascular Risk Factors** 

**Myocardial Infarct Patients** 

*Management and Science University,* 

**in Elderly Normolipidaemic Acute** 

*Department Of Biochemistry, International Medical School,* 

Cardiovascular diseases (CVD) are a group of disorders of the heart and blood vessels, and include coronary heart disease (CHD), cerebrovascular disease (stroke), raised blood pressure (hypertension), peripheral artery disease, rheumatic heart disease, congenital heart disease and heart failure. According to the World Health Organization, CVD are the number one cause of death globally and claim 17 million lives each year. By 2030, almost 24 million people will die from CVD, mainly from heart disease and stroke. These are projected to remain the single leading causes of death (World Health Organization. Cardiovascular diseases (CVDs). Available from: http://www.who.int/mediacentre/factsheets/fs 317/en/index.html). In the United States, CVD account for more than one-third (34.3%) of deaths annually, and responsible for nearly 3 million Americans reporting disability. The costs of CVD are also staggering. In 2010, the total cost including health care services, medications and lost productivity, is estimated to be over \$503 billion in the United States (Centers for Disease Control and Prevention. Heart Disease and Stroke Prevention. Addressing the nation's leading killers: at a glance 2010. Available from: http://www.cdc.gov/chronicdisease/resources/publications/AAG/dhdsp.htm). Similarly, the National Heart Foundation of Australia reported that CVD are the leading cause of mortality and morbidity in Australia, killing one person nearly every 10 minutes (National Heart Foundation of Australia. Data and statistics. Available from: http:// www.heartfoundation.org.au/information-for-professionals/data-and-statistics). Despite improvements over the last few decades, CVD remain as the second largest disease burden to our society after cancers. As the population ages, the economic impact of CVD on the health care system will become even greater. Tobacco smoking, an unhealthy diet, physical inactivity and high alcohol consumption increase the risk of CVD. Indeed, behavioral and dietary risk factors are responsible for about 80% of coronary heart disease and cerebrovascular disease (World Health Organization. Cardiovascular diseases (CVDs). Available from: http://www.who.int/mediacentre/factsheets/fs317/en/index.html).

Interestingly, both the incidence and mortality rate of CVD are much lower in Japan than other countries (Mozaffarian D) which may be attributed to the high consumption of

**1. Introduction** 


### **Cardiovascular Risk Factors in Elderly Normolipidaemic Acute Myocardial Infarct Patients**

#### Arun Kumar

*Department Of Biochemistry, International Medical School, Management and Science University, Malaysia* 

#### **1. Introduction**

82 Recent Advances in Cardiovascular Risk Factors

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Cardiovascular diseases (CVD) are a group of disorders of the heart and blood vessels, and include coronary heart disease (CHD), cerebrovascular disease (stroke), raised blood pressure (hypertension), peripheral artery disease, rheumatic heart disease, congenital heart disease and heart failure. According to the World Health Organization, CVD are the number one cause of death globally and claim 17 million lives each year. By 2030, almost 24 million people will die from CVD, mainly from heart disease and stroke. These are projected to remain the single leading causes of death (World Health Organization. Cardiovascular diseases (CVDs). Available from: http://www.who.int/mediacentre/factsheets/fs 317/en/index.html). In the United States, CVD account for more than one-third (34.3%) of deaths annually, and responsible for nearly 3 million Americans reporting disability. The costs of CVD are also staggering. In 2010, the total cost including health care services, medications and lost productivity, is estimated to be over \$503 billion in the United States (Centers for Disease Control and Prevention. Heart Disease and Stroke Prevention. Addressing the nation's leading killers: at a glance 2010. Available from: http://www.cdc.gov/chronicdisease/resources/publications/AAG/dhdsp.htm). Similarly, the National Heart Foundation of Australia reported that CVD are the leading cause of mortality and morbidity in Australia, killing one person nearly every 10 minutes (National Heart Foundation of Australia. Data and statistics. Available from: http:// www.heartfoundation.org.au/information-for-professionals/data-and-statistics). Despite improvements over the last few decades, CVD remain as the second largest disease burden to our society after cancers. As the population ages, the economic impact of CVD on the health care system will become even greater. Tobacco smoking, an unhealthy diet, physical inactivity and high alcohol consumption increase the risk of CVD. Indeed, behavioral and dietary risk factors are responsible for about 80% of coronary heart disease and cerebrovascular disease (World Health Organization. Cardiovascular diseases (CVDs). Available from: http://www.who.int/mediacentre/factsheets/fs317/en/index.html).

Interestingly, both the incidence and mortality rate of CVD are much lower in Japan than other countries (Mozaffarian D) which may be attributed to the high consumption of

Cardiovascular Risk Factors in Elderly Normolipidaemic Acute Myocardial Infarct Patients 85

institutional ethical committee board and informed consent was obtained from the patients and controls. Inclusion criteria were patients with a diagnosis of acute myocardial infarction (AMI) with normal lipid profile. Patients with diabetes mellitus, renal insufficiency, current and past smokers, hepatic disease or taking lipid lowering drugs or antioxidant vitamin supplements were excluded from the study. Normolipidemic status was judged by the following criteria: LDL160 mg/dl; HDL, 35 mg/dl; total cholesterol (TC), <200 mg/dl; and triglycerides (TG), <150 mg/dl (NCEP, ATP-III, 2001). Ten milliliters of blood was collected after overnight fasting for lipid profile assay. For ischemia-modified albumin (IscMA) analysis, 2 ml of blood was collected from the patients immediately after admission

**Lipid Profile** Total cholesterol (TC), triglyceride (TG) and high density lipoproteincholesterol (HDL-c) were analyzed enzymatically using kit obtained from Randox Laboratories Limited, Crumlin, UK. Plasma low density lipoprotein -cholesterol was determined from the values of total cholesterol and high density lipoprotein-cholesterol

( ) TG LDL-cholesterol=TC - - HDL-cholesterol mg/dl <sup>5</sup>

**Other assays -** Serum albumin was measured by Bromocresol green binding method (Perry *et al*, 1979). Serum uric acid was estimated by the method of Brown based on the development of a blue color due to tungsten blue as phosphotungstic acid is reduced by uric acid in alkaline medium (Brown ,1945). Serum total bilirubin was estimated by the method

The glutathione peroxidase (GPx) activity was determined by the procedure of Paglia and Valentine (Paglia and Valentine,1967). Superoxide dismutase (SOD) enzyme activity was measured by SOD assay kit using rate of inhibition of 2-(4-indophenyl)-(4-Nitrophenol)-5 phenyltetrazolium chloride (I.N.T) reduction method modified by Sun et al (Sun *et al*, 1988). Catalase activity was measured spectrophotometrically as described by Beutler (Beutler, 1984). MDA levels were estimated by thiobarbituric acid (TBA) reaction (Bernheim *et al*, 1948).Conjugated diene (CD) levels were measured by Recknagel and Glende method (Recknagel and Glende, 1984) with little modification. Caeruloplasmin assay was done by *p*phenylene diamine method (Ravin, 1961). Ischemia-modified albumin (IscMA) concentration was determined by addition of a known amount of cobalt (II) to a serum sample and measurement of the unbound cobalt (II) by the intensity of colored complex formed after reacting with dithiothreitol (DTT) by colorimeter (Libby ,2003). Lipoprotein (a), levels were determined by Latex- Enhanced turbidimetric method. Serum paraoxonase was estimated using Zeptometrix Assay Kit obtained from Zeptometrix Corp, New York, 14202 based on the cleavage of phenyl acetate resulting in phenol formation. The rate of formation

of phenol is measured by monitoring the increase in absorbance at 270 nm at 25°C.

Estimation of ascorbic acid was carried out by Roe and Kuether method (Roe and Kuether, 1943). The C-reactive protein were determined using high sensitivity enzyme Immunoassay kit manufactured by Life Diagnostics,inc., Catalog Number: 2210. The principle of the assay was based on a solid phase enzyme-linked immunosorbent assay (Kumar and Sivakanesan,

to intensive care unit.

using the following formula:

of Jendrassik and Grof (Jendrassik and Grof, 1938).

fish/seafood by the Japanese population (Meyer BJ).The fact that fish is abundant in omega-3 polyunsaturated fatty acids has opened an effective venue to the prevention and treatment of this disease by either dietary modifications or pharmacological supplementation.

Cardiovascular disease is multi-factorial which is associated with factors like hereditary, hyperlipidemia, obesity, hypertension, environmental and life style variables like stress, smoking, alcohol consumption, etc (Chopra and Wasir 1998). Lipoprotein profile has been investigated extensively in recent years, which is deranged in large proportion of coronary artery disease (CAD) patients; especially Asians showing a mixed picture of dyslipidemia (Vasisht *et al*, 1990). Literature survey reveals dyslipidaemic subjects are more prone to myocardial infarction, due to increased free radical generation and ischemia as it is a conventional risk factor. (Malhotra *et al*, 2003; Mishra *et al*, 2005; Ghosh *et al*, 2006; Patil *et al*, 2007; Rajasekhar *et al*, 2004; Rani *et al*, 2005; Gomez *et al*,1996). Lowering of high density lipoprotein- cholesterol (HDL-C) is a common phenomenon observed in MI patients supported by previous studies (Malhotra *et al*, 2003; Mishra *et al*, 2005; Ghosh *et al*, 2006; Patil *et al*, 2007; Rajasekhar *et al*, 2004; Rani *et al*, 2005). High density lipoprotein- cholesterol (HDL-c) is the most important independent protective factor for arteriosclerosis which underlies coronary heart disease (CHD). High density lipoprotein- cholesterol (HDL-c) associated paraoxonase-1 (PON1) enzyme is protective against lipid peroxidation (Singh *et al*, 2007). Numerous cohort studies and clinical trials have confirmed the association between a low high density lipoprotein- cholesterol (HDL-c) and increased risk of coronary heart disease (CHD). Low density lipoprotein-cholesterol (LDL-C) is considered as the most important risk factor of coronary artery disease (CAD). Its oxidized form promotes foam cells formation which initiates the process of atherosclerosis by accumulating in subendothelium cells leading to fatty streaks and complex fibro fatty or atheromatous plaques formation (Berliner *et al*, 1995). The oxidation of low-density lipoprotein (LDL) can be limited by antioxidant enzyme system, including superoxide dismutase, catalase, glutathione peroxidase and antioxidant vitamins C, A, E and other carotenoids. Among the endogenous antioxidant system, includes albumin, uric acid, and total bilirubin. Imbalance of this reaction either due to excess free radical formation or insufficient removal by antioxidants leads to oxidative stress (Frei *et al*, 1998; Shrinivas *et al*, 2000; Maritim *et al*, 2003).

Various other risk factors have been identified apart from dyslipidemia are caeruloplasmin, C-reactive proteins, Lipoprotein (a), plasma fibrinogen, etc. Since we have encountered myocardial infarct patients with normal serum lipid concentration, we conducted a prospective case-control study to evaluate the concentration of antioxidant enzymes, degree of lipid peroxidation and other risk factors associated with acute myocardial infarction.

#### **2. Materials and methods**

The prospective case-control study consisted of 165 patients (123 men and 42 women) with AMI, admitted to the Intensive Cardiac Care Unit (ICCU), Sharda Hospital, India. The diagnosis of AMI was established according to diagnostic criteria: chest pain lasting for 3 hours, electrocardiographic (ECG) changes (ST elevation 2 mm in at least two leads) and elevation in enzymatic activities of serum creatine phosphokinase (CPK) and aspartate aminotransferase (AST). The control group consisted of 165 age/sex-matched healthy volunteers (123 men and 42 women). The design of this study was pre-approved by the

fish/seafood by the Japanese population (Meyer BJ).The fact that fish is abundant in omega-3 polyunsaturated fatty acids has opened an effective venue to the prevention and treatment

Cardiovascular disease is multi-factorial which is associated with factors like hereditary, hyperlipidemia, obesity, hypertension, environmental and life style variables like stress, smoking, alcohol consumption, etc (Chopra and Wasir 1998). Lipoprotein profile has been investigated extensively in recent years, which is deranged in large proportion of coronary artery disease (CAD) patients; especially Asians showing a mixed picture of dyslipidemia (Vasisht *et al*, 1990). Literature survey reveals dyslipidaemic subjects are more prone to myocardial infarction, due to increased free radical generation and ischemia as it is a conventional risk factor. (Malhotra *et al*, 2003; Mishra *et al*, 2005; Ghosh *et al*, 2006; Patil *et al*, 2007; Rajasekhar *et al*, 2004; Rani *et al*, 2005; Gomez *et al*,1996). Lowering of high density lipoprotein- cholesterol (HDL-C) is a common phenomenon observed in MI patients supported by previous studies (Malhotra *et al*, 2003; Mishra *et al*, 2005; Ghosh *et al*, 2006; Patil *et al*, 2007; Rajasekhar *et al*, 2004; Rani *et al*, 2005). High density lipoprotein- cholesterol (HDL-c) is the most important independent protective factor for arteriosclerosis which underlies coronary heart disease (CHD). High density lipoprotein- cholesterol (HDL-c) associated paraoxonase-1 (PON1) enzyme is protective against lipid peroxidation (Singh *et al*, 2007). Numerous cohort studies and clinical trials have confirmed the association between a low high density lipoprotein- cholesterol (HDL-c) and increased risk of coronary heart disease (CHD). Low density lipoprotein-cholesterol (LDL-C) is considered as the most important risk factor of coronary artery disease (CAD). Its oxidized form promotes foam cells formation which initiates the process of atherosclerosis by accumulating in subendothelium cells leading to fatty streaks and complex fibro fatty or atheromatous plaques formation (Berliner *et al*, 1995). The oxidation of low-density lipoprotein (LDL) can be limited by antioxidant enzyme system, including superoxide dismutase, catalase, glutathione peroxidase and antioxidant vitamins C, A, E and other carotenoids. Among the endogenous antioxidant system, includes albumin, uric acid, and total bilirubin. Imbalance of this reaction either due to excess free radical formation or insufficient removal by antioxidants leads to oxidative stress (Frei *et al*, 1998; Shrinivas *et al*, 2000; Maritim *et al*,

Various other risk factors have been identified apart from dyslipidemia are caeruloplasmin, C-reactive proteins, Lipoprotein (a), plasma fibrinogen, etc. Since we have encountered myocardial infarct patients with normal serum lipid concentration, we conducted a prospective case-control study to evaluate the concentration of antioxidant enzymes, degree of lipid peroxidation and other risk factors associated with acute myocardial infarction.

The prospective case-control study consisted of 165 patients (123 men and 42 women) with AMI, admitted to the Intensive Cardiac Care Unit (ICCU), Sharda Hospital, India. The diagnosis of AMI was established according to diagnostic criteria: chest pain lasting for 3 hours, electrocardiographic (ECG) changes (ST elevation 2 mm in at least two leads) and elevation in enzymatic activities of serum creatine phosphokinase (CPK) and aspartate aminotransferase (AST). The control group consisted of 165 age/sex-matched healthy volunteers (123 men and 42 women). The design of this study was pre-approved by the

of this disease by either dietary modifications or pharmacological supplementation.

2003).

**2. Materials and methods** 

institutional ethical committee board and informed consent was obtained from the patients and controls. Inclusion criteria were patients with a diagnosis of acute myocardial infarction (AMI) with normal lipid profile. Patients with diabetes mellitus, renal insufficiency, current and past smokers, hepatic disease or taking lipid lowering drugs or antioxidant vitamin supplements were excluded from the study. Normolipidemic status was judged by the following criteria: LDL160 mg/dl; HDL, 35 mg/dl; total cholesterol (TC), <200 mg/dl; and triglycerides (TG), <150 mg/dl (NCEP, ATP-III, 2001). Ten milliliters of blood was collected after overnight fasting for lipid profile assay. For ischemia-modified albumin (IscMA) analysis, 2 ml of blood was collected from the patients immediately after admission to intensive care unit.

**Lipid Profile** Total cholesterol (TC), triglyceride (TG) and high density lipoproteincholesterol (HDL-c) were analyzed enzymatically using kit obtained from Randox Laboratories Limited, Crumlin, UK. Plasma low density lipoprotein -cholesterol was determined from the values of total cholesterol and high density lipoprotein-cholesterol using the following formula:

$$\text{LDL-cholesky=TC} \cdot \text{\textdegree TC - HDL-cholesterol} \,(\text{mg/dl})$$

**Other assays -** Serum albumin was measured by Bromocresol green binding method (Perry *et al*, 1979). Serum uric acid was estimated by the method of Brown based on the development of a blue color due to tungsten blue as phosphotungstic acid is reduced by uric acid in alkaline medium (Brown ,1945). Serum total bilirubin was estimated by the method of Jendrassik and Grof (Jendrassik and Grof, 1938).

The glutathione peroxidase (GPx) activity was determined by the procedure of Paglia and Valentine (Paglia and Valentine,1967). Superoxide dismutase (SOD) enzyme activity was measured by SOD assay kit using rate of inhibition of 2-(4-indophenyl)-(4-Nitrophenol)-5 phenyltetrazolium chloride (I.N.T) reduction method modified by Sun et al (Sun *et al*, 1988). Catalase activity was measured spectrophotometrically as described by Beutler (Beutler, 1984). MDA levels were estimated by thiobarbituric acid (TBA) reaction (Bernheim *et al*, 1948).Conjugated diene (CD) levels were measured by Recknagel and Glende method (Recknagel and Glende, 1984) with little modification. Caeruloplasmin assay was done by *p*phenylene diamine method (Ravin, 1961). Ischemia-modified albumin (IscMA) concentration was determined by addition of a known amount of cobalt (II) to a serum sample and measurement of the unbound cobalt (II) by the intensity of colored complex formed after reacting with dithiothreitol (DTT) by colorimeter (Libby ,2003). Lipoprotein (a), levels were determined by Latex- Enhanced turbidimetric method. Serum paraoxonase was estimated using Zeptometrix Assay Kit obtained from Zeptometrix Corp, New York, 14202 based on the cleavage of phenyl acetate resulting in phenol formation. The rate of formation of phenol is measured by monitoring the increase in absorbance at 270 nm at 25°C.

Estimation of ascorbic acid was carried out by Roe and Kuether method (Roe and Kuether, 1943). The C-reactive protein were determined using high sensitivity enzyme Immunoassay kit manufactured by Life Diagnostics,inc., Catalog Number: 2210. The principle of the assay was based on a solid phase enzyme-linked immunosorbent assay (Kumar and Sivakanesan,

Cardiovascular Risk Factors in Elderly Normolipidaemic Acute Myocardial Infarct Patients 87

Age 60.55 ± 3.98 61.84 ± 3.80 0.0037( 61.26-62.42) Total Cholesterol (mg/dl) 168.58 ± 12.16 186.44 ± 13.95 <0.001(184.31-188.56) HDL-Cholesterol (mg/dl) 50.51 ± 6.78 41.27 ± 4.62 <0.001(40.56-41.97) Triglycerides (mg/dl) 107.84 ± 11.51 128.96 ± 12.19 <0.001(127.10-130.82) LDL-Cholesterol (mg/dl) 83.59 ± 11.95 119.37 ± 14.05 <0.001(17.22-21.51) TC: HDL-C 3.39 ± 0.36 4.57 ± 0.58 <0.001(4.48-4.65) LDL:HDL-C 1.90 ± 0.31 2.93 ± 0.51 <0.001(2.85-3.00) TG: HDL-C 2.17 ± 0.35 3.16 ± 0.49 0.3149(3.086-3.234)

*Patients (n=165)* 

> Patients (n=165)

3.70 ± 0.20 ( n=31)

4.53 ± 0.27 ( n=90)

2.65 ± 0.27 (n=59)

(n=99)

(n=7)

1.86 ± 0.15 (n=5)

2.57 ± 0.27 (n=81)

(n=74)

(n=5)

(n=44)



*P-value (95%CI)* 

*(n=165)* 

Table 2. Lipid profile in patients and healthy controls (mean ± SD)

5-6 - 5.26 ± 0.23

3-4 - 3.42 ± 0.26

4-5 - 4.22 ± 0.19

3-4 - 3.32 ± 0.21

4-5 - 4.11 ± 0.12

Table 3. Distribution of Lipid ratios in patients and healthy controls (mean ± SD)

Variables *Controls* 

Ratio Controls

2-3 2.90 ± 0.09

3-4 3.44 ± 0.25

4-5 4.19 ± 0.22

1-2 1.77 ± 0.13

2-3 2.38 ± 0.23

1-2 1.71 ± 0.17

2-3 2.23 ± 0.21

TC/HDL-C

TG/HDL-C

LDL-C/HDL-C

(n=165)

(n=28)

(n=129)

(n=8)

(n=56)

(n=109)

(n=106)

( n=59)

2008). The plasma fibrinogen was determined using kit which was obtained from TEClot Fib Kit 10 Catalog No: 050-500, manufactured by TECO GmbH, Dieselstr. 1, 84088 Neufahrn NB Germany (Kumar and Sivakanesan, 2008).

All chemicals of analytical grade were obtained from Sigma-Aldrich Company, New Delhi.

#### **3. Results**

Anthropometric parameters in acute myocardial infarction (AMI) patients and control are shown in Table 1. Total cholesterol, its ratio to high density lipoprotein -cholesterol (TC/HDL-C) and triglyceride were significantly higher in both sexes of patients compared with control (Table 2 and 3). The low density lipoprotein –cholesterol (LDL-c) and its ratio to high density lipoprotein –cholesterol (LDL-c) (LDL-C/HDL-C) were higher in acute myocardial infarction (AMI) subjects than in control (Table-3). The behavioral pattern and familial history of cardiovascular disease is presented in Table 4. The distribution of risk factors and relative risk according to potential risk factors among cases and controls are presented in Table 5 and Table 6. The status of antioxidants and lipid peroxidation are shown in Tables 7. All antioxidants were significantly decreased in patients compared with controls. In agreement with this serum malondialdehyde (MDA) and conjugated diene (CD) were more abundant in patients compared with controls. Ischemia-modified albumin (IscMA) levels were also greater in both male and female patients compared with control (Table 7).Serum fibrinogen, caeruloplasmin, ischemia- modified albumin and C-reactive protein were significantly higher where as arylesterase activity were significantly lowered in cases compared with controls ( Table 8).


Table 1. Anthropometric data of control and patients (mean ± SD)

2008). The plasma fibrinogen was determined using kit which was obtained from TEClot Fib Kit 10 Catalog No: 050-500, manufactured by TECO GmbH, Dieselstr. 1, 84088 Neufahrn NB

All chemicals of analytical grade were obtained from Sigma-Aldrich Company, New Delhi.

Anthropometric parameters in acute myocardial infarction (AMI) patients and control are shown in Table 1. Total cholesterol, its ratio to high density lipoprotein -cholesterol (TC/HDL-C) and triglyceride were significantly higher in both sexes of patients compared with control (Table 2 and 3). The low density lipoprotein –cholesterol (LDL-c) and its ratio to high density lipoprotein –cholesterol (LDL-c) (LDL-C/HDL-C) were higher in acute myocardial infarction (AMI) subjects than in control (Table-3). The behavioral pattern and familial history of cardiovascular disease is presented in Table 4. The distribution of risk factors and relative risk according to potential risk factors among cases and controls are presented in Table 5 and Table 6. The status of antioxidants and lipid peroxidation are shown in Tables 7. All antioxidants were significantly decreased in patients compared with controls. In agreement with this serum malondialdehyde (MDA) and conjugated diene (CD) were more abundant in patients compared with controls. Ischemia-modified albumin (IscMA) levels were also greater in both male and female patients compared with control (Table 7).Serum fibrinogen, caeruloplasmin, ischemia- modified albumin and C-reactive protein were significantly higher where as arylesterase activity were significantly lowered in

Germany (Kumar and Sivakanesan, 2008).

cases compared with controls ( Table 8).

Control *(n=165)* 

60.5 ± 3.4 *(48-69)*

Table 1. Anthropometric data of control and patients (mean ± SD)

Height (m) 1.63 ± 0.04 1.64 ± 0.59 0.2919 (1.55-1.72) Weight ( kg) 68.34 ± 3.97 72.01 ± 5.37 <0.01 (71.25-72.76) BMI ( kg/m2) 25.40 ± 1.20 26.16 ± 1.45 <0.01 (25.95-26.36) Waist Circumference (cm) 93.70 ± 3.63 100.77 ± 6.06 <0.01 (99.91-101.62) Hip Circumference (cm) 100.01 ± 3.16 105.72 ± 5.23 <0.01 (104.82-106.45) Waist-Hip ratio 0.93 ± 0.01 0.95 ± 0.01 <0.001 (0.94-0.95) Mid Arm Circumference (cm) 29.70 ± 1.47 30.63 ± 1.87 <0.01 (30.36-30.89)

MI patients *(n=165)* 

61.8 ± 3.8 *(48-69)* 

6.95 ± 1.05 7.5 ± 1.38 <0.001 (7.30-7.69)

11.97 ± 1.27 12.89 ± 1.69 <0.001 (12.65-13.12)

121.06 ± 4.19 134.32 ± 11.65 <0.05 (132.67-135.96)

79.90 ± 3.64 86.04 ± 4.25 <0.05 (85.44-86.63)

P- value (95%CI)

0.0037 (61.26- 62.33)

**3. Results** 

Age (years) Range (years)

(mm)

(mm)

(mmHg)

(mmHg)

Biceps skin fold thickness

Triceps skin fold thickness

Systolic blood pressure

Diastolic blood pressure


Table 2. Lipid profile in patients and healthy controls (mean ± SD)


Table 3. Distribution of Lipid ratios in patients and healthy controls (mean ± SD)

Cardiovascular Risk Factors in Elderly Normolipidaemic Acute Myocardial Infarct Patients 89

25 135 114 2.7 ( 1.8,4.1) 2.9 ( 1.6, 5.1)

> 1.0 113 28 3.9 (2.1, 6.3) 2.8 (1.6, 5.7)

Yes 62 14 2.1( 1.6, 2.7) 2.7 ( 1.8, 3.8)

Yes 29 23 2.1 ( 1.7, 3.2) 1.9 ( 1.4, 2.9)

None 101 132 3.1 ( 1.3, 5.1) 3.6 (1.0, 6.2)

Joint 145 101 4.5 ( 1.5- 2.9) 3.9( 1.2-2.6)

Middle Class 119 131 3.4 ( 4.3, 6.7) 2.8 ( 3.7, 5.9) Higher Class 36 15 4.7 ( 4.9, 7.2) 3.8 ( 3.1, 4.7)

145 MET-min/d 83 107 0.76 ( 0.4, 0.8) 0.68 (0.4, 0.7)

<5000 rupees/month 10 19 1.8 ( 1.2, 2.7) 1.7 ( 1.0, 3.1)

Yes 132 153 0.8 (0.6, 1.1) 0.9 ( 0.7, 1.3) *<sup>a</sup>*MET, metabolic equivalent. RR estimates were obtained by using conditional logistic regression analysis controlled for the matching factors (age, sex, and hospital) and then additional potential risk

 Covariates controlled for in the multivariate model were as follows: age; sex; hospital; cigarette smoking never, current (10 cigarettes/d, >10 cigarettes/d)]; bidi smoking [never, current (10 bidis/d, >10 bidis/d)]; BMI, in kg/m2 (20-24.9, 25); waist-to-hip ratio (0.95, >1.0); leisure time physical exercise (none, < 145 MET-min/d, 145 MET-min/d); history of hypertension (no, yes); history of diabetes (no, yes); history of high cholesterol (no, yes); family history of IHD (no, yes); education (none, primary school, middle, secondary, higher secondary, college, graduate or professional); household income (<5000, 5000-

*\** Bidis (pronounced bee-dees) are small hand-rolled cigarettes manufactured in India and other southeast Asian countries. They are exported to as many as 122 countries, according to one bidi manufacturer. Bidi

Table 6. Relative risk (RR) of Acute Myocardial Infarction (AMI) according to potential risk

cigarettes are made of tobacco wrapped in tendu or temburni leaf *(Diospyros melanxylon)*.

20-24.9 30 51 1.0 1.0

0.95 52 137 1.0 1.0

No 97 151 1.0 1.0

No 136 142 1.0 1.0

Highest level of education 25 27 1.0 1.0

Split 20 64 1.0 1.0

Lower Class 10 19 1.0 1.0

Non-exerciser 82 58 1.0 1.0

>10 000 rupees/month 155 146 1.0 1.0

No 33 12 1.0 1.0

10000, 10000-15000,>10000 rupees/mo); and Hindu religion (no, yes).

**BMI (kg/m2)** 

**Waist –to-hip ratio**

**Family history of MI**

**Education level**

**Type of Family**

**Civil Status** 

**Leisure –time exercise**

**Household income**

**Hindu religion** 

*<sup>b</sup>* Also adjusted for hospital.

factors.

factorsa

*c*

**History of Hypertension**


Numbers in parentheses are percent unless mentioned otherwise

\*Triffle thinker: subjects who thinks and worries on unnecessary small things

Table 4. Behavioral Pattern in AMI patients and control


Values are in Mean ± SD

a,b,c,d,e,f Significantly different from cases (*t* test for matched data): a,b,c *P* 0.001, d,e *P* 0.0001, f *P* 0.003

Table 5. Distribution of risk factors among AMI patients and control



*<sup>a</sup>*MET, metabolic equivalent. RR estimates were obtained by using conditional logistic regression analysis controlled for the matching factors (age, sex, and hospital) and then additional potential risk factors.

*<sup>b</sup>* Also adjusted for hospital.

88 Recent Advances in Cardiovascular Risk Factors

68 *(41.21)*

97 *(58.78)*

99 *(60.00)*

66 *(40.00)*

106 *(64.24)*

59 *(35.75)*

AMI Cases *( n=165)* Controls ( *n= 165)*

Age- and sex adjusted RR (95% CI)b

Multivariate RR (95% CI)c

Control Group Study Group

39 *(23.63)*

126 *(76.36)*

30 *(18.18)*

135 *(81.81)*

50 *(30.30)*

115 *(69.69)*

Age (y) 61.84 ± 3.80 60.55 ± 3.98 BMI (kg/m2) 26.16 ± 1.45 25.40 ± 1.20 Waist-to-hip ratio 0.95 ± 0.11 0.93 ± 0.08a Alcohol intake (servings/d) 0.36 ± 0.68 0.15 ± 0.34a Physical activity ( MET-min/d) 56.23 ± 123.8 97.83 ± 174.8a

a,b,c,d,e,f Significantly different from cases (*t* test for matched data): a,b,c *P* 0.001, d,e *P* 0.0001, f *P* 0.003

*N* 

>10 cigarettes/d 36 6 7.8 (4.9, 13.5) 7.4 (4.3, 15.2)

> 10 bidis/d 49 8 8.2 (5.2, 14.2) 6.5 (3.9, 12.9)

Never smoker 120 136 1.0 1.0

Never smoker 120 136 1.0 1.0

No. of controls

\*Triffle thinker: subjects who thinks and worries on unnecessary small things

Current cigarette smokers (%) 14.45 3.6b Current bidi smokers (%) 23.67 12.31c Family history of MI (%) 37.57 8.48d Hypertension (%) 49.09 1.8e Alcoholics (%) 47.87 20.60f

Table 5. Distribution of risk factors among AMI patients and control

No. of cases

*N* 

Numbers in parentheses are percent unless mentioned otherwise

Table 4. Behavioral Pattern in AMI patients and control

 *Yes*

 *no*

 *yes*

 *yes* **Irrelevant thinker**   *No*

Values are in Mean ± SD

**Cigarette smoking** 

**Bidi smoking\*** 

**Hyperactive** 

**Triffle thinker \***   *no* 

> *c* Covariates controlled for in the multivariate model were as follows: age; sex; hospital; cigarette smoking never, current (10 cigarettes/d, >10 cigarettes/d)]; bidi smoking [never, current (10 bidis/d, >10 bidis/d)]; BMI, in kg/m2 (20-24.9, 25); waist-to-hip ratio (0.95, >1.0); leisure time physical exercise (none, < 145 MET-min/d, 145 MET-min/d); history of hypertension (no, yes); history of diabetes (no, yes); history of high cholesterol (no, yes); family history of IHD (no, yes); education (none, primary school, middle, secondary, higher secondary, college, graduate or professional); household income (<5000, 5000- 10000, 10000-15000,>10000 rupees/mo); and Hindu religion (no, yes).

*\** Bidis (pronounced bee-dees) are small hand-rolled cigarettes manufactured in India and other southeast Asian countries. They are exported to as many as 122 countries, according to one bidi manufacturer. Bidi cigarettes are made of tobacco wrapped in tendu or temburni leaf *(Diospyros melanxylon)*.

Table 6. Relative risk (RR) of Acute Myocardial Infarction (AMI) according to potential risk factorsa

Cardiovascular Risk Factors in Elderly Normolipidaemic Acute Myocardial Infarct Patients 91

The search for the newer risk factors continues and researchers are investigating the role of inflammatory markers and other potential risks factors which could link with acute

In this prospective case-control study in India, only normolipidaemic acute myocardial infarction (AMI) patients were selected. The study was designed to identify and evaluate potential risk factors in normolipidaemic acute myocardial infarction (AMI) patients. The subjects selected for the study comprised of 165 controls, 48-69 y and 165 acute MI patients,

Anthropometric variables in acute myocardial infarction (AMI) patients showed highly significant differences in waist/hip ratio and biceps skin fold thickness. Study reported (Heitman *et al*, 2004) that waist /hip ratio is a dominant, independent and predictive variable of cardiovascular disease and coronary heart disease deaths in Australian men and women. Megnien *et al*, 1999 also reported high hip circumference relative to weight and waist circumference is a better predictor of low incidence of cardiovascular disease and coronary heart disease. The present study is in good agreement with the observations of the above studies. Among Indians the cardiovascular risk is high even the prevalence of obesity is minimal (Megnien *et al*, 1999). In the present study the mean body mass index and waist /hip ratio in all subjects was 26.56 and 0.96 respectively, showing a significantly higher

Based on the observations of the aforementioned studies and further supported by the present study it could be concluded that weight/hip ratio is a better predictor of cardiovascular disease (CVD) than body mass index. So it is better tool for indentifying the future risk of acute myocardial infarction (AMI) in subjects by non-invasive procedures.

The mean total cholesterol level of the controls compared with acute myocardial infarction patients (186.44 ± 13.95 mg/dl) was significantly (p<0.001) higher compared with controls (168.58 ± 12.16 mg/dl). The mean high density lipoprotein-cholesterol level in the patients was significantly lower (p<0.001) compared with controls. Triglyceride (TG) values observed in acute myocardial infarction (AMI) patients was (129mg/dl) significantly higher than controls (107.8mg/dl). The mean low density lipoprotein-cholesterol (LDL-c) levels in patients was (119.4mg/dl) significantly higher than controls (83.6 mg/dl). The total cholesterol / high density lipoprotein – cholesterol ratio in acute myocardial infarct patients (4.6) was significantly (p<0.001) higher compared with controls (3.4). The present study observed significantly higher ratio (2.9) in acute myocardial infarction patients compared

Earlier studies in lipid profile analysis conducted on acute myocardial infarction patients (Mishra *et al*, 2001; Das *et al*, 2002; Goswami *et al*, 2003; Kharb *et al*, 2003; Malhotra *et al*, 2003; Burman *et al*, 2004; Rajashekhar *et al*, 2004; Sivaraman *et al*, 2004; Rani *et al*, 2005; Shindhe, *et al*, 2005; Yadhav *et al*, 2006; Patil *et al*, 2007) observed higher total cholesterol, triglyceride, low-density lipoprotein –cholesterol and lower levels of high-density lipoprotein-cholesterol

Also higher ratio of total cholesterol to high density lipoprotein-cholesterol, low-density lipoprotein-cholesterol to high-density cholesterol-lipoprotein and higher triglyceride to

body mass index and weight /hip ratio in patients compared with control.

myocardial infarction (AMI).

Observations of lipid profile

with control (1.9).

in patients compared to controls.

48-69 y.


Table 7. Antioxidant status and Lipid Peroxidation in Control and AMI patients (mean ± SD)


Table 8. Other Biochemical parameters in Control and AMI patients (mean ± SD)

#### **4. Dicussion**

Coronary artery disease (CAD) remains the major cause of morbidity and mortality in all developed and developing countries in the world including India (Reddy and Yusuf, 1998). Dyslipidemia is one of the major modifiable risk factors for CAD (Chopra *et al*, 1998; Vasisht *et al*, 2000; Malhotra *et al*, 2003).

The coronary artery disease (CAD) risk factors do not predict the occurrence of acute myocardial infarction (AMI) as variation in risk factors is observed in South Asian population due to varied dietary habits and life style (Mishra *et al*, 2005). The search for various conventional risk factors among Asians could be helpful in recognizing the future events of stroke. These curiosities prompted us to identify the newer risk factors, with respect to Indian population.

The search for the newer risk factors continues and researchers are investigating the role of inflammatory markers and other potential risks factors which could link with acute myocardial infarction (AMI).

In this prospective case-control study in India, only normolipidaemic acute myocardial infarction (AMI) patients were selected. The study was designed to identify and evaluate potential risk factors in normolipidaemic acute myocardial infarction (AMI) patients. The subjects selected for the study comprised of 165 controls, 48-69 y and 165 acute MI patients, 48-69 y.

Anthropometric variables in acute myocardial infarction (AMI) patients showed highly significant differences in waist/hip ratio and biceps skin fold thickness. Study reported (Heitman *et al*, 2004) that waist /hip ratio is a dominant, independent and predictive variable of cardiovascular disease and coronary heart disease deaths in Australian men and women. Megnien *et al*, 1999 also reported high hip circumference relative to weight and waist circumference is a better predictor of low incidence of cardiovascular disease and coronary heart disease. The present study is in good agreement with the observations of the above studies. Among Indians the cardiovascular risk is high even the prevalence of obesity is minimal (Megnien *et al*, 1999). In the present study the mean body mass index and waist /hip ratio in all subjects was 26.56 and 0.96 respectively, showing a significantly higher body mass index and weight /hip ratio in patients compared with control.

Based on the observations of the aforementioned studies and further supported by the present study it could be concluded that weight/hip ratio is a better predictor of cardiovascular disease (CVD) than body mass index. So it is better tool for indentifying the future risk of acute myocardial infarction (AMI) in subjects by non-invasive procedures.

#### Observations of lipid profile

90 Recent Advances in Cardiovascular Risk Factors

AMI patients *(n=165)* 

1826.5 ± 31.9 813.9 ± 208.9 <0.02 (784.42-843.37)

61.3 ± 3.9 42.6 ± 6.3 <0.001(41.71- 43.48)

5.7 ± 1.0 14.8 ± 1.7 <0.02(11.55-15.06)

31.0 ± 2.7 48.3 ± 5.5 <0.001(47.44-49.11)

AMI patients *(n=165)* 

98.4 ± 6.2 69.7 ± 10.0 <0.0001(68.28-71.11)

81.9 ± 3.9 97.5 ± 11.7 <0.001(95.84-99.15)

P value (95%CI)

*P* value (95% CI)

Control *(n=165)* 

Serum superoxide dismutase

Serum glutathione peroxidase

Serum malondialdehyde

Serum conjugated dienes

Serum Arylesterase activity (

*et al*, 2000; Malhotra *et al*, 2003).

respect to Indian population.

Serum Ischemia modified albumin

(U/gHb)

(U/gHb)

(nmol/L)

(µmol/L)

kU/L)

(U/ml)

**4. Dicussion** 

Serum albumin ( mg/dl) 4.4 ± 0.3 4.2 ± 0.3 <0.001(4.17-4.28) Serum uric acid ( mg/dl) 5.8 ± 1.2 4.3 ± 0.9 <0.01(4.18-4.45) Serum ascorbic acid ( mg/dl) 5.3 ± 1.2 2.8 ± 0.7 <0.0001(2.70-2.89) Serum Total bilirubin ( mg/dl) 0.8 ± 0.2 0.7 ± 0.2 <0.001(0.62-0.69)

Serum catalase (k/gHb) 256.2 ± 26.7 193.1 ± 35.9 <0.001(188.03-198.16) Serum Lipoprotein (a) (mg/dl) 3.0 ± 1.1 10.9 ± 2.2 <0.0001 (10.58-11.21)

Table 7. Antioxidant status and Lipid Peroxidation in Control and AMI patients (mean ± SD)

Plasma fibrinogen (mg/dl) 237.5 ± 17.4 357.8 ± 23.2 <0.0001 (354.52 -361.07 Serum caeruloplasmin ( mg/dl) 20.4 ± 2.3 51.5 ± 2.4 <0.0001 (51.16-51.83)

Serum C-reactive protein (mg/dl) 1.1 ± 0.3 3.0 ± 1.1 <0.0001(2.84-3.15)

Coronary artery disease (CAD) remains the major cause of morbidity and mortality in all developed and developing countries in the world including India (Reddy and Yusuf, 1998). Dyslipidemia is one of the major modifiable risk factors for CAD (Chopra *et al*, 1998; Vasisht

The coronary artery disease (CAD) risk factors do not predict the occurrence of acute myocardial infarction (AMI) as variation in risk factors is observed in South Asian population due to varied dietary habits and life style (Mishra *et al*, 2005). The search for various conventional risk factors among Asians could be helpful in recognizing the future events of stroke. These curiosities prompted us to identify the newer risk factors, with

Table 8. Other Biochemical parameters in Control and AMI patients (mean ± SD)

Control *(n=165)* 

> The mean total cholesterol level of the controls compared with acute myocardial infarction patients (186.44 ± 13.95 mg/dl) was significantly (p<0.001) higher compared with controls (168.58 ± 12.16 mg/dl). The mean high density lipoprotein-cholesterol level in the patients was significantly lower (p<0.001) compared with controls. Triglyceride (TG) values observed in acute myocardial infarction (AMI) patients was (129mg/dl) significantly higher than controls (107.8mg/dl). The mean low density lipoprotein-cholesterol (LDL-c) levels in patients was (119.4mg/dl) significantly higher than controls (83.6 mg/dl). The total cholesterol / high density lipoprotein – cholesterol ratio in acute myocardial infarct patients (4.6) was significantly (p<0.001) higher compared with controls (3.4). The present study observed significantly higher ratio (2.9) in acute myocardial infarction patients compared with control (1.9).

> Earlier studies in lipid profile analysis conducted on acute myocardial infarction patients (Mishra *et al*, 2001; Das *et al*, 2002; Goswami *et al*, 2003; Kharb *et al*, 2003; Malhotra *et al*, 2003; Burman *et al*, 2004; Rajashekhar *et al*, 2004; Sivaraman *et al*, 2004; Rani *et al*, 2005; Shindhe, *et al*, 2005; Yadhav *et al*, 2006; Patil *et al*, 2007) observed higher total cholesterol, triglyceride, low-density lipoprotein –cholesterol and lower levels of high-density lipoprotein-cholesterol in patients compared to controls.

> Also higher ratio of total cholesterol to high density lipoprotein-cholesterol, low-density lipoprotein-cholesterol to high-density cholesterol-lipoprotein and higher triglyceride to

Cardiovascular Risk Factors in Elderly Normolipidaemic Acute Myocardial Infarct Patients 93

The mean serum Lipoprotein (a) malondialdehyde (MDA) and conjugated diene (CD) levels in MI patients were higher compared with controls. Earlier studies conducted (Burman *et al*, 2004; Guha *et al*, 2001; Bal *et al*, 2001; Rajashekhar *et al*, 2004) also observed higher Lipoprotein (a) in AMI patients where as Nascetti *et al*, (1996) did not observed any change in Lipoprotein (a) levels in cardiovascular disease (CVD) patients and concluded lipoprotein (a) not to be considered as an independent risk factor in cardiovascular disease (CVD)

Studies conducted (Senthil *et al*, 2004; Das *et al*, 2002; Kharb 2003; Bhakuni *et al*, 2006; Shindhe *et al*, 2005; Gupta *et al*, 2006) reported higher levels of malondialdehyde (MDA) in

The levels of caeruloplasmin, C-reactive protein, fibrinogen, ischemia-modified albumin were higher and arylesterase activities were lowered in patients. Studies conducted (Grobusch *et al*, 1999; El-Badry *et al*, 1995; Giurgie, 2005; Awadallah *et al*, 2006) observed significantly higher (p<0.001) levels of caeruloplasmin where as (Berton *et al*, 2003; Bhagat *et al*, 2003; Sivaraman *et al*, 2004; Kulsoom *et al*, 2006; Boncler *et al*, 2006) observed higher levels of C-reactive protein in patients. Shukla *et al*, (2006) stated elevated levels of caeruloplasmin as a risk factor for acute myocardial infarct patients. The reactive oxygen species disrupts copper binding to caeruloplasmin thus impairing its antioxidant property and further promoting oxidative pathology. Studies conducted on plasma fibrinogen levels in acute myocardial infarct patients (Harkut *et al*, 2004; Coppola *et al*, 2005; Beg *et al*, 2007; Sivaraman *et al*, 2004) reported rise in plasma fibrinogen as the present study. Earlier study conducted (Chawla *et al*, 2006; Auxter, 2003; Bar-Or *et al*, 2001) in acute myocardial infarct patients also reported higher levels in patients as observed by the present study. Studies on arylesterase activities in acute myocardial infarct patients (Aviram *et al*, 1999; Ayub *et al*, 1999; Richard *et al*, 2000; Jarvik *et al*, 2002; Azizi *et al*, 2002; Singh *et al*, 2007; Sarkar *et al*, 2006) also observed lower activities as concurrent to the current study. Increased C-reactive protein (CRP) concentrations in patients with unstable angina and acute myocardial infarct might induce the production by the monocytes of the tissue factor which initiates the coagulation process. C-reactive protein together with fibrinogen acts as a chemotactic factor. Fibrinogen is responsible for the adhesion of macrophages to the endothelial surface for their migration into the intima. The elevated c-reactive protein levels have been found to be related to the occurrence of cardiovascular complications such as sudden cardiac death or AMI (Pepys

Our study concluded apart from lipid profile, other variables which could be a probable risk for the future myocardial events have to be equally monitored. It is also recommended to increase dietary antioxidant intake in persons who already have known risk factors so that to some extent the myocardial infarction could be delayed. It is also important to check inflammatory markers like c-reactive protein and ischemia-modified albumin in a regular period of time after stepping early forties as they could be a cost effective mode of diagnosis and the subjects can be efficiently monitored and complications of myocardial infarction can

Lipoprotein (a) and lipid peroxidation

patients.

myocardial infarct patients.

and Hirschfield, 2003).

be prolonged.

Other biochemical parameters

high-density cholesterol-lipoprotein was observed in the present study. The present study concludes the importance of assessing the lipid ratios even in normolipidaemic subjects as it is one of the atherogenic factors for development of myocardial infarction and other coronary complications. The practice of computing the ratio should be implemented even in a normal health check up packages. In the final analysis it appears that myocardial infarction and coronary artery disease are not always associated with an elevated serum total cholesterol concentration. The major concern of this observation is that subjects who maintain desirable total cholesterol concentration also are targets for myocardial infarction (MI) and coronary artery disease (CAD) and therefore analysis of other risk factors that are non-conventional and newly emerging will be of immense important in the eventual assessment of the risk status. The existing literature and the results of the present study all point out that acute myocardial infarction and coronary artery disease patients have significantly higher total cholesterol concentration whether the values are in the desirable range or elevated.

#### Antioxidant status

The serum endogenous antioxidants were decreased in acute myocardial infarction compared to controls. Similarly the enzyme antioxidants were also significantly lowered in patients.

Study conducted (Olusi *et al*, 1999; Djousse *et al* 2003) in acute myocardial infarction patients, reported significantly lower (p<0.0001) albumin and bilirubin (p<0.0001), where as lower levels of uric acid (Jing *et al*, 2000; Brand *et al*, 1985; Niskanen *et al*, 2004) and ascorbic acid (Nyossen *et al*, 1997; Bhakuni *et al*, 2006; Das *et al*, 2002; Kurl *et al*, 2002) in acute myocardial infarct patients were reported.

The aforementioned studies suggested the expected risk of acute myocardial infarction is increased where these endogenous antioxidants are lowered due to enhanced utilization during oxidative stress in patients. Though, uric acid is well established antioxidant, but at times it can also act as a pro-oxidant, which might increase the risk of myocardial infarction. Aulinskas *et al*, (1983) established the role of ascorbic acid as up regulator of low density –lipoprotein (LDL) receptors, facilitating the clearance of low density – lipoprotein (LDL). The low levels of ascorbic acid in acute myocardial infarction (AMI) patients in the present study might be due to enhanced utilization of ascorbic acid during oxidative stress in patients.

The enzymatic antioxidants namely superoxide dismutase, catalase and glutathione peroxidase are also lowered in patients compared with controls. The findings of the present study concurs to earlier studies (Senthil *et al*, 2004; Bhakuni *et al*, 2006; Jain *et al*, 2000; Rajashekhar *et al*, 2004; Das *et al*, 2002; Gupta *et al*, 2006; Patil *et al*, 2007) where lower activities of superoxide dismutase, catalase and glutathione peroxidase. Studies conducted (Senthil *et al*, 2004; Shindhe *et al*, 2005; Rajasekhar *et al*, 2004; El-Badry *et al*, 1995; Gupta *et al*, 2006 and Kharb 2003) also reported reduced activities of glutathione peroxidase in patients compared with controls. These studies are based on the hypothesis of decreased antioxidants due to oxidative insult in myocardial infarct patients. Thus it is indicative that low levels of both endogenous and enzyme antioxidants in circulation may be due to its increased utilization to scavenge toxic lipid peroxides.

#### Lipoprotein (a) and lipid peroxidation

92 Recent Advances in Cardiovascular Risk Factors

high-density cholesterol-lipoprotein was observed in the present study. The present study concludes the importance of assessing the lipid ratios even in normolipidaemic subjects as it is one of the atherogenic factors for development of myocardial infarction and other coronary complications. The practice of computing the ratio should be implemented even in a normal health check up packages. In the final analysis it appears that myocardial infarction and coronary artery disease are not always associated with an elevated serum total cholesterol concentration. The major concern of this observation is that subjects who maintain desirable total cholesterol concentration also are targets for myocardial infarction (MI) and coronary artery disease (CAD) and therefore analysis of other risk factors that are non-conventional and newly emerging will be of immense important in the eventual assessment of the risk status. The existing literature and the results of the present study all point out that acute myocardial infarction and coronary artery disease patients have significantly higher total cholesterol concentration whether the values are in the desirable

The serum endogenous antioxidants were decreased in acute myocardial infarction compared to controls. Similarly the enzyme antioxidants were also significantly lowered in

Study conducted (Olusi *et al*, 1999; Djousse *et al* 2003) in acute myocardial infarction patients, reported significantly lower (p<0.0001) albumin and bilirubin (p<0.0001), where as lower levels of uric acid (Jing *et al*, 2000; Brand *et al*, 1985; Niskanen *et al*, 2004) and ascorbic acid (Nyossen *et al*, 1997; Bhakuni *et al*, 2006; Das *et al*, 2002; Kurl *et al*, 2002) in acute

The aforementioned studies suggested the expected risk of acute myocardial infarction is increased where these endogenous antioxidants are lowered due to enhanced utilization during oxidative stress in patients. Though, uric acid is well established antioxidant, but at times it can also act as a pro-oxidant, which might increase the risk of myocardial infarction. Aulinskas *et al*, (1983) established the role of ascorbic acid as up regulator of low density –lipoprotein (LDL) receptors, facilitating the clearance of low density – lipoprotein (LDL). The low levels of ascorbic acid in acute myocardial infarction (AMI) patients in the present study might be due to enhanced utilization of ascorbic acid during

The enzymatic antioxidants namely superoxide dismutase, catalase and glutathione peroxidase are also lowered in patients compared with controls. The findings of the present study concurs to earlier studies (Senthil *et al*, 2004; Bhakuni *et al*, 2006; Jain *et al*, 2000; Rajashekhar *et al*, 2004; Das *et al*, 2002; Gupta *et al*, 2006; Patil *et al*, 2007) where lower activities of superoxide dismutase, catalase and glutathione peroxidase. Studies conducted (Senthil *et al*, 2004; Shindhe *et al*, 2005; Rajasekhar *et al*, 2004; El-Badry *et al*, 1995; Gupta *et al*, 2006 and Kharb 2003) also reported reduced activities of glutathione peroxidase in patients compared with controls. These studies are based on the hypothesis of decreased antioxidants due to oxidative insult in myocardial infarct patients. Thus it is indicative that low levels of both endogenous and enzyme antioxidants in circulation may be due to its

range or elevated. Antioxidant status

myocardial infarct patients were reported.

increased utilization to scavenge toxic lipid peroxides.

oxidative stress in patients.

patients.

The mean serum Lipoprotein (a) malondialdehyde (MDA) and conjugated diene (CD) levels in MI patients were higher compared with controls. Earlier studies conducted (Burman *et al*, 2004; Guha *et al*, 2001; Bal *et al*, 2001; Rajashekhar *et al*, 2004) also observed higher Lipoprotein (a) in AMI patients where as Nascetti *et al*, (1996) did not observed any change in Lipoprotein (a) levels in cardiovascular disease (CVD) patients and concluded lipoprotein (a) not to be considered as an independent risk factor in cardiovascular disease (CVD) patients.

Studies conducted (Senthil *et al*, 2004; Das *et al*, 2002; Kharb 2003; Bhakuni *et al*, 2006; Shindhe *et al*, 2005; Gupta *et al*, 2006) reported higher levels of malondialdehyde (MDA) in myocardial infarct patients.

#### Other biochemical parameters

The levels of caeruloplasmin, C-reactive protein, fibrinogen, ischemia-modified albumin were higher and arylesterase activities were lowered in patients. Studies conducted (Grobusch *et al*, 1999; El-Badry *et al*, 1995; Giurgie, 2005; Awadallah *et al*, 2006) observed significantly higher (p<0.001) levels of caeruloplasmin where as (Berton *et al*, 2003; Bhagat *et al*, 2003; Sivaraman *et al*, 2004; Kulsoom *et al*, 2006; Boncler *et al*, 2006) observed higher levels of C-reactive protein in patients. Shukla *et al*, (2006) stated elevated levels of caeruloplasmin as a risk factor for acute myocardial infarct patients. The reactive oxygen species disrupts copper binding to caeruloplasmin thus impairing its antioxidant property and further promoting oxidative pathology. Studies conducted on plasma fibrinogen levels in acute myocardial infarct patients (Harkut *et al*, 2004; Coppola *et al*, 2005; Beg *et al*, 2007; Sivaraman *et al*, 2004) reported rise in plasma fibrinogen as the present study. Earlier study conducted (Chawla *et al*, 2006; Auxter, 2003; Bar-Or *et al*, 2001) in acute myocardial infarct patients also reported higher levels in patients as observed by the present study. Studies on arylesterase activities in acute myocardial infarct patients (Aviram *et al*, 1999; Ayub *et al*, 1999; Richard *et al*, 2000; Jarvik *et al*, 2002; Azizi *et al*, 2002; Singh *et al*, 2007; Sarkar *et al*, 2006) also observed lower activities as concurrent to the current study. Increased C-reactive protein (CRP) concentrations in patients with unstable angina and acute myocardial infarct might induce the production by the monocytes of the tissue factor which initiates the coagulation process. C-reactive protein together with fibrinogen acts as a chemotactic factor. Fibrinogen is responsible for the adhesion of macrophages to the endothelial surface for their migration into the intima. The elevated c-reactive protein levels have been found to be related to the occurrence of cardiovascular complications such as sudden cardiac death or AMI (Pepys and Hirschfield, 2003).

Our study concluded apart from lipid profile, other variables which could be a probable risk for the future myocardial events have to be equally monitored. It is also recommended to increase dietary antioxidant intake in persons who already have known risk factors so that to some extent the myocardial infarction could be delayed. It is also important to check inflammatory markers like c-reactive protein and ischemia-modified albumin in a regular period of time after stepping early forties as they could be a cost effective mode of diagnosis and the subjects can be efficiently monitored and complications of myocardial infarction can be prolonged.

Cardiovascular Risk Factors in Elderly Normolipidaemic Acute Myocardial Infarct Patients 95

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**7** 

*Nigeria* 

**Erectile Dysfunction Complicating** 

Irekpita Eshiobo, Emeka Kesieme and Taofik Salami

*Ambrose Alli University, Ekpoma,* 

**Cardiovascular Risk Factors and Disease** 

Erectile Dysfunction (ED) is the persistent inability to achieve and or maintain penile erection sufficient for satisfactory sexual Intercourse. 1 *In 1995 it was estimated that 152 million men world-wide experienced ED and it was projected that by 2025, the prevalence worldwide would be 320 million*.2 In the United states alone, 34 million men suffer from ED3 while in China, according to Yang et al, 4 prevalence is reported to be 73.1% in the general population. Shaeer et al 5 in their study of the prevalence of ED in diverse nationalities representing a wide range of cultural, religious, racial and socio-economic backgrounds concluded that prevalence rates from various countries are difficult to compare because of variable definition and age range. So far, the most comprehensive epidemiological study of ED has been the Massachusetts male aging study6 which reported ED to be present in 10% of men aged between forty and fifty years and almost 70% in Men aged 70% years and above.

**COUNTRIES PREVALENCE** 

**1. Malaysia 17.0% 2. Germany 19.0% 3. Japan 34.0%** 

**5. Morocco 53.6% 6. Nigeria 57.4% 7. Egypt 63.6% 8. Turkey 64.3% 9. China 73.1% 10. Pakistan 80.8%** 

**of America 52.0%** 

**4. United States** 

Table 1. Reported Prevalence of ED in selected Countries.

**1. Introduction** 


### **Erectile Dysfunction Complicating Cardiovascular Risk Factors and Disease**

Irekpita Eshiobo, Emeka Kesieme and Taofik Salami *Ambrose Alli University, Ekpoma, Nigeria* 

#### **1. Introduction**

98 Recent Advances in Cardiovascular Risk Factors

Shrinivas K, Vijaya Bhaskar M, Aruna Kumari M, Nagaraj K, Reddy KK. Antioxidants, lipid

Shinde S, Kumar P, Patil N. Decreased Levels Of Erythrocyte Glutathione In Patients With Myocardial Infarction. *The Internet Journal of Alternative Medicine* 2005; 2:1. Singh S, Venketesh S, Verma JS, Verma M, Lellamma CO, Goel RC. Paraoxonase (PON11)

Sivaraman S K, Zachariah G, Annamalai PT. Evaluation of C - reactive protein and other

Sun Y, Oberly LW, Li Y. A simple method for clinical assay of superoxide dismutase. *Clin* 

Vasisht S, Narula J, Awtade A, Tandon R, Srivastava LM. Lipids and lipoproteins in normal

World Health Organization. Cardiovascular diseases (CVDs). Available from: http://www.who.int/mediacentre/factsheets/fs317/en/index.html Yadhav AS, Bhagwat VR, Rathod IM. Relationship of Plasma homocysteine with lipid

diabetes mellitus. *Indian J Med Res* 2007; 125:783-7.

88.

36(1):35-37.

*Chem* 1988; 34: 497-500.

2006; 21(1):106-110.

*Med Sci (India)* 1990; 26:57-66.

peroxidation and lipoproteins in primary hypertension. *Indian Heart J* 2000; 52:285-

activity in northwest Indian Punjabis with coronary artery disease & type II

Inflammatory Markers in Acute Coronary Syndromes. *Kuwait Medical Journal* 2004;

controls and clinically documented coronary heart disease patients. *Ann Natl Acad* 

profile parameters in Ischemic Heart disease. *Indian Journal of Clinical Biochemistry*

Erectile Dysfunction (ED) is the persistent inability to achieve and or maintain penile erection sufficient for satisfactory sexual Intercourse. 1 *In 1995 it was estimated that 152 million men world-wide experienced ED and it was projected that by 2025, the prevalence worldwide would be 320 million*.2 In the United states alone, 34 million men suffer from ED3 while in China, according to Yang et al, 4 prevalence is reported to be 73.1% in the general population. Shaeer et al 5 in their study of the prevalence of ED in diverse nationalities representing a wide range of cultural, religious, racial and socio-economic backgrounds concluded that prevalence rates from various countries are difficult to compare because of variable definition and age range. So far, the most comprehensive epidemiological study of ED has been the Massachusetts male aging study6 which reported ED to be present in 10% of men aged between forty and fifty years and almost 70% in Men aged 70% years and above.


Table 1. Reported Prevalence of ED in selected Countries.

Erectile Dysfunction Complicating Cardiovascular Risk Factors and Disease 101

men have been treated and have returned to normal life especially with the advent of the phosphodiesterase 5 inhibitors. 20 This chapter focuses on erectile dysfunction and its

The human penis is made up of two dorsally located corpora cavernosa and one ventrally placed corpus spongiosum.21 The corpus spongiosum expands proximally to form the bulb of the penis while distally it expands to form the glans penis, the most sensitive part of the organ. Proximally, the two corpora cavernrnosa diverge laterally and are attached, one on each side, to the inferior surface of the ischio pubic rami.22 The erectile tissue proper is located in the corpora cavernosa and surrounded by a tough fibrous tissue called the tunica

The corpora cavernosa consist of sinusoids with smooth muscle lined internally by endothelium similar to that of blood vessels. The helicine arteries, the terminal branches of the cavernosal arteries, open directly into the sinusoids. The blood is drained by veins which course beneath the tunica albuginea before piercing it. These join the dorsal vein complex. The penis is innervated by both autonomic and somatic nerves; the latter supplies the skin while the former sub serves the erectile tissue proper.23 Somatic supply consist of free nerve endings (receptors) in the skin of the penis, usually up to ten times as numerous in the penile skin and the glans particularly. These receptors coalescence to form the dorsal nerve of the penis, a branch of the pudendal nerve which originates from the dorsal rami of S2 -4. 24 The nerve fibres are mainly unmyelinated C and A delta fibres which sub serve the sensation of pain, pressure and touch (tactile stimulus), the stimulus responsible for reflexogenic erection.The para sympathetic pathway which serves the cavernosa arises from neurons in the S2- 425 and follow the ventral rami. This group of cells constitutes the Onuf nucleus and their preganglionic fibres join the sympathetic fibers from the hypogastric plexus to form the pelvic plexus (pelvic splachnic nerves). The post ganglonic fibres (nervi erigentis ) arise from the pelvic plexus and passing anterior to the rectum and posteriolateral to the prostate, they pierce the pelvic membrane to reach the corpora cavernosa. They are often damaged during surgical procedures which involve total removal of the prostate and the rectum because of which these

The sympathetic pathway whose fibres are mainly inhibitory originate from T11 - L2 spinal segment. The fibres pass through the white rami to the sympathetic ganglion. Some of these fibres reach the inferior mesenteric and superior hypogastric plexus through the lumbar splanchnic nerves. From the hypogastric plexus, some fibres reach the pelvis and join the parasympathetic fibres to form the pelvic plexus. The fibres from the pelvic plexus which reach the carvenosa along with those from S2-4 are mainly from T10 -12. 27 These fibres also carry impulses which control ejaculation and may be damaged during radical retro

The central control of penile erection depends on the input from somatic and autonomic pathways and environmental factors, which include smell, sight and thought of Sex. Tactile impulses from the dorsal nerve of the penis travel via the spino thalamic and spino reticular pathways to the thalamus and sensory cortex for sensory interpretation. Studies have shown that the medial pre-optic area and the para ventricular nucleus of the hypothalamus are the

cardiovascular system correlates.

albuginea.

**2. Neuroanatomy and physiology of penile erection** 

procedures are often complicated by erectile dysfunction.26

peritoneal dissection as they course behind the peritoneum. 28

In the early 1980s, issues of quality of life ( Qol) were brought into the fore of medical practice prompting the rise of ED into prominence as a diagnostic entity. According to Pomerville 7 '20 years ago, ED did not exist as a diagnostic term. Its former name 'Impotence' carried a heavy connotation – an impotent man was powerless, worthless, less than a man. Impotence was seldom discussed in the locker rooms or bed rooms of nations" Impotence was relegated and considered to be due to the wears and tears of the aging process for which there is no treatment. This is strongly supported by the following statement by Wyne et al:8 "for centuries, sexual medicine was a taboo subject practiced by quasiscientists, back alley charlatans and village shamans. Because of a paucity of basic knowledge about the anatomy, Physiology and Pharmacology of the erectile process, many myths on causation and therapy were promulgated through time."

This perception has changed remarkably due to research into ED in the last century which led to the discovery that certain factors are associated with ED with a cause- effect relationship.9 10 Currently, there is a better understanding of the incidence, prevalence, etiology and risk factors for ED.

ED is classified as psychogenic, neurological and vasculogenic. Vasculogenic erectile dysfunction, similar to coronary artery disease, is usually due to atherosclerosis, in the case of ED, arthrosclerosis of the branches of the pudendal artery. Cardiovascular risk factors are so classified because of their propensity for causing artherosclersis12 which may involve the coronary artery thereby predisposing the heart to myocardial hypoperfussion disease. The common denominator in ED and CAD therefore, is arthrosclerosis whose main causes are these life style abnormities referred to as cardiovascular risk factors. The implication of this is that the risk factors for ED and CAD are similar or are in fact the same.13 14 15

Hypertension, Diabetes mellitus, dyslipidemia and cigarette smoking have been well documented as predisposing to atherosclerosis of the coronary artery which has earned them the term 'cardiovascular risk factors". Currently, reduced androgen level, particularly testosterone, is under intense research as another risk factor because of the vascular changes associated with it.16 Several epidemiological studies have demonstrated that ED is more prevalent in men with atherosclerotic disease than the general population. 17 It is now generally accepted that ED, like CAD, results from the endothelial dysfunction which usually co-exist with or predates true atherosclerotic vascular changes.18


Table 2. CVS risk factors.

In spite of the adverse effects of ED on quality of life of the affected men, the disease is generally under-reported and under-treated19 due to poor self reporting as a result of cultural taboos, fear of stigmatization, ignorance on the part of the patient or physician and unavailability of specialists in the area. Erectile dysfunction is a treatable disease and many

In the early 1980s, issues of quality of life ( Qol) were brought into the fore of medical practice prompting the rise of ED into prominence as a diagnostic entity. According to Pomerville 7 '20 years ago, ED did not exist as a diagnostic term. Its former name 'Impotence' carried a heavy connotation – an impotent man was powerless, worthless, less than a man. Impotence was seldom discussed in the locker rooms or bed rooms of nations" Impotence was relegated and considered to be due to the wears and tears of the aging process for which there is no treatment. This is strongly supported by the following statement by Wyne et al:8 "for centuries, sexual medicine was a taboo subject practiced by quasiscientists, back alley charlatans and village shamans. Because of a paucity of basic knowledge about the anatomy, Physiology and Pharmacology of the erectile process, many

This perception has changed remarkably due to research into ED in the last century which led to the discovery that certain factors are associated with ED with a cause- effect relationship.9 10 Currently, there is a better understanding of the incidence, prevalence,

ED is classified as psychogenic, neurological and vasculogenic. Vasculogenic erectile dysfunction, similar to coronary artery disease, is usually due to atherosclerosis, in the case of ED, arthrosclerosis of the branches of the pudendal artery. Cardiovascular risk factors are so classified because of their propensity for causing artherosclersis12 which may involve the coronary artery thereby predisposing the heart to myocardial hypoperfussion disease. The common denominator in ED and CAD therefore, is arthrosclerosis whose main causes are these life style abnormities referred to as cardiovascular risk factors. The implication of this

Hypertension, Diabetes mellitus, dyslipidemia and cigarette smoking have been well documented as predisposing to atherosclerosis of the coronary artery which has earned them the term 'cardiovascular risk factors". Currently, reduced androgen level, particularly testosterone, is under intense research as another risk factor because of the vascular changes associated with it.16 Several epidemiological studies have demonstrated that ED is more prevalent in men with atherosclerotic disease than the general population. 17 It is now generally accepted that ED, like CAD, results from the endothelial dysfunction which

In spite of the adverse effects of ED on quality of life of the affected men, the disease is generally under-reported and under-treated19 due to poor self reporting as a result of cultural taboos, fear of stigmatization, ignorance on the part of the patient or physician and unavailability of specialists in the area. Erectile dysfunction is a treatable disease and many

**EVALUATED** 

**Low testosterone level** 

is that the risk factors for ED and CAD are similar or are in fact the same.13 14 15

usually co-exist with or predates true atherosclerotic vascular changes.18

**Documented Risk Factors CVS RISK FACTOR BEING** 

myths on causation and therapy were promulgated through time."

etiology and risk factors for ED.

**Cigarette smoking Dyslipidemia Diabetes Mellitus Hypertension** 

Table 2. CVS risk factors.

men have been treated and have returned to normal life especially with the advent of the phosphodiesterase 5 inhibitors. 20 This chapter focuses on erectile dysfunction and its cardiovascular system correlates.

#### **2. Neuroanatomy and physiology of penile erection**

The human penis is made up of two dorsally located corpora cavernosa and one ventrally placed corpus spongiosum.21 The corpus spongiosum expands proximally to form the bulb of the penis while distally it expands to form the glans penis, the most sensitive part of the organ. Proximally, the two corpora cavernrnosa diverge laterally and are attached, one on each side, to the inferior surface of the ischio pubic rami.22 The erectile tissue proper is located in the corpora cavernosa and surrounded by a tough fibrous tissue called the tunica albuginea.

The corpora cavernosa consist of sinusoids with smooth muscle lined internally by endothelium similar to that of blood vessels. The helicine arteries, the terminal branches of the cavernosal arteries, open directly into the sinusoids. The blood is drained by veins which course beneath the tunica albuginea before piercing it. These join the dorsal vein complex.

The penis is innervated by both autonomic and somatic nerves; the latter supplies the skin while the former sub serves the erectile tissue proper.23 Somatic supply consist of free nerve endings (receptors) in the skin of the penis, usually up to ten times as numerous in the penile skin and the glans particularly. These receptors coalescence to form the dorsal nerve of the penis, a branch of the pudendal nerve which originates from the dorsal rami of S2 -4. 24 The nerve fibres are mainly unmyelinated C and A delta fibres which sub serve the sensation of pain, pressure and touch (tactile stimulus), the stimulus responsible for reflexogenic erection.The para sympathetic pathway which serves the cavernosa arises from neurons in the S2- 425 and follow the ventral rami. This group of cells constitutes the Onuf nucleus and their preganglionic fibres join the sympathetic fibers from the hypogastric plexus to form the pelvic plexus (pelvic splachnic nerves). The post ganglonic fibres (nervi erigentis ) arise from the pelvic plexus and passing anterior to the rectum and posteriolateral to the prostate, they pierce the pelvic membrane to reach the corpora cavernosa. They are often damaged during surgical procedures which involve total removal of the prostate and the rectum because of which these procedures are often complicated by erectile dysfunction.26

The sympathetic pathway whose fibres are mainly inhibitory originate from T11 - L2 spinal segment. The fibres pass through the white rami to the sympathetic ganglion. Some of these fibres reach the inferior mesenteric and superior hypogastric plexus through the lumbar splanchnic nerves. From the hypogastric plexus, some fibres reach the pelvis and join the parasympathetic fibres to form the pelvic plexus. The fibres from the pelvic plexus which reach the carvenosa along with those from S2-4 are mainly from T10 -12. 27 These fibres also carry impulses which control ejaculation and may be damaged during radical retro peritoneal dissection as they course behind the peritoneum. 28

The central control of penile erection depends on the input from somatic and autonomic pathways and environmental factors, which include smell, sight and thought of Sex. Tactile impulses from the dorsal nerve of the penis travel via the spino thalamic and spino reticular pathways to the thalamus and sensory cortex for sensory interpretation. Studies have shown that the medial pre-optic area and the para ventricular nucleus of the hypothalamus are the

Erectile Dysfunction Complicating Cardiovascular Risk Factors and Disease 103

Kinase G leading to a decrease in calcium influx into the cell which in turn causes activation of myokinase and finally, relaxation of corpora smooth muscle, a sine qua non in the erectile

Penile erection is classified as: Central, reflexogenic and Nocturnal. 43 In central erection, the stimulus is from thought, site and smell related to Sexual intercourse which activates the spinal centres, the impulses of which travel through the parasympathetic to the corpora. Reflexogenic erection results from tactile stimulation of the dorsal nerve of the penis. The afferent pathway reaches the onuf nucleus which then activates the erectile parasympathetic pathway via the same process as in central erection. Nocturnal Penile tumescence (NPT) is currently poorly understood. It occurs during rapid eye movement sleep and it is presently

Following central or reflexogenic stimulus, there is activation of the parasympathetic erectile pathway with the release of nitric oxide and the consequent cascade of events which lead to relaxation of cavernous smooth muscle and the helicine arteries. The ensuing rapid influx of blood into the sinusoids leads to increased intra cavernosal pressure and compression of the sub tunical venous system against the tunica abulginea. There is reduced outflow in the presence of markedly increased inflow and the penis becomes firm and increased in size – phase of full erection. Through the pudendal nerve, sexual impulses also reach the ischio carveranosus and bulbospongiosus muscles which contract vigorously, further impeding

Penile detumescense is a consequence of the intense, diffuse sympathetic discharge which herald orgasm, emission and ejaculation. The catecholamines released in the process cause smooth muscle contraction, thereby terminating erection. The smooth muscle contraction leads to reduced blood flow into the sinusoids, reduced intra cavernosa pressure and the opening of the venous channels. There is therefore increased venous outflow in the presence of reduced arterial inflow. The trapped blood in the sinusoids is expelled and the penis

thought to play a key role in keeping the erectile tissue per fussed. 3

venous drainage - phase of rigid erection.

Fig. 1. Penile tumescence and detumescence

becomes flaccid23.

process 42.

integration centre for erection and sexual activity.29 It is currently suggested that there may be projections from the hypothalamic nuclei to the sacral erection centre, S2 – 4, as is found in animals. Other centres which contribute to control of sexual activity also abound in the mid brain and medulla.30

Several neuro transmitters are involved in penile erection and control of sexual activity. The neuro transmitter traditionally associated with the parasympathetic (parasympathetic finally control erection) is acetylcholine while nor– adrenaline and adrenaline are involved in sympathetic transmission. Other neuro-transmitters secreted at the level of the autonomic nervous system in the genito-urinary tract are vaso- active intestinal peptide (VIP) and several prostaglandins. These neurotransmitters do not however, completely account for the events that lead to penile erection. Currently, the substance which is considered to act as the neurotransmitter involved in penile erection is a non adrenergic, non cholinergic (NANC) and it is now known to be nitric oxide (NO). 31

Nitric oxide is a ubiquitous neuro-transmitter in the lower urinary tract 32 and its role in penile erection process is presently well documented. It is formed from L-arginine under the control of Nitric oxide synthase. 33 There are three isoforms of Nitric oxide synthase; eNos, nNos and iNos depending on the cell in which it is synthesized. Neural stimulation causes release of Nitric oxide from neurons while its release from endothelium is in response to sheer stress. 34 The neurotransmitters involved in the central control of penile erection and sexual activity include oxytocin, gama amminobutyric acid and serotonin.

In the flaccid state, the smooth muscle of the cavernosa is semi contracted under the tonic influence of the sympathetic system, its intrinsic myogenic activity and endothelium derived factors such as prostaglandin F2 alpha. 37 In this state, the blood flow to the penis is kept at the barest minimum required to meet its nutritional needs at a pCo2 of 35mmHg.38 Following sexual stimulation, the non adrenergic non cholinergic fibres which accompany the parasympathetic to the cavernosa, release nitric oxide which triggers the erectile process 39.


Table 3. Summary of physiology of penile erection.

Nitric oxide is a potent vasodilator which readily diffuses into the cell to initiate a series of bio-chemical events. Nitric oxide modulates the activities of guanylate cyclase, 40 an action which leads to the conversion of guanosine triphosphate (GTP) to cyclic guanosine monophosphate (cGMP). The latter, acting as a second messenger, regulates calcium channel activities including those of intracellular contractile proteins that lead to the relaxation of corpus cavernosal smooth muscle.41 Specifically, cGMP activates protein

integration centre for erection and sexual activity.29 It is currently suggested that there may be projections from the hypothalamic nuclei to the sacral erection centre, S2 – 4, as is found in animals. Other centres which contribute to control of sexual activity also abound in the

Several neuro transmitters are involved in penile erection and control of sexual activity. The neuro transmitter traditionally associated with the parasympathetic (parasympathetic finally control erection) is acetylcholine while nor– adrenaline and adrenaline are involved in sympathetic transmission. Other neuro-transmitters secreted at the level of the autonomic nervous system in the genito-urinary tract are vaso- active intestinal peptide (VIP) and several prostaglandins. These neurotransmitters do not however, completely account for the events that lead to penile erection. Currently, the substance which is considered to act as the neurotransmitter involved in penile erection is a non adrenergic, non cholinergic (NANC)

Nitric oxide is a ubiquitous neuro-transmitter in the lower urinary tract 32 and its role in penile erection process is presently well documented. It is formed from L-arginine under the control of Nitric oxide synthase. 33 There are three isoforms of Nitric oxide synthase; eNos, nNos and iNos depending on the cell in which it is synthesized. Neural stimulation causes release of Nitric oxide from neurons while its release from endothelium is in response to sheer stress. 34 The neurotransmitters involved in the central control of penile erection and

In the flaccid state, the smooth muscle of the cavernosa is semi contracted under the tonic influence of the sympathetic system, its intrinsic myogenic activity and endothelium derived factors such as prostaglandin F2 alpha. 37 In this state, the blood flow to the penis is kept at the barest minimum required to meet its nutritional needs at a pCo2 of 35mmHg.38 Following sexual stimulation, the non adrenergic non cholinergic fibres which accompany the parasympathetic to the cavernosa, release nitric oxide which triggers the erectile process 39.

Nitric oxide is a potent vasodilator which readily diffuses into the cell to initiate a series of bio-chemical events. Nitric oxide modulates the activities of guanylate cyclase, 40 an action which leads to the conversion of guanosine triphosphate (GTP) to cyclic guanosine monophosphate (cGMP). The latter, acting as a second messenger, regulates calcium channel activities including those of intracellular contractile proteins that lead to the relaxation of corpus cavernosal smooth muscle.41 Specifically, cGMP activates protein

sexual activity include oxytocin, gama amminobutyric acid and serotonin.

 Central (smell, thought) and tactile (penile skin) stimulus Processing of resulting impulses by the Onuf nucleus. Activation of the effector pathway (nervi-erigentes).

Increase blood flow into the cavernous sinus-penile erection.

Activation of the NO-cGMP pathway.

Table 3. Summary of physiology of penile erection.

Release of neuronal NO following neuronal NO Synthase activation.

Increase flow stimulates release of endothelial NO- sustained erection

mid brain and medulla.30

and it is now known to be nitric oxide (NO). 31

Kinase G leading to a decrease in calcium influx into the cell which in turn causes activation of myokinase and finally, relaxation of corpora smooth muscle, a sine qua non in the erectile process 42.

Penile erection is classified as: Central, reflexogenic and Nocturnal. 43 In central erection, the stimulus is from thought, site and smell related to Sexual intercourse which activates the spinal centres, the impulses of which travel through the parasympathetic to the corpora. Reflexogenic erection results from tactile stimulation of the dorsal nerve of the penis. The afferent pathway reaches the onuf nucleus which then activates the erectile parasympathetic pathway via the same process as in central erection. Nocturnal Penile tumescence (NPT) is currently poorly understood. It occurs during rapid eye movement sleep and it is presently thought to play a key role in keeping the erectile tissue per fussed. 3

Following central or reflexogenic stimulus, there is activation of the parasympathetic erectile pathway with the release of nitric oxide and the consequent cascade of events which lead to relaxation of cavernous smooth muscle and the helicine arteries. The ensuing rapid influx of blood into the sinusoids leads to increased intra cavernosal pressure and compression of the sub tunical venous system against the tunica abulginea. There is reduced outflow in the presence of markedly increased inflow and the penis becomes firm and increased in size – phase of full erection. Through the pudendal nerve, sexual impulses also reach the ischio carveranosus and bulbospongiosus muscles which contract vigorously, further impeding venous drainage - phase of rigid erection.

Penile detumescense is a consequence of the intense, diffuse sympathetic discharge which herald orgasm, emission and ejaculation. The catecholamines released in the process cause smooth muscle contraction, thereby terminating erection. The smooth muscle contraction leads to reduced blood flow into the sinusoids, reduced intra cavernosa pressure and the opening of the venous channels. There is therefore increased venous outflow in the presence of reduced arterial inflow. The trapped blood in the sinusoids is expelled and the penis becomes flaccid23.

Fig. 1. Penile tumescence and detumescence

Erectile Dysfunction Complicating Cardiovascular Risk Factors and Disease 105

and macrophages into the intima, a sin-qua non for atherosclerotic changes.

inflammatory nuclear factor Kappa B and hence cellular inflammation.51

Fig. 2. Mechnisms by which CVS risk factors cause ED and CAD

**4. Cardiovascular risk factors and erectile dysfunction** 

**4.1 Diabetes mellitus** 

 Atherosclerosis is characterized by intimal lesion called atheroma or atheromatous plagues or fibrofatty plagues which project into the lumen of the vessel. It forms the common pathway through which the well documented cardiovascular risk factors cause their deleterious effects on the heart, cerebrum and the erectile tissues. Endothelial dysfunction results from inhibition of dimethyl arginine dimethyl amino hydrolase which catalyses the hydrolysis of asymmetric dimethyl arginine, an inhibitor of endothelial nitric oxide synthase. The subsequent uncoupling of eNos leads to endothelial oxidative stress and formation of peroxy nitrates, oxidation of pro-

Endothelial inflammation causes increase permeability of the endothelial cells and the tight junctions. The subsequent increased movement of lipids, protein and inflammatory cells, particularly macrophages into the intima, initiates the process of atherosclerosis which affects all the arterial bed. Timing of clinical appearance however depends on the severity and size of the affected vessel as up to 75% of the vessel lumen maybe occluded before effects become obvious.52 Smaller vessels such as the pudendal artery may be occluded up to ten years before bigger vessels like the coronary arteries are affected. This explains why erectile dysfunction usually precedes the occurrence of coronary artery diseases or stroke.

Diabetes mellitus(DM) is defined as persistent hyperglycemia secondary to relative or absolute insulin deficiency. This has a profound effect on protein, lipid and carbohydrate metabolism with a myriad of complications of which ED is one. These complications can broadly be classified as vascular or neurologic and ED has been documented in both classes. In DM, neurologic damage which may predispose to ED usually result from peripheral

Endothelial dysfunction is a systemic disease and it is usually as a result of diverse injury including sheer stress, trauma and inflammation. It occurs in the initial and subsequent phases of atherosclerosis. Though all arterial beds are affected, the effects manifests earlier in the smaller vessel beds50 such as the pudendal artery which supply the erectile tissue of the penis. In the presence of endothelial dysfunction, the endothelium becomes unduly permeable, allowing substances such as lipids, proteins

### **3. Atherosclerosis**

Arteries vary in wall thickness and size and based on these, they are classified as45 (A) Elastic or large arteries. A typical example is the aorta, Innorminate, subclavian and illiac arteries (b) Medium sized arteries typical of which are coronary arteries (c) Small arteries. These are usually less than 2mm in diameter, coursing for most of their path through the substance of the issue or organ. A good example is the pudendal and cavernosa arteries which supply the penile erectile tissue and upon which erection depends. The arterial wall, independent of class, is made up of three layers namely;


The endothelium is physiologically endowed to control the vessel in order to vary blood flow according to tissue, organ and regional requirements. It is in most part, responsible for the smooth flow of blood and therefore has profound effect on vascular reactivity and thrombogenesis. It inhibits platelet aggregation, reduces the recruitment of inflammatory cells into the intima and the entry of lipids into the arterial wall.

The endothelium secretes a wide variety of substances with paracrine effect while also expressing receptors for various substances in the blood stream, the latter including hormones, local mediators and vasoactive substances. Based on these, the endothelium is capable of sensing and responding to local changes either by causing vasodilatation or constriction. The overall effect of endothelial reactivity is however vasodilatation or a tendency to vasodilatation reaching up to 80% in the penile arterial bed as against 15% in other tissues.47 48

Nitric oxide (NO) is a major neurotransmitter produced by the endothelium and is responsible for the endothelium derived vasodilatation. It is produced, as a neurotransmitter, in most parts of the lower urinary tract either normally or pathologically.49 It inhibits cytochrome C oxidase, reduces oxygen consumption by the vasculature and inhibits or modifies the endothelial cells for circulating white blood cells. Its release is stimulated by acetyl choline, brandykin, substance P and sheer stress on the arterial wall.

 Endothelial dysfunction is defined as a state of altered phenotype which leads to impairment of vascular reactivity or an induced surface which is thrombogenic or abnormally adhesive for inflammatory cells.45 This change may predate or co-exist with atherosclerosis and is characterized by loss of vascular reactivity. The initial phase is characterized by decreased bioavailability of nitric oxide either due to reduced production or increased breakdown. There is therefore loss of the endothelium derived smooth muscle relaxation with a tendency to vasoconstriction and increased peripheral resistance. These effects have been implicated in hypertension, diabetes mellitus, cigarette smoking and hyperlipidemia, all of which are risk factors for ED and CAD.

Arteries vary in wall thickness and size and based on these, they are classified as45 (A) Elastic or large arteries. A typical example is the aorta, Innorminate, subclavian and illiac arteries (b) Medium sized arteries typical of which are coronary arteries (c) Small arteries. These are usually less than 2mm in diameter, coursing for most of their path through the substance of the issue or organ. A good example is the pudendal and cavernosa arteries which supply the penile erectile tissue and upon which erection depends. The arterial wall,

1. Adventitia. This is a fibrous tissue which covers the artery externally and separates it

2. The media. This is responsible for most of the wall thickness and consists mostly of smooth muscle cells (SMC) which are under the tonic control of the endothelium.46 It is

3. The intima. This consists of a single layer of endothelium which rests on a basement membraine. The endothelial cells are joined together by tight junctions which are

The endothelium is physiologically endowed to control the vessel in order to vary blood flow according to tissue, organ and regional requirements. It is in most part, responsible for the smooth flow of blood and therefore has profound effect on vascular reactivity and thrombogenesis. It inhibits platelet aggregation, reduces the recruitment of inflammatory

The endothelium secretes a wide variety of substances with paracrine effect while also expressing receptors for various substances in the blood stream, the latter including hormones, local mediators and vasoactive substances. Based on these, the endothelium is capable of sensing and responding to local changes either by causing vasodilatation or constriction. The overall effect of endothelial reactivity is however vasodilatation or a tendency to vasodilatation reaching up to 80% in the penile arterial bed as against 15% in

Nitric oxide (NO) is a major neurotransmitter produced by the endothelium and is responsible for the endothelium derived vasodilatation. It is produced, as a neurotransmitter, in most parts of the lower urinary tract either normally or pathologically.49 It inhibits cytochrome C oxidase, reduces oxygen consumption by the vasculature and inhibits or modifies the endothelial cells for circulating white blood cells. Its release is stimulated by acetyl choline, brandykin,

 Endothelial dysfunction is defined as a state of altered phenotype which leads to impairment of vascular reactivity or an induced surface which is thrombogenic or abnormally adhesive for inflammatory cells.45 This change may predate or co-exist with atherosclerosis and is characterized by loss of vascular reactivity. The initial phase is characterized by decreased bioavailability of nitric oxide either due to reduced production or increased breakdown. There is therefore loss of the endothelium derived smooth muscle relaxation with a tendency to vasoconstriction and increased peripheral resistance. These effects have been implicated in hypertension, diabetes mellitus, cigarette smoking and hyperlipidemia, all of which are risk factors for ED and CAD.

from this layer that SMC are recruited in the formation of atheroma.

normally impermeable to most substances in the blood.

cells into the intima and the entry of lipids into the arterial wall.

substance P and sheer stress on the arterial wall.

**3. Atherosclerosis** 

other tissues.47 48

independent of class, is made up of three layers namely;

from the surrounding structures or tissues.

Endothelial dysfunction is a systemic disease and it is usually as a result of diverse injury including sheer stress, trauma and inflammation. It occurs in the initial and subsequent phases of atherosclerosis. Though all arterial beds are affected, the effects manifests earlier in the smaller vessel beds50 such as the pudendal artery which supply the erectile tissue of the penis. In the presence of endothelial dysfunction, the endothelium becomes unduly permeable, allowing substances such as lipids, proteins and macrophages into the intima, a sin-qua non for atherosclerotic changes.

 Atherosclerosis is characterized by intimal lesion called atheroma or atheromatous plagues or fibrofatty plagues which project into the lumen of the vessel. It forms the common pathway through which the well documented cardiovascular risk factors cause their deleterious effects on the heart, cerebrum and the erectile tissues. Endothelial dysfunction results from inhibition of dimethyl arginine dimethyl amino hydrolase which catalyses the hydrolysis of asymmetric dimethyl arginine, an inhibitor of endothelial nitric oxide synthase. The subsequent uncoupling of eNos leads to endothelial oxidative stress and formation of peroxy nitrates, oxidation of proinflammatory nuclear factor Kappa B and hence cellular inflammation.51

Endothelial inflammation causes increase permeability of the endothelial cells and the tight junctions. The subsequent increased movement of lipids, protein and inflammatory cells, particularly macrophages into the intima, initiates the process of atherosclerosis which affects all the arterial bed. Timing of clinical appearance however depends on the severity and size of the affected vessel as up to 75% of the vessel lumen maybe occluded before effects become obvious.52 Smaller vessels such as the pudendal artery may be occluded up to ten years before bigger vessels like the coronary arteries are affected. This explains why erectile dysfunction usually precedes the occurrence of coronary artery diseases or stroke.

Fig. 2. Mechnisms by which CVS risk factors cause ED and CAD

#### **4. Cardiovascular risk factors and erectile dysfunction**

#### **4.1 Diabetes mellitus**

Diabetes mellitus(DM) is defined as persistent hyperglycemia secondary to relative or absolute insulin deficiency. This has a profound effect on protein, lipid and carbohydrate metabolism with a myriad of complications of which ED is one. These complications can broadly be classified as vascular or neurologic and ED has been documented in both classes. In DM, neurologic damage which may predispose to ED usually result from peripheral

Erectile Dysfunction Complicating Cardiovascular Risk Factors and Disease 107

ED is highly common in type 2 DM (T2DM) patients and the duration of DM is usually longer in ED patients. The atherosclerotic process is usually ongoing, dynamic and progressive 61making the clinical course of ED in DM gradual and ED may be the only sign62. As DM, especially type 2, maybe present for years before diagnosis, many patients already have complications before or at presentation and this may include ED. Based on these, it has been proposed that ED should be regarded as an observable marker of DM. In a study of ED In diabetics, Sun et al 63 concluded that men with ED are more than twice likely to have DM, strongly so for men 45 years and below but not for men older than 66 years. Twelve percent of men studied by Deutsch et al 64 were found to have unrecognized DM while the Massachusetts Male Aging Study (MMAS) 65 showed that the probability of ED is three times more common in men who reported being treated for DM and figures as high as three quarters of all diabetics have been documented. DM has other complications such as leg ulcers which impact negatively on psyche and quality of life and in such patients, ED may be partly psychogenic. This should not be underestimated in the course of evaluation.

Essential hypertension is the leading risk factor for mortality worldwide accounting for 13% of all deaths globally.66 It is the most common non communicable disease in Nigeria67 and according to Essien et al,68 the mean venous blood glucose level of hypertensive adult Nigerians is higher than their normotensive counterparts. According to recent studies, approximately 67-68% of men with hypertension have some degree of erectile dysfunction.69 Erectile dysfunction in hypertension, similar to diabetes mellitus, results from endothelial dysfunction and or atherosclerosis. Endothelial dysfunction in hypertension is caused by the sheer stress of elevated blood pressure on the vessel wall.70 This endothelial damage causes the impairment of endothelium derived relaxation. The inflammation which accompanies endothelial dysfunction leads to altered NO metabolism, formation of free oxygen radicals and an increased movement of lipids into the intima, a necessity for atheromatous vascular damage. Hypertension is associated with structural and functional changes in the arterial

wall and this is responsible for the mortality and morbidity associated with it.71

disease than in those with the untreated condition

In a number of cardiovascular pathologies, such as hypertension and heart failure, according to Boulauger et al,72 the balance in the endothelial production of vasodialating mediators is altered. The underlying dysfunction is likely to be the consequence of the high blood pressure and could facilitate the maintenance of elevated peripheral resistance with subsequent development of atherosclerosis. The ensuing arterial stiffness is an independent cardiovascular risk factor.73 From this perspective, it can be deduced that hypertension is an independent risk factor for coronary heart disease and vasculogenic erectile dysfunction. In the work of Modebe74 in Nigeria, 8% of the untreated hypertension population had erectile dysfunction while this was 61% in the treated group. Treatment, rather than the hypertension therefore, may be responsible for the ED and may account for the non compliance with treatment frequently seen in this group of patients who may want to maintain their potency. This agrees with the of opinion of Shiri et al75 who in their study concluded that the risk of ED is higher in men suffering from treated hypertension and heart

Erectile function and neuromuscular transmission are calcium dependent phenomena, which to a large extent, depends on the general physical well being of the man. Erectile

**4.2 Hypertension** 

neuropathy and ED is well known to be associated with both somatic and autonomic neuropathies. 53 Both types of autonomic neuropathies ie, axonal and demyelinating, occur in DM and are responsible for the 'failure to initiate' type of ED.

Vascular damage from DM causes the 'failure to fill' type of erectile dysfunction. This results from endothelial dysfunction and or atherosclerosis both of which are companions of diabetes mellitus. Diabetes mellitus is associated with low grade inflammation, dyslipidemia, hypertension and the metabolic syndrome all of which, as independent entities, are risk factors for ED. This low grade inflammatory state in DM has been well documented by many authors who have demonstrated elevated c-Reactive protein, Tumor Necrosis Factor alpha (TNF) and interleukin 6 (IL-6) in diabetic patients with ED who have no other risk factors. 54 55 56

The low grade inflammation in DM causes endothelial dysfunction which leads to an endothelium that is more thrombogenic and permeable to lipids, inflammatory cells such as macrophages. This initiates the process of atherosclerosis which is usually diffuse and occurs earlier in diabetic patients. (The reader is referred to standard vascular texts for the pathology of atherosclerosis) The ensuing diffuse atherosclerosis and micro-angiopathy is responsible for the nephropathy, retinopathy, stroke, coronary artery disease and ED that accompany DM. According to Meng et al, 57 higher glucose levels induces apoptosis in endothelial cells while Di Filippo et al 58 conclude that DM is associated with increased cardiovasular disease due to established risk factors such as dyslipdemia , hypertension and atherosclerosis as a result of increased inflammation. This leads to the production of free oxygen radicals, impaired NO metabolism and increased movement of lipids into the intima leading to early and diffuse atherosclerosis. Through this atherosclerotic damage, cerebral, cardiac, renal and erectile function maybe impaired.

The pathophysiologic relationship between diabetes mellitus and erectile dysfunction, according to Moore et al, 59 is multifactorial. They proposed the mechanism of ED in DM to include elevated advance glycation end products, increased level of free oxygen radicals, impaired NO synthesis, increased endothelin B binding sites and ultrastructural changes, up regulated Rhoa/ Rho-Kinase pathway, NO dependent selective nitregic nerve degeneration and impaired c-GMP dependent kinase. Overall, there is impaired flow mediated dilatation in DM patients which is in part secondary to increased inflammation and endothelial and platelet activation60


Table 4. Summary of Causes of ED in DM.

ED is highly common in type 2 DM (T2DM) patients and the duration of DM is usually longer in ED patients. The atherosclerotic process is usually ongoing, dynamic and progressive 61making the clinical course of ED in DM gradual and ED may be the only sign62. As DM, especially type 2, maybe present for years before diagnosis, many patients already have complications before or at presentation and this may include ED. Based on these, it has been proposed that ED should be regarded as an observable marker of DM. In a study of ED In diabetics, Sun et al 63 concluded that men with ED are more than twice likely to have DM, strongly so for men 45 years and below but not for men older than 66 years. Twelve percent of men studied by Deutsch et al 64 were found to have unrecognized DM while the Massachusetts Male Aging Study (MMAS) 65 showed that the probability of ED is three times more common in men who reported being treated for DM and figures as high as three quarters of all diabetics have been documented. DM has other complications such as leg ulcers which impact negatively on psyche and quality of life and in such patients, ED may be partly psychogenic. This should not be underestimated in the course of evaluation.

#### **4.2 Hypertension**

106 Recent Advances in Cardiovascular Risk Factors

neuropathy and ED is well known to be associated with both somatic and autonomic neuropathies. 53 Both types of autonomic neuropathies ie, axonal and demyelinating, occur

Vascular damage from DM causes the 'failure to fill' type of erectile dysfunction. This results from endothelial dysfunction and or atherosclerosis both of which are companions of diabetes mellitus. Diabetes mellitus is associated with low grade inflammation, dyslipidemia, hypertension and the metabolic syndrome all of which, as independent entities, are risk factors for ED. This low grade inflammatory state in DM has been well documented by many authors who have demonstrated elevated c-Reactive protein, Tumor Necrosis Factor alpha (TNF) and interleukin 6 (IL-6) in diabetic patients with ED who have

The low grade inflammation in DM causes endothelial dysfunction which leads to an endothelium that is more thrombogenic and permeable to lipids, inflammatory cells such as macrophages. This initiates the process of atherosclerosis which is usually diffuse and occurs earlier in diabetic patients. (The reader is referred to standard vascular texts for the pathology of atherosclerosis) The ensuing diffuse atherosclerosis and micro-angiopathy is responsible for the nephropathy, retinopathy, stroke, coronary artery disease and ED that accompany DM. According to Meng et al, 57 higher glucose levels induces apoptosis in endothelial cells while Di Filippo et al 58 conclude that DM is associated with increased cardiovasular disease due to established risk factors such as dyslipdemia , hypertension and atherosclerosis as a result of increased inflammation. This leads to the production of free oxygen radicals, impaired NO metabolism and increased movement of lipids into the intima leading to early and diffuse atherosclerosis. Through this atherosclerotic damage, cerebral,

The pathophysiologic relationship between diabetes mellitus and erectile dysfunction, according to Moore et al, 59 is multifactorial. They proposed the mechanism of ED in DM to include elevated advance glycation end products, increased level of free oxygen radicals, impaired NO synthesis, increased endothelin B binding sites and ultrastructural changes, up regulated Rhoa/ Rho-Kinase pathway, NO dependent selective nitregic nerve degeneration and impaired c-GMP dependent kinase. Overall, there is impaired flow mediated dilatation in DM patients which is in part secondary to increased inflammation and endothelial and

in DM and are responsible for the 'failure to initiate' type of ED.

cardiac, renal and erectile function maybe impaired.

Other associated CVS risk factors e.g. hypertension, dyslipidemia.

 Low grade inflammation causing endothelial dysfunction. Production of free O2 radicals, impaired NO metabolism.

Nitric oxide dependent selective nitregic nerve degeneration.

 Elevated advance glycation end products. Increased endothelin B receptor binding sites. Up regulation of RhOA/Rho-Kinase Pathway.

Table 4. Summary of Causes of ED in DM.

no other risk factors. 54 55 56

platelet activation60

Essential hypertension is the leading risk factor for mortality worldwide accounting for 13% of all deaths globally.66 It is the most common non communicable disease in Nigeria67 and according to Essien et al,68 the mean venous blood glucose level of hypertensive adult Nigerians is higher than their normotensive counterparts. According to recent studies, approximately 67-68% of men with hypertension have some degree of erectile dysfunction.69

Erectile dysfunction in hypertension, similar to diabetes mellitus, results from endothelial dysfunction and or atherosclerosis. Endothelial dysfunction in hypertension is caused by the sheer stress of elevated blood pressure on the vessel wall.70 This endothelial damage causes the impairment of endothelium derived relaxation. The inflammation which accompanies endothelial dysfunction leads to altered NO metabolism, formation of free oxygen radicals and an increased movement of lipids into the intima, a necessity for atheromatous vascular damage. Hypertension is associated with structural and functional changes in the arterial wall and this is responsible for the mortality and morbidity associated with it.71

In a number of cardiovascular pathologies, such as hypertension and heart failure, according to Boulauger et al,72 the balance in the endothelial production of vasodialating mediators is altered. The underlying dysfunction is likely to be the consequence of the high blood pressure and could facilitate the maintenance of elevated peripheral resistance with subsequent development of atherosclerosis. The ensuing arterial stiffness is an independent cardiovascular risk factor.73 From this perspective, it can be deduced that hypertension is an independent risk factor for coronary heart disease and vasculogenic erectile dysfunction. In the work of Modebe74 in Nigeria, 8% of the untreated hypertension population had erectile dysfunction while this was 61% in the treated group. Treatment, rather than the hypertension therefore, may be responsible for the ED and may account for the non compliance with treatment frequently seen in this group of patients who may want to maintain their potency. This agrees with the of opinion of Shiri et al75 who in their study concluded that the risk of ED is higher in men suffering from treated hypertension and heart disease than in those with the untreated condition

Erectile function and neuromuscular transmission are calcium dependent phenomena, which to a large extent, depends on the general physical well being of the man. Erectile

Erectile Dysfunction Complicating Cardiovascular Risk Factors and Disease 109

accompanied by low level chronic inflammation, elevated c-Reactive protein and may be followed by diffuse endothelial dysfunction and atherosclerosis.80 This low grade inflammatory state allow increase in low density lipoprotein transport across the

Dyslipidemia and obesity are major components of the metabolic syndrome (MS) which is currently well documented as being accompanied by a low systemic inflammatory state which predisposes to vascular endothelial dysfunction.81This is the underlying mechanism of ED in men who have the metabolic syndrome and concomitant ED. This is evidenced by the presence of raised level of inflammatory markers in the affected men. MS and obesity are linked to lowered serum testosterone82 in a double edge manner and low serum testosterone as an independent cardiovascular risk factor, is presently being examined. It is considered to be due to the reduced level of sex hormone binding globulin. Additionally, in obesity which is often present in MS, there is increased level of low density lipoprotein as a

A third of citizens of the United States are presently considered, by current definition, to be obese83 and the prevalence of MS in ED population is 45% compared with 24% in matched control.84 Conversely, the prevalence of ED in MS population is 34 – 43% and this depends on the number of independent risk factors-85 dyslipidemia, hypertension and diabetes mellitus. Dyshpidemia is also often an accompaniment of DM and hypertension in different combinations. However, dyslipidemia is an independent risk factor for ED and CAD.86 Hypercholesterolemia has a well established link with endothelial dysfunction with oxidized low density lipoprotein being a key mediator.87 According to Brunner et al, 77 in familial hypercholesterolemia, endothelial dysfunction is present prior to clinical arterial disease. Endothelial dysfunction is related to particle size and concentration, 88 transport across the endothelium being inversely related to the particle size and directly to the concentration. This explains why low density lipoprotein, and not high density lipoproteins, 89 is incriminated in endothelial dysfunction. Low density lipoprotein leads to reduction in endogenous NO synthase90 which in turn causes a reduced bioavailability of the endothelium derived relaxation factor, NO, probably by enhancing super oxide anion. Lipoproteins are transported across the endothelium by the process of transcytosis and in the presence of endothelial dysfunction, reduced NO bioavailability and increased concentration of LDL, this process is enhanced leading to atherosclerotic vascular changes. In contrast, HDLs are presently considered to enhance endothelial function and a low

endothelium into the intima, thereby initiating the process of atherosclerosis.

Table 6. Summary of causes of ED in cigarette smoking and Dyslipidemia

Direct toxicity to vascular endothelium.

Chronic obstructive airway disease.

result of abnormal insulin metabolism.82

concentration may predispose to endothelial dysfunction.91

Functional and structural changes in vessel wall.

 Endothelial dysfunction and atherosclerosis. Reduction in concentration of NO synthase.

dysfunction is associated with the use of calcium channel blockers, angiotensin II antagonist, non selective alpha blockers and diuretics. The continuous use of diuretics, particularly thiazide diuretics, is often accompanied by dehydration, electrolyte imbalance and elevated blood sugar all of which may lead to ED or aggravate an already existing mild disease. ED is however not currently known to be associated with the use of organic nitrates, angiotensin converting enzymes (ACE) inhibitors, selective alpha blockers and drugs that lower serum lipid levels.


Table 5. Summary of causes of ED in hypertension

The preliminary report of the telmisartan alone, and in combination with ramipril global end point trial/telmisartan randomized assessment study in ACE – intolerant subjects with cardiovascular disease (ONTARGET/TRACEND) study76 has shown that on the contrary, calcium channel blockers tend to have a significant adverse effect on erectile function whereas diuretics, beta blockers, ACE inhibitors, ATI antagonist and alpha blockers do not. Treatment with ACE inhibitors and ATI antagonist or a combination of both is suggested to improve erectile function in cardiovascular high risk patients.

On the overall, hypertension is complicated by numerous vascular conditions which impact negatively on the quality of life of the affected men. Apart from coronary heart disease and erectile dysfunction, conditions such as hypertensive renal damage, intermittent claudication, retinal damage and heart failure often complicate hypertension and lower the quality of life of these men, with severe effect on their psyche. Like DM therefore, the contribution of psychogenic erectile dysfunction in hypertensives should not be underestimated in the course of treatment.

#### **5. Cigarrette smoking/dylipidemia**

Cigarette smoking is currently a major health concern and in spite of the vigorous campaign against it, more people continue to take to smoking in many countries of the world. The most well mentioned and documented toxin of cigarette smoke is nicotine but others exist. Cigarette smoke is directly toxic to vascular endothelium77 and this culminates in vascular endothelial dysfunction, functional and architectural changes in the vessel wall.78 The cascade of events which follow this endothelial dysfunction is similar to that in DM and hypertension. The end result is atherosclerosis and its deleterious effect on penile erectile, cardiac and cerebral function.

According to Chen et al, 79 an increased ED prevalence has been reported in patients with chronic obstructive airway diseases and sustained inflammation seem to play a central role in this linkage. Cigarette smoking has traditionally been associated to chronic obstructive airway disease (chronic bronchitis, emphysema) and through this mechanism, it may contribute significantly to the burden of ED. These conditions, similar to DM, are accompanied by low level chronic inflammation, elevated c-Reactive protein and may be followed by diffuse endothelial dysfunction and atherosclerosis.80 This low grade inflammatory state allow increase in low density lipoprotein transport across the endothelium into the intima, thereby initiating the process of atherosclerosis.


108 Recent Advances in Cardiovascular Risk Factors

dysfunction is associated with the use of calcium channel blockers, angiotensin II antagonist, non selective alpha blockers and diuretics. The continuous use of diuretics, particularly thiazide diuretics, is often accompanied by dehydration, electrolyte imbalance and elevated blood sugar all of which may lead to ED or aggravate an already existing mild disease. ED is however not currently known to be associated with the use of organic nitrates, angiotensin converting enzymes (ACE) inhibitors, selective alpha blockers and drugs that lower serum

The preliminary report of the telmisartan alone, and in combination with ramipril global end point trial/telmisartan randomized assessment study in ACE – intolerant subjects with cardiovascular disease (ONTARGET/TRACEND) study76 has shown that on the contrary, calcium channel blockers tend to have a significant adverse effect on erectile function whereas diuretics, beta blockers, ACE inhibitors, ATI antagonist and alpha blockers do not. Treatment with ACE inhibitors and ATI antagonist or a combination of both is suggested to

On the overall, hypertension is complicated by numerous vascular conditions which impact negatively on the quality of life of the affected men. Apart from coronary heart disease and erectile dysfunction, conditions such as hypertensive renal damage, intermittent claudication, retinal damage and heart failure often complicate hypertension and lower the quality of life of these men, with severe effect on their psyche. Like DM therefore, the contribution of psychogenic erectile dysfunction in hypertensives should not be

Cigarette smoking is currently a major health concern and in spite of the vigorous campaign against it, more people continue to take to smoking in many countries of the world. The most well mentioned and documented toxin of cigarette smoke is nicotine but others exist. Cigarette smoke is directly toxic to vascular endothelium77 and this culminates in vascular endothelial dysfunction, functional and architectural changes in the vessel wall.78 The cascade of events which follow this endothelial dysfunction is similar to that in DM and hypertension. The end result is atherosclerosis and its deleterious effect on penile erectile,

According to Chen et al, 79 an increased ED prevalence has been reported in patients with chronic obstructive airway diseases and sustained inflammation seem to play a central role in this linkage. Cigarette smoking has traditionally been associated to chronic obstructive airway disease (chronic bronchitis, emphysema) and through this mechanism, it may contribute significantly to the burden of ED. These conditions, similar to DM, are

lipid levels.

 Ultrastructural changes in the vessel wall. Endothelial dysfunction and atherosclerosis.

Target organ damage e.g. stroke and heart failure.

improve erectile function in cardiovascular high risk patients.

Table 5. Summary of causes of ED in hypertension

Medication side effect e.g. diuretics.

underestimated in the course of treatment.

**5. Cigarrette smoking/dylipidemia** 

cardiac and cerebral function.


Table 6. Summary of causes of ED in cigarette smoking and Dyslipidemia

Dyslipidemia and obesity are major components of the metabolic syndrome (MS) which is currently well documented as being accompanied by a low systemic inflammatory state which predisposes to vascular endothelial dysfunction.81This is the underlying mechanism of ED in men who have the metabolic syndrome and concomitant ED. This is evidenced by the presence of raised level of inflammatory markers in the affected men. MS and obesity are linked to lowered serum testosterone82 in a double edge manner and low serum testosterone as an independent cardiovascular risk factor, is presently being examined. It is considered to be due to the reduced level of sex hormone binding globulin. Additionally, in obesity which is often present in MS, there is increased level of low density lipoprotein as a result of abnormal insulin metabolism.82

A third of citizens of the United States are presently considered, by current definition, to be obese83 and the prevalence of MS in ED population is 45% compared with 24% in matched control.84 Conversely, the prevalence of ED in MS population is 34 – 43% and this depends on the number of independent risk factors-85 dyslipidemia, hypertension and diabetes mellitus. Dyshpidemia is also often an accompaniment of DM and hypertension in different combinations. However, dyslipidemia is an independent risk factor for ED and CAD.86 Hypercholesterolemia has a well established link with endothelial dysfunction with oxidized low density lipoprotein being a key mediator.87 According to Brunner et al, 77 in familial hypercholesterolemia, endothelial dysfunction is present prior to clinical arterial disease. Endothelial dysfunction is related to particle size and concentration, 88 transport across the endothelium being inversely related to the particle size and directly to the concentration. This explains why low density lipoprotein, and not high density lipoproteins, 89 is incriminated in endothelial dysfunction. Low density lipoprotein leads to reduction in endogenous NO synthase90 which in turn causes a reduced bioavailability of the endothelium derived relaxation factor, NO, probably by enhancing super oxide anion. Lipoproteins are transported across the endothelium by the process of transcytosis and in the presence of endothelial dysfunction, reduced NO bioavailability and increased concentration of LDL, this process is enhanced leading to atherosclerotic vascular changes. In contrast, HDLs are presently considered to enhance endothelial function and a low concentration may predispose to endothelial dysfunction.91

Erectile Dysfunction Complicating Cardiovascular Risk Factors and Disease 111

activation. The absolute risk of sexual activity triggering a myocardial infarction (MI) is low. Men with CAD or previous MI have a 10-fold higher risk, which means that during sexual

Several cardiovascular disorders cause heart failure which may be left or right sided failure. These include hypertension, congenital and acquired heart defects and cardiomyophathies. Left side failure usually results from hypertension, mitral and aortic valvular abnormalities which may be acquired or congenital. Eventually, left sided failure leads to pulmonary hypertension and with continued back pressure, right sided failure which is referred to as congestive cardiac failure (CCF). Heart failure may occur independent of traditional cardiovascular risk factors or in association with one or more of them. For instance, Ukoh103 et al in their study observed hyperlipidemia in three groups of patients. (1) Hypertensive's with or without heart disease (2) patients with ischemic heart disease and (3) those with hypertensive cardiomyopathy. ED may therefore occur in men with heart failure not because of the failure itself, but because of the background cardiovascular risk factors

The prevalence of heart failure (HF) in the United States, according to the American Heart Association, is estimated at 5.3 million.104 ED is present, estimatedly, in 60.8% of heart failure patients and prevalence ranging from 81 to 91% has been documented in this group.105 In CCF, there is peripheral venous stasis, decreased venous return, reduced stroke volume and cardiac output, a must situation for physical in-activity or under-activity. Most patients with chronic cardiovascular disease experience decreased libido and frequency of

The ED in HF patients is multifactorial in origin. Anxiety or depression may lead to performance fears and therefore psychogenic ED. The reduced pulmonary and cardiac reserve in CCF patients makes exercise intolerable, bearing in mind that sexual activity involve mild to moderate exercise and that most of erectile function is CVS event. Treatment of heart failure may lead to ED as a side effect of the drugs used. Diuretics, particular thiazides,106are associated with ED through unknown mechanism which may not be unconnected with the accompanying electrolyte imbalance. Beta blockers, Digoxin and aldosterone are also documented as causing ED. Heart failure is accompanied by endothelial

dysfunction which as an independent factor, may cause ED in these men.107

inter-course, the probability of such a man having MI is 20/Million/hour".

**7. Heart failture (HF)** 

sexual activity as well as ED.

 Endothelial dysfunction. Reduced cardiac output. Exercise in tolerance from PH. Associated CVS Risk factors.

Table 7. Causes of ED in Heart Failure

Drug side effect.

present.

#### **6. Coronary Artery Disease (CAD)**

In Britain, approximately a quarter of all deaths among men and one-fifth of all deaths among women are due to ischemic heart diseases.92 In England and Wales, 30% of all deaths among men and 22% among women are as a result of ischemic heart diseases. 93 Through atherosclerotic narrowing, cardiac ischemia precipitates cardiac malfunction and sometimes infarction of the myocardium. The major risk factors for endothelial dysfunction and atherosclerosis, which are also the cause of CAD and ED, are hypertension DM, dyslipidemia and cigarette smoking and these life style abnormalities have been traditionally documented as cardiovascular risk factors.

ED is related to CAD in a double edged manner. ED represents an independent risk factor for feature CVS event independent of classic risk factors such as hypertension, DM and dyslipidema 94. The common denominator for ED, CAD and these cardiovascular risk factors is endothelial dysfunction which is usually diffuse, affecting several arterial beds such as the pudendal and coronary arteries and thereafter culminating in atherosclerosis. In 2003 Montorsi et al 95 proposed the small artery theory to explain the earlier occurrence of ED than CAD. Coronary arteries, being larger than the pudendal arteries, take a longer period for occlusion from endothelial dysfunction and atherosclerotic vacscular narrowing to occur. Eventually, as the atherosclerosis progresses, larger vessels such as the cerebral, coronary and renal arteries become involved and this precipitates target organ damage.

Several studies have documented this with clear evidence. In the study by Pritzter, 96 of 20 men, out of a study group of 50 who were investigated angiographically following self reported ED without any other complaints, all have demonstrable disease. They compared sexual function to the penile stress test as a window to the hearts of men. Vlachopoulos et al 97 investigated men whose only complaint was ED and found angiographically silent CAD in 19%. Bensal et al 98 also in their work documented 56% asymptomatic CAD in an ED population. Ultimately, in some of these men, CAD become overt, and as documented by Rodriguez et al 99 in DM, taking an average period of 38.8 months. According to their study, 100% of DM men experienced ED prior to onset of CAD. Based on this premise, ED is currently considered a sentinel event for CAD as both have their origin from endothelial dysfunction and atherosclerosis.100 These are mounting evidence therefore, that ED is an early predictor of CAD and that there is need for physicians to evaluate men who present with ED, with no other cardiovascular symptom, for CAD and its risk factors. 101

CAD is an independent risk factor for ED and the prevalence of ED in CAD population is remarkable. James et al102 studied the relation between ED and CAD in men referred for stress myocardial perfusion single-photon emission computed tomography (MPS). They concluded that men sent for MPS have a higher prevalence of ED. Also, men with ED exhibited a higher prevalence of severe CAD and left ventricular dysfunction than those without ED. In the work of Bensal, 98 75% of men with CAD have symptoms of ED and 91% of their ED populations have cardiovascular risks. This has implication for men who have CAD and who intend to engage in sexual activity. Herschorn100 in his work on cardiovascular safety of phosphodiestares 5 inhibitors said that "in general, sexual activity has an effect similar to mild-moderate exercise in increasing heart rate, blood pressure, cardiac output and respiratory rate. The degree of change in these physiologic parameters however, is greater than expected because of disproportionate increase in sympathetic activation. The absolute risk of sexual activity triggering a myocardial infarction (MI) is low. Men with CAD or previous MI have a 10-fold higher risk, which means that during sexual inter-course, the probability of such a man having MI is 20/Million/hour".

#### **7. Heart failture (HF)**

110 Recent Advances in Cardiovascular Risk Factors

In Britain, approximately a quarter of all deaths among men and one-fifth of all deaths among women are due to ischemic heart diseases.92 In England and Wales, 30% of all deaths among men and 22% among women are as a result of ischemic heart diseases. 93 Through atherosclerotic narrowing, cardiac ischemia precipitates cardiac malfunction and sometimes infarction of the myocardium. The major risk factors for endothelial dysfunction and atherosclerosis, which are also the cause of CAD and ED, are hypertension DM, dyslipidemia and cigarette smoking and these life style abnormalities have been

ED is related to CAD in a double edged manner. ED represents an independent risk factor for feature CVS event independent of classic risk factors such as hypertension, DM and dyslipidema 94. The common denominator for ED, CAD and these cardiovascular risk factors is endothelial dysfunction which is usually diffuse, affecting several arterial beds such as the pudendal and coronary arteries and thereafter culminating in atherosclerosis. In 2003 Montorsi et al 95 proposed the small artery theory to explain the earlier occurrence of ED than CAD. Coronary arteries, being larger than the pudendal arteries, take a longer period for occlusion from endothelial dysfunction and atherosclerotic vacscular narrowing to occur. Eventually, as the atherosclerosis progresses, larger vessels such as the cerebral, coronary and renal arteries become involved and this precipitates target organ damage.

Several studies have documented this with clear evidence. In the study by Pritzter, 96 of 20 men, out of a study group of 50 who were investigated angiographically following self reported ED without any other complaints, all have demonstrable disease. They compared sexual function to the penile stress test as a window to the hearts of men. Vlachopoulos et al 97 investigated men whose only complaint was ED and found angiographically silent CAD in 19%. Bensal et al 98 also in their work documented 56% asymptomatic CAD in an ED population. Ultimately, in some of these men, CAD become overt, and as documented by Rodriguez et al 99 in DM, taking an average period of 38.8 months. According to their study, 100% of DM men experienced ED prior to onset of CAD. Based on this premise, ED is currently considered a sentinel event for CAD as both have their origin from endothelial dysfunction and atherosclerosis.100 These are mounting evidence therefore, that ED is an early predictor of CAD and that there is need for physicians to evaluate men who present

with ED, with no other cardiovascular symptom, for CAD and its risk factors. 101

CAD is an independent risk factor for ED and the prevalence of ED in CAD population is remarkable. James et al102 studied the relation between ED and CAD in men referred for stress myocardial perfusion single-photon emission computed tomography (MPS). They concluded that men sent for MPS have a higher prevalence of ED. Also, men with ED exhibited a higher prevalence of severe CAD and left ventricular dysfunction than those without ED. In the work of Bensal, 98 75% of men with CAD have symptoms of ED and 91% of their ED populations have cardiovascular risks. This has implication for men who have CAD and who intend to engage in sexual activity. Herschorn100 in his work on cardiovascular safety of phosphodiestares 5 inhibitors said that "in general, sexual activity has an effect similar to mild-moderate exercise in increasing heart rate, blood pressure, cardiac output and respiratory rate. The degree of change in these physiologic parameters however, is greater than expected because of disproportionate increase in sympathetic

**6. Coronary Artery Disease (CAD)** 

traditionally documented as cardiovascular risk factors.

Several cardiovascular disorders cause heart failure which may be left or right sided failure. These include hypertension, congenital and acquired heart defects and cardiomyophathies. Left side failure usually results from hypertension, mitral and aortic valvular abnormalities which may be acquired or congenital. Eventually, left sided failure leads to pulmonary hypertension and with continued back pressure, right sided failure which is referred to as congestive cardiac failure (CCF). Heart failure may occur independent of traditional cardiovascular risk factors or in association with one or more of them. For instance, Ukoh103 et al in their study observed hyperlipidemia in three groups of patients. (1) Hypertensive's with or without heart disease (2) patients with ischemic heart disease and (3) those with hypertensive cardiomyopathy. ED may therefore occur in men with heart failure not because of the failure itself, but because of the background cardiovascular risk factors present.

The prevalence of heart failure (HF) in the United States, according to the American Heart Association, is estimated at 5.3 million.104 ED is present, estimatedly, in 60.8% of heart failure patients and prevalence ranging from 81 to 91% has been documented in this group.105 In CCF, there is peripheral venous stasis, decreased venous return, reduced stroke volume and cardiac output, a must situation for physical in-activity or under-activity. Most patients with chronic cardiovascular disease experience decreased libido and frequency of sexual activity as well as ED.


#### Table 7. Causes of ED in Heart Failure

The ED in HF patients is multifactorial in origin. Anxiety or depression may lead to performance fears and therefore psychogenic ED. The reduced pulmonary and cardiac reserve in CCF patients makes exercise intolerable, bearing in mind that sexual activity involve mild to moderate exercise and that most of erectile function is CVS event. Treatment of heart failure may lead to ED as a side effect of the drugs used. Diuretics, particular thiazides,106are associated with ED through unknown mechanism which may not be unconnected with the accompanying electrolyte imbalance. Beta blockers, Digoxin and aldosterone are also documented as causing ED. Heart failure is accompanied by endothelial dysfunction which as an independent factor, may cause ED in these men.107

Erectile Dysfunction Complicating Cardiovascular Risk Factors and Disease 113

Diet has a profound effect on the general well being and it is part of general healthy living that men should be mindful of what they eat. Diet modification can help control weight gain, glucose level in diabetes and cholesterol level in dyslipidemic men. When combined with regular exercise, the effect may be synergistic with remarkable weight loss and it has been documented that about one-third of obese men with ED are able to improve sexual function through life style changes.115 Dietary modification should involve the services of a trained dietician who should set a target from the beginning, bearing in mind existing co-

The medical treatment for erectile dysfunction complicating CVS risk factors and disease should necessarily include treating for the underlying risk factors107. These include the control of hypertension, diabetes mellitus, dyslipidemia, heart failure and coronary artery disease. Although this therapeutic approach, according to Tikkanen et al116, appears justified, relatively few intervention studies have investigated the effect of risk factor reduction on established ED. However, in the work by Saltzman et al117, artovastatin treatment of men with hyperlipidemia as the only risk factor, for instance, led to an improvement in erectile function. The medical treatment of these risk factors is beyond this

a. Phosphodiesterase 5 inhibitors

b. Peripheral alpha 1 blockers 1. Phentolamine Mesylate c. Central Alpha2 antagonist 1. Yohimbine d. Dopamine agonists 1. Apomorpine

1. Aprostodil (PGEI)

a. Phosphodiesterase Inhibitor

c. Direct smooth muscle relaxant 1. Aprostodil (PGEI)

2. Vaso-active intestinal peptide (VIP).

1. Papaverine b. Peripheral alpha 1 blocker 1. Phentolamine

1. Sildenafil 2. Vardanefil 3. Tadalafil

**9.3 Modification of diet** 

morbidities and the age of the patients.

**9.4 Principles of medical treatment** 

chapter but a few guiding principles need to be explained.

2. Intra-Urethral a. Direct smooth muscle relaxant

Table 8. Summary of Drugs used in Pharmacotherapy for ED

Route of Administration Drugs Used

1. Oral

3. Intra-cavernosal (injectable)

#### **8. Treatment of ED complicating CVS risk factors and disease**

Following the evaluation of these men (this is beyond the scope of this chapter), there is need for treatment in order to improve their quality of life. Gerald et al108 evaluated the sexual attitudes and beliefs of middle aged and older adults in non-European 'Westernized' countries in their work. Approximately 85% of men and women felt that satisfactory sexual intercourse is essential for the maintenance of a relationship. Most respondents felt that it is acceptable for older men to use medication to enable them to continue to enjoy sexual activity. By extension, this is necessary if they are to continue to maintain spousal relationship which at this age has profound effect on quality of life and longevity.

ED is multifactorial in terms of etiology, the implication of which is that treatment should be holistic. It is based on this premise that David et al109 proposed a multifaceted approach with the argument that no single agent has proven to be highly efficacious. He supported this with the work of Goldstein et al110 in which though, 84% of men had improved function following treatment with sildenafil, they achieved a fully rigid erection only four times per month. This is limiting for a couple and may therefore not be enough to maintain a satisfactory relationship.

#### **9. Life style modification**

#### **9.1 Smoking and alcohol**

According to Feldman et al, 6 smoking doubles the risk of developing ED. Cigarette smoking increases the risk of death from CAD and according to Derling et al,111 also the incidence of ED. Traditionally, alcohol has been associated with the capacity to lower serum cholesterol and therefore improve erectile function. Alcohol has a central sedating effect which may be over powering. It is also associated with lowered NO production.112 Men who smoke and drink alcohol should be advised to cease smoking and alcohol consumption. The later may be restricted to a single daily drink, preferably, red wine which has been documented to improve NO production.

#### **9.2 Regular exercise**

Regular exercise is beneficial to all body tissues and organs particularly in healthy individuals. During exercise, the cardiac output, the chronotropic and inotropic activity of the heart increase and the circulation therefore becomes more rapid. The blood supply to different organs and tissues, particularly the heart and muscles, increase with remarkable improvement in tissue perfusion. A sedentary life style is three times as likely to lead to ED where as moderate physical activity reduces the risk of ED by two-thirds.113 The sheer stress of increase blood flow stimulates the production of NO, an effect which lasts for up to 2-7days with a magnitude in the order of fourfold.114 During penile erection, the increase in flow is more than this, implying a more increased production of NO. On this premise, some studies have shown improved erectile function in men with regular and frequent sexual activity.

Regular exercise also may help control weight gain, obesity, dyslipidimia and the metabolic syndrome. Reduction in weight in the already obese men may lead to regularization of testosterone concentration and correct the loss of libido often associated with ED in the obese. Men with ED should therefore be advised on regular physical exercise and sexual activity.

#### **9.3 Modification of diet**

112 Recent Advances in Cardiovascular Risk Factors

Following the evaluation of these men (this is beyond the scope of this chapter), there is need for treatment in order to improve their quality of life. Gerald et al108 evaluated the sexual attitudes and beliefs of middle aged and older adults in non-European 'Westernized' countries in their work. Approximately 85% of men and women felt that satisfactory sexual intercourse is essential for the maintenance of a relationship. Most respondents felt that it is acceptable for older men to use medication to enable them to continue to enjoy sexual activity. By extension, this is necessary if they are to continue to maintain spousal

ED is multifactorial in terms of etiology, the implication of which is that treatment should be holistic. It is based on this premise that David et al109 proposed a multifaceted approach with the argument that no single agent has proven to be highly efficacious. He supported this with the work of Goldstein et al110 in which though, 84% of men had improved function following treatment with sildenafil, they achieved a fully rigid erection only four times per month. This is limiting for a couple and may therefore not be enough to maintain a

According to Feldman et al, 6 smoking doubles the risk of developing ED. Cigarette smoking increases the risk of death from CAD and according to Derling et al,111 also the incidence of ED. Traditionally, alcohol has been associated with the capacity to lower serum cholesterol and therefore improve erectile function. Alcohol has a central sedating effect which may be over powering. It is also associated with lowered NO production.112 Men who smoke and drink alcohol should be advised to cease smoking and alcohol consumption. The later may be restricted to a single daily drink, preferably, red wine which has been documented to

Regular exercise is beneficial to all body tissues and organs particularly in healthy individuals. During exercise, the cardiac output, the chronotropic and inotropic activity of the heart increase and the circulation therefore becomes more rapid. The blood supply to different organs and tissues, particularly the heart and muscles, increase with remarkable improvement in tissue perfusion. A sedentary life style is three times as likely to lead to ED where as moderate physical activity reduces the risk of ED by two-thirds.113 The sheer stress of increase blood flow stimulates the production of NO, an effect which lasts for up to 2-7days with a magnitude in the order of fourfold.114 During penile erection, the increase in flow is more than this, implying a more increased production of NO. On this premise, some studies have shown

Regular exercise also may help control weight gain, obesity, dyslipidimia and the metabolic syndrome. Reduction in weight in the already obese men may lead to regularization of testosterone concentration and correct the loss of libido often associated with ED in the obese. Men with ED should therefore be advised on regular physical exercise and sexual

improved erectile function in men with regular and frequent sexual activity.

**8. Treatment of ED complicating CVS risk factors and disease** 

relationship which at this age has profound effect on quality of life and longevity.

satisfactory relationship.

improve NO production.

**9.2 Regular exercise** 

activity.

**9. Life style modification 9.1 Smoking and alcohol** 

Diet has a profound effect on the general well being and it is part of general healthy living that men should be mindful of what they eat. Diet modification can help control weight gain, glucose level in diabetes and cholesterol level in dyslipidemic men. When combined with regular exercise, the effect may be synergistic with remarkable weight loss and it has been documented that about one-third of obese men with ED are able to improve sexual function through life style changes.115 Dietary modification should involve the services of a trained dietician who should set a target from the beginning, bearing in mind existing comorbidities and the age of the patients.

#### **9.4 Principles of medical treatment**

The medical treatment for erectile dysfunction complicating CVS risk factors and disease should necessarily include treating for the underlying risk factors107. These include the control of hypertension, diabetes mellitus, dyslipidemia, heart failure and coronary artery disease. Although this therapeutic approach, according to Tikkanen et al116, appears justified, relatively few intervention studies have investigated the effect of risk factor reduction on established ED. However, in the work by Saltzman et al117, artovastatin treatment of men with hyperlipidemia as the only risk factor, for instance, led to an improvement in erectile function. The medical treatment of these risk factors is beyond this chapter but a few guiding principles need to be explained.


Table 8. Summary of Drugs used in Pharmacotherapy for ED

Erectile Dysfunction Complicating Cardiovascular Risk Factors and Disease 115

failure provided that aortic stenosis and obstructive valvular cardiomyophathy have been

Opinions are varied as to whether the phosphodiesterase 5 inhibitors should be used in patients who have coronary artery disease and concomitant erectile dysfunction. According to George et al121, on the basis of the pharmacokinetic profile of sildenafil for instance, the co-administration of a nitrate within the first 24 hours is likely to produce a severe, potentially, life threatening hypotensive response and it is therefore contradicted. However, Parker et al122 insist in their work that though contradicted, there are occasions when a patient who has recently taken a phosphodiesterase 5 inhibitor might need intravenous nitroglycerin treatment with the proviso that such patients should be closely monitored and should have stable CAD. In the work by Webb et al,123 when sublingual nitroglycerin was administered, there was a fourfold decrease in systolic blood pressure In patients with sildenafil treatment. Their conclusion was that sildenafil potentiated the hypotensive effect of nitrates and their concomitant use was absolutely contra indicated. According to Velasquez et al124 adverse cardiac event associated to sildenafil use, for instance, include MI, angina, ventricular tachycardia and death. Therefore, the use of phosphodiesterase 5 inhibitors and nitrates in men with ED and concomitant CAD should be done with caution, individualized

and in consultation between the physician and Urologist in order to optimize care.

surgery. Penile venous surgery is presently considered historical.

**11. Use of vacuum erection devices** 

phosphodiesterase 5 inhibitors calls for caution.

Surgical treatment of ED has been relegated to the background as the last option with the advent of effective medical therapy. The main-stay of surgical treatment includes the use of penile implants and penile arterial revascularization procedures.125 Infection is a major setback in the use of penile implants particularly in DM.Penile revascularization procedures may be complicated by numbness of the glans and penile skin, defeating the aim of the

These devices induce erection by increasing corporal perfusion and or impeding venous return.126 Their efficacy profile is good but non compliance by patients is high due to difficulty with operating them and the associated ejaculatory problems. In truth, vacuum

Endothelial dysfunction and atherosclerosis are the pathways through which cardiovascular risk factors predispose to coronary artery disease, stroke and erectile dysfunction, the ED occurring earlier because of the small size of the pudendal artery which supplies the cavernous bed. As atherosclerosis is a progressive and dynamic disease, larger vessels are eventually involved and this often includes the coronary artery with the development of CAD. ED is presently regarded as a sentinel event for CAD and patients who present with ED with no other risk factors should be evaluated for silent CAD. The drugs used for the treatment of CVS risk factors may have ED as side effect and patients should therefore be questioned directly for the occurrence of ED. The concomitant use of nitrates and

erection devices cause a hinged erection and the penis is rather truly not rigid.

excluded.

**10. Surgical treatment** 

**12. Conclusion** 

There are conflicting reports on the effects of the drugs used for specific therapy of these risk factors and disease on ED. Statins have been reported to improve erectile function due to their positive effect on endothelial function.118 There are however observational reports associating statin use with ED.119 Diuretics, calcium channel blockers and beta blockers used for the treatment of hypertension and digoxin used for heart-failure have all been reportedly linked with ED. The preliminary report of the telmisartan alone, and in combination with ramipril global end point trial/telmisartan randomized assessment study in ACE-intolerant subjects with cardiovascular Disease (CONTARGET/TRANCEND) study76 shows that calcium channel blockers tend to have a significant adverse effect on erectile function while treatment with beta blockers, diuretics, ACE inhibitors, ATI antagonist and alpha antagonist do not. Treatment with ACE inhibitors, ATI antagonist or a combination of both is suggested to improve erectile function in cardiovascular high risk patients. Based on this premise, the treating physician is advised to document the base-line erectile function at the commencement of treatment as subsequent development of ED is often quoted as responsible for non compliance by the affected men who may wish to maintain their potency. In the course of treatment, these men should be questioned directly about erectile function, and if this wanes or the patients complain, the drugs should be withdrawn. If erectile function returns, the drug/drugs can then be documented as the culprit and permanently withdrawn. This emphasizes the need for individualization of the management of these men.

Erectile dysfunction has social implications for the couple and any treatment regimen should necessarily take the female partner into consideration114. Some modalities of treatment may require the co-operation of the female partner due to their psychosocial and medical side effects which may be distressful to the couple. For instance, apormorphine may cause nausea and or vomiting sometimes necessitating the use of anti-emetics. Table 8 shows some of the drugs for the treatment of ED, their mode of administration and some of their side effects. Also, the use of phosphodiesterase 5 inhibitors is associated with diverse reaction which may threaten life.

#### **9.5 Treatment of ED in CAD and HF patients; Use of PDE 5 inhibitors**

The development and approval for use of the phosphodiesterase 5 inhibitors has revolutionized the treatment of erectile dysfunction, but this is not without short-comings especially in men with HF and CAD. The main stay of the treatment of CAD is the use of nitrates which dilate the coronary arterial bed. The active factor in the nitrates is the nitricoxide which it releases and which is also the endothelium dependent vasodilator and neurotransmitter mostly responsible for penile erection in man. The phosphodiesterase 5 inhibitors inhibit the breakdown of nitric oxide, an action which potentiates that of the nitrates used for the treatment of CAD.

Patients with heart disease have a reduced exercise tolerance because of which there is need to assess and advise them on fitness to undergo sexual activity. They should be stratified into low risk, intermediate risk and high risk based on the presence and number of risk factors, angina, previous MI and their New York Heart Association classification status121. The amount of energy required for sexual activity has been shown to be about 20-40 metabolic equivalent of the tasks which is equivalent to doing easy house hood work or climbing a flight of stairs. This should guide the physician in advising patients with heart failure provided that aortic stenosis and obstructive valvular cardiomyophathy have been excluded.

Opinions are varied as to whether the phosphodiesterase 5 inhibitors should be used in patients who have coronary artery disease and concomitant erectile dysfunction. According to George et al121, on the basis of the pharmacokinetic profile of sildenafil for instance, the co-administration of a nitrate within the first 24 hours is likely to produce a severe, potentially, life threatening hypotensive response and it is therefore contradicted. However, Parker et al122 insist in their work that though contradicted, there are occasions when a patient who has recently taken a phosphodiesterase 5 inhibitor might need intravenous nitroglycerin treatment with the proviso that such patients should be closely monitored and should have stable CAD. In the work by Webb et al,123 when sublingual nitroglycerin was administered, there was a fourfold decrease in systolic blood pressure In patients with sildenafil treatment. Their conclusion was that sildenafil potentiated the hypotensive effect of nitrates and their concomitant use was absolutely contra indicated. According to Velasquez et al124 adverse cardiac event associated to sildenafil use, for instance, include MI, angina, ventricular tachycardia and death. Therefore, the use of phosphodiesterase 5 inhibitors and nitrates in men with ED and concomitant CAD should be done with caution, individualized and in consultation between the physician and Urologist in order to optimize care.

#### **10. Surgical treatment**

114 Recent Advances in Cardiovascular Risk Factors

There are conflicting reports on the effects of the drugs used for specific therapy of these risk factors and disease on ED. Statins have been reported to improve erectile function due to their positive effect on endothelial function.118 There are however observational reports associating statin use with ED.119 Diuretics, calcium channel blockers and beta blockers used for the treatment of hypertension and digoxin used for heart-failure have all been reportedly linked with ED. The preliminary report of the telmisartan alone, and in combination with ramipril global end point trial/telmisartan randomized assessment study in ACE-intolerant subjects with cardiovascular Disease (CONTARGET/TRANCEND) study76 shows that calcium channel blockers tend to have a significant adverse effect on erectile function while treatment with beta blockers, diuretics, ACE inhibitors, ATI antagonist and alpha antagonist do not. Treatment with ACE inhibitors, ATI antagonist or a combination of both is suggested to improve erectile function in cardiovascular high risk patients. Based on this premise, the treating physician is advised to document the base-line erectile function at the commencement of treatment as subsequent development of ED is often quoted as responsible for non compliance by the affected men who may wish to maintain their potency. In the course of treatment, these men should be questioned directly about erectile function, and if this wanes or the patients complain, the drugs should be withdrawn. If erectile function returns, the drug/drugs can then be documented as the culprit and permanently withdrawn. This emphasizes the need for individualization of the

Erectile dysfunction has social implications for the couple and any treatment regimen should necessarily take the female partner into consideration114. Some modalities of treatment may require the co-operation of the female partner due to their psychosocial and medical side effects which may be distressful to the couple. For instance, apormorphine may cause nausea and or vomiting sometimes necessitating the use of anti-emetics. Table 8 shows some of the drugs for the treatment of ED, their mode of administration and some of their side effects. Also, the use of phosphodiesterase 5 inhibitors is associated with diverse

The development and approval for use of the phosphodiesterase 5 inhibitors has revolutionized the treatment of erectile dysfunction, but this is not without short-comings especially in men with HF and CAD. The main stay of the treatment of CAD is the use of nitrates which dilate the coronary arterial bed. The active factor in the nitrates is the nitricoxide which it releases and which is also the endothelium dependent vasodilator and neurotransmitter mostly responsible for penile erection in man. The phosphodiesterase 5 inhibitors inhibit the breakdown of nitric oxide, an action which potentiates that of the

Patients with heart disease have a reduced exercise tolerance because of which there is need to assess and advise them on fitness to undergo sexual activity. They should be stratified into low risk, intermediate risk and high risk based on the presence and number of risk factors, angina, previous MI and their New York Heart Association classification status121. The amount of energy required for sexual activity has been shown to be about 20-40 metabolic equivalent of the tasks which is equivalent to doing easy house hood work or climbing a flight of stairs. This should guide the physician in advising patients with heart

**9.5 Treatment of ED in CAD and HF patients; Use of PDE 5 inhibitors** 

management of these men.

reaction which may threaten life.

nitrates used for the treatment of CAD.

Surgical treatment of ED has been relegated to the background as the last option with the advent of effective medical therapy. The main-stay of surgical treatment includes the use of penile implants and penile arterial revascularization procedures.125 Infection is a major setback in the use of penile implants particularly in DM.Penile revascularization procedures may be complicated by numbness of the glans and penile skin, defeating the aim of the surgery. Penile venous surgery is presently considered historical.

#### **11. Use of vacuum erection devices**

These devices induce erection by increasing corporal perfusion and or impeding venous return.126 Their efficacy profile is good but non compliance by patients is high due to difficulty with operating them and the associated ejaculatory problems. In truth, vacuum erection devices cause a hinged erection and the penis is rather truly not rigid.

#### **12. Conclusion**

Endothelial dysfunction and atherosclerosis are the pathways through which cardiovascular risk factors predispose to coronary artery disease, stroke and erectile dysfunction, the ED occurring earlier because of the small size of the pudendal artery which supplies the cavernous bed. As atherosclerosis is a progressive and dynamic disease, larger vessels are eventually involved and this often includes the coronary artery with the development of CAD. ED is presently regarded as a sentinel event for CAD and patients who present with ED with no other risk factors should be evaluated for silent CAD. The drugs used for the treatment of CVS risk factors may have ED as side effect and patients should therefore be questioned directly for the occurrence of ED. The concomitant use of nitrates and phosphodiesterase 5 inhibitors calls for caution.

Erectile Dysfunction Complicating Cardiovascular Risk Factors and Disease 117

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**8** 

*Japan* 

Mikiya Nakastuka

**Vascular Dysfunction in Women** 

**with Recurrent Pregnancy Loss** 

*Graduate School of Health Sciences, Okayama University* 

*Department of Obstetrics and Gynecology, Okayama University Hospital* 

Women have unique risk factors for cardiovascular diseases and cerebrovascular diseases such as pregnancy and hormone replacement therapy. Pregnancy provides an opportunity to reveal various cardiovascular disease risk factors and estimate a woman's lifetime risk

The causes of recurrent pregnancy loss (RPL) are classified as genetic, anatomic, endocrinologic, immunologic, microbiologic, environmental, and further more (Kutteh, 1999; Christiansen et al., 2005). Several lines of study have suggested that certain coagulation abnormalities such as antiphospholipid antibodies or Factor V Leiden, the genetic defect underlying resistance to activated protein C, are causes of RPL (Kutteh, 1999). New diagnostic methods have improved the clinical triage of RPL (Kutteh, 1999; Li, 1998). Transvaginal pulsed Doppler ultrasonography allows noninvasive evaluation of uterine circulation. The introduction of pulsed Doppler ultrasonography has provided noninvasive means for the evaluation of uterine impedance, and gives physiologic data, rather than anatomic information alone. It is known that resistance to uterine arterial blood flow is associated with poor obstetrical outcome such as preeclampsia and fetal growth restriction

We have reported that impaired uterine perfusion is observed in a portion of women with RPL (Habara et al., 2002; Nakatsuka et al., 2003a, 2003b). Pulsed Doppler ultrasonography in the uterine artery may be useful in distinguishing women with RPL caused by vascular dysfunction from women with unexplained RPL (Habara et al., 2002). Furthermore, the plasma level of adrenomedullin, which is often associated with pathological processes of the vasculature (Hinson, 2000), is elevated in women with RPL (Nakatsuka et al., 2003a).

Antiphospholipid antibody syndrome (APS), which is an autoimmune disease associated with coagulopathy, is a well-known cause of RPL (Nakatsuka et al., 2003b). Antiphospholipid antibodies refer to several groups of autoantibodies with specificity for a number of negatively-charged phospholipids such as cardiolipin, phosphatidylserine, and phosphatidylethanolamine, or phospholipid-binding glycoproteins such as ,2 glycoprotein I

However, vascular changes in women with RPL have not been fully elucidated.

because of its unique cardiovascular and metabolic stress.

(Nakatsuka et al, 1999b; Nakatsuka et al, 2002; Takata et al, 200).

**1. Introduction** 


### **Vascular Dysfunction in Women with Recurrent Pregnancy Loss**

Mikiya Nakastuka

*Graduate School of Health Sciences, Okayama University Department of Obstetrics and Gynecology, Okayama University Hospital Japan* 

#### **1. Introduction**

122 Recent Advances in Cardiovascular Risk Factors

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lowering drugs: results of the drug interaction study with an organic nitrate and a

(Acute myocardial infarction associated to the sildenafil consumption. A case

inflatable penile prosthesis implantation for impotence treatment. *J Urol* 1997; 157:

Women have unique risk factors for cardiovascular diseases and cerebrovascular diseases such as pregnancy and hormone replacement therapy. Pregnancy provides an opportunity to reveal various cardiovascular disease risk factors and estimate a woman's lifetime risk because of its unique cardiovascular and metabolic stress.

The causes of recurrent pregnancy loss (RPL) are classified as genetic, anatomic, endocrinologic, immunologic, microbiologic, environmental, and further more (Kutteh, 1999; Christiansen et al., 2005). Several lines of study have suggested that certain coagulation abnormalities such as antiphospholipid antibodies or Factor V Leiden, the genetic defect underlying resistance to activated protein C, are causes of RPL (Kutteh, 1999).

New diagnostic methods have improved the clinical triage of RPL (Kutteh, 1999; Li, 1998). Transvaginal pulsed Doppler ultrasonography allows noninvasive evaluation of uterine circulation. The introduction of pulsed Doppler ultrasonography has provided noninvasive means for the evaluation of uterine impedance, and gives physiologic data, rather than anatomic information alone. It is known that resistance to uterine arterial blood flow is associated with poor obstetrical outcome such as preeclampsia and fetal growth restriction (Nakatsuka et al, 1999b; Nakatsuka et al, 2002; Takata et al, 200).

We have reported that impaired uterine perfusion is observed in a portion of women with RPL (Habara et al., 2002; Nakatsuka et al., 2003a, 2003b). Pulsed Doppler ultrasonography in the uterine artery may be useful in distinguishing women with RPL caused by vascular dysfunction from women with unexplained RPL (Habara et al., 2002). Furthermore, the plasma level of adrenomedullin, which is often associated with pathological processes of the vasculature (Hinson, 2000), is elevated in women with RPL (Nakatsuka et al., 2003a). However, vascular changes in women with RPL have not been fully elucidated.

Antiphospholipid antibody syndrome (APS), which is an autoimmune disease associated with coagulopathy, is a well-known cause of RPL (Nakatsuka et al., 2003b). Antiphospholipid antibodies refer to several groups of autoantibodies with specificity for a number of negatively-charged phospholipids such as cardiolipin, phosphatidylserine, and phosphatidylethanolamine, or phospholipid-binding glycoproteins such as ,2 glycoprotein I

Vascular Dysfunction in Women with Recurrent Pregnancy Loss 125

the uterine artery has been reported to predict preeclampsia, fetal growth restriction, or gestational diabetes (van den Elzen et al., 1995). However, predictive value of uterine arterial pulsed Doppler velocimetry in pregnancy loss is controversial (van den Elzen et al., 1993; Jauniaux, et al., 1994; Kurjak et al., 1994; Alcázar, 2000; Nakatsuka et al., 2003a)..

We measured the resistance in the uterine arteries of 104 pregnant women with and without RPL at 4 to 5 weeks' gestation and evaluated association of autoantibodies including antiphospholipid antibodies (Nakatsuka et al., 2003a). In this study, uterine arterial pulsatility index (PI) in the RPL group was significantly higher than that in the control group (Figure 1). Women with antiphospholipid antibodies had an elevated PI in the uterine artery, which is prominent in women with RPL (Table 1). Coagulopathy and vascular

**2.1 Pregnant women with RPL associated with antiphospholipid antibodies** 

dysfunction caused by antiphospholipid antibodies may impair uterine perfusion.

Fig. 1. Resistance in uterine arterial blood flow of women with recurrent pregnancy loss

Table 1. Antiphospholipid antibodies and pulsatility index in the uterine artery

(Nakatsuka et al., 2003a).

APA : Antiphospholipid antibodies, n.a.: not available, Student's t-test\* : p<0.007.

(Nakatsuka et al., 2003a).

(,2GPI) and prothrombin. There are currently data supporting an association between various types of antiphospholipid antibodies and vascular diseases (Nayak & Komatireddy et al., 2002). Previous studies suggest that predominantly IgG and to lesser extent IgM isotype of antiphospholipid antibodies and lupus anticoagulant (LAC) are associated with arterial and venous thrombosis, thrombocytopenia, and livedo reticularis (Nayak & Komatireddy et al., 2002). Anti-cardiolipin and anti-,2GPI antibodies are elevated in patients with coronary artery disease. Anti-cardiolipin antibodies are also associated with typical chest pain, significant coronary artery stenosis on angiography and prediction of myocardial infarction (Sherer &Shoenfeld, 2003).

Premature atherosclerosis is a clinical feature of thrombotic patients with primary APS (Ames et al., 2009). Early data from the Italian Antiphospholipid Registry calculated a 2.5% patient/year incidence of recurrent thrombosis often fatal; a prospective Spanish study demonstrated that 5.2% of primary APS patients died of recurrent arterial occlusions; Italian longitudinal study showed a 5.2% patient/year mortality rate for recurrent arterial thrombosis and a Russian group recently reported a 17% 8-year vascular mortality for primary APS (Ames et al., 2009).

Polycystic ovary syndrome (PCOS) is one of the most common endocrinological disorders among reproductive-age women (Franks, 1995; Dunaif, 1997, The Rotterdam ESHRE/ASRM-Sponsored PCOS consensus workshop group, 2004). Using a combination of clinical, ultrasonographic, and biochemical criteria, the diagnosis of PCOS is usually reserved for those women who display one or more clinical symptoms including chronic anovulation, an ultrasonographical morphology of polycystic ovaries, inappropriate gonadotropin secretion, and hyperandrogenism (Franks, 1995; Dunaif, 1997). It is reported that women with PCOS have adverse pregnancy outcome including miscarriage (Abbott et al., 2002; Doldi et al., 1998; Glueck et al., 1999; Wang et al., 2001; Diejomaoh et al., 2003).

We and the other researchers demonstrated an impaired uterine perfusion in women with PCOS (Ajossa et al., 2002; Chekir C et al., 2005). Abnormal sex steroid hormones have been suggested as the cause for the elevated blood flow resistance in the uterine artery of women with PCOS (Zaidi et al., 1998). Furthermore, risk factors for cardiovascular disease including central obesity, hyperandrogenism, hyperinsulinemia, and dyslipidemia, which are commonly observed in women with PCOS, may lead to impairment of uterine perfusion and vascular dysfunction (Slowinska-Srzednicka et al., 1991; Legro ,2003; Sabuncu et al., 2001; Fenkci et al., 2003; Setji & Brown, 2007).

In the light of these studies, vascular dysfunction may be the key to the pathophysiology of pregnancy loss. This chapter reviews association between RPL and vascular dysfunction.

#### **2. Impaired uterine arterial blood flow in women with RPL**

Pregnancy loss in LPS-treated rats is associated with coagulopathy, decreased placental blood flow, and placental and fetal hypoxia. This impairment in uteroplacental hemodynamics in LPS-treated rats is linked to increased uterine artery resistance (Graham et al., 2011). In Human, peripheral vascular resistance in normal pregnancy decreases as early as 5 weeks of gestation (Robson, 1989). Resistance in uterine arterial blood flow also exhibits a progressive decrease after implantation while it increases in women with preeclampsia or fetal growth restriction (Steel et al., 1990). Pulsed Doppler velocimetry of

(,2GPI) and prothrombin. There are currently data supporting an association between various types of antiphospholipid antibodies and vascular diseases (Nayak & Komatireddy et al., 2002). Previous studies suggest that predominantly IgG and to lesser extent IgM isotype of antiphospholipid antibodies and lupus anticoagulant (LAC) are associated with arterial and venous thrombosis, thrombocytopenia, and livedo reticularis (Nayak & Komatireddy et al., 2002). Anti-cardiolipin and anti-,2GPI antibodies are elevated in patients with coronary artery disease. Anti-cardiolipin antibodies are also associated with typical chest pain, significant coronary artery stenosis on angiography and prediction of

Premature atherosclerosis is a clinical feature of thrombotic patients with primary APS (Ames et al., 2009). Early data from the Italian Antiphospholipid Registry calculated a 2.5% patient/year incidence of recurrent thrombosis often fatal; a prospective Spanish study demonstrated that 5.2% of primary APS patients died of recurrent arterial occlusions; Italian longitudinal study showed a 5.2% patient/year mortality rate for recurrent arterial thrombosis and a Russian group recently reported a 17% 8-year vascular mortality for

Polycystic ovary syndrome (PCOS) is one of the most common endocrinological disorders among reproductive-age women (Franks, 1995; Dunaif, 1997, The Rotterdam ESHRE/ASRM-Sponsored PCOS consensus workshop group, 2004). Using a combination of clinical, ultrasonographic, and biochemical criteria, the diagnosis of PCOS is usually reserved for those women who display one or more clinical symptoms including chronic anovulation, an ultrasonographical morphology of polycystic ovaries, inappropriate gonadotropin secretion, and hyperandrogenism (Franks, 1995; Dunaif, 1997). It is reported that women with PCOS have adverse pregnancy outcome including miscarriage (Abbott et al., 2002; Doldi et al., 1998; Glueck et al., 1999; Wang et al., 2001; Diejomaoh et al., 2003).

We and the other researchers demonstrated an impaired uterine perfusion in women with PCOS (Ajossa et al., 2002; Chekir C et al., 2005). Abnormal sex steroid hormones have been suggested as the cause for the elevated blood flow resistance in the uterine artery of women with PCOS (Zaidi et al., 1998). Furthermore, risk factors for cardiovascular disease including central obesity, hyperandrogenism, hyperinsulinemia, and dyslipidemia, which are commonly observed in women with PCOS, may lead to impairment of uterine perfusion and vascular dysfunction (Slowinska-Srzednicka et al., 1991; Legro ,2003; Sabuncu et al.,

In the light of these studies, vascular dysfunction may be the key to the pathophysiology of pregnancy loss. This chapter reviews association between RPL and vascular dysfunction.

Pregnancy loss in LPS-treated rats is associated with coagulopathy, decreased placental blood flow, and placental and fetal hypoxia. This impairment in uteroplacental hemodynamics in LPS-treated rats is linked to increased uterine artery resistance (Graham et al., 2011). In Human, peripheral vascular resistance in normal pregnancy decreases as early as 5 weeks of gestation (Robson, 1989). Resistance in uterine arterial blood flow also exhibits a progressive decrease after implantation while it increases in women with preeclampsia or fetal growth restriction (Steel et al., 1990). Pulsed Doppler velocimetry of

**2. Impaired uterine arterial blood flow in women with RPL** 

myocardial infarction (Sherer &Shoenfeld, 2003).

2001; Fenkci et al., 2003; Setji & Brown, 2007).

primary APS (Ames et al., 2009).

the uterine artery has been reported to predict preeclampsia, fetal growth restriction, or gestational diabetes (van den Elzen et al., 1995). However, predictive value of uterine arterial pulsed Doppler velocimetry in pregnancy loss is controversial (van den Elzen et al., 1993; Jauniaux, et al., 1994; Kurjak et al., 1994; Alcázar, 2000; Nakatsuka et al., 2003a)..

#### **2.1 Pregnant women with RPL associated with antiphospholipid antibodies**

We measured the resistance in the uterine arteries of 104 pregnant women with and without RPL at 4 to 5 weeks' gestation and evaluated association of autoantibodies including antiphospholipid antibodies (Nakatsuka et al., 2003a). In this study, uterine arterial pulsatility index (PI) in the RPL group was significantly higher than that in the control group (Figure 1). Women with antiphospholipid antibodies had an elevated PI in the uterine artery, which is prominent in women with RPL (Table 1). Coagulopathy and vascular dysfunction caused by antiphospholipid antibodies may impair uterine perfusion.

Fig. 1. Resistance in uterine arterial blood flow of women with recurrent pregnancy loss (Nakatsuka et al., 2003a).


APA : Antiphospholipid antibodies,

n.a.: not available, Student's t-test\* : p<0.007.

(Nakatsuka et al., 2003a).

Table 1. Antiphospholipid antibodies and pulsatility index in the uterine artery

Vascular Dysfunction in Women with Recurrent Pregnancy Loss 127

and 45 control women with regular menstrual cycles (Chekir et al.,2005). Among the control group, the uterine arterial PI in the luteal phase was significantly lower than that in the follicular phase (Figure 3). Among women with PCOS, the uterine arterial PI in the luteal phase tended to be lower than that in the follicular phase. The PI in the uterine artery in women with PCOS was significantly higher than that for the control group both in the follicular phase and in the luteal phase. Among women with PCOS, women with amenorrhea had a significantly higher uterine arterial PI than that of women with

Fig. 2. Pulsatility index in the uterine artery of women with or without antinuclear antibodies in control or RPL group. Bars, Mean. Values are expressed as mean ±S.D. (Habara et al., 2002)

Fig. 3. Pulsatility index in the uterine artery in control women and women with PCOS. Left panel: Uterine arterial PI in control women. Data from control women during the follicular phase and the luteal phase are indicated by open circles. Right panel: Uterine arterial PI in women with PCOS. Data from women with amenorrhea during the follicular phase are indicated by closed circles. Data from women with oligomenorrhea during the follicular phase and the luteal phase are indicated by open circles (Chekir et al, 2005).

oligomenorrhea.

Pregnancies complicated with hypertensive disorders and/or fetal growth restriction are known to be associated with a defective trophoblastic invasion. Antiphospholipid antibodies are known to interfere with syncytialization of the trophoblasts in early pregnancy and cause decidual vasculopathy, thrombosis, and placental infarction later in pregnancy. However, the elevation of uterine arterial PI that we observed is more likely to be associated with vascular dysfunction rather than impaired trophoblastic invasion. Trophoblastic invasion affects little on uterine arterial blood flow at 4-5 weeks of gestation because decrease in blood flow resistance in the uterine artery is very slow until 8 weeks of gestation (Dickey et al., 1995).

Pregnant women with antiphospholipid antibodies have vascular dysfunctions in the uterine artery although the prediction of adverse pregnancy outcome is not conclusive (Caruso et al, 1993; Venkat-Raman et al, 2001; Nakatsuka et al., 2003a).

Interestingly, the uterine arterial PI in RPL women without antiphospholipid antibodies is significantly higher than that in the control pregnant women even among women without antiphospholipid antibodies in our study (Nakatsuka et al., 2003a).

#### **2.2 Non-pregnant women with unexplained RPL**

Pulsed Doppler ultrasonography demonstrated blood flow changes in the uterus and ovaries during the menstrual cycle (Goswamy and Steptoe, 1988a; Chekir et al., 2005). The uterine arterial PI has been known to diminish progressively during the luteal phase, during which implantation occurs. Differences in uterine blood flow impedance between fertile and infertile women (Goswamy et al., 1988b; Steer et al., 1994). Based on studies from the IVF-ET programme, impedance of blood flow through the uterine arteries is a good indicator of the probability of subsequent pregnancy (Salle et al., 1998).

We investigated whether women with unexplained RPL have impaired uterine perfusion in the mid-luteal phase of non-conception cycles (Habara et al., 2002). The uterine arterial PI of 121 women including 49 women with unexplained RPL was measured by transvaginal pulsed Doppler ultrasonography. The uterine artery PI in RPL group (2.54±0.45, mean ±S.D.) was significantly higher than that in the control group (2.20±0.35). In the RPL group, the PI in the uterine artery of women with antinuclear antibodies (ANA) was significantly higher than that of women without ANA (Figure 2). There is no significant difference between the PI in the uterine artery of women with ANA and that of women without ANA in control group. Among women without ANA, the uterine artery PI in RPL group was also significantly higher than that in the control group.

Although ANA are not specific pathogens for pregnancy loss (Ogasawara, et al., 1996), women with positive ANA may have other autoimmune antibodies causing vasculopathy or coagulopathy. These pathological changes are likely to cause elevation of the uterine arterial blood flow resistance and lead to RPL, early onset preeclampsia, or fetal growth restriction. Although the apparent underlying pathophysiology of these cases was not elucidated, they may have subclinical vasculopathy, which was not diagnosed by routine screening tests for RPL.

#### **2.3 Women with PCOS**

Relatively high rate of pregnancy loss has been reported in women with PCOS. We performed a pulsed Doppler study on uterine arterial blood flow in 25 women with PCOS

Pregnancies complicated with hypertensive disorders and/or fetal growth restriction are known to be associated with a defective trophoblastic invasion. Antiphospholipid antibodies are known to interfere with syncytialization of the trophoblasts in early pregnancy and cause decidual vasculopathy, thrombosis, and placental infarction later in pregnancy. However, the elevation of uterine arterial PI that we observed is more likely to be associated with vascular dysfunction rather than impaired trophoblastic invasion. Trophoblastic invasion affects little on uterine arterial blood flow at 4-5 weeks of gestation because decrease in blood flow resistance in the uterine artery is very slow until 8 weeks of gestation (Dickey et al., 1995).

Pregnant women with antiphospholipid antibodies have vascular dysfunctions in the uterine artery although the prediction of adverse pregnancy outcome is not conclusive

Interestingly, the uterine arterial PI in RPL women without antiphospholipid antibodies is significantly higher than that in the control pregnant women even among women without

Pulsed Doppler ultrasonography demonstrated blood flow changes in the uterus and ovaries during the menstrual cycle (Goswamy and Steptoe, 1988a; Chekir et al., 2005). The uterine arterial PI has been known to diminish progressively during the luteal phase, during which implantation occurs. Differences in uterine blood flow impedance between fertile and infertile women (Goswamy et al., 1988b; Steer et al., 1994). Based on studies from the IVF-ET programme, impedance of blood flow through the uterine arteries is a good indicator of the

We investigated whether women with unexplained RPL have impaired uterine perfusion in the mid-luteal phase of non-conception cycles (Habara et al., 2002). The uterine arterial PI of 121 women including 49 women with unexplained RPL was measured by transvaginal pulsed Doppler ultrasonography. The uterine artery PI in RPL group (2.54±0.45, mean ±S.D.) was significantly higher than that in the control group (2.20±0.35). In the RPL group, the PI in the uterine artery of women with antinuclear antibodies (ANA) was significantly higher than that of women without ANA (Figure 2). There is no significant difference between the PI in the uterine artery of women with ANA and that of women without ANA in control group. Among women without ANA, the uterine artery PI in RPL group was also

Although ANA are not specific pathogens for pregnancy loss (Ogasawara, et al., 1996), women with positive ANA may have other autoimmune antibodies causing vasculopathy or coagulopathy. These pathological changes are likely to cause elevation of the uterine arterial blood flow resistance and lead to RPL, early onset preeclampsia, or fetal growth restriction. Although the apparent underlying pathophysiology of these cases was not elucidated, they may have subclinical vasculopathy, which was not diagnosed by routine

Relatively high rate of pregnancy loss has been reported in women with PCOS. We performed a pulsed Doppler study on uterine arterial blood flow in 25 women with PCOS

(Caruso et al, 1993; Venkat-Raman et al, 2001; Nakatsuka et al., 2003a).

antiphospholipid antibodies in our study (Nakatsuka et al., 2003a).

**2.2 Non-pregnant women with unexplained RPL** 

probability of subsequent pregnancy (Salle et al., 1998).

significantly higher than that in the control group.

screening tests for RPL.

**2.3 Women with PCOS** 

and 45 control women with regular menstrual cycles (Chekir et al.,2005). Among the control group, the uterine arterial PI in the luteal phase was significantly lower than that in the follicular phase (Figure 3). Among women with PCOS, the uterine arterial PI in the luteal phase tended to be lower than that in the follicular phase. The PI in the uterine artery in women with PCOS was significantly higher than that for the control group both in the follicular phase and in the luteal phase. Among women with PCOS, women with amenorrhea had a significantly higher uterine arterial PI than that of women with oligomenorrhea.

Fig. 2. Pulsatility index in the uterine artery of women with or without antinuclear antibodies in control or RPL group. Bars, Mean. Values are expressed as mean ±S.D. (Habara et al., 2002)

Fig. 3. Pulsatility index in the uterine artery in control women and women with PCOS. Left panel: Uterine arterial PI in control women. Data from control women during the follicular phase and the luteal phase are indicated by open circles. Right panel: Uterine arterial PI in women with PCOS. Data from women with amenorrhea during the follicular phase are indicated by closed circles. Data from women with oligomenorrhea during the follicular phase and the luteal phase are indicated by open circles (Chekir et al, 2005).

Vascular Dysfunction in Women with Recurrent Pregnancy Loss 129

Adrenomedullin has interaction with various bioactive molecules including nitric oxide, prostaglandins, atrial natriuretic peptide, renin, aldosterone, norepinephrine, arginine vasopressin, endothelin-1, and adrenocorticotropic hormone (Jougasaki & Burnett, 2000). The plasma level of adrenomedullin is elevated in various diseases including hypertension, diabetes, cardiac failure, septic shock, or SLE, which are often associated with pathologic

We measured plasma levels of adrenomedullin of 100 pregnant women in the midluteal phase of a nonpregnant cycle (Nakatsuka et al., 2003b). We also measured the PI in the uterine arteries by transvaginal pulsed Doppler ultrasonography at the same time. The plasma level of adrenomedullin in women with RPL was significantly higher than that in control women. Uterine arterial PI of women with RPL was significantly higher than that in control women. Plasma level of adrenomedullin had a significant positive correlation with uterine arterial PI both in the control group (r=0.58, p < 0.001) and in the RPL group (r=0.78, p < 0.001) (Figure 4). Both plasma adrenomedullin concentration and uterine arterial PI were

processes of the vasculature (Jougasaki & Burnett, 2000; Hinson et al., 2000).

significantly high in women with antiphospholipid antibodies.

Fig. 4. Plasma adrenomedullin and uterine arterial pulsatility index.

systemic vasculatures.

A) Plasma adrenomedullin concentration and uterine arterial pulsatility index in control women. A significant positive correlation was determined by Pearson correlation coefficient (r =0.58, p<0.001). B) Plasma adrenomedullin concentration and uterine arterial pulsatility index in women with recurrent pregnancy loss. A significant positive correlation was determined by Pearson correlation coefficient (r =0.78,p <0.001). (Nakatsuka et al., 2003b)

Increased plasma adrenomedullin in women with RPL is likely to be a response to vascular damage and increased vascular tone. Plasma adrenomedullin levels observed in women with recurrent pregnancy loss were similar to the values reported in patients with hypertension, mitral stenosis, primary aldosteronism, or SLE (Nakatsuka et al., 2003b).

Although the pathophysiologic roles of adrenomedullin in RPL have not been fully elucidated, this peptide may serve as a biochemical marker to identify women with impaired uterine perfusion (Nakatsuka et al., 2003b; Ashraf et al., 2011) and also impaired

The uterine arterial PI was correlated with body mass index, luteinizing hormone /folliclestimulating hormone ratio, or low-density lipoprotein-cholesterol (LDL-C)/high-density lipoprotein-cholesterol (HDL-C) ratio while it was inversely correlated with the HDL-C level.

### **3. Biomarkers for cardiovascular risk assessment in women with RPL**

The introduction of pulsed Doppler ultrasonography has provided the means for the noninvasive evaluation of uterine impedance, thus providing physiologic data on hemodynamic abnormalities in early pregnancy failures. Are these vascular changes in women with PRL observed solely in the uterine artery? Biomarkers for cardiovascular risk assessment are substances that are released into the blood when the vasculature is damaged. Some biomarkers cause vascular dysfunction directly or indirectly. It may be worth measuring biomarkers for cardiovascular risk assessment in women with RPL.

#### **3.1 Thrombomodulin**

Thrombomodulin binds thrombin, changes thrombin conformation and allows thrombin to activate protein C, which inhibits coagulation and thrombin-activatable fibrinolysis inhibitor, which inhibits fibrinolysis. Increased serum thrombomodulin, cleaved products of cellular thrombomodulin, has been demonstrated previously in preeclampsia (Dusse et al., 2011). Endothelial thrombomodulin is known to be a major vasoprotective molecule. However, elevated serum thrombomodulin is also likely to be a response to vascular activation.

We measured serum levels of soluble thrombomodulin of 54 pregnant women at 4-8 weeks of gestation (Nakatsuka et al., 2004). Serum thrombomodulin was significantly elevated in women with antiphospholipid antibodies in RPL group compared with control women and with RPL women without antiphospholipid antibodies. Among women with APS, serum thrombomodulin in women who subsequently had a growth-restricted fetus had been significantly higher than that in women who subsequently had an appropriate-for-date fetus. Elevation of thrombomodulin is likely to indicate impaired uterine blood flow in women with RPL.

It is controversial whether the soluble thrombomodulin level is an independent risk factor for coronary heart disease (Wu, 2003; Huang et al., 2008; Karakas et al., 2011). However, anti-cardiolipin antibodies are reported to be important not only in the pathogenesis of mixed connective tissue disease (MCTD) but in the induction of endothelial cell causing elevation of soluble thrombomodulin, and may play crucial roles in the development of early atherosclerosis in MCTD (Soltesz, 2010). It is also known that development of atherosclerosis and elevation of soluble thrombomodulin in serum of systemic lupus erythematosus (SLE) patients with metabolic syndrome (Mok et al., 2010).

#### **3.2 Adrenomedullin**

Adrenomedullin, a 52-amino acids-ringed, structured peptide, mediates vasodilatory properties through the second messenger cyclic adenosine, 3,5-monophosphate (Jougasaki & Burnett, 2000). The main source of plasma adrenomedullin is considered to be vascular endothelial cells and vascular smooth muscle cells.

The uterine arterial PI was correlated with body mass index, luteinizing hormone /folliclestimulating hormone ratio, or low-density lipoprotein-cholesterol (LDL-C)/high-density lipoprotein-cholesterol (HDL-C) ratio while it was inversely correlated with the HDL-C

The introduction of pulsed Doppler ultrasonography has provided the means for the noninvasive evaluation of uterine impedance, thus providing physiologic data on hemodynamic abnormalities in early pregnancy failures. Are these vascular changes in women with PRL observed solely in the uterine artery? Biomarkers for cardiovascular risk assessment are substances that are released into the blood when the vasculature is damaged. Some biomarkers cause vascular dysfunction directly or indirectly. It may be worth

Thrombomodulin binds thrombin, changes thrombin conformation and allows thrombin to activate protein C, which inhibits coagulation and thrombin-activatable fibrinolysis inhibitor, which inhibits fibrinolysis. Increased serum thrombomodulin, cleaved products of cellular thrombomodulin, has been demonstrated previously in preeclampsia (Dusse et al., 2011). Endothelial thrombomodulin is known to be a major vasoprotective molecule. However,

We measured serum levels of soluble thrombomodulin of 54 pregnant women at 4-8 weeks of gestation (Nakatsuka et al., 2004). Serum thrombomodulin was significantly elevated in women with antiphospholipid antibodies in RPL group compared with control women and with RPL women without antiphospholipid antibodies. Among women with APS, serum thrombomodulin in women who subsequently had a growth-restricted fetus had been significantly higher than that in women who subsequently had an appropriate-for-date fetus. Elevation of thrombomodulin is likely to indicate impaired uterine blood flow in

It is controversial whether the soluble thrombomodulin level is an independent risk factor for coronary heart disease (Wu, 2003; Huang et al., 2008; Karakas et al., 2011). However, anti-cardiolipin antibodies are reported to be important not only in the pathogenesis of mixed connective tissue disease (MCTD) but in the induction of endothelial cell causing elevation of soluble thrombomodulin, and may play crucial roles in the development of early atherosclerosis in MCTD (Soltesz, 2010). It is also known that development of atherosclerosis and elevation of soluble thrombomodulin in serum of systemic lupus

Adrenomedullin, a 52-amino acids-ringed, structured peptide, mediates vasodilatory properties through the second messenger cyclic adenosine, 3,5-monophosphate (Jougasaki & Burnett, 2000). The main source of plasma adrenomedullin is considered to be vascular

erythematosus (SLE) patients with metabolic syndrome (Mok et al., 2010).

endothelial cells and vascular smooth muscle cells.

**3. Biomarkers for cardiovascular risk assessment in women with RPL** 

measuring biomarkers for cardiovascular risk assessment in women with RPL.

elevated serum thrombomodulin is also likely to be a response to vascular activation.

level.

**3.1 Thrombomodulin** 

women with RPL.

**3.2 Adrenomedullin** 

Adrenomedullin has interaction with various bioactive molecules including nitric oxide, prostaglandins, atrial natriuretic peptide, renin, aldosterone, norepinephrine, arginine vasopressin, endothelin-1, and adrenocorticotropic hormone (Jougasaki & Burnett, 2000). The plasma level of adrenomedullin is elevated in various diseases including hypertension, diabetes, cardiac failure, septic shock, or SLE, which are often associated with pathologic processes of the vasculature (Jougasaki & Burnett, 2000; Hinson et al., 2000).

We measured plasma levels of adrenomedullin of 100 pregnant women in the midluteal phase of a nonpregnant cycle (Nakatsuka et al., 2003b). We also measured the PI in the uterine arteries by transvaginal pulsed Doppler ultrasonography at the same time. The plasma level of adrenomedullin in women with RPL was significantly higher than that in control women. Uterine arterial PI of women with RPL was significantly higher than that in control women. Plasma level of adrenomedullin had a significant positive correlation with uterine arterial PI both in the control group (r=0.58, p < 0.001) and in the RPL group (r=0.78, p < 0.001) (Figure 4). Both plasma adrenomedullin concentration and uterine arterial PI were significantly high in women with antiphospholipid antibodies.

Fig. 4. Plasma adrenomedullin and uterine arterial pulsatility index.

A) Plasma adrenomedullin concentration and uterine arterial pulsatility index in control women. A significant positive correlation was determined by Pearson correlation coefficient (r =0.58, p<0.001). B) Plasma adrenomedullin concentration and uterine arterial pulsatility index in women with recurrent pregnancy loss. A significant positive correlation was determined by Pearson correlation coefficient (r =0.78,p <0.001). (Nakatsuka et al., 2003b)

Increased plasma adrenomedullin in women with RPL is likely to be a response to vascular damage and increased vascular tone. Plasma adrenomedullin levels observed in women with recurrent pregnancy loss were similar to the values reported in patients with hypertension, mitral stenosis, primary aldosteronism, or SLE (Nakatsuka et al., 2003b).

Although the pathophysiologic roles of adrenomedullin in RPL have not been fully elucidated, this peptide may serve as a biochemical marker to identify women with impaired uterine perfusion (Nakatsuka et al., 2003b; Ashraf et al., 2011) and also impaired systemic vasculatures.

Vascular Dysfunction in Women with Recurrent Pregnancy Loss 131

There are various methods in evaluation of atherosclerosis. Intima media thickness (IMT) of carotid arteries is used to evaluate early atherosclerosis and the risk of associated cardiovascular disease (Burke, et al., 1995). The The ankle-brachial index (ABI) for each leg was calculated as the ratio of the systolic pressures in the leg and the systolic pressure of either the left or right arm. An ABI < 1.0 in either leg was considered abnormal, suggesting peripheral arterial disease; progressively lower ABI values indicate more severe obstruction

Pulse wave velocity (PWV) and the augmentation index (AI) are widely used as arterial stiffness indices. Recently, brachial-ankle PWV (baPWV) measurement can be performed easily by simultaneous oscillometric measurement of pulse waves in all four extremities. Brachial-ankle PWV was used as a substitute for aortic PWV because baPWV is known to be strongly correlated with aortic PWV (Matsui et al., 2004). PWV are known to be a marker of both the severity of vascular damage and the prognosis of atherosclerotic vascular diseases in patients with hypertension (Blacher et al., 1999), end-stage renal failure (London & Cohn, 2002), and diabetes (Yokoyama et al., 2003). Increased PWV is known to be an independent predictor of the prognosis in hypertension, including in subjects under anti-hypertensive

Carotid AI (cAI) was assessed by the proportion of the central pulse pressure resulting from peripheral arterial wave reflection. The AI is known to be an independent predictor of allcause and cardiovascular mortality in end-stage renal failure patients (London et al., 2001). It has been also reported that increased AI is associated with the presence and severity of coronary artery disease, particularly in younger and middle-aged male patients (Weber et al., 2004). Although PWV and AI have been known to be useful indices of atherosclerotic

Antiphospholipid antibodies is known to play a central role in both pregnancy loss and cardiovascular diseases (Mackworth-Young, 2004). Antiphospholipid antibodies is a risk factor for incident stroke, however, the evidence to support the role of antiphospholipid antibodies in recurrent stroke is conflicting (Brey, 2004). Neither anti-cardiolipin antibodies nor anti-,2GPI antibodies is reported to be associated with atherosclerosis in premenopausal women with APS and SLE, who have an increased prevalence of carotid and femoral plaque (Vlachoyiannopoulos et al., 2003). Furthermore, previous studies have paradoxically proposed a beneficial role for some antiphospholipid antibodies in

We assessed arterial stiffness of 153 women with RPL and 66 healthy women with one or less pregnancy loss. It is reported that abnormal ABIs are more common in primary APS than in healthy controls (Baron et al., 2005). However, we did not observe significant difference in ABI value or incidence of abnormal ABI between RPL women with antiphospholipid antibodies and control women. More sensitive methods may be necessary to detect early changes of vascular system in younger patients with antiphospholipid antibodies. Women with RPL had significantly higher baPWV than control women (Figure 5). Mean value of baPWV of RPL women with antiphospholipid antibodies is significantly higher than that in control women. None in control women or women with unexplained RPL

(Sacks et al., 2002).

medication (Laurent et al., 2001).

vascular diseases, data in young females are scarce.

atherosclerosis (Nicolo & Monestier, 2004).

**4.2 Women with PRL associated with antiphospholipid antibodies** 

#### **3.3 The other markers associated with cardiovascular diseases**

We have previously reported that there is no significant difference in serum nitric oxide metabolite level between control women and women with recurrent pregnancy loss (Habara et al., 2002). However, anti-cardiolipin antibodies induce nitric oxide and superoxide production from vascular vessels, resulting in enhanced local levels of plasma peroxynitrite (Alves & Grima, 2003), which is a powerful pro-oxidant molecule (Nakatsuka et al, 1999). These oxidative damages in the cardiovascular system may be involved in atherosclerosis.

PCOS has deserved major attention because it is linked to the same cluster of events that promote the metabolic syndrome. We have reported that women with PCOS had significantly higher total cholesterol, triglyceride, and β-lipoprotein levels than those of the control group. Significantly lower HDL-C, higher LDL-C, and consequently a higher LDL-C/HDL-C ratio were observed in women with PCOS as compared to those of the control women. Fasting serum insulin and homeostasis model assessment-R (HOMA-R), which are indexes of insulin resistance, in women with PCOS were significantly higher than those for the control group.

It has been reported that isolated adipocytes from women with PCOS express higher mRNA concentrations of some adipokines involved in cardiovascular risk and insulin resistance (Garruti et al., 2009). The actions of adipokines and adipocytokines on platelets and vascular smooth muscle cells, both of which are deeply involved in atherothrombosis, have been reported (Anfossi et al, 2010). Adipose tissue from individuals with central obesity synthesizes and releases increased amount of proinflammatory chemokines and cytokines, such as monocyte chemoattractant protein-1 (MCP-1), macrophage migration inhibitory factor (MIF), tumor necrosis factor-+ (TNF-+), and interleukins, including interleukin-1, (IL-1,) and interleukin-6 (IL-6); procoagulant and proinflammatory mediators such as tissue factor (TF) and plasminogen activator inhibitor-1 (PAI-1); vasoactive substances such as angiotensinogen and endothelin-1 (ET-1); molecules involved in the pathogenesis of insulin resistance, such as TNF-+ and resistin (Anfossi et al, 2010). Elevated levels of PAI-1, the major natural antifibrinolytic agent, are involved in atherothrombosis in women with PCOS (Ehrmann, 2005). Women with RPL, who are characterized as PCOS might be monitored by measuring these bioactive molecules to predict development of cardiovascular diseases.

#### **4. Arterial stiffness in women with RPL**

Impaired uterine arterial blood flow and elevated cardiovascular disease risk markers suggest that early changes of systemic vasculature may be progressing in women with RPL. These changes lead to athrosclerosis and atherothrombosis and may cause coronary heart disease and/or stroke.

#### **4.1 Evaluation methods of arterial stiffness**

Reductions in the elasticity of central arteries may act as a marker of early changes that predispose to the development of major vascular disease. Arterial stiffness has been known as a major contributory factor to cardiovascular morbidity and mortality in patients with hypertension. Independent studies have shown that central arterial stiffness is increased in older individuals and in those with coronary artery disease, myocardial infarction, heart failure, hypertension, stroke, diabetes mellitus, end-stage renal disease, hypercholesterolemia, and inflammation (Nichols, 2005).

We have previously reported that there is no significant difference in serum nitric oxide metabolite level between control women and women with recurrent pregnancy loss (Habara et al., 2002). However, anti-cardiolipin antibodies induce nitric oxide and superoxide production from vascular vessels, resulting in enhanced local levels of plasma peroxynitrite (Alves & Grima, 2003), which is a powerful pro-oxidant molecule (Nakatsuka et al, 1999). These oxidative damages in the cardiovascular system may be involved in atherosclerosis. PCOS has deserved major attention because it is linked to the same cluster of events that promote the metabolic syndrome. We have reported that women with PCOS had significantly higher total cholesterol, triglyceride, and β-lipoprotein levels than those of the control group. Significantly lower HDL-C, higher LDL-C, and consequently a higher LDL-C/HDL-C ratio were observed in women with PCOS as compared to those of the control women. Fasting serum insulin and homeostasis model assessment-R (HOMA-R), which are indexes of insulin resistance, in women with PCOS were significantly higher than those for the control group.

It has been reported that isolated adipocytes from women with PCOS express higher mRNA concentrations of some adipokines involved in cardiovascular risk and insulin resistance (Garruti et al., 2009). The actions of adipokines and adipocytokines on platelets and vascular smooth muscle cells, both of which are deeply involved in atherothrombosis, have been reported (Anfossi et al, 2010). Adipose tissue from individuals with central obesity synthesizes and releases increased amount of proinflammatory chemokines and cytokines, such as monocyte chemoattractant protein-1 (MCP-1), macrophage migration inhibitory factor (MIF), tumor necrosis factor-+ (TNF-+), and interleukins, including interleukin-1, (IL-1,) and interleukin-6 (IL-6); procoagulant and proinflammatory mediators such as tissue factor (TF) and plasminogen activator inhibitor-1 (PAI-1); vasoactive substances such as angiotensinogen and endothelin-1 (ET-1); molecules involved in the pathogenesis of insulin resistance, such as TNF-+ and resistin (Anfossi et al, 2010). Elevated levels of PAI-1, the major natural antifibrinolytic agent, are involved in atherothrombosis in women with PCOS (Ehrmann, 2005). Women with RPL, who are characterized as PCOS might be monitored by measuring these bioactive molecules to predict development of cardiovascular diseases.

Impaired uterine arterial blood flow and elevated cardiovascular disease risk markers suggest that early changes of systemic vasculature may be progressing in women with RPL. These changes lead to athrosclerosis and atherothrombosis and may cause coronary heart

Reductions in the elasticity of central arteries may act as a marker of early changes that predispose to the development of major vascular disease. Arterial stiffness has been known as a major contributory factor to cardiovascular morbidity and mortality in patients with hypertension. Independent studies have shown that central arterial stiffness is increased in older individuals and in those with coronary artery disease, myocardial infarction, heart failure, hypertension, stroke, diabetes mellitus, end-stage renal disease,

**3.3 The other markers associated with cardiovascular diseases** 

**4. Arterial stiffness in women with RPL** 

**4.1 Evaluation methods of arterial stiffness** 

hypercholesterolemia, and inflammation (Nichols, 2005).

disease and/or stroke.

(Sacks et al., 2002). Pulse wave velocity (PWV) and the augmentation index (AI) are widely used as arterial stiffness indices. Recently, brachial-ankle PWV (baPWV) measurement can be performed easily by simultaneous oscillometric measurement of pulse waves in all four extremities. Brachial-ankle PWV was used as a substitute for aortic PWV because baPWV is known to be strongly correlated with aortic PWV (Matsui et al., 2004). PWV are known to be a marker of both the severity of vascular damage and the prognosis of atherosclerotic vascular diseases in patients with hypertension (Blacher et al., 1999), end-stage renal failure (London & Cohn, 2002), and diabetes (Yokoyama et al., 2003). Increased PWV is known to be an independent predictor of the prognosis in hypertension, including in subjects under anti-hypertensive

medication (Laurent et al., 2001).

Carotid AI (cAI) was assessed by the proportion of the central pulse pressure resulting from peripheral arterial wave reflection. The AI is known to be an independent predictor of allcause and cardiovascular mortality in end-stage renal failure patients (London et al., 2001). It has been also reported that increased AI is associated with the presence and severity of coronary artery disease, particularly in younger and middle-aged male patients (Weber et al., 2004). Although PWV and AI have been known to be useful indices of atherosclerotic vascular diseases, data in young females are scarce.

#### **4.2 Women with PRL associated with antiphospholipid antibodies**

Antiphospholipid antibodies is known to play a central role in both pregnancy loss and cardiovascular diseases (Mackworth-Young, 2004). Antiphospholipid antibodies is a risk factor for incident stroke, however, the evidence to support the role of antiphospholipid antibodies in recurrent stroke is conflicting (Brey, 2004). Neither anti-cardiolipin antibodies nor anti-,2GPI antibodies is reported to be associated with atherosclerosis in premenopausal women with APS and SLE, who have an increased prevalence of carotid and femoral plaque (Vlachoyiannopoulos et al., 2003). Furthermore, previous studies have paradoxically proposed a beneficial role for some antiphospholipid antibodies in atherosclerosis (Nicolo & Monestier, 2004).

We assessed arterial stiffness of 153 women with RPL and 66 healthy women with one or less pregnancy loss. It is reported that abnormal ABIs are more common in primary APS than in healthy controls (Baron et al., 2005). However, we did not observe significant difference in ABI value or incidence of abnormal ABI between RPL women with antiphospholipid antibodies and control women. More sensitive methods may be necessary to detect early changes of vascular system in younger patients with antiphospholipid antibodies.

Women with RPL had significantly higher baPWV than control women (Figure 5). Mean value of baPWV of RPL women with antiphospholipid antibodies is significantly higher than that in control women. None in control women or women with unexplained RPL

Vascular Dysfunction in Women with Recurrent Pregnancy Loss 133

antibodies to define the patient's risk of arterial and venous thrombosis (Galli et al., 2005)

Petri have reported that twenty years after diagnosis, SLE patients with lupus anticoagulant have a 50% chance of a venous thrombotic event and myocardial infarction occurs significantly more often (22%) in those with lupus anticoagulant (Petri, 2004). However, neither anti-cardiolipin nor lupus anticoagulant is associated with an increase of carotid IMT, carotid plaque, nor coronary calcium by helical CT, which are signs of subclinical atherosclerosis (Petri, 2004). In our study, we could not find any significant differences in baPWV or cAI between women with lupus anticoagulant and control women (Figure 7).

\*:p<0.05 vs. Control, a,2GPI: anti-,2GPI antibodies, aCL: anti-cardiolipin antibodies, LAC: lupus anticoagulant, aPS: anti-phosphatidylserine antibodies, aPS/PT: antibodies against phosphatidylserine/prothrombin complex, K(-)aPE: kininogen independent anti

Fig. 7. cAI among women with various types of APA

women with anti-,2GPI antibodies were small population.

phosphatidylethanolamine antibodies, K(+)aPE: kininogen dependent anti-phosphatidylethanolamine

\*\*

There is some evidence that high anti-,2GPI antibodies can present a risk factor for atherosclerosis, but more epidemiological data are required in order to confirm whether the pro-atherogenic properties of anti-phospholipid antibodies signifies an independent risk factor for atherosclerosis and its complications. We observed that women with anti-,2GPI antibodies had high cAI. Unfortunately, we could not perform statistical analysis because

It has been reported that a significantly high prevalence of anti-phosphatidylserine IgG was found in stroke patients (57.7%) (Kahles et al., 2005). Anti-phosphatidylserine antibodies have a strong predictive value and association for arterial thrombosis (Lopez et al., 2004). Antibodies against phosphatidylserine/prothrombin complex have been reported to be closely associated with clinical features of APS rather than antibodies against prothrombin alone (Atsumi et al., 2004). However, there are no reports on atherosclerosis in women with these antibodies. We observed no significant association between anti-phosphatidylserine antibodies

Recent studies have shown that some patients with unexplained thrombophilic disorders may have anti-phosphatidylethanolamine antibodies as the sole basis for their hypercoagulable

or antibodies against phosphatidylserine-prothrombin complex and arterial stiffness.

although it is inconclusive.

antibodies

showed abnormal baPWV while five in RPL women with antiphospholipid antibodies showed abnormal baPWV (baPWV > 1,400 m/sec, American Heart Association Medical/Scientific Statement, 1993). Women with RPL had significantly higher cAI (22.5 ± 171.2 %) than control women (-67.3 ± 151.7 %) (p<0.0005). Mean value of cAI of women with unexplained RPL or that of RPL women with antiphospholipid antibodies is significantly higher than that in control women (Figure 6).

Fig. 5. baPWV of women with RPL

Fig. 6. cAI of women with RPL

#### **4.3 Types of autoimmune antibodies and arterial stiffness in women with RPL**

Antiphospholipid antibodies are a heterogeneous group of autoantibodies directed against phospholipid binding proteins, such as anti-cardiolipin antibodies, anti-,2GPI antibodies, and anti-phosphatidylserine/prothrombin antibodies, lupus anticoagulant, antiphosphatidylserine antibodies and anti-phosphatidylethanolamine antibodies. Pathogenesis of antiphospholipid antibodies may vary depending on types and target phospholipid. There is an increasing interest in clinical significance of various types of antiphospholipid

showed abnormal baPWV while five in RPL women with antiphospholipid antibodies showed abnormal baPWV (baPWV > 1,400 m/sec, American Heart Association Medical/Scientific Statement, 1993). Women with RPL had significantly higher cAI (22.5 ± 171.2 %) than control women (-67.3 ± 151.7 %) (p<0.0005). Mean value of cAI of women with unexplained RPL or that of RPL women with antiphospholipid antibodies is significantly

**4.3 Types of autoimmune antibodies and arterial stiffness in women with RPL** 

Antiphospholipid antibodies are a heterogeneous group of autoantibodies directed against phospholipid binding proteins, such as anti-cardiolipin antibodies, anti-,2GPI antibodies, and anti-phosphatidylserine/prothrombin antibodies, lupus anticoagulant, antiphosphatidylserine antibodies and anti-phosphatidylethanolamine antibodies. Pathogenesis of antiphospholipid antibodies may vary depending on types and target phospholipid. There is an increasing interest in clinical significance of various types of antiphospholipid

higher than that in control women (Figure 6).

Fig. 5. baPWV of women with RPL

Fig. 6. cAI of women with RPL

antibodies to define the patient's risk of arterial and venous thrombosis (Galli et al., 2005) although it is inconclusive.

Petri have reported that twenty years after diagnosis, SLE patients with lupus anticoagulant have a 50% chance of a venous thrombotic event and myocardial infarction occurs significantly more often (22%) in those with lupus anticoagulant (Petri, 2004). However, neither anti-cardiolipin nor lupus anticoagulant is associated with an increase of carotid IMT, carotid plaque, nor coronary calcium by helical CT, which are signs of subclinical atherosclerosis (Petri, 2004). In our study, we could not find any significant differences in baPWV or cAI between women with lupus anticoagulant and control women (Figure 7).

\*:p<0.05 vs. Control, a,2GPI: anti-,2GPI antibodies, aCL: anti-cardiolipin antibodies, LAC: lupus anticoagulant, aPS: anti-phosphatidylserine antibodies, aPS/PT: antibodies against phosphatidylserine/prothrombin complex, K(-)aPE: kininogen independent anti phosphatidylethanolamine antibodies, K(+)aPE: kininogen dependent anti-phosphatidylethanolamine antibodies

Fig. 7. cAI among women with various types of APA

There is some evidence that high anti-,2GPI antibodies can present a risk factor for atherosclerosis, but more epidemiological data are required in order to confirm whether the pro-atherogenic properties of anti-phospholipid antibodies signifies an independent risk factor for atherosclerosis and its complications. We observed that women with anti-,2GPI antibodies had high cAI. Unfortunately, we could not perform statistical analysis because women with anti-,2GPI antibodies were small population.

It has been reported that a significantly high prevalence of anti-phosphatidylserine IgG was found in stroke patients (57.7%) (Kahles et al., 2005). Anti-phosphatidylserine antibodies have a strong predictive value and association for arterial thrombosis (Lopez et al., 2004). Antibodies against phosphatidylserine/prothrombin complex have been reported to be closely associated with clinical features of APS rather than antibodies against prothrombin alone (Atsumi et al., 2004). However, there are no reports on atherosclerosis in women with these antibodies. We observed no significant association between anti-phosphatidylserine antibodies or antibodies against phosphatidylserine-prothrombin complex and arterial stiffness.

Recent studies have shown that some patients with unexplained thrombophilic disorders may have anti-phosphatidylethanolamine antibodies as the sole basis for their hypercoagulable

Vascular Dysfunction in Women with Recurrent Pregnancy Loss 135

women with unexplained RPL and those in control women were overlapped, at least a portion of women with unexplained RPL showed increased baPWV and cAI. These observations suggest that antiphospholipid antibodies or endocrinological disorders may not be the sole cause for arterial stiffness in women with RPL. Vascular dysfunction caused by various factors may be involved in at least a portion of women with unexplained RPL.

A portion of women with unexplained RPL should be considered as a high risk group for atherosclerosis and cardiovascular diseases. Measurement of baPWV or AI is a promising

In pregnant women with RPL, who have early changes of systemic vasculatures, may suffer vascular complications during pregnancy because of physiological hypercoagulability and

Thrombophilia is a risk factor for venous thromboembolism (VTE) in pregnancy because of the hypercoagulability of pregnancy, which is further increased in the presence of thrombophilia. Vascular reactivity, which is believed to be increased during pregnancy, may

Pregnant women are at increased risk of venous thromboembolism (VTE). Estimated incidence of VTE during pregnancy is about 1 event per 1000 pregnancies (Chauleur et al., 2007). Despite this low incidence, thromboembolic complications occurring during pregnancy and post-partum remain a major cause of maternal death. Although pregnant women have a higher risk of developing thromboembolic complications than non-pregnant

A scoring system for VTE risk in pregnant women (Chauleur et al., 2007) includes antiphospholipid antibodies and reproductive history of one stillbirth or at least three recurrent miscarriages, which are clinical features of women with APS (Bobba et al., 2007).

The risk of acute myocardial infarction is known to be approximately 3 to 4 times higher in pregnancy. The incidence of pregnancy-related acute myocardial infarction is in the broad range of 3 to 10 per 100 000 deliveries that has been reported previously (James et al., 2006). Hypertension (odds ratio (OR) 21.7), thrombophilia including history of thrombosis and APS (OR 22.3), diabetes mellitus (OR 3.6), smoking (OR 8.4), transfusion (OR 5.1), postpartum infection (OR 3.2), and age 30 years and older remain as significant risk factors for pregnancy-related acute myocardial infarction in the multivariable analysis (James et al., 2006). The odds of acute myocardial infarction are 30-fold higher for women aged 40 years

Thrombophilia, gestational diabetes mellitus associated with PCOS, or age 30 years and

women, treating all pregnant women to prevent these events is not recommended.

technique to assess vascular dysfunction in women with RPL.

hemodynamic changes associated with pregnancy.

**5.2 Coronary heart disease and stroke in pregnancy** 

older may increase the risk further in women with RPL.

and older than for women 20 years of age.

**5.1 Venous thromboembolism (VTE)** 

also compound the risk.

**5. Possible cardiovascular diseases in pregnant women with RPL** 

state (Sanmarco, et al., 2001). It is noteworthy that anti-phosphatidylethanolamine antibodies have been described as the sole antiphospholipid antibodies in patients with thrombotic diseases (Staub, et al., 1989; Karmochkine, et al., 1992; Berard, et al., 1996). However, there are little studies on association of anti-phosphatidylethanolamine antibodies and arterial stiffness or atherosclerosis.

We observed that cAI was significantly increased in RPL women with antiphosphatidylethanolamine antibodies. Anti-phosphatidylethanolamine antibodies may be a risk factor for atherosclerosis in women with RPL. It has been reported that kininogendependent IgG anti-phosphatidylethanolamine antibodies markedly increases thrombininduced platelet aggregation in vitro while kininogen independent IgG antiphosphatidylethanolamine antibodies do not augment thrombin-induced platelet aggregation (Sugi et al, 1999). However, we observed that both two types of antiphosphatidylethanolamine antibodies were associated with arterial stiffness.

Physical distribution of phosphatidylethanolamine is known to be at the blood-endothelium interface. The luminal phosphatidylethanolamine is a vulnerable to antiphosphatidylethanolamine autoimmunity, which is consistent with the association between anti- phosphatidylethanolamine antibodies and elevated risk for idiopathic thrombosis (Zhixin et al., 2011).

Risk factors for atherosclerosis in SLE include traditional risk factors (mainly the Framingham risk factors). Moreover, specific antibodies to ,2GPI; anticardiolipin antibodies; anti-oxidized low-density lipoprotein (oxLDL); and antibodies to heat shock proteins may be cardiovascular disease risk factors (Sherer et al., 2010). Immune complexes containing oxLDL, ,2GPI, and/or CRP are known to be involved in atherosclerosis (Matsuura et al., 2006, Chekir et al, 2009). Autoantibodies to oxLDL/,2GPI complex were detected in SLE and APS patients, and were strongly associated with arterial thrombosis (Christodoulou et al., 2007). Further studies may help evaluating clinical usefullness of these autoimmune antibodies involving in progression of atherosclerosis in women RPL.

#### **4.4 Women with PCOS**

Previous study on arterial stiffness has shown that both baPWV and cAI are useful for risk stratification of hypertensive patients (Matsui et al., 2004). In this study, both of these indices are significantly correlated with age and systolic blood pressure and cAI is reported to be correlated with total cholesterol and LDL-C in hypertensive patients.

We have reported that women with PCOS in reproductive age have a significantly higher baPWV than that for the control women (Sasaki et al., 2011). Arterial stiffness evaluated using the baPWV and cAI in mildly-hypertensive women (systolic blood pressure 120 mmHg or diastolic blood pressure 90 mmHg) with PCOS was significantly higher than that in the control women or normotensive women with PCOS. Early changes in vascular function were detected in mildly-hypertensive women with PCOS.

#### **4.5 Women with unexplained RPL**

We observed that women with unexplained RPL showed increased baPWV and cAI in average as compared with control women (Figure 5, 6). Although ranges of these indices in

state (Sanmarco, et al., 2001). It is noteworthy that anti-phosphatidylethanolamine antibodies have been described as the sole antiphospholipid antibodies in patients with thrombotic diseases (Staub, et al., 1989; Karmochkine, et al., 1992; Berard, et al., 1996). However, there are little studies on association of anti-phosphatidylethanolamine antibodies and arterial stiffness

We observed that cAI was significantly increased in RPL women with antiphosphatidylethanolamine antibodies. Anti-phosphatidylethanolamine antibodies may be a risk factor for atherosclerosis in women with RPL. It has been reported that kininogendependent IgG anti-phosphatidylethanolamine antibodies markedly increases thrombininduced platelet aggregation in vitro while kininogen independent IgG antiphosphatidylethanolamine antibodies do not augment thrombin-induced platelet aggregation (Sugi et al, 1999). However, we observed that both two types of anti-

Physical distribution of phosphatidylethanolamine is known to be at the blood-endothelium interface. The luminal phosphatidylethanolamine is a vulnerable to antiphosphatidylethanolamine autoimmunity, which is consistent with the association between anti- phosphatidylethanolamine antibodies and elevated risk for idiopathic thrombosis

Risk factors for atherosclerosis in SLE include traditional risk factors (mainly the Framingham risk factors). Moreover, specific antibodies to ,2GPI; anticardiolipin antibodies; anti-oxidized low-density lipoprotein (oxLDL); and antibodies to heat shock proteins may be cardiovascular disease risk factors (Sherer et al., 2010). Immune complexes containing oxLDL, ,2GPI, and/or CRP are known to be involved in atherosclerosis (Matsuura et al., 2006, Chekir et al, 2009). Autoantibodies to oxLDL/,2GPI complex were detected in SLE and APS patients, and were strongly associated with arterial thrombosis (Christodoulou et al., 2007). Further studies may help evaluating clinical usefullness of these

Previous study on arterial stiffness has shown that both baPWV and cAI are useful for risk stratification of hypertensive patients (Matsui et al., 2004). In this study, both of these indices are significantly correlated with age and systolic blood pressure and cAI is reported

We have reported that women with PCOS in reproductive age have a significantly higher baPWV than that for the control women (Sasaki et al., 2011). Arterial stiffness evaluated using the baPWV and cAI in mildly-hypertensive women (systolic blood pressure 120 mmHg or diastolic blood pressure 90 mmHg) with PCOS was significantly higher than that in the control women or normotensive women with PCOS. Early changes in vascular

We observed that women with unexplained RPL showed increased baPWV and cAI in average as compared with control women (Figure 5, 6). Although ranges of these indices in

autoimmune antibodies involving in progression of atherosclerosis in women RPL.

to be correlated with total cholesterol and LDL-C in hypertensive patients.

function were detected in mildly-hypertensive women with PCOS.

phosphatidylethanolamine antibodies were associated with arterial stiffness.

or atherosclerosis.

(Zhixin et al., 2011).

**4.4 Women with PCOS** 

**4.5 Women with unexplained RPL** 

women with unexplained RPL and those in control women were overlapped, at least a portion of women with unexplained RPL showed increased baPWV and cAI. These observations suggest that antiphospholipid antibodies or endocrinological disorders may not be the sole cause for arterial stiffness in women with RPL. Vascular dysfunction caused by various factors may be involved in at least a portion of women with unexplained RPL.

A portion of women with unexplained RPL should be considered as a high risk group for atherosclerosis and cardiovascular diseases. Measurement of baPWV or AI is a promising technique to assess vascular dysfunction in women with RPL.

#### **5. Possible cardiovascular diseases in pregnant women with RPL**

In pregnant women with RPL, who have early changes of systemic vasculatures, may suffer vascular complications during pregnancy because of physiological hypercoagulability and hemodynamic changes associated with pregnancy.

#### **5.1 Venous thromboembolism (VTE)**

Thrombophilia is a risk factor for venous thromboembolism (VTE) in pregnancy because of the hypercoagulability of pregnancy, which is further increased in the presence of thrombophilia. Vascular reactivity, which is believed to be increased during pregnancy, may also compound the risk.

Pregnant women are at increased risk of venous thromboembolism (VTE). Estimated incidence of VTE during pregnancy is about 1 event per 1000 pregnancies (Chauleur et al., 2007). Despite this low incidence, thromboembolic complications occurring during pregnancy and post-partum remain a major cause of maternal death. Although pregnant women have a higher risk of developing thromboembolic complications than non-pregnant women, treating all pregnant women to prevent these events is not recommended.

A scoring system for VTE risk in pregnant women (Chauleur et al., 2007) includes antiphospholipid antibodies and reproductive history of one stillbirth or at least three recurrent miscarriages, which are clinical features of women with APS (Bobba et al., 2007).

#### **5.2 Coronary heart disease and stroke in pregnancy**

The risk of acute myocardial infarction is known to be approximately 3 to 4 times higher in pregnancy. The incidence of pregnancy-related acute myocardial infarction is in the broad range of 3 to 10 per 100 000 deliveries that has been reported previously (James et al., 2006). Hypertension (odds ratio (OR) 21.7), thrombophilia including history of thrombosis and APS (OR 22.3), diabetes mellitus (OR 3.6), smoking (OR 8.4), transfusion (OR 5.1), postpartum infection (OR 3.2), and age 30 years and older remain as significant risk factors for pregnancy-related acute myocardial infarction in the multivariable analysis (James et al., 2006). The odds of acute myocardial infarction are 30-fold higher for women aged 40 years and older than for women 20 years of age.

Thrombophilia, gestational diabetes mellitus associated with PCOS, or age 30 years and older may increase the risk further in women with RPL.

Vascular Dysfunction in Women with Recurrent Pregnancy Loss 137

Lifestyle risk factors associated with child-rearing lead to obesity and result in increased coronary heart diseass in both sexes but biological responses of pregnancy may have

It is suggested that the association between childbirth and concurrent changes in IMT may be independent of traditional cardiovascular risk factors (Skilton et al., 2010). However, another study described no association between parity and either IMT or presence of plaques after adjustment for age in a population based cohort of 746 Finnish women (Kharazmi et al., 2007). The causality of the link between parity and early atherosclerosis is

Women who suffered one or more pregnancy losses have had pregnancies of shorter duration than usual and consequently they have received less/shorter estrogen exposure during pregnancy. The protective effect of estrogen, therefore, is potentially less than in the case of a full-term pregnancy (Kleijn & Schouw, 1999). However, it is more likely that cardiovascular disease risk in women with history of RPL may reflect common determinants, such as thrombophilic genetic defects, antiphospholipid antibodies, and

Women with subclinical cardiovascular disease could have a higher risk of pregnancy loss and cardiovascular events later in life. There have been various reports on association between pregnancy loss and coronary heart disease risk or pregnancy loss and stroke risk, which are inconclusive (de Kleijn & Schouw, 1999; La Vecchia et al., 1987; Smith et al, 2001). It has been reported that a history of any spontaneous loss of early pregnancy before the first live birth was associated with an increased risk of ischemic heart diseases (Smith et al., 2003). The association was independent of maternal age at the time of first birth, height, socioeconomic deprivation, essential hypertension, and complications during the first pregnancy. By contrast, there was no association between therapeutic abortion and subsequent risk of ischemic heart diseases. Women who had experienced at least one spontaneous or induced abortion had either increased or similar risk of coronary heart

There is a report describing that abortions, either spontaneous or induced, are not related to myocardial infarction risk, although underreporting cannot be excluded, because some women do not realize that early abortion may have occurred and because induced abortion

In women 50–74 years of age who had experienced pregnancy, history of pregnancy loss tended to be associated with a higher risk of myocardial infarction (age-adjusted OR 2.1), and the risk increased significantly with the number of pregnancy loss (age-adjusted OR 1.4) (Kharazmi et al., 2010). This result suggests that women who experience RPL are likely to be at an increased risk of vascular disease later in life. Spontaneous RPL (>3) is associated with about five times higher risk of myocardial infarction after full adjustment (Kharazmi et al.,

disease than women who had never had an abortion (Bertuccioa et al., 2007).

additional adverse effects in women (Lawlor et al., 2003).

not concluded.

**6.2 Pregnancy loss** 

endocrinological or metabolic disorders.

may not be reported (Bertuccioa et al., 2007).

**6.3 Recurrent pregnancy loss** 

There are few data on the risk of stroke in relation to the full range of outcomes of pregnancy (spontaneous or induced abortion, stillbirth, and live birth) (Kittner & Stern, 1996). One of the most important risks factors for stroke is advanced maternal age, which suggests arterial stiffness and atherosclerosis may affect on the incidence of stroke (Bushnell, 2008). The majority (48%) of pregnancy-related strokes occur in the postpartum period, versus 41% at delivery, and 11% antepartum (James et al., 2005). The Baltimore Washington Cooperative Young Stroke Study found that stroke rate was not increased in pregnant compared with nonpregnant women, but during the postpartum period, there was a fivefold increased risk of ischemic stroke (Kittner et al., 1996). Risk factors found to be associated with pregnancy-related stroke in the most recent analysis include thrombophilia (OR 16.0) and lupus (OR 15.2) (James et al., 2005). Preeclampsia and gestational hypertension, which are sometimes observed in women with RPL even during treatments, increase the risk of stroke during pregnancy as a result of severe hypertension and disturbed cerebral autoregulation.

#### **6. Reproductive history and cardiovascular disease risk later in life**

Adverse reproductive history and complications during pregnancy in women with RPL should to be considered as cardiovascular risk factors later in life. Physiological hypercoagulability, hemodynamic changes, and metabolic syndrome during pregnancy may provoke pregnancy complications including pregnancy loss, preeclampsia, placental abruption, preterm birth, or birth of an infant small for gestational age, or gestational diabetes mellitus in women with subclinical thrombophilia such as antiphospholipid antibodies or Factor V Leiden and/or subclinical endcrinological abnormalities such or PCOS. They could be considered a "failed stress test," possibly unmasking early or preexisting endothelial dysfunction and vascular or metabolic disease (Mosca, 2011).

#### **6.1 Parity**

Endothelial function is improved and asymmetrical dimethylarginine, an endogenous nitric oxide synthase inhibitor, decreased during pregnancy, both of which would be expected to slow the progression of atherosclerosis. In contrast, childbirth modified cardiovascular risk factors, most notably a redistribution of body fat to a phenotype characterized by increased abdominal adiposity and marked reductions in HDL-C and apoA-I.

There is an emerging body of literature examining the association between parity and cardiovascular disease. After adjustment for age, obesity, and family history of diabetes, increased parity was associated with a significantly increased risk of both non insulin dependent diabetes mellitus (NIDDM) (OR 1.16 per pregnancy) and impaired glucose tolerance (OR 1.10 per pregnancy) (Kritz-Silverstein et al., 1989).

Most of the studies on the association between parity and coronary heart disease have included only women. However, comparisons between men and women distinguish whether the mechanisms for the association between parity and atherosclerosis involve biological processes related to pregnancy or socioeconomic or lifestyle factors that are related to family size and child-rearing. It has been reported that increasing parity is associated with carotid atherosclerosis in women but not in men among a population with at least one risk factor for cardiovascular disease (Lawlor et al., 2003; Skilton et al., 2009; Skilton et al., 2010).

Lifestyle risk factors associated with child-rearing lead to obesity and result in increased coronary heart diseass in both sexes but biological responses of pregnancy may have additional adverse effects in women (Lawlor et al., 2003).

It is suggested that the association between childbirth and concurrent changes in IMT may be independent of traditional cardiovascular risk factors (Skilton et al., 2010). However, another study described no association between parity and either IMT or presence of plaques after adjustment for age in a population based cohort of 746 Finnish women (Kharazmi et al., 2007). The causality of the link between parity and early atherosclerosis is not concluded.

#### **6.2 Pregnancy loss**

136 Recent Advances in Cardiovascular Risk Factors

There are few data on the risk of stroke in relation to the full range of outcomes of pregnancy (spontaneous or induced abortion, stillbirth, and live birth) (Kittner & Stern, 1996). One of the most important risks factors for stroke is advanced maternal age, which suggests arterial stiffness and atherosclerosis may affect on the incidence of stroke (Bushnell, 2008). The majority (48%) of pregnancy-related strokes occur in the postpartum period, versus 41% at delivery, and 11% antepartum (James et al., 2005). The Baltimore Washington Cooperative Young Stroke Study found that stroke rate was not increased in pregnant compared with nonpregnant women, but during the postpartum period, there was a fivefold increased risk of ischemic stroke (Kittner et al., 1996). Risk factors found to be associated with pregnancy-related stroke in the most recent analysis include thrombophilia (OR 16.0) and lupus (OR 15.2) (James et al., 2005). Preeclampsia and gestational hypertension, which are sometimes observed in women with RPL even during treatments, increase the risk of stroke during pregnancy as a result of severe hypertension and

**6. Reproductive history and cardiovascular disease risk later in life** 

preexisting endothelial dysfunction and vascular or metabolic disease (Mosca, 2011).

abdominal adiposity and marked reductions in HDL-C and apoA-I.

tolerance (OR 1.10 per pregnancy) (Kritz-Silverstein et al., 1989).

Endothelial function is improved and asymmetrical dimethylarginine, an endogenous nitric oxide synthase inhibitor, decreased during pregnancy, both of which would be expected to slow the progression of atherosclerosis. In contrast, childbirth modified cardiovascular risk factors, most notably a redistribution of body fat to a phenotype characterized by increased

There is an emerging body of literature examining the association between parity and cardiovascular disease. After adjustment for age, obesity, and family history of diabetes, increased parity was associated with a significantly increased risk of both non insulin dependent diabetes mellitus (NIDDM) (OR 1.16 per pregnancy) and impaired glucose

Most of the studies on the association between parity and coronary heart disease have included only women. However, comparisons between men and women distinguish whether the mechanisms for the association between parity and atherosclerosis involve biological processes related to pregnancy or socioeconomic or lifestyle factors that are related to family size and child-rearing. It has been reported that increasing parity is associated with carotid atherosclerosis in women but not in men among a population with at least one risk factor for

cardiovascular disease (Lawlor et al., 2003; Skilton et al., 2009; Skilton et al., 2010).

Adverse reproductive history and complications during pregnancy in women with RPL should to be considered as cardiovascular risk factors later in life. Physiological hypercoagulability, hemodynamic changes, and metabolic syndrome during pregnancy may provoke pregnancy complications including pregnancy loss, preeclampsia, placental abruption, preterm birth, or birth of an infant small for gestational age, or gestational diabetes mellitus in women with subclinical thrombophilia such as antiphospholipid antibodies or Factor V Leiden and/or subclinical endcrinological abnormalities such or PCOS. They could be considered a "failed stress test," possibly unmasking early or

disturbed cerebral autoregulation.

**6.1 Parity** 

Women who suffered one or more pregnancy losses have had pregnancies of shorter duration than usual and consequently they have received less/shorter estrogen exposure during pregnancy. The protective effect of estrogen, therefore, is potentially less than in the case of a full-term pregnancy (Kleijn & Schouw, 1999). However, it is more likely that cardiovascular disease risk in women with history of RPL may reflect common determinants, such as thrombophilic genetic defects, antiphospholipid antibodies, and endocrinological or metabolic disorders.

Women with subclinical cardiovascular disease could have a higher risk of pregnancy loss and cardiovascular events later in life. There have been various reports on association between pregnancy loss and coronary heart disease risk or pregnancy loss and stroke risk, which are inconclusive (de Kleijn & Schouw, 1999; La Vecchia et al., 1987; Smith et al, 2001).

It has been reported that a history of any spontaneous loss of early pregnancy before the first live birth was associated with an increased risk of ischemic heart diseases (Smith et al., 2003). The association was independent of maternal age at the time of first birth, height, socioeconomic deprivation, essential hypertension, and complications during the first pregnancy. By contrast, there was no association between therapeutic abortion and subsequent risk of ischemic heart diseases. Women who had experienced at least one spontaneous or induced abortion had either increased or similar risk of coronary heart disease than women who had never had an abortion (Bertuccioa et al., 2007).

There is a report describing that abortions, either spontaneous or induced, are not related to myocardial infarction risk, although underreporting cannot be excluded, because some women do not realize that early abortion may have occurred and because induced abortion may not be reported (Bertuccioa et al., 2007).

#### **6.3 Recurrent pregnancy loss**

In women 50–74 years of age who had experienced pregnancy, history of pregnancy loss tended to be associated with a higher risk of myocardial infarction (age-adjusted OR 2.1), and the risk increased significantly with the number of pregnancy loss (age-adjusted OR 1.4) (Kharazmi et al., 2010). This result suggests that women who experience RPL are likely to be at an increased risk of vascular disease later in life. Spontaneous RPL (>3) is associated with about five times higher risk of myocardial infarction after full adjustment (Kharazmi et al.,

Vascular Dysfunction in Women with Recurrent Pregnancy Loss 139

Preeclampsia affects about 5–8% of all first pregnancies and is a major cause of maternal and fetal morbidity and mortality worldwide. Preeclampsia is associated with vascular dysfunction manifesting hypertension (Nakatsuka et al., 2002) and one of common complications observed in women with RPL even during treatment. We have reported that uterine, orbital, and brachial circulations are impaired in women with preeclampsia (Takata et al., 2002). Several studies focused on an attenuated vasodilatory response in large blood vessels by evaluating flow-mediated dilatation or venous occlusion plethysmography (Spaana et al., 2010). One study evaluated microvascular function several years after preeclampsia, observing a lower response to both endothelium-dependent and independent vasodilatation using laser Doppler imaging of the forearm 20 years after preeclampsia (Ramsay et al. 2003). One of the most common risk factors for stroke in pregnancy, particularly postpartum, is preeclampsia/eclampsia (Bushnell & Chireau, 2011). A history of preeclampsia during pregnancy lead to an increased risk of stroke later in life (Bellamy et al, 2007). Biomarkers of endothelial dysfunction such as intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) are known to be elevated in women preeclampsia. Women with a history of preeclampsia are more likely to have higher insulin levels

Although the symptoms of preeclampsia typically regress within a few days post partum, impaired vascular dilatation is still present several years after preeclampsia, suggesting persistent endothelial dysfunction, which may contribute to the development of cardiovascular disease in these women (Spaana et al., 2010). A recent large meta-analysis found that women with a history of preeclampsia have approximately double the risk for subsequent ischemic heart disease, stroke, and venous thromboembolic events over the 5 to 15 years after pregnancy (Moska, 2011). Preeclampsia, particularly in association with preterm delivery, has been identified as a risk factor for myocardial infarction and mortality

As described in the meta-analysis and other longitudinal studies, hypertension is the risk factor for cerebrovascular disease that women with a history of preeclampsia and

Other factors, which are prevalent among women with RPL and may make special contributions to cardiovascular disease risk, include anxiety, depression, and other

Anxiety, which is often observed in women with RPL, is also suggested to be an independent predictor of adverse cardiovascular events (Olafiranye et al., 2011). Individuals with high levels of anxiety are at increased risk of coronary heart disease, congestive heart failure, stroke, fatal ventricular arrhythmias, and sudden cardiac death. Anxiety following a major cardiac event can impede recovery, and is associated with a higher morbidity and

Risk for an episode of major depressive disorder among miscarrying women in the 6 months following loss is compared with the 6-month risk among community women who

from cardiovascular disease later in life (James et al., 2006).

psychosocial risk factors (Blackmore et al., 2011).

gestational hypertension are most likely to develop (Bellamy et al, 2007).

**7.1 Preeclampsia** 

compared to controls.

**7.2 Anxiety and depression** 

mortality.

2011). Women who experience spontaneous pregnancy loss are at a substantially higher risk of myocardial infarction later in life. Although women who had history of RPL (>3) tended to have a higher risk of stroke (adjusted OR 1.43), associations between RPL and cerebrovascular events including stroke are also inconclusive.

Several studies have shown associations between acquired and inherited thrombophilias and both spontaneous loss of early pregnancy and ischemic heart disease (Smith et al., 2003). High homocysteine levels in early pregnancy are another risk factor for pregnancy loss and preecclampsia (Dodds, et al., 2008). Elevated levels of homocysteine in the bloodstream can irritate the blood vessels, which may eventually lead to hardening of the arteries, stroke or heart attack.

Miscarriage can sometimes lead to infections which may also have some links with cardiovascular diseases (Kharazmi et al., 2011). For instance, chlamydia infection has been found to be associated with occurrence of miscarriage and also with atherogenesis. Inflammation and infection as known risk factors for cardiovascular diseases might be the underlying mechanisms that explain the association between miscarriage and cardiovascular disease.

It is possible that the cause of pregnancy loss is related to hemodynamic factors, such as preeclampsia, and therefore to cardiovascular risk or disease (de Kleijn & Schouw, 1999). This means that the causal relationship could be reversed: women with a cardiovascular disease risk could have a higher risk of pregnancy loss. Occult cardiovascular, microvascular, or haemostatic dysfunction result in pregnancy complications during reproductive years and in overt cardiovascular disease later in life (Smith et al., 2003).

#### **6.4 Still birth**

Stillbirth is known to be associated with an increased risk of death from coronary heart disease, all circulatory and renal causes (Calderon-Margalit et al., 2007). A history of stillbirth is reported to be associated with an increased age-adjusted risk of plaque (OR 3.43), but it lost its statistical significance in the fully adjusted model (Kharazmi et al., 2007). Recent study have reported that each stillbirth increased the risk of myocardial infarction 2.32 times after adjustment for age, smoking, alcohol consumption, body mass index, waist to hip ratio, physical activity, education, number of pregnancies, hypertension, hyperlipidaemia and diabetes mellitus (Kharazmi et al., 2011). Stillbirth is a strong sexspecific predictor for myocardial infarction and thus should be considered as important indicators for cardiovascular risk factors monitoring and preventive measures (Kharazmi et al., 2011).

#### **7. Complications in women with RPL and cardiovascular diseases**

Pregnancy complications such as preeclampsia, placental abruption, preterm birth, or birth of an infant small for gestational age, or gestational diabetes mellitus are characteristic in women with RPL. Women with RPL are also at increased risk for recurrent episode of major depressive disorder. These complications may be associated with vascular dysfunction in women with RPL.

#### **7.1 Preeclampsia**

138 Recent Advances in Cardiovascular Risk Factors

2011). Women who experience spontaneous pregnancy loss are at a substantially higher risk of myocardial infarction later in life. Although women who had history of RPL (>3) tended to have a higher risk of stroke (adjusted OR 1.43), associations between RPL and

Several studies have shown associations between acquired and inherited thrombophilias and both spontaneous loss of early pregnancy and ischemic heart disease (Smith et al., 2003). High homocysteine levels in early pregnancy are another risk factor for pregnancy loss and preecclampsia (Dodds, et al., 2008). Elevated levels of homocysteine in the bloodstream can irritate the blood vessels, which may eventually lead to hardening of the arteries, stroke or

Miscarriage can sometimes lead to infections which may also have some links with cardiovascular diseases (Kharazmi et al., 2011). For instance, chlamydia infection has been found to be associated with occurrence of miscarriage and also with atherogenesis. Inflammation and infection as known risk factors for cardiovascular diseases might be the underlying mechanisms that explain the association between miscarriage and

It is possible that the cause of pregnancy loss is related to hemodynamic factors, such as preeclampsia, and therefore to cardiovascular risk or disease (de Kleijn & Schouw, 1999). This means that the causal relationship could be reversed: women with a cardiovascular disease risk could have a higher risk of pregnancy loss. Occult cardiovascular, microvascular, or haemostatic dysfunction result in pregnancy complications during reproductive years and in overt cardiovascular disease later in life (Smith et al., 2003).

Stillbirth is known to be associated with an increased risk of death from coronary heart disease, all circulatory and renal causes (Calderon-Margalit et al., 2007). A history of stillbirth is reported to be associated with an increased age-adjusted risk of plaque (OR 3.43), but it lost its statistical significance in the fully adjusted model (Kharazmi et al., 2007). Recent study have reported that each stillbirth increased the risk of myocardial infarction 2.32 times after adjustment for age, smoking, alcohol consumption, body mass index, waist to hip ratio, physical activity, education, number of pregnancies, hypertension, hyperlipidaemia and diabetes mellitus (Kharazmi et al., 2011). Stillbirth is a strong sexspecific predictor for myocardial infarction and thus should be considered as important indicators for cardiovascular risk factors monitoring and preventive measures (Kharazmi et

**7. Complications in women with RPL and cardiovascular diseases** 

Pregnancy complications such as preeclampsia, placental abruption, preterm birth, or birth of an infant small for gestational age, or gestational diabetes mellitus are characteristic in women with RPL. Women with RPL are also at increased risk for recurrent episode of major depressive disorder. These complications may be associated with vascular dysfunction in

cerebrovascular events including stroke are also inconclusive.

heart attack.

**6.4 Still birth** 

al., 2011).

women with RPL.

cardiovascular disease.

Preeclampsia affects about 5–8% of all first pregnancies and is a major cause of maternal and fetal morbidity and mortality worldwide. Preeclampsia is associated with vascular dysfunction manifesting hypertension (Nakatsuka et al., 2002) and one of common complications observed in women with RPL even during treatment. We have reported that uterine, orbital, and brachial circulations are impaired in women with preeclampsia (Takata et al., 2002). Several studies focused on an attenuated vasodilatory response in large blood vessels by evaluating flow-mediated dilatation or venous occlusion plethysmography (Spaana et al., 2010). One study evaluated microvascular function several years after preeclampsia, observing a lower response to both endothelium-dependent and independent vasodilatation using laser Doppler imaging of the forearm 20 years after preeclampsia (Ramsay et al. 2003).

One of the most common risk factors for stroke in pregnancy, particularly postpartum, is preeclampsia/eclampsia (Bushnell & Chireau, 2011). A history of preeclampsia during pregnancy lead to an increased risk of stroke later in life (Bellamy et al, 2007). Biomarkers of endothelial dysfunction such as intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) are known to be elevated in women preeclampsia. Women with a history of preeclampsia are more likely to have higher insulin levels compared to controls.

Although the symptoms of preeclampsia typically regress within a few days post partum, impaired vascular dilatation is still present several years after preeclampsia, suggesting persistent endothelial dysfunction, which may contribute to the development of cardiovascular disease in these women (Spaana et al., 2010). A recent large meta-analysis found that women with a history of preeclampsia have approximately double the risk for subsequent ischemic heart disease, stroke, and venous thromboembolic events over the 5 to 15 years after pregnancy (Moska, 2011). Preeclampsia, particularly in association with preterm delivery, has been identified as a risk factor for myocardial infarction and mortality from cardiovascular disease later in life (James et al., 2006).

As described in the meta-analysis and other longitudinal studies, hypertension is the risk factor for cerebrovascular disease that women with a history of preeclampsia and gestational hypertension are most likely to develop (Bellamy et al, 2007).

#### **7.2 Anxiety and depression**

Other factors, which are prevalent among women with RPL and may make special contributions to cardiovascular disease risk, include anxiety, depression, and other psychosocial risk factors (Blackmore et al., 2011).

Anxiety, which is often observed in women with RPL, is also suggested to be an independent predictor of adverse cardiovascular events (Olafiranye et al., 2011). Individuals with high levels of anxiety are at increased risk of coronary heart disease, congestive heart failure, stroke, fatal ventricular arrhythmias, and sudden cardiac death. Anxiety following a major cardiac event can impede recovery, and is associated with a higher morbidity and mortality.

Risk for an episode of major depressive disorder among miscarrying women in the 6 months following loss is compared with the 6-month risk among community women who

Vascular Dysfunction in Women with Recurrent Pregnancy Loss 141

Antiphospholipid antibodies in women RPL may pass from mother to child via the placenta, can alter the placenta itself and may be a risk for PAS. The inherited thrombophilias both in mothers and fetuses may cause RPL and PAS in neonates (Nelson, 2007). It seems likely that maternal and perhaps infant thrombophilias can lead to complications of pregnancy, such as RPL, preeclampsia, placental abruption, placental vasculopathy, and fetal growth restriction, which are in turn risk factors for neonatal encephalopathy, stroke, or cerebral

Complications of pregnancy such as preeclampsia, which are observed in untreated and treated women with PRL, link to low birth weight. Previous studies have shown an association between an individual's birth weight and his or her subsequent risk of ischemic heart disease, hypertension, and diabetes mellitus (Hübinettea et al., 2001). Barker and colleagues have postulated that fetal adaptation to inadequate intrauterine nutrition, due to poor maternal diet or placental dysfunction, results in physiological programming of a "thrifty phenotype", which increases the risk of hypertension and ischemic heart disease in

An alternative hypothesis is that common genetic factors predispose to fetal growth restriction, preterm birth, and ischemic heart diseases. Common genetic risk factors might explain the link between birthweight and risk of ischemic heart disease in both the mother and the child (Smith et al., 2001). A genetic link would be consistent with the much stronger association between birthweight and ischemic heart disease in the mother (11-fold) (Smith et al., 2001) than in the offspring (1.5–2.0-fold) (Barker et al., 1989). Maternal genes might modulate fetal growth both by affecting the intrauterine environment, for instance by effects on uterine blood flow, and by inheritance of genes from the mother that regulate fetal

Epidemiological studies also provide evidence for common genetic links. Fathers of lowbirthweight babies are at increased risk of coronary heart disease, hypertension, and diabetes (Smith et al., 2001). Children and their mothers, who experienced RPL, may have

**9. Treatment of pregnancy loss and prevention of cardiovascular diseases**  Currently, low-dose aspirin treatment is used as an effective therapy for women with RPL associated with anti-phospholipid antibodies (Coulam et al., 1997). It has been reported that low-dose aspirin is effective in improving implantation and pregnancy rates in the IVF programme (Rubinstein et al., 1999). Two trials demonstrated that women without hereditary thrombophilia and at least three unexplained consecutive losses randomized to prophylactic low molecular weight heparin had higher live birth rates than those assigned

Antiphospholipid antibodies are more common in patients with thrombosis but a causal association is unproven and the clinical relevance of transient or low titer antiphospholipid antibodies remains uncertain (Lim et al., 2006). In patients with APS, moderate-intensity warfarin is effective for preventing recurrent venous thrombosis and perhaps also arterial thrombosis. Aspirin appears to be as effective as moderate-intensity warfarin for preventing

common genetic factors linked to cardiovascular diseases.

to placebo or no treatment (Bates, 2010).

palsy (Nelson, 2007).

**8.2 Genetic risk factors** 

later life (Barker et al., 1989).

growth directly.

have not been pregnant in the preceding year (Neugebauer et al., 1997). Among miscarrying women, 10.9% experience an episode of major depressive disorder, compared with 4.3% of community women. The overall relative risk (RR) for an episode of major depressive disorder for miscarrying women is 2.5 and is substantially higher for childless women (RR 5.0) than for women with children (RR 1.3).

Midlife women are particularly vulnerable to depressive mood; the changing hormonal milieu during the menopausal transition contributes to increased prevalence of depressive symptoms and to the worsening of the cardiovascular disease profile (Janssen et al., 2011). Among miscarrying women with a history of major depressive disorder, 54% experience a recurrence later in life.

Symptoms of depression and major depressive disorder have been identified as potential risk factors for coronary heart disease (Janssen et al., 2011). Longitudinal studies have consistently shown that persons with high levels of depressive symptoms, or with a history of major depressive disorders, are more likely to have clinical coronary events than persons without depression. Major depression and depressive symptoms are associated with cardiovascular disease, but the impact of depression on early atherogenesis is less well known (Janssen et al., 2011). Anxiety and/or depression may be a risk factor for cardiovascular diseases in women with RPL.

#### **8. Vascular dysfunction in children of women with PRL**

Previous studies suggested that the atherogenic process in humans has already started during fetal development (Napoli et al, 1997). Intrauterine exposure to maternal atherosclerotic risk factors may increases the susceptibility to atherosclerosis in adult life (Alkemade et al., 2007). In a morphometric postmortem analysis of atherosclerosis in fetuses and children (Fate of Early Lesions in Children Study), it is demonstrated that specifically maternal hypercholesterolemia is associated with a higher incidence of atherosclerotic lesions during the fetal period and a faster progression of these atherosclerotic lesions after birth even under conditions of normocholesterolemia in the offspring (Napoli et al, 1999).

#### **8.1 Perinatal arterial ischemic stroke (PAS)**

Perinatal arterial ischemic stroke (PAS), defined as a thromboembolic event occurring before age 28 days, is an increasingly recognized cause of neurological disabilities such as cerebral palsy, epilepsy, and cognitive abnormalities (Lee et al., 2005). PAS occurs at a frequency of 1/1600 to 1/5000 live births (Chabriera et al., 2010). Previous fetal loss, first pregnancy, primiparity, twin-gestation, cesarean and traumatic delivery, neonatal distress, male sex and premature rupture of membranes in PAS were statistically more common than in the general population (Chabriera et al., 2010).

PAS may result from thrombosis of intracranial vessels or from embolism from another site such as extracranial vessels, heart, umbilical vein, or placenta (Nelson, 2007). Although the site of origin is usually not clearly established, it is suspected that the fetal side of the placenta may often be the source. In addition to cerebral infarction, thrombosis in other sites, including kidney, heart, aorta, and limb arteries, is more common in neonates than at other times in childhood.

Antiphospholipid antibodies in women RPL may pass from mother to child via the placenta, can alter the placenta itself and may be a risk for PAS. The inherited thrombophilias both in mothers and fetuses may cause RPL and PAS in neonates (Nelson, 2007). It seems likely that maternal and perhaps infant thrombophilias can lead to complications of pregnancy, such as RPL, preeclampsia, placental abruption, placental vasculopathy, and fetal growth restriction, which are in turn risk factors for neonatal encephalopathy, stroke, or cerebral palsy (Nelson, 2007).

#### **8.2 Genetic risk factors**

140 Recent Advances in Cardiovascular Risk Factors

have not been pregnant in the preceding year (Neugebauer et al., 1997). Among miscarrying women, 10.9% experience an episode of major depressive disorder, compared with 4.3% of community women. The overall relative risk (RR) for an episode of major depressive disorder for miscarrying women is 2.5 and is substantially higher for childless women (RR

Midlife women are particularly vulnerable to depressive mood; the changing hormonal milieu during the menopausal transition contributes to increased prevalence of depressive symptoms and to the worsening of the cardiovascular disease profile (Janssen et al., 2011). Among miscarrying women with a history of major depressive disorder, 54% experience a

Symptoms of depression and major depressive disorder have been identified as potential risk factors for coronary heart disease (Janssen et al., 2011). Longitudinal studies have consistently shown that persons with high levels of depressive symptoms, or with a history of major depressive disorders, are more likely to have clinical coronary events than persons without depression. Major depression and depressive symptoms are associated with cardiovascular disease, but the impact of depression on early atherogenesis is less well known (Janssen et al., 2011). Anxiety and/or depression may be a risk factor for

Previous studies suggested that the atherogenic process in humans has already started during fetal development (Napoli et al, 1997). Intrauterine exposure to maternal atherosclerotic risk factors may increases the susceptibility to atherosclerosis in adult life (Alkemade et al., 2007). In a morphometric postmortem analysis of atherosclerosis in fetuses and children (Fate of Early Lesions in Children Study), it is demonstrated that specifically maternal hypercholesterolemia is associated with a higher incidence of atherosclerotic lesions during the fetal period and a faster progression of these atherosclerotic lesions after birth even under conditions of normocholesterolemia in the offspring (Napoli et al, 1999).

Perinatal arterial ischemic stroke (PAS), defined as a thromboembolic event occurring before age 28 days, is an increasingly recognized cause of neurological disabilities such as cerebral palsy, epilepsy, and cognitive abnormalities (Lee et al., 2005). PAS occurs at a frequency of 1/1600 to 1/5000 live births (Chabriera et al., 2010). Previous fetal loss, first pregnancy, primiparity, twin-gestation, cesarean and traumatic delivery, neonatal distress, male sex and premature rupture of membranes in PAS were statistically more common than in the

PAS may result from thrombosis of intracranial vessels or from embolism from another site such as extracranial vessels, heart, umbilical vein, or placenta (Nelson, 2007). Although the site of origin is usually not clearly established, it is suspected that the fetal side of the placenta may often be the source. In addition to cerebral infarction, thrombosis in other sites, including kidney, heart, aorta, and limb arteries, is more common in neonates than at

5.0) than for women with children (RR 1.3).

cardiovascular diseases in women with RPL.

**8.1 Perinatal arterial ischemic stroke (PAS)** 

general population (Chabriera et al., 2010).

other times in childhood.

**8. Vascular dysfunction in children of women with PRL** 

recurrence later in life.

Complications of pregnancy such as preeclampsia, which are observed in untreated and treated women with PRL, link to low birth weight. Previous studies have shown an association between an individual's birth weight and his or her subsequent risk of ischemic heart disease, hypertension, and diabetes mellitus (Hübinettea et al., 2001). Barker and colleagues have postulated that fetal adaptation to inadequate intrauterine nutrition, due to poor maternal diet or placental dysfunction, results in physiological programming of a "thrifty phenotype", which increases the risk of hypertension and ischemic heart disease in later life (Barker et al., 1989).

An alternative hypothesis is that common genetic factors predispose to fetal growth restriction, preterm birth, and ischemic heart diseases. Common genetic risk factors might explain the link between birthweight and risk of ischemic heart disease in both the mother and the child (Smith et al., 2001). A genetic link would be consistent with the much stronger association between birthweight and ischemic heart disease in the mother (11-fold) (Smith et al., 2001) than in the offspring (1.5–2.0-fold) (Barker et al., 1989). Maternal genes might modulate fetal growth both by affecting the intrauterine environment, for instance by effects on uterine blood flow, and by inheritance of genes from the mother that regulate fetal growth directly.

Epidemiological studies also provide evidence for common genetic links. Fathers of lowbirthweight babies are at increased risk of coronary heart disease, hypertension, and diabetes (Smith et al., 2001). Children and their mothers, who experienced RPL, may have common genetic factors linked to cardiovascular diseases.

#### **9. Treatment of pregnancy loss and prevention of cardiovascular diseases**

Currently, low-dose aspirin treatment is used as an effective therapy for women with RPL associated with anti-phospholipid antibodies (Coulam et al., 1997). It has been reported that low-dose aspirin is effective in improving implantation and pregnancy rates in the IVF programme (Rubinstein et al., 1999). Two trials demonstrated that women without hereditary thrombophilia and at least three unexplained consecutive losses randomized to prophylactic low molecular weight heparin had higher live birth rates than those assigned to placebo or no treatment (Bates, 2010).

Antiphospholipid antibodies are more common in patients with thrombosis but a causal association is unproven and the clinical relevance of transient or low titer antiphospholipid antibodies remains uncertain (Lim et al., 2006). In patients with APS, moderate-intensity warfarin is effective for preventing recurrent venous thrombosis and perhaps also arterial thrombosis. Aspirin appears to be as effective as moderate-intensity warfarin for preventing

Vascular Dysfunction in Women with Recurrent Pregnancy Loss 143

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The relationship between preeclampsia and stroke involves shared risk factors for both disorders, including chronic endothelial dysfunction and increased risk for long-term hypertension following preeclampsia, one of the major risk factors for stroke (Bushnell & Chireau, 2010). Thrombophilic conditions and Vitamin D deficiency (Grant, 2009) has emerged as an important potentially modifiable risk factor for both preeclampsia and stroke. These overlaps provide insights into underlying pathophysiology and potential preventive strategies for both preeclampsia and stroke. For example, aspirin or Vitamin D may prevent both disorders.

Early changes in vascular function are detected in mildly-hypertensive women with PCOS (Sasaki et al., 2011). All women diagnosed with PCOS are not likely to share the same cardiovascular risk profiles and increased mortality and morbidity rates from cardiovascular disease in the PCOS population. However, lifestyle intervention such as diet and exercise should be the first-line of treatment in women with PCOS, particularly if they are hypertensive or overweight.

It is unlikely to treat all young women with aspirin following the occurrence of PRL without any other risk factors. Pharmacological therapies for hypertension, insulin resistance, or dyslipidemia are also available but should be tailored on an individual basis. It is important to evaluate arterial stiffness to identify women at early risk of cardiovascular disease and stroke and ultimately assess the risks and benefits of various prevention approaches.

#### **10. Conclusion**

Women who experienced RPL are at a substantially higher risk of vascular dysfunction, which leads to coronary heart disease, stroke, or VTE later in life. Reproductive history of obstetrical complications associated with RPL such as preeclampsia, premature delivery, fetal growth restriction, placental abruption, and gestational diabetes mellitus could be considered a "failed stress test," possibly unmasking early or preexisting vascular dysfunction and vascular or metabolic diseases. Therefore, these women should be carefully monitored and controlled. Healthcare professionals who meet women for the first time later in their lives should take a careful and detailed history of RPL and characteristic pregnancy complications.

Further studies will provide more information on association between PRL and vascular dysfunction, effective treatment for both women with PRL and their fetuses.

#### **11. References**

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The relationship between preeclampsia and stroke involves shared risk factors for both disorders, including chronic endothelial dysfunction and increased risk for long-term hypertension following preeclampsia, one of the major risk factors for stroke (Bushnell & Chireau, 2010). Thrombophilic conditions and Vitamin D deficiency (Grant, 2009) has emerged as an important potentially modifiable risk factor for both preeclampsia and stroke. These overlaps provide insights into underlying pathophysiology and potential preventive strategies for both preeclampsia and stroke. For example, aspirin or Vitamin D

Early changes in vascular function are detected in mildly-hypertensive women with PCOS (Sasaki et al., 2011). All women diagnosed with PCOS are not likely to share the same cardiovascular risk profiles and increased mortality and morbidity rates from cardiovascular disease in the PCOS population. However, lifestyle intervention such as diet and exercise should be the first-line of treatment in women with PCOS, particularly if they

It is unlikely to treat all young women with aspirin following the occurrence of PRL without any other risk factors. Pharmacological therapies for hypertension, insulin resistance, or dyslipidemia are also available but should be tailored on an individual basis. It is important to evaluate arterial stiffness to identify women at early risk of cardiovascular disease and

Women who experienced RPL are at a substantially higher risk of vascular dysfunction, which leads to coronary heart disease, stroke, or VTE later in life. Reproductive history of obstetrical complications associated with RPL such as preeclampsia, premature delivery, fetal growth restriction, placental abruption, and gestational diabetes mellitus could be considered a "failed stress test," possibly unmasking early or preexisting vascular dysfunction and vascular or metabolic diseases. Therefore, these women should be carefully monitored and controlled. Healthcare professionals who meet women for the first time later in their lives should take a careful and detailed history of RPL and characteristic pregnancy

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**10. Conclusion** 

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**9** 

*Romania* 

**The Polycystic Ovary Syndrome Status –** 

*"Iuliu Hatieganu" University of Medicine and Pharmacy Cluj-Napoca,* 

Teodora Mocan, Steliana Ghibu and Cornel Iancu

**A Risk Factor for Future Cardiovascular Disease** 

Ioana Ilie, Razvan Ilie, Lucian Mocan, Carmen Georgescu, Ileana Duncea,

Polycystic ovary syndrome (PCOS) is a common endocrine disorder in women of reproductive age with a prevalence estimated at 4–8% (Azziz et al., 2004; Moran & Teede, 2009). It is associated with a range of reproductive, obstetric, metabolic and psychological features. Reproductive and obstetric manifestations include hyperandrogenism, menstrual dysfunction, infertility and pregnancy complications, such as early pregnancy loss, gestational diabetes, pregnancy-induced hypertensive disorders and neonatal complications (Boomsma et al., 2006). Additionally, women with PCOS cluster risk factors for cardiovascular disease (CVD) and type 2 diabetes mellitus (DM) as well as the metabolic syndrome (MS). As a consequence, metabolic complications and potential major long-term sequelaes include: an elevated risk of impaired glucose tolerance (IGT), type 2 DM (Legro, 1999, as cited in Moran & Teede, 2009), as well as an increased rate of hypertension and CVD (Shaw et al., 2008). Although there are still aspects to be cleared regarding the longterm consequences of these well-known cardiovascular (CV) risk factors, the research that has been conducted so far seems to indicate that patients with PCOS are at increased risk for adverse CV morbidity and mortality. Of utmost importance is the fact that these women are likely to develop CV disease early, and that even very young and nonobese patients may be

However, there is a wide variety of diagnostic criteria generating several reproductive diagnostic phenotypes and the best diagnostic criteria for PCOS as well as the metabolic implications of newer non-National Institute of Health (NIH) PCOS phenotypes are still under intense debate. Although the prevalence of the MS is likely to vary according to PCOS and MS definition and ethnicity, its occurrence is estimated to be substantially higher in women with PCOS compared with the general population. Endothelial dysfunction and increased arterial stiffness are early markers of atherosclerosis, which has been recognized as a chronic inflammatory state. Increased carotid intima-media thickness (IMT) has been reported to occur relatively early in the atherosclerotic process and to represent a powerful predictor of coronary and cerebrovascular events. These alterations were first found in middle-aged women with PCOS and then demonstrated also in young ones. Moreover, coronary artery calcium, a radiographic marker for atherosclerosis, correlates with the extent of coronary atherosclerotic plaque. Up to the present, the results of the literature have

**1. Introduction** 

affected (Moran & Teede, 2009; Lorenz & Wild, 2007).


### **The Polycystic Ovary Syndrome Status – A Risk Factor for Future Cardiovascular Disease**

Ioana Ilie, Razvan Ilie, Lucian Mocan, Carmen Georgescu, Ileana Duncea, Teodora Mocan, Steliana Ghibu and Cornel Iancu *"Iuliu Hatieganu" University of Medicine and Pharmacy Cluj-Napoca, Romania* 

#### **1. Introduction**

150 Recent Advances in Cardiovascular Risk Factors

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antiphosphatidylethanolamine antibodies in patients with recurrent early

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predicting pregnancy outcome in women with antiphospholipid syndrome. Obstet

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abortion following assisted reproductive technology treatment. Hum Reprod

Pulse wave velocity in lower-limb arteries among diabetic patients with peripheral

in women with polycystic ovary syndrome who respond to clomiphene citrate: correlation with serum hormone concentrations. Ultrasound Obstet Gynecol.

Phosphatidylethanolamine at the luminal endothelial surface--implications for hemostasis and thrombotic autoimmunity. Clin Appl Thromb Hemost 17: 158-163, Polycystic ovary syndrome (PCOS) is a common endocrine disorder in women of reproductive age with a prevalence estimated at 4–8% (Azziz et al., 2004; Moran & Teede, 2009). It is associated with a range of reproductive, obstetric, metabolic and psychological features. Reproductive and obstetric manifestations include hyperandrogenism, menstrual dysfunction, infertility and pregnancy complications, such as early pregnancy loss, gestational diabetes, pregnancy-induced hypertensive disorders and neonatal complications (Boomsma et al., 2006). Additionally, women with PCOS cluster risk factors for cardiovascular disease (CVD) and type 2 diabetes mellitus (DM) as well as the metabolic syndrome (MS). As a consequence, metabolic complications and potential major long-term sequelaes include: an elevated risk of impaired glucose tolerance (IGT), type 2 DM (Legro, 1999, as cited in Moran & Teede, 2009), as well as an increased rate of hypertension and CVD (Shaw et al., 2008). Although there are still aspects to be cleared regarding the longterm consequences of these well-known cardiovascular (CV) risk factors, the research that has been conducted so far seems to indicate that patients with PCOS are at increased risk for adverse CV morbidity and mortality. Of utmost importance is the fact that these women are likely to develop CV disease early, and that even very young and nonobese patients may be affected (Moran & Teede, 2009; Lorenz & Wild, 2007).

However, there is a wide variety of diagnostic criteria generating several reproductive diagnostic phenotypes and the best diagnostic criteria for PCOS as well as the metabolic implications of newer non-National Institute of Health (NIH) PCOS phenotypes are still under intense debate. Although the prevalence of the MS is likely to vary according to PCOS and MS definition and ethnicity, its occurrence is estimated to be substantially higher in women with PCOS compared with the general population. Endothelial dysfunction and increased arterial stiffness are early markers of atherosclerosis, which has been recognized as a chronic inflammatory state. Increased carotid intima-media thickness (IMT) has been reported to occur relatively early in the atherosclerotic process and to represent a powerful predictor of coronary and cerebrovascular events. These alterations were first found in middle-aged women with PCOS and then demonstrated also in young ones. Moreover, coronary artery calcium, a radiographic marker for atherosclerosis, correlates with the extent of coronary atherosclerotic plaque. Up to the present, the results of the literature have

The Polycystic Ovary Syndrome Status – A Risk Factor for Future Cardiovascular Disease 153

heterogeneity into PCOS from a reproductive and possibly from a metabolic perspective. In fact, according to these guidelines, the diagnosis of PCOS may present in patients with four different phenotypes: [1] hyperandrogenism, chronic anovulation, and PCO; [2] hyperandrogenism and chronic anovulation but normal ovaries; [3] hyperandrogenism and PCO but ovulatory cycles; and [4] chronic anovulation and PCO but no clinical or biochemical hyperandrogenism (Guastella et al., 2010). In 2006, the AES published a position statement which suggested that androgen excess is the key component of PCOS related to clinical presentation and long-term morbidity. The proposal of the AES was, therefore, to include in the diagnosis of PCOS only the first three phenotypes, excluding the phenotype of PCO and irregular cycles without hyperandrogenism (Azziz et al., 2006, 2009;

For simplification, PCOS can be subdivided into four reproductive phenotypes: NIHdiagnosed PCOS either with (phenotype A) or without PCO (phenotype B); biochemical/clinical hyperandrogenism with PCO but no oligo/anovulation (phenotype C), or no biochemical/clinical hyperandrogenism with PCO and oligo/anovulation (phenotype

There is an increasing body of literature devoted to examining the metabolic implications of the reproductive diagnostic phenotypes of PCOS. The majority of literature to date has

Hence, a very recent study performed in Brazil in order to assess the NIH PCOS phenotypes (A and B) for metabolic features indicated that PCOS diagnosis based on the presence of hyperandrogenism and ovulatory dysfunction, with or without PCO, is associated with a worse metabolic profile and more insulin resistance than that observed in ovulatory women with the hyperandrogenism +PCO phenotype or with isolated hirsutism, and in ovulatory control women without hirsutism, even after adjustment for BMI (because the prevalence of obesity was higher in the classic PCOS group) (Wiltgen & Spritzer, 2010). However, when weight-matched, the most of data suggest that the metabolic profile of the newer phenotypes is similar to the profile seen in NIH phenotypes (Moran & Teede, 2009). Interestingly, the adiponectin levels, which have been proposed as possible links between reproductive and metabolic anomalies in PCOS (Yilmaz et al., 2009), were significantly lower in groups 1 and 2 (classic PCOS) in comparison with the concentrations in women with the newer phenotype groups 3 and 4, added in accordance with the 2003 criteria. (phenotypes C and D), which were much higher, reaching those of control levels. It is worth mentioning that there was no significant difference in the waist circumference (WC), the waist-to-hip ratio (WHR) and, consequently, the amount of visceral fat between the five groups of women in the study. Thus, adiponectin levels could reflect the distinct PCOS phenotype (Karkanaki et al., 2009). Furthermore, the few studies specifically comparing NIH PCOS with or without PCO (phenotype A versus B) have generally shown similar risk of metabolic disease for the two phenotypes of NIH PCOS. (Dewailly et al., 2006; Diamanti-Kandarakis and Panidis, 2007; Hsu et al., 2007; Shroff et al., 2007, as cited in Moran & Teede,

Research on the metabolic implications of the newer phenotypes of PCOS introduced by the

Depending on the population recruited from, up to 18% of women with PCOS by ESHRE/ASRM criteria can have non-hyperandrogenic PCOS (D) and up to 25% of women

Guastella et al., 2010; Moran & Teede, 2009).

focused on the NIH diagnosis of PCOS.

D) (Moran & Teede, 2009).

2009; Moran & Teede, 2009).

ESHRE/ASRM is only emerging.

been controversial as far as the presence of arterial structural and functional alterations or of chronic inflammation in PCOS are concerned. However, most lines of investigation point to increased CV risk and sustain the presence of sub-clinic CVD among women with PCOS. Therefore, medical intervention should target the reduction of the risk factors that cluster in women with this disorder. It has even been suggested that if pathologic values of IMT are found, these patients should be aggressively treated, starting with lifestyle programs and eventually with insulin-sensitizing or also statins (Carmina, 2009). Furthermore, the oral contraceptive pills (OCPs), the most common drugs used in PCOS, may exacerbate the metabolic profile of women with this disorder, as they were associated in some studies with a deterioration of carbohydrate metabolism and lipid profile. Women using OCPs have higher highly sensitive C-reactive protein (hsCRP) concentrations than non-users and the ethinyl-estradiol –cyproterne acetate pill has been shown to significantly increase serum CRP levels in PCOS subjects. Though controversies still exist, there are important reports providing suggestions that increased CV risk among women with PCOS indeed increases likelihood of them developing CVD and CV events (Shaw et al., 2008). PCOS thus constitutes a significant health and economic burden estimated at over \$4 billion in the USA with -40.5% of costs related to treatment of type 2 DM. (Azziz et al., 2005). If PCOS status were recognized as an independent risk factor for subsequent CV events, this would, then, justify earlier risk-factor intervention.

#### **2. Phenotypes of the polycystic ovary syndrome and the risk for cardiovascular disease**

Although it is widely recognised that PCOS is a diagnosis of exclusion, the optimal diagnostic criteria for PCOS remain controversial. Several definitions are in use today: one arising from an expert conference sponsored by the National Institute of Health (NIH1990 criteria) and the other from another expert conference sponsored by the European Society for Human Reproduction and Embryology (ESHRE) and the American Society for Reproductive Medicine (ASRM) in 2003 in Rotterdam (Rotterdam 2003 criteria). To date, there is limited understanding of the relative prevalence of risk factors for metabolic diseases (type 2 DM and CVD) across the reproductive diagnostic phenotypes of PCOS and clearly the prevalence and the long-term morbidity of PCOS will depend, to some degree, on the criteria used to define this disorder. Clarification of this will aid in determining whether to include non-NIH phenotypes as part of the complex condition of PCOS and in identifying if specific reproductive PCOS phenotypes have elevated metabolic risks (Moran & Teede, 2009).

NIH diagnostic criteria have been used for the past 15 years based on biochemical or clinical hyperandrogenism and anovulation (excluding other secondary causes including thyroid dysfunction, non-congenital adrenal hyperplasia or hyperprolactinaemia) (Zawdaki & Dunaif, 1992). According to these criteria, 4–8% of women in a general population have PCOS ( Asuncion et al., 2000; Azziz et al., 2004; Diamanti-Kandarakis et al., 1999; Knochenhauer et al., 1998, as cited in Moran & Teede, 2009). The Rotterdam criteria (ESHRE/ASRM, 2004), which are more extensive, were formulated as two of the three criteria of hyperandrogenism, polycystic ovaries (PCO) on ultrasound and irregular anovulatory periods (Guastella et al., 2010). This introduced two new PCOS phenotypes (non-NIH PCOS) of hyperandrogenic ovulatory women with PCO or non-hyperandrogenic anovulatory women with PCO (2004) (Moran & Teede, 2009). The development of both the ESHRE/ASRM and the Androgen Excess Society (AES) criteria has introduced greater

been controversial as far as the presence of arterial structural and functional alterations or of chronic inflammation in PCOS are concerned. However, most lines of investigation point to increased CV risk and sustain the presence of sub-clinic CVD among women with PCOS. Therefore, medical intervention should target the reduction of the risk factors that cluster in women with this disorder. It has even been suggested that if pathologic values of IMT are found, these patients should be aggressively treated, starting with lifestyle programs and eventually with insulin-sensitizing or also statins (Carmina, 2009). Furthermore, the oral contraceptive pills (OCPs), the most common drugs used in PCOS, may exacerbate the metabolic profile of women with this disorder, as they were associated in some studies with a deterioration of carbohydrate metabolism and lipid profile. Women using OCPs have higher highly sensitive C-reactive protein (hsCRP) concentrations than non-users and the ethinyl-estradiol –cyproterne acetate pill has been shown to significantly increase serum CRP levels in PCOS subjects. Though controversies still exist, there are important reports providing suggestions that increased CV risk among women with PCOS indeed increases likelihood of them developing CVD and CV events (Shaw et al., 2008). PCOS thus constitutes a significant health and economic burden estimated at over \$4 billion in the USA

40.5% of costs related to treatment of type 2 DM. (Azziz et al., 2005). If PCOS status were recognized as an independent risk factor for subsequent CV events, this would, then,

Although it is widely recognised that PCOS is a diagnosis of exclusion, the optimal diagnostic criteria for PCOS remain controversial. Several definitions are in use today: one arising from an expert conference sponsored by the National Institute of Health (NIH1990 criteria) and the other from another expert conference sponsored by the European Society for Human Reproduction and Embryology (ESHRE) and the American Society for Reproductive Medicine (ASRM) in 2003 in Rotterdam (Rotterdam 2003 criteria). To date, there is limited understanding of the relative prevalence of risk factors for metabolic diseases (type 2 DM and CVD) across the reproductive diagnostic phenotypes of PCOS and clearly the prevalence and the long-term morbidity of PCOS will depend, to some degree, on the criteria used to define this disorder. Clarification of this will aid in determining whether to include non-NIH phenotypes as part of the complex condition of PCOS and in identifying if specific

reproductive PCOS phenotypes have elevated metabolic risks (Moran & Teede, 2009).

NIH diagnostic criteria have been used for the past 15 years based on biochemical or clinical hyperandrogenism and anovulation (excluding other secondary causes including thyroid dysfunction, non-congenital adrenal hyperplasia or hyperprolactinaemia) (Zawdaki & Dunaif, 1992). According to these criteria, 4–8% of women in a general population have PCOS ( Asuncion et al., 2000; Azziz et al., 2004; Diamanti-Kandarakis et al., 1999; Knochenhauer et al., 1998, as cited in Moran & Teede, 2009). The Rotterdam criteria (ESHRE/ASRM, 2004), which are more extensive, were formulated as two of the three criteria of hyperandrogenism, polycystic ovaries (PCO) on ultrasound and irregular anovulatory periods (Guastella et al., 2010). This introduced two new PCOS phenotypes (non-NIH PCOS) of hyperandrogenic ovulatory women with PCO or non-hyperandrogenic anovulatory women with PCO (2004) (Moran & Teede, 2009). The development of both the ESHRE/ASRM and the Androgen Excess Society (AES) criteria has introduced greater

**2. Phenotypes of the polycystic ovary syndrome and the risk for** 

with -

justify earlier risk-factor intervention.

**cardiovascular disease** 

heterogeneity into PCOS from a reproductive and possibly from a metabolic perspective. In fact, according to these guidelines, the diagnosis of PCOS may present in patients with four different phenotypes: [1] hyperandrogenism, chronic anovulation, and PCO; [2] hyperandrogenism and chronic anovulation but normal ovaries; [3] hyperandrogenism and PCO but ovulatory cycles; and [4] chronic anovulation and PCO but no clinical or biochemical hyperandrogenism (Guastella et al., 2010). In 2006, the AES published a position statement which suggested that androgen excess is the key component of PCOS related to clinical presentation and long-term morbidity. The proposal of the AES was, therefore, to include in the diagnosis of PCOS only the first three phenotypes, excluding the phenotype of PCO and irregular cycles without hyperandrogenism (Azziz et al., 2006, 2009; Guastella et al., 2010; Moran & Teede, 2009).

For simplification, PCOS can be subdivided into four reproductive phenotypes: NIHdiagnosed PCOS either with (phenotype A) or without PCO (phenotype B); biochemical/clinical hyperandrogenism with PCO but no oligo/anovulation (phenotype C), or no biochemical/clinical hyperandrogenism with PCO and oligo/anovulation (phenotype D) (Moran & Teede, 2009).

There is an increasing body of literature devoted to examining the metabolic implications of the reproductive diagnostic phenotypes of PCOS. The majority of literature to date has focused on the NIH diagnosis of PCOS.

Hence, a very recent study performed in Brazil in order to assess the NIH PCOS phenotypes (A and B) for metabolic features indicated that PCOS diagnosis based on the presence of hyperandrogenism and ovulatory dysfunction, with or without PCO, is associated with a worse metabolic profile and more insulin resistance than that observed in ovulatory women with the hyperandrogenism +PCO phenotype or with isolated hirsutism, and in ovulatory control women without hirsutism, even after adjustment for BMI (because the prevalence of obesity was higher in the classic PCOS group) (Wiltgen & Spritzer, 2010). However, when weight-matched, the most of data suggest that the metabolic profile of the newer phenotypes is similar to the profile seen in NIH phenotypes (Moran & Teede, 2009). Interestingly, the adiponectin levels, which have been proposed as possible links between reproductive and metabolic anomalies in PCOS (Yilmaz et al., 2009), were significantly lower in groups 1 and 2 (classic PCOS) in comparison with the concentrations in women with the newer phenotype groups 3 and 4, added in accordance with the 2003 criteria. (phenotypes C and D), which were much higher, reaching those of control levels. It is worth mentioning that there was no significant difference in the waist circumference (WC), the waist-to-hip ratio (WHR) and, consequently, the amount of visceral fat between the five groups of women in the study. Thus, adiponectin levels could reflect the distinct PCOS phenotype (Karkanaki et al., 2009). Furthermore, the few studies specifically comparing NIH PCOS with or without PCO (phenotype A versus B) have generally shown similar risk of metabolic disease for the two phenotypes of NIH PCOS. (Dewailly et al., 2006; Diamanti-Kandarakis and Panidis, 2007; Hsu et al., 2007; Shroff et al., 2007, as cited in Moran & Teede, 2009; Moran & Teede, 2009).

Research on the metabolic implications of the newer phenotypes of PCOS introduced by the ESHRE/ASRM is only emerging.

Depending on the population recruited from, up to 18% of women with PCOS by ESHRE/ASRM criteria can have non-hyperandrogenic PCOS (D) and up to 25% of women

The Polycystic Ovary Syndrome Status – A Risk Factor for Future Cardiovascular Disease 155

abdominal obesity. The metabolic profile of newer reproductive phenotypes appears to be milder than that of NIH PCOS, but more adverse than that of controls and, again, strongly related to abdominal adiposity. While the ovulatory subgroup is more hyperandrogenic, evidence suggests non-hyperandrogenic women as having a similar metabolic profile to ovulatory PCOS with limited evidence even suggesting they present with more severe IR and dyslipidaemia (Norman et al., 1995a, as cited in Moran & Teede, 2009). In the setting of either hyperandrogenism or insulin resistance, metabolic abnormalities are observed. Consequently, the literature has been, so far, sustaining the inclusion of both newer phenotypes of PCOS based on the ESHRE/ASRM Rotterdam diagnostic criteria, suggesting

There are, however, serious limitations of the literature on the metabolic features of PCOS, such as: ethnic diversity, recruitment sources of participants, consistency in the use of endpoints and inconsistently defined controls or the use of CV risk factors instead of clinical disease outcomes (e.g. type 2 DM, coronary artery disease, subclinical or clinical atherosclerosis), to name but a few. Taking all these into account, it has become obvious that we need rigorous, well designed studies with well-defined controls and longitudinal followup to efficiently grasp clinical outcomes in order to clarify many of the ambiguous aspects of

Several studies have examined the presence of CV risk factors in premenopausal women with PCOS. It has long been established that women with PCOS have an unfavorable cardiometabolic risk profile. Based on National Cholesterol Education Program guidelines, traditional CV risk factors include age > 55 years, current cigarette smoking, diabetes, history of premature coronary artery disease in first-degree relatives (men < 55 years, women < 65 years), hypertension, and dyslipidemia. (Birdsall et al., 1997; Dahlgren et al.,

The patient's risk is determined by the levels of both systolic (SBP) and diastolic blood pressure (DBP). In fact, of the two readings, the SBP may best predict all the complications

In spite of the fact that blood pressures (BP) are generally within the normal range in young women with PCOS and not very different from that of age and weight matched controls (Dokras, 2008) abnormalities in the regulation of BP are common in these patients. Prehypertension, defined as SBP 120 to 139 mm Hg or DBP 80 to 89 mm Hg, is associated with a twofold increased risk of CV mortality (Chobanian et al., 2003, as cited in Dokras, 2008). 24-hour ambulatory BP readings showed that women with PCOS have an increased risk of prehypertension (Holte et al., 1996, as cited in Dokras, 2008; Lo et al.,2006). Moreover, although both SBP and DBP are normal in PCOS women in many studies, there are reports in which mean arterial pressures and ambulatory SBP (e.g. Holte et al., 1996, as cited in Cho et al., 2007) or the prevalence of hypertension (Cho et al., 2007) are increased in women with PCOS compared with controls (Hoffman & Ehrmann, 2008). Confirming these results, both

that these phenotypes are milder forms of PCOS.

the metabolic phenotype of PCOS (Moran & Teede, 2009).

**3. Cardiovascular risk factors in PCOS 3.1 Traditional cardiovascular risk factors** 

1992; Solomon et al., 2002, as cited in Dokras, 2008).

**3.1.1 High blood pressure** 

related to hypertension.

can have ovulatory PCOS (C). This indicates an increasing number of women with PCOS who may experience different reproductive and metabolic risks, when compared with those who have NIH PCOS with potential implications for research, screening and clinical practice. There is emerging evidence that these two phenotypes have a less adverse metabolic profile than NIH PCOS (Moran & Teede, 2009).

Regarding the phenotype C and D, particularly, compared with NIH PCOS, the literature, although scarce, suggests less adverse metabolic profiles for both hyperandrogenic ovulatory PCOS and non-hyperandrogenic anovulatory PCOS. Reduced adiposity and abdominal adiposity contribute to a more favourable metabolic profile in these two non-NIH phenotypes. These patients presented with most of the PCOS characteristics but in a milder form. In fact, patients with ovulatory PCOS had intermediate values (between classic PCOS and controls) of BMI, WC, testosterone, insulin, and quantitative insulin sensitivity index (QUICKI) (Guastella et al., 2010; Moran & Teede, 2009). As far as the specific PCOS phenotype- hirsute ovulatory patients with PCO are concerned, there are data indicating a lower prevalence of CV risk factors in hirsute ovulatory patients with PCO and normal androgen levels than with the classic PCOS phenotype, being similar in that regard to women with isolated hirsutism. However, obesity might be implicated in increased susceptibility to insulin resistance in hirsute women, and the monitoring of this group for metabolic comorbidities and CV risk factors is warranted even in the presence of androgen levels within the normal range (Wiltgen & Spritzer, 2010). All in all, both non-NIH (hyperandrogenic ovulatory) PCOS (phenotype C) and non-hyperandrogenic PCOS (phenotype D) generally have lower body weight and body mass index (BMI) and better metabolic profiles compared with NIH PCOS (phenotypes A/B). However, non-NIH (phenotype C and D) PCOS and weight-matched NIH PCOS appear to present with similar metabolic risk profiles, particularly where abdominal fat and total fat are similar between subjects (Moran & Teede, 2009).

In comparison to controls, although not universally observed, women with non-NIH (hyperandrogenic ovulatory) PCOS (phenotype C) seem to be more adversely metabolically affected and this appears to be strongly related to the presence of adiposity and specifically abdominal adiposity. Additionally, some evidence exists to suggest that nonhyperandrogenic anovulatory PCOS matched for abdominal obesity have an adverse metabolic profile compared with controls.

There are even fewer studies comparing the non-NIH phenotypes of hyperandrogenic ovulatory PCOS (phenotype C) and non-hyperandrogenic anovulatory PCOS (phenotype D) and from this limited and conflicting literature, non-hyperandrogenic anovulatory PCOS seems not to display improved metabolic risk factors compared with hyperandrogenic ovulatory PCOS. There is, thus, currently limited evidence to support the exclusion of nonhyperandrogenic PCOS as a phenotype of PCOS based on metabolic presentation (Moran & Teede, 2009).

The current evidence shows that patients with NIH PCOS, who are hyperandrogenic and generally insulin resistant, have the most severe metabolic features. The adverse metabolic profile is strongly connected to obesity and abdominal obesity, the latter being more severe in NIH PCOS than in other non-NIH phenotypes. Both hyperandrogenism and insulin resistance play a role in the adverse metabolic profiles and both may establish the metabolic phenotypes of women with PCOS, either directly or through a high inclination towards abdominal obesity. The metabolic profile of newer reproductive phenotypes appears to be milder than that of NIH PCOS, but more adverse than that of controls and, again, strongly related to abdominal adiposity. While the ovulatory subgroup is more hyperandrogenic, evidence suggests non-hyperandrogenic women as having a similar metabolic profile to ovulatory PCOS with limited evidence even suggesting they present with more severe IR and dyslipidaemia (Norman et al., 1995a, as cited in Moran & Teede, 2009). In the setting of either hyperandrogenism or insulin resistance, metabolic abnormalities are observed. Consequently, the literature has been, so far, sustaining the inclusion of both newer phenotypes of PCOS based on the ESHRE/ASRM Rotterdam diagnostic criteria, suggesting that these phenotypes are milder forms of PCOS.

There are, however, serious limitations of the literature on the metabolic features of PCOS, such as: ethnic diversity, recruitment sources of participants, consistency in the use of endpoints and inconsistently defined controls or the use of CV risk factors instead of clinical disease outcomes (e.g. type 2 DM, coronary artery disease, subclinical or clinical atherosclerosis), to name but a few. Taking all these into account, it has become obvious that we need rigorous, well designed studies with well-defined controls and longitudinal followup to efficiently grasp clinical outcomes in order to clarify many of the ambiguous aspects of the metabolic phenotype of PCOS (Moran & Teede, 2009).

#### **3. Cardiovascular risk factors in PCOS**

#### **3.1 Traditional cardiovascular risk factors**

Several studies have examined the presence of CV risk factors in premenopausal women with PCOS. It has long been established that women with PCOS have an unfavorable cardiometabolic risk profile. Based on National Cholesterol Education Program guidelines, traditional CV risk factors include age > 55 years, current cigarette smoking, diabetes, history of premature coronary artery disease in first-degree relatives (men < 55 years, women < 65 years), hypertension, and dyslipidemia. (Birdsall et al., 1997; Dahlgren et al., 1992; Solomon et al., 2002, as cited in Dokras, 2008).

#### **3.1.1 High blood pressure**

154 Recent Advances in Cardiovascular Risk Factors

can have ovulatory PCOS (C). This indicates an increasing number of women with PCOS who may experience different reproductive and metabolic risks, when compared with those who have NIH PCOS with potential implications for research, screening and clinical practice. There is emerging evidence that these two phenotypes have a less adverse

Regarding the phenotype C and D, particularly, compared with NIH PCOS, the literature, although scarce, suggests less adverse metabolic profiles for both hyperandrogenic ovulatory PCOS and non-hyperandrogenic anovulatory PCOS. Reduced adiposity and abdominal adiposity contribute to a more favourable metabolic profile in these two non-NIH phenotypes. These patients presented with most of the PCOS characteristics but in a milder form. In fact, patients with ovulatory PCOS had intermediate values (between classic PCOS and controls) of BMI, WC, testosterone, insulin, and quantitative insulin sensitivity index (QUICKI) (Guastella et al., 2010; Moran & Teede, 2009). As far as the specific PCOS phenotype- hirsute ovulatory patients with PCO are concerned, there are data indicating a lower prevalence of CV risk factors in hirsute ovulatory patients with PCO and normal androgen levels than with the classic PCOS phenotype, being similar in that regard to women with isolated hirsutism. However, obesity might be implicated in increased susceptibility to insulin resistance in hirsute women, and the monitoring of this group for metabolic comorbidities and CV risk factors is warranted even in the presence of androgen levels within the normal range (Wiltgen & Spritzer, 2010). All in all, both non-NIH (hyperandrogenic ovulatory) PCOS (phenotype C) and non-hyperandrogenic PCOS (phenotype D) generally have lower body weight and body mass index (BMI) and better metabolic profiles compared with NIH PCOS (phenotypes A/B). However, non-NIH (phenotype C and D) PCOS and weight-matched NIH PCOS appear to present with similar metabolic risk profiles, particularly where abdominal fat and total fat are similar between

In comparison to controls, although not universally observed, women with non-NIH (hyperandrogenic ovulatory) PCOS (phenotype C) seem to be more adversely metabolically affected and this appears to be strongly related to the presence of adiposity and specifically abdominal adiposity. Additionally, some evidence exists to suggest that nonhyperandrogenic anovulatory PCOS matched for abdominal obesity have an adverse

There are even fewer studies comparing the non-NIH phenotypes of hyperandrogenic ovulatory PCOS (phenotype C) and non-hyperandrogenic anovulatory PCOS (phenotype D) and from this limited and conflicting literature, non-hyperandrogenic anovulatory PCOS seems not to display improved metabolic risk factors compared with hyperandrogenic ovulatory PCOS. There is, thus, currently limited evidence to support the exclusion of nonhyperandrogenic PCOS as a phenotype of PCOS based on metabolic presentation (Moran &

The current evidence shows that patients with NIH PCOS, who are hyperandrogenic and generally insulin resistant, have the most severe metabolic features. The adverse metabolic profile is strongly connected to obesity and abdominal obesity, the latter being more severe in NIH PCOS than in other non-NIH phenotypes. Both hyperandrogenism and insulin resistance play a role in the adverse metabolic profiles and both may establish the metabolic phenotypes of women with PCOS, either directly or through a high inclination towards

metabolic profile than NIH PCOS (Moran & Teede, 2009).

subjects (Moran & Teede, 2009).

Teede, 2009).

metabolic profile compared with controls.

The patient's risk is determined by the levels of both systolic (SBP) and diastolic blood pressure (DBP). In fact, of the two readings, the SBP may best predict all the complications related to hypertension.

In spite of the fact that blood pressures (BP) are generally within the normal range in young women with PCOS and not very different from that of age and weight matched controls (Dokras, 2008) abnormalities in the regulation of BP are common in these patients. Prehypertension, defined as SBP 120 to 139 mm Hg or DBP 80 to 89 mm Hg, is associated with a twofold increased risk of CV mortality (Chobanian et al., 2003, as cited in Dokras, 2008). 24-hour ambulatory BP readings showed that women with PCOS have an increased risk of prehypertension (Holte et al., 1996, as cited in Dokras, 2008; Lo et al.,2006). Moreover, although both SBP and DBP are normal in PCOS women in many studies, there are reports in which mean arterial pressures and ambulatory SBP (e.g. Holte et al., 1996, as cited in Cho et al., 2007) or the prevalence of hypertension (Cho et al., 2007) are increased in women with PCOS compared with controls (Hoffman & Ehrmann, 2008). Confirming these results, both

The Polycystic Ovary Syndrome Status – A Risk Factor for Future Cardiovascular Disease 157

In conclusion, PCOS women seem to be at increased risk for hypertension development, if not during their reproductive years, then at least later in life (Hoffman & Ehrmann, 2008). Accordingly, very recently (2010), the AE-PCOS Board committee *has been recommending* a BP routine check at each visit. Ideal BP is 120 mm Hg systolic and 80 mm Hg diastolic or lower, and prehypertension should be detected and treated (Cushman, 2007). BP control has

Dyslipidemia is a major determinant of progression of atherosclerosis. Atherosclerosis starts at a very young age, and PCOS may represent an important model of lipid alterations starting during adolescence or fertile age (Wild et al., 1985; Wild et al., 2011). Actually, lipid abnormalities have been reported in up to 70% of PCOS patients and displayed different patterns, depending on several factors such as: the PCOS phenotype, the presence of obesity and the associated effects of IR and hyperandrogenism that combine with environmental (diet, physical exercise) and genetic factors (Essah et al., 2008; Valkenburg et al., 2008; Wild et al., 2011). The dyslipidaemia occurs independent of BMI (Wild et al., 1985; Wild & Bartholomew, 1988, as cited in Teede et al., 2010a; Talbott et al., 1995, as cited in Sukalich & Guzick, 2003; Teede et al., 2010a) and several studies have confirmed adverse lipid alterations in both obese and nonobese women with PCOS compared with weight-matched control women (Sukalich & Guzick, 2003). Consequently, PCOS might be considered the

Wild and colleagues (Wild et al., 1985) were the first to present data suggesting that women with PCOS had a more adverse lipid profile than control subjects and there are many studies that have analyzed this aspect ever since. Lipid abnormalities include low highdensity lipoproteins cholesterol (HDL-C), high low-density lipoprotein cholesterol (LDL-C), high TG levels and small dense LDL particles (Lambrinoudaki, 2011; Wild et al., 2011), a combination closely linked to insulin resistance and an independent predictor of myocardial infarction (MI) and CVD. Increased TG/HDL-C ratio is also a marker for atherogenic, small, dense LDL-C particles and it may be also used as a simple metabolic marker to identify overweight individuals who are insulin-resistant (McLaughlin et al., 2003, Brehm et al., 2004, as cited in Dokras, 2008). This relationship has been demonstrated in women with PCOS by identifying a negative correlation between TG/HDL-C and QUICKI (Dokras et al., 2005). Recently, it has been proved that TG/HDL C > 3.2 and respectively > 3.5 identify both the MS as well as insulin-resistant and dyslipidemic patients whose chances to be at an increased risk for CVD are extremely high (Dokras, 2008). As mentioned earlier, prevalent MS in women with PCOS (Apridonidze et al., 2005; Dokras et al., 2005; Moran et al., 2010) has concentrated attention of most authors on changes in TG and HDL-C (that are components of the metabolic syndrome) with relatively little attention to other lipid changes, although LDL-C and nonHDL-C are considered to be the primary and secondary targets to reduce CVD and atherosclerosis as described by the National Cholesterol Education Program Adult Treatment Panel III (NCEP-ATP III) guidelines (NCEP-ATP III, 2002, as cited in Wild et al., 2011). However, qualitative disorders of LDL-C have also been identified in women who have PCOS and there have been a large number of studies during the past decade who found increased LDL-C levels in women with this disorder (Carmina et al., 2005; Legro et al., 2001; Rizzo et al., 2009; Wild et al., 2011) and LDL particles smaller than in matched controls (Dejager et al., 2001). Small, dense LDL particles are associated

the largest benefit for reducing CVD (Wild et al., 2010).

**3.1.2 High blood cholesterol and related lipid problems** 

most common cause of dyslipidemia in women under 40.

mean arterial BP and the risk of preeclampsia have been found to be higher in women with PCOS (Akram et al., 2010; Boomsma et al., 2006).

According to a study carried out in 2005, there is no difference in ambulatory or office-based BP measurements between women with PCOS and control individuals; however, women with PCOS had a less significant overnight drop in mean arterial BP, a phenomenon also noted in obese adolescents with PCOS (Wild et al., 2005, as cited in Hoffman & Ehrmann, 2008). A Taiwanese study showed that the characteristic hyperandrogenemia in PCOS is associated with an elevated SBP and DBP independent of age, insulin resistance, obesity, or dyslipidemia (Chen et al., 2003). Overall, it appears that elevated SBP is detected after the third decade of life and may be independent of obesity (Dokras, 2008). Moreover, in a very recent study that targeted 113 PCOS women, the frequency of women with BP values above the normal limit was significantly higher in the PCOS group than in the control group. In the PCOS group, the values of SBP and DBP were positively correlated with age, BMI, WC, and triglycerides (TG) (p<0.05) to a significant degree. These results underline the importance of preventive strategies in PCOS women in anticipating pathological events related to the cardiovascular system (Azevedo et al., 2011).

Women with PCOS are under an increased risk of developing hypertension later in life, as proved by retrospective data. Follow-up data from the Pittsburgh case-control study proved physician-diagnosed hypertension in 23% of women with PCOS whereas the percentage of cycling control women in this situation was of only 6.9% (Talbott et al., 2001). In an earlier retrospective cohort study of 33 older women with histopathology consistent with PCOS on wedge resection 22 to 31 years earlier, Dahlgren et al. (1992) found the diagnosis of hypertension to be three times more common in women with PCOS than in normal agematched controls (Dahlgren et al., 1992, as cited in Sukalich & Guzick, 2003). Preliminary data from the Nurses' Health Study demonstrate that the risk of developing hypertension was twice higher in women with increased or highly irregular menstrual cycle length (a possible surrogate for a clinical diagnosis of PCOS) than in women with regular cycles (Rich-Edwards et al., 1998, as cited in Sukalich & Guzick, 2003). A significant difference in the prevalence of hypertension was still noted after correction for BMI.

In spite of noted characteristics that typically accompany PCOS (e.g. insulin resistance, obesity etc.) the exact mechanisms responsible for hypertension in women with PCOS are yet to be clarified. Insulin resistance causes secondary hyperinsulinemia. Hyperinsulinemia may produce enhanced sodium retention (Zavaroni et al., 1995, as cited in Cho et al., 2007), increasing intracellular sodium and calcium and augmenting sympathetic activity (Reaven et al., 1996, as cited in Sukalich & Guzick, 2003) which may have a role in the development of hypertension (Sukalich & Guzick, 2003). Insulin also stimulates the release of insulin-like growth factor (IGF-1) that may contribute to the development of hypertension by determining vascular smooth muscle hypertrophy (Cho et al., 2007). Furthermore, the obesity that is common in PCOS adds to the risk of hypertension. The higher level of androgens seems to be strongly related to BP in women with PCOS who are not obese. Although the mechanisms by which hyperandrogenemia mediates the higher BP in women with PCOS remain to be determined, it is possible that androgens may directly stimulate endothelin-1 (ET-1) or may stimulate the rennin-angiotensin system (RAS) to increase ET-1, thus leading to the expression of two powerful vasoconstrictors that could impact BP in these women (Reckelhoff, 2007).

In conclusion, PCOS women seem to be at increased risk for hypertension development, if not during their reproductive years, then at least later in life (Hoffman & Ehrmann, 2008). Accordingly, very recently (2010), the AE-PCOS Board committee *has been recommending* a BP routine check at each visit. Ideal BP is 120 mm Hg systolic and 80 mm Hg diastolic or lower, and prehypertension should be detected and treated (Cushman, 2007). BP control has the largest benefit for reducing CVD (Wild et al., 2010).

#### **3.1.2 High blood cholesterol and related lipid problems**

156 Recent Advances in Cardiovascular Risk Factors

mean arterial BP and the risk of preeclampsia have been found to be higher in women with

According to a study carried out in 2005, there is no difference in ambulatory or office-based BP measurements between women with PCOS and control individuals; however, women with PCOS had a less significant overnight drop in mean arterial BP, a phenomenon also noted in obese adolescents with PCOS (Wild et al., 2005, as cited in Hoffman & Ehrmann, 2008). A Taiwanese study showed that the characteristic hyperandrogenemia in PCOS is associated with an elevated SBP and DBP independent of age, insulin resistance, obesity, or dyslipidemia (Chen et al., 2003). Overall, it appears that elevated SBP is detected after the third decade of life and may be independent of obesity (Dokras, 2008). Moreover, in a very recent study that targeted 113 PCOS women, the frequency of women with BP values above the normal limit was significantly higher in the PCOS group than in the control group. In the PCOS group, the values of SBP and DBP were positively correlated with age, BMI, WC, and triglycerides (TG) (p<0.05) to a significant degree. These results underline the importance of preventive strategies in PCOS women in anticipating pathological events

Women with PCOS are under an increased risk of developing hypertension later in life, as proved by retrospective data. Follow-up data from the Pittsburgh case-control study proved physician-diagnosed hypertension in 23% of women with PCOS whereas the percentage of cycling control women in this situation was of only 6.9% (Talbott et al., 2001). In an earlier retrospective cohort study of 33 older women with histopathology consistent with PCOS on wedge resection 22 to 31 years earlier, Dahlgren et al. (1992) found the diagnosis of hypertension to be three times more common in women with PCOS than in normal agematched controls (Dahlgren et al., 1992, as cited in Sukalich & Guzick, 2003). Preliminary data from the Nurses' Health Study demonstrate that the risk of developing hypertension was twice higher in women with increased or highly irregular menstrual cycle length (a possible surrogate for a clinical diagnosis of PCOS) than in women with regular cycles (Rich-Edwards et al., 1998, as cited in Sukalich & Guzick, 2003). A significant difference in

In spite of noted characteristics that typically accompany PCOS (e.g. insulin resistance, obesity etc.) the exact mechanisms responsible for hypertension in women with PCOS are yet to be clarified. Insulin resistance causes secondary hyperinsulinemia. Hyperinsulinemia may produce enhanced sodium retention (Zavaroni et al., 1995, as cited in Cho et al., 2007), increasing intracellular sodium and calcium and augmenting sympathetic activity (Reaven et al., 1996, as cited in Sukalich & Guzick, 2003) which may have a role in the development of hypertension (Sukalich & Guzick, 2003). Insulin also stimulates the release of insulin-like growth factor (IGF-1) that may contribute to the development of hypertension by determining vascular smooth muscle hypertrophy (Cho et al., 2007). Furthermore, the obesity that is common in PCOS adds to the risk of hypertension. The higher level of androgens seems to be strongly related to BP in women with PCOS who are not obese. Although the mechanisms by which hyperandrogenemia mediates the higher BP in women with PCOS remain to be determined, it is possible that androgens may directly stimulate endothelin-1 (ET-1) or may stimulate the rennin-angiotensin system (RAS) to increase ET-1, thus leading to the expression of two powerful vasoconstrictors that could impact BP in

PCOS (Akram et al., 2010; Boomsma et al., 2006).

related to the cardiovascular system (Azevedo et al., 2011).

the prevalence of hypertension was still noted after correction for BMI.

these women (Reckelhoff, 2007).

Dyslipidemia is a major determinant of progression of atherosclerosis. Atherosclerosis starts at a very young age, and PCOS may represent an important model of lipid alterations starting during adolescence or fertile age (Wild et al., 1985; Wild et al., 2011). Actually, lipid abnormalities have been reported in up to 70% of PCOS patients and displayed different patterns, depending on several factors such as: the PCOS phenotype, the presence of obesity and the associated effects of IR and hyperandrogenism that combine with environmental (diet, physical exercise) and genetic factors (Essah et al., 2008; Valkenburg et al., 2008; Wild et al., 2011). The dyslipidaemia occurs independent of BMI (Wild et al., 1985; Wild & Bartholomew, 1988, as cited in Teede et al., 2010a; Talbott et al., 1995, as cited in Sukalich & Guzick, 2003; Teede et al., 2010a) and several studies have confirmed adverse lipid alterations in both obese and nonobese women with PCOS compared with weight-matched control women (Sukalich & Guzick, 2003). Consequently, PCOS might be considered the most common cause of dyslipidemia in women under 40.

Wild and colleagues (Wild et al., 1985) were the first to present data suggesting that women with PCOS had a more adverse lipid profile than control subjects and there are many studies that have analyzed this aspect ever since. Lipid abnormalities include low highdensity lipoproteins cholesterol (HDL-C), high low-density lipoprotein cholesterol (LDL-C), high TG levels and small dense LDL particles (Lambrinoudaki, 2011; Wild et al., 2011), a combination closely linked to insulin resistance and an independent predictor of myocardial infarction (MI) and CVD. Increased TG/HDL-C ratio is also a marker for atherogenic, small, dense LDL-C particles and it may be also used as a simple metabolic marker to identify overweight individuals who are insulin-resistant (McLaughlin et al., 2003, Brehm et al., 2004, as cited in Dokras, 2008). This relationship has been demonstrated in women with PCOS by identifying a negative correlation between TG/HDL-C and QUICKI (Dokras et al., 2005). Recently, it has been proved that TG/HDL C > 3.2 and respectively > 3.5 identify both the MS as well as insulin-resistant and dyslipidemic patients whose chances to be at an increased risk for CVD are extremely high (Dokras, 2008). As mentioned earlier, prevalent MS in women with PCOS (Apridonidze et al., 2005; Dokras et al., 2005; Moran et al., 2010) has concentrated attention of most authors on changes in TG and HDL-C (that are components of the metabolic syndrome) with relatively little attention to other lipid changes, although LDL-C and nonHDL-C are considered to be the primary and secondary targets to reduce CVD and atherosclerosis as described by the National Cholesterol Education Program Adult Treatment Panel III (NCEP-ATP III) guidelines (NCEP-ATP III, 2002, as cited in Wild et al., 2011). However, qualitative disorders of LDL-C have also been identified in women who have PCOS and there have been a large number of studies during the past decade who found increased LDL-C levels in women with this disorder (Carmina et al., 2005; Legro et al., 2001; Rizzo et al., 2009; Wild et al., 2011) and LDL particles smaller than in matched controls (Dejager et al., 2001). Small, dense LDL particles are associated

The Polycystic Ovary Syndrome Status – A Risk Factor for Future Cardiovascular Disease 159

ethnicity are considered additional modifiers of dyslipidemia in women with PCOS (Ehrmann et al., 2006, as cited in Hoffman & Ehrmann, 2008). The lipid profile of both lean and obese women with PCOS is improved by treatment with the androgen-receptor blocker flutamide. Moreover, both in vitro and in vivo studies have included hyperandrogenemia in the pathogenesis of low HDL-C levels, possibly by upregulating the genes involved in the catabolism of HDL-C (Hoffman & Ehrmann, 2008). However, Wild et al. found that hyperinsulinemia has a more significant effect on lipids than hyperandrogenemia in hirsute women (Wild et al., 1992, as cited in Sukalich & Guzick, 2003). This lipid pattern was reported in about 70% of American women with classic PCOS but is less commonly met in other countries where mean body weight is lower (Essah et al., 2008). However, also in Mediterranean countries, about one half of women with PCOS have low HDL-C and a small dense LDL phenotype. However, because of the common increase of LDL-C, a second mechanism related to altered LDL quality has been demonstrated (Wild et al., 2010). Many studies have also demonstrated an increase in LDL-C in women with PCOS (Essah et al., 2008; Legro et al., 2001; Valkenburg et al., 2008). However, its prevalence in PCOS is generally lower than that found for the atherogenic dyslipidemia and ranges from 24 to 40% (Essah et al., 2008; Valkenburg et al., 2008). It depends on body weight to a lesser extent and may be at least partially related to the hyperandrogenism (Wild et al., 2010). In fact, several studies have shown that in female postmenopausal populations, increased circulating values of LDL are related to higher testosterone and free androgen index (Mudali et al.,

In conclusion, dyslipidemia is likely to be found in women with PCOS. Consequently both the American College of Obstetricians and Gynecologists (ACOG) (ACOG, 2009 as cited in Wild et al., 2011) and the Androgen Excess and PCOS Society (Wild et al., 2010) guidelines have recently pointed to the fact that an individual CV risk assessment in women with PCOS is highly necessary and that the CV risk assessment of patients with this disorder should include a complete fasting lipid and lipoprotein evaluation including LDL-C and nonHDL-C, as well as for TG and HDL-C. In addition, the Androgen Excess and PCOS Society guidelines have indicated different LDL-C cutoff values depending on the degree of CV risk of PCOS patients (Wild et al., 2010). In the prevention of CV risk, the first goal is to bring LDL-C to normal levels by using lifestyle intervention and medication if necessary.

Obesity and excess weight are among the most widely met chronic diseases in the Western world countries. The prevalence of increased BMI ranges between 30% and 80% among women with PCOS (Vribkova & Hainer, 2009). Moreover, it has been widely proved that 50– 60% of women with PCOS have a body fat distribution of the android type irrespective of their BMI (Barber et al., 2006) and that patients with PCOS had a central fat excess independent of total fat mass (Lambrinoudaki, 2011; Puder et al., 2005; Ilie et al., 2008).

Obesity also increases hyperandrogenism, hirsutism, insulin resistance and metabolic disorders, infertility and pregnancy complications both independently and by exacerbating PCOS phenotypic expression (Balen et al., 1995, Kiddy et al., 1990, as cited in Teede et al., 2010a). Therefore, in spite of the lack of epidemiological data, the increasing epidemic of obesity worldwide is thought to facilitate the high prevalence of PCOS in the general

2005, Liu et al., 2001, as cited in Wild et al., 2011).

**3.1.3 Obesity** 

The second goal is keeping nonHDL-C reduced (Wild et al., 2011).

with increase in cardiovascular risk independent of total LDL (Austin et al., 1988, as cited in Sukalich & Guzick, 2003). However, the wide variability of LDL-C values between individual studies indicate that LDL-C values are influenced by many variables, including ethnic groups, severity of the syndrome (anovulatory versus ovulatory forms), quality of food, and body weight (Essah et al., 2008; Wild et al., 2011). When subset analyses including only BMI-matched patients were performed, LDL-C was still higher in women with PCOS. It clearly indicates that PCOS per se is responsible for increased lipid values, although the absolute value and the related cardiovascular risk may be different among individual patients (Wild et al., 2011).

Only one study reported mean values of LDL-C <100 mg/dL. Four studies reported mean concentrations in PCOS women 130 mg/dL, whereas no study showed mean LDL-C values >130 mg/dL in control subjects. Considering the reported variances, it is not uncommon for individual PCOS patients to have LDL concentrations >160 mg/dL. Elevated nonHDL-C was reported as a common abnormality in PCOS women included in the studies. 21 of the 31 comparisons, registered nonHDL-C >130 mg/dL and the mean difference was still considerably higher with BMI matching (16 mg/dL vs. control subjects). Similarly to what was found for LDL-C, higher nonHDL-C levels were generally reported in PCOS subjects whose mean BMI was higher. There were clear differences with PCOS even when the BMI was in the nonobese or nonoverweight categories. This suggests that obesity or overweight (and higher insulin resistance) is not the only factor that accounts for elevated LDL-C and nonHDL-C in PCOS (Carmina, 2009; Wild et al., 1985).

However, though decreased HDL-C (particularly HDL2, the most antiatherogenic HDL subtype) (Legro et al., 2001; Diamanti-Kandarakis et al., 2007, as cited in Hoffman & Ehrmann, 2008) may represent the most common lipid alteration in PCOS (Essah et al., 2008), and many studies have reported elevated levels of TG as compared to controls, a very recent meta-analysis performed by Wild et al. (2011) shows that altered levels of TG are not commonly identified in many populations (Carmina et al., 2003, as cited in Wild et al., 2011; Essah et al., 2008). Only three studies reported TG concentrations that exceeded 150 mg/dL, and these were found in women who were overweight or obese (Wild et al., 2011).

However, two main patterns of lipid alteration were described separately or combined (Wild et al., 2011). The most common pattern is probably classic atherogenic dyslipidemia (increased TG, low HDL-C, and increased small dense LDL subclasses + increased nonHDL-C). This lipid pattern is similar to that found in type 2 DM, and it is mainly the consequence of insulin resistance that impairs the ability of insulin to suppress lipolysis, thereby increasing mobilization of free fatty acids (FFA) from adipose stores. Consequently, increased hepatic delivery of FFAs impairs insulin inhibition of hepatic very low-density lipoprotein 1 synthesis, causing altered catabolism of very low-density lipoprotein (Brunzell et al., 2003 as cited in Wild et al., 2010). Additionally, the ability of insulin resistance to alter the expression of lipoprotein lipase and hepatic lipase may also contribute to this lipid pattern (Wild et al., 1985). Sustaining this hypothesis, insulin resistance was linked with hyperlipidemia in most of the studies including women with PCOS (Slowinska-Srzednicka et al., 1991, as cited in Sukalich & Guzick, 2003). Due to the fact that excessive adipose tissue increases insulin resistance, this pattern is likely to be found in obese patients with PCOS. These lipid abnormalities are further augmented among those women who develop glucose intolerance in association with PCOS. The magnitude of androgen elevation, race and ethnicity are considered additional modifiers of dyslipidemia in women with PCOS (Ehrmann et al., 2006, as cited in Hoffman & Ehrmann, 2008). The lipid profile of both lean and obese women with PCOS is improved by treatment with the androgen-receptor blocker flutamide. Moreover, both in vitro and in vivo studies have included hyperandrogenemia in the pathogenesis of low HDL-C levels, possibly by upregulating the genes involved in the catabolism of HDL-C (Hoffman & Ehrmann, 2008). However, Wild et al. found that hyperinsulinemia has a more significant effect on lipids than hyperandrogenemia in hirsute women (Wild et al., 1992, as cited in Sukalich & Guzick, 2003). This lipid pattern was reported in about 70% of American women with classic PCOS but is less commonly met in other countries where mean body weight is lower (Essah et al., 2008). However, also in Mediterranean countries, about one half of women with PCOS have low HDL-C and a small dense LDL phenotype. However, because of the common increase of LDL-C, a second mechanism related to altered LDL quality has been demonstrated (Wild et al., 2010). Many studies have also demonstrated an increase in LDL-C in women with PCOS (Essah et al., 2008; Legro et al., 2001; Valkenburg et al., 2008). However, its prevalence in PCOS is generally lower than that found for the atherogenic dyslipidemia and ranges from 24 to 40% (Essah et al., 2008; Valkenburg et al., 2008). It depends on body weight to a lesser extent and may be at least partially related to the hyperandrogenism (Wild et al., 2010). In fact, several studies have shown that in female postmenopausal populations, increased circulating values of LDL are related to higher testosterone and free androgen index (Mudali et al., 2005, Liu et al., 2001, as cited in Wild et al., 2011).

In conclusion, dyslipidemia is likely to be found in women with PCOS. Consequently both the American College of Obstetricians and Gynecologists (ACOG) (ACOG, 2009 as cited in Wild et al., 2011) and the Androgen Excess and PCOS Society (Wild et al., 2010) guidelines have recently pointed to the fact that an individual CV risk assessment in women with PCOS is highly necessary and that the CV risk assessment of patients with this disorder should include a complete fasting lipid and lipoprotein evaluation including LDL-C and nonHDL-C, as well as for TG and HDL-C. In addition, the Androgen Excess and PCOS Society guidelines have indicated different LDL-C cutoff values depending on the degree of CV risk of PCOS patients (Wild et al., 2010). In the prevention of CV risk, the first goal is to bring LDL-C to normal levels by using lifestyle intervention and medication if necessary. The second goal is keeping nonHDL-C reduced (Wild et al., 2011).

#### **3.1.3 Obesity**

158 Recent Advances in Cardiovascular Risk Factors

with increase in cardiovascular risk independent of total LDL (Austin et al., 1988, as cited in Sukalich & Guzick, 2003). However, the wide variability of LDL-C values between individual studies indicate that LDL-C values are influenced by many variables, including ethnic groups, severity of the syndrome (anovulatory versus ovulatory forms), quality of food, and body weight (Essah et al., 2008; Wild et al., 2011). When subset analyses including only BMI-matched patients were performed, LDL-C was still higher in women with PCOS. It clearly indicates that PCOS per se is responsible for increased lipid values, although the absolute value and the related cardiovascular risk may be different among individual

Only one study reported mean values of LDL-C <100 mg/dL. Four studies reported mean concentrations in PCOS women 130 mg/dL, whereas no study showed mean LDL-C values >130 mg/dL in control subjects. Considering the reported variances, it is not uncommon for individual PCOS patients to have LDL concentrations >160 mg/dL. Elevated nonHDL-C was reported as a common abnormality in PCOS women included in the studies. 21 of the 31 comparisons, registered nonHDL-C >130 mg/dL and the mean difference was still considerably higher with BMI matching (16 mg/dL vs. control subjects). Similarly to what was found for LDL-C, higher nonHDL-C levels were generally reported in PCOS subjects whose mean BMI was higher. There were clear differences with PCOS even when the BMI was in the nonobese or nonoverweight categories. This suggests that obesity or overweight (and higher insulin resistance) is not the only factor that accounts for elevated LDL-C and nonHDL-C in PCOS (Carmina, 2009; Wild et al., 1985).

However, though decreased HDL-C (particularly HDL2, the most antiatherogenic HDL subtype) (Legro et al., 2001; Diamanti-Kandarakis et al., 2007, as cited in Hoffman & Ehrmann, 2008) may represent the most common lipid alteration in PCOS (Essah et al., 2008), and many studies have reported elevated levels of TG as compared to controls, a very recent meta-analysis performed by Wild et al. (2011) shows that altered levels of TG are not commonly identified in many populations (Carmina et al., 2003, as cited in Wild et al., 2011; Essah et al., 2008). Only three studies reported TG concentrations that exceeded 150 mg/dL,

However, two main patterns of lipid alteration were described separately or combined (Wild et al., 2011). The most common pattern is probably classic atherogenic dyslipidemia (increased TG, low HDL-C, and increased small dense LDL subclasses + increased nonHDL-C). This lipid pattern is similar to that found in type 2 DM, and it is mainly the consequence of insulin resistance that impairs the ability of insulin to suppress lipolysis, thereby increasing mobilization of free fatty acids (FFA) from adipose stores. Consequently, increased hepatic delivery of FFAs impairs insulin inhibition of hepatic very low-density lipoprotein 1 synthesis, causing altered catabolism of very low-density lipoprotein (Brunzell et al., 2003 as cited in Wild et al., 2010). Additionally, the ability of insulin resistance to alter the expression of lipoprotein lipase and hepatic lipase may also contribute to this lipid pattern (Wild et al., 1985). Sustaining this hypothesis, insulin resistance was linked with hyperlipidemia in most of the studies including women with PCOS (Slowinska-Srzednicka et al., 1991, as cited in Sukalich & Guzick, 2003). Due to the fact that excessive adipose tissue increases insulin resistance, this pattern is likely to be found in obese patients with PCOS. These lipid abnormalities are further augmented among those women who develop glucose intolerance in association with PCOS. The magnitude of androgen elevation, race and

and these were found in women who were overweight or obese (Wild et al., 2011).

patients (Wild et al., 2011).

Obesity and excess weight are among the most widely met chronic diseases in the Western world countries. The prevalence of increased BMI ranges between 30% and 80% among women with PCOS (Vribkova & Hainer, 2009). Moreover, it has been widely proved that 50– 60% of women with PCOS have a body fat distribution of the android type irrespective of their BMI (Barber et al., 2006) and that patients with PCOS had a central fat excess independent of total fat mass (Lambrinoudaki, 2011; Puder et al., 2005; Ilie et al., 2008).

Obesity also increases hyperandrogenism, hirsutism, insulin resistance and metabolic disorders, infertility and pregnancy complications both independently and by exacerbating PCOS phenotypic expression (Balen et al., 1995, Kiddy et al., 1990, as cited in Teede et al., 2010a). Therefore, in spite of the lack of epidemiological data, the increasing epidemic of obesity worldwide is thought to facilitate the high prevalence of PCOS in the general

The Polycystic Ovary Syndrome Status – A Risk Factor for Future Cardiovascular Disease 161

Insulin resistance occurs in around 50% to 80% of women with PCOS (Wild et al., 2000, as cited in Wild et al., 2010), and in 95% of obese women with PCOS (Carmina & Lobo, 2004, DeUgarte et al., 2005, as cited in Wild et al., 2010), primarily in the more severe NIH diagnosed PCOS. Lean women and milder Rotterdam diagnosed PCOS seem to be less affected by severe insulin resistance. Ethnicity may also be an independent factor that contributes to the risk for insulin resistance and glucose intolerance. For example, though there are few data related to the relatively higher prevalence of PCOS among women of South Asian origin, there is some evidence that the latter group of women are more likely to be affected by insulin resistance than Caucasian women (Rodin et al., 1998, Wijeyaratne et al., 2002, Balen et al., 2005, Bhathena, 2007, as cited in Bathena, 2011). Women with PCOS manifest IR independently and additively with obesity, with PCOS and obesity acting synergistically to impair insulin sensitivity (Dunaif et al., 1989, as cited in Wild et al., 2010). A family history of type 2 diabetes is also more widely met among PCOS women with IGT or type 2 DM compared with those with normal glucose tolerance (Ehrmann et al., 2005, as cited in Dokras, 2008). Furthermore, in spite of the variability in reports concerning the prevalence of prediabetes and DM among women with PCOS, most studies confirm that women with PCOS, especially obese PCOS women, have a higher prevalence of impaired fasting glucose (IFG), IGT and DM (Chang & Wild, 2009), and a risk to develop the disease at an earlier age than the general population (Sukalich & Guzick, 2003). PCOS is now recognized by the ADA as a leading risk factor for DM screening in adolescent girls (Palmert et al., 2002, as cited in Dokras, 2008) and premenopausal women irrespective of

Mechanisms involved in insulin resistance are likely to be complex with genetic and environmental contributors. Specific abnormalities of insulin metabolism identified in PCOS include reductions in secretion, reduced hepatic extraction, impaired suppression of hepatic gluconeogenesis and abnormalities in insulin receptor signaling. Interestingly, there is a paradoxical expression of insulin resistance in PCOS whereby insulin-stimulated androgen production persists while its role in glucose metabolism is impaired (Dunaif, 1997). Therefore, insulin resistance in PCOS results in hyperinsulinaemia with its associated diverse and complex effects on regulating lipid metabolism, protein synthesis and

Lean women with PCOS often have abnormalities of insulin secretion and action compared to weight-matched control subjects. An overweight woman with PCOS may also demonstrate extrinsic insulin resistance associated with adiposity, which can be mechanistically distinct from the insulin resistance present in lean women with PCOS. Only a subgroup of women with insulin resistance and PCOS develops coexistent pancreatic insufficiency with , cell failure followed by type 2 (non-insulin-dependent) DM. In this setting, insulin output cannot overcome resistance and hyperglycaemia develops (Dunaif et al., 1989, Dunaif and Finegood, 1996, Ehrmann et al., 1999, Legro et al., 1999, Kelly et al., 2000, Goodarzi and Korenman, 2003,

Overall, the risk of IGT in premenopausal PCOS women in the United States may be 25 to 40% and that of type 2 DM 4 to 10%, irrespective of race (Dokras, 2008). Solomon et al.

**3.1.4.1 Insulin resistance and abnormal glucose metabolism** 

race and ethnicity (Dokras, 2009).

modulation of androgen production.

Balen et al., 2005, Ehrmann, 2005, as cited in Bahthena, 2011).

**3.1.4.2 Impaired glucose tolerance (IGT) and type 2 diabetes mellitus** 

population. It is of utmost importance to underline the fact that the defining characteristic of PCOS is visceral fat, rather than subcutaneous one, as visceral fat plays an important role in the proinflammatory response. Accumulation of visceral fat leads to insulin resistance, endothelial dysfunction and a proinflammatory status through fat-derived metabolic products, hormones (adiponectin, resistin, FFA) and cytokines (interleukin-1 (IL-1), interleukin-6 (IL-6), interleukin-18 (IL-18), tumor necrosis factor-+ (TNF-+). Although many researches (Puder et al., 2005; Cascella et al*.*, 2008; Svendsen et al*.*, 2008, as cited in Penaforte et al., 2011) have advanced the idea that women with PCOS accumulate fat mainly in the upper body compared to controls matched for weight and age (Gambineri et al*.*, 2002, as cited in Penaforte et al., 2011), other studies, including ours, did not detect any differences in total body fat, abdominal fat, visceral fat or trunk fat (Faloia et al., 2004; Glintborg et al., 2006; Barber et al*.*, 2008, as cited in Penaforte et al., 2011; Carmina et al*.,* 2007; Ilie et al., 2011), or trunk to peripheral fat (arm fat + leg fat + head fat) ratio in obese women with and without the syndrome (Svendsen et al*.*, 2008, as cited in Penaforte et al., 2011), either.

We have already underlined the fact that the presence of obesity has a big impact on the development and expression of PCOS (for instance, in a woman who has PCO but does not meet diagnostic criteria for PCOS, obesity can cause disorders such as: menstrual irregularity and hirsutism, thus completing the PCOS diagnosis) (Kiddy et al., 1990 as cited in Farrel &.Antoni, 2010). There is evidence that molecules secreted by adipose tissue such as leptin, TNF-+, IL-6 might also influence adrenal and ovarian function (Escobar-Morreale et al., 2007). In fact, PCO is present in approximately 20% of all women and many of these women do not display other syndrome features (i.e., hirsutism, irregular menstrual cycles, and elevated testosterone), which would likely appear if a PCO woman were to become overweight or obese (Polson et al., 1998 as cited in Farrel &.Antoni, 2010). The prevention of obesity, then, can be a crucial factor against the increased incidence of PCOS and its associated physiological abnormalities, especially during childhood and preadolescent years (Farrel & Antoni, 2010). Whether obesity represents a factor amplifying intrinsic hormonal and metabolic components of PCOS or, alternatively, whether it has a direct pathophysiological role is still a matter of debate. However, overall, all the reports suggest that obesity (especially abdominal obesity) and PCOS interact to promote premature atherosclerosis and increase CV mortality (Guzick et al., 1996; Birdsall et al., 1997; Christian et al., 2003, as cited in Amato et al., 2011; Pierpoint et al., 1998; Wild et al., 2000; Shaw et al., 2008).

#### **3.1.4 Insulin resistance, glucose intolerance, diabetes mellitus**

The American Heart Association and American Diabetes Association (ADA) consider the diagnosis of DM a CV disease equivalent to a prior MI. Insulin resistance is considered one of the main pathogenic factors behind the development of PCOS (Svendsen et al., 2010). Hyperandrogenism and insulin resistance were first linked in 1921 when Achard and Thiers (1921) published their classic description of a bearded woman with diabetes. This link has been confirmed ever since by many investigators (Legro, 2006; Svenden et al., 2010) and insulin resistance is now considered the main pathogenic factor in the development of PCOS. Many of the late complications of PCOS, primarily diabetes, dyslipidemia, and CVD also seem to be connected to insulin resistance (Dunaif, 1997, as cited in Svendson et al., 2010). Moreover, IGT and MS, as predictors of type 2 DM and premature CVD mortality, are more widely met in women with PCOS (odds ratio, approximately 4:1) (Ehrmann et al., 1999, Legro et al., 1999, as cited in Wild et al., 2010).

#### **3.1.4.1 Insulin resistance and abnormal glucose metabolism**

160 Recent Advances in Cardiovascular Risk Factors

population. It is of utmost importance to underline the fact that the defining characteristic of PCOS is visceral fat, rather than subcutaneous one, as visceral fat plays an important role in the proinflammatory response. Accumulation of visceral fat leads to insulin resistance, endothelial dysfunction and a proinflammatory status through fat-derived metabolic products, hormones (adiponectin, resistin, FFA) and cytokines (interleukin-1 (IL-1), interleukin-6 (IL-6), interleukin-18 (IL-18), tumor necrosis factor-+ (TNF-+). Although many researches (Puder et al., 2005; Cascella et al*.*, 2008; Svendsen et al*.*, 2008, as cited in Penaforte et al., 2011) have advanced the idea that women with PCOS accumulate fat mainly in the upper body compared to controls matched for weight and age (Gambineri et al*.*, 2002, as cited in Penaforte et al., 2011), other studies, including ours, did not detect any differences in total body fat, abdominal fat, visceral fat or trunk fat (Faloia et al., 2004; Glintborg et al., 2006; Barber et al*.*, 2008, as cited in Penaforte et al., 2011; Carmina et al*.,* 2007; Ilie et al., 2011), or trunk to peripheral fat (arm fat + leg fat + head fat) ratio in obese women with and

without the syndrome (Svendsen et al*.*, 2008, as cited in Penaforte et al., 2011), either.

2011; Pierpoint et al., 1998; Wild et al., 2000; Shaw et al., 2008).

1999, Legro et al., 1999, as cited in Wild et al., 2010).

**3.1.4 Insulin resistance, glucose intolerance, diabetes mellitus** 

We have already underlined the fact that the presence of obesity has a big impact on the development and expression of PCOS (for instance, in a woman who has PCO but does not meet diagnostic criteria for PCOS, obesity can cause disorders such as: menstrual irregularity and hirsutism, thus completing the PCOS diagnosis) (Kiddy et al., 1990 as cited in Farrel &.Antoni, 2010). There is evidence that molecules secreted by adipose tissue such as leptin, TNF-+, IL-6 might also influence adrenal and ovarian function (Escobar-Morreale et al., 2007). In fact, PCO is present in approximately 20% of all women and many of these women do not display other syndrome features (i.e., hirsutism, irregular menstrual cycles, and elevated testosterone), which would likely appear if a PCO woman were to become overweight or obese (Polson et al., 1998 as cited in Farrel &.Antoni, 2010). The prevention of obesity, then, can be a crucial factor against the increased incidence of PCOS and its associated physiological abnormalities, especially during childhood and preadolescent years (Farrel & Antoni, 2010). Whether obesity represents a factor amplifying intrinsic hormonal and metabolic components of PCOS or, alternatively, whether it has a direct pathophysiological role is still a matter of debate. However, overall, all the reports suggest that obesity (especially abdominal obesity) and PCOS interact to promote premature atherosclerosis and increase CV mortality (Guzick et al., 1996; Birdsall et al., 1997; Christian et al., 2003, as cited in Amato et al.,

The American Heart Association and American Diabetes Association (ADA) consider the diagnosis of DM a CV disease equivalent to a prior MI. Insulin resistance is considered one of the main pathogenic factors behind the development of PCOS (Svendsen et al., 2010). Hyperandrogenism and insulin resistance were first linked in 1921 when Achard and Thiers (1921) published their classic description of a bearded woman with diabetes. This link has been confirmed ever since by many investigators (Legro, 2006; Svenden et al., 2010) and insulin resistance is now considered the main pathogenic factor in the development of PCOS. Many of the late complications of PCOS, primarily diabetes, dyslipidemia, and CVD also seem to be connected to insulin resistance (Dunaif, 1997, as cited in Svendson et al., 2010). Moreover, IGT and MS, as predictors of type 2 DM and premature CVD mortality, are more widely met in women with PCOS (odds ratio, approximately 4:1) (Ehrmann et al., Insulin resistance occurs in around 50% to 80% of women with PCOS (Wild et al., 2000, as cited in Wild et al., 2010), and in 95% of obese women with PCOS (Carmina & Lobo, 2004, DeUgarte et al., 2005, as cited in Wild et al., 2010), primarily in the more severe NIH diagnosed PCOS. Lean women and milder Rotterdam diagnosed PCOS seem to be less affected by severe insulin resistance. Ethnicity may also be an independent factor that contributes to the risk for insulin resistance and glucose intolerance. For example, though there are few data related to the relatively higher prevalence of PCOS among women of South Asian origin, there is some evidence that the latter group of women are more likely to be affected by insulin resistance than Caucasian women (Rodin et al., 1998, Wijeyaratne et al., 2002, Balen et al., 2005, Bhathena, 2007, as cited in Bathena, 2011). Women with PCOS manifest IR independently and additively with obesity, with PCOS and obesity acting synergistically to impair insulin sensitivity (Dunaif et al., 1989, as cited in Wild et al., 2010).

A family history of type 2 diabetes is also more widely met among PCOS women with IGT or type 2 DM compared with those with normal glucose tolerance (Ehrmann et al., 2005, as cited in Dokras, 2008). Furthermore, in spite of the variability in reports concerning the prevalence of prediabetes and DM among women with PCOS, most studies confirm that women with PCOS, especially obese PCOS women, have a higher prevalence of impaired fasting glucose (IFG), IGT and DM (Chang & Wild, 2009), and a risk to develop the disease at an earlier age than the general population (Sukalich & Guzick, 2003). PCOS is now recognized by the ADA as a leading risk factor for DM screening in adolescent girls (Palmert et al., 2002, as cited in Dokras, 2008) and premenopausal women irrespective of race and ethnicity (Dokras, 2009).

Mechanisms involved in insulin resistance are likely to be complex with genetic and environmental contributors. Specific abnormalities of insulin metabolism identified in PCOS include reductions in secretion, reduced hepatic extraction, impaired suppression of hepatic gluconeogenesis and abnormalities in insulin receptor signaling. Interestingly, there is a paradoxical expression of insulin resistance in PCOS whereby insulin-stimulated androgen production persists while its role in glucose metabolism is impaired (Dunaif, 1997). Therefore, insulin resistance in PCOS results in hyperinsulinaemia with its associated diverse and complex effects on regulating lipid metabolism, protein synthesis and modulation of androgen production.

Lean women with PCOS often have abnormalities of insulin secretion and action compared to weight-matched control subjects. An overweight woman with PCOS may also demonstrate extrinsic insulin resistance associated with adiposity, which can be mechanistically distinct from the insulin resistance present in lean women with PCOS. Only a subgroup of women with insulin resistance and PCOS develops coexistent pancreatic insufficiency with , cell failure followed by type 2 (non-insulin-dependent) DM. In this setting, insulin output cannot overcome resistance and hyperglycaemia develops (Dunaif et al., 1989, Dunaif and Finegood, 1996, Ehrmann et al., 1999, Legro et al., 1999, Kelly et al., 2000, Goodarzi and Korenman, 2003, Balen et al., 2005, Ehrmann, 2005, as cited in Bahthena, 2011).

#### **3.1.4.2 Impaired glucose tolerance (IGT) and type 2 diabetes mellitus**

Overall, the risk of IGT in premenopausal PCOS women in the United States may be 25 to 40% and that of type 2 DM 4 to 10%, irrespective of race (Dokras, 2008). Solomon et al.

The Polycystic Ovary Syndrome Status – A Risk Factor for Future Cardiovascular Disease 163

history of IGT, DM2 and CVD in PCOS and further research is needed, the International Diabetes Federation (IDF) has identified PCOS as a major non-modifiable risk factor

It has become more and more obvious that IGT has significant clinical relevance and its early identification and intervention improve long-term outcomes and can prevent IGT

There are currently no generic guidelines for IGT screening, only for type 2 DM based on fasting glucose or more recently on HbA1c as a first line. However, impaired fasting glucose cannot accurately predict IGT in women, in general, and in PCOS (Teede et al., 2010a). As a consequence, and taking into account the high prevalence of insulin resistance, IGT and type 2 DM in PCOS, as well as their implications in the pathogenesis of this disease and especially in the onset of type 2 DM and of CVD, recently, the AE-PCOS Board recommend that a 2-h post 75-g oral glucose challenge be performed in PCOS women with a BMI greater than 30 kg/m2, or alternatively in lean PCOS women with advanced age (>40 yr), personal history of GDM, or family history of type 2 DM (Salley et al., 2007). Those with IGT should be screened annually for developing type 2 DM, acknowledging efficacy of treating IGT, but not necessarily impaired fasting glucose, to prevent type 2 DM (Salley et al., 2007). Hemoglobin A1c above 6.5% has been proposed as the defining criterion for diabetes (Lorenzo & Haffner, 2010). This criterion was proposed for risk assessment, but further studies will be needed to determine whether this criterion is useful in implementing lifestyle

associated with type 2 DM (Alberti et al., 2007, as cited in Teede et al., 2010a).

interventions and medical management for CVD prevention (Wild et al., 2010).

Besides the traditional CVD risk factors, a large number of markers that have been proposed as "nontraditional" CVD risk factors (adiponectin, leptin, CRP, IL-6, plasminogen activator inhibitor 1 (PAI-1) or serum amyloid A) and were shown to contribute to accelerated atherosclerosis in diabetes might also be involved in the pathogenesis of vascular disease in PCOS. Platelet function abnormalities, alterations in the coagulation cascade or a prothrombotic state (reduced fibrinolysis and raised level of PAI-1 or elevated clotting factors such as fibrinogen) can be seen with diabetes and hyperinsulinemia. Elevated levels of CRP, or of inflammatory cytokines such as TNF-+ and IL-6, IL-18, of matrix metalloproteinases, fibrinogen, ET-1, of white blood cell and platelet counts have been reported in women with PCOS (Wild et al., 2010; Jovanovic et al., as cited in Lambrinoudaki, 2011; Lorenz & Wild et al., 2007; Essah et al., 2007). Talbott et al. also identified significantly increased mean PAI-1 levels in women with PCOS (28 ng/mL) compared with controls (19 ng/mL), a relationship that remained after adjustments for BMI and insulin levels (Talbott et al., 2000b). Moreover, the increased level of PAI activity in PCOS was directly correlated with insulin resistance and it decreased with improvement in insulin sensitivity, either through weight loss or through the use of sensitizing agents, thus implicating it as a contributing cardiovascular risk factor (Cho et al., 2007). No differences, however, in PAI-1 between women with PCOS and controls have been identified by other studies (Atiomo et al., 2000, Yarali et al., 2001, as cited in Sukalich & Guzick, 2003). Further research regarding

Elevated concentrations of circulating homocysteine have been identified as another cardiovascular risk factor, causing endothelial oxidative stress and platelet aggregation and

**3.2 Other risk factors-nontraditional cardiovascular risk factors** 

PAI-1 in women with PCOS is needed to resolve this discrepancy.

progression to DM, including in high-risk PCOS women.

examined the risk for type 2 DM development in the 106,052 women enrolled in the Nurses' Health Study. Women with menstrual cycles greater than 40 days or irregular cycles had an age-adjusted relative risk of type 2 DM of 2.42 (95% confidence interval, 1.81-3.24) compared with normally cycling controls (Solomon et al., 1998, as cited in Sukalich & Guzick, 2003). But do women with PCOS switch to DM at higher rates?

Women with PCOS in the United States who have IGT have been reported to convert to DM anywhere from 6% over 3 years to 13.4% over 8 years in older women (Ehrmann et al., 1999, Legro et al., 2005, as cited in Chang & Wild, 2009). Smaller samples have reported rates of 29% (4 of 14) over 2 years and 54% (7 of 13) after 6 years (Norman et al., 2001, as cited in Chang & Wild, 2009).

Additionally, the rate of conversion from IGT to type 2 DM in a general Australian population was estimated in the large cohort Australian Diabetes, Obesity and Lifestyle (AusDiab) study at 2.9% per year for young females (Barr et al., 2005, as cited in Teede et al., 2010a). Another Australian study has reported a substantially higher conversion rate (8.7% per year over 6.2 years) in women with PCOS (Norman et al., 2001, as cited in Teede et al., 2010a). However, this has not been uniformly reported (Legro et al., 2005, as cited in Teede et al., 2010a). In the United States, women with normal glucose tolerance at baseline had a 16% conversion to IGT per year, and those with baseline IGT had a 6% conversion rate over ~3 years, or 2% per year (Legro et al., 2005, as cited in Dokras, 2009). In another study, classic PCOS patients had a 5-fold risk of developing type 2 DM over 8 yr *vs.* age- and weight-matched controls, although only 12% of PCOS patients without obesity developed glucose abnormalities (Wild et al., 2010).

Comparatively, other studies reported conversion rates to DM of 25% and 66% over 5 and 10 years in the high-risk Pima Indian population (Saad et al., 1988, as cited in Chang & Wild, 2009) and 50% over 5 years in Latina women with a previous history of gestational diabetes (Kim et al., 2002, as cited in Chang & Wild, 2009). Conversion rates for normoglycemic women to IGT vary from 9 to 16%, to as high as 40% (Chang & Wild, 2009). Collectively, these data may support high rates of conversion from IGT to DM and normal glucose tolerance to IGT, but the rates are not higher than those of other at-risk populations. These data are also incomplete due to the fact that the presence or absence of PCOS status according to race has not been defined in the populations referred to. Finally, with the RR of developing DM 6.90 (95% CI, 4.35 to 10.94) over 8 years in women with the MS (established in much larger cohorts though also among older women), the assessment of the MS and the prevention of conversion to DM are of high clinical relevance for women with or without PCOS (Chang & Wild, 2009).

Alarmingly, IGT and type 2 DM are highly prevalent among PCOS adolescents (Palmert et al., 2002, as cited in Wild et al., 2010). Although incident data are not rigorous, up to 40% of women with classic PCOS develop IGT or type 2 DM by the fourth decade of life and their glycemic control is seriously affected by age and weight gain. (Boudreaux et al., 2006, Legro et al., 2005, Norman et al., 2001, as cited in Wild et al., 2010).

Women with PCOS also have higher gestational diabetes (GDM) risk, with a recent metaanalysis reporting an odds ratio (OR) of 2.94 (Boomsma et al., 2006). The risk of GDM occurs both independent of and is exacerbated by obesity (Boudreaux et al., 2006, Legro et al., 1999, as cited in Teede et al., 2010a). Though there are few studies to adequately assess the natural

examined the risk for type 2 DM development in the 106,052 women enrolled in the Nurses' Health Study. Women with menstrual cycles greater than 40 days or irregular cycles had an age-adjusted relative risk of type 2 DM of 2.42 (95% confidence interval, 1.81-3.24) compared with normally cycling controls (Solomon et al., 1998, as cited in Sukalich & Guzick, 2003).

Women with PCOS in the United States who have IGT have been reported to convert to DM anywhere from 6% over 3 years to 13.4% over 8 years in older women (Ehrmann et al., 1999, Legro et al., 2005, as cited in Chang & Wild, 2009). Smaller samples have reported rates of 29% (4 of 14) over 2 years and 54% (7 of 13) after 6 years (Norman et al., 2001, as cited in

Additionally, the rate of conversion from IGT to type 2 DM in a general Australian population was estimated in the large cohort Australian Diabetes, Obesity and Lifestyle (AusDiab) study at 2.9% per year for young females (Barr et al., 2005, as cited in Teede et al., 2010a). Another Australian study has reported a substantially higher conversion rate (8.7% per year over 6.2 years) in women with PCOS (Norman et al., 2001, as cited in Teede et al., 2010a). However, this has not been uniformly reported (Legro et al., 2005, as cited in Teede et al., 2010a). In the United States, women with normal glucose tolerance at baseline had a 16% conversion to IGT per year, and those with baseline IGT had a 6% conversion rate over ~3 years, or 2% per year (Legro et al., 2005, as cited in Dokras, 2009). In another study, classic PCOS patients had a 5-fold risk of developing type 2 DM over 8 yr *vs.* age- and weight-matched controls, although only 12% of PCOS patients without obesity developed

Comparatively, other studies reported conversion rates to DM of 25% and 66% over 5 and 10 years in the high-risk Pima Indian population (Saad et al., 1988, as cited in Chang & Wild, 2009) and 50% over 5 years in Latina women with a previous history of gestational diabetes (Kim et al., 2002, as cited in Chang & Wild, 2009). Conversion rates for normoglycemic women to IGT vary from 9 to 16%, to as high as 40% (Chang & Wild, 2009). Collectively, these data may support high rates of conversion from IGT to DM and normal glucose tolerance to IGT, but the rates are not higher than those of other at-risk populations. These data are also incomplete due to the fact that the presence or absence of PCOS status according to race has not been defined in the populations referred to. Finally, with the RR of developing DM 6.90 (95% CI, 4.35 to 10.94) over 8 years in women with the MS (established in much larger cohorts though also among older women), the assessment of the MS and the prevention of conversion to DM are of high clinical relevance for women with or without

Alarmingly, IGT and type 2 DM are highly prevalent among PCOS adolescents (Palmert et al., 2002, as cited in Wild et al., 2010). Although incident data are not rigorous, up to 40% of women with classic PCOS develop IGT or type 2 DM by the fourth decade of life and their glycemic control is seriously affected by age and weight gain. (Boudreaux et al., 2006, Legro

Women with PCOS also have higher gestational diabetes (GDM) risk, with a recent metaanalysis reporting an odds ratio (OR) of 2.94 (Boomsma et al., 2006). The risk of GDM occurs both independent of and is exacerbated by obesity (Boudreaux et al., 2006, Legro et al., 1999, as cited in Teede et al., 2010a). Though there are few studies to adequately assess the natural

et al., 2005, Norman et al., 2001, as cited in Wild et al., 2010).

But do women with PCOS switch to DM at higher rates?

Chang & Wild, 2009).

glucose abnormalities (Wild et al., 2010).

PCOS (Chang & Wild, 2009).

history of IGT, DM2 and CVD in PCOS and further research is needed, the International Diabetes Federation (IDF) has identified PCOS as a major non-modifiable risk factor associated with type 2 DM (Alberti et al., 2007, as cited in Teede et al., 2010a).

It has become more and more obvious that IGT has significant clinical relevance and its early identification and intervention improve long-term outcomes and can prevent IGT progression to DM, including in high-risk PCOS women.

There are currently no generic guidelines for IGT screening, only for type 2 DM based on fasting glucose or more recently on HbA1c as a first line. However, impaired fasting glucose cannot accurately predict IGT in women, in general, and in PCOS (Teede et al., 2010a). As a consequence, and taking into account the high prevalence of insulin resistance, IGT and type 2 DM in PCOS, as well as their implications in the pathogenesis of this disease and especially in the onset of type 2 DM and of CVD, recently, the AE-PCOS Board recommend that a 2-h post 75-g oral glucose challenge be performed in PCOS women with a BMI greater than 30 kg/m2, or alternatively in lean PCOS women with advanced age (>40 yr), personal history of GDM, or family history of type 2 DM (Salley et al., 2007). Those with IGT should be screened annually for developing type 2 DM, acknowledging efficacy of treating IGT, but not necessarily impaired fasting glucose, to prevent type 2 DM (Salley et al., 2007). Hemoglobin A1c above 6.5% has been proposed as the defining criterion for diabetes (Lorenzo & Haffner, 2010). This criterion was proposed for risk assessment, but further studies will be needed to determine whether this criterion is useful in implementing lifestyle interventions and medical management for CVD prevention (Wild et al., 2010).

#### **3.2 Other risk factors-nontraditional cardiovascular risk factors**

Besides the traditional CVD risk factors, a large number of markers that have been proposed as "nontraditional" CVD risk factors (adiponectin, leptin, CRP, IL-6, plasminogen activator inhibitor 1 (PAI-1) or serum amyloid A) and were shown to contribute to accelerated atherosclerosis in diabetes might also be involved in the pathogenesis of vascular disease in PCOS. Platelet function abnormalities, alterations in the coagulation cascade or a prothrombotic state (reduced fibrinolysis and raised level of PAI-1 or elevated clotting factors such as fibrinogen) can be seen with diabetes and hyperinsulinemia. Elevated levels of CRP, or of inflammatory cytokines such as TNF-+ and IL-6, IL-18, of matrix metalloproteinases, fibrinogen, ET-1, of white blood cell and platelet counts have been reported in women with PCOS (Wild et al., 2010; Jovanovic et al., as cited in Lambrinoudaki, 2011; Lorenz & Wild et al., 2007; Essah et al., 2007). Talbott et al. also identified significantly increased mean PAI-1 levels in women with PCOS (28 ng/mL) compared with controls (19 ng/mL), a relationship that remained after adjustments for BMI and insulin levels (Talbott et al., 2000b). Moreover, the increased level of PAI activity in PCOS was directly correlated with insulin resistance and it decreased with improvement in insulin sensitivity, either through weight loss or through the use of sensitizing agents, thus implicating it as a contributing cardiovascular risk factor (Cho et al., 2007). No differences, however, in PAI-1 between women with PCOS and controls have been identified by other studies (Atiomo et al., 2000, Yarali et al., 2001, as cited in Sukalich & Guzick, 2003). Further research regarding PAI-1 in women with PCOS is needed to resolve this discrepancy.

Elevated concentrations of circulating homocysteine have been identified as another cardiovascular risk factor, causing endothelial oxidative stress and platelet aggregation and

The Polycystic Ovary Syndrome Status – A Risk Factor for Future Cardiovascular Disease 165

high prevalence. These factors are particularly relevant to the pathogenesis of OSA in women with PCOS (Nitsche & Ehrmann, 2010). Both WHR, a measure of central obesity, and circulating total testosterone were correlated with AHI. The androgenization of the PCOS women promotes development of central obesity, which has been established in other studies as a predictor for OSA. However, others have suggested that insulin resistance was the strongest predictor of OSA, stronger than were age, BMI or circulating testosterone

Nitsche (2010) has even proposed that there may be two "subtypes" of PCOS, i.e. PCOS with or without OSA, and these two subtypes may be associated with distinct metabolic and endocrine abnormalities. PCOS women with OSA may be at much higher risk for diabetes and CVD than PCOS women without OSA and may benefit from therapeutic interventions

Metabolic syndrome is a conglomeration of multiple interrelated risk factors for CVD and type 2 DM, occurring 'more often than by chance alone'. These include atherogenic dyslipidemia, elevated blood pressure and blood glucose levels, along with central obesity (Bhattacharya, 2011). Although there is a general agreement regarding the main components of the MS, at least six diagnostic definitions have been proposed, including those formulated by the Adult Treatment Panel-III (ATP-III), the IDF and the World Health Organization (WHO). This variation requires different cut-off points and inclusion criteria (Day, 2007, as cited in Kandaraki et al., 2009). The definition proposed by the National Cholesterol Education Program Adult Treatment Panel III (NCEP ATPIII) (Grundi et al., 2004, as cited in Kandaraki et al., 2009) is the most commonly used for clinical and research purposes. The IDF has proposed the most recent criteria, which resemble the NCEP ones, with the exception that central obesity, assessed according to ethnicity-specific cut-offs, is an integral

Along with the epidemic of obesity, the prevalence of MS is increasing worldwide, both in the developing and developed countries. As noted previously, MS is associated with a risk of CVD and is a common early abnormality in the development of type 2 DM. In addition, MS plays a well-recognised role in the development of OSA, erectile dysfunction, PCOS and

MS and PCOS are undoubtedly common afflictions in women of reproductive age in the general population. The significant interconnection between MS and PCOS when studied in combination is noteworthy. Namely, MS is significantly more prevalent in women with PCOS than in their age-matched counterparts from the general population. This PCOS-MS overlap is not singularly met in Caucasian women with PCOS. A highly prevalent MS has also been identified in Brazilian, Chinese, Korean, Indian and in multiracial PCOS populations, at least in overweight/obese patients, in spite of the fact that there are variations in the MS prevalence rates dependent on ethnic/racial regions, age, the diagnostic criteria and the comparison group studied (Fig.1) (Soares et al., 2008; Cheung et al., 2008; Park et al., 2007; Bhattacharya, 2008; Glueck et al., 2003; Apridonidze et al., 2005;

(Vgontzas et al., 2001, as cited in Cho et al., 2007).

part of the IDF definition (Kandaraki et al., 2009).

malignant tumours (Baranova et al., 2011).

Ehrmann et al., 2006; Dokras et al., 2005).

**4. Metabolic syndrome in PCOS** 

targeted to decrease the severity of OSA (Nitsche & Ehrmann, 2010).

consequently leading to atherosclerosis. Significantly elevated concentrations of homocysteine have been documented in both lean and obese women with PCOS versus normal controls (Yarali et al., 2001, as cited in Sukalich & Guzick, 2003; Lorenz & Wild, 2007). Several but not all studies have found insulin resistance to be the most important predictor of increased homocysteine (Lorenz & Wild, 2007).

Adiponectin, a crucial adipocytokine, may also have a protective role in vascular damage in PCOS. Adiponectin has been shown to inhibit endothelial inflammation, to stimulate the production of NO in the endothelium, protecting blood vessels from the damage associated with insulin resistance. The majority of studies performed on women with PCOS demonstrate decreased level of adiponectin, compared to controls (Escobar-Morreale et al., 2006; Ardawi et al., 2005; Carmina et al., 2006b). Moreover, it was shown that hypoadiponectinemia is present in PCOS independent of BMI, with WHR, WC and free testosterone levels as the major determinants of decreased concentrations of adiponectin (Escobar-Morreale et al., 2006; Gulcelik et al., 2008; Ilie et al., 2008). These studies suggest that abdominal adiposity is characteristically associated with hypoadiponectinemia; hyperandrogenemia may also have a role, probably by facilitating an abdominal deposition of fat. A negative correlation was found between levels of adiponectin and IMT in a PCOS group compared with matched controls, apparently independent of the well known association of carotid change with insulin resistance and BMI, suggesting that the decrease in adiponectin may be an independent risk factor for the development of endothelial damage in PCOS (Carmina et al., 2006b).

Obstructive sleep apnea (OSA) is an independent CV risk factor and women with PCOS are 5-30 times more likely to have this disorder than are controls (Hoffman & Ehrmann, 2008). Moreover, it was suggested that the prevalence of OSA among women with PCOS is equal to, or may even exceed that in men. As previously noted, OSA is characterized by the combination of episodic sleep disruption and hypoxemia, each of which can trigger at least three major hormonal responses: activation of the hypothalamic-pituitary-adrenal (HPA) axis with increased cortisol production/secretion, increased catecholamine output from sympathetic nervous system stimulation, and increased release of adipokines from adipose tissue. These responses appear to contribute to the metabolic abnormalities associated with OSA, particularly to the decline in insulin sensitivity and glucose tolerance (Nitsche & Ehrmann, 2010).

The risk of OSA is increased as a function of both total body fat mass as well as body fat distribution. Visceral fat appears to be more metabolically active and the quantity of visceral fat has been shown to highly correlate with OSA risk (Nitsche & Ehrmann, 2010). Furthermore, disordered breathing during sleep has been found to be more common in PCOS than controls, even when controlled for BMI (Gopal et al., 2002; Fogel et al., 2001, as cited in Cho et al., 2007). In two studies (Vgontzas et al., 2001, Gopal et al., 2002, as cited in Nitsche & Ehrmann, 2010), the severity of sleep apnea did not correlate with BMI and in a third one (Fogel et al., 2001, as cited in Nitsche & Ehrmann, 2010), even after controlling for BMI, PCOS women were as much as 30 times more likely to have sleep disordered breathing and 9 times more likely than controls to have daytime sleepiness (Nitsche & Ehrmann, 2010). Overall, all these data indicate that the high prevalence of OSA in women with this disorder cannot be fully attributed to excess adiposity and that additional factors (e.g. hyperandrogenemia that is characteristic of PCOS or insulin resistance) might explain the high prevalence. These factors are particularly relevant to the pathogenesis of OSA in women with PCOS (Nitsche & Ehrmann, 2010). Both WHR, a measure of central obesity, and circulating total testosterone were correlated with AHI. The androgenization of the PCOS women promotes development of central obesity, which has been established in other studies as a predictor for OSA. However, others have suggested that insulin resistance was the strongest predictor of OSA, stronger than were age, BMI or circulating testosterone (Vgontzas et al., 2001, as cited in Cho et al., 2007).

Nitsche (2010) has even proposed that there may be two "subtypes" of PCOS, i.e. PCOS with or without OSA, and these two subtypes may be associated with distinct metabolic and endocrine abnormalities. PCOS women with OSA may be at much higher risk for diabetes and CVD than PCOS women without OSA and may benefit from therapeutic interventions targeted to decrease the severity of OSA (Nitsche & Ehrmann, 2010).

#### **4. Metabolic syndrome in PCOS**

164 Recent Advances in Cardiovascular Risk Factors

consequently leading to atherosclerosis. Significantly elevated concentrations of homocysteine have been documented in both lean and obese women with PCOS versus normal controls (Yarali et al., 2001, as cited in Sukalich & Guzick, 2003; Lorenz & Wild, 2007). Several but not all studies have found insulin resistance to be the most important

Adiponectin, a crucial adipocytokine, may also have a protective role in vascular damage in PCOS. Adiponectin has been shown to inhibit endothelial inflammation, to stimulate the production of NO in the endothelium, protecting blood vessels from the damage associated with insulin resistance. The majority of studies performed on women with PCOS demonstrate decreased level of adiponectin, compared to controls (Escobar-Morreale et al., 2006; Ardawi et al., 2005; Carmina et al., 2006b). Moreover, it was shown that hypoadiponectinemia is present in PCOS independent of BMI, with WHR, WC and free testosterone levels as the major determinants of decreased concentrations of adiponectin (Escobar-Morreale et al., 2006; Gulcelik et al., 2008; Ilie et al., 2008). These studies suggest that abdominal adiposity is characteristically associated with hypoadiponectinemia; hyperandrogenemia may also have a role, probably by facilitating an abdominal deposition of fat. A negative correlation was found between levels of adiponectin and IMT in a PCOS group compared with matched controls, apparently independent of the well known association of carotid change with insulin resistance and BMI, suggesting that the decrease in adiponectin may be an independent risk factor for the development of endothelial

Obstructive sleep apnea (OSA) is an independent CV risk factor and women with PCOS are 5-30 times more likely to have this disorder than are controls (Hoffman & Ehrmann, 2008). Moreover, it was suggested that the prevalence of OSA among women with PCOS is equal to, or may even exceed that in men. As previously noted, OSA is characterized by the combination of episodic sleep disruption and hypoxemia, each of which can trigger at least three major hormonal responses: activation of the hypothalamic-pituitary-adrenal (HPA) axis with increased cortisol production/secretion, increased catecholamine output from sympathetic nervous system stimulation, and increased release of adipokines from adipose tissue. These responses appear to contribute to the metabolic abnormalities associated with OSA, particularly to the decline in insulin sensitivity and glucose tolerance (Nitsche &

The risk of OSA is increased as a function of both total body fat mass as well as body fat distribution. Visceral fat appears to be more metabolically active and the quantity of visceral fat has been shown to highly correlate with OSA risk (Nitsche & Ehrmann, 2010). Furthermore, disordered breathing during sleep has been found to be more common in PCOS than controls, even when controlled for BMI (Gopal et al., 2002; Fogel et al., 2001, as cited in Cho et al., 2007). In two studies (Vgontzas et al., 2001, Gopal et al., 2002, as cited in Nitsche & Ehrmann, 2010), the severity of sleep apnea did not correlate with BMI and in a third one (Fogel et al., 2001, as cited in Nitsche & Ehrmann, 2010), even after controlling for BMI, PCOS women were as much as 30 times more likely to have sleep disordered breathing and 9 times more likely than controls to have daytime sleepiness (Nitsche & Ehrmann, 2010). Overall, all these data indicate that the high prevalence of OSA in women with this disorder cannot be fully attributed to excess adiposity and that additional factors (e.g. hyperandrogenemia that is characteristic of PCOS or insulin resistance) might explain the

predictor of increased homocysteine (Lorenz & Wild, 2007).

damage in PCOS (Carmina et al., 2006b).

Ehrmann, 2010).

Metabolic syndrome is a conglomeration of multiple interrelated risk factors for CVD and type 2 DM, occurring 'more often than by chance alone'. These include atherogenic dyslipidemia, elevated blood pressure and blood glucose levels, along with central obesity (Bhattacharya, 2011). Although there is a general agreement regarding the main components of the MS, at least six diagnostic definitions have been proposed, including those formulated by the Adult Treatment Panel-III (ATP-III), the IDF and the World Health Organization (WHO). This variation requires different cut-off points and inclusion criteria (Day, 2007, as cited in Kandaraki et al., 2009). The definition proposed by the National Cholesterol Education Program Adult Treatment Panel III (NCEP ATPIII) (Grundi et al., 2004, as cited in Kandaraki et al., 2009) is the most commonly used for clinical and research purposes. The IDF has proposed the most recent criteria, which resemble the NCEP ones, with the exception that central obesity, assessed according to ethnicity-specific cut-offs, is an integral part of the IDF definition (Kandaraki et al., 2009).

Along with the epidemic of obesity, the prevalence of MS is increasing worldwide, both in the developing and developed countries. As noted previously, MS is associated with a risk of CVD and is a common early abnormality in the development of type 2 DM. In addition, MS plays a well-recognised role in the development of OSA, erectile dysfunction, PCOS and malignant tumours (Baranova et al., 2011).

MS and PCOS are undoubtedly common afflictions in women of reproductive age in the general population. The significant interconnection between MS and PCOS when studied in combination is noteworthy. Namely, MS is significantly more prevalent in women with PCOS than in their age-matched counterparts from the general population. This PCOS-MS overlap is not singularly met in Caucasian women with PCOS. A highly prevalent MS has also been identified in Brazilian, Chinese, Korean, Indian and in multiracial PCOS populations, at least in overweight/obese patients, in spite of the fact that there are variations in the MS prevalence rates dependent on ethnic/racial regions, age, the diagnostic criteria and the comparison group studied (Fig.1) (Soares et al., 2008; Cheung et al., 2008; Park et al., 2007; Bhattacharya, 2008; Glueck et al., 2003; Apridonidze et al., 2005; Ehrmann et al., 2006; Dokras et al., 2005).

The Polycystic Ovary Syndrome Status – A Risk Factor for Future Cardiovascular Disease 167

2007). Despite being obviously higher than the US population-based estimates, these prevalence rates may, to some extent, reflect the impact of the high prevalence of obesity in the above populations – independently of PCOS per se (Glueck et al., 2003; Apridonidze et al., 2005; Ehrmann et al., 2006; Dokras et al., 2005). Furthermore, in three of the above US studies, the included control groups were not specifically selected (Glueck et al., 2003; Apridonidze et al., 2005; Ehrmann et al., 2006). In another US study (Dokras et al., 2005), the apparent preponderance of MS in the PCOS group was eliminated when non-obese PCOS patients were compared with age and BMI- matched controls, while the difference between the obese subgroups of patients and controls was reduced to non-significant levels. Thus, the actual impact of MS in women with PCOS as compared to controls, evenly matched for

Moreover, European studies among PCOS populations with lower BMI and also other non-US studies have reported significantly lower prevalence rates of MS than the ones reported by the US studies (Carmina et al., 2006a; Vural et al., 2005; Vrbikova et al., 2005). In spite of these different results, in some studies (Carmina et al., 2006a, c; Vural et al., 2005), but not all (Vrbikova et al., 2005), the MS has been shown to be more prevalent in European PCOS

For instance, in Italy, where women with PCOS have a lower mean body weight and less frequently increased serum TG than US PCOS, MS is less common but still 4 times more frequent in PCOS patients than in the general female population of similar age. Patients with mild PCOS phenotype (ovulatory PCOS) have a lower prevalence of MS but, in these patients too, MS is twice more frequent than in the normal population (Carmina, 2006c). Carmina et al. (2006a) sought to determine the prevalence of MS in Italian women using both the ATP-III and the WHO criteria. Using ATP-III criteria, the prevalence of MS was 8.2% and, using WHO criteria, it was 16% in Italian women with PCOS, higher than in controls, where the prevalence was 2.4% using both methods. Regarding the influence of the way in which PCOS is diagnosed, the MS prevalence was higher (8.9% by ATP-III, 17.3% by WHO) in classic PCOS patients than in ovulatory PCOS (5% and 10.6% respectively). Body weight significantly modified prevalence rates (Carmina et al., 2006a). Furthermore, in a very recent study, the same researchers found a relatively low prevalence of MS (7.1%) in Mediterranean PCOS, but higher than in normoweight and BMI-matched controls (2.4% and 3.5%, respectively) (Rizzo et al., 2011). Additionally, in a Czech study, MS (defined by IDF adolescent criteria) was present in five adolescents (11,62%) with PCOS (defined according to the ESHRE criteria) in comparison with one healthy girl. When comparing the prevalence of adolescents with at least one feature of MS, there was no difference between PCOS (17 out of 43) and healthy controls (27 out of 48) (VrbÍková et al., 2010a). Moreover, using ATP III criteria this time, MS was detected in 28.7% of those 179 women with PCOS. The most frequent features were an increased WC, decreased concentration of HDL - C (both in 96%), and increased BP (88%). Increased TG (49%) and impaired fasting blood glucose or type 2

Another study performed on Caucasian women also found an approximate 4-fold increase in the prevalence of MS in women with PCOS compared with the age-matched female population (33% by WHO, 37% by NCEP-ATP-III and 40% by IDF criteria in PCOS subjects, compared with 10% by NCEP-ATP-III and 13% by IDF in controls) (Cussons et al., 2008). A similar prevalence was found also in a German study using IDF criteria (Hahn et al., 2007, as

age and BMI, awaits further investigation.

patients than in controls of similar ethnicity and age.

DM (37.3%) were less common (VrbÍková et al., 2010b).

Fig. 1. The prevalence of metabolic syndrome in studies of PCOS

Data are presented as %. Studies are listed on the pattern: first author - the definition of the MS - the number of participants included in the study. Wherever unspecified (in the brackets), the criteria used for the diagnosis of PCOS were the NIH criteria. Except for the studies of Carmina, who examined both NIH and ESHRE/ASRM criteria, the rest of the mentioned researches used either NIH or ESHRE criteria (as mentioned). NIH, National Institute of Health; ESHRE/ASRM, European Society for Human Reproduction and Embryology/American Society for Reproductive Medicine

Four US studies in predominantly obese women with PCOS have reported that 33.4% to 47.3% of these women fulfill the NCEP ATPIII criteria for MS (Glueck et al., 2003; Apridonidze et al., 2005; Ehrmann et al., 2006; Dokras et al., 2005). Apridonidze et al. (2005) performed a study on 106 women with PCOS and recorded a 43% prevalence rate, which is twice higher than the age-adjusted rate of 24% in women of all ages in the general population based on data from the Third National Health and Human Examination Survey III (NHANES III). The prevalence of MS in PCOS women, divided by decade of life registered the following values: women between ages 20 and 29 - 45% and women between ages 30 and 39 - 53%, compared with 6% and 15%, respectively, in women in the general US population of the same age ranges. Notably, the prevalence rate of the MS in women with PCOS between ages 30 and 39 (53%) was even higher than the reported 44% rate observed in women aged 60 to 69 years from the NHANES III study. Similar results were reported by Dokras et al. (2005) who discovered, in a retrospective study carried on 129 women with PCOS and 177 normal controls, that the age-adjusted prevalence rate of the MS was 47.3% compared with a 4.3% rate in controls. They also observed that when compared by age group, MS was significantly more prevalent in PCOS subjects than in controls (Essah et al.,

46.2 47.3

14.5

43.0

4.7

16.0

8.9

17.3

8.2

33.8

20.0

33.4

Fig. 1. The prevalence of metabolic syndrome in studies of PCOS

28.7

0.0 5.0 10.0 15.0 20.0 25.0 30.0 35.0 40.0 45.0 50.0

40.0

33.0 37.0

46.4

11.6

Embryology/American Society for Reproductive Medicine

Data are presented as %. Studies are listed on the pattern: first author - the definition of the MS - the number of participants included in the study. Wherever unspecified (in the brackets), the criteria used for the diagnosis of PCOS were the NIH criteria. Except for the studies of Carmina, who examined both NIH and ESHRE/ASRM criteria, the rest of the mentioned researches used either NIH or ESHRE criteria (as mentioned). NIH, National Institute of Health; ESHRE/ASRM, European Society for Human Reproduction and

Four US studies in predominantly obese women with PCOS have reported that 33.4% to 47.3% of these women fulfill the NCEP ATPIII criteria for MS (Glueck et al., 2003; Apridonidze et al., 2005; Ehrmann et al., 2006; Dokras et al., 2005). Apridonidze et al. (2005) performed a study on 106 women with PCOS and recorded a 43% prevalence rate, which is twice higher than the age-adjusted rate of 24% in women of all ages in the general population based on data from the Third National Health and Human Examination Survey III (NHANES III). The prevalence of MS in PCOS women, divided by decade of life registered the following values: women between ages 20 and 29 - 45% and women between ages 30 and 39 - 53%, compared with 6% and 15%, respectively, in women in the general US population of the same age ranges. Notably, the prevalence rate of the MS in women with PCOS between ages 30 and 39 (53%) was even higher than the reported 44% rate observed in women aged 60 to 69 years from the NHANES III study. Similar results were reported by Dokras et al. (2005) who discovered, in a retrospective study carried on 129 women with PCOS and 177 normal controls, that the age-adjusted prevalence rate of the MS was 47.3% compared with a 4.3% rate in controls. They also observed that when compared by age group, MS was significantly more prevalent in PCOS subjects than in controls (Essah et al., 2007). Despite being obviously higher than the US population-based estimates, these prevalence rates may, to some extent, reflect the impact of the high prevalence of obesity in the above populations – independently of PCOS per se (Glueck et al., 2003; Apridonidze et al., 2005; Ehrmann et al., 2006; Dokras et al., 2005). Furthermore, in three of the above US studies, the included control groups were not specifically selected (Glueck et al., 2003; Apridonidze et al., 2005; Ehrmann et al., 2006). In another US study (Dokras et al., 2005), the apparent preponderance of MS in the PCOS group was eliminated when non-obese PCOS patients were compared with age and BMI- matched controls, while the difference between the obese subgroups of patients and controls was reduced to non-significant levels. Thus, the actual impact of MS in women with PCOS as compared to controls, evenly matched for age and BMI, awaits further investigation.

Moreover, European studies among PCOS populations with lower BMI and also other non-US studies have reported significantly lower prevalence rates of MS than the ones reported by the US studies (Carmina et al., 2006a; Vural et al., 2005; Vrbikova et al., 2005). In spite of these different results, in some studies (Carmina et al., 2006a, c; Vural et al., 2005), but not all (Vrbikova et al., 2005), the MS has been shown to be more prevalent in European PCOS patients than in controls of similar ethnicity and age.

For instance, in Italy, where women with PCOS have a lower mean body weight and less frequently increased serum TG than US PCOS, MS is less common but still 4 times more frequent in PCOS patients than in the general female population of similar age. Patients with mild PCOS phenotype (ovulatory PCOS) have a lower prevalence of MS but, in these patients too, MS is twice more frequent than in the normal population (Carmina, 2006c). Carmina et al. (2006a) sought to determine the prevalence of MS in Italian women using both the ATP-III and the WHO criteria. Using ATP-III criteria, the prevalence of MS was 8.2% and, using WHO criteria, it was 16% in Italian women with PCOS, higher than in controls, where the prevalence was 2.4% using both methods. Regarding the influence of the way in which PCOS is diagnosed, the MS prevalence was higher (8.9% by ATP-III, 17.3% by WHO) in classic PCOS patients than in ovulatory PCOS (5% and 10.6% respectively). Body weight significantly modified prevalence rates (Carmina et al., 2006a). Furthermore, in a very recent study, the same researchers found a relatively low prevalence of MS (7.1%) in Mediterranean PCOS, but higher than in normoweight and BMI-matched controls (2.4% and 3.5%, respectively) (Rizzo et al., 2011). Additionally, in a Czech study, MS (defined by IDF adolescent criteria) was present in five adolescents (11,62%) with PCOS (defined according to the ESHRE criteria) in comparison with one healthy girl. When comparing the prevalence of adolescents with at least one feature of MS, there was no difference between PCOS (17 out of 43) and healthy controls (27 out of 48) (VrbÍková et al., 2010a). Moreover, using ATP III criteria this time, MS was detected in 28.7% of those 179 women with PCOS. The most frequent features were an increased WC, decreased concentration of HDL - C (both in 96%), and increased BP (88%). Increased TG (49%) and impaired fasting blood glucose or type 2 DM (37.3%) were less common (VrbÍková et al., 2010b).

Another study performed on Caucasian women also found an approximate 4-fold increase in the prevalence of MS in women with PCOS compared with the age-matched female population (33% by WHO, 37% by NCEP-ATP-III and 40% by IDF criteria in PCOS subjects, compared with 10% by NCEP-ATP-III and 13% by IDF in controls) (Cussons et al., 2008). A similar prevalence was found also in a German study using IDF criteria (Hahn et al., 2007, as

The Polycystic Ovary Syndrome Status – A Risk Factor for Future Cardiovascular Disease 169

However, in a more recent study carried on a mixed population of obese and non-obese Caucasian PCOS women, comparable rates of MS have been reported, irrespective of the criteria used for MS diagnosis (NCEP, the WHO or the IDF criteria) (Cussons et al., 2008). Last but not least, the use of the 2009 "joint interim criteria" has just begun. The retrospective design is another weakness of the majority of the available studies. Differences in the exclusion criteria of each study should be also considered in the interpretation of their

It has been shown that women with PCOS have high CV risk factors. Whether this risk


means an increased clinical disease is still debatable (Sukalich & Guzick, 2003).




Note: ET-1 = endothelin-1, sVCAM = soluble vascular cell adhesion molecules; sICAM = soluble intercellular adhesion molecule; ADMA = asymmetric dimethylarginine; PAI = plasminogen activator inhibitor-1; tPA = tissue plasminogen activator, vWF = vonWillebrand factor; hs-CRP = highly sensitive C reactive protein; TNF-+ = tumor necrosis factor alpha; IL-1 = interleukin 1; IL-6 = interleukin 6;

Atherosclerosis is a systemic process the main characteristics of which is chronic inflammation, a disorder that affects all vascular territories and modifies their structure and function, ultimately leading to arterial thrombosis. Due to the fact that there is a long latency phase before clinical symptoms manifest during the progression of this disorder, the possibility of assessing arterial function before angiographically detectable atherosclerotic plaques appear could have a significant role in the early detection and evaluation of the risk

varying results (Kandaraki et al., 2009).

Functional studies

Morphological studies

for CVD (Soares et al., 2009).

FFA = free fatty acid

 Carotid wall thickness Arterial calcification

 Ventricular function Arterial stiffness Endothelial function Vascular tone

> - Vascular reactivity - Vasoconstrictors: ET-1

Markers of inflammation

Markers of endothelial activation

Markers of coagulation/fibrinolysis

Table 1. Studies of markers of subclinical cardiovascular disease

**5. Markers of sub-clinical cardiovascular disease** 

cited in Cussons et al., 2008). The most common individual component of the MS present in the PCOS group was an elevated WC, followed by reduced HDL, then insulin resistance indicated by the homeostasis model assessment of insulin resistance (HOMA-IR). The least prevalent individual component was an elevated fasting glucose (Cussons et al., 2008). The adverse metabolic milieu of the syndrome can affect not only adults but also adolescents with PCOS (Diamanti-Kandarakis et al., 2008, as cited in Kandaraki et al., 2009). This is extremely likely to generate an increased prevalence of MS in this age group of patients, too. To explore this intuitively plausible concept, a few studies have addressed the prevalence of MS in adolescents with PCOS and the results are contradictory (Coviello et al., 2006, Rossi et al., 2008, as cited in Kandaraki et al., 2009). In an Indian study, MS (defined by IDF criteria) was found in 46.2% of females with PCOS, with both adolescent and adults being similarly affected. Regarding the individual component of the MS, dyslipidaemia and elevated BP were more common than fasting glucose abnormalities in both the adolescents and adults groups (Bhattacharya, 2008). Furthermore, the first study from India utilizing the 2009 "joint interim criteria" reported recently that the adolescents with PCOS were reported to have 4.26 times more chances of developing MS compared to those without (Bhattacharya, 2011). The prevalence of MS, diagnosed by IDF criteria, was 4.7% in the studied Chinese adolescents with PCOS. Metabolic disorders were common in these adolescents as more than one third of them exhibited at least one component of MS. Central obesity and dyslipidemia were the two most common metabolic features, which was also the case in adult PCOS patients according to a previous study (Huang et al., 2010). And finally, compared with Caucasians and Chinese women in Westernized societies, mainland Chinese women with PCOS were found to have a low risk of MS, as, overall, the prevalence of MS was 6.4% and its presence does not vary across the specific four PCOS phenotypes, according to the 2003 Rotterdam consensus criteria (range of 2.3-12.2%) (Guo et al., 2010).

However, there are some important limitations that should be mentioned. Most of these studies have not included patients and controls properly matched for age and BMI. Thus, not only the presence of PCOS but also BMI differences may have contributed to the above findings. The ethnic variations in the rates of MS reported by different studies may be attributable not only to anthropometric differences between diverse ethnicity, but also to differences in the criteria used for PCOS diagnosis. The selection of PCOS patients was based on NIH criteria in studies from the United States, but mostly on Rotterdam criteria in European or other non-US countries. The patients fulfilling the NIH definition of PCOS are expected to be more severely metabolically affected than the patients selected by Rotterdam criteria. Consequently, the MS rates were lower in women with hyperandrogenism and polycystic ovarian morphology, but normal ovulation, as well as in those with anovulation and PCO, but normal androgen levels when compared with women with classic PCOS (Kandaraki et al., 2009). The NCEP-ATP III criteria have been implemented for the diagnosis of MS by the majority of worldwide PCOS studies. However, there was one study that has employed both the WHO (gives more importance to the presence of insulin resistance or glucose intolerance) and the NCEP ATP III criteria, and has proposed the former set as more discriminating and more appropriate for the evaluation of MS in PCOS women with lower degrees of obesity (Carmina et al., 2006a). Accordingly, another study reported a significantly higher MS prevalence in PCOS women than in controls when using the WHO criteria, while the NCEP criteria identified a considerably lower MS in PCOS women, and thus could not show any difference between PCOS women and controls (Vural et al., 2005). However, in a more recent study carried on a mixed population of obese and non-obese Caucasian PCOS women, comparable rates of MS have been reported, irrespective of the criteria used for MS diagnosis (NCEP, the WHO or the IDF criteria) (Cussons et al., 2008). Last but not least, the use of the 2009 "joint interim criteria" has just begun. The retrospective design is another weakness of the majority of the available studies. Differences in the exclusion criteria of each study should be also considered in the interpretation of their varying results (Kandaraki et al., 2009).

#### **5. Markers of sub-clinical cardiovascular disease**

It has been shown that women with PCOS have high CV risk factors. Whether this risk means an increased clinical disease is still debatable (Sukalich & Guzick, 2003).

Functional studies

168 Recent Advances in Cardiovascular Risk Factors

cited in Cussons et al., 2008). The most common individual component of the MS present in the PCOS group was an elevated WC, followed by reduced HDL, then insulin resistance indicated by the homeostasis model assessment of insulin resistance (HOMA-IR). The least prevalent individual component was an elevated fasting glucose (Cussons et al., 2008). The adverse metabolic milieu of the syndrome can affect not only adults but also adolescents with PCOS (Diamanti-Kandarakis et al., 2008, as cited in Kandaraki et al., 2009). This is extremely likely to generate an increased prevalence of MS in this age group of patients, too. To explore this intuitively plausible concept, a few studies have addressed the prevalence of MS in adolescents with PCOS and the results are contradictory (Coviello et al., 2006, Rossi et al., 2008, as cited in Kandaraki et al., 2009). In an Indian study, MS (defined by IDF criteria) was found in 46.2% of females with PCOS, with both adolescent and adults being similarly affected. Regarding the individual component of the MS, dyslipidaemia and elevated BP were more common than fasting glucose abnormalities in both the adolescents and adults groups (Bhattacharya, 2008). Furthermore, the first study from India utilizing the 2009 "joint interim criteria" reported recently that the adolescents with PCOS were reported to have 4.26 times more chances of developing MS compared to those without (Bhattacharya, 2011). The prevalence of MS, diagnosed by IDF criteria, was 4.7% in the studied Chinese adolescents with PCOS. Metabolic disorders were common in these adolescents as more than one third of them exhibited at least one component of MS. Central obesity and dyslipidemia were the two most common metabolic features, which was also the case in adult PCOS patients according to a previous study (Huang et al., 2010). And finally, compared with Caucasians and Chinese women in Westernized societies, mainland Chinese women with PCOS were found to have a low risk of MS, as, overall, the prevalence of MS was 6.4% and its presence does not vary across the specific four PCOS phenotypes, according to the 2003 Rotterdam consensus criteria (range of 2.3-12.2%) (Guo et al., 2010).

However, there are some important limitations that should be mentioned. Most of these studies have not included patients and controls properly matched for age and BMI. Thus, not only the presence of PCOS but also BMI differences may have contributed to the above findings. The ethnic variations in the rates of MS reported by different studies may be attributable not only to anthropometric differences between diverse ethnicity, but also to differences in the criteria used for PCOS diagnosis. The selection of PCOS patients was based on NIH criteria in studies from the United States, but mostly on Rotterdam criteria in European or other non-US countries. The patients fulfilling the NIH definition of PCOS are expected to be more severely metabolically affected than the patients selected by Rotterdam criteria. Consequently, the MS rates were lower in women with hyperandrogenism and polycystic ovarian morphology, but normal ovulation, as well as in those with anovulation and PCO, but normal androgen levels when compared with women with classic PCOS (Kandaraki et al., 2009). The NCEP-ATP III criteria have been implemented for the diagnosis of MS by the majority of worldwide PCOS studies. However, there was one study that has employed both the WHO (gives more importance to the presence of insulin resistance or glucose intolerance) and the NCEP ATP III criteria, and has proposed the former set as more discriminating and more appropriate for the evaluation of MS in PCOS women with lower degrees of obesity (Carmina et al., 2006a). Accordingly, another study reported a significantly higher MS prevalence in PCOS women than in controls when using the WHO criteria, while the NCEP criteria identified a considerably lower MS in PCOS women, and thus could not show any difference between PCOS women and controls (Vural et al., 2005).

	- Vascular tone
		- Vascular reactivity
		- Vasoconstrictors: ET-1
		- Vadsodilators: products of nitric oxide
	- Markers of endothelial activation
		- Adhesion molecules (sVCAM, sICAM, E- and P-selectin), ADMA
	- Markers of coagulation/fibrinolysis
		- PAI-1, tPA, fibrinogen, trombomodulin, vWF
	- Markers of inflammation
		- hs-CRP, TNF-+ , IL-1, IL-6, ferritine, homocysteine
	- Hormones and substrates with known vascular effects
		- Adiponectin, resistin, visfatin, FFA, leptin, ghrelin
	- Carotid wall thickness
	- Arterial calcification

Note: ET-1 = endothelin-1, sVCAM = soluble vascular cell adhesion molecules; sICAM = soluble intercellular adhesion molecule; ADMA = asymmetric dimethylarginine; PAI = plasminogen activator inhibitor-1; tPA = tissue plasminogen activator, vWF = vonWillebrand factor; hs-CRP = highly sensitive C reactive protein; TNF-+ = tumor necrosis factor alpha; IL-1 = interleukin 1; IL-6 = interleukin 6; FFA = free fatty acid

Table 1. Studies of markers of subclinical cardiovascular disease

Atherosclerosis is a systemic process the main characteristics of which is chronic inflammation, a disorder that affects all vascular territories and modifies their structure and function, ultimately leading to arterial thrombosis. Due to the fact that there is a long latency phase before clinical symptoms manifest during the progression of this disorder, the possibility of assessing arterial function before angiographically detectable atherosclerotic plaques appear could have a significant role in the early detection and evaluation of the risk for CVD (Soares et al., 2009).

The Polycystic Ovary Syndrome Status – A Risk Factor for Future Cardiovascular Disease 171

echocardiography (Yarali et al., 2001, as cited in Sukalich & Guzick, 2003). They reported that patients with PCOS had slower mitral E velocity, a lower mitral E/A ratio and shorter IVRT than a group of healthy controls, suggesting that LV diastolic function was impaired in PCOS. However, in this study, patients with PCOS were older and had higher LDL cholesterol and BMI than those found in other patients (Tekin et al., 2009). Of note, they did not report BPs measures of the study population. Thus, it is unclear whether the impaired diastolic function could be attributed to presence of PCOS, per se, or to the inclusion of sicker subjects in PCOS group. On the contrary, Tekin et al. (2009) reported that there were no significant differences between patients with PCOS and control subject with respect to EF, mitral E/A, DT, IVRT and pulmonary velocity. They showed that the tissue doppler profiles of patients with PCOS and controls were also not significantly different. Topcu et al. assessed coronary flow reserve in patients with PCOS, similar in terms of age, BMI, total cholesterol, smoking status and BP with the women in the study of Tekin et al. (2009), by using echocardiographically determined colour Doppler flow mapping of left anterior descending artery. They ended up with normal coronary flow reserve in patients with PCOS who had no associated CV risk factors. They also noticed that mitral E velocity, mitral A velocity and E/A were not significantly different between patients with PCOS and controls (Topcu et al., 2006, as cited in Tekin et al., 2009). Selcoki et al.(2010) also suggest that there are no significant differences in certain conventional and tissue Doppler echocardiographic measures of cardiac function between patients with PCOS and control groups. However, their PCOS populations have low BMI, normal BP, low TG, LDL and total cholesterol. Furthermore, no significant differences were found in insulin, HOMA-IR, HDL, LDL and total cholesterol, and TG levels between the two groups. Hence, these echocardiographic findings support previous reports (Topcu et al., 2006, as cited in Tekin et al., 2009) Tekin et al., (2009) indicating that diastolic function is preserved in young patients with PCOS who have no associated CV risk factors. Risk reduction regimens may be particularly important to those with other recognized risk factors for CVD to herald the progression of the disease.

Further studies with obese, older and dyslipidemic subjects are needed.

One of the major contributing factors to CV morbidity and mortality in patients with hypertension is arterial stiffness. The reduced elasticity of central arteries may play an important role in the early changes that predispose to the development of major vascular disease (Sasaki et al., 2011). Reduced arterial compliance and increased arterial stiffness represent independent risk factors for CVD and may favour the development and progression of hypertension, LV hypertrophy, MI and heart failure (Safar &London, 2000, as

The ankle–brachial index (ABI) – the ratio of ankle-to-brachial systolic blood pressure (SBP) – is a simple, non-invasive, reliable method to estimate the presence of peripheral arterial occlusive disease. The brachial–ankle pulse wave velocity (baPWV) is known to be a marker for both the severity of vascular damage and the prognosis of atherosclerotic vascular diseases in patients with hypertension, end-stage renal failure and DM. The carotid augmentation index (cAI), which is augmentation expressed as a percentage of the pulse pressure and influenced by the vascular tone of the small muscular arteries, is associated with the presence and severity of coronary artery disease, particularly in younger and

**5.1.2 Arterial stiffness** 

cited in Soares et al., 2008).

This early detection of CVD risk can be performed with the help of noninvasive methods that permit the assessment of arterial structure and function. Currently, several such approaches exist; echographic measurements of arterial elasticity, IMT and endothelial function are clinically useful techniques for assessing arterial structure and function (Table 1). As a first approximation to studying the translation of risk factors into disease, data are accumulating on "preclinical" CVD of the carotid and coronary arteries as well as changes in vascular function (Sukalich & Guzick, 2003).

#### **5.1 Functional studies**

Vascular damage plays a key role in the development and progression of atherosclerosis and has been evaluated as an early risk marker of atherosclerosis and an independent predictor of future cardiac events (Sasaki et al., 2011).

#### **5.1.1 Ventricular function**

Left ventricular (LV) diastolic dysfunction is an early manifestation of diabetic cardiomyopathy and atherosclerotic CVD and has been shown to identify hypertensive patients at increased risk of CV events (Cussons et al., 2006). Its aetiology is multifactorial and relates to coronary artery disease, hypertension, autonomic neuropathy, microangiopathy, dyslipidaemia, insulin resistance, endothelial dysfunction and oxidative stress (Aurigemma et al., 2004, as cited in Cussons et al., 2006). Specific myocardial mechanisms include altered cardiomyocyte substrate metabolism and bioenergetics, altered collagen metabolism, inflammation and fibrosis (Watts et al., 2003, as cited in Cussons et al., 2006). Ventricular systolic and diastolic dysfunction may be an early finding for coronary heart disease in patients with PCOS (Kosmala et al., 2008).

In a case-control, echocardiographic study, women with PCOS were found to have an increased isovolumetric relaxation time (IVRT), an index of early LV diastolic dysfunction, and lower ejection fraction (EF) compared with weight matched controls (Tiras et al., 1999, as cited in Cussons et al., 2006). These changes were linked to the presence of a nonrestrictive type of diastolic dysfunction and LV stiffness in patients with PCOS. However, E/A ratio, and DT were not significantly different between patients with PCOS and control. A significant direct relationship between plasma insulin levels and IVRT was demonstrated in the PCOS group. These findings were consistent with another report showing an independent correlation between hyperinsulinaemia and LV mass (Orio et al., 2004, as cited in Cussons et al., 2006). The existing studies sustain the hypothesis that insulin resistance may contribute to myocardial dysfunction in PCOS. Moreover, one study also predicted impaired coronary flow reserve and decreased myocardial glucose utilisation in PCOS related to insulin resistance and type 2 DM (Iozzo et al., as cited in Cussons et al., 2006).

Orio et al. studied the prevalence of LV hypertrophy and diastolic filling and systolic performance by echocardiography in a selected cohort of young women with PCOS. These authors found that patients with PCOS had significantly higher interventricular septum, left ventricle posterior wall thickness, end-systolic volume and lower LV EF, E/A ratio than controls. Nevertheless, all patients had normal LV EF and only two out of 30 patients with PCOS had abnormal E/A ratio (Orio et al., 2004, as cited in Tekin et al., 2009). Yarali et al. assessed systolic and diastolic function in 30 women with PCOS and 30 controls with echocardiography (Yarali et al., 2001, as cited in Sukalich & Guzick, 2003). They reported that patients with PCOS had slower mitral E velocity, a lower mitral E/A ratio and shorter IVRT than a group of healthy controls, suggesting that LV diastolic function was impaired in PCOS. However, in this study, patients with PCOS were older and had higher LDL cholesterol and BMI than those found in other patients (Tekin et al., 2009). Of note, they did not report BPs measures of the study population. Thus, it is unclear whether the impaired diastolic function could be attributed to presence of PCOS, per se, or to the inclusion of sicker subjects in PCOS group. On the contrary, Tekin et al. (2009) reported that there were no significant differences between patients with PCOS and control subject with respect to EF, mitral E/A, DT, IVRT and pulmonary velocity. They showed that the tissue doppler profiles of patients with PCOS and controls were also not significantly different. Topcu et al. assessed coronary flow reserve in patients with PCOS, similar in terms of age, BMI, total cholesterol, smoking status and BP with the women in the study of Tekin et al. (2009), by using echocardiographically determined colour Doppler flow mapping of left anterior descending artery. They ended up with normal coronary flow reserve in patients with PCOS who had no associated CV risk factors. They also noticed that mitral E velocity, mitral A velocity and E/A were not significantly different between patients with PCOS and controls (Topcu et al., 2006, as cited in Tekin et al., 2009). Selcoki et al.(2010) also suggest that there are no significant differences in certain conventional and tissue Doppler echocardiographic measures of cardiac function between patients with PCOS and control groups. However, their PCOS populations have low BMI, normal BP, low TG, LDL and total cholesterol. Furthermore, no significant differences were found in insulin, HOMA-IR, HDL, LDL and total cholesterol, and TG levels between the two groups. Hence, these echocardiographic findings support previous reports (Topcu et al., 2006, as cited in Tekin et al., 2009) Tekin et al., (2009) indicating that diastolic function is preserved in young patients with PCOS who have no associated CV risk factors. Risk reduction regimens may be particularly important to those with other recognized risk factors for CVD to herald the progression of the disease. Further studies with obese, older and dyslipidemic subjects are needed.

#### **5.1.2 Arterial stiffness**

170 Recent Advances in Cardiovascular Risk Factors

This early detection of CVD risk can be performed with the help of noninvasive methods that permit the assessment of arterial structure and function. Currently, several such approaches exist; echographic measurements of arterial elasticity, IMT and endothelial function are clinically useful techniques for assessing arterial structure and function (Table 1). As a first approximation to studying the translation of risk factors into disease, data are accumulating on "preclinical" CVD of the carotid and coronary arteries as well as changes in

Vascular damage plays a key role in the development and progression of atherosclerosis and has been evaluated as an early risk marker of atherosclerosis and an independent

Left ventricular (LV) diastolic dysfunction is an early manifestation of diabetic cardiomyopathy and atherosclerotic CVD and has been shown to identify hypertensive patients at increased risk of CV events (Cussons et al., 2006). Its aetiology is multifactorial and relates to coronary artery disease, hypertension, autonomic neuropathy, microangiopathy, dyslipidaemia, insulin resistance, endothelial dysfunction and oxidative stress (Aurigemma et al., 2004, as cited in Cussons et al., 2006). Specific myocardial mechanisms include altered cardiomyocyte substrate metabolism and bioenergetics, altered collagen metabolism, inflammation and fibrosis (Watts et al., 2003, as cited in Cussons et al., 2006). Ventricular systolic and diastolic dysfunction may be an early finding for coronary

In a case-control, echocardiographic study, women with PCOS were found to have an increased isovolumetric relaxation time (IVRT), an index of early LV diastolic dysfunction, and lower ejection fraction (EF) compared with weight matched controls (Tiras et al., 1999, as cited in Cussons et al., 2006). These changes were linked to the presence of a nonrestrictive type of diastolic dysfunction and LV stiffness in patients with PCOS. However, E/A ratio, and DT were not significantly different between patients with PCOS and control. A significant direct relationship between plasma insulin levels and IVRT was demonstrated in the PCOS group. These findings were consistent with another report showing an independent correlation between hyperinsulinaemia and LV mass (Orio et al., 2004, as cited in Cussons et al., 2006). The existing studies sustain the hypothesis that insulin resistance may contribute to myocardial dysfunction in PCOS. Moreover, one study also predicted impaired coronary flow reserve and decreased myocardial glucose utilisation in PCOS related to insulin resistance and type 2 DM (Iozzo et al., as cited in Cussons et al., 2006).

Orio et al. studied the prevalence of LV hypertrophy and diastolic filling and systolic performance by echocardiography in a selected cohort of young women with PCOS. These authors found that patients with PCOS had significantly higher interventricular septum, left ventricle posterior wall thickness, end-systolic volume and lower LV EF, E/A ratio than controls. Nevertheless, all patients had normal LV EF and only two out of 30 patients with PCOS had abnormal E/A ratio (Orio et al., 2004, as cited in Tekin et al., 2009). Yarali et al. assessed systolic and diastolic function in 30 women with PCOS and 30 controls with

vascular function (Sukalich & Guzick, 2003).

predictor of future cardiac events (Sasaki et al., 2011).

heart disease in patients with PCOS (Kosmala et al., 2008).

**5.1 Functional studies** 

**5.1.1 Ventricular function** 

One of the major contributing factors to CV morbidity and mortality in patients with hypertension is arterial stiffness. The reduced elasticity of central arteries may play an important role in the early changes that predispose to the development of major vascular disease (Sasaki et al., 2011). Reduced arterial compliance and increased arterial stiffness represent independent risk factors for CVD and may favour the development and progression of hypertension, LV hypertrophy, MI and heart failure (Safar &London, 2000, as cited in Soares et al., 2008).

The ankle–brachial index (ABI) – the ratio of ankle-to-brachial systolic blood pressure (SBP) – is a simple, non-invasive, reliable method to estimate the presence of peripheral arterial occlusive disease. The brachial–ankle pulse wave velocity (baPWV) is known to be a marker for both the severity of vascular damage and the prognosis of atherosclerotic vascular diseases in patients with hypertension, end-stage renal failure and DM. The carotid augmentation index (cAI), which is augmentation expressed as a percentage of the pulse pressure and influenced by the vascular tone of the small muscular arteries, is associated with the presence and severity of coronary artery disease, particularly in younger and

The Polycystic Ovary Syndrome Status – A Risk Factor for Future Cardiovascular Disease 173

index (,) (CCA ,) was higher in PCOS than in control women and CCA distensibility was lower, indicating that young women with PCOS exhibit changes in vascular elasticity even in the absence of classical risk factors for CVD, such as hypertension, obesity and type 2 DM. These data suggest that patients with PCOS may be at higher risk for CVD, because their arterial distensibility is already reduced during atheroma formation and thus represents a subclinical sign of atherosclerosis (Mattsson et al., 2008, as cited in Soares et al., 2008). However, as the women from this study did not present with hyperglycaemia or hyperinsulinaemia, the increased stiffness index and the reduced distensibility of the carotid artery detected in PCOS women might be attributed to the hyperandrogenism inherent to PCOS and not to comorbid conditions associated with the syndrome (Soares et al., 2008).

On the contrary, and sustaining the results of Muneyyirci-Delate et al., Ketel et al. (2010) demonstrated in a small study that (central) obesity, but not PCOS, is associated with greater arterial stiffness (Muneyyirci-Delate et al., 2007, as cited in Ketel et al., 2010). Beside the fact that most of the studies have not distinguished the effects of obesity from those of PCOS, they also have not taken into consideration the fact that stiffness of elastic (e.g. the carotid) and muscular (e.g. the femoral) arteries may differ in their association with obesity and potentially PCOS. The study of Ketel et al. (2010) is the first comprehensive study taking into account stiffness in both elastic and muscular arteries in both lean and obese women with and without PCOS. Previous investigations, which investigated one type of artery, pulse wave velocity over elastic and (or) muscular vascular regions, or small and large arterial compliance, showed somehow discordant results. Nevertheless, in their research, Ketel et al. (2010) demonstrated that PCOS was not associated with stiffening of either

In conclusion, current studies suggest that differences in vascular function between normal women and women with PCOS may be very small if both are young, of normal weight and have normal BP. However, they showed that slightly increased BP, which weakly correlated with BMI, can be rightfully considered a risk factor for arterial stiffness in young women with PCOS. It has not been yet established whether these adverse effects of mild hypertension on arterial stiffness are characteristic to women with PCOS. We thus need further research on arterial stiffness in young women with mild hypertension caused by the other diseases (Sasaki et al., 2011). The mechanism for increased arterial stiffness reported in some studies of PCOS have not been cleared but, as in the metabolic syndrome and insulin resistance, may involve endothelial dysfunction and altered artery wall collagen metabolism (Cussons et al., 2006).

Endothelial dysfunction can be defined as the partial or complete loss of balance between vasoconstrictors and vasodilators, growth promoting and inhibiting factors, pro-atherogenic and anti-atherogenic factors, and pro-coagulant and anti-coagulant factors. Endothelial dysfunction is the initiating event in the development of atherogenesis and has been shown to precede the development of clinically detectable atherosclerotic plaques in the coronary arteries, being also implicated in the development of microvascular complications in diabetes (Caballero, 2005). Therefore, the assessment of endothelial function by different methods has emerged as a tool for detection of preclinical CVD. A common approach to the evaluation of endothelial function is the assessment of blood flow and vascular reactivity which allows the investigator to evaluate the status of the endothelial cells as well as that of

muscular or elastic arteries.

**5.1.3 Endothelial dysfunction** 

middle-aged male patients. Recently, these indices can be measured easily by simultaneous oscillometric measurement of pulse waves and they are widely used as arterial stiffness indices (Sasaki et al., 2011).

In a small case-control study, Kelly et al. reported increased pulse wave velocity of the brachial artery, but not of the aorta, in a young obese PCOS group (Kelly et al., 2002, as cited in Cussons et al., 2006). Similarly, Lakhani et al. demonstrated increased stiffness of both internal and external carotid arteries in woman with both PCOS and PCO (ultrasonographic polycystic ovaries alone) compared with controls (Lakhani et al., 2000, as cited in Cussons et al., 2006).), even if BP and BMI were taken into account. However, this study did not show an independent relationship between insulin resistance or other CV risk factors and arterial stiffness and, as the patients with PCOS studied by these investigators had higher BMIs and arterial pressures and basal insulinaemia than the remaining participants, it was unclear whether the presence of PCOS *per se*, as opposed to the comorbidities of this syndrome, was responsible for this difference. Pulse wave velocity over the carotid-femoral tract, another method for the assessment of arterial distensibility, also demonstrated unfavourable results for young obese women with PCOS in a previous study. A significant relationship between PWV and BP and between PWV and both insulin and glucose has been detected during a glucose tolerance test (Meyer et al., 2005a). However, some possible confounding factors, such as smoking, insulin resistance and hypertriglyceridaemia, were not excluded in this research and no adjustments were made for BP or BMI.

Moreover, in the study by Sasaki et al. (2011), women with PCOS had a significantly higher brachial–ankle pulse wave velocity (baPWV) than that of the controls whereas there was no significant difference in the carotid augmentation index (cAI) as well as ABI between the two groups. There was no significant difference in age or BMI between the controls and the women with PCOS. These women with PCOS had a significantly higher serum testosterone and CRP levels and showed insulin resistance and dyslipidemia. The mean BP in women with PCOS was within the normal range, but still significantly higher than those in the controls. Stepwise multiple regression analysis revealed that BP influences the baPWV in women with PCOS. Arterial stiffness evaluated using the baPWV in mildly-hypertensive women (SBP 120 mmHg or DBP 90 mmHg) with PCOS was significantly higher than that in the controls or normotensive women with PCOS, suggesting early changes in vascular function in mildly-hypertensive women with PCOS. Furthermore, among the women with PCOS, obese women had significantly higher baPWV as compared to normal-weight women although no significant difference was observed in the ABI and cAI. Hence, it appears that obesity and elevation of BP within subclinical levels in women with PCOS may have adverse effects on vascular functions (Sasaki et al., 2011).

In view of these results, PCOS has been suggested to be associated with arterial stiffening. The question remains whether the risks conferred by obesity and other CV risk factors, commonly found in PCOS subjects and PCOS, are additive. The drawback of other studies that have evaluated nonobese women with PCOS (Arikan et al., 2009, Orio et al., 2004) is the fact that they did not analyse these markers simultaneously so they did not identify those linked specifically with the presence of PCOS and not simply triggered by the metabolic dysfunctions so common in these patients. Thus, Soares et al. (2008) evaluated the subclinical markers of CVD (echographic and serum markers of chronic inflammation) in nonobese women with PCOS but without comorbidities. Common carotid artery stiffness index (,) (CCA ,) was higher in PCOS than in control women and CCA distensibility was lower, indicating that young women with PCOS exhibit changes in vascular elasticity even in the absence of classical risk factors for CVD, such as hypertension, obesity and type 2 DM. These data suggest that patients with PCOS may be at higher risk for CVD, because their arterial distensibility is already reduced during atheroma formation and thus represents a subclinical sign of atherosclerosis (Mattsson et al., 2008, as cited in Soares et al., 2008). However, as the women from this study did not present with hyperglycaemia or hyperinsulinaemia, the increased stiffness index and the reduced distensibility of the carotid artery detected in PCOS women might be attributed to the hyperandrogenism inherent to PCOS and not to comorbid conditions associated with the syndrome (Soares et al., 2008).

On the contrary, and sustaining the results of Muneyyirci-Delate et al., Ketel et al. (2010) demonstrated in a small study that (central) obesity, but not PCOS, is associated with greater arterial stiffness (Muneyyirci-Delate et al., 2007, as cited in Ketel et al., 2010). Beside the fact that most of the studies have not distinguished the effects of obesity from those of PCOS, they also have not taken into consideration the fact that stiffness of elastic (e.g. the carotid) and muscular (e.g. the femoral) arteries may differ in their association with obesity and potentially PCOS. The study of Ketel et al. (2010) is the first comprehensive study taking into account stiffness in both elastic and muscular arteries in both lean and obese women with and without PCOS. Previous investigations, which investigated one type of artery, pulse wave velocity over elastic and (or) muscular vascular regions, or small and large arterial compliance, showed somehow discordant results. Nevertheless, in their research, Ketel et al. (2010) demonstrated that PCOS was not associated with stiffening of either muscular or elastic arteries.

In conclusion, current studies suggest that differences in vascular function between normal women and women with PCOS may be very small if both are young, of normal weight and have normal BP. However, they showed that slightly increased BP, which weakly correlated with BMI, can be rightfully considered a risk factor for arterial stiffness in young women with PCOS. It has not been yet established whether these adverse effects of mild hypertension on arterial stiffness are characteristic to women with PCOS. We thus need further research on arterial stiffness in young women with mild hypertension caused by the other diseases (Sasaki et al., 2011). The mechanism for increased arterial stiffness reported in some studies of PCOS have not been cleared but, as in the metabolic syndrome and insulin resistance, may involve endothelial dysfunction and altered artery wall collagen metabolism (Cussons et al., 2006).

#### **5.1.3 Endothelial dysfunction**

172 Recent Advances in Cardiovascular Risk Factors

middle-aged male patients. Recently, these indices can be measured easily by simultaneous oscillometric measurement of pulse waves and they are widely used as arterial stiffness

In a small case-control study, Kelly et al. reported increased pulse wave velocity of the brachial artery, but not of the aorta, in a young obese PCOS group (Kelly et al., 2002, as cited in Cussons et al., 2006). Similarly, Lakhani et al. demonstrated increased stiffness of both internal and external carotid arteries in woman with both PCOS and PCO (ultrasonographic polycystic ovaries alone) compared with controls (Lakhani et al., 2000, as cited in Cussons et al., 2006).), even if BP and BMI were taken into account. However, this study did not show an independent relationship between insulin resistance or other CV risk factors and arterial stiffness and, as the patients with PCOS studied by these investigators had higher BMIs and arterial pressures and basal insulinaemia than the remaining participants, it was unclear whether the presence of PCOS *per se*, as opposed to the comorbidities of this syndrome, was responsible for this difference. Pulse wave velocity over the carotid-femoral tract, another method for the assessment of arterial distensibility, also demonstrated unfavourable results for young obese women with PCOS in a previous study. A significant relationship between PWV and BP and between PWV and both insulin and glucose has been detected during a glucose tolerance test (Meyer et al., 2005a). However, some possible confounding factors, such as smoking, insulin resistance and hypertriglyceridaemia, were not excluded in this

Moreover, in the study by Sasaki et al. (2011), women with PCOS had a significantly higher brachial–ankle pulse wave velocity (baPWV) than that of the controls whereas there was no significant difference in the carotid augmentation index (cAI) as well as ABI between the two groups. There was no significant difference in age or BMI between the controls and the women with PCOS. These women with PCOS had a significantly higher serum testosterone and CRP levels and showed insulin resistance and dyslipidemia. The mean BP in women with PCOS was within the normal range, but still significantly higher than those in the controls. Stepwise multiple regression analysis revealed that BP influences the baPWV in women with PCOS. Arterial stiffness evaluated using the baPWV in mildly-hypertensive women (SBP 120 mmHg or DBP 90 mmHg) with PCOS was significantly higher than that in the controls or normotensive women with PCOS, suggesting early changes in vascular function in mildly-hypertensive women with PCOS. Furthermore, among the women with PCOS, obese women had significantly higher baPWV as compared to normal-weight women although no significant difference was observed in the ABI and cAI. Hence, it appears that obesity and elevation of BP within subclinical levels in women with PCOS may

In view of these results, PCOS has been suggested to be associated with arterial stiffening. The question remains whether the risks conferred by obesity and other CV risk factors, commonly found in PCOS subjects and PCOS, are additive. The drawback of other studies that have evaluated nonobese women with PCOS (Arikan et al., 2009, Orio et al., 2004) is the fact that they did not analyse these markers simultaneously so they did not identify those linked specifically with the presence of PCOS and not simply triggered by the metabolic dysfunctions so common in these patients. Thus, Soares et al. (2008) evaluated the subclinical markers of CVD (echographic and serum markers of chronic inflammation) in nonobese women with PCOS but without comorbidities. Common carotid artery stiffness

indices (Sasaki et al., 2011).

research and no adjustments were made for BP or BMI.

have adverse effects on vascular functions (Sasaki et al., 2011).

Endothelial dysfunction can be defined as the partial or complete loss of balance between vasoconstrictors and vasodilators, growth promoting and inhibiting factors, pro-atherogenic and anti-atherogenic factors, and pro-coagulant and anti-coagulant factors. Endothelial dysfunction is the initiating event in the development of atherogenesis and has been shown to precede the development of clinically detectable atherosclerotic plaques in the coronary arteries, being also implicated in the development of microvascular complications in diabetes (Caballero, 2005). Therefore, the assessment of endothelial function by different methods has emerged as a tool for detection of preclinical CVD. A common approach to the evaluation of endothelial function is the assessment of blood flow and vascular reactivity which allows the investigator to evaluate the status of the endothelial cells as well as that of

The Polycystic Ovary Syndrome Status – A Risk Factor for Future Cardiovascular Disease 175

associated with a decrease in insulin resistance, testosterone and PAI-1 level (Paradisi et al., 2003). In an ex vivo study, the contractile response of gluteal resistance arteries to norepinephrine before and after incubation with insulin was measured, thus confirming the above results, namely that there is a resistance to the vasodilator effects of insulin in PCOS (Kelly et al., 2002), which proved to be present even in the absence of other risk factors. The defect in the resistance arterioles revealed in this study appears to be specific to the action of insulin, as constriction to norepinephrine and relaxation to Ach were not different between the PCOS group and the control group. In contrast, Bickerton et al.(2005), using venous occlusion plethysmography to assess differences in reactive hyperemia of the forearm microcirculation, found no evidence of endothelial dysfunction in women with PCOS compared with age- and weight-matched controls without PCOS. Carmassi et al. (2005), who studied PCOS women with and without insulin resistance, demonstrated a blunted vasodilatory response to insulin and an abolished expression of tissue plasminogen activator (t-PA) exclusively in insulin-resistant patients, suggesting that insulin resistance,

rather than PCOS status, confers endothelial dysfunction in this vascular bed.

brachial artery FMD and free testosterone (Pepene, 2011).

The measurement of plasma levels of several markers of endothelial activation (e.g. soluble vascular cell adhesion molecules (sVCAM), soluble intercellular adhesion molecule (sICAM), E- and P-selectin), coagulation and/or fibrinolysis (e.g. PAI-1, tPA, fibrinogen, von Willebrand factor), vascular tone (e.g. ET-1, products of nitric oxide), inflammation and other products, cytokines and hormones associated with vascular function also represent common approaches to evaluate endothelial function in humans (Caballero, 2005) (Table 1). Endothelin-1 has endothelial mitogenic effects (Dubin et al., 1989, as cited in Ilie et al., 2008) and seems to play a role in the early events of endothelial dysfunction. Therefore, it can be used as a marker of abnormal vascular reactivity. Diamanti-Kandarakis et al. were the first ones to demonstrate elevated ET-1 levels in PCOS compared with controls. A positive correlation of ET-1 with free testosterone levels was shown, as well as a negative correlation of ET-1 with glucose utilization, which might indicate an involvement of hyperinsulinemia and insulin resistance as well as of hyperandrogenemia in the abnormal endothelial status (Diamanti-Kandarakis et al., 2001). It appears that early impairment of endothelial structure and function is present even in young women with PCOS as suggested by significantly higher ET-1 levels in normal-weight, non-dyslipidemic and non-hypertensive women with PCOS than in BMI-matched healthy controls (Orio et al., 2004). The metformin therapy appeared to have beneficial effects, decreasing ET-1 levels (Diamanti-Kandarakis et al., 2001; Orio et al., 2005, as cited in Ilie et al., 2008). Consistent with others results, we also clearly demonstrated in a previous report that young, euglycaemic, eulipidemic and normotensive women with PCOS have evidence of endothelial dysfunction as assessed by both hemodynamic (FMD) and biochemical methods (ET-1). Impaired vascular reactivity and increased level of ET-1 were linked to markers of hyperandrogenemia in our study (Ilie et al., 2011). Interestingly, visfatin, an adipokine predominantly expressed in, and secreted from visceral adipose tissue, has also been shown to play a role in the pathogenesis of endothelial dysfunction in PCOS, independent of additional risk factors, since circulating visfatin was significantly related to

In healthy vessels, an infusion of an endothelium-dependent vasodilatator, such as acetylcholine, induces a nitric oxide (NO)-mediated vasodilatory response; however, in patients with endothelial dysfunction, this effect is blunted or paradoxical vasoconstriction

the underlying vascular smooth muscle cells. A lot of invasive and noninvasive techniques are available for evaluating these functions such as catheterization, ultrasound, positron emission tomography, laser Doppler flowmetry, and plethysmography (Caballero, 2005).

Measurement of post-ischemic flow mediated dilatation (FMD) of the brachial artery with high resolution ultrasonography is an established method to assess endothelial function of conduit arteries and correlates with measures of coronary endothelial dysfunction (Meyer et al., 2005b). Most of the studies performed in women with PCOS showed altered endothelial function in women with this disorder either using non-invasive methods (endotheliumdependent FMD or endothelium-independent, glyceroltrinitrate (GTN)-mediated dilatation vascular responses of the brachial artery) or invasive methods (Orio et al., 2004; Kravariti et al., 2005; Diamanti-Kandarakis et al., 2005, 2006a; Tarkun et al., 2004; Sorensen et al., 2006; Dagre et al., 2006) as compared to the control group. Impaired FMD is associated with insulin resistance, total cholesterol and CRP levels, while relationship to testosterone levels is controversial (Cussons et al., 2006; Kravariti et al.; Tarkun et al., 2004). However, it is not clear yet if the endothelial dysfunction found in women with PCOS is independent of obesity and insulin resistance that characterize so often this disorder. Therefore, Cussons et al. (2009) performed a study in nonobese, noninsulin resistant women with PCOS, assessing endothelial function (with FMD of the brachial artery) and arterial stiffness (with PWV and augmentation index (AI)). Although there were no significant differences between PCOS and control subjects in terms of BMI, BP, HOMA-IR, lipids, oestradiol and markers of arterial stiffness, the PCOS subjects had significantly lower FMD of the brachial artery compared with the controls. On the contrary, others suggested that middle-aged patients with PCOS display signs of endothelial dysfunction in comparison to age-matched controls, but that this is largely due to the increased prevalence of independent risk factors for CVD found in this group (Hudecova et al., 2010). On the other hand, there are also reports which do not sustain an association between PCOS and impaired endothelial function (assessed by FMD of the brachial artery) (Arikan et al., 2009; Brinckworth et al., 2006; Mather et al., 2000). Hence, Arikan et al. (2009) could not find a deteriorated endothelial function in young nonobese women with PCOS in spite of a significant insulin resistance in PCOS patients. They suggest that the existence of insulin resistance alone may not be an adequate factor for deterioration of endothelial function and carotid IMT in young, nonobese patients with PCOS. Other factors such as duration of insulin resistance, older age, presence of obesity, and inflammatory markers may play an important role in this process.

Endothelial dysfunction of the microcirculation and resistance vessels in PCOS has also been proved by invasive studies employing intra-arterial infusion of vasoactive agents with thermodilutional or plethysmographic assessment of limb blood flow. In a study by Paradisi et al., leg blood flow (LBF) was measured by means of an intravenous thermodilution catheter in a group of obese women with PCOS showing impaired LBF responses to methacoline and to hyperinsulinemia during an euglycaemic clamp compared with the control group. However, it should be mentioned that LBF response to sodium nitroprusside, an endothelium independent vasodilator, was not assessed, meaning that the demonstrated defect could not necessarily be localized to the endothelial cell as opposed to smooth muscle. Impaired endothelium-dependent vasodilatation was related to both androgen levels and insulin resistance (Paradisi et al., 2001). Using the same techniques, an improvement in the endothelial dysfunction was noted after 3 months of treatment with rosiglitazone in PCOS subjects compared with controls and this improvement was

the underlying vascular smooth muscle cells. A lot of invasive and noninvasive techniques are available for evaluating these functions such as catheterization, ultrasound, positron emission tomography, laser Doppler flowmetry, and plethysmography (Caballero, 2005). Measurement of post-ischemic flow mediated dilatation (FMD) of the brachial artery with high resolution ultrasonography is an established method to assess endothelial function of conduit arteries and correlates with measures of coronary endothelial dysfunction (Meyer et al., 2005b). Most of the studies performed in women with PCOS showed altered endothelial function in women with this disorder either using non-invasive methods (endotheliumdependent FMD or endothelium-independent, glyceroltrinitrate (GTN)-mediated dilatation vascular responses of the brachial artery) or invasive methods (Orio et al., 2004; Kravariti et al., 2005; Diamanti-Kandarakis et al., 2005, 2006a; Tarkun et al., 2004; Sorensen et al., 2006; Dagre et al., 2006) as compared to the control group. Impaired FMD is associated with insulin resistance, total cholesterol and CRP levels, while relationship to testosterone levels is controversial (Cussons et al., 2006; Kravariti et al.; Tarkun et al., 2004). However, it is not clear yet if the endothelial dysfunction found in women with PCOS is independent of obesity and insulin resistance that characterize so often this disorder. Therefore, Cussons et al. (2009) performed a study in nonobese, noninsulin resistant women with PCOS, assessing endothelial function (with FMD of the brachial artery) and arterial stiffness (with PWV and augmentation index (AI)). Although there were no significant differences between PCOS and control subjects in terms of BMI, BP, HOMA-IR, lipids, oestradiol and markers of arterial stiffness, the PCOS subjects had significantly lower FMD of the brachial artery compared with the controls. On the contrary, others suggested that middle-aged patients with PCOS display signs of endothelial dysfunction in comparison to age-matched controls, but that this is largely due to the increased prevalence of independent risk factors for CVD found in this group (Hudecova et al., 2010). On the other hand, there are also reports which do not sustain an association between PCOS and impaired endothelial function (assessed by FMD of the brachial artery) (Arikan et al., 2009; Brinckworth et al., 2006; Mather et al., 2000). Hence, Arikan et al. (2009) could not find a deteriorated endothelial function in young nonobese women with PCOS in spite of a significant insulin resistance in PCOS patients. They suggest that the existence of insulin resistance alone may not be an adequate factor for deterioration of endothelial function and carotid IMT in young, nonobese patients with PCOS. Other factors such as duration of insulin resistance, older age, presence of obesity,

and inflammatory markers may play an important role in this process.

Endothelial dysfunction of the microcirculation and resistance vessels in PCOS has also been proved by invasive studies employing intra-arterial infusion of vasoactive agents with thermodilutional or plethysmographic assessment of limb blood flow. In a study by Paradisi et al., leg blood flow (LBF) was measured by means of an intravenous thermodilution catheter in a group of obese women with PCOS showing impaired LBF responses to methacoline and to hyperinsulinemia during an euglycaemic clamp compared with the control group. However, it should be mentioned that LBF response to sodium nitroprusside, an endothelium independent vasodilator, was not assessed, meaning that the demonstrated defect could not necessarily be localized to the endothelial cell as opposed to smooth muscle. Impaired endothelium-dependent vasodilatation was related to both androgen levels and insulin resistance (Paradisi et al., 2001). Using the same techniques, an improvement in the endothelial dysfunction was noted after 3 months of treatment with rosiglitazone in PCOS subjects compared with controls and this improvement was associated with a decrease in insulin resistance, testosterone and PAI-1 level (Paradisi et al., 2003). In an ex vivo study, the contractile response of gluteal resistance arteries to norepinephrine before and after incubation with insulin was measured, thus confirming the above results, namely that there is a resistance to the vasodilator effects of insulin in PCOS (Kelly et al., 2002), which proved to be present even in the absence of other risk factors. The defect in the resistance arterioles revealed in this study appears to be specific to the action of insulin, as constriction to norepinephrine and relaxation to Ach were not different between the PCOS group and the control group. In contrast, Bickerton et al.(2005), using venous occlusion plethysmography to assess differences in reactive hyperemia of the forearm microcirculation, found no evidence of endothelial dysfunction in women with PCOS compared with age- and weight-matched controls without PCOS. Carmassi et al. (2005), who studied PCOS women with and without insulin resistance, demonstrated a blunted vasodilatory response to insulin and an abolished expression of tissue plasminogen activator (t-PA) exclusively in insulin-resistant patients, suggesting that insulin resistance, rather than PCOS status, confers endothelial dysfunction in this vascular bed.

The measurement of plasma levels of several markers of endothelial activation (e.g. soluble vascular cell adhesion molecules (sVCAM), soluble intercellular adhesion molecule (sICAM), E- and P-selectin), coagulation and/or fibrinolysis (e.g. PAI-1, tPA, fibrinogen, von Willebrand factor), vascular tone (e.g. ET-1, products of nitric oxide), inflammation and other products, cytokines and hormones associated with vascular function also represent common approaches to evaluate endothelial function in humans (Caballero, 2005) (Table 1).

Endothelin-1 has endothelial mitogenic effects (Dubin et al., 1989, as cited in Ilie et al., 2008) and seems to play a role in the early events of endothelial dysfunction. Therefore, it can be used as a marker of abnormal vascular reactivity. Diamanti-Kandarakis et al. were the first ones to demonstrate elevated ET-1 levels in PCOS compared with controls. A positive correlation of ET-1 with free testosterone levels was shown, as well as a negative correlation of ET-1 with glucose utilization, which might indicate an involvement of hyperinsulinemia and insulin resistance as well as of hyperandrogenemia in the abnormal endothelial status (Diamanti-Kandarakis et al., 2001). It appears that early impairment of endothelial structure and function is present even in young women with PCOS as suggested by significantly higher ET-1 levels in normal-weight, non-dyslipidemic and non-hypertensive women with PCOS than in BMI-matched healthy controls (Orio et al., 2004). The metformin therapy appeared to have beneficial effects, decreasing ET-1 levels (Diamanti-Kandarakis et al., 2001; Orio et al., 2005, as cited in Ilie et al., 2008). Consistent with others results, we also clearly demonstrated in a previous report that young, euglycaemic, eulipidemic and normotensive women with PCOS have evidence of endothelial dysfunction as assessed by both hemodynamic (FMD) and biochemical methods (ET-1). Impaired vascular reactivity and increased level of ET-1 were linked to markers of hyperandrogenemia in our study (Ilie et al., 2011). Interestingly, visfatin, an adipokine predominantly expressed in, and secreted from visceral adipose tissue, has also been shown to play a role in the pathogenesis of endothelial dysfunction in PCOS, independent of additional risk factors, since circulating visfatin was significantly related to brachial artery FMD and free testosterone (Pepene, 2011).

In healthy vessels, an infusion of an endothelium-dependent vasodilatator, such as acetylcholine, induces a nitric oxide (NO)-mediated vasodilatory response; however, in patients with endothelial dysfunction, this effect is blunted or paradoxical vasoconstriction

The Polycystic Ovary Syndrome Status – A Risk Factor for Future Cardiovascular Disease 177

vasculoprotective effect of hyperandrogenemia in PCOS. However, further researches are needed to confirm whether elevated DHEAS actually protects against atherogenesis in PCOS (Cussons et al., 2006). As inflammation has been implicated as a novel risk factor in the development and progression of atherosclerosis, the same Talbott et al. investigated the elevated CRP, as a marker of inflammation, as a possible determinant of increased IMT in middle-aged women with PCOS. They noted, however, that although CRP was significantly higher in PCOS patients than in controls, it did not appear to appreciably mediate the influence of PCOS on IMT (Talbott et al., 2004b). On the contrary, in another study, elevated serum IL-18 levels were associated with greater carotid IMT in patients with PCOS, suggesting a link between IL-18 and carotid atherosclerosis in patients with this disorder (Kaya et al., 2009). In line with other studies (Arikan et al., 2009; Costa et al., 2008; Meyer et al., 2005a), we could not find evidence of altered arterial structure in our group of young

Coronary artery calcifications, both radiographic markers for coronary atherosclerotic plaque and predictors of clinical events were found more prevalent in premenopausal obese women with PCOS aged 30-45 years in comparison to controls (Christian et al., 2003, as cited in Ilie et al., 2008). In a nine year follow-up study, an increased incidence of CAC was described in middle-aged women with PCOS; the degree of calcification was dependent on central obesity, elevated BP, dyslipidemia and thus insulin resistance (Talbott et al., 2004a). Furthermore, the prevalence of coronary artery calcium (CAC) was significantly higher also in young, obese women with PCOS, as compared with age and weight matched controls, even though the majority of subjects with detectable coronary artery calcification did not have traditional CV risk factors. The finding supports the idea that the presence of PCOS status per se appeared to contribute to this increased risk of CAC (Shroff et al., 2007). These reports suggest that women with PCOS have morphological evidence of coronary atherosclerosis and that insulin resistance, in particular, is the major causal factor. While the morphological changes in the vascular wall seem to be more evident in middle-aged and older women with PCOS, endothelial dysfunction is more likely a feature of the vasculopathy of young women with PCOS. This is not surprising, as endothelial dysfunction has been shown to occur earlier in the atherosclerosis development and

progression, preceding the onset of increased carotid IMT (Meyer et al., 2005a, b).

Recent evidence has been focused on a state of low-grade chronic inflammation, which has been described in PCOS as a potential link between hyperandrogenism, insulin resistance or abdominal adiposity and the metabolic and CV long-term complications of the syndrome. Low-grade inflammation can be defined as a condition characterized by increased circulation levels of several mediators of inflammation triggered by a noxious stimulus, such as classic molecules (TNF-+, IL-1, IL-6, CRP), or white blood cell (WBC) count (Repaci et al., 2011). Obesity, particularly the visceral phenotype, has been defined as a state of low-grade inflammation because visceral adipose tissue is able to produce cytokines (TNF-+, IL-6, and IL-1), chemokines (interferon-inducible protein-10 (IP-10), IL-8, IL-18, monocyte chemotactic protein-1 (MCP-1)) and other adipokines (FFA, PAI-1, leptin, resistin, visfatin, and adiponectin) that act, directly or indirectly, as mediators of systemic inflammation. Interestingly, the adipo-cytokines and, overall, the chronic inflammatory state associated

**5.3 Low-grade chronic inflammation** 

subjects with PCOS, as compared to controls (Ilie et al., 2011).

may occur (Verma et al., 2003, as cited in Ilie et al., 2008). Nacul et al. are the first who evaluated the concentration of nitrite/nitrate (as index of endothelium-derived NO) in a PCOS group compared to an age-matched control group and found similar levels of nitrite/nitrate and fibrinogen in the two groups. A negative, BMI-independent correlation between NO and insulin resistance was seen in PCOS patients only, but no association was observed between NO and BMI, WC, WHR or androgens suggesting that in PCOS endothelial dysfunction is related to the presence of insulin resistance (Nacul et al., 2007). Moreover, in a very recent study, Battaglia et al. (2010) demonstrated that young women with PCOS displayed levels of nitrite/nitrate within normal range and similar with those found in controls, which preserved the endothelium-dependent vasodilatation, which was also similar in both groups. There are also reports which, on the contrary, found lower levels of nitrites/nitrates in PCOS and subjects with polycystic ovaries (Battaglia et al., 2008) as well as in normal weight, eumenorrheic, nonhirsute daughters of patients with PCOS (Battaglia et al., 2009) compared with controls, which suggests that PCOS is a condition associated with an increased vascular risk.

Overall, data from the literature support the presence of endothelial dysfunction in women with PCOS, including those who are young and apparently not displaying CV risk factors. So far, insulin resistance appears to be the major responsible factor related to endothelial dysfunction in PCOS subjects. Additionally, there are also reports indicating an association of endothelial dysfunction with obesity, hsCRP and, less consistently, with hyperandrogenemia. The slight differences among the obtained results might be explained either through the different methodology used for the investigation of vascular reactivity and endothelial dysfunction, or through the different metabolic status of the recruited patients. Additionally, small sample sizes, bias in case-control designs, and the use of different criteria to define PCOS might also contribute to discrepancies amongst some studies.

#### **5.2 Morphological studies**

The two major anatomic markers for sub-clinical cardiovascular disease are carotid IMT, assessed by ultrasonography or angiography and coronary artery calcifications (CAC) evaluated by electron beam tomography (Essah et al., 2007). Increased carotid IMT has been reported to occur relatively early in the atherosclerotic process and has been linked with traditional CV risk factors, such as increasing age, obesity and adverse lipid profile, as commonly observed in PCOS (Talbott et al., 2004a). Several studies demonstrated the role of carotid IMT in predicting coronary and cerebrovascular events, with increasing carotid IMT associated with an elevated age-adjusted cardiovascular risk (Cussons et al., 2006). In particular, Talbott et al. demonstrated a difference in carotid IMT between middle-aged women with PCOS (45 years) and age-matched controls, but not in younger women (group aged 30 to 45 years) (Talbott et al., 2000a). However, there are also reports showing significantly higher carotid IMT values also in young, normal weight PCOS women compared with controls (Carmina et al., 2006b; Orio et al., 2004, Luque-Ramirez et al., 2007). Increased carotid IMT was found to be related to insulin resistance and lower adiponectin levels (Carmina et al., 2006b) or correlate with free androgen index or TT (Costa et al., 2008; Orio et al., 2004; Luque-Ramirez et al., 2007) and DHEAS (Pamuk et al., 2008), thus suggesting a contribution of hyperandrogenemia to the progression of atherosclerosis in PCOS (Ilie et al., 2008). By contrast, other studies showed that increased carotid IMT was inversely correlated with plasma DHEAS and androstendion levels suggesting an intriguing vasculoprotective effect of hyperandrogenemia in PCOS. However, further researches are needed to confirm whether elevated DHEAS actually protects against atherogenesis in PCOS (Cussons et al., 2006). As inflammation has been implicated as a novel risk factor in the development and progression of atherosclerosis, the same Talbott et al. investigated the elevated CRP, as a marker of inflammation, as a possible determinant of increased IMT in middle-aged women with PCOS. They noted, however, that although CRP was significantly higher in PCOS patients than in controls, it did not appear to appreciably mediate the influence of PCOS on IMT (Talbott et al., 2004b). On the contrary, in another study, elevated serum IL-18 levels were associated with greater carotid IMT in patients with PCOS, suggesting a link between IL-18 and carotid atherosclerosis in patients with this disorder (Kaya et al., 2009). In line with other studies (Arikan et al., 2009; Costa et al., 2008; Meyer et al., 2005a), we could not find evidence of altered arterial structure in our group of young subjects with PCOS, as compared to controls (Ilie et al., 2011).

Coronary artery calcifications, both radiographic markers for coronary atherosclerotic plaque and predictors of clinical events were found more prevalent in premenopausal obese women with PCOS aged 30-45 years in comparison to controls (Christian et al., 2003, as cited in Ilie et al., 2008). In a nine year follow-up study, an increased incidence of CAC was described in middle-aged women with PCOS; the degree of calcification was dependent on central obesity, elevated BP, dyslipidemia and thus insulin resistance (Talbott et al., 2004a). Furthermore, the prevalence of coronary artery calcium (CAC) was significantly higher also in young, obese women with PCOS, as compared with age and weight matched controls, even though the majority of subjects with detectable coronary artery calcification did not have traditional CV risk factors. The finding supports the idea that the presence of PCOS status per se appeared to contribute to this increased risk of CAC (Shroff et al., 2007). These reports suggest that women with PCOS have morphological evidence of coronary atherosclerosis and that insulin resistance, in particular, is the major causal factor. While the morphological changes in the vascular wall seem to be more evident in middle-aged and older women with PCOS, endothelial dysfunction is more likely a feature of the vasculopathy of young women with PCOS. This is not surprising, as endothelial dysfunction has been shown to occur earlier in the atherosclerosis development and progression, preceding the onset of increased carotid IMT (Meyer et al., 2005a, b).

#### **5.3 Low-grade chronic inflammation**

176 Recent Advances in Cardiovascular Risk Factors

may occur (Verma et al., 2003, as cited in Ilie et al., 2008). Nacul et al. are the first who evaluated the concentration of nitrite/nitrate (as index of endothelium-derived NO) in a PCOS group compared to an age-matched control group and found similar levels of nitrite/nitrate and fibrinogen in the two groups. A negative, BMI-independent correlation between NO and insulin resistance was seen in PCOS patients only, but no association was observed between NO and BMI, WC, WHR or androgens suggesting that in PCOS endothelial dysfunction is related to the presence of insulin resistance (Nacul et al., 2007). Moreover, in a very recent study, Battaglia et al. (2010) demonstrated that young women with PCOS displayed levels of nitrite/nitrate within normal range and similar with those found in controls, which preserved the endothelium-dependent vasodilatation, which was also similar in both groups. There are also reports which, on the contrary, found lower levels of nitrites/nitrates in PCOS and subjects with polycystic ovaries (Battaglia et al., 2008) as well as in normal weight, eumenorrheic, nonhirsute daughters of patients with PCOS (Battaglia et al., 2009) compared with controls, which suggests that PCOS is a condition

Overall, data from the literature support the presence of endothelial dysfunction in women with PCOS, including those who are young and apparently not displaying CV risk factors. So far, insulin resistance appears to be the major responsible factor related to endothelial dysfunction in PCOS subjects. Additionally, there are also reports indicating an association of endothelial dysfunction with obesity, hsCRP and, less consistently, with hyperandrogenemia. The slight differences among the obtained results might be explained either through the different methodology used for the investigation of vascular reactivity and endothelial dysfunction, or through the different metabolic status of the recruited patients. Additionally, small sample sizes, bias in case-control designs, and the use of different criteria to define PCOS

The two major anatomic markers for sub-clinical cardiovascular disease are carotid IMT, assessed by ultrasonography or angiography and coronary artery calcifications (CAC) evaluated by electron beam tomography (Essah et al., 2007). Increased carotid IMT has been reported to occur relatively early in the atherosclerotic process and has been linked with traditional CV risk factors, such as increasing age, obesity and adverse lipid profile, as commonly observed in PCOS (Talbott et al., 2004a). Several studies demonstrated the role of carotid IMT in predicting coronary and cerebrovascular events, with increasing carotid IMT associated with an elevated age-adjusted cardiovascular risk (Cussons et al., 2006). In particular, Talbott et al. demonstrated a difference in carotid IMT between middle-aged women with PCOS (45 years) and age-matched controls, but not in younger women (group aged 30 to 45 years) (Talbott et al., 2000a). However, there are also reports showing significantly higher carotid IMT values also in young, normal weight PCOS women compared with controls (Carmina et al., 2006b; Orio et al., 2004, Luque-Ramirez et al., 2007). Increased carotid IMT was found to be related to insulin resistance and lower adiponectin levels (Carmina et al., 2006b) or correlate with free androgen index or TT (Costa et al., 2008; Orio et al., 2004; Luque-Ramirez et al., 2007) and DHEAS (Pamuk et al., 2008), thus suggesting a contribution of hyperandrogenemia to the progression of atherosclerosis in PCOS (Ilie et al., 2008). By contrast, other studies showed that increased carotid IMT was inversely correlated with plasma DHEAS and androstendion levels suggesting an intriguing

associated with an increased vascular risk.

**5.2 Morphological studies** 

might also contribute to discrepancies amongst some studies.

Recent evidence has been focused on a state of low-grade chronic inflammation, which has been described in PCOS as a potential link between hyperandrogenism, insulin resistance or abdominal adiposity and the metabolic and CV long-term complications of the syndrome.

Low-grade inflammation can be defined as a condition characterized by increased circulation levels of several mediators of inflammation triggered by a noxious stimulus, such as classic molecules (TNF-+, IL-1, IL-6, CRP), or white blood cell (WBC) count (Repaci et al., 2011). Obesity, particularly the visceral phenotype, has been defined as a state of low-grade inflammation because visceral adipose tissue is able to produce cytokines (TNF-+, IL-6, and IL-1), chemokines (interferon-inducible protein-10 (IP-10), IL-8, IL-18, monocyte chemotactic protein-1 (MCP-1)) and other adipokines (FFA, PAI-1, leptin, resistin, visfatin, and adiponectin) that act, directly or indirectly, as mediators of systemic inflammation. Interestingly, the adipo-cytokines and, overall, the chronic inflammatory state associated

The Polycystic Ovary Syndrome Status – A Risk Factor for Future Cardiovascular Disease 179

inflammation (Diamanti-Kandarakis et al., 2006a, b) were described. Diamanti-Kandarakis et al. (2006a) demonstrated that serum CRP concentrations were positively related to BMI and negatively related to FMD values and insulin sensitivity indices. Furthermore, FMD was statistically higher in PCOS population with CRP1mg -1 when compared with PCOS population with CRP1mg -1. The finding that the increased levels of CRP are consistent with the severity of endothelial dysfunction supports the hypothesis that chronic inflammation contributes to endothelial dysfunction (Diamanti-Kandarakis et al., 2006a).

Other classic markers of low-grade inflammation, which are significantly increased in PCOS, although not constantly (Shroff et al., 2007) are IL-18 (Escobar-Morreale et al., 2004, as cited in Repaci et al., 2011; Kaya et al., 2008), TNF-+ and IL-6 (Gonzalez et al., 1999, as cited in Repaci et al., 2011; Tarkun et al., 2006). Like CRP, these cytokines have been related to the risk of developing type 2 DM and CVD and are strongly associated with insulin

Accordingly, an increase of WBCs, particularly lymphocytes and monocytes (Orio et al., 2005a, as cited in ) or neutrophils (Ibanez et al., 2005, as cited in Ilie et al., 2008) which have been demonstrated to be strongly associated with insulin resistance (Orio et al., 2007; Ibanez et al., 2005) has also been noted in PCOS. Metformin and the combination flutamidemetformin had a beneficial effect on low-grade inflammation, attenuating WBC (Orio et al., 2007, as cited in Ilie et al., 2008) and hyperneutrophilia (Ibanez et al., 2005, as cited in Ilie et al., 2008), that further supports a link between hyperleukocytosis and insulin resistance (Ilie et al., 2008). A high expression of other classic markers, such as IL-6, MCP-1 and matrix metallopeptidase-2 (MMP2), have also been described in PCOS. Whether this relationship may be attributable to increased adiposity, particularly to the visceral phenotype, or, conversely, may depend on other factors is, however, still debatable (Vgontzas et al., 2006; Hu et al., 2006; Lewandowki et al., 2006; Orio et al., 2004b; Gonzalez et al., 2006a,b, 2009;

Endothelial dysfunction triggers the expression of cell adhesion molecules on the surface of endothelial cells and leukocytes to handle the intricate process of leukocyte rolling, adhesion, and transmigration into the sub-intimal space. Last but not least, in several studies, PCOS has been shown to associate with high levels of adhesion molecules, such as sICAM-1, sVCAM-1, ET-1, and sE-selectin, (Ley and Huo, 2001; Pai et al., 2004; Meigs et al., 2004; Roldan et al., 2003; Corti et al., 2004; Kowalska et al., 2002; Kadoand Nagata, 1999; Diamanti-Kandarakis et al., 2006b, as cited in Repaci et al., 2011). An interesting fact reported was that all these adhesion molecules were positively associated not only with insulin resistance (Diamanti-Kandarakis et al., 2006b), but also with the degree of hyperandrogenemia in PCOS (Diamanti-Kandarakis et al., 2001, 2006a). This association makes androgen excess a potential factor in the development of endothelial dysfunction. This hypothesis is confirmed by a recent in vitro study which proved that high levels of dihydrotestosterone favored the development of atherosclerotic lesions by inducing the expression of sVCAM on endothelial cell surface through a NF-.B dependent mechanism, which finally induces the adhesion of monocytes to endothelial cells (Death et al., 2004).

Among the non-classic markers of low-grade inflammation, increased levels of ferritin have been described in obesity, type 2 DM and the MS (Fernandez-Real et al., 2002, as cited in Repaci et al., 2011) as well as in PCOS, where it was associated with obesity and insulin excess (Escobar-Morreale et al., 2005, Botella-Carretero et al., 2006; Luque-Ramirez et al.,

resistance and body fat amount (Repaci et al., 2011; Tarkun et al., 2006).

Glintborg et al., 2009, as cited in Repaci et al., 2011).

with obesity are also related to the insulin resistance state (Sell & Eckel, 2009, as cited in Repaci et al., 2011), type 2 DM or CVD due to the development of hypertension, dyslipidemia, and endothelial dysfunction (Yudkin et al., 1999, as cited in Repaci et al., 2011) (Fig. 2). Furthermore, accumulating evidence suggests that atherosclerosis represents a chronic inflammatory process and that the inflammation at the level of the vessel wall plays a pivotal role in atherosclerotic lesion formation, progression, and eventual rupture.

There is much evidence sustaining that high levels of IL-6, TNF-+, and leptin, and reduced levels of adiponectin inhibit the expression and activity of nitric oxide synthase (eNOS), leading to decreased NO synthesis and reduced vasodilation and consequently promoting endothelial dysfunction (Rizvi, 2007, 2009, as cited in Repaci et al., 2011) (Fig. 2). The release of cytokines and chemokines, such as TNF-+, MCP-1, and IL-18 has been demonstrated to promote the recruitment of macrophages from the bloodstream which, through the production of adhesion molecules, in turn accelerate adhesion, arrest, and diapedesis of inflammatory cells through the endothelium with the consequent infiltration of the lipid core. These inflammation mediators also promote the proliferation of smooth muscle cells and their migration from the tunica media to the tunica intima (Sengenès et al., 2007, as cited in Reapaci et al., 2011), leading to the formation of atherosclerotic plaque. Hence, chronic inflammation is responsible of early and late atherosclerotic processes.

#### **5.3.1 Classic and non-classic markers of low-grade inflammation in PCOS**

A classic and widely studied marker of low-grade inflammation is CRP, which has been shown to significantly predict the incidence of peripheral vascular diseases, endothelial dysfunction, MI, stroke, and sudden death (Ridker, 2003, as cited in Repaci et al., 2011). Recent data suggests that CRP is not only an inflammatory marker of atherosclerosis, but it may have some direct deleterious effects in the vascular wall, contributing to the pathogenesis of lesion formation, plaque rupture, and coronary thrombosis by interacting with and altering the endothelial cell phenotype (Verma et al., 2003, as cited in Ilie et al., 2008). CRP may directly promote endothelial dysfunction by increasing the expression of endothelial cell adhesion molecules (CAMs), the MCP-1 secretion and macrophage LDLuptake. Also it appears that CRP directly influences the production of endothelium-derived vasoactive factors: it decreases the NO production while augmenting production of the potent endothelium-derived vasoconstrictor ET-1 as well as of PAI-1 (Devaraj et al., 2003, as cited in Ilie et al., 2008). Additionally, CRP facilitates endothelial cell apoptosis and attenuates angiogenesis. The most published data, but not all (Capoglu et al., 2009; Mohlig et al., 2005; Shroff et al., 2007), demonstrate increased levels of hsCRP in women with PCOS (Diamanti-Kandarakis et al., 2006a, b; Talbott et al., 2004b; Tarkun et al., 2004). However, many studies, including our previous report, have demonstrated that in women with PCOS the CRP values are primarily dependent to upon co-existent obesity and fatty mass or insulin resistance (Kelly et al., 2001, as cited in Ilie et al., 2008; Benson et al., 2008, as cited in Repaci et al., 2011; Ilie et al., 2011), rather than to PCOS status per se. Hence, increased serum CRP levels were found in obese and non-obese PCOS compared to controls (Tarkun et al., 2004; Talbott et al., 2004b; Kelly et al., 2001, Orio et al., 2005, as cited in Ilie et al., 2008) with significantly higher values in obese than in non-obese PCOS subjects (Morin-Papunen et al., 2003), decreasing after metformin (Morin-Papunen et al., 2003). Regarding hyperandrogenemia, in PCOS subjects, either no correlation (Tarkun et al., 2004; Puder et al., 2005) or a positive association between increased androgen levels and indices of chronic

with obesity are also related to the insulin resistance state (Sell & Eckel, 2009, as cited in Repaci et al., 2011), type 2 DM or CVD due to the development of hypertension, dyslipidemia, and endothelial dysfunction (Yudkin et al., 1999, as cited in Repaci et al., 2011) (Fig. 2). Furthermore, accumulating evidence suggests that atherosclerosis represents a chronic inflammatory process and that the inflammation at the level of the vessel wall plays

There is much evidence sustaining that high levels of IL-6, TNF-+, and leptin, and reduced levels of adiponectin inhibit the expression and activity of nitric oxide synthase (eNOS), leading to decreased NO synthesis and reduced vasodilation and consequently promoting endothelial dysfunction (Rizvi, 2007, 2009, as cited in Repaci et al., 2011) (Fig. 2). The release of cytokines and chemokines, such as TNF-+, MCP-1, and IL-18 has been demonstrated to promote the recruitment of macrophages from the bloodstream which, through the production of adhesion molecules, in turn accelerate adhesion, arrest, and diapedesis of inflammatory cells through the endothelium with the consequent infiltration of the lipid core. These inflammation mediators also promote the proliferation of smooth muscle cells and their migration from the tunica media to the tunica intima (Sengenès et al., 2007, as cited in Reapaci et al., 2011), leading to the formation of atherosclerotic plaque. Hence, chronic

a pivotal role in atherosclerotic lesion formation, progression, and eventual rupture.

inflammation is responsible of early and late atherosclerotic processes.

**5.3.1 Classic and non-classic markers of low-grade inflammation in PCOS** 

A classic and widely studied marker of low-grade inflammation is CRP, which has been shown to significantly predict the incidence of peripheral vascular diseases, endothelial dysfunction, MI, stroke, and sudden death (Ridker, 2003, as cited in Repaci et al., 2011). Recent data suggests that CRP is not only an inflammatory marker of atherosclerosis, but it may have some direct deleterious effects in the vascular wall, contributing to the pathogenesis of lesion formation, plaque rupture, and coronary thrombosis by interacting with and altering the endothelial cell phenotype (Verma et al., 2003, as cited in Ilie et al., 2008). CRP may directly promote endothelial dysfunction by increasing the expression of endothelial cell adhesion molecules (CAMs), the MCP-1 secretion and macrophage LDLuptake. Also it appears that CRP directly influences the production of endothelium-derived vasoactive factors: it decreases the NO production while augmenting production of the potent endothelium-derived vasoconstrictor ET-1 as well as of PAI-1 (Devaraj et al., 2003, as cited in Ilie et al., 2008). Additionally, CRP facilitates endothelial cell apoptosis and attenuates angiogenesis. The most published data, but not all (Capoglu et al., 2009; Mohlig et al., 2005; Shroff et al., 2007), demonstrate increased levels of hsCRP in women with PCOS (Diamanti-Kandarakis et al., 2006a, b; Talbott et al., 2004b; Tarkun et al., 2004). However, many studies, including our previous report, have demonstrated that in women with PCOS the CRP values are primarily dependent to upon co-existent obesity and fatty mass or insulin resistance (Kelly et al., 2001, as cited in Ilie et al., 2008; Benson et al., 2008, as cited in Repaci et al., 2011; Ilie et al., 2011), rather than to PCOS status per se. Hence, increased serum CRP levels were found in obese and non-obese PCOS compared to controls (Tarkun et al., 2004; Talbott et al., 2004b; Kelly et al., 2001, Orio et al., 2005, as cited in Ilie et al., 2008) with significantly higher values in obese than in non-obese PCOS subjects (Morin-Papunen et al., 2003), decreasing after metformin (Morin-Papunen et al., 2003). Regarding hyperandrogenemia, in PCOS subjects, either no correlation (Tarkun et al., 2004; Puder et al., 2005) or a positive association between increased androgen levels and indices of chronic inflammation (Diamanti-Kandarakis et al., 2006a, b) were described. Diamanti-Kandarakis et al. (2006a) demonstrated that serum CRP concentrations were positively related to BMI and negatively related to FMD values and insulin sensitivity indices. Furthermore, FMD was statistically higher in PCOS population with CRP1mg -1 when compared with PCOS population with CRP1mg -1. The finding that the increased levels of CRP are consistent with the severity of endothelial dysfunction supports the hypothesis that chronic inflammation contributes to endothelial dysfunction (Diamanti-Kandarakis et al., 2006a).

Other classic markers of low-grade inflammation, which are significantly increased in PCOS, although not constantly (Shroff et al., 2007) are IL-18 (Escobar-Morreale et al., 2004, as cited in Repaci et al., 2011; Kaya et al., 2008), TNF-+ and IL-6 (Gonzalez et al., 1999, as cited in Repaci et al., 2011; Tarkun et al., 2006). Like CRP, these cytokines have been related to the risk of developing type 2 DM and CVD and are strongly associated with insulin resistance and body fat amount (Repaci et al., 2011; Tarkun et al., 2006).

Accordingly, an increase of WBCs, particularly lymphocytes and monocytes (Orio et al., 2005a, as cited in ) or neutrophils (Ibanez et al., 2005, as cited in Ilie et al., 2008) which have been demonstrated to be strongly associated with insulin resistance (Orio et al., 2007; Ibanez et al., 2005) has also been noted in PCOS. Metformin and the combination flutamidemetformin had a beneficial effect on low-grade inflammation, attenuating WBC (Orio et al., 2007, as cited in Ilie et al., 2008) and hyperneutrophilia (Ibanez et al., 2005, as cited in Ilie et al., 2008), that further supports a link between hyperleukocytosis and insulin resistance (Ilie et al., 2008). A high expression of other classic markers, such as IL-6, MCP-1 and matrix metallopeptidase-2 (MMP2), have also been described in PCOS. Whether this relationship may be attributable to increased adiposity, particularly to the visceral phenotype, or, conversely, may depend on other factors is, however, still debatable (Vgontzas et al., 2006; Hu et al., 2006; Lewandowki et al., 2006; Orio et al., 2004b; Gonzalez et al., 2006a,b, 2009; Glintborg et al., 2009, as cited in Repaci et al., 2011).

Endothelial dysfunction triggers the expression of cell adhesion molecules on the surface of endothelial cells and leukocytes to handle the intricate process of leukocyte rolling, adhesion, and transmigration into the sub-intimal space. Last but not least, in several studies, PCOS has been shown to associate with high levels of adhesion molecules, such as sICAM-1, sVCAM-1, ET-1, and sE-selectin, (Ley and Huo, 2001; Pai et al., 2004; Meigs et al., 2004; Roldan et al., 2003; Corti et al., 2004; Kowalska et al., 2002; Kadoand Nagata, 1999; Diamanti-Kandarakis et al., 2006b, as cited in Repaci et al., 2011). An interesting fact reported was that all these adhesion molecules were positively associated not only with insulin resistance (Diamanti-Kandarakis et al., 2006b), but also with the degree of hyperandrogenemia in PCOS (Diamanti-Kandarakis et al., 2001, 2006a). This association makes androgen excess a potential factor in the development of endothelial dysfunction. This hypothesis is confirmed by a recent in vitro study which proved that high levels of dihydrotestosterone favored the development of atherosclerotic lesions by inducing the expression of sVCAM on endothelial cell surface through a NF-.B dependent mechanism, which finally induces the adhesion of monocytes to endothelial cells (Death et al., 2004).

Among the non-classic markers of low-grade inflammation, increased levels of ferritin have been described in obesity, type 2 DM and the MS (Fernandez-Real et al., 2002, as cited in Repaci et al., 2011) as well as in PCOS, where it was associated with obesity and insulin excess (Escobar-Morreale et al., 2005, Botella-Carretero et al., 2006; Luque-Ramirez et al.,

The Polycystic Ovary Syndrome Status – A Risk Factor for Future Cardiovascular Disease 181

carbohydrate metabolism, in oxidative stress and in the differentiation of pre-adipocytes into mature adipocytes. Additionally, androgens enhance lipolysis, determining an increased release of FFA (Cortón et al., 2007, Xu et al., 1991, as cited in Repaci et al., 2011). Briefly, there still are aspects that need to be cleared regarding the bidirectional relationship between low-grade inflammation and androgen excess in PCOS, and much more research

Several polymorphisms of cytokines have been studied for the possible association with PCOS. In particular, Escobar-Morreale et al. showed the association between one polymorphism at the level of the promoter of TNF-+ [308 A] and increased androgen circulating levels in both hyperandrogenic and healthy women, independent of the presence

Additionally, some studies have demonstrated the association between adipokines and androgens. One of these adipokines is visfatin, whose mRNA levels are increased in the adipose tissue of women with PCOS (Panidis et al., 2008, as cited in Repci et al., 2011; Carmina et al., 2009) and who has recently been shown to be positively associated with LH, androstenedione, and free testosterone levels, and negatively with SHBG (Panidis et al., 2008; Kowalska et al., 2007; Gen et al., 2009, as cited in Repaci et al., 2011; Pepene CE, 2011). Adiponectin and leptin are another two adipokines involved in the mediation of systemic inflammation, whose relationship with androgens is the subject of conflicting results (Page et al., 2005; Seftel, 2005; Yilmaz et al., 2009, as cited in Repaci et al., 2011). Regarding leptin in particular, some studies failed to demonstrate any relationship with androgens (Vicennati et al., 1998; Pehlivanov and Mitkov, 2009; El Orabi et al., 1999, as cited in Repaci et al., 2011), whereas others showed a positive relationship between leptin circulating levels and androgens (Hislop et al., 1999; Castrogiovanni et al., 2003; Pardo et al., 2004, as cited in Repaci et al., 2011). More convincing results have been obtained by the analysis of the ratio adiponectin/leptin, considered a good marker of a state of inflammation in PCOS (Xita et al., 2007), that was found to be positively related with SHBG. Reduced adiponectin (Carmina et al., 2006b), and increased leptin (Gomez-Ambrosi et al., 2002, as cited in Repaci et al., 2011) may also contribute to the development of endothelial dysfunction in PCOS, as shown by some reports. Finally, circulating levels of ghrelin and androgens, particularly androstenedione, have been found to be negatively connected in women with PCOS. These results have been further confirmed by the observation that a short-term treatment with flutamide, a drug with anti-androgenic properties, triggered a significant increase in ghrelin circulating levels (Glintborg et al., 2006; Pagotto et al., 2002; Panidis et al., 2005; Gambineri et

In conclusion, PCOS is associated with a low-grade of chronic inflammation mainly attributable to the accumulation of visceral fat, although an effect of insulin resistance cannot be excluded. The impact of hyperandrogenemia can be restricted so far to the influence of androgens on the development and distribution of the adipose tissue, which is, in fact, a true endocrine gland. A vicious circle is established with a continuous release of inflammatory mediators that are responsible for the development of insulin resistance, dyslipidemia, endothelial dysfunction, and metabolic and CV long-term complications. In addition, through the impact on the regulation of the synthesis and secretion of androgens in the ovary and in the adrenal, the state of low-grade inflammation also seems able to

contribute to the maintenance of the syndrome (Repaci et al., 2011).

should, thus, be performed in this exciting area.

al., 2003, as cited in Repaci et al., 2011).

of obesity and insulin resistance (Escobar-Morreale et al., 2001).

2007b, as cited in Repaci et al., 2011). Osteoprotegerin (OPG) has both anti-inflammatory and antiapoptotic effects, particularly at the level of endothelial cells, where it exerts a protective role in the development of atherosclerotic plaque and plaque rupture (Scatena and Giachelli, 2002; Schoppert et al., 2002; Kiechl et al., 2007; Kim et al., 2005, as cited in Repaci et al., 2011). Reduced levels of OPG have been observed in obesity and in the presence of insulin resistance (Ugur-Altun et al., 2005; Holecki et al., 2007, as cited in Repaci et al., 2011), as well as in PCOS (Escobar-Morreale et al., 2008), where they were related to the androgen excess (Escobar-Morreale et al., 2008). Interestingly, we have demonstrated very recently that circulating OPG was negatively and significantly correlated with FMD and, moreover, that PCOS women in the upper OPG quartile group (> 2.65 pmol/l) presented significantly altered endothelial function compared to all the rest. Nevertheless, it is unclear whether the relationship between OPG and FMD found here reflects a compensatory response of the endothelium to injury or if it should rather be regarded as a direct involvement of OPG in the pathogenesis of endothelial dysfunction. In previous studies, elevated plasma OPG concentrations, reported in several states associated with vascular damage, were negatively related to endothelium-dependent arterial dilation in newly diagnosed diabetes and hypothyroidism (Shin et al., 2006, Rasmussen et al., 2006, Guang-da et al., 2008, as cited in Pepene et al., 2011). Coming back to our results, since patients with important insulin resistance and severely impaired FMD presented with the highest OPG levels, we might speculate that in PCOS, OPG may link disturbances in insulin sensitivity to impaired endothelial dysfunction and progression of atherosclerosis, independently from hyperandrogenemia. Moreover, confirming previous results (Escobar-Morreale et al., 2008) that suggest a deleterious effect of androgen excess on OPG, the latter was negatively related to FT in our study (Pepene et al., 2011). Finally, PCOS has been associated with high levels of advanced glycation endproducts (AGEs), which is described to act as oxidants and inflammatory mediators as well as increased levels of platelet mean volume (MPV), an indicator of platelet activation and aggregation (Repaci et al., 2011).

#### **5.3.2 Androgen excess and low-grade chronic inflammation**

As far as the relationship between androgen excess and low-grade chronic inflammation is concerned, there are still debates whether molecules involved in the low-grade inflammatory state are involved in the pathogenesis of hyperandrogenemia or, conversely, whether androgen excess might promote the inflammatory state. Either no correlation (Tarkun et al., 2004; Puder et al., 2005; Cascella et al., 2008; Tosi et al., 2009) or a positive association between increased androgen levels and indices of chronic inflammation (Diamanti-Kandarakis et al., 2006a, b) were described in PCOS subjects. The in vitro studies support the hypothesis that the state of low-grade inflammation influences androgens, showing that TNF-+ stimulates the proliferation and steroidogenesis of theca cells (Spaczynski et al., 1999, as cited in Repaci et al., 2011), whereas IL-6 is capable of stimulating human adrenal cells, thereby increasing adrenal steroidogenesis (Path et al., 1997, as cited in Repaci et al., 2011).

In spite of the fact that the role of androgens in low-grade inflammation is yet to be demonstrated, vast evidence has accumulated to suggest that androgens could, however, play an indirect role in the development of low-grade inflammation through the impact on the adipose tissue. In fact, androgens seem to have a stimulatory effect on the hypertrophy of adipocytes by influencing the expression of enzymes and proteins involved in lipid and

2007b, as cited in Repaci et al., 2011). Osteoprotegerin (OPG) has both anti-inflammatory and antiapoptotic effects, particularly at the level of endothelial cells, where it exerts a protective role in the development of atherosclerotic plaque and plaque rupture (Scatena and Giachelli, 2002; Schoppert et al., 2002; Kiechl et al., 2007; Kim et al., 2005, as cited in Repaci et al., 2011). Reduced levels of OPG have been observed in obesity and in the presence of insulin resistance (Ugur-Altun et al., 2005; Holecki et al., 2007, as cited in Repaci et al., 2011), as well as in PCOS (Escobar-Morreale et al., 2008), where they were related to the androgen excess (Escobar-Morreale et al., 2008). Interestingly, we have demonstrated very recently that circulating OPG was negatively and significantly correlated with FMD and, moreover, that PCOS women in the upper OPG quartile group (> 2.65 pmol/l) presented significantly altered endothelial function compared to all the rest. Nevertheless, it is unclear whether the relationship between OPG and FMD found here reflects a compensatory response of the endothelium to injury or if it should rather be regarded as a direct involvement of OPG in the pathogenesis of endothelial dysfunction. In previous studies, elevated plasma OPG concentrations, reported in several states associated with vascular damage, were negatively related to endothelium-dependent arterial dilation in newly diagnosed diabetes and hypothyroidism (Shin et al., 2006, Rasmussen et al., 2006, Guang-da et al., 2008, as cited in Pepene et al., 2011). Coming back to our results, since patients with important insulin resistance and severely impaired FMD presented with the highest OPG levels, we might speculate that in PCOS, OPG may link disturbances in insulin sensitivity to impaired endothelial dysfunction and progression of atherosclerosis, independently from hyperandrogenemia. Moreover, confirming previous results (Escobar-Morreale et al., 2008) that suggest a deleterious effect of androgen excess on OPG, the latter was negatively related to FT in our study (Pepene et al., 2011). Finally, PCOS has been associated with high levels of advanced glycation endproducts (AGEs), which is described to act as oxidants and inflammatory mediators as well as increased levels of platelet mean volume (MPV), an indicator of platelet activation and aggregation (Repaci et al., 2011).

**5.3.2 Androgen excess and low-grade chronic inflammation** 

Repaci et al., 2011).

As far as the relationship between androgen excess and low-grade chronic inflammation is concerned, there are still debates whether molecules involved in the low-grade inflammatory state are involved in the pathogenesis of hyperandrogenemia or, conversely, whether androgen excess might promote the inflammatory state. Either no correlation (Tarkun et al., 2004; Puder et al., 2005; Cascella et al., 2008; Tosi et al., 2009) or a positive association between increased androgen levels and indices of chronic inflammation (Diamanti-Kandarakis et al., 2006a, b) were described in PCOS subjects. The in vitro studies support the hypothesis that the state of low-grade inflammation influences androgens, showing that TNF-+ stimulates the proliferation and steroidogenesis of theca cells (Spaczynski et al., 1999, as cited in Repaci et al., 2011), whereas IL-6 is capable of stimulating human adrenal cells, thereby increasing adrenal steroidogenesis (Path et al., 1997, as cited in

In spite of the fact that the role of androgens in low-grade inflammation is yet to be demonstrated, vast evidence has accumulated to suggest that androgens could, however, play an indirect role in the development of low-grade inflammation through the impact on the adipose tissue. In fact, androgens seem to have a stimulatory effect on the hypertrophy of adipocytes by influencing the expression of enzymes and proteins involved in lipid and carbohydrate metabolism, in oxidative stress and in the differentiation of pre-adipocytes into mature adipocytes. Additionally, androgens enhance lipolysis, determining an increased release of FFA (Cortón et al., 2007, Xu et al., 1991, as cited in Repaci et al., 2011). Briefly, there still are aspects that need to be cleared regarding the bidirectional relationship between low-grade inflammation and androgen excess in PCOS, and much more research should, thus, be performed in this exciting area.

Several polymorphisms of cytokines have been studied for the possible association with PCOS. In particular, Escobar-Morreale et al. showed the association between one polymorphism at the level of the promoter of TNF-+ [308 A] and increased androgen circulating levels in both hyperandrogenic and healthy women, independent of the presence of obesity and insulin resistance (Escobar-Morreale et al., 2001).

Additionally, some studies have demonstrated the association between adipokines and androgens. One of these adipokines is visfatin, whose mRNA levels are increased in the adipose tissue of women with PCOS (Panidis et al., 2008, as cited in Repci et al., 2011; Carmina et al., 2009) and who has recently been shown to be positively associated with LH, androstenedione, and free testosterone levels, and negatively with SHBG (Panidis et al., 2008; Kowalska et al., 2007; Gen et al., 2009, as cited in Repaci et al., 2011; Pepene CE, 2011). Adiponectin and leptin are another two adipokines involved in the mediation of systemic inflammation, whose relationship with androgens is the subject of conflicting results (Page et al., 2005; Seftel, 2005; Yilmaz et al., 2009, as cited in Repaci et al., 2011). Regarding leptin in particular, some studies failed to demonstrate any relationship with androgens (Vicennati et al., 1998; Pehlivanov and Mitkov, 2009; El Orabi et al., 1999, as cited in Repaci et al., 2011), whereas others showed a positive relationship between leptin circulating levels and androgens (Hislop et al., 1999; Castrogiovanni et al., 2003; Pardo et al., 2004, as cited in Repaci et al., 2011). More convincing results have been obtained by the analysis of the ratio adiponectin/leptin, considered a good marker of a state of inflammation in PCOS (Xita et al., 2007), that was found to be positively related with SHBG. Reduced adiponectin (Carmina et al., 2006b), and increased leptin (Gomez-Ambrosi et al., 2002, as cited in Repaci et al., 2011) may also contribute to the development of endothelial dysfunction in PCOS, as shown by some reports. Finally, circulating levels of ghrelin and androgens, particularly androstenedione, have been found to be negatively connected in women with PCOS. These results have been further confirmed by the observation that a short-term treatment with flutamide, a drug with anti-androgenic properties, triggered a significant increase in ghrelin circulating levels (Glintborg et al., 2006; Pagotto et al., 2002; Panidis et al., 2005; Gambineri et al., 2003, as cited in Repaci et al., 2011).

In conclusion, PCOS is associated with a low-grade of chronic inflammation mainly attributable to the accumulation of visceral fat, although an effect of insulin resistance cannot be excluded. The impact of hyperandrogenemia can be restricted so far to the influence of androgens on the development and distribution of the adipose tissue, which is, in fact, a true endocrine gland. A vicious circle is established with a continuous release of inflammatory mediators that are responsible for the development of insulin resistance, dyslipidemia, endothelial dysfunction, and metabolic and CV long-term complications. In addition, through the impact on the regulation of the synthesis and secretion of androgens in the ovary and in the adrenal, the state of low-grade inflammation also seems able to contribute to the maintenance of the syndrome (Repaci et al., 2011).

The Polycystic Ovary Syndrome Status – A Risk Factor for Future Cardiovascular Disease 183

menstrual cycle and for improving the clinical signs of hyperandrogenism. Although highly effective, COC, in particular early formulations, were associated with significant adverse effects and unacceptable CV risk. In addition, a decade after the advent of oral hormonal contraception, epidemiologic research confirmed an increased risk for cardiovascular events, particularly venous thromboembolism (VTE), in women using the high-dose COCs or COCs containing the newer gonane progestins desogestrel and gestodene (commonly referred to as "third-generation" progestins) (Burkmana et al., 2011). Research indicates also that COC use is associated with an increased risk of MI, particularly in women who smoke and are older than 35 years and in those who have underlying risk factors for coronary artery disease, such as hypertension (Burkman et al., 2011). Different estrogens and progestins exert different metabolic and CV effects. Estrogen component is almost always ethinyl estradiol (EE) in doses ranging from 15 to 50 /g. The progestin component is of variable potency and androgenicity. Hence, while in combined COCs the EE component seems to be responsible for the slight changes in the procoagulation and fibrinolytic balance, most progestins appear not to affect the coagulation factors and liver proteins when given alone (Sitruk-Ware & Nath, 2011). However, the metabolic effects of pills are extremely variable; for example estrogens impair insulin action dose-dependently and the associated progestins may modify these effects (Nader & Diamanti-Kandarakis, 2007). It was suggested that when a dose of EE < 50 µg/day is used, the effects of the COCs on the lipid and glycoinsulinemic metabolism is related to the progestin used in the combination (Mancini et al., 2010). Hence, the estrogen component of hormonal contraceptives when administered orally tends to increase the production of lipoproteins such as, the very low density lipoprotein (VLDL) and HDL levels while decreasing LDL levels (Sitruk-Ware & Nath, 2011). Non-androgenic or anti-androgenic progestins exert minimal influence on the lipid profile and carbohydrate metabolism, which lessens the risk of developing coronary heart disease while androgenic progestins have shown an adverse effect over total and HDL cholesterol (Nath & Sitruk-Ware, 2009, as cited in Sitruk-Ware & Nath, 2011). Over the past half century, COCs have undergone a number of evolutionary steps, involving modifications of hormone doses and types, dosage regimens and administration schedules (Burkmana et al., 2011) in order to improve the safety profile, particularly the metabolic and cardiovascular safety. In young women, the risk of CVD is very low but can be strongly influenced by smoking and the presence of other risks factors, such as hypertension, obesity, and DM or PCOS, specially for women over 35 (Farley et al., 1998, 1999, as cited in Sitruk-

As a special subgroup of COCs users, women with PCOS have an increased risk for IGT and as COCs are often prescribed as first-line treatment, a worsening of the metabolic outcomes in this population (e.g. insulin resistance, glucose tolerance, lipid profile or chronic inflammation) would be of concern. In the literature, there are conflicting reports about the CV effects of OCPs on PCOS patients (Battaglia et al., 2010; Meyer et al., 2007). Some of these reports support the beneficial effects of OCPs on the CV system in PCOS patients (Luque-Ramirez et al., 2009; Mancini et al., 2010). However, a recent meta-analysis suggested that OCPs may have negative effects on CV events in healthy individuals and these effects may be aggravated in patients displaying increased CV risk, such as PCOS (Baillargeon et al., 2005, as cited in Gode et al., 2010; Nader & Diamanti-Kandarakis, 2007). The outcome of the studies evaluating the effects of OCPs on carbohydrate metabolism in PCOS has been extremely variable, their results covering all possibilities: improvement, no change,

Ware & Nath, 2011).

Fig. 2. Some hypothetical pathways for the development of cardiovascular disease in women with PCOS

Several traditional and non-traditional cardiovascular risk factors commonly met in PCOS play a role either directly or indirectly by developing insulin resistance (in the liver and the muscles). The latter definitely has a major influence, beside other mechanisms, in the development of subclinic cardiovascular disease, eventually leading to cardiovascular disease. The hypertrophic adipocytes secrete various molecules, including inflammatory markers and adipokines, which may, in turn, determine the development of insulin resistance, an increase of the hepatic production of hsCRP, an increase of ovarian and adrenal androgens production which may further aggravate central adiposity distribution and may even directly act to determine vascular injury. It is not, yet clear if androgen excess (the marker of PCOS) has a direct influence in the development of atherosclerosis and preclinic cardiovascular disease. TNF-+ = tumor necrosis factor alpha; IL-1 = interleukin 1; IL-6 = interleukin 6; PAI-1 = plasminogen activator inhibitor-1; IL-18 = interleukin 18; FFA = free fatty acids; hsCRP = highly sensitive C reactive protein; ET-1=endothelin-1; AT II= angiotensin II; ADMA= asymmetric dimethylarginine; vWF= vonWillebrand factor; NO=nitric oxide; PG I2 = prostacyclin; CVD=cardiovascular disease.

#### **6. Oral contraceptives may further exacerbate the metabolic profile of women with PCOS**

Since its introduction in 1960, the combined oral contraceptive (COC) pill has become one of the most widely and frequently used methods of contraception worldwide. COC also have a long history of use in patients with PCOS, where they are prescribed for obtaining a regular

Fig. 2. Some hypothetical pathways for the development of cardiovascular disease in

NO=nitric oxide; PG I2 = prostacyclin; CVD=cardiovascular disease.

Several traditional and non-traditional cardiovascular risk factors commonly met in PCOS play a role either directly or indirectly by developing insulin resistance (in the liver and the muscles). The latter definitely has a major influence, beside other mechanisms, in the development of subclinic cardiovascular disease, eventually leading to cardiovascular disease. The hypertrophic adipocytes secrete various molecules, including inflammatory markers and adipokines, which may, in turn, determine the development of insulin resistance, an increase of the hepatic production of hsCRP, an increase of ovarian and adrenal androgens production which may further aggravate central adiposity distribution and may even directly act to determine vascular injury. It is not, yet clear if androgen excess (the marker of PCOS) has a direct influence in the development of atherosclerosis and preclinic cardiovascular disease. TNF-+ = tumor necrosis factor alpha; IL-1 = interleukin 1; IL-6 = interleukin 6; PAI-1 = plasminogen activator inhibitor-1; IL-18 = interleukin 18; FFA = free fatty acids; hsCRP = highly sensitive C reactive protein; ET-1=endothelin-1; AT II= angiotensin II; ADMA= asymmetric dimethylarginine; vWF= vonWillebrand factor;

**6. Oral contraceptives may further exacerbate the metabolic profile of women** 

Since its introduction in 1960, the combined oral contraceptive (COC) pill has become one of the most widely and frequently used methods of contraception worldwide. COC also have a long history of use in patients with PCOS, where they are prescribed for obtaining a regular

women with PCOS

**with PCOS** 

menstrual cycle and for improving the clinical signs of hyperandrogenism. Although highly effective, COC, in particular early formulations, were associated with significant adverse effects and unacceptable CV risk. In addition, a decade after the advent of oral hormonal contraception, epidemiologic research confirmed an increased risk for cardiovascular events, particularly venous thromboembolism (VTE), in women using the high-dose COCs or COCs containing the newer gonane progestins desogestrel and gestodene (commonly referred to as "third-generation" progestins) (Burkmana et al., 2011). Research indicates also that COC use is associated with an increased risk of MI, particularly in women who smoke and are older than 35 years and in those who have underlying risk factors for coronary artery disease, such as hypertension (Burkman et al., 2011). Different estrogens and progestins exert different metabolic and CV effects. Estrogen component is almost always ethinyl estradiol (EE) in doses ranging from 15 to 50 /g. The progestin component is of variable potency and androgenicity. Hence, while in combined COCs the EE component seems to be responsible for the slight changes in the procoagulation and fibrinolytic balance, most progestins appear not to affect the coagulation factors and liver proteins when given alone (Sitruk-Ware & Nath, 2011). However, the metabolic effects of pills are extremely variable; for example estrogens impair insulin action dose-dependently and the associated progestins may modify these effects (Nader & Diamanti-Kandarakis, 2007). It was suggested that when a dose of EE < 50 µg/day is used, the effects of the COCs on the lipid and glycoinsulinemic metabolism is related to the progestin used in the combination (Mancini et al., 2010). Hence, the estrogen component of hormonal contraceptives when administered orally tends to increase the production of lipoproteins such as, the very low density lipoprotein (VLDL) and HDL levels while decreasing LDL levels (Sitruk-Ware & Nath, 2011). Non-androgenic or anti-androgenic progestins exert minimal influence on the lipid profile and carbohydrate metabolism, which lessens the risk of developing coronary heart disease while androgenic progestins have shown an adverse effect over total and HDL cholesterol (Nath & Sitruk-Ware, 2009, as cited in Sitruk-Ware & Nath, 2011). Over the past half century, COCs have undergone a number of evolutionary steps, involving modifications of hormone doses and types, dosage regimens and administration schedules (Burkmana et al., 2011) in order to improve the safety profile, particularly the metabolic and cardiovascular safety. In young women, the risk of CVD is very low but can be strongly influenced by smoking and the presence of other risks factors, such as hypertension, obesity, and DM or PCOS, specially for women over 35 (Farley et al., 1998, 1999, as cited in Sitruk-Ware & Nath, 2011).

As a special subgroup of COCs users, women with PCOS have an increased risk for IGT and as COCs are often prescribed as first-line treatment, a worsening of the metabolic outcomes in this population (e.g. insulin resistance, glucose tolerance, lipid profile or chronic inflammation) would be of concern. In the literature, there are conflicting reports about the CV effects of OCPs on PCOS patients (Battaglia et al., 2010; Meyer et al., 2007). Some of these reports support the beneficial effects of OCPs on the CV system in PCOS patients (Luque-Ramirez et al., 2009; Mancini et al., 2010). However, a recent meta-analysis suggested that OCPs may have negative effects on CV events in healthy individuals and these effects may be aggravated in patients displaying increased CV risk, such as PCOS (Baillargeon et al., 2005, as cited in Gode et al., 2010; Nader & Diamanti-Kandarakis, 2007). The outcome of the studies evaluating the effects of OCPs on carbohydrate metabolism in PCOS has been extremely variable, their results covering all possibilities: improvement, no change,

The Polycystic Ovary Syndrome Status – A Risk Factor for Future Cardiovascular Disease 185

Regarding different OCPs components, FMD appears to increase when circulating levels of estrogens augment naturally or synthetically and this beneficial vasodilator effect of estrogens on the arterial function might be antagonized by some certain types of progestins e.g. levonorgestrel, desogestrel (Torgrimson et al., 2007), but not by DRP, a progestin with antiandrogenic and antimineralocorticoid activity (Meendering et al., 2010). Consistent with these findings, it has been demonstrated that endothelial function was less in women using either a levonorgestrel combined OCP or medroxyprogesterone acetate (MPA) (a highly androgenic progestin) compared to nonusers (Meendering et al., 2010). However, controversial results have been reported in PCOS, suggesting that OCPs may have deteriorating, neutral or improving effects on endothelial function (Battaglia et al., 2010; Mancini et al., 2010; Meyer et al., 2007). In the study of Mancini et al. (2010), hypocaloric diet was prescribed to the obese patients in addition to OCPs and a significant weight reduction was observed after the treatment. In lean patients with PCOS, the DRP/EE30µg did not seem to affect endothelial function, whereas in overweight PCOS women it did not seem to counteract the loss of weight due to healthier lifestyle changes, which was associated with an improvement of insulin sensitivity and FMD. Therefore, it may be hypothesized that life style implementation may hinder the negative effects of treatment or natural progression of PCOS. In another recent study, there was a tendency of reduction in FMD in PCOS women during the 6-month treatment with EE and CPA combination. In addition, the significant alteration, which was observed particularly in the overweight group, supports the previous reports, which emphasize the independent role of obesity in CV risk (Vural et al., 2005, Mancini et al., 2009, as cited in Gode et al., 2010). Raised plasma concentrations of the endogenous nitric oxide synthase inhibitor asymmetric dimethylarginine (ADMA) have been associated with a higher prevalence of CV risk factors and CVD in different populations, including PCOS women (Dahlgren et al., 1992). Interestingly, ADMA levels were significantly decreased in PCOS subjects, after the administration for a longer period of time-12 months -of natural or synthetic estrogens, combined with anti-androgens (CPA or desogestrel), to levels comparable with those in the control group, in accordance with previous reports on ADMA concentrations in young and healthy women. A borderline but interesting decrease in ET-1 was also observed, possibly further indicating the beneficiary effect estrogens, combined with antiandrogens, have on endothelial function in women with this syndrome. The results of the present study are in agreement with previously published evidence regarding an inhibitory effect of estrogenic compounds on ADMA production

Regarding carotid IMT, a non-significant tendency toward a decrease in carotid IMT was reported with EE/CPA treatment in PCOS subjects (Luque-Ramirez et al., 2009). The remarkable aspect of the study was that a diet and physical activity were prescribed to all patients in addition to the ongoing treatment. The results of Gode et al. (2010) were contrary to the above mentioned study, and reported a significant increase in both right and left carotid IMTs in both the normal weight and the overweight groups after the 6-month follow-up of PCOS patients given OCP containing CPA. It is possible that the significant change in carotid IMT was triggered by increased lipid taking into account the fact that, with the exception of HDL, all lipid profile components were significantly augmented in the normal-weight patients while only TG levels were significantly increased in the overweight patients and endothelial dysfunction and increased IMT coexist with abnormal lipid profile (Karasek et al., 2006, as cited in Gode et al., 2010). Moreover, a recent study reported a CIMT

(Charitidou et al., 2008).

deterioration (Nader & Diamanti-Kandarakis, 2007). Different OCPs used with different dosage of estrogen, type of progestin and formulation of the pill, different laboratory methods used to assess these effects represent, however, few but important limitations of these studies which should be mentioned.

Hence, Nader & Diamanti-Kandarkis (2007) concluded that the effects of OCPs on carbohydrate metabolism in PCOS will be determined by the degree of androgenicity of the woman and the androgen-lowering effect of the pill, genetically determined endogenous insulin sensitivity of the individual, anthropometric differences that can affect insulin action and by the natural history of PCOS or environmental influences and by other factors, such as puberty, which is associated with decreased insulin sensitivity (Moran et al., 1999, as cited in Nader & Diamanti-Kandarakis, 2007).

These concepts are represented in a few studies. Thus, Cagnacci et al. compared a monophasic 35-µg EE/cyproterone acetate (CPA) pill, widely used in PCOS with a biphasic 40/30 EE/desogestrel pill. The results were contradictory: insulin sensitivity improved with the CPA pill (lowering of androgens with a pill containing the most potent antiandrogenic progesterone) and deteriorated with the desogestrel pill (a more androgenic pill and/or a different formulation with a higher initial dose of estrogen) (Cagnacci et al., 2003, as cited in Nader & Diamanti-Kandarakis, 2007). Ibanez & de Zegher (2004a) also demonstrated the effect of different progestins of potentially different androgenicity. They included postadolescent PCOS subjects who were already taking flutamide and metformin along with a 20-µg EE-/gestodene-containing OCP in their study and, after some months of this treatment, they randomized subjects to replacement of the gestodene OCP with a drospirenone (DRP) OCP containing 30 µg of EE. The patients who were switched DRP presented a reduced total and abdominal fat and an increased lean body mass (a less androgenic OCP reduced the abdominal fat and potentially improved the metabolic profile in spite of the higher dose of estrogen in the drospirenone pill) as compared with those who remained on gestodene.

As far as lipid metabolism is concerned, HDL -C and TG generally increase after OCP treatment in PCOS and this effect varies with the progestin. Higher TG concentrations are seen with less androgenic progestins (Mastorakos et al., 2002, as cited in Nader & Diamanti-Kandarakis, 2007). Ibanez & de Zegher (2004b) demonstrated that abnormal adipocytokines (increased IL-6 levels and decreased adiponectin levels), hypertriglyceridaemia and body adiposity deteriorated in a group of adolescents and young women with PCOS who were administered EE/DRP, and improved towards the norm after flutamide and metformin were added. They also showed that flutamide and metformin have beneficial effects on these adverse factors either taken individually or together (Ibanez et al., 2004; Ibanez & de Zegher, 2005, as cited in Nader & Diamanti-Kandarakis, 2007).

Furthermore, Halperin et al. (2011) undertook a meta-analysis of published observational studies to investigate the association between COC use and dysglycemia, dyslipidemia and insulin resistance in women with PCOS. COC use was significantly associated with an increase in HDL-C and TG, but was not associated with changes in fasting glucose or fasting insulin. The authors concluded that the use of COCs in women with PCOS was not associated with clinically significant adverse metabolic consequences (Halperin et al., 2011). Additionally, according to the findings in the literature, OCPs seem to have neutral or negative effects on endothelial function among healthy individuals (Gode et al., 2010).

deterioration (Nader & Diamanti-Kandarakis, 2007). Different OCPs used with different dosage of estrogen, type of progestin and formulation of the pill, different laboratory methods used to assess these effects represent, however, few but important limitations of

Hence, Nader & Diamanti-Kandarkis (2007) concluded that the effects of OCPs on carbohydrate metabolism in PCOS will be determined by the degree of androgenicity of the woman and the androgen-lowering effect of the pill, genetically determined endogenous insulin sensitivity of the individual, anthropometric differences that can affect insulin action and by the natural history of PCOS or environmental influences and by other factors, such as puberty, which is associated with decreased insulin sensitivity (Moran et al., 1999, as

These concepts are represented in a few studies. Thus, Cagnacci et al. compared a monophasic 35-µg EE/cyproterone acetate (CPA) pill, widely used in PCOS with a biphasic 40/30 EE/desogestrel pill. The results were contradictory: insulin sensitivity improved with the CPA pill (lowering of androgens with a pill containing the most potent antiandrogenic progesterone) and deteriorated with the desogestrel pill (a more androgenic pill and/or a different formulation with a higher initial dose of estrogen) (Cagnacci et al., 2003, as cited in Nader & Diamanti-Kandarakis, 2007). Ibanez & de Zegher (2004a) also demonstrated the effect of different progestins of potentially different androgenicity. They included postadolescent PCOS subjects who were already taking flutamide and metformin along with a 20-µg EE-/gestodene-containing OCP in their study and, after some months of this treatment, they randomized subjects to replacement of the gestodene OCP with a drospirenone (DRP) OCP containing 30 µg of EE. The patients who were switched DRP presented a reduced total and abdominal fat and an increased lean body mass (a less androgenic OCP reduced the abdominal fat and potentially improved the metabolic profile in spite of the higher dose of estrogen in the drospirenone pill) as compared with those who

As far as lipid metabolism is concerned, HDL -C and TG generally increase after OCP treatment in PCOS and this effect varies with the progestin. Higher TG concentrations are seen with less androgenic progestins (Mastorakos et al., 2002, as cited in Nader & Diamanti-Kandarakis, 2007). Ibanez & de Zegher (2004b) demonstrated that abnormal adipocytokines (increased IL-6 levels and decreased adiponectin levels), hypertriglyceridaemia and body adiposity deteriorated in a group of adolescents and young women with PCOS who were administered EE/DRP, and improved towards the norm after flutamide and metformin were added. They also showed that flutamide and metformin have beneficial effects on these adverse factors either taken individually or together (Ibanez et al., 2004; Ibanez & de

Furthermore, Halperin et al. (2011) undertook a meta-analysis of published observational studies to investigate the association between COC use and dysglycemia, dyslipidemia and insulin resistance in women with PCOS. COC use was significantly associated with an increase in HDL-C and TG, but was not associated with changes in fasting glucose or fasting insulin. The authors concluded that the use of COCs in women with PCOS was not associated with clinically significant adverse metabolic consequences (Halperin et al., 2011). Additionally, according to the findings in the literature, OCPs seem to have neutral or negative effects on endothelial function among healthy individuals (Gode et al., 2010).

Zegher, 2005, as cited in Nader & Diamanti-Kandarakis, 2007).

these studies which should be mentioned.

cited in Nader & Diamanti-Kandarakis, 2007).

remained on gestodene.

Regarding different OCPs components, FMD appears to increase when circulating levels of estrogens augment naturally or synthetically and this beneficial vasodilator effect of estrogens on the arterial function might be antagonized by some certain types of progestins e.g. levonorgestrel, desogestrel (Torgrimson et al., 2007), but not by DRP, a progestin with antiandrogenic and antimineralocorticoid activity (Meendering et al., 2010). Consistent with these findings, it has been demonstrated that endothelial function was less in women using either a levonorgestrel combined OCP or medroxyprogesterone acetate (MPA) (a highly androgenic progestin) compared to nonusers (Meendering et al., 2010). However, controversial results have been reported in PCOS, suggesting that OCPs may have deteriorating, neutral or improving effects on endothelial function (Battaglia et al., 2010; Mancini et al., 2010; Meyer et al., 2007). In the study of Mancini et al. (2010), hypocaloric diet was prescribed to the obese patients in addition to OCPs and a significant weight reduction was observed after the treatment. In lean patients with PCOS, the DRP/EE30µg did not seem to affect endothelial function, whereas in overweight PCOS women it did not seem to counteract the loss of weight due to healthier lifestyle changes, which was associated with an improvement of insulin sensitivity and FMD. Therefore, it may be hypothesized that life style implementation may hinder the negative effects of treatment or natural progression of PCOS. In another recent study, there was a tendency of reduction in FMD in PCOS women during the 6-month treatment with EE and CPA combination. In addition, the significant alteration, which was observed particularly in the overweight group, supports the previous reports, which emphasize the independent role of obesity in CV risk (Vural et al., 2005, Mancini et al., 2009, as cited in Gode et al., 2010). Raised plasma concentrations of the endogenous nitric oxide synthase inhibitor asymmetric dimethylarginine (ADMA) have been associated with a higher prevalence of CV risk factors and CVD in different populations, including PCOS women (Dahlgren et al., 1992). Interestingly, ADMA levels were significantly decreased in PCOS subjects, after the administration for a longer period of time-12 months -of natural or synthetic estrogens, combined with anti-androgens (CPA or desogestrel), to levels comparable with those in the control group, in accordance with previous reports on ADMA concentrations in young and healthy women. A borderline but interesting decrease in ET-1 was also observed, possibly further indicating the beneficiary effect estrogens, combined with antiandrogens, have on endothelial function in women with this syndrome. The results of the present study are in agreement with previously published evidence regarding an inhibitory effect of estrogenic compounds on ADMA production (Charitidou et al., 2008).

Regarding carotid IMT, a non-significant tendency toward a decrease in carotid IMT was reported with EE/CPA treatment in PCOS subjects (Luque-Ramirez et al., 2009). The remarkable aspect of the study was that a diet and physical activity were prescribed to all patients in addition to the ongoing treatment. The results of Gode et al. (2010) were contrary to the above mentioned study, and reported a significant increase in both right and left carotid IMTs in both the normal weight and the overweight groups after the 6-month follow-up of PCOS patients given OCP containing CPA. It is possible that the significant change in carotid IMT was triggered by increased lipid taking into account the fact that, with the exception of HDL, all lipid profile components were significantly augmented in the normal-weight patients while only TG levels were significantly increased in the overweight patients and endothelial dysfunction and increased IMT coexist with abnormal lipid profile (Karasek et al., 2006, as cited in Gode et al., 2010). Moreover, a recent study reported a CIMT

The Polycystic Ovary Syndrome Status – A Risk Factor for Future Cardiovascular Disease 187

rate of CVDs in postmenopausal women affected by PCOS. However, in spite of the fact that risk factors have accumulated, we still need evidence to indicate that the high risk increases events and available studies have not ascertained, yet, a uniform association between PCOS and CVD. This is because of the lack of adequate PCOS characterization, appropriate CV disease measurement, and sufficient duration of follow-up or a true lack of association.

Initial studies on the prevalence of CVDs in postmenopausal women who were probably affected by PCOS indicated an increased risk for developing MI (Dahlgren et al., 1992). It was calculated that postmenopausal women with previous PCOS have a 7.1 higher risk than non-PCOS women of developing MI (Carmina, 2009). In a Pittsburgh cohort of 162 Caucasian women with PCOS who were followed up for CV events for up to 12 years, 5 women reported MI, angina pectoris, and/or coronary bypass or angioplasty, whereas no events were observed among 142 control women who were similarly followed (Talbott et al., 2004c). Additionally, a 4-fold risk in cardiac events among women with PCOS was reported in a cohort from the Czech Republic (Cibula et al., 2000). On the other hand, reviewing death certificates of 786 women in the United Kingdom who were diagnosed with PCOS at an average age of 26.4 years and followed for an average duration of 30 years, Pierpoint et al. (1998) failed to show a statistically significant increase in CV mortality. This study has been criticized because the diagnosis of PCOS was based on historical records over a very long period and was not supported by hormonal studies or ovarian morphology. Additionally, interpretation of their data is constrained by the fact that PCOS cases were diagnosed mainly on the basis of hospital records related to wedge resection and by the absence of a matched control cohort. Wedge resection can correct the anovulation and metabolic changes that are observed in PCOS for long periods of time, and this method of case identification may under-ascertain PCOS cases as defined by clinical characteristics. Probably, a more important criticism is that the study evaluated the causes of death of relatively young women. However, in a later report, the same authors (Wild et al., 2000) noted a higher prevalence of cerebrovascular accidents in women who had PCOS during their fertile age. The history of coronary heart disease was, on the contrary, not significantly more common in women with PCOS. On the other hand, in the Nurses' Health Study, which followed 82 439 women for 14 years, it was noted that the women with very irregular menses, which can be considered a surrogate marker for PCOS, had a significantly increased relative risk of 1.5 (95% confidence interval (CI) 1.3–1.9) for coronary heart disease and 1.9 (95% CI 1.3–2.7) for fatal MI compared with eumenorrheic women (Dawber et al., 1951, as cited in Carmina, 2009). More recently, a sub-study of the Women's Ischemia Evaluation Study (WISE) confirmed that women with PCOS have a larger number of CV events. In this study, 104 postmenopausal women with PCOS and a control group of 286 matched normal postmenopausal women were followed prospectively for close to 10 years; multi-vessel angiographic coronary artery disease was observed in 32% of PCOS women compared to 25% of non-PCOS women (odds ratio 1.7) and correlated with several factors, including increased free testosterone. In addition, the event-free survival (including fatal and non-fatal events) was significantly lower in PCOS compared to non- PCOS women. The difference between the two groups was higher when cerebrovascular accidents were also considered, confirming the association of PCOS with stroke. Moreover, women with clinical features of PCOS with increased hsCRP concentrations had a 12.2-fold higher risk of CV death or nonfatal MI than women without clinical features of PCOS and lower levels of hsCRP, suggesting that the independent adverse association between PCOS status and postmenopause CV events may act vis-à-vis inflammatory pathway (Shaw et al., 2008).

progression rate of 0.015 ± 0.024 mm per year among healthy young women (Johnson et al., 2007, as cited in Gode et al., 2010). However, the increase in carotid IMT in PCOS patients was 0.03 ± 0.01 mm at the end of the 6 month-period, a value which is four times higher than that reported in the healthy young women of that study. The quick advance of these CV risk parameters draws the attention to the importance of close follow-up in PCOS patients. However, due to the fact that the study included only one type of pill, to explain the deterioration of arterial structure through CPA would be a rushed conclusion. Another possibility is that EE plays a role in this fast progression of atherosclerosis. Therefore, we need additional studies to include different types of OCPs to provide clear and accurate results.

In previous studies, a CRP greater than 3 mg/L was accepted to be correlated with CVD (Boulman et al., 2004, as cited in Gode et al., 2010). In the same study performed by Gode et al. (2010), there was also a tendency toward increment in hCRP after 6 months treatment with EE/CPA. Sustaining these observations, the use of OCPs (EEDesogestrel combination) in obese and nonobese patients with PCOS with impaired glucose tolerance resulted in significantly higher ADMA and hs-CRP levels compared to pretreatment values, thus creating an increase in the metabolic risk (Kilic et al., 2010). The hsPCR levels increased after a 6 month-treatment with COC in young, obese PCOS patients even though CO included progestatives with antiandrogenic activity (Morin-Papunen et al., 2003; Teede et al., 2010b). Both estrogens and progestin content and dosage appear to be implicated in CRP regulation, even though the role of oestrogen might be more important than that of progestin (Haarala et al., 2009). It was noted that COCs stimulate hepatocytes directly to synthesize CRP, and not via IL-6-mediated inflammation. Furthermore, there are studies that demonstrate that DRP/EE30µg monotherapy additionally deteriorated the already increased levels of IL-6 and the decreased levels of adiponectin as well as the adiposity of young PCOS female patients, whereas the treatment with flutamide-metformin reduced all studied indices towards normal levels (Ibanez L & de Zegher, 2006).

To date combined oral contraceptive pills (OCPs) have been the first-line treatment for PCOS. They induce predictable cyclic menses, reduce luteinizing hormone secretion, lower ovarian androgen production and ameliorate androgenic symptoms, increase sex hormone binding globulin thus reducing free androgens and protecting the endometrium. Taken together, until further definitive results are available, these findings revealed that OCPs may not be protective against the progression of CV risk parameters in PCOS. Thus, in some PCOS patients, such as the obese or pubertal, this additional metabolic risk should be considered when OCPs are prescribed, and appropriate surveillance is advisable, as is concomitant use of agents that modify these effects, such as metformin. Some additional treatment modalities such as life style implementation may be added to the treatment, especially in overweight PCOS patients too. Additionally, it is preferable to evaluate CV risk factors of PCOS patients under treatment with OCP, especially the obese ones. Long-term studies with larger populations are needed to confirm how specific OCP formulations affect vascular function of the arterial system in young women.

#### **7. Do cardiovascular risk factors in PCOS result in more cardiovascular events?**

The high prevalence of CV risk factors and signs of early atherosclerosis (e.g. increased IMT or altered endothelial reactivity) in young women with PCOS may determine an increased

progression rate of 0.015 ± 0.024 mm per year among healthy young women (Johnson et al., 2007, as cited in Gode et al., 2010). However, the increase in carotid IMT in PCOS patients was 0.03 ± 0.01 mm at the end of the 6 month-period, a value which is four times higher than that reported in the healthy young women of that study. The quick advance of these CV risk parameters draws the attention to the importance of close follow-up in PCOS patients. However, due to the fact that the study included only one type of pill, to explain the deterioration of arterial structure through CPA would be a rushed conclusion. Another possibility is that EE plays a role in this fast progression of atherosclerosis. Therefore, we need additional studies to include different types of OCPs to provide clear and accurate results.

In previous studies, a CRP greater than 3 mg/L was accepted to be correlated with CVD (Boulman et al., 2004, as cited in Gode et al., 2010). In the same study performed by Gode et al. (2010), there was also a tendency toward increment in hCRP after 6 months treatment with EE/CPA. Sustaining these observations, the use of OCPs (EEDesogestrel combination) in obese and nonobese patients with PCOS with impaired glucose tolerance resulted in significantly higher ADMA and hs-CRP levels compared to pretreatment values, thus creating an increase in the metabolic risk (Kilic et al., 2010). The hsPCR levels increased after a 6 month-treatment with COC in young, obese PCOS patients even though CO included progestatives with antiandrogenic activity (Morin-Papunen et al., 2003; Teede et al., 2010b). Both estrogens and progestin content and dosage appear to be implicated in CRP regulation, even though the role of oestrogen might be more important than that of progestin (Haarala et al., 2009). It was noted that COCs stimulate hepatocytes directly to synthesize CRP, and not via IL-6-mediated inflammation. Furthermore, there are studies that demonstrate that DRP/EE30µg monotherapy additionally deteriorated the already increased levels of IL-6 and the decreased levels of adiponectin as well as the adiposity of young PCOS female patients, whereas the treatment with flutamide-metformin reduced all

To date combined oral contraceptive pills (OCPs) have been the first-line treatment for PCOS. They induce predictable cyclic menses, reduce luteinizing hormone secretion, lower ovarian androgen production and ameliorate androgenic symptoms, increase sex hormone binding globulin thus reducing free androgens and protecting the endometrium. Taken together, until further definitive results are available, these findings revealed that OCPs may not be protective against the progression of CV risk parameters in PCOS. Thus, in some PCOS patients, such as the obese or pubertal, this additional metabolic risk should be considered when OCPs are prescribed, and appropriate surveillance is advisable, as is concomitant use of agents that modify these effects, such as metformin. Some additional treatment modalities such as life style implementation may be added to the treatment, especially in overweight PCOS patients too. Additionally, it is preferable to evaluate CV risk factors of PCOS patients under treatment with OCP, especially the obese ones. Long-term studies with larger populations are needed to confirm how specific OCP formulations affect

**7. Do cardiovascular risk factors in PCOS result in more cardiovascular** 

The high prevalence of CV risk factors and signs of early atherosclerosis (e.g. increased IMT or altered endothelial reactivity) in young women with PCOS may determine an increased

studied indices towards normal levels (Ibanez L & de Zegher, 2006).

vascular function of the arterial system in young women.

**events?** 

rate of CVDs in postmenopausal women affected by PCOS. However, in spite of the fact that risk factors have accumulated, we still need evidence to indicate that the high risk increases events and available studies have not ascertained, yet, a uniform association between PCOS and CVD. This is because of the lack of adequate PCOS characterization, appropriate CV disease measurement, and sufficient duration of follow-up or a true lack of association.

Initial studies on the prevalence of CVDs in postmenopausal women who were probably affected by PCOS indicated an increased risk for developing MI (Dahlgren et al., 1992). It was calculated that postmenopausal women with previous PCOS have a 7.1 higher risk than non-PCOS women of developing MI (Carmina, 2009). In a Pittsburgh cohort of 162 Caucasian women with PCOS who were followed up for CV events for up to 12 years, 5 women reported MI, angina pectoris, and/or coronary bypass or angioplasty, whereas no events were observed among 142 control women who were similarly followed (Talbott et al., 2004c). Additionally, a 4-fold risk in cardiac events among women with PCOS was reported in a cohort from the Czech Republic (Cibula et al., 2000). On the other hand, reviewing death certificates of 786 women in the United Kingdom who were diagnosed with PCOS at an average age of 26.4 years and followed for an average duration of 30 years, Pierpoint et al. (1998) failed to show a statistically significant increase in CV mortality. This study has been criticized because the diagnosis of PCOS was based on historical records over a very long period and was not supported by hormonal studies or ovarian morphology. Additionally, interpretation of their data is constrained by the fact that PCOS cases were diagnosed mainly on the basis of hospital records related to wedge resection and by the absence of a matched control cohort. Wedge resection can correct the anovulation and metabolic changes that are observed in PCOS for long periods of time, and this method of case identification may under-ascertain PCOS cases as defined by clinical characteristics. Probably, a more important criticism is that the study evaluated the causes of death of relatively young women. However, in a later report, the same authors (Wild et al., 2000) noted a higher prevalence of cerebrovascular accidents in women who had PCOS during their fertile age. The history of coronary heart disease was, on the contrary, not significantly more common in women with PCOS. On the other hand, in the Nurses' Health Study, which followed 82 439 women for 14 years, it was noted that the women with very irregular menses, which can be considered a surrogate marker for PCOS, had a significantly increased relative risk of 1.5 (95% confidence interval (CI) 1.3–1.9) for coronary heart disease and 1.9 (95% CI 1.3–2.7) for fatal MI compared with eumenorrheic women (Dawber et al., 1951, as cited in Carmina, 2009). More recently, a sub-study of the Women's Ischemia Evaluation Study (WISE) confirmed that women with PCOS have a larger number of CV events. In this study, 104 postmenopausal women with PCOS and a control group of 286 matched normal postmenopausal women were followed prospectively for close to 10 years; multi-vessel angiographic coronary artery disease was observed in 32% of PCOS women compared to 25% of non-PCOS women (odds ratio 1.7) and correlated with several factors, including increased free testosterone. In addition, the event-free survival (including fatal and non-fatal events) was significantly lower in PCOS compared to non- PCOS women. The difference between the two groups was higher when cerebrovascular accidents were also considered, confirming the association of PCOS with stroke. Moreover, women with clinical features of PCOS with increased hsCRP concentrations had a 12.2-fold higher risk of CV death or nonfatal MI than women without clinical features of PCOS and lower levels of hsCRP, suggesting that the independent adverse association between PCOS status and postmenopause CV events may act vis-à-vis inflammatory pathway (Shaw et al., 2008).

The Polycystic Ovary Syndrome Status – A Risk Factor for Future Cardiovascular Disease 189

PCOS. Early CV screening and treatment of all modifiable CV risk factors may be clinically considered a secondary preventing intervention. Recognition of PCOS as an independent risk factor for subsequent CV events would then justify earlier risk-factor intervention.

Achard C & Thiers J. (1921). Le virilisme pilaire et son association á l`insuffisance glycotique (diabete des femmes á barbe). BullAcad Natl Méd (Paris) Vol. 86, pp. 51–56. Akram T, Hasan S, Imran M, Karim A & Arslan M. (2010). Association of polycystic ovary

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While other studies are needed to confirm and expand the available information, PCOS seems, however, to be associated with an increased risk for cerebrovascular events (stroke) and probably also for fatal and non-fatal coronary heart disease, that is independent of their underlying clinical risk, indicating that PCOS-related protracted hyperandrogenism may be one possible mechanism for their CV risk. Consequently, identification of clinical features of PCOS in postmenopausal women may provide an opportunity for risk factor intervention for the prevention of CV disease and CV events.

Nevertheless, there is a clear need for longitudinal, prospective study of a large cohort of well-characterized premenopausal PCOS, along with an appropriate cohort of matched control women that can be followed into menopause. Studies like these will provide a more categorical analysis of the increased CV risk in PCOS and a more accurate assessment of the risk for CV events and mortality in a way that could apply to the overall population of women with PCOS.

#### **8. PCOS status - A major cardiovascular risk factor in women**

Taken all together, there is no doubt that the diagnosis of PCOS implies increased cardiac risk. Moreover, in a previous research, we noted that the regression analysis performed on our subjects (women with PCOS and healthy age-matched controls) revealed a predictive value of PCOS status on both FMD and ET-1 values (Ilie et al., 2011). Therefore, in line with others who have suggested PCOS as a major unrecognized CV risk factor in women (Alexander et al., 2009), we proposed that PCOS status should be regarded as a predictor marker of CV risk, beside other well-known CV risk factors, e.g. insulin resistance, dyslipidemia.

The common clustering of CV risk factors with PCOS in reproductive young women might become a public health concern if proved to trigger an increased risk for the development of CVD. Hence, there is a need for longitudinal, prospective, case-control, more rigorous, large sample size studies in young women with PCOS, all of which should be not only long term so as to go beyond menopause, but also homogeneous in terms of both prevalence and degree of obesity as well as the methodology used for evaluating vascular injury or insulin resistance in order to document the early presence of vascular abnormalities and chronic inflammation and to establish whether these CV risk factors subsequently lead to CV events. These are also especially important because it was hypothesized that differences in risk factors between women with PCOS and controls may diminish with increasing age as a consequence of both spontaneous decrease in androgen secretion after the age of 35 years in normal women and in those with PCOS as well as decrease in the polycystic ovary prevalence with age (Labrie et al., 1997, Koivunen et al., 1999, as cited in Carmina, 2009). This might account for the possible overestimation of CV risk in young women with PCOS or for failure to detect excess risk of CV mortality among women with this disorder. However, these is very unlikely since at least one-third of PCOS patients present with increased levels of non-HDL C (Berneis et al., 2009, as cited in Carmina, 2009) and/or display several other traditional and nontraditional CV risk factors. Moreover, short-term trials of pertinent interventions, for example OCPs, insulin sensitizers, on surrogate CV outcomes are warranted, since their effect on CV risk factors and events are not clearly settled. We need accurate studies to establish CV screening guidelines for women with PCOS. Early CV screening and treatment of all modifiable CV risk factors may be clinically considered a secondary preventing intervention. Recognition of PCOS as an independent risk factor for subsequent CV events would then justify earlier risk-factor intervention.

#### **9. References**

188 Recent Advances in Cardiovascular Risk Factors

While other studies are needed to confirm and expand the available information, PCOS seems, however, to be associated with an increased risk for cerebrovascular events (stroke) and probably also for fatal and non-fatal coronary heart disease, that is independent of their underlying clinical risk, indicating that PCOS-related protracted hyperandrogenism may be one possible mechanism for their CV risk. Consequently, identification of clinical features of PCOS in postmenopausal women may provide an opportunity for risk factor intervention

Nevertheless, there is a clear need for longitudinal, prospective study of a large cohort of well-characterized premenopausal PCOS, along with an appropriate cohort of matched control women that can be followed into menopause. Studies like these will provide a more categorical analysis of the increased CV risk in PCOS and a more accurate assessment of the risk for CV events and mortality in a way that could apply to the overall population of

Taken all together, there is no doubt that the diagnosis of PCOS implies increased cardiac risk. Moreover, in a previous research, we noted that the regression analysis performed on our subjects (women with PCOS and healthy age-matched controls) revealed a predictive value of PCOS status on both FMD and ET-1 values (Ilie et al., 2011). Therefore, in line with others who have suggested PCOS as a major unrecognized CV risk factor in women (Alexander et al., 2009), we proposed that PCOS status should be regarded as a predictor marker of CV risk, beside other well-known CV risk factors, e.g. insulin resistance,

The common clustering of CV risk factors with PCOS in reproductive young women might become a public health concern if proved to trigger an increased risk for the development of CVD. Hence, there is a need for longitudinal, prospective, case-control, more rigorous, large sample size studies in young women with PCOS, all of which should be not only long term so as to go beyond menopause, but also homogeneous in terms of both prevalence and degree of obesity as well as the methodology used for evaluating vascular injury or insulin resistance in order to document the early presence of vascular abnormalities and chronic inflammation and to establish whether these CV risk factors subsequently lead to CV events. These are also especially important because it was hypothesized that differences in risk factors between women with PCOS and controls may diminish with increasing age as a consequence of both spontaneous decrease in androgen secretion after the age of 35 years in normal women and in those with PCOS as well as decrease in the polycystic ovary prevalence with age (Labrie et al., 1997, Koivunen et al., 1999, as cited in Carmina, 2009). This might account for the possible overestimation of CV risk in young women with PCOS or for failure to detect excess risk of CV mortality among women with this disorder. However, these is very unlikely since at least one-third of PCOS patients present with increased levels of non-HDL C (Berneis et al., 2009, as cited in Carmina, 2009) and/or display several other traditional and nontraditional CV risk factors. Moreover, short-term trials of pertinent interventions, for example OCPs, insulin sensitizers, on surrogate CV outcomes are warranted, since their effect on CV risk factors and events are not clearly settled. We need accurate studies to establish CV screening guidelines for women with

**8. PCOS status - A major cardiovascular risk factor in women** 

for the prevention of CV disease and CV events.

women with PCOS.

dyslipidemia.


The Polycystic Ovary Syndrome Status – A Risk Factor for Future Cardiovascular Disease 191

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(2004b). The relationship between C-reactive protein and carotid intima-media wall thickness in middle-aged women with polycystic ovary syndrome. J Clin

Evidence for an association between metabolic cardiovascular syndrome and coronary and aortic calcification among women with polycystic ovary syndrome. J

syndrome have an increased risk of cardiovascular disease? Review of the


**10** 

*Japan* 

**Premature Atherosclerosis** 

Nobutaka Noto and Tomoo Okada

**Long After Kawasaki Disease** 

*Department of Pediatrics and Child Health, Nihon University School of Medicine, Tokyo,* 

Kawasaki disease (KD) is a systemic vasculitis of unknown etiology in infants and children. First described in Japan in 1967, KD has been described worldwide among children of all races and ethnicities (Kawasaki, 1967). More than 4000 hospitalizations associated with KD were reported in 2000 and KD is the leading cause of acquired heart disease in childhood in the United State (Taubert et al., 1991). In Japan, the national survey of KD has performed every 2 years since 1970. From the most recent 20th survey dealt with the years 2007 and 2008, the number of new cases of KD in those years was 11,581 and 11,756, respectively. As of 2008, the total number of patients since 1970 was 249,019 (Nakamura et al., 2010). Therefore, patients diagnosed with KD in the sixties and seventies have already reached adulthood. The increased incidence of young adults who experience KD during childhood has been accompanied by a new problem of an association between post-KD lesions and

In this chapter, we would like to review and verify with regarding to the propensity of "Premature atherosclerosis long after Kawasaki disease" from several cutting edges such as epidemiologic features, dyslipidemia, non-invasive diagnostic methods for evaluating atherosclerosis such as flow-mediated dilatation (FMD) of brachial artery, carotid intimamedia thickness (IMT), pulse wave velocity (PWV), invasive diagnostic method such as intravascular ultrasound imaging (IVUS) and pathological features along with the findings

The incidence rate and number of patients with KD in Japan continue to increase. The 20th nationwide survey of KD in Japan was conducted in 2009, and included patients treated for the disease in 2007 and 2008 (Nakamura et al., 2010). Hospitals specializing in pediatrics, and hospitals with pediatric departments and 100 or more beds, were asked to report all patients with KD during the 2 survey years. From a total of 1540 departments and hospitals, 23,337 patients (11 581 in 2007 and 11 756 in 2008) were reported: 13,523 boys and 9814 girls. The annual incidence rates were 215.3 and 218.6 per 100,000 children aged 0-4 years in 2007 and 2008, respectively. This incidence was approximately 10 to 25 times higher than that of the United States and the United Kingdom. These were the highest annual KD incidence

**1. Introduction** 

atherosclerosis.

of our recent studies.

**2. Epidemiology of Kawasaki disease** 


## **Premature Atherosclerosis Long After Kawasaki Disease**

Nobutaka Noto and Tomoo Okada

*Department of Pediatrics and Child Health, Nihon University School of Medicine, Tokyo, Japan* 

#### **1. Introduction**

200 Recent Advances in Cardiovascular Risk Factors

Vrbíková J, Zamrazilová H, Sedláková B, Snajderová M. (2010a). Metabolic syndrome in adolescents with polycystic ovary syndrome, Gynecol Endocrinol, nd Vural B, Caliskan E, Turkoz E, Kilic T & Demirci A. (2005). Evaluation of metabolic

Wild RA, Rizzo M, Clifton S & Carmina E. (2011). .Lipid levels in polycystic ovary

Wild RA, Painter PC, Coulson PB, Carruth KB & Ranney GB. (1985). Lipoprotein lipid

Wild S, Pierpoint T, McKeigue P & Jacobs H. (2000). Cardiovascular disease in women with

Wiltgen D & Spritzer PM. (2010). Variation in metabolic and cardiovascular risk in women

Xita, N, Papassotiriou I, Georgiou I, Vounatsou M, Margeli A & Tsatsoulis A. (2007). The

Yilmaz M, Bukan N, Demirci H, Oztürk C, Kan E, Ayvaz G & Arslan M. (2009). Serum

Zawdaki J, Dunaif A. Diagnostic criteria for polycystic ovary syndrome: towards a rational

No. issue 5, pp. 2038-2049

J Clin Endocrinol Metab, Vol. 61, pp. 946-951

Clin Endocrinol (Oxf), Vol.52, No. issue 5, pp. 595-600.

pp. 1073-9.e1-11

issue 6, pp. 2493-2496

Endocrinol, Vol. 25, pp. 246–252

Blackwell Scientific; 1992. pp. 377-384.

6, pp. 766–771

syndrome frequency and premature carotid atherosclerosis in young women with polycystic ovary syndrome. Hum Reprod, Vol. 20, No. issue 9, pp. 2409-2413 Wild RA, Carmina E, Diamanti-Kandarakis E, Dokras A, Escobar-Morreale HF, Futterweit

W, Lobo R, Norman RJ, Talbott E & Dumesic DA. (2010). Assessment of cardiovascular risk and prevention of cardiovascular disease in women with the polycystic ovary syndrome: a consensus statement by the Androgen Excess and Polycystic Ovary Syndrome (AE-PCOS) Society. J Clin Endocrinol Metab, Vol. 95,

syndrome: systematic review and meta-analysis. Fertil Steril, Vol. 95, No. issue 3,

concentrations and cardiovascular risk in women with polycystic ovary syndrome.

polycystic ovary syndrome at long-term follow-up: a retrospective cohort study.

with different polycystic ovary syndrome phenotypes. Fertil Steril, Vol 94, No.

adiponectin-to-leptin ratio inwomenwith polycystic ovary syndrome: relation to insulin resistance and proinflammatory markers. Metab Clin Exp, Vol. 56, No.issue

resistin and adiponectin levels in women with polycystic ovary syndrome. Gynecol

approach. In: Dunaif A, Givens J, Haseltine F, Marrian G, editors. Polycystic Ovary Syndrome. Current Issues in Endocrinology and Metabolism. Vol. 4. Boston: Kawasaki disease (KD) is a systemic vasculitis of unknown etiology in infants and children. First described in Japan in 1967, KD has been described worldwide among children of all races and ethnicities (Kawasaki, 1967). More than 4000 hospitalizations associated with KD were reported in 2000 and KD is the leading cause of acquired heart disease in childhood in the United State (Taubert et al., 1991). In Japan, the national survey of KD has performed every 2 years since 1970. From the most recent 20th survey dealt with the years 2007 and 2008, the number of new cases of KD in those years was 11,581 and 11,756, respectively. As of 2008, the total number of patients since 1970 was 249,019 (Nakamura et al., 2010). Therefore, patients diagnosed with KD in the sixties and seventies have already reached adulthood. The increased incidence of young adults who experience KD during childhood has been accompanied by a new problem of an association between post-KD lesions and atherosclerosis.

In this chapter, we would like to review and verify with regarding to the propensity of "Premature atherosclerosis long after Kawasaki disease" from several cutting edges such as epidemiologic features, dyslipidemia, non-invasive diagnostic methods for evaluating atherosclerosis such as flow-mediated dilatation (FMD) of brachial artery, carotid intimamedia thickness (IMT), pulse wave velocity (PWV), invasive diagnostic method such as intravascular ultrasound imaging (IVUS) and pathological features along with the findings of our recent studies.

#### **2. Epidemiology of Kawasaki disease**

The incidence rate and number of patients with KD in Japan continue to increase. The 20th nationwide survey of KD in Japan was conducted in 2009, and included patients treated for the disease in 2007 and 2008 (Nakamura et al., 2010). Hospitals specializing in pediatrics, and hospitals with pediatric departments and 100 or more beds, were asked to report all patients with KD during the 2 survey years. From a total of 1540 departments and hospitals, 23,337 patients (11 581 in 2007 and 11 756 in 2008) were reported: 13,523 boys and 9814 girls. The annual incidence rates were 215.3 and 218.6 per 100,000 children aged 0-4 years in 2007 and 2008, respectively. This incidence was approximately 10 to 25 times higher than that of the United States and the United Kingdom. These were the highest annual KD incidence

Premature Atherosclerosis Long After Kawasaki Disease 203

infarction and sudden death have been caused by coronary artery occlusion, two-thirds of cases with occlusion have no symptomatic episodes due to the consequence of postocclusion recanalization and development of collateral vessels. Tsuda et al. reported the clinical features of 50 adult patients (mean age 28 years old) who had an acute coronary syndrome caused by coronary artery lesions due to KD from 1980 to 2008. Of the 50 patients, 43 had thrombotic occlusion of an aneurysm and 40 had giant coronary aneurysms. Based on these evidences, it is likely that patients with known aneurysms during the acute phase of KD will have some cardiovascular morbidity and mortality as young adults (Tsuda et al., 2011).

Fig. 1. Transition of the number of patients with cardiac sequelae in KD

New vessels after occlusion are called as segmental stenosis. Segmental stenosis has been observed in 14.8% of patients with KD sequelae, 90% of which occurred in the right coronary artery. The right coronary artery is occluded or recanalized more easily than the

At coronary angiography, localized stenosis is reported to occur at a frequency from 12% to 4.7% (Kato et al.,1996). Localized stenosis is often seen especially in the territory of the left anterior descending coronary artery, and usually occurs in both the inflow and outflow

shoulder of the aneurysm. It is caused mainly by inward luminal intimal thickening.

An, coronary aneurysm; Dilatation, coronary dilatation.

**3.3 Recanalization (segmental stenosis)** 

left coronary artery (Suzuki et al., 1997).

**3.4 Localized stenosis** 

rates ever recorded in Japan. The age-specific incidence rate showed a monomodal distribution with a peak at age 9-11 months. The prevalence of both cardiac lesions during the acute phase of the disease and cardiac sequelae were higher among infants and older age groups. During the acute phase, 2577 (11.0%) patients had a cardiac lesion(s): 58 (0.25%) had giant coronary aneurysms, 282 (1.21%) had coronary aneurysms less than 8 mm in diameter, 1992 (8.54%) had coronary dilatations, 8 (0.03%) had coronary stenoses, 3 (0.01%) had myocardial infarction, and 383 (1.64%) had valvular lesions. A total of 746 patients (3.2%) had cardiac sequelae 1 month after the onset of KD: 59 (0.25%) had giant coronary aneurysms, 188 (0.81%) had coronary aneurysms less than 8mm in diameter, 435 (1.86%) had coronary dilatations, 5 (0.02%) had coronary stenoses, 2 (0.01%) had myocardial infarction, and 114 (0.49%) had valvular lesions. The proportion of patients with cardiac sequelae has decreased year by year. The proportion was 7.0% in the 15th nationwide survey in 1997–1998, 5.9% in the 16th (1999–2000), 5.0% in the 17th (2001–2002), 4.4% in the 18th (2003–2004), and 3.8% in the 19th survey. 87% of patients received intravenous immunoglobulin therapy (IVIG). Of those, 16.5% of patients received additional IVIG therapy, 5.0% of patients were treated with steroids, 0.35%of patients received infliximab, and 0.23% of patients were treated with immunosuppressive agents. Although the incidence of coronary sequelae has gradually been decreasing, the rate of severe sequelae, such as giant aneurysms, has not been decreasing as expected (Figure 1). Patients with persistent coronary aneurysms, stenoses, and regressed aneurysms have suffered the most severe arterial insult. In such patients, the possibility of early progression to atherosclerosis occurs. It is therefore important to determine the factors concerning with how to reduce the patients with severe sequelae.

#### **3. Fate of coronary sequelae in Kawasaki disease**

Kato et al. demonstrated the angiographic outcome of coronary aneurysms in KD (Kato et al., 1996). This fundamental work has led to the current understanding that coronary artery lesions (CALs) change markedly with time into a variety of forms. These lesions may regress, stay unchanged, progress to stenosis or obstructive lesions with or without recanalization, and on very rare occasions, rupture, develop new lesions, or expand. These structural changes may modified the early progression of atherosclerosis long after KD.

#### **3.1 Regression**

Many aneurysms formed in the acute phase demonstrate a shrinking tendency in the convalescent phase and thereafter. The phenomenon in which the aneurysm resolves normally and disappears on coronary angiography is described as regression, and commonly occurs 1 to 2 years after disease onset. Regression frequently occurs for small-tomedium-sized aneurysms, and 32-50% of patients with CALs (Sasaguri& Kato, 1982) (Suzuki et al., 1994). Regression is mainly the results of smooth muscle cell infiltration and proliferation in the intima.

#### **3.2 Occlusion**

Thrombotic occlusion is often seen in medium-sized or large coronary aneurysms. Suzuki et al. have reported that 16% of coronary aneurysms are occluded at follow-up, 78% of those became occluded within 2 years from onset (Suzuki et al., 1997). Whereas, acute myocardial

rates ever recorded in Japan. The age-specific incidence rate showed a monomodal distribution with a peak at age 9-11 months. The prevalence of both cardiac lesions during the acute phase of the disease and cardiac sequelae were higher among infants and older age groups. During the acute phase, 2577 (11.0%) patients had a cardiac lesion(s): 58 (0.25%) had giant coronary aneurysms, 282 (1.21%) had coronary aneurysms less than 8 mm in diameter, 1992 (8.54%) had coronary dilatations, 8 (0.03%) had coronary stenoses, 3 (0.01%) had myocardial infarction, and 383 (1.64%) had valvular lesions. A total of 746 patients (3.2%) had cardiac sequelae 1 month after the onset of KD: 59 (0.25%) had giant coronary aneurysms, 188 (0.81%) had coronary aneurysms less than 8mm in diameter, 435 (1.86%) had coronary dilatations, 5 (0.02%) had coronary stenoses, 2 (0.01%) had myocardial infarction, and 114 (0.49%) had valvular lesions. The proportion of patients with cardiac sequelae has decreased year by year. The proportion was 7.0% in the 15th nationwide survey in 1997–1998, 5.9% in the 16th (1999–2000), 5.0% in the 17th (2001–2002), 4.4% in the 18th (2003–2004), and 3.8% in the 19th survey. 87% of patients received intravenous immunoglobulin therapy (IVIG). Of those, 16.5% of patients received additional IVIG therapy, 5.0% of patients were treated with steroids, 0.35%of patients received infliximab, and 0.23% of patients were treated with immunosuppressive agents. Although the incidence of coronary sequelae has gradually been decreasing, the rate of severe sequelae, such as giant aneurysms, has not been decreasing as expected (Figure 1). Patients with persistent coronary aneurysms, stenoses, and regressed aneurysms have suffered the most severe arterial insult. In such patients, the possibility of early progression to atherosclerosis occurs. It is therefore important to determine the factors concerning with how to reduce the patients with severe sequelae.

Kato et al. demonstrated the angiographic outcome of coronary aneurysms in KD (Kato et al., 1996). This fundamental work has led to the current understanding that coronary artery lesions (CALs) change markedly with time into a variety of forms. These lesions may regress, stay unchanged, progress to stenosis or obstructive lesions with or without recanalization, and on very rare occasions, rupture, develop new lesions, or expand. These structural changes may modified the early progression of atherosclerosis long after KD.

Many aneurysms formed in the acute phase demonstrate a shrinking tendency in the convalescent phase and thereafter. The phenomenon in which the aneurysm resolves normally and disappears on coronary angiography is described as regression, and commonly occurs 1 to 2 years after disease onset. Regression frequently occurs for small-tomedium-sized aneurysms, and 32-50% of patients with CALs (Sasaguri& Kato, 1982) (Suzuki et al., 1994). Regression is mainly the results of smooth muscle cell infiltration and

Thrombotic occlusion is often seen in medium-sized or large coronary aneurysms. Suzuki et al. have reported that 16% of coronary aneurysms are occluded at follow-up, 78% of those became occluded within 2 years from onset (Suzuki et al., 1997). Whereas, acute myocardial

**3. Fate of coronary sequelae in Kawasaki disease** 

**3.1 Regression** 

**3.2 Occlusion** 

proliferation in the intima.

infarction and sudden death have been caused by coronary artery occlusion, two-thirds of cases with occlusion have no symptomatic episodes due to the consequence of postocclusion recanalization and development of collateral vessels. Tsuda et al. reported the clinical features of 50 adult patients (mean age 28 years old) who had an acute coronary syndrome caused by coronary artery lesions due to KD from 1980 to 2008. Of the 50 patients, 43 had thrombotic occlusion of an aneurysm and 40 had giant coronary aneurysms. Based on these evidences, it is likely that patients with known aneurysms during the acute phase of KD will have some cardiovascular morbidity and mortality as young adults (Tsuda et al., 2011).

Fig. 1. Transition of the number of patients with cardiac sequelae in KD An, coronary aneurysm; Dilatation, coronary dilatation.

#### **3.3 Recanalization (segmental stenosis)**

New vessels after occlusion are called as segmental stenosis. Segmental stenosis has been observed in 14.8% of patients with KD sequelae, 90% of which occurred in the right coronary artery. The right coronary artery is occluded or recanalized more easily than the left coronary artery (Suzuki et al., 1997).

#### **3.4 Localized stenosis**

At coronary angiography, localized stenosis is reported to occur at a frequency from 12% to 4.7% (Kato et al.,1996). Localized stenosis is often seen especially in the territory of the left anterior descending coronary artery, and usually occurs in both the inflow and outflow shoulder of the aneurysm. It is caused mainly by inward luminal intimal thickening.

Premature Atherosclerosis Long After Kawasaki Disease 205

endothelial cell and increases the endothelial expression of adhesion molecules. Inflammatory mediators, such as hs-CRP, may themselves promote atherosclerosis. Therefore, we suspect that in patients with CALs chronic low grade inflammation may continue after the acute phase of KD. Whereas, several studies suggest that oxidative stress may promote endothelial dysfunction through increased production of reactive oxygen species (ROS) (Griendling et al., 2003). Urinary 8-iso-prostaglandin F2+ is increased long after the onset of KD, as well as after acute phase KD. Oxidative stress, evidenced by significant higher levels of malonyldialdehyde (MDA) and hydroperoxides, is increased in KD patients with CALs compared with those in control subjects and is associated with

A number of noninvasive methods have been developed to study endothelial function and structural changes suggestive of atherosclerosis. Brachial artery flow-mediated dilatation (FMD) has been studied widely and applied to varied group of patients including children. The %FMD reflects endothelial NO-dependent vasodilatation. Decreased %FMD reflects endothelial cell dysfunction, and a significant decrease in %FMD is a common feature of early marker in adult atherosclerosis. Decreased %FMD in patients with KD has been reported by many facilities (Niboshi et al., 2008). Ikemoto et al. demonstrated endothelial dysfunction, as indicated by decreased %FMD, only among patients with persistent CALs (Ikemoto et al., 2005). Endothelial dysfunction is worst among patients with coronary artery aneurysms (Dhillon et al.,1996) (Noto et al., 2009) (Figure 2). Abnormalities of systemic endothelial function are present many years after resolution of acute KD, even in patients without detectable early coronary involvement. In contrast, McCrindle et al. reported that patients with KD, compared with healthy control subjects, had similar systemic endothelial function, assessed by %FMD. Furthermore, FMD was not significantly related to either patient or KD characteristics (McCrindle et al., 2007). With respect to the noninvasive

method investigating endothelial dysfunction after KD, data are not consistent.

dilatation or even constriction (Yamakawa et al., 1998)(Iemura et al., 2000) .

**7. Diagnostic methods for assessment of atherosclerosis in Kawasaki** 

Structural arterial abnormalities are indicated by cartid intima-media thickness (IMT) measured by B-mode ultrasonography. Increased IMT has been shown to reliably indicate

The coronary artery response to acetylcholine or isosorbide has been well documented as a marker for endothelial dysfunction. Isosorbide induces dilatation of arteries in an endothelium-independent manner. On the other hand, acetylcholine dilates arteries in an endothelium-dependent manner. The reaction to acetylcholine of normal-appearing coronary arteries in patients with KD is equivalent to that of the control. However, the response to acetylcholine of coronary arteries with aneurysms or stenoses or both is poor

**6.2 Invasive method for evaluating endothelial function** 

**disease** 

**7.1 Carotid intima-media thickness** 

carotid intima-media thickness and stiffness (Cheung et al., 2008).

**6.1 Non-invasive method for evaluating endothelial function** 

**6. Endothelial function in Kawasaki disease** 

#### **3.5 Coronary artery with no aneurysm formation**

Coronary arteries that appear normal from the onset of KD have been regarded as normal as yet. Even in coronary arteries in which there is no aneurysm formation in the acute phase, mild intimal thickening may be seen in some patients (Takahashi et al.,2001). Therefore, there is some debate about the possibility that a history of KD might be a risk factor in the progression of atherosclerosis.

#### **4. Dyslipidemia in Kawasaki disease**

It is well known that the process of atherosclerosis is enhanced in the presence of cardiovascular risk factors (CRFs). Undoubtedly, one of the strongest CRFs for atherosclerosis is dyslipidemia. Among patients characteristics that influence vascular health, dyslipidemia is prevalent in patients with KD with or without overt coronary artery sequelae well beyond the time that the clinical disease has resolved. Earlier studies have shown that high-density lipoprotein cholesterol (HDL-C) levels may be depressed in the acute phase of KD (Okada T et al, 1990) (Newberger JW et al., 1991). However, there is controversy whether this decrease persists over the long time. In the convalescent phase, McCrindle et al. showed that KD patients 10 to 20 years of age had lower apolipoprotein A1 levels than the healthy control subjects (McCrindle et al., 2007). Mitani et al. showed that there were no differences between patients with KD 10 years after the onset and the control subjects with regard to total cholesterol (T-C) and HDL-C levels (Mitani et al., 2005). Cheung et al. reported that KD patients around 10 years old with or without coronary artery lesion (CAL) had significantly higher levels of apolipoprotein B and lower levels of HDL-C compared with control subjects (Cheung et al., 2004). Therefore, no consensus has been reached whether dyslipidemia in KD patients over the term after clinical resolution causes a higher risk of atherosclerosis than that in the general population. In epidemiological studies of children, the best correlation with adult cholesterol levels was obtained from cholesterol levels measured during the late teen years, suggesting that subtle adverse lipoprotein profiles obtained at this age could be predictive of future dyslipidemia in young adulthood (Strong WB et al., 1992). Since young adults often become much less active once they leave school and physical activities are generally restricted in KD patients at AHA risk levels IV to V with giant coronary aneurysms and stenoses, it is likely that subtle risk factors cluster in individual patients. Hence, we suggest that even in subtle abnormalities of lipid profile in post-KD patients with CALs may contribute to further endothelial dysfunction and the propensity for subclinical atherosclerosis in the future.

#### **5. Inflammatory examinations in Kawasaki disease**

Atherosclerosis is an inflammatory disease as demonstrated in animal models and human. Clinical or subclinical inflammation of the coronary and systemic arteries may form the substrate for long-term functional and structural abnormalities and increase the risk of premature atherosclerosis. Serum high-sensitivity C-reactive protein (hs-CRP) and serum amyloid-A (SAA), have recently been regarded as reliable clinical markers for the prediction of coronary events, independent of other known CRFs (Mitani et al., 2005). In fact, serum high-sensitivity C-reactive protein (hs-CRP) level in patients with CALs is significantly higher than those seen in normal age-matched control or among patients with KD without aneurysms or regressed aneurysms. C-reactive protein inhibits NO production by

Coronary arteries that appear normal from the onset of KD have been regarded as normal as yet. Even in coronary arteries in which there is no aneurysm formation in the acute phase, mild intimal thickening may be seen in some patients (Takahashi et al.,2001). Therefore, there is some debate about the possibility that a history of KD might be a risk factor in the

It is well known that the process of atherosclerosis is enhanced in the presence of cardiovascular risk factors (CRFs). Undoubtedly, one of the strongest CRFs for atherosclerosis is dyslipidemia. Among patients characteristics that influence vascular health, dyslipidemia is prevalent in patients with KD with or without overt coronary artery sequelae well beyond the time that the clinical disease has resolved. Earlier studies have shown that high-density lipoprotein cholesterol (HDL-C) levels may be depressed in the acute phase of KD (Okada T et al, 1990) (Newberger JW et al., 1991). However, there is controversy whether this decrease persists over the long time. In the convalescent phase, McCrindle et al. showed that KD patients 10 to 20 years of age had lower apolipoprotein A1 levels than the healthy control subjects (McCrindle et al., 2007). Mitani et al. showed that there were no differences between patients with KD 10 years after the onset and the control subjects with regard to total cholesterol (T-C) and HDL-C levels (Mitani et al., 2005). Cheung et al. reported that KD patients around 10 years old with or without coronary artery lesion (CAL) had significantly higher levels of apolipoprotein B and lower levels of HDL-C compared with control subjects (Cheung et al., 2004). Therefore, no consensus has been reached whether dyslipidemia in KD patients over the term after clinical resolution causes a higher risk of atherosclerosis than that in the general population. In epidemiological studies of children, the best correlation with adult cholesterol levels was obtained from cholesterol levels measured during the late teen years, suggesting that subtle adverse lipoprotein profiles obtained at this age could be predictive of future dyslipidemia in young adulthood (Strong WB et al., 1992). Since young adults often become much less active once they leave school and physical activities are generally restricted in KD patients at AHA risk levels IV to V with giant coronary aneurysms and stenoses, it is likely that subtle risk factors cluster in individual patients. Hence, we suggest that even in subtle abnormalities of lipid profile in post-KD patients with CALs may contribute to further endothelial dysfunction and the

Atherosclerosis is an inflammatory disease as demonstrated in animal models and human. Clinical or subclinical inflammation of the coronary and systemic arteries may form the substrate for long-term functional and structural abnormalities and increase the risk of premature atherosclerosis. Serum high-sensitivity C-reactive protein (hs-CRP) and serum amyloid-A (SAA), have recently been regarded as reliable clinical markers for the prediction of coronary events, independent of other known CRFs (Mitani et al., 2005). In fact, serum high-sensitivity C-reactive protein (hs-CRP) level in patients with CALs is significantly higher than those seen in normal age-matched control or among patients with KD without aneurysms or regressed aneurysms. C-reactive protein inhibits NO production by

**3.5 Coronary artery with no aneurysm formation** 

progression of atherosclerosis.

**4. Dyslipidemia in Kawasaki disease** 

propensity for subclinical atherosclerosis in the future.

**5. Inflammatory examinations in Kawasaki disease** 

endothelial cell and increases the endothelial expression of adhesion molecules. Inflammatory mediators, such as hs-CRP, may themselves promote atherosclerosis. Therefore, we suspect that in patients with CALs chronic low grade inflammation may continue after the acute phase of KD. Whereas, several studies suggest that oxidative stress may promote endothelial dysfunction through increased production of reactive oxygen species (ROS) (Griendling et al., 2003). Urinary 8-iso-prostaglandin F2+ is increased long after the onset of KD, as well as after acute phase KD. Oxidative stress, evidenced by significant higher levels of malonyldialdehyde (MDA) and hydroperoxides, is increased in KD patients with CALs compared with those in control subjects and is associated with carotid intima-media thickness and stiffness (Cheung et al., 2008).

#### **6. Endothelial function in Kawasaki disease**

#### **6.1 Non-invasive method for evaluating endothelial function**

A number of noninvasive methods have been developed to study endothelial function and structural changes suggestive of atherosclerosis. Brachial artery flow-mediated dilatation (FMD) has been studied widely and applied to varied group of patients including children. The %FMD reflects endothelial NO-dependent vasodilatation. Decreased %FMD reflects endothelial cell dysfunction, and a significant decrease in %FMD is a common feature of early marker in adult atherosclerosis. Decreased %FMD in patients with KD has been reported by many facilities (Niboshi et al., 2008). Ikemoto et al. demonstrated endothelial dysfunction, as indicated by decreased %FMD, only among patients with persistent CALs (Ikemoto et al., 2005). Endothelial dysfunction is worst among patients with coronary artery aneurysms (Dhillon et al.,1996) (Noto et al., 2009) (Figure 2). Abnormalities of systemic endothelial function are present many years after resolution of acute KD, even in patients without detectable early coronary involvement. In contrast, McCrindle et al. reported that patients with KD, compared with healthy control subjects, had similar systemic endothelial function, assessed by %FMD. Furthermore, FMD was not significantly related to either patient or KD characteristics (McCrindle et al., 2007). With respect to the noninvasive method investigating endothelial dysfunction after KD, data are not consistent.

#### **6.2 Invasive method for evaluating endothelial function**

The coronary artery response to acetylcholine or isosorbide has been well documented as a marker for endothelial dysfunction. Isosorbide induces dilatation of arteries in an endothelium-independent manner. On the other hand, acetylcholine dilates arteries in an endothelium-dependent manner. The reaction to acetylcholine of normal-appearing coronary arteries in patients with KD is equivalent to that of the control. However, the response to acetylcholine of coronary arteries with aneurysms or stenoses or both is poor dilatation or even constriction (Yamakawa et al., 1998)(Iemura et al., 2000) .

#### **7. Diagnostic methods for assessment of atherosclerosis in Kawasaki disease**

#### **7.1 Carotid intima-media thickness**

Structural arterial abnormalities are indicated by cartid intima-media thickness (IMT) measured by B-mode ultrasonography. Increased IMT has been shown to reliably indicate

Premature Atherosclerosis Long After Kawasaki Disease 207

Fig. 3. Intima-media thickness (IMT) and % normal predicted IMT (%N IMT) in KD patients and control subjects. (Left) Scatter plot of IMT versus age for subjects with KD(() and control subjects (1). Regression lines for subjects with KD and controls are represented by a solid and dashed line, respectively. A significant correlation (r=0.61, p<0.001) between age and IMT was observed. (Right) Bar graphs of IMT and % N IMT in KD patients (black bar) and control subjects (white bar). The normal predicted IMT value (np-IMT) and percent normal predicted IMT value (%N IMT) were calculated from the following formula: np-

An alternative noninvasive method is measurement of arterial stiffness by pulse wave velocity (PWV). The PWV is simple to measure and is a powerful marker for the risk of atherosclerosis in adults. It has been reported that PWV is especially high in patients with KD who have CALs. Patients with a history of KD have a higher PWV than control subjects, moreover there is no significant difference in the PWV between the KD patients with or without CALs (Cheung et al., 2004). However, in other study PWV does not differ significantly between KD patients with normal-appearing coronary arteries and healthy

Iemura et al have shown the evidence of persisting abnormal vascular wall morphology and vascular dysfunction at the site of regressed coronary aneurysms in patients with KD by intravascular ultrasound imaging using intracoronary infusion of acetylcholine and isosorbide dinitrate (Iemura et al., 2000). More recently, Mitani et al have demonstrated the

IMT=0.381+0.004×age, %N IMT=100×measured IMT/np-IMT.

controls (Ooyanagi et al., 2004). Data are conflicting.

**7.3 Intravascular ultrasound imaging** 

**7.2 Pulse wave velocity** 

the presence of atherosclerosis. In KD patients with persistent CALs, compared with control patients, the carotid arterial wall has been reported to have a higher IMT and lower distensibility (Noto et al., 2001&2007) (Figure 3). Carotid IMT, expressed as both unadjusted dimension and z-score, was greater among patients with KD than control subjects; within the KD group, patients with CALs had greater carotid IMT than those without CALs (Della Pozza et al., 2007). Cheung et al. found increased carotid IMT even among patients with KD with normal coronary arteries, compared with control subjects (Cheung et al., 2007). However, some investigators have found no difference in carotid IMT between patients with KD and control subjects, consistent with the hypothesis that functional abnormalities might precede those of structure (McCrindle et al., 2007).

Fig. 2. Endothelial dysfunction in KD patients with CALs . (Left) Scatter plot of FMD% versus age in the subjects with KD(() and the control subjects(1). Regression lines for subjects with KD and controls are represented by a solid and dashed line, respectively. (Right) %FMD and %GTN in KD (black bar) and control subjects (white bar). FMD, flow mediated dilatation;

GTN, sublingual glyceryl trinitrate.

the presence of atherosclerosis. In KD patients with persistent CALs, compared with control patients, the carotid arterial wall has been reported to have a higher IMT and lower distensibility (Noto et al., 2001&2007) (Figure 3). Carotid IMT, expressed as both unadjusted dimension and z-score, was greater among patients with KD than control subjects; within the KD group, patients with CALs had greater carotid IMT than those without CALs (Della Pozza et al., 2007). Cheung et al. found increased carotid IMT even among patients with KD with normal coronary arteries, compared with control subjects (Cheung et al., 2007). However, some investigators have found no difference in carotid IMT between patients with KD and control subjects, consistent with the hypothesis that functional abnormalities might

Fig. 2. Endothelial dysfunction in KD patients with CALs . (Left) Scatter plot of FMD% versus age in the subjects with KD(() and the control subjects(1). Regression lines for subjects with KD and controls are represented by a solid and dashed line, respectively. (Right) %FMD and %GTN in KD (black bar) and control subjects (white bar). FMD, flow

mediated dilatation;

GTN, sublingual glyceryl trinitrate.

precede those of structure (McCrindle et al., 2007).

Fig. 3. Intima-media thickness (IMT) and % normal predicted IMT (%N IMT) in KD patients and control subjects. (Left) Scatter plot of IMT versus age for subjects with KD(() and control subjects (1). Regression lines for subjects with KD and controls are represented by a solid and dashed line, respectively. A significant correlation (r=0.61, p<0.001) between age and IMT was observed. (Right) Bar graphs of IMT and % N IMT in KD patients (black bar) and control subjects (white bar). The normal predicted IMT value (np-IMT) and percent normal predicted IMT value (%N IMT) were calculated from the following formula: np-IMT=0.381+0.004×age, %N IMT=100×measured IMT/np-IMT.

#### **7.2 Pulse wave velocity**

An alternative noninvasive method is measurement of arterial stiffness by pulse wave velocity (PWV). The PWV is simple to measure and is a powerful marker for the risk of atherosclerosis in adults. It has been reported that PWV is especially high in patients with KD who have CALs. Patients with a history of KD have a higher PWV than control subjects, moreover there is no significant difference in the PWV between the KD patients with or without CALs (Cheung et al., 2004). However, in other study PWV does not differ significantly between KD patients with normal-appearing coronary arteries and healthy controls (Ooyanagi et al., 2004). Data are conflicting.

#### **7.3 Intravascular ultrasound imaging**

Iemura et al have shown the evidence of persisting abnormal vascular wall morphology and vascular dysfunction at the site of regressed coronary aneurysms in patients with KD by intravascular ultrasound imaging using intracoronary infusion of acetylcholine and isosorbide dinitrate (Iemura et al., 2000). More recently, Mitani et al have demonstrated the

Premature Atherosclerosis Long After Kawasaki Disease 209

expressed in adventitial vasa vasorum in the localized and recanalized vessels of KD children with CALs (Suzuki et al., 2000). Furthermore, vascular senescence, demonstrated as increased ,-galactosidase activity, adhesion molecules and pro-inflammatory cytokines, as well as a reduction of normal physiological vascular proteins, such as eNOS, was increased in KD with CALs. These findings were more remarkable in intima as well as in vasculature of vasa vasorum (Fukazawa et al., 2007). Inevitably, adult atherosclerosis originates from the intimal side of the arteries. In contrast, arteriosclerotic changes characterized by active vascular remodeling including with luxuriant intimal proliferation and neoangiogenesis in KD with CALs may develop earlier from the both sides of the intima and adventitia of the

Fig. 4. Complicated atherosclerotic lesion in the orifice of the left coronary artery. (Elastica–

Recently, Ozawa et al. developed an animal model of KD, which is similar to the model of vasculitis in juvenile rabbits. Allergic coronary arteritis in rabbits induced by serial horse serum injections showed typical panarteritis characterized by inflammatory cell invasion of both sides from intima and adventitia, medial edema, and destruction of internal elastic lamina. When a high fat diet was being fed to this allergic arteritis rabbits models, typical atherosclerotic plaque appeared significantly (Ozawa et al., 2006). This experimental result

HE, ×10). Right side is the orifice of the left coronary artery. Left side is aorta.

**9. Experimental model of coronary arteritis** 

Takahashi K, et al. Pediatr Cardiol, 2001.

arteries.

plaque composition and morphology in CALs after KD using a virtual histologyintravascular ultrasound imaging (VH-IVUS). Qualitatively, the normal coronary segment had no or trivial intravascular ultrasound-visible plaque area. While, the CALs exhibited a heterogeneous plaque area with the components (fibrous, fibrofatty, necrotic core, and dense calcium) in different amounts and proportions. VH-IVUS findings in KD suggest that arteriosclerotic lesions found in CALs but not in the normal segment in patients with KD are characterized by a heterogeneous intimal area, distinct from a purely fibrotic area (Mitani et al., 2009). These findings may give new insight into the potential role of atherosclerosis in CALs long after KD.

#### **8. Histological examinations in Kawasaki disease**

#### **8.1 Pathological features**

In the acute phase of KD, histopathological investigations has shown that inflammatory cells invade the intima and destroy the internal elastic lamina and continue to infiltrated the tunica media. With inflammatory cell invasion from the adventitia, panvasculitis develops (Naoe et al., 1991).In the convalescent phase, even in clinically normal coronary artery of a child, who died of unrelated causes, pathological studies in autopsy cases showed a slightly thickened intima and the disrupted lamina interna in some parts and many +-actin-positive smooth muscle cells were observed in the intima (Suzuki et al., 2004). In fact, the lamina interna serves as a barrier for cells and macromolecules migration between the intima and the media in the vascular wall. Therefore, one can easily expect that KD patients with CALs might have a vulnerability to more easily develop atherosclerotic changes in early adulthood according to these structural alterations. Whereas, current histological studies showed that there was no significant macrophage infiltration or foamy cell formation in the intima in KD coronary arteries with localized stenosis several years after the onset of KD (Suzuki et al., 2000). Likewise, no atheromatous plaques were found in seemingly normal coronary arteries distal to aneurysmsin 10 autopsied adults KD patients (Burns et al., 1996). On the contrary, Takahashi et al. reported that 3 patients who were 15, 20, and 39 years of age had new intimal thickening, including atheroma-like bright areas and foamy macrophages in CALs (Takahashi et al., 2001). Also, Negoro et al. showed that a substantial lipid core with cholesterol crystals and macrophages was found in an atherectomy specimen from a stenotic lesion in a 32-year-old man who presented with acute coronary syndrome (Negoro et al., 2003). Due to conflicting results, we speculate that there is some heterogeneity on the propensity of atherosclerosis in individual including such as age, the latent CRFs and the given degree of structural changes after vascular remodeling in KD with CALs. At least, aneurysms are present, a history of KD in children become a risk factor for atherosclerosis later in life. Thus, a study of coronary arteries more than a decade after being afflicted with KD is greatly needed.

#### **8.2 Immunohistological features**

Current immunohistological studies using antibodies against vascular growth factors (GFs), such as transforming growth factor ,1, platelet-derived growth factor-A, basic fibroblast growth factor, and vascular endothelial growth factor, demonstrated that GFs were strongly expressed in the newly formed microvessels within the intima and some GFs were also

plaque composition and morphology in CALs after KD using a virtual histologyintravascular ultrasound imaging (VH-IVUS). Qualitatively, the normal coronary segment had no or trivial intravascular ultrasound-visible plaque area. While, the CALs exhibited a heterogeneous plaque area with the components (fibrous, fibrofatty, necrotic core, and dense calcium) in different amounts and proportions. VH-IVUS findings in KD suggest that arteriosclerotic lesions found in CALs but not in the normal segment in patients with KD are characterized by a heterogeneous intimal area, distinct from a purely fibrotic area (Mitani et al., 2009). These findings may give new insight into the potential role of atherosclerosis in

In the acute phase of KD, histopathological investigations has shown that inflammatory cells invade the intima and destroy the internal elastic lamina and continue to infiltrated the tunica media. With inflammatory cell invasion from the adventitia, panvasculitis develops (Naoe et al., 1991).In the convalescent phase, even in clinically normal coronary artery of a child, who died of unrelated causes, pathological studies in autopsy cases showed a slightly thickened intima and the disrupted lamina interna in some parts and many +-actin-positive smooth muscle cells were observed in the intima (Suzuki et al., 2004). In fact, the lamina interna serves as a barrier for cells and macromolecules migration between the intima and the media in the vascular wall. Therefore, one can easily expect that KD patients with CALs might have a vulnerability to more easily develop atherosclerotic changes in early adulthood according to these structural alterations. Whereas, current histological studies showed that there was no significant macrophage infiltration or foamy cell formation in the intima in KD coronary arteries with localized stenosis several years after the onset of KD (Suzuki et al., 2000). Likewise, no atheromatous plaques were found in seemingly normal coronary arteries distal to aneurysmsin 10 autopsied adults KD patients (Burns et al., 1996). On the contrary, Takahashi et al. reported that 3 patients who were 15, 20, and 39 years of age had new intimal thickening, including atheroma-like bright areas and foamy macrophages in CALs (Takahashi et al., 2001). Also, Negoro et al. showed that a substantial lipid core with cholesterol crystals and macrophages was found in an atherectomy specimen from a stenotic lesion in a 32-year-old man who presented with acute coronary syndrome (Negoro et al., 2003). Due to conflicting results, we speculate that there is some heterogeneity on the propensity of atherosclerosis in individual including such as age, the latent CRFs and the given degree of structural changes after vascular remodeling in KD with CALs. At least, aneurysms are present, a history of KD in children become a risk factor for atherosclerosis later in life. Thus, a study of coronary arteries more than a decade after being

Current immunohistological studies using antibodies against vascular growth factors (GFs), such as transforming growth factor ,1, platelet-derived growth factor-A, basic fibroblast growth factor, and vascular endothelial growth factor, demonstrated that GFs were strongly expressed in the newly formed microvessels within the intima and some GFs were also

CALs long after KD.

**8.1 Pathological features** 

afflicted with KD is greatly needed.

**8.2 Immunohistological features** 

**8. Histological examinations in Kawasaki disease** 

expressed in adventitial vasa vasorum in the localized and recanalized vessels of KD children with CALs (Suzuki et al., 2000). Furthermore, vascular senescence, demonstrated as increased ,-galactosidase activity, adhesion molecules and pro-inflammatory cytokines, as well as a reduction of normal physiological vascular proteins, such as eNOS, was increased in KD with CALs. These findings were more remarkable in intima as well as in vasculature of vasa vasorum (Fukazawa et al., 2007). Inevitably, adult atherosclerosis originates from the intimal side of the arteries. In contrast, arteriosclerotic changes characterized by active vascular remodeling including with luxuriant intimal proliferation and neoangiogenesis in KD with CALs may develop earlier from the both sides of the intima and adventitia of the arteries.

Takahashi K, et al. Pediatr Cardiol, 2001.

Fig. 4. Complicated atherosclerotic lesion in the orifice of the left coronary artery. (Elastica– HE, ×10). Right side is the orifice of the left coronary artery. Left side is aorta.

#### **9. Experimental model of coronary arteritis**

Recently, Ozawa et al. developed an animal model of KD, which is similar to the model of vasculitis in juvenile rabbits. Allergic coronary arteritis in rabbits induced by serial horse serum injections showed typical panarteritis characterized by inflammatory cell invasion of both sides from intima and adventitia, medial edema, and destruction of internal elastic lamina. When a high fat diet was being fed to this allergic arteritis rabbits models, typical atherosclerotic plaque appeared significantly (Ozawa et al., 2006). This experimental result

Premature Atherosclerosis Long After Kawasaki Disease 211

Fig. 5. Relationship between IMT and the age in KD patients and control subjects. (Left) Scatter plot of IMT versus age for subjects with KD(() and control subjects(1). Regression lines for subjects with KD and controls are represented by a solid and dashed line, respectively. (Right) Difference in IMT()IMT) and 95% CI between subjects with KD

Fig. 6. Relationship between Ep and the age in KD patients and control subjects. (Left) Scatter plot of Ep versus age for subjects with KD(() and control subjects(1). Regression lines for subjects with KD and controls are represented by a solid and dashed line, respectively. (Right) Difference in Ep()Ep) and 95% CI between subjects with KD and

controls plotted against age. Mean=solid line, 95%CI=dashed lines.

and controls plotted against age. Mean=solid line, 95% CI=dashed lines.

suggests that post arteritis tissue may more easily develop atherosclerosis. Intriguingly, they also demonstrated the anti-inflammatory activities of HMG-CoA reductase inhibitors against the progress of acute arteritis (Hamaoka et al., 2010).

#### **10. The propensity for atherosclerosis in KD**

Due to conflicting previous reports dealing with the endothelial function and non-invasive diagnostic methods in KD, concerns have been raised as to whether KD patients are really at risk for premature atherosclerosis later in adulthood. Although the exact reasons for these conflicting results remain undetermined, differences in study populations, methodology, and latent CRFs including age, pubertal status, systemic inflammation may play a role. Age is of particular interest because the atherosclerotic process begins in childhood. Additionally, a strong association exists between the adverse lipoprotein levels and the initial stages of atherosclerosis in adolescents and young adults, and advanced atherosclerotic lesions are enhanced with age. We speculated that endothelial dysfunction and the propensity for subclinical atherosclerosis gradually appear during adolescence, and then rapidly increase with age, particularly in post-KD patients with CALs, who show diffuse vascular inflammation during the acute phase of KD. To test this hypothesis, we examined the relationship between age and the progression of endothelial dysfunction and subclinical atherosclerosis in case-control study using post-KD patients with CALs and healthy control subjects across a wide age range (Noto et al 2009).

#### **10.1 Study design**

A case-control study was performed that included 35 post-KD subjects across a wide age range (range 8-42 years) without traditional cardiovascular risk factors (CRFs) and 35 ageand sex-matched healthy control subjects (Cont). Flow-mediated dilatation (FMD) of the brachial artery induced by reactive hyperemia, intima-media thickness (IMT), and the elastic modulus (Ep) of the common carotid artery were compared between KD and Cont subjects assessed against age.

#### **10.2 Results**

KD subjects had slightly higher levels of BMI, lipid profile, and HbA1c than Cont subjects, but the differences were not significant. The mean IMT (p<0.001), age-adjusted % normal IMT (%N IMT) (p<0.0001), and Ep (p<0.001), were significantly higher in KD than Cont subjects, and the peak FMD% (p<0.01) was significantly lower in KD than Cont subjects. There were significant correlations between FMD% and age (r= -0.51 p<0.0001), IMT and age (R=0.68 p<0.001), and Ep and age (r=0.58 p<0.01) in KD but not Cont subjects. When the difference in FMD% between KD and matched Cont subjects ()FMD%) was plotted against age, no significant relationship was found, although significant correlations between )IMT and age (r=0.52, p<0.01) as well as )Ep and age (r=0.46, p<0.05) were observed. When we defined values that were +2.0 SD over the mean control values (i.e. %N IMT 120% and or Ep 50KPa) as markers of subclinical atherosclerosis, 15 subjects met the criteria. Subjects over the age of 22 years were more likely to be (OR=16.54, p=0.0001) subclinical atherosclerosis in this cohort (Figure 5)(Figure 6)(Figure 7). Our results suggest that endothelial dysfunction and the development of premature atherosclerosis were accelerated in adult post-KD than Cont subjects.

suggests that post arteritis tissue may more easily develop atherosclerosis. Intriguingly, they also demonstrated the anti-inflammatory activities of HMG-CoA reductase inhibitors

Due to conflicting previous reports dealing with the endothelial function and non-invasive diagnostic methods in KD, concerns have been raised as to whether KD patients are really at risk for premature atherosclerosis later in adulthood. Although the exact reasons for these conflicting results remain undetermined, differences in study populations, methodology, and latent CRFs including age, pubertal status, systemic inflammation may play a role. Age is of particular interest because the atherosclerotic process begins in childhood. Additionally, a strong association exists between the adverse lipoprotein levels and the initial stages of atherosclerosis in adolescents and young adults, and advanced atherosclerotic lesions are enhanced with age. We speculated that endothelial dysfunction and the propensity for subclinical atherosclerosis gradually appear during adolescence, and then rapidly increase with age, particularly in post-KD patients with CALs, who show diffuse vascular inflammation during the acute phase of KD. To test this hypothesis, we examined the relationship between age and the progression of endothelial dysfunction and subclinical atherosclerosis in case-control study using post-KD patients with CALs and

A case-control study was performed that included 35 post-KD subjects across a wide age range (range 8-42 years) without traditional cardiovascular risk factors (CRFs) and 35 ageand sex-matched healthy control subjects (Cont). Flow-mediated dilatation (FMD) of the brachial artery induced by reactive hyperemia, intima-media thickness (IMT), and the elastic modulus (Ep) of the common carotid artery were compared between KD and Cont subjects

KD subjects had slightly higher levels of BMI, lipid profile, and HbA1c than Cont subjects, but the differences were not significant. The mean IMT (p<0.001), age-adjusted % normal IMT (%N IMT) (p<0.0001), and Ep (p<0.001), were significantly higher in KD than Cont subjects, and the peak FMD% (p<0.01) was significantly lower in KD than Cont subjects. There were significant correlations between FMD% and age (r= -0.51 p<0.0001), IMT and age (R=0.68 p<0.001), and Ep and age (r=0.58 p<0.01) in KD but not Cont subjects. When the difference in FMD% between KD and matched Cont subjects ()FMD%) was plotted against age, no significant relationship was found, although significant correlations between )IMT and age (r=0.52, p<0.01) as well as )Ep and age (r=0.46, p<0.05) were observed. When we defined values that were +2.0 SD over the mean control values (i.e. %N IMT 120% and or Ep 50KPa) as markers of subclinical atherosclerosis, 15 subjects met the criteria. Subjects over the age of 22 years were more likely to be (OR=16.54, p=0.0001) subclinical atherosclerosis in this cohort (Figure 5)(Figure 6)(Figure 7). Our results suggest that endothelial dysfunction and the development of premature atherosclerosis were accelerated

against the progress of acute arteritis (Hamaoka et al., 2010).

healthy control subjects across a wide age range (Noto et al 2009).

**10.1 Study design** 

assessed against age.

in adult post-KD than Cont subjects.

**10.2 Results** 

**10. The propensity for atherosclerosis in KD** 

Fig. 5. Relationship between IMT and the age in KD patients and control subjects. (Left) Scatter plot of IMT versus age for subjects with KD(() and control subjects(1). Regression lines for subjects with KD and controls are represented by a solid and dashed line, respectively. (Right) Difference in IMT()IMT) and 95% CI between subjects with KD and controls plotted against age. Mean=solid line, 95% CI=dashed lines.

Fig. 6. Relationship between Ep and the age in KD patients and control subjects. (Left) Scatter plot of Ep versus age for subjects with KD(() and control subjects(1). Regression lines for subjects with KD and controls are represented by a solid and dashed line, respectively. (Right) Difference in Ep()Ep) and 95% CI between subjects with KD and controls plotted against age. Mean=solid line, 95%CI=dashed lines.

Premature Atherosclerosis Long After Kawasaki Disease 213

Fig. 8. Representative typical Visual Scoring (VS) by magnified B-mode ultrasound images of the intima-media complex (IMC) in heterozygous familial hypercholesterolemia (FH). Visual Scoring 1: normal wall (intima media and adventitia clearly separated); Visual Scoring 2: proximal interface disrupted by hypoechoic components in the proximal echogenic layer; Visual Scoring 3: intima-media granulation with mixed hypoechoic and echogenic components. According to the increased IMT with medial infiltration of lipids, VS progresses from proximal interface disruption to intima-media granulation (Noto et al.,

J Am Soc Echocardiog 2011).

Fig. 7. Relationship between %N IMT and Ep and ROC Curve in KD patients and Control Subjects. (Left) Regression line for all subjects is represented by a solid line. Arrows represent the cut-off points of %N IMT 120% and or Ep 50KPa as markers of subclinical atherosclerosis. (Right) Receiver-operating characteristic curve for the prediction of subclinical atherosclerosis against age in all subjects. Arrow indicates 22 years of age with a high odds ratio (OR=16.54, p=0.0001).

#### **11. Future perspective: Detection of earlier atherosclerotic involvement in KD patients with CALs**

It remains unclear whether the vascular remodeling process of atherosclerosis in KD differs from that in adults. In particular, major concern has been raised as to whether the vessels with predisposed arteriosclerotic changes in KD patients with CALs may more easily develop atherosclerotic changes. Several studies showed that carotid IMT, a surrogate of atherosclerotic vessel wall change, is sensitive to risk intervention and constitutes a reliable indicator of clinical outcome in adult subjects, even in children with familial hypercholesterolemia (FH) (Jarvisalo et al., 2001). Furthermore, these changes in the arterial wall occur earlier in the carotid arteries than in the coronary circulation, making the carotid arteries an ideal site for detection of premature atherosclerotic disease (Bland et al., 1986). In addition, current study has demonstrated that textural changes in the carotid intima-media complex (IMC) by B-mode ultrasound were associated with early atherosclerotic involvement in patients with FH (Noto et al., 2011) (Figure 8). We speculated that changes in the textural characteristics of carotid IMC by B-mode ultrasound could be indicative of the earlier involvement of carotid atherosclerosis in KD patients with CALs. To test this hypothesis, we assessed the ultrasonic textural parameters of the IMC by first-order statistics, second-order statistics in KD patients with CALs without CRFs, age-matched healthy control subjects and age-matched FH subjects and findings were compared among the 3 groups.

Fig. 7. Relationship between %N IMT and Ep and ROC Curve in KD patients and Control Subjects. (Left) Regression line for all subjects is represented by a solid line. Arrows

represent the cut-off points of %N IMT 120% and or Ep 50KPa as markers of subclinical

subclinical atherosclerosis against age in all subjects. Arrow indicates 22 years of age with a

**11. Future perspective: Detection of earlier atherosclerotic involvement in KD** 

It remains unclear whether the vascular remodeling process of atherosclerosis in KD differs from that in adults. In particular, major concern has been raised as to whether the vessels with predisposed arteriosclerotic changes in KD patients with CALs may more easily develop atherosclerotic changes. Several studies showed that carotid IMT, a surrogate of atherosclerotic vessel wall change, is sensitive to risk intervention and constitutes a reliable indicator of clinical outcome in adult subjects, even in children with familial hypercholesterolemia (FH) (Jarvisalo et al., 2001). Furthermore, these changes in the arterial wall occur earlier in the carotid arteries than in the coronary circulation, making the carotid arteries an ideal site for detection of premature atherosclerotic disease (Bland et al., 1986). In addition, current study has demonstrated that textural changes in the carotid intima-media complex (IMC) by B-mode ultrasound were associated with early atherosclerotic involvement in patients with FH (Noto et al., 2011) (Figure 8). We speculated that changes in the textural characteristics of carotid IMC by B-mode ultrasound could be indicative of the earlier involvement of carotid atherosclerosis in KD patients with CALs. To test this hypothesis, we assessed the ultrasonic textural parameters of the IMC by first-order statistics, second-order statistics in KD patients with CALs without CRFs, age-matched healthy control subjects and age-matched FH subjects and findings were compared among

atherosclerosis. (Right) Receiver-operating characteristic curve for the prediction of

high odds ratio (OR=16.54, p=0.0001).

**patients with CALs** 

the 3 groups.

Fig. 8. Representative typical Visual Scoring (VS) by magnified B-mode ultrasound images of the intima-media complex (IMC) in heterozygous familial hypercholesterolemia (FH). Visual Scoring 1: normal wall (intima media and adventitia clearly separated); Visual Scoring 2: proximal interface disrupted by hypoechoic components in the proximal echogenic layer; Visual Scoring 3: intima-media granulation with mixed hypoechoic and echogenic components. According to the increased IMT with medial infiltration of lipids, VS progresses from proximal interface disruption to intima-media granulation (Noto et al., J Am Soc Echocardiog 2011).

Premature Atherosclerosis Long After Kawasaki Disease 215

The mean IMT was significantly higher in FH (p<0.0001) and KD (p=0.005) than Cont. KD showed significantly higher gray-scale median (GSM) than FH (p=0.027) and Cont (p=0.048), however, there were no significant differences in Skewness, Kurtosis and Contrast among 3 groups. While, KD (p<0.0001) and FH (p=0.004) showed significantly higher Entropy than Cont. Furthermore, KD (p=0.0041) and FH (p=0.001) showed significantly lower Angular second moment (ASM) than Cont, but no significant difference was found between KD and FH. As for PC, KD showed significantly higher gray value both at the minimum point in IMC (p<0.01) and at the adventitia-media junction (p<0.01) than FH and Cont. These findings demonstrate that higher GSM in KD may indicate alteration of tissue component and heterogeneity IMC, suggesting sclerotic vascular remodeling after vasculitis. In contrast, decreased GSM may indicate atherotic vascular remodeling often

Fig. 10. Histogram variables among KD patients, Familial hypercholesterolemia, and control

We reviewed the recent several problems concerning with the propensity of atherosclerosis in patients long after KD. Among KD patients with CALs, impairments of coronary artery structure and function have been well documented. However, it is still too early to determine the conclusion whether post-KD patients in whom coronary abnormalities were never detected are at risk for premature atherosclerosis later in adulthood until the early

**11.2 Results** 

observed in FH patients.

subjects.

**12. Conclusion** 

#### **11.1 Study design**

To test the hypothesis that textural changes in the carotid intima-media complex (IMC) by Bmode ultrasound are associated with the earlier atherosclerotic involvement, 12 patients with KD and CALs without cardiovascular risk factors (mean age 20.5 years), 12 patients with heterozygous familial hypercholesterolemia (FH) (mean age 18.6 years) and age-matched 9 healthy control subjects (Cont) were assessed for intima-media thickness (IMT), first- and second-order statistics, and plot profile curve (PC) for regional analysis of the alteration of gray value in IMC, and were compared among 3 groups.

Fig. 9. Measurement of the textural parameters and gray value by plot profile curve (a) Conventional B-mode image of common carotid artery (CCA). Rectangle indicates the magnified portion around the far wall of CCA. (b) Maximal magnified B-mode image (×8.6). Rectangle indicates the region of interest (ROI) in the far wall of the intima-media complex (IMC). (c) Histogram of ROI. This feature was automatically extracted by Image J 1.38x software. (d) Maximal magnified B-mode image (8.6). *Line* indicates the portion of measurement of plot profile curve located center of ROI in the far wall of the intima-media complex (IMC). (e) Plot profile curve of gray values of the pixels along a 0.7mm line over the IMC.

#### **11.2 Results**

214 Recent Advances in Cardiovascular Risk Factors

To test the hypothesis that textural changes in the carotid intima-media complex (IMC) by Bmode ultrasound are associated with the earlier atherosclerotic involvement, 12 patients with KD and CALs without cardiovascular risk factors (mean age 20.5 years), 12 patients with heterozygous familial hypercholesterolemia (FH) (mean age 18.6 years) and age-matched 9 healthy control subjects (Cont) were assessed for intima-media thickness (IMT), first- and second-order statistics, and plot profile curve (PC) for regional analysis of the alteration of

Fig. 9. Measurement of the textural parameters and gray value by plot profile curve (a) Conventional B-mode image of common carotid artery (CCA). Rectangle indicates the magnified portion around the far wall of CCA. (b) Maximal magnified B-mode image (×8.6). Rectangle indicates the region of interest (ROI) in the far wall of the intima-media complex (IMC). (c) Histogram of ROI. This feature was automatically extracted by Image J 1.38x software. (d) Maximal magnified B-mode image (8.6). *Line* indicates the portion of measurement of plot profile curve located center of ROI in the far wall of the intima-media complex (IMC). (e) Plot profile curve of gray values of the pixels along a 0.7mm line over the

gray value in IMC, and were compared among 3 groups.

**11.1 Study design** 

IMC.

The mean IMT was significantly higher in FH (p<0.0001) and KD (p=0.005) than Cont. KD showed significantly higher gray-scale median (GSM) than FH (p=0.027) and Cont (p=0.048), however, there were no significant differences in Skewness, Kurtosis and Contrast among 3 groups. While, KD (p<0.0001) and FH (p=0.004) showed significantly higher Entropy than Cont. Furthermore, KD (p=0.0041) and FH (p=0.001) showed significantly lower Angular second moment (ASM) than Cont, but no significant difference was found between KD and FH. As for PC, KD showed significantly higher gray value both at the minimum point in IMC (p<0.01) and at the adventitia-media junction (p<0.01) than FH and Cont. These findings demonstrate that higher GSM in KD may indicate alteration of tissue component and heterogeneity IMC, suggesting sclerotic vascular remodeling after vasculitis. In contrast, decreased GSM may indicate atherotic vascular remodeling often observed in FH patients.

Fig. 10. Histogram variables among KD patients, Familial hypercholesterolemia, and control subjects.

#### **12. Conclusion**

We reviewed the recent several problems concerning with the propensity of atherosclerosis in patients long after KD. Among KD patients with CALs, impairments of coronary artery structure and function have been well documented. However, it is still too early to determine the conclusion whether post-KD patients in whom coronary abnormalities were never detected are at risk for premature atherosclerosis later in adulthood until the early

Premature Atherosclerosis Long After Kawasaki Disease 217

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levels of hight-sensitivity C-reactive protein and serum amyloid-A late after Kawasaki disease: association between inflammation and coronary sequelae in

Shimpo H, & Komada Y. (2009). In vivo plaque composition and morphology in coronary artery lesions in adolescents and young adults long after Kawasaki disease: a virtual histology intra vascular ultrasound study. Circulation, Vol. 119,

Yanagawa H. (2010). Epidemiologic features of Kawasaki disease in Japan: results

Hoshiga M, Morita H, Isihara T, & Kanafusa T. (2003). Successful catheter interventional therapy for acute coronary syndrome secondary to Kawasaki disease

Harada K. (2001). Noninvasive assessment of the early progression of atherosclerosis in adolescents with Kawasaki disease and coronary artery lesions.

(2009). Age-related acceleration of endothelial dysfunction and subclinical atherosclerosis in subjects with coronary artery lesions after Kawasaki disease.

N, & Mugishima H. (2011). Changes in the textural characteristics of intima-media complex in young patients with familial hypercholesterolemia: Implication for visual inspection on B-mode ultrasound. J Am Soc Echocardiogr, Vol. 24, pp. 438-

Kawasaki disease (acute mococutaneous lymph node syndrome). Jpn Circ J, Vol.

Japanese cohort reaches middle and older age. To date, all patients with a history of KD should be carefully assessed for factors promoting atherosclerosis, including hypercholesterolemia, obesity, systemic hypertension, and smoking.

#### **13. References**


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Burns JC, Shike H, Gordon JB, Malhotra A, Schoenwetter M, & Kawasaki T. (1996). Sequelae

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Dhillon R, Clarkson P, Donald AE, Powe AJ, Nash M, Novelli V, Dillon MJ, & Deanfield JE.

Fukazawa R, Ikeman E, Watanabe M, Hajikano M, Kamisago M, Katsube Y, Yamauchi H,

Hamaoka A, Hamaoka K, Yahata T, Fujii M, Ozawa S, Toiyama K, Nishida M, & Itoi T.

Ikemoto Y, Ogini H, Teraguchi M, & Kobayashi Y. (2005). Evaluation of preclinical

Jarvisalo MJ, Jartti L, Nanto-Saloren K, Irjala K, Ronnemaa T, & Hartiala JJ. (2001). Increased

Animal and human studies. Circulation, Vol. 108, pp.2034-2040.

and function. Heart, Vol. 83, pp. 307-311.

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of Kawasaki disease in adolescents and young adults. J Am Coll Cardiol, Vol. 28,

risk factors in children after Kawasaki disease. J Am Coll Cardiol, Vol. 43, pp. 120-

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thickness and arterial stiffness in children after Kawasaki disease. Arch Dis Child,

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atherosclerosis, but normal autonomic function after Kawasaki disease. J Pediatr,

(1996). Endothelial dysfunction late after Kawasaki disease. Circulation, Vol. 94,

Ochi M, & Ogawa S. (2007). Coronary artery aneurysm induced by Kawasaki disease in children show features typical senescence. Circ J, Vol. 71, pp. 709-715. Griending KK, & FitzGerald GA. (2003). Oxidative stress and cardiovascular injury: Part 2:

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regressed coronary aneurysms after Kawasaki disease: vascular wall morphology

atherosclerosis by flow-mediated dilatation of the brachial artery and carotid artery analysis in patients with a history Kawasaki disease. Pediatr Cardiol, Vol. 26, pp.

aortic intima-media thickness: a marker of preclinical atherosclerosis in high-risk

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atherosclerosis and stiffness. BMJ Pediatrcs, Vol.8, pp. 20.

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pp. 2103-2106.

782-786.


**11** 

*Romania* 

**Dysmetabolic Syndrome** 

Maxim Razvan and Parepa Irinel

Craiu Elvira, Cojocaru Lucia, Rusali Andrei,

*Ovidius University of Constantza, Faculty of Medicine,* 

The dysmetabolic syndrome (DMS) reunites a cluster of interrelated and important risk factors and/or medical conditions or disorders which act and worsen each other, aggravate and provoke each other, promoting the development of atherosclerotic vascular disease and

Although it has been termed "a syndrome or a disease-state", the prevalence of metabolic syndrome has risen dramatically in all societies over the past two decades; therefore, DMS should be analyzed as "an educational concept that focuses attention on complex multifactorial health problems", but in relation "to four key areas: pathophysiology, epidemiology, clinical work and public health" (Ford et all., 2002; Simmons et all., 2010). This aspect must be well deepened by doctors and understood by patients, because the patients with DMS are at a much higher risk for many and serious medical conditions (atherogenic dyslipidemia, elevated blood pressure, elevated plasma glucose,

This review evaluates the multiple similarities and differences between several concepts and definitions of DMS, in an attempt to clarify its practical and clinical usefulness, amid many exciting controversies. The clinical significance of DMS, as a distinct entity, has been debated in the past years. Initially, DMS was scarcely used as a practical tool for clinical management, educational concept or pre-morbid condition, until 1988, when GM Reaven brought it to the attention of clinicians and theorists in "Banting Lecture"; thus, GM Reaven remains the main author who has developed and strengthened this "clinical and pathological concept", identifying insulin resistance as the central pathophysiologic feature

DMS is known under many names: "Metabolic syndrome" (World Health Organisation [WHO], 1998; National Cholesterol Education Program [NCEP], 2002; International Diabetes Federation [IDF], 2005), "Insulin resistance syndrome", "Dysmetabolic syndrome", "Cardiometabolic syndrome", "Syndrome X" (Reaven, 1988), "Plurametabolic syndrome", "Deadly Quartet: upper-body obesity, glucose intolerance, hypertriglyceridemia, and hypertension" (Kaplan, 1989), "Atherometabolic Syndrome", "Sindrom dismetabopres"

**1. Introduction** 

type 2 diabetes (DM).

(Reaven, 1988).

proinflammatory and prothrombotic states, and so on).

(Buoi G, 2005), "CHAOS" (in Australia), and so on.


## **Dysmetabolic Syndrome**

Craiu Elvira, Cojocaru Lucia, Rusali Andrei, Maxim Razvan and Parepa Irinel *Ovidius University of Constantza, Faculty of Medicine, Romania* 

#### **1. Introduction**

218 Recent Advances in Cardiovascular Risk Factors

Ooyanagi R, Fuse S, Tomita H, Takamuro M, Horita N, Mori M, & Tsutsumi H. (2004). Pulse

Ozawa S, & Hamaoka K. (2006). HMG-CoA refuctase inhibitors are effective for prevention

Sasaguri Y, & Kato H. (1982). Regression of aneurysms in Kawasaki disease: a pathological

Suzuki A, Kamiya T, Arakaki Y, Kinoshita Y, & Kimura K. (1994). Fate of coronary arterrial

Suzuki A, Kamiya T, Tsuda E, & Shinya T. (1997). Natural history of coronary arterial lesions in Kawasaki disease. Prog Pediatr Cardiol, Vol. 6, pp. 211-218. Suzuki A, Miyagawa-Tomita S, Komatsu K, Nishikawa T, Sakomura Y, Horie T, &

Suzuki A, Miyagawa-Tomita S, Komatsu K, Nakazawa M, Fukaya T, Baba K, & Yutani C.

Takahashi K, Oharaseki T, & Naoe S. (2001). Pathological study of postcoronary arteritis in

Tsuda E, Abe T, Tamaki W. (2011). Acute coronary syndrome in adult patients with

Yamakawa R, Ishii M, Sugimura T, Akagi T, Eto G, Iemura M, Tsutsumi T, & Kato H. (1998).

after Kawasaki disease. Pediatrics International, Vol. 46, pp. 590-596. Strong WB, Deckelbaum RJ, Gidding SS, Kavey RE, Washinton R, Wilmore JH & Perry CL.

aneurysms in Kawasaki disease. Am J Cardiol, Vol. 74, pp. 822-824.

Int, Vol. 46, pp. 398-402.

Vol. 46(Suppl A), pp.244A.

2935-41.

study. J Pediatr, Vol. 100, pp. 225-231.

Association. Circulation., Vol. 85, pp. 1638-50.

Young, Vol 21, pp.74-82.

and acute rheumatic fever. J Pediatr, Vol. 119, pp. 279-282.

wave velocity and ankle brachial index in patients with Kawasaki disease. Peditr

of acute coronary injury in a rabbit model of Kawasaki disease. J Am Coll Cadiol,

Nakazawa M. (2000). Active remodeling of the coronary arterial lesions in the late phase of Kawasaki disease: Immunohistochemical study. Circulation, Vol. 101, pp.

(2004). Immunohistochemical study of apparently intact coronary artery in a child

(1992). Integreted cardiovascular health promotion in childhood. A statement for health profrssionals from the subcommittee on atherosclerosis and hypertension in childhood of the council on cardiovascular disease in the young, American Heart

adolescents and young adults: with reference to the relationship between sequelae of Kawasaki disease and atherosclerosis. Pediatr Cardiol, Vol. 22, pp. 138-142. Taubert KA, Rowley AH, & Shulman ST. (1991). Nationwide survey of Kawasaki disease

coronary artery lesions caused by Kawasaki disease: review of case reports. Cardiol

Coronary endothelial dysfunction after Kawasaki disease: evaluation by intracoronary injection of acetylcholine. J Am Coll Cardiol, Vol.31, pp.1074-1080.

The dysmetabolic syndrome (DMS) reunites a cluster of interrelated and important risk factors and/or medical conditions or disorders which act and worsen each other, aggravate and provoke each other, promoting the development of atherosclerotic vascular disease and type 2 diabetes (DM).

Although it has been termed "a syndrome or a disease-state", the prevalence of metabolic syndrome has risen dramatically in all societies over the past two decades; therefore, DMS should be analyzed as "an educational concept that focuses attention on complex multifactorial health problems", but in relation "to four key areas: pathophysiology, epidemiology, clinical work and public health" (Ford et all., 2002; Simmons et all., 2010). This aspect must be well deepened by doctors and understood by patients, because the patients with DMS are at a much higher risk for many and serious medical conditions (atherogenic dyslipidemia, elevated blood pressure, elevated plasma glucose, proinflammatory and prothrombotic states, and so on).

This review evaluates the multiple similarities and differences between several concepts and definitions of DMS, in an attempt to clarify its practical and clinical usefulness, amid many exciting controversies. The clinical significance of DMS, as a distinct entity, has been debated in the past years. Initially, DMS was scarcely used as a practical tool for clinical management, educational concept or pre-morbid condition, until 1988, when GM Reaven brought it to the attention of clinicians and theorists in "Banting Lecture"; thus, GM Reaven remains the main author who has developed and strengthened this "clinical and pathological concept", identifying insulin resistance as the central pathophysiologic feature (Reaven, 1988).

DMS is known under many names: "Metabolic syndrome" (World Health Organisation [WHO], 1998; National Cholesterol Education Program [NCEP], 2002; International Diabetes Federation [IDF], 2005), "Insulin resistance syndrome", "Dysmetabolic syndrome", "Cardiometabolic syndrome", "Syndrome X" (Reaven, 1988), "Plurametabolic syndrome", "Deadly Quartet: upper-body obesity, glucose intolerance, hypertriglyceridemia, and hypertension" (Kaplan, 1989), "Atherometabolic Syndrome", "Sindrom dismetabopres" (Buoi G, 2005), "CHAOS" (in Australia), and so on.

Dysmetabolic Syndrome 221

fasting

Insulin resistance or

Insulin resistance in top 25 %•; glucose 6.1 mmol/L (110 mg/dL); 2 hour glucose 7.8 mmol/L (140 mg/dL)

High risk of insulin resistance) or BMI 25 kg/m2 or waist 102 cm (men) or 88 cm (women)

(110 mg/dL); 2 hour glucose 7.8 mmol/L (140 mg/dL)

<1.0 mmol/L (40 mg/dL) (men); <1.3 mmol/L (50 mg/dL) (women)

1.7 mmol/L (150 mg/dL)

130/85 mmHg

6.1 mmol/L

<0.9 mmol/L (35 mg/dL) (men); <1.0 mmol/L (40 mg/dL) (women)

or 1.7 mmol/L (150 mg/dL)

Waist/hip ratio >0.9 (men) or >0.85 (women) or BMI 30 kg/m2

140/90 mmHg

hyperinsulinemia in top 25 percent

3 of: And 2 of: And 2 of: And 2 of: And 2 of:

<1.0 mmol/L (40 mg/dL)

or 2.0 mmol/L (180 mg/dL) or drug treatment for dyslipidemia

Waist 94 cm (men) or 80 cm (women)

140/90 mmHg or drug treatment for hypertension

\* For South Asia and Chinese patients, waist 90 cm (men) or 80 cm (women); for Japanese patients,

Treatment with one or more of fibrates or niacin. § In Asian patients, waist 90 cm (men) or 80 cm

) High risk of being insulin resistant is indicated by the presence of at least one of the following: diagnosis of CVD, hypertension, polycystic ovary syndrome, non-alcoholic fatty liver disease or acanthosis nigricans; family history of Type 2 diabetes, hypertension of CVD; history of gestational diabetes or glucose intolerance; nonwhite ethnicity; sedentary lifestyle; BMI 25 kb/m2 or waist

mg/dL)

6.1-6.9 mmol/ (110-125

**Parameters NCEP ATP3 2005 IDF 2005 EGIR 1999 WHO 1999 AACE 2003** 

(men) or 80 cm (women)\*

5.6 mmol/L (100 mg/dL)

diagnosediabe

<1.0 mmol/L (40 mg/dL) (men); <1.3 mmol/L (50 mg/dL) (women) or drug treatment for low HDL-C

1.7 mmol/L (150 mg/dL) or drug treatment for high TGs

130/85 mmHg or drug treatment for HTA

circumference 94 cm for men and 80 cm for women; and age 40 years.

Table 1. Five current definitions of the metabolic syndrome (Meigs, 2006).

or

tes

Required Waist 94 cm

mg/dL) or drug treatment for elevated blood glucose

mg/dL) (men); <1.3 mmol/L (50 mg/dL) (women) or drug treatment for low HDL-C

mg/dL) or drug treatment for elevated triglycerides

(men) or 88 cm (women)§

drug treatment for

• Insulin resistance measured using insulin clamp.

Glucose 5.6 mmol/L (100

HDL cholesterol <1.0 mmol/L (40

TGs 1.7 mmol/L (150

Obesity Waist 102 cm

HTA 130/85 mmHg or

HTA

waist 90 cm (men) or 80 cm (women).

(women).

Nr. of abnormalities

### **2. History**

The history of DMS was not an easy one; we will present, in chronological order, the most important events (Isomaa et al., 2001).

Between 1920-1923, Kylin (as cited in Lau, 2009), a Swedish physician, described for the first time a constellation of metabolic disturbance and the clustering of hypertension, hyperglycemia, and gout. After Kylin, J Vague, from the University of Marseille, reported that body fat topography, respectively upper-body obesity, causes the predisposition to diabetes mellitus, atherosclerosis, gout, and renal calculi, and that its anatomical distribution differs according to gender. Vague used the term "android obesity" to define the pattern of fat distribution with an accumulation of adipose tissue over the trunk and the term "gynoid obesity" for adipose tissue that accumulates mostly around the hips and thighs, commonly found in women (Vague et al., 1947). As the research continued, after 1960, a link between obesity, insulin-resistance and related complications was suggested. Albrink and Meigs reported an association between trunk fat and hypertriglyceridemia (Meigs et al., 2003). In 1975-1977, Haller used the term "metabolic syndrome" for an association of obesity, diabetes mellitus, hyperlipoproteinemia, hyperuricemia, and hepatic steatosis, and describes the additional role of these risk factors on the cardiovascular disease (CVD) (Haller, 1977). In 1977-1978, Singer and Phillips developed the concept that risk factors for myocardial infarction, respectively a "constellation of associated abnormalities" (i.e. glucose intolerance, hyperinsulinemia, hypercholesterolemia, hypertriglyceridemia, and hypertension) are associated with CVD, aging, obesity, sex hormones, and other clinical states (Singer, 1977; Phillips, 1978). In 1988, Reaven, describes "Syndrome X" in his famous Banting Lecture; this is a critical moment, subject to many controversies in the literature; more and more risk factors (RF) (hypertension, hyperglycemia, glucose intolerance, elevated triglycerides, and low HDL-cholesterol), as well as many metabolic disturbances, especially insulin resistance, are incriminated in the pathogeny of CVD (Reaven, 1988). In 2005, Kahn draws attention to several unresolved questions about the metabolic syndrome, many of them still unresolved even now:


#### **3. Definitions of dysmetabolic syndrome**

In 1998-1999, WHO defines metabolic syndrome (MS) as a clustering of arterial hypertension, dyslipidemia, obesity with high waist to hip ratio, microalbuminuria, glucose intolerance or insulin resistance, or type 2 diabetes; at the same time, it recognizes "CVD as the primary outcome of the metabolic syndrome'' (table 1).

The criteria for the diagnosis of MS are: the presence of one of diabetes mellitus, impaired glucose tolerance, impaired fasting glucose or insulin resistance, AND two of the following:


The history of DMS was not an easy one; we will present, in chronological order, the most

Between 1920-1923, Kylin (as cited in Lau, 2009), a Swedish physician, described for the first time a constellation of metabolic disturbance and the clustering of hypertension, hyperglycemia, and gout. After Kylin, J Vague, from the University of Marseille, reported that body fat topography, respectively upper-body obesity, causes the predisposition to diabetes mellitus, atherosclerosis, gout, and renal calculi, and that its anatomical distribution differs according to gender. Vague used the term "android obesity" to define the pattern of fat distribution with an accumulation of adipose tissue over the trunk and the term "gynoid obesity" for adipose tissue that accumulates mostly around the hips and thighs, commonly found in women (Vague et al., 1947). As the research continued, after 1960, a link between obesity, insulin-resistance and related complications was suggested. Albrink and Meigs reported an association between trunk fat and hypertriglyceridemia (Meigs et al., 2003). In 1975-1977, Haller used the term "metabolic syndrome" for an association of obesity, diabetes mellitus, hyperlipoproteinemia, hyperuricemia, and hepatic steatosis, and describes the additional role of these risk factors on the cardiovascular disease (CVD) (Haller, 1977). In 1977-1978, Singer and Phillips developed the concept that risk factors for myocardial infarction, respectively a "constellation of associated abnormalities" (i.e. glucose intolerance, hyperinsulinemia, hypercholesterolemia, hypertriglyceridemia, and hypertension) are associated with CVD, aging, obesity, sex hormones, and other clinical states (Singer, 1977; Phillips, 1978). In 1988, Reaven, describes "Syndrome X" in his famous Banting Lecture; this is a critical moment, subject to many controversies in the literature; more and more risk factors (RF) (hypertension, hyperglycemia, glucose intolerance, elevated triglycerides, and low HDL-cholesterol), as well as many metabolic disturbances, especially insulin resistance, are incriminated in the pathogeny of CVD (Reaven, 1988). In 2005, Kahn draws attention to several unresolved questions about the metabolic syndrome, many of

In 1998-1999, WHO defines metabolic syndrome (MS) as a clustering of arterial hypertension, dyslipidemia, obesity with high waist to hip ratio, microalbuminuria, glucose intolerance or insulin resistance, or type 2 diabetes; at the same time, it recognizes "CVD as

The criteria for the diagnosis of MS are: the presence of one of diabetes mellitus, impaired glucose tolerance, impaired fasting glucose or insulin resistance, AND two of the following:

2. dyslipidemia: triglycerides (TG) 1.695 mmol/L and high-density lipoprotein

cholesterol (HDL-C) 0.9 mmol/L (in males) or 1.0 mmol/L (in females);

**2. History** 

important events (Isomaa et al., 2001).

them still unresolved even now: - Metabolic syndrome name? - Existence of metabolic syndrome? - More than some of its parts?

1. blood pressure 140/90 mmH


the primary outcome of the metabolic syndrome'' (table 1).

**3. Definitions of dysmetabolic syndrome** 


\* For South Asia and Chinese patients, waist 90 cm (men) or 80 cm (women); for Japanese patients, waist 90 cm (men) or 80 cm (women).

• Insulin resistance measured using insulin clamp.

) High risk of being insulin resistant is indicated by the presence of at least one of the following: diagnosis of CVD, hypertension, polycystic ovary syndrome, non-alcoholic fatty liver disease or acanthosis nigricans; family history of Type 2 diabetes, hypertension of CVD; history of gestational diabetes or glucose intolerance; nonwhite ethnicity; sedentary lifestyle; BMI 25 kb/m2 or waist circumference 94 cm for men and 80 cm for women; and age 40 years.

 Treatment with one or more of fibrates or niacin. § In Asian patients, waist 90 cm (men) or 80 cm (women).

Table 1. Five current definitions of the metabolic syndrome (Meigs, 2006).

Dysmetabolic Syndrome 223

The IDF (International Diabetes Federation) releases in 2005 the "Consensus worldwide definition" of the metabolic syndrome, which mentions the following criteria needed for the

1. central obesity is an essential element (defined as waist circumference with

2. raised triglycerides: > 150 mg/dL (1.7 mmol/L) or specific treatment for this lipid

3. reduced HDL cholesterol: < 40 mg/dL (1.03 mmol/L) in males, < 50 mg/dL (1.29

4. raised blood pressure: systolic BP > 130 mmHg or diastolic BP >85 mmHg, or treatment

5. raised fasting plasma glucose : > 100 mg/dL (5.6 mmol/L), or previously diagnosed



race/ethnicity specific values) and any two of the following:

of previously diagnosed hypertension;

need to be measured (Alberti et al., 2005, 2006).

**ADDITIONAL METABOLIC IDF CRITERIA (for research)** 




Table 2. IDF: Additional metabolic criteria (for research) -"platinum standard" definition




type 2 diabetes. (table 1 and 2).

**Abnormal body fat distribution:** 




**Proinflammatory state:**

**Prothrombotic state:**

(www.idf,org)



**Dysglycaemia:** Oral glucose tolerance test (OGTT). **Insulin resistance (other than elevated fasting glucose):**


mmol/L) in females, or specific treatment for this lipid abnormality;

recommended, but is not necessary to define presence of the MS;

**Atherogenic dyslipidaemia (beyond elevated triglyceride and low HDL):**

diagnosis:

abnormality;

It can be noted that:


We can see a potential disadvantage of the WHO criteria, namely the need for the routine assessment of the glycemic metabolism. (Simmons et al., 2010; Alberti & Zimmet, 1998).

The European Group for the Study of Insulin Resistance (EGIR, 1999), designed to be used in non diabetics only, requires two or more of the following:


In 2001-2002, the National Cholesterol Education Program and Adult Treatment Panel (ATPIII) provides a clinical definition of metabolic syndrome, "a multiplex risk factor for cardiovascular disease (CVD) and identifies" six components of the metabolic syndrome that relate to CVD: abdominal obesity, atherogenic dyslipidemia, raised blood pressure, insulin resistance ± glucose intolerance, proinflammatory state, prothrombotic state". It also states that "the presence of type 2 DM does not exclude a diagnosis of metabolic syndrome" and the definition does not require evidence of insulin or glucose abnormalities, although "abnormal glycemia is one of the criteria". The US NCEP and ATP III require at least three of the following:

	- TG 150 mg/dL (1.7 mmol/L), or drug treatment for elevated triglycerides;
	- HDL-C < 40 mg/dL (1 mmol/L) (male), < 50 mg/dL (1,3 mmol/L) (female), or drug treatment for low HDL-C;

We can see that:


In 2005, ADA (American Diabetes Association) and EASD (European Association for the Study of Diabetes) discourage the use of the term "metabolic syndrome" in the field literature, questioning "whether this constellation of clinical findings constitutes a syndrome" and "whether that constellation, in and of itself, is an entity of medical concern above and beyond the individual components", and so on (Beaser & Levy, 2007).

3. central obesity: waist:hip ratio > 0.90 (in males) and > 0.85 (in females), or body mass

4. microalbuminuria: urinary albumin excretion ratio 20 µg/min. or albumin:creatinine

We can see a potential disadvantage of the WHO criteria, namely the need for the routine assessment of the glycemic metabolism. (Simmons et al., 2010; Alberti & Zimmet, 1998).

The European Group for the Study of Insulin Resistance (EGIR, 1999), designed to be used

2. dyslipidemia: TG 2.0 mmol/L and/or HDL-C < 1.0 mmol/L or treated for

In 2001-2002, the National Cholesterol Education Program and Adult Treatment Panel (ATPIII) provides a clinical definition of metabolic syndrome, "a multiplex risk factor for cardiovascular disease (CVD) and identifies" six components of the metabolic syndrome that relate to CVD: abdominal obesity, atherogenic dyslipidemia, raised blood pressure, insulin resistance ± glucose intolerance, proinflammatory state, prothrombotic state". It also states that "the presence of type 2 DM does not exclude a diagnosis of metabolic syndrome" and the definition does not require evidence of insulin or glucose abnormalities, although "abnormal glycemia is one of the criteria". The US NCEP and ATP III require at least three

1. central (abdominal) obesity: waist circumference 102 cm (40 inches) (male), 88 cm



In 2005, ADA (American Diabetes Association) and EASD (European Association for the Study of Diabetes) discourage the use of the term "metabolic syndrome" in the field literature, questioning "whether this constellation of clinical findings constitutes a syndrome" and "whether that constellation, in and of itself, is an entity of medical concern

3. blood pressure 130/85 mmHg, or drug treatment for elevated blood pressure; 4. FPG 100 mg/dL (5.6 mmol/L) or drug treatment for elevated blood glucose (table 1).


above and beyond the individual components", and so on (Beaser & Levy, 2007).

considered for NCEP ATP III (NCEPT ATPIII, 2001, 2002).


3. hypertension: blood pressure 140/90 mmHg or antihypertensive medication;

in non diabetics only, requires two or more of the following:

4. fasting plasma glucose (FPG) 6.1 mmol/L (table 1).

1. central obesity: waist circumference 94 cm (male), 80 cm (female);

index > 30 kg/m2 ;

ratio 30 mg/g.

dyslipidemia;

of the following:

2. dyslipidemia:

We can see that:

(35 inches) (female);

(defined by the "BMI");

drug treatment for low HDL-C;

The IDF (International Diabetes Federation) releases in 2005 the "Consensus worldwide definition" of the metabolic syndrome, which mentions the following criteria needed for the diagnosis:


It can be noted that:



**Hormonal factors:** pituitary-adrenal axis.

Table 2. IDF: Additional metabolic criteria (for research) -"platinum standard" definition (www.idf,org)

Dysmetabolic Syndrome 225

Now the DMS has been recognized as a proinflammatory, prothrombotic state, with elevated levels of C-reactive protein, interleukin (IL)-6, and plasminogen activator inhibitor (PAI)-1, and so on. Therefore, in the near future, many other diagnostic criteria will arise; for example, if high-sensitivity c-reactive protein (hs-CRP) can be used as a marker to predict coronary vascular diseases in DMS and as a predictor for non-alcoholic fatty liver disease, in correlation with serum markers that indicate the disturbance of the lipid and glucose

These sets of defining criteria for DMS are similar but they also have many important differences (clinical, etiopathogenic, ethnical, geographical, and so on) (tables 1,2,3). In fact, these five definitions of DMS illustrate "its complexity and heterogeneity", although they

**ATP III / IDF** 

**ADA / EASD** 

Table 3. Clustering or constellation of the metabolic risk factors (3+) in definition of the

The DMS is an insulin-resistant state with a cluster of cardiovascular risk factors, including various combinations and substantial additional CV risk, which occur in the same individual, but the question is "how to integrate the DMS into concepts of insulin resistance, pre-diabetes, and type-2 diabetes". Insulin resistance is a component of obesity, it favors the onset of type 2 DM and is found in many cases of hypertension and hypertriglyceridaemia with low levels of HDL-cholesterol (Reaven, 1988), in association with correlated metabolic abnormalities recognized as CV risk factors that are present prior to the onset of diabetes. If insulin resistance evolves with many other characteristics (abdominal obesity, dyslipidemia, hypertension, non-dipper pattern of blood pressure, salt sensitivity, glucose intolerance, history of gestational DM, increased PAI-1/ platelets and so on), can all of these be seen as cardiovascular RF in various combinations? Which and how many of these RF carry the

In all definitions of DMS, the abdominal adiposity is underlined and not the body weight. It is indeed the visceral adipocytes that are metabolically active, leading "to elevated plasma free fatty acids that result in elevated triglycerides and lower HDL cholesterol, and contribute to elevated plasma glucose"; is this explication enough to consider that this type


metabolism (Kogiso et al., 2009).

are not unanimously accepted.

Atherogenic dyslipidemia Elevated blood pressure Elevated plasma glucose Prothrombotic state Proinflammatory state

Atherogenic dyslipidemia

Elevated plasma glucose Prothrombotic state Proinflammatory state

greatest impact? (Johnson & Weinsstock, 2006).

of obesity is the RF with the greatest impact? (Beaser & Levy, 2005).

metabolic syndrome

Prediabetes

After a brief period of time, AHA (American Heart Association) and NHLB (National Heart, Lung, and Blood Institute) state the opposite and consider MS:


The confusion generated by AHA/NHLBI is clarified by AHA/Updated NCEP ATP III, which gives the following criteria of diagnosis:

	- men greater than 40 inches (102 cm);
	- women greater than 35 inches (88 cm);
	- men less than 40 mg/dL (1.03 mmol/L);
	- women less than 50 mg/dL (1.29 mmol/L);
	- equal to or greater than 130/85 mm Hg or
	- use of medication for hypertension;
	- equal to or greater than 100 mg/dL (5.6 mmol/L) or use of medication for hyperglycemia.

The following notes are made:


Following this statement, AACE (American Association of Clinical Endocrinologists) propose a "third set of clinical criteria for the insulin resistance syndrome", in fact ''a hybrid of those of ATP III and WHO metabolic syndrome'', but with "clinical value as a diagnosis".

As a disease entity, MS is recognized by the American Society of Endocrinology, NCEP, and WHO, among others. The above mentioned set has some complicated aspects:

	- diagnosis of CVD, hypertension, polycystic ovary syndrome, non-alcoholic fatty liver disease or acanthosis nigricans;
	- family history of type 2 diabetes, hypertension or CVD;
	- history of gestational diabetes or glucose intolerance;
	- non-white ethnicity; sedentary lifestyle; BMI 25 kb/m2 or waist circumference 94 cm for men and 80 cm for women; and

After a brief period of time, AHA (American Heart Association) and NHLB (National Heart,


The confusion generated by AHA/NHLBI is clarified by AHA/Updated NCEP ATP III,




Following this statement, AACE (American Association of Clinical Endocrinologists) propose a "third set of clinical criteria for the insulin resistance syndrome", in fact ''a hybrid of those of ATP III and WHO metabolic syndrome'', but with "clinical value as a diagnosis". As a disease entity, MS is recognized by the American Society of Endocrinology, NCEP, and




WHO, among others. The above mentioned set has some complicated aspects:


2. elevated triglycerides - equal to or greater than 150 mg/dL (1.7 mmol/L);

Lung, and Blood Institute) state the opposite and consider MS:


(ASCVD) and type 2 diabetes;

1. elevated waist circumference:

4. elevated blood pressure:

5. elevated fasting glucose:

hyperglycemia. The following notes are made:

al., 2004, 2005; Beilby, 2004).


liver disease or acanthosis nigricans;

cm for men and 80 cm for women; and


following:

3. reduced HDL ("good") cholesterol:

which gives the following criteria of diagnosis:




the ADA criteria for impaired fasting plasma glucose; - the inclusion of diabetes in the hyperglycemia trait definition; Now the DMS has been recognized as a proinflammatory, prothrombotic state, with elevated levels of C-reactive protein, interleukin (IL)-6, and plasminogen activator inhibitor (PAI)-1, and so on. Therefore, in the near future, many other diagnostic criteria will arise; for example, if high-sensitivity c-reactive protein (hs-CRP) can be used as a marker to predict coronary vascular diseases in DMS and as a predictor for non-alcoholic fatty liver disease, in correlation with serum markers that indicate the disturbance of the lipid and glucose metabolism (Kogiso et al., 2009).

These sets of defining criteria for DMS are similar but they also have many important differences (clinical, etiopathogenic, ethnical, geographical, and so on) (tables 1,2,3). In fact, these five definitions of DMS illustrate "its complexity and heterogeneity", although they are not unanimously accepted.


Table 3. Clustering or constellation of the metabolic risk factors (3+) in definition of the metabolic syndrome

The DMS is an insulin-resistant state with a cluster of cardiovascular risk factors, including various combinations and substantial additional CV risk, which occur in the same individual, but the question is "how to integrate the DMS into concepts of insulin resistance, pre-diabetes, and type-2 diabetes". Insulin resistance is a component of obesity, it favors the onset of type 2 DM and is found in many cases of hypertension and hypertriglyceridaemia with low levels of HDL-cholesterol (Reaven, 1988), in association with correlated metabolic abnormalities recognized as CV risk factors that are present prior to the onset of diabetes. If insulin resistance evolves with many other characteristics (abdominal obesity, dyslipidemia, hypertension, non-dipper pattern of blood pressure, salt sensitivity, glucose intolerance, history of gestational DM, increased PAI-1/ platelets and so on), can all of these be seen as cardiovascular RF in various combinations? Which and how many of these RF carry the greatest impact? (Johnson & Weinsstock, 2006).

In all definitions of DMS, the abdominal adiposity is underlined and not the body weight. It is indeed the visceral adipocytes that are metabolically active, leading "to elevated plasma free fatty acids that result in elevated triglycerides and lower HDL cholesterol, and contribute to elevated plasma glucose"; is this explication enough to consider that this type of obesity is the RF with the greatest impact? (Beaser & Levy, 2005).

Dysmetabolic Syndrome 227


Endothelial dysfunction (ED) is a key event in the pathogenesis of atherosclerosis. The possibility of early identification of individuals at risk and achieving an objective control of the effectiveness of treatment in clinical practice become an attractive goal of therapeutic strategies useful to reduce cardiovascular morbid-mortality, and the endothelium is the

Given that DMS includes "an atherogenic dyslipidaemia, an insulin resistance state leading to a disturbed plasma glucose/insulin homeostasis, a abdominal obesity, especially visceral obesity, a thrombotic and inflammatory profile, as well as an endothelial dysfunction could substantially increase the risk of coronary heart disease (CHD) and type 2 diabetes"

K. Watson draws attention to the practicality of cardiometabolic risk management, particularly attractive to lower morbidity and mortality but also the economic costs for health, particularly if the disease or diseases and/or it's complications are identified early,

The key to identification of cardiometabolic risk is the recognition that a patient with one or 2 clinically evident risk factors likely has additional factors, as these risk factors have been shown to "cluster"; this cluster effect is not specific for DMS; it remains a concept on the

Meigs et al. refined the concept of the metabolic syndrome "by outlining the function of distinct clusters of risk factors", actually "three factors underlie the clustering of risk

1. a "metabolic" factor, including BMI, waist circumference, 2-hour glucose tolerance,

basis of which the results of future research will show new perspectives.

triglycerides, insulin sensitivity, and plasminogen activator inhibitor;


**CLINICAL SYNDROMES ASSOCIATED WITH INSULIN RESISTANCE** 

Table 4. Clinical syndromes associated with insulin resistance

**4. Etiopathogeny of dysmetabolic syndrome** 

logical "window" for the next evolution of atherosclerosis.

(Alexander et al., 2003; Kahn R, et al., 2005; Grundy, 2006).

especially in the subclinical phase (Watson, 2007).

variables", risk factors that are still topical:

trials.

Type 2 diabetes

Sleep apnea

Essential hypertension Polycystic ovary syndrome Nonalcoholic fatty liver disease

Certain forms of cancer

CVD

In addition to age, race, and weight are there other RF associated with an increased risk of DMS? Indeed, in NHANES III trial, other RF have emerged (postmenopausal status, smoking, low household income, high carbohydrate diet, no alcohol consumption, and physical inactivity); should these RF be included in the DMS? And when other RF for CVD will arise, which RF will not be components of the DMS? We should not forget that the notion of DMS ignores "other several strong risk factors" for cardiovascular disease (like cigarette smoking and elevated levels of low-density lipoprotein cholesterol, for example). (Palaniappan et al., 2004).

Is the treatment of the DMS different, next to the treatment for each of its components? It is obvious that the presence of a DMS component will lead to its evaluation and optimal treatment, but it is also important to look for and evaluate all the individual components of DMS from all the definitions. If a patient has certain characteristics included in one of the definitions for DMS (large waist circumference, high triglycerides and high fasting glucose) and another patient has other characteristics (high blood pressure, low HDL, and high triglycerides), both of them will be diagnosed with DMS, but will they benefit from different therapeutic strategies? Because there is no unique mechanism for DMS, thereby there won't be a unique treatment (Bayturan et al., 2010).

If we accept that "the Framingham score for the risk" is a better "short-term" (10 year) risk tool, does it mean that the metabolic syndrome was meant to identify individuals at "higher long-term risk"? Are there other risk factors for CVD, which are not components of the metabolic syndrome, such as inflammatory markers, which may have equal or greater bearing on risk? (Grundy, 2006). Is the CVD risk associated with the metabolic syndrome higher than the sum of its individual components? (Sundstorm et al., 2006).

The setting of diagnostic criteria for DMS is very difficult with numerous controversies and uncertainties. Therefore, is the DMS:


This research showed that the SM is a most complex problem, a focus of much research and clinical interest, involving:


Metabolic syndrome affects 44% of the U.S. population older than age 50; the percentage of women having the syndrome is higher than that of men; the age dependency of the syndrome's prevalence is seen in most populations around the world. The "clustering" of CV and metabolic abnormalities in the same person will lead to an additional CV risk "over and above the sum of the risks associated with each abnormality" (Golden et al, 2002).

Thus, it is necessary:

In addition to age, race, and weight are there other RF associated with an increased risk of DMS? Indeed, in NHANES III trial, other RF have emerged (postmenopausal status, smoking, low household income, high carbohydrate diet, no alcohol consumption, and physical inactivity); should these RF be included in the DMS? And when other RF for CVD will arise, which RF will not be components of the DMS? We should not forget that the notion of DMS ignores "other several strong risk factors" for cardiovascular disease (like cigarette smoking and elevated levels of low-density lipoprotein cholesterol, for example).

Is the treatment of the DMS different, next to the treatment for each of its components? It is obvious that the presence of a DMS component will lead to its evaluation and optimal treatment, but it is also important to look for and evaluate all the individual components of DMS from all the definitions. If a patient has certain characteristics included in one of the definitions for DMS (large waist circumference, high triglycerides and high fasting glucose) and another patient has other characteristics (high blood pressure, low HDL, and high triglycerides), both of them will be diagnosed with DMS, but will they benefit from different therapeutic strategies? Because there is no unique mechanism for DMS, thereby there won't

If we accept that "the Framingham score for the risk" is a better "short-term" (10 year) risk tool, does it mean that the metabolic syndrome was meant to identify individuals at "higher long-term risk"? Are there other risk factors for CVD, which are not components of the metabolic syndrome, such as inflammatory markers, which may have equal or greater bearing on risk? (Grundy, 2006). Is the CVD risk associated with the metabolic syndrome

The setting of diagnostic criteria for DMS is very difficult with numerous controversies and

3. a clustering of certain signs and symptoms that tend to occur under certain

4. three or more related diagnostic entities associated with any morbid process? And so on

This research showed that the SM is a most complex problem, a focus of much research and

Metabolic syndrome affects 44% of the U.S. population older than age 50; the percentage of women having the syndrome is higher than that of men; the age dependency of the syndrome's prevalence is seen in most populations around the world. The "clustering" of CV and metabolic abnormalities in the same person will lead to an additional CV risk "over and above the sum of the risks associated with each abnormality" (Golden et al, 2002).

higher than the sum of its individual components? (Sundstorm et al., 2006).


2. a simple collection of things with "an identifiable pattern" ?

(Palaniappan et al., 2004).

be a unique treatment (Bayturan et al., 2010).

uncertainties. Therefore, is the DMS:

(Kahn et al., 2005; Balta, 2010).



1. a true syndrome?

circumstances?

clinical interest, involving:

Thus, it is necessary:


#### **CLINICAL SYNDROMES ASSOCIATED WITH INSULIN RESISTANCE**

Type 2 diabetes CVD Essential hypertension Polycystic ovary syndrome Nonalcoholic fatty liver disease Certain forms of cancer Sleep apnea

Table 4. Clinical syndromes associated with insulin resistance

#### **4. Etiopathogeny of dysmetabolic syndrome**

Endothelial dysfunction (ED) is a key event in the pathogenesis of atherosclerosis. The possibility of early identification of individuals at risk and achieving an objective control of the effectiveness of treatment in clinical practice become an attractive goal of therapeutic strategies useful to reduce cardiovascular morbid-mortality, and the endothelium is the logical "window" for the next evolution of atherosclerosis.

Given that DMS includes "an atherogenic dyslipidaemia, an insulin resistance state leading to a disturbed plasma glucose/insulin homeostasis, a abdominal obesity, especially visceral obesity, a thrombotic and inflammatory profile, as well as an endothelial dysfunction could substantially increase the risk of coronary heart disease (CHD) and type 2 diabetes" (Alexander et al., 2003; Kahn R, et al., 2005; Grundy, 2006).

K. Watson draws attention to the practicality of cardiometabolic risk management, particularly attractive to lower morbidity and mortality but also the economic costs for health, particularly if the disease or diseases and/or it's complications are identified early, especially in the subclinical phase (Watson, 2007).

The key to identification of cardiometabolic risk is the recognition that a patient with one or 2 clinically evident risk factors likely has additional factors, as these risk factors have been shown to "cluster"; this cluster effect is not specific for DMS; it remains a concept on the basis of which the results of future research will show new perspectives.

Meigs et al. refined the concept of the metabolic syndrome "by outlining the function of distinct clusters of risk factors", actually "three factors underlie the clustering of risk variables", risk factors that are still topical:

1. a "metabolic" factor, including BMI, waist circumference, 2-hour glucose tolerance, triglycerides, insulin sensitivity, and plasminogen activator inhibitor;

Dysmetabolic Syndrome 229

There is debate "whether obesity or insulin resistance are the causes of the DMS or if they

The multiple risk factors represent factors of metabolic origin, and can be grouped in several

Both insulin resistance and central obesity are considered significant factors. It is necessary to specify that insulin resistance is not synonymous with type 2 diabetes mellitus. Insulin resistance is not a disease, but remains the "primary mediator of metabolic syndrome". Insulin resistance does not necessarily lead to the clinical syndromes or to obesity; obesity is a "physiologic variable that increases the likelihood that an individual will be insulin resistant". It appears long before the diagnosis of diabetes and suggests an increased risk for the latter. Unlike type 2 diabetes, in the case of insulin resistance, the pancreas produces too much insulin as a compensatory mechanism and does not require drug treatment, but only diet and exercise. The combination of insulin resistance with compensatory hyperinsulinemia will determine an increased risk for CVD (table 4, 5) (Nakamura et al., 1994; Nesto, 2003, Matsuzawa et al., 2002). Present studies maintain "the central obesity and

insulin resistance" as "main etiological factors" in DMS (Matsuzawa et al., 2002).

Triglycerides: 50-100 mg/dL is optimal,

Fasting glucose: Normal is 74-106 mg/dL.

(PAI -1): Greater than 31

Table 5. Lab exams in DMS (Grundy et al., 2005)

Homocysteine: Less than 6 /mol.L is optimal;

10 IU/mL and below is optimal;

Less than 1.0 /U/ml is optimal.

100-150 mg/dL is moderate and over 150 mg/dL is high.

greater than 9 /mol/L is high.

indicative of Type 2 Diabetes.

high and too low are problematic.

LDL of less than 100 mg/dL is good.

In studies, women with HDL of 70 mg/dL had low

Total cholesterol should be less than 200 mg/dL or

Values greater than or equal to 126 mg/dL are

Values of 100-125 mg/dL are indicative of Pre-Diabetes.

Results greater than or equal to 200 at 2 hours following the oral glucose tolerance test indicate Type 2 Diabetes.

processes; the results vary greatly with the patient's age, gender and test method. Results that are both too

Cholesterol: HDL 40 mg/dL is good, although higher is even better.

cardiac risk.

under.

Fibrinogen: This test is a general measure of inflammatory

over 10 IU/mL is high.

are consequences of a more far-reaching metabolic derangement".

syndromes, each of which are metabolic risk factors;

Fasting insulin (Insulin Assay):

High sensitive CRP (C-Reactive Protein):

Oral glucose tolerance (with 75 gr. Glucose load):


Wilcox, presenting the much-disputed "Z syndrome" still in the concept of "cluster", cautions that "in populations at risk of vascular disease, many patients who experience a cardiovascular event either do not have identifiable risk factors or have disease severity which appears to be out of proportion to their known risk factors. Since these risk factors have been shown to be independent predictors of adverse events, they will show at least additive effects in combination and possibly potentiate each other" (Wilcox et al., 1998).

The metabolic syndrome "is a cluster of the most dangerous heart-attack risk factors:


The clustering of CVD risk factors that marks the metabolic syndrome is now considered to be "the driving force for a CVD epidemic" (Stern et al., 2004).

The exact mechanisms of the complex syndrome are not yet completely known and elucidated. Presently, the main etiologic factors for DMS obesity and the dysfunctional adipose tissue are present in clinical situations determined by insulin resistance; this process also "prevents the efficient conversion of food into energy because of a vastly reduced number of insulin receptors on the cell wall", thus inducing "an increase in blood levels of insulin". In addition, there is a multiple "set of risk factors" that commonly appear together in MS, but confer "a substantial additional CV risk, over and above the sum of the risks associated with each abnormality" (Golden et al., 2002).

The etiopathogenesis of the DMS is not entirely known and understood.

There are three potential etiological categories: obesity and disorders of adipose tissue, insulin resistance and a number of independent factors that mediate specific components of the DMS;

It is an established fact that "all components of metabolic syndrome are strongly interconnected and so they cannot be treated separately".

Questioning if and when "the whole is greater than the sum of its parts?" and "what factors comprise the syndrome?", Kahn argue with several answers:


2. an "inflammation" factor, including BMI, waist circumference, fibrinogen, C-reactive

3. a "blood pressure" factor, including systolic and diastolic blood pressure)" (Meigs et al.,

Wilcox, presenting the much-disputed "Z syndrome" still in the concept of "cluster", cautions that "in populations at risk of vascular disease, many patients who experience a cardiovascular event either do not have identifiable risk factors or have disease severity which appears to be out of proportion to their known risk factors. Since these risk factors have been shown to be independent predictors of adverse events, they will show at least additive effects in combination and possibly potentiate each other" (Wilcox et al., 1998). The metabolic syndrome "is a cluster of the most dangerous heart-attack risk factors:

The clustering of CVD risk factors that marks the metabolic syndrome is now considered to

The exact mechanisms of the complex syndrome are not yet completely known and elucidated. Presently, the main etiologic factors for DMS obesity and the dysfunctional adipose tissue are present in clinical situations determined by insulin resistance; this process also "prevents the efficient conversion of food into energy because of a vastly reduced number of insulin receptors on the cell wall", thus inducing "an increase in blood levels of insulin". In addition, there is a multiple "set of risk factors" that commonly appear together in MS, but confer "a substantial additional CV risk, over and above the sum of the risks

There are three potential etiological categories: obesity and disorders of adipose tissue, insulin resistance and a number of independent factors that mediate specific components of

It is an established fact that "all components of metabolic syndrome are strongly

Questioning if and when "the whole is greater than the sum of its parts?" and "what factors




be "the driving force for a CVD epidemic" (Stern et al., 2004).

associated with each abnormality" (Golden et al., 2002).

interconnected and so they cannot be treated separately".

comprise the syndrome?", Kahn argue with several answers:

for predicting cardiovascular disease or diabetes";

The etiopathogenesis of the DMS is not entirely known and understood.

protein, and insulin sensitivity ; and

1997).

the DMS;

clustering";

2005).

• diabetes and pre-diabetes, • abdominal obesity, • high cholesterol and • high blood pressure".

There is debate "whether obesity or insulin resistance are the causes of the DMS or if they are consequences of a more far-reaching metabolic derangement".

The multiple risk factors represent factors of metabolic origin, and can be grouped in several syndromes, each of which are metabolic risk factors;

Both insulin resistance and central obesity are considered significant factors. It is necessary to specify that insulin resistance is not synonymous with type 2 diabetes mellitus. Insulin resistance is not a disease, but remains the "primary mediator of metabolic syndrome". Insulin resistance does not necessarily lead to the clinical syndromes or to obesity; obesity is a "physiologic variable that increases the likelihood that an individual will be insulin resistant". It appears long before the diagnosis of diabetes and suggests an increased risk for the latter. Unlike type 2 diabetes, in the case of insulin resistance, the pancreas produces too much insulin as a compensatory mechanism and does not require drug treatment, but only diet and exercise. The combination of insulin resistance with compensatory hyperinsulinemia will determine an increased risk for CVD (table 4, 5) (Nakamura et al., 1994; Nesto, 2003, Matsuzawa et al., 2002). Present studies maintain "the central obesity and insulin resistance" as "main etiological factors" in DMS (Matsuzawa et al., 2002).


Table 5. Lab exams in DMS (Grundy et al., 2005)

Dysmetabolic Syndrome 231

1. is independently associated with each of the other metabolic syndrome components,

2. contributes to hypertension, dyslipidemia (high serum cholesterol, low HDL-c) and hyperglycemia, and is independently associated with higher CVD risk. (Anderson et al.,

The increased flow of free fatty acids through the liver leads to accelerated synthesis of VLDL-C, hypertriglyceridemia, endothelial dysfunction, and vasoconstriction, leading to an increase in blood pressure; also, through this mechanism, insulin resistance may exert an

A number of markers of systemic inflammation, including C-reactive protein, are often increased, as are fibrinogen, interleukin 6 (IL–6), tumor necrosis factor-alpha (TNF+), and

CVD and diabetes are associated with elevated levels of inflammatory biomarkers,

At the same time, CRP is the best-studied inflammatory marker of atherothrombotic risk,

C-reactive protein is present in the MS; its plasma levels increase with the number of metabolic risk factors, and also with other inflammatory markers; these associations are

The mechanisms that lead to the increase of CRP are complex and only partially understood; we take this opportunity to remind that only excessive fatty tissue releases inflammatory cytokines and determines higher CRP levels; adipose cell enlargement and infiltration of macrophages into adipose tissue will lead to the release of proinflammatory cytokines and promote insulin resistance. It increases the thrombogenicity of circulating blood, in part by raising plasminogen activator type 1 and adipokine levels, and it causes endothelial

The prothrombotic and proinflammatory states may be metabolically interconnected by "plasma plasminogen activator inhibitor (PAI)-1 and fibrinogen", "in response to a high-

Atherogenic dyslipidemia represents the combination of raised triglycerides (TG), low HDL-C, elevated apolipoprotein B (ApoB), small dense LDL and small HDL particles, and so on; all are independently atherogenic and are observed in type 2 DM and DMS. Low HDL-c is considered to be "a particularly key risk factor for CVD in both non-diabetic and diabetic individuals", "an independent contributor to CVD, in both men and women". Low HDL-c

"purely correlative, not causative, and do not imply a mechanistic action" (table 2)

placed among the parameters used in "Framingham Risk Score".

dysfunction (Grundy et al., 2004; Ridker et al., 2004).

**4.3 The prothrombotic and proinflammatory states** 

cytokine state" (Grundy et al., 2004)

**4.4 Atherogenic dyslipidemia** 

In conclusion, central obesity:

including insulin resistance

2001; Zimmet et al., 2001).

atherogenic effect (Fonseca, 2005).

including C-reactive protein (CRP).

**4.2 Inflammation** 

others.

#### **4.1 Central obesity**

It has been demonstrated that the central obesity is by far the most prevalent form of the MS and a major component of the MS. Reaven points out that insulin resistance does not cause obesity; rather, obesity causes insulin resistance and has a key role in the pathophysiology of metabolic disorders; but insulin resistance also occurs in 10% to 15% of people who are not overweight. Basically, abdominal (visceral) obesity:


These affirmations are sustained by the following arguments:


The distribution of adipose tissue appears to affect its role in metabolic syndrome. While visceral, intra-abdominal fat correlates with inflammation, subcutaneous fat does not. Only abdominal fat produces potentially harmful levels of cytokines (tumor necrosis factor, adiponectin, leptin, resistin, and plasminogen activator inhibitor). Only visceral fat accumulation and insulin-resistance have been associated with a cluster of dyslipidaemic features (i.e., elevated plasma triglyceride, increased very low density lipoprotein /VLDL, presence of small dense LDL particles, with decreased of HDL-cholesterol, and so on).

It has been demonstrated that the central obesity is by far the most prevalent form of the MS and a major component of the MS. Reaven points out that insulin resistance does not cause obesity; rather, obesity causes insulin resistance and has a key role in the pathophysiology of metabolic disorders; but insulin resistance also occurs in 10% to 15% of people who are









The distribution of adipose tissue appears to affect its role in metabolic syndrome. While visceral, intra-abdominal fat correlates with inflammation, subcutaneous fat does not. Only abdominal fat produces potentially harmful levels of cytokines (tumor necrosis factor, adiponectin, leptin, resistin, and plasminogen activator inhibitor). Only visceral fat accumulation and insulin-resistance have been associated with a cluster of dyslipidaemic features (i.e., elevated plasma triglyceride, increased very low density lipoprotein /VLDL, presence of small dense LDL particles, with decreased of HDL-cholesterol, and so on).

**4.1 Central obesity** 

not overweight. Basically, abdominal (visceral) obesity:



other MS components, including insulin resistance,

These affirmations are sustained by the following arguments:

1, etc.), which contributes to a prothrombotic state;

to be a risk factor for developing and worsening MS;

anti diabetic, anti-atherosclerotic and anti-inflammatory functions;

and inflammation, complex phenomenon representing DMS ;



protective function;

2004a, 2004b).


In conclusion, central obesity:


The increased flow of free fatty acids through the liver leads to accelerated synthesis of VLDL-C, hypertriglyceridemia, endothelial dysfunction, and vasoconstriction, leading to an increase in blood pressure; also, through this mechanism, insulin resistance may exert an atherogenic effect (Fonseca, 2005).

#### **4.2 Inflammation**

A number of markers of systemic inflammation, including C-reactive protein, are often increased, as are fibrinogen, interleukin 6 (IL–6), tumor necrosis factor-alpha (TNF+), and others.

CVD and diabetes are associated with elevated levels of inflammatory biomarkers, including C-reactive protein (CRP).

At the same time, CRP is the best-studied inflammatory marker of atherothrombotic risk, placed among the parameters used in "Framingham Risk Score".

C-reactive protein is present in the MS; its plasma levels increase with the number of metabolic risk factors, and also with other inflammatory markers; these associations are "purely correlative, not causative, and do not imply a mechanistic action" (table 2)

The mechanisms that lead to the increase of CRP are complex and only partially understood; we take this opportunity to remind that only excessive fatty tissue releases inflammatory cytokines and determines higher CRP levels; adipose cell enlargement and infiltration of macrophages into adipose tissue will lead to the release of proinflammatory cytokines and promote insulin resistance. It increases the thrombogenicity of circulating blood, in part by raising plasminogen activator type 1 and adipokine levels, and it causes endothelial dysfunction (Grundy et al., 2004; Ridker et al., 2004).

#### **4.3 The prothrombotic and proinflammatory states**

The prothrombotic and proinflammatory states may be metabolically interconnected by "plasma plasminogen activator inhibitor (PAI)-1 and fibrinogen", "in response to a highcytokine state" (Grundy et al., 2004)

#### **4.4 Atherogenic dyslipidemia**

Atherogenic dyslipidemia represents the combination of raised triglycerides (TG), low HDL-C, elevated apolipoprotein B (ApoB), small dense LDL and small HDL particles, and so on; all are independently atherogenic and are observed in type 2 DM and DMS. Low HDL-c is considered to be "a particularly key risk factor for CVD in both non-diabetic and diabetic individuals", "an independent contributor to CVD, in both men and women". Low HDL-c

Dysmetabolic Syndrome 233

…"it remains to be demonstrated that the impact of the syndrome exceeds that of the sum of

David Nathan (Massachusetts General Hospital, Boston) described the situation as "a


**Hypertriglyceridaemic waist phenotype** estimated prevalence: 20-25% - Atherogenic metabolic triad (fasting hyperinsulinaemia, elevated apolipoprotein B levels

Table 6. Hypertriglyceridaemic waist phenotupe association with features of metabolic




Even if now we assign to DMS multiple metabolic risk factors and/or a complex of interrelated risk factors for CVD and DM, although we know that DMS substantially increases the risk of CHD, we do not know which of its defining characteristics (insulin resistance/ hyperinsulinaemia, small LDL particles, reduced adiponectin levels, increased CRP, etc.) are "critical therapeutic targets for the optimal management of CHD and type 2 diabetic risk" (Hu et al., 2001). We need to globally asses the individual risk of these patients

We note some compelling conclusions regarding the current global state of DMS:


multiple metabolic risk factors for CVD and DM. - The metabolic syndrome is not an absolute risk indicator.

many primary populations"(Alberti et al., 2009).


its individual components" and so on (Kahn et al., 2005).

firestorm about nothing" and adds about DMS:

conditions had a single underling cause".

and increased proportion of small LDL particles - Elevated total cholesterol/HDL cholesterol ratio


– Increased risk of type 2 diabetes – Increased cardiovascular risk

elevated plasma glucose.

– Glucose intolerance

syndrome


and high TG levels are frequently found in insulin resistance, with or without type 2 diabetes, and both are risk factors for CVD (table 3) (Robins et al., 2003; Brunzell & Ayyobi, 2003).

#### **5. Diagnosis of DMS**

Although the present review does not have as purpose the detailed presentation of the way in which the diagnosis of DMS is made, we consider that few remarks are necessary in order to manage it correctly.

The literature of the past years regarding the management of this concept agrees upon the necessity of a team research, especially in the population of high risk, on national criteria, with subsequent establishment of a realistic management programme.

S Julius (and not only him) supports this point of view because "the clinical spectrum of the Metabolic Syndrome is variable and influenced by gender, ethnicity, and genetic susceptibility", especially in arterial hypertension, and even in borderline hypertension (Julius & Nesbitt, 1996). Paul Zimmet, from Australia, draws attention to the need of a "careful definition and management of the "tick test" in the DMS", which must rely on "evidence-based criteria"; he adds: "Tackling diabetes and obesity is likely to be the single most important challenge for Australia's public health in the 21st century. It is a battle that we can and must win!" (Eckel et al., 2005; Barr et al., 2006). More recently, a Joint Scientific Statement was necessary "in an attempt to unify criteria", to underline the fact that DMS is a "multifaceted, but distinct entity", and that "further progress depends in part on interdisciplinary dissemination of knowledge" (Table 6); moreover, "various diagnostic criteria have been proposed by different organizations over the past decade", which often led to confusion.

Simmons RK and collaborators evaluated the utility of metabolic syndrome from several points of view: pathophysiology, epidemiology, clinical work and public health, but also educational; the authors conclude that they agree with this "concept that focuses attention on complex multifactorial health problems", but they accept it only as "a diagnostic or management tool" with a limited practical utility (Simmons et al., 2010).

To gave up the widespread term of DMS that Sindrom metabolic, which is used for many years for this "cluster of risk factors", " is an unrealistic act, even impossible to fulfill, because the term is rooted in the medical literature"; DMS is: "a heterogeneous entity, composed of abnormal situations involved in the production of a metabolic imbalance with common metabolic links, but also with important differences in the etiopathogenesis of its components" (Balta, 2010).

Despite numerous criticisms of the concept by many authors, Kahn and collegues express "our recommendations to clinicians":

"All CVD risk factors should be individually and aggressively treated"

"Pharmacological therapy to reduce insulin resistance will be beneficial to patients with the metabolic syndrome"

"An aggressive research agenda to identify the underlying cause(s) of the CVD risk factor clustering"

and high TG levels are frequently found in insulin resistance, with or without type 2 diabetes, and both are risk factors for CVD (table 3) (Robins et al., 2003; Brunzell & Ayyobi,

Although the present review does not have as purpose the detailed presentation of the way in which the diagnosis of DMS is made, we consider that few remarks are necessary in order

The literature of the past years regarding the management of this concept agrees upon the necessity of a team research, especially in the population of high risk, on national criteria,

S Julius (and not only him) supports this point of view because "the clinical spectrum of the Metabolic Syndrome is variable and influenced by gender, ethnicity, and genetic susceptibility", especially in arterial hypertension, and even in borderline hypertension (Julius & Nesbitt, 1996). Paul Zimmet, from Australia, draws attention to the need of a "careful definition and management of the "tick test" in the DMS", which must rely on "evidence-based criteria"; he adds: "Tackling diabetes and obesity is likely to be the single most important challenge for Australia's public health in the 21st century. It is a battle that we can and must win!" (Eckel et al., 2005; Barr et al., 2006). More recently, a Joint Scientific Statement was necessary "in an attempt to unify criteria", to underline the fact that DMS is a "multifaceted, but distinct entity", and that "further progress depends in part on interdisciplinary dissemination of knowledge" (Table 6); moreover, "various diagnostic criteria have been proposed by different organizations over the past decade", which often

Simmons RK and collaborators evaluated the utility of metabolic syndrome from several points of view: pathophysiology, epidemiology, clinical work and public health, but also educational; the authors conclude that they agree with this "concept that focuses attention on complex multifactorial health problems", but they accept it only as "a diagnostic or

To gave up the widespread term of DMS that Sindrom metabolic, which is used for many years for this "cluster of risk factors", " is an unrealistic act, even impossible to fulfill, because the term is rooted in the medical literature"; DMS is: "a heterogeneous entity, composed of abnormal situations involved in the production of a metabolic imbalance with common metabolic links, but also with important differences in the etiopathogenesis of its

Despite numerous criticisms of the concept by many authors, Kahn and collegues express

"Pharmacological therapy to reduce insulin resistance will be beneficial to patients with the

"An aggressive research agenda to identify the underlying cause(s) of the CVD risk factor

with subsequent establishment of a realistic management programme.

management tool" with a limited practical utility (Simmons et al., 2010).

"All CVD risk factors should be individually and aggressively treated"

2003).

**5. Diagnosis of DMS** 

to manage it correctly.

led to confusion.

components" (Balta, 2010).

metabolic syndrome"

clustering"

"our recommendations to clinicians":

…"it remains to be demonstrated that the impact of the syndrome exceeds that of the sum of its individual components" and so on (Kahn et al., 2005).

David Nathan (Massachusetts General Hospital, Boston) described the situation as "a firestorm about nothing" and adds about DMS:


### **Hypertriglyceridaemic waist phenotype**

estimated prevalence: 20-25%





Table 6. Hypertriglyceridaemic waist phenotupe association with features of metabolic syndrome

We note some compelling conclusions regarding the current global state of DMS:


Even if now we assign to DMS multiple metabolic risk factors and/or a complex of interrelated risk factors for CVD and DM, although we know that DMS substantially increases the risk of CHD, we do not know which of its defining characteristics (insulin resistance/ hyperinsulinaemia, small LDL particles, reduced adiponectin levels, increased CRP, etc.) are "critical therapeutic targets for the optimal management of CHD and type 2 diabetic risk" (Hu et al., 2001). We need to globally asses the individual risk of these patients

Dysmetabolic Syndrome 235

There are multiple laboratory tests and many of them are vital (glycaemic profile, lipids, inflammatory tests); we must not forget about exploring the thyroid function or investigating other systems (cerebrovascular, hepatic or renal). The new guidelines recognize the importance of elevated triglycerides and of reduced HDL cholesterol concentrations as useful lipid markers of the presence of an atherogenic "dysmetabolic" milieu. In DMS, the lipidic profile can vary, and so can the therapeutic options; they can be




Interventions which can improve insulin sensitivity, especially lifestyle modifications, weight loss, Mediterranean diet, and increased physical activity, remain the elements of choice in DMS, because of their favorable impact on DMS components. (Hu et al., 2001)

As an example for the complexity of what we call DMS, Zeller, Steg et al. insist that "fasting hyperglycaemia is the most important risk factor for development of severe heart failure in patients with metabolic syndrome"; these situations are associated with "a higher inhospital case fatality rate"; strict control of glycemic levels in patients with DMS with or without a critical state is recommended by many authors and by the algorithm of the American Diabetes Association and the European Association for the Study of Diabetes

Similarly, for a correct and complete diagnosis, Enzo Bonora et al. proposed a short list of novel (non-traditional) risk factors in order to emphasise the fact that, in the "metabolic syndrome approach", it is necessary to prove the "existence of underlying pathogenic disorders of the cluster, i.e. central obesity and insulin resistance"; to this end, the authors present a "systematization of biomarkers" that are useful in DMS and in clinical trials:



The authors consider that a better diagnosis and treatment of "several classic and ancillary components of the metabolic syndrome" will accomplish "a substantial reduction of the


dysfunction (e.g. E-selectin, intercellular adhesion molecule-1, ICAM-1,


prescribed together with dietary changes or sustained physical effort.

preferred to reduce the risk of a first or recurrent CHD event;

preferred as the first therapeutic option;

"atherogenic dyslipidemia" (Sacks, 2002).

(2006) (Zeller et al. 2005; Nathan et al., 2006).

erythrocyte sedimentation rate, ESR),


cardiovascular risk" (Bonora et al., 2003).

Here are a few patterns:

in order to optimally manage the dyslipidaemic state in this high-risk population. In the context of the current knowledge regarding the DMS, the patients diagnosed with arterial hypertension, DLP, or hyperglycemia will be actively investigated for DMS. We must insist upon the realisation of a complete diagnosis, specifying the risk score for CV and/or metabolic disorders that we found (table 5, 6).

Given the susceptibility to many other pathological conditions, we must use all clinical and paraclinical methods needed in order to establish a positive and differential diagnosis (ex. polycystic ovary syndrome, fatty liver, cholesterol gallstones, asthma, sleep disturbances, some forms of cancer), especially if they are sugested by the familial genetic aspect, by the signs and simptoms and so on. The mesurement that are needed (weight, height, waist circumference or waist/hip ratio) for initial diagnosis of DMS and for follow-up will be taken in a proper manner, following the current guidelines (by gender, etnicity, etc.). It is estimated that the introduction of "waist circumference rather than the body mass index has been "a major conceptual leap", because it recognizes the much greater causal role of abdominal obesity than general obesity. (www.metabolicsyndromeinstitut/information /screeningdiagnosis/procedures-for-the-measurement-of-the-waist-circumference.php).

The canadian researchers Lemieux I, Pascot A, Couillard C et al., introduce a new notion, namely: "HYPERTRIGLYCERIDEMIC WAIST": a marker of the atherogenic metabolic triad: hyperinsulinemia, hyperapolipoprotein B, and small, dense LDL, in men. We note the depth of this interesting concept, its validation in a trial (Quebec Health Survey) and its practical usefulness, at least from two points of view:


"The hypertriglyceridaemic waist phenotype significantly increased the odds of finding CAD in men, whereas impaired fasting glucose was not predictive of CAD in the absence of hypertriglyceridaemic waist":


Identified by the simultaneous measurement of waist circumference and fasting triglyceride levels, this approach can be "a simple and inexpensive marker to better identify individuals at high risk of CVD and/or type-2 diabetes and to evaluate CHD risk in individuals with abdominal obesity"(Lemieux et al., 2002; Blackburn et al. 2003).

in order to optimally manage the dyslipidaemic state in this high-risk population. In the context of the current knowledge regarding the DMS, the patients diagnosed with arterial hypertension, DLP, or hyperglycemia will be actively investigated for DMS. We must insist upon the realisation of a complete diagnosis, specifying the risk score for CV and/or

Given the susceptibility to many other pathological conditions, we must use all clinical and paraclinical methods needed in order to establish a positive and differential diagnosis (ex. polycystic ovary syndrome, fatty liver, cholesterol gallstones, asthma, sleep disturbances, some forms of cancer), especially if they are sugested by the familial genetic aspect, by the signs and simptoms and so on. The mesurement that are needed (weight, height, waist circumference or waist/hip ratio) for initial diagnosis of DMS and for follow-up will be taken in a proper manner, following the current guidelines (by gender, etnicity, etc.). It is estimated that the introduction of "waist circumference rather than the body mass index has been "a major conceptual leap", because it recognizes the much greater causal role of abdominal obesity than general obesity. (www.metabolicsyndromeinstitut/information /screeningdiagnosis/procedures-for-the-measurement-of-the-waist-circumference.php).

The canadian researchers Lemieux I, Pascot A, Couillard C et al., introduce a new notion, namely: "HYPERTRIGLYCERIDEMIC WAIST": a marker of the atherogenic metabolic triad: hyperinsulinemia, hyperapolipoprotein B, and small, dense LDL, in men. We note the depth



"The hypertriglyceridaemic waist phenotype significantly increased the odds of finding CAD in men, whereas impaired fasting glucose was not predictive of CAD in the absence of




Identified by the simultaneous measurement of waist circumference and fasting triglyceride levels, this approach can be "a simple and inexpensive marker to better identify individuals at high risk of CVD and/or type-2 diabetes and to evaluate CHD risk in individuals with

bec Health Survey) and its practical

metabolic disorders that we found (table 5, 6).

of this interesting concept, its validation in a trial (Que-

hyperinsulinaemia and additional risk factors.

abdominal obesity"(Lemieux et al., 2002; Blackburn et al. 2003).

usefulness, at least from two points of view:

impaired fasting glucose state;

tomography.

phenotype";

hypertriglyceridaemic waist":

individuals at high risk";

There are multiple laboratory tests and many of them are vital (glycaemic profile, lipids, inflammatory tests); we must not forget about exploring the thyroid function or investigating other systems (cerebrovascular, hepatic or renal). The new guidelines recognize the importance of elevated triglycerides and of reduced HDL cholesterol concentrations as useful lipid markers of the presence of an atherogenic "dysmetabolic" milieu. In DMS, the lipidic profile can vary, and so can the therapeutic options; they can be prescribed together with dietary changes or sustained physical effort.

Here are a few patterns:


Interventions which can improve insulin sensitivity, especially lifestyle modifications, weight loss, Mediterranean diet, and increased physical activity, remain the elements of choice in DMS, because of their favorable impact on DMS components. (Hu et al., 2001)

As an example for the complexity of what we call DMS, Zeller, Steg et al. insist that "fasting hyperglycaemia is the most important risk factor for development of severe heart failure in patients with metabolic syndrome"; these situations are associated with "a higher inhospital case fatality rate"; strict control of glycemic levels in patients with DMS with or without a critical state is recommended by many authors and by the algorithm of the American Diabetes Association and the European Association for the Study of Diabetes (2006) (Zeller et al. 2005; Nathan et al., 2006).

Similarly, for a correct and complete diagnosis, Enzo Bonora et al. proposed a short list of novel (non-traditional) risk factors in order to emphasise the fact that, in the "metabolic syndrome approach", it is necessary to prove the "existence of underlying pathogenic disorders of the cluster, i.e. central obesity and insulin resistance"; to this end, the authors present a "systematization of biomarkers" that are useful in DMS and in clinical trials:


The authors consider that a better diagnosis and treatment of "several classic and ancillary components of the metabolic syndrome" will accomplish "a substantial reduction of the cardiovascular risk" (Bonora et al., 2003).

Dysmetabolic Syndrome 237

Because DMS is associated with dramatically higher risk of DCV, diabetes, and so on, its

According to the DMS concept and the recognition of its defining combinations (hypertriglyceridemia, hyperglycemia, hypertension, low HDL-C level, and greater waist



These elements represent the foundation of DMS complex management. (Ding et al., 2010).

Primary prevention consists in a healthy life style, smoking cessation, caloric restriction, and a modified daily diet. A moderate increase of physical activity and a 7-10% weight loss over

Low caloric diet and moderate but sustained exertion are considered "the most important

If we analyze each individual component of DMS reporting it to the DMS diagnosis, a greater chance of progression was observed, especially if we consider that hyperglycemia, hypertension, and low HDL-C level are the main risk factors; consequently, these risk factors represent a potential target for active and individual cardiovascular prevention in

Secondary prevention is addressed to patients for whom lifestyle change is not enough and who are considered to be at high risk for CVD; these patients will receive medical treatment

All these will act "as a whole" on the basic mechanisms of DMS in order to reduce the evolutionary impact of all risk factors and all "metabolic and cardiovascular consequences",

Separate, incomplete or inconsistent approach of individual components of DMS is to be avoided; the emphasis will be laid on sustained reduction of individual risk, especially in patients with several DMS components and complications; only, in this way, we can really reduce "the overall impact on CVD and DM risk". (Lindström et al., 2003; Tuomilehto et al.

After the complete diagnosis is established, the management of DMS must be more comprehensive and more aggressive compared to other clinical situations, in order to reduce the risk for CVD and DM; thereby, a complete evaluation of cardiovascular risk

according to present guidelines of medical practice is of outmost importance.

recognition and follow-up have become a major issue in preventive cardiology.

components are present, their duration, and existing complications.

initial steps in treating metabolic syndrome" (Ford et al., 2002).

**6. Management of DMS** 

condition;

**6.1 Primary prevention** 

6-12 months are also indicated.

these patients (Després et al., 2008).

**6.2 Secondary prevention** 

together with lifestyle changes.

on short and long-term evolution.

2001).

circumference /adiposity) there are:

Matsuzawa Y, Funahashi T, and many others emphasize the importance of adiponectin; "one of these adipocytokines which we identified in human adipose tissue"; it circulates abundantly in human plasma, and has both anti-atherogenic and anti-diabetic effects. In addition, a series of clinical and experimental studies suggest that adiponectin may become a novel 'hot' marker of the Metabolic Syndrome (Matsuzawa et al., 2004).

Based on complicated lab research, Kumada M, Kihara S, Ouchi N et al. suggest the results above and conclude that "plasma adiponectin may become a novel biomarker for atherosclerotic vascular diseases, as well as plasma cholesterol and glucose levels" (Kumada et al., 2004).

The significance of adiponectin as "a negative risk for diabetes and its dual protective capacity", both against diabetes and atherosclerosis, makes the subject of much prestigious research; we are talking about "adipocytokines", considering the remark that "reduction of adiponectin may facilitate coronary plaque rupture" but, at the same time, adiponectin "suppresses both the atherosclerotic process and the production of an inflammatory cytokine," and so on.

Based on all these observations, it is obvious why "body weight reduction, physical exercise, and lifestyle changes" can raise plasma adiponectin levels. In addition, agents such as "thiazolidinediones, renin-angiotensin system blockers and glimepiride" have been reported to increase adiponectin concentrations (Weyer et al., 2001).

Wiecek et al. reported that plasma adiponectin levels are negatively correlated with mean blood pressure (BP) in patients with essential hypertension (Adamczak et al., 2003).

Some well known Japanese authors point out the association of obesity with increased risk for breast and endometrial cancers; also, high serum adiponectin levels are associated with an increased risk for breast cancer (Miyoshi et al., 2003).

Imaging tests are not routinely indicated, but they can be performed when previous examinations suggest cardiovascular complications.

Adverse clinical consequences and/or target organ damage appear during long term evolution of DMS; all these elements will be periodically investigated and quantified by specific methods. The most frequent example is represented by arterial hypertension, which evolves with very important target organ damage (left ventricular hypertrophy, progressive peripheral arterial disease, and renal dysfunction).

Using the NCEP/ATP III definition, Mulè et al. investigated the efect of SXM on markers of target organ damage, and they have demonstrated that these lesions can explain "the enhanced cardiovascular risk associated with the Metabolic Syndrome". The authors also conclude that there must be a global evaluation of the "influence of the SXM on some cardiac, renal, and retinal markers of target organ impairment" (Mulè et al., 2005).

We must not overlook the complications associated with DMS, representing short and longterm prognosis factors; there can be cardiovascular (coronary heart disease, atrial fibrillation, heart failure, aortic stenosis , ischemic stroke, and so on) or extra cardiac complications (nonalcoholic fatty liver disease, obstructive sleep apnea, breast cancer, cancer of the colon, gallbladder, kidney, prostate gland, and so on).

Matsuzawa Y, Funahashi T, and many others emphasize the importance of adiponectin; "one of these adipocytokines which we identified in human adipose tissue"; it circulates abundantly in human plasma, and has both anti-atherogenic and anti-diabetic effects. In addition, a series of clinical and experimental studies suggest that adiponectin may become

Based on complicated lab research, Kumada M, Kihara S, Ouchi N et al. suggest the results above and conclude that "plasma adiponectin may become a novel biomarker for atherosclerotic vascular diseases, as well as plasma cholesterol and glucose levels" (Kumada

The significance of adiponectin as "a negative risk for diabetes and its dual protective capacity", both against diabetes and atherosclerosis, makes the subject of much prestigious research; we are talking about "adipocytokines", considering the remark that "reduction of adiponectin may facilitate coronary plaque rupture" but, at the same time, adiponectin "suppresses both the atherosclerotic process and the production of an inflammatory

Based on all these observations, it is obvious why "body weight reduction, physical exercise, and lifestyle changes" can raise plasma adiponectin levels. In addition, agents such as "thiazolidinediones, renin-angiotensin system blockers and glimepiride" have been

Wiecek et al. reported that plasma adiponectin levels are negatively correlated with mean

Some well known Japanese authors point out the association of obesity with increased risk for breast and endometrial cancers; also, high serum adiponectin levels are associated with

Imaging tests are not routinely indicated, but they can be performed when previous

Adverse clinical consequences and/or target organ damage appear during long term evolution of DMS; all these elements will be periodically investigated and quantified by specific methods. The most frequent example is represented by arterial hypertension, which evolves with very important target organ damage (left ventricular hypertrophy, progressive

Using the NCEP/ATP III definition, Mulè et al. investigated the efect of SXM on markers of target organ damage, and they have demonstrated that these lesions can explain "the enhanced cardiovascular risk associated with the Metabolic Syndrome". The authors also conclude that there must be a global evaluation of the "influence of the SXM on some

We must not overlook the complications associated with DMS, representing short and longterm prognosis factors; there can be cardiovascular (coronary heart disease, atrial fibrillation, heart failure, aortic stenosis , ischemic stroke, and so on) or extra cardiac complications (nonalcoholic fatty liver disease, obstructive sleep apnea, breast cancer, cancer

cardiac, renal, and retinal markers of target organ impairment" (Mulè et al., 2005).

blood pressure (BP) in patients with essential hypertension (Adamczak et al., 2003).

a novel 'hot' marker of the Metabolic Syndrome (Matsuzawa et al., 2004).

reported to increase adiponectin concentrations (Weyer et al., 2001).

an increased risk for breast cancer (Miyoshi et al., 2003).

examinations suggest cardiovascular complications.

peripheral arterial disease, and renal dysfunction).

of the colon, gallbladder, kidney, prostate gland, and so on).

et al., 2004).

cytokine," and so on.

#### **6. Management of DMS**

Because DMS is associated with dramatically higher risk of DCV, diabetes, and so on, its recognition and follow-up have become a major issue in preventive cardiology.

According to the DMS concept and the recognition of its defining combinations (hypertriglyceridemia, hyperglycemia, hypertension, low HDL-C level, and greater waist circumference /adiposity) there are:


These elements represent the foundation of DMS complex management. (Ding et al., 2010).

#### **6.1 Primary prevention**

Primary prevention consists in a healthy life style, smoking cessation, caloric restriction, and a modified daily diet. A moderate increase of physical activity and a 7-10% weight loss over 6-12 months are also indicated.

Low caloric diet and moderate but sustained exertion are considered "the most important initial steps in treating metabolic syndrome" (Ford et al., 2002).

If we analyze each individual component of DMS reporting it to the DMS diagnosis, a greater chance of progression was observed, especially if we consider that hyperglycemia, hypertension, and low HDL-C level are the main risk factors; consequently, these risk factors represent a potential target for active and individual cardiovascular prevention in these patients (Després et al., 2008).

#### **6.2 Secondary prevention**

Secondary prevention is addressed to patients for whom lifestyle change is not enough and who are considered to be at high risk for CVD; these patients will receive medical treatment together with lifestyle changes.

All these will act "as a whole" on the basic mechanisms of DMS in order to reduce the evolutionary impact of all risk factors and all "metabolic and cardiovascular consequences", on short and long-term evolution.

Separate, incomplete or inconsistent approach of individual components of DMS is to be avoided; the emphasis will be laid on sustained reduction of individual risk, especially in patients with several DMS components and complications; only, in this way, we can really reduce "the overall impact on CVD and DM risk". (Lindström et al., 2003; Tuomilehto et al. 2001).

After the complete diagnosis is established, the management of DMS must be more comprehensive and more aggressive compared to other clinical situations, in order to reduce the risk for CVD and DM; thereby, a complete evaluation of cardiovascular risk according to present guidelines of medical practice is of outmost importance.

Dysmetabolic Syndrome 239


3. reduce LDL-c levels (elevated levels represent a high risk in the metabolic syndrome). Fibrates (PPAR alpha agonists) improve all components of atherogenic dyslipidaemia (elevated triglyceride and low HDL-C levels), especially in overweight patients, and reduce

For patients with elevated triglyceride levels, the addition of omega-3 fatty acids is likely to

Statins are administered in order to reduce all apoB-containing lipoproteins and to achieve ATP III goals for LDL-cholesterol, as well as for non-HDL-Cholesterol; the multiple benefits are confirmed through many clinical trials "at all indicated ranges", the pleiotropic and

Management of reduced high-density lipoprotein cholesterol (HDL-C) remains controversial, not yet having a specific treatment; some measures have proved a positive influence: dietary changes, sustained physical efort, some statins (ex. Rosuvatatin), etc.

The latest guidelines insist on LDL-cholesterol being "the primary target of treatment by adequate use of statins"; although high levels of LDL-cholesterol are not necessarily associated with DMS, they will be properly quantified and treated, for reducing the

Fibric-acid derivatives, bile acid sequestrants, and ezetimibe may be useful in patients who have not achieved target LDL with statin therapy or cannot tolerate statins (Eckel et al., 2005; Alberti et al., 2009; Robins et al., 2003; Heart Protection Study Collaborative Group, 2003; The Task Force for the management of dyslipidaemias of the European Society of

Hypertension remains a clinical target according to present guidelines; regular blood pressure screening, lifestyle modification, and drug therapy are recommended. The focus must be on clinical situations associated with target organ damage, diabetes, or renal complications. Hypertension and diabetes, both components of metabolic syndrome, are known to be associated with renal dysfunction; oxidative stress and inflammation mediated by renin-angiotensin system activation are the most frequently involved mechanisms. Other mechanisms acting singly or in combination, linking obesity to chronic kidney disease, have been proposed: renal adaptation to increased body mass, with an increased excretory load,

Microalbuminuria, however, was more common in subjects who had a constellation of all 3 components of metabolic syndrome than in those without. Presently, microalbuminuria is an early marker of renal dysfunction due to hypertension, predicts the onset of kidney

Cardiology (ESC) and the European Atherosclerosis Society 2011).

sodium retention, insulin resistance, renal lipotoxicity, etc. (Praga, 2002).

inflammatory agents (Antithrombotic Trialists' Collaboration, 2002).

1. lower TG (as well as lowering ApoB and non-HDL cholesterol),

In dyslipidemia, primary aims for therapy are:

the risk for cardiovascular disease in DMS,

produce added benefit (according to clinical trials).

metabolic effects being an "undisputed reality".

2. raise HDL-c levels,

cardiovascular risk.

Framingham risk scoring (or other risk scores, although they include different components) will be used to estimate "10-year atherosclerotic disease risk" and may guide the use of medication therapy (Spellman & Chemitiganti, 2010; Nicholls et al., 2007).

#### **6.3 Medical care**

The initial management of metabolic syndrome involves lifestyle modifications (changes in diet and exercise habits).

The choice of drug and dose should be individualized to the patient and titrated to achieve guideline-recommended goals.

Diets that promote the consumption of fruits, vegetables, and low-fat dairy products ("DASH-style diet") help lower blood pressure and may lower risk of stroke. The consumption of products with high-glycemic-index will be avoided.

Increasing physical activity is associated with a reduction in the risk of stroke, at least 30 minutes of moderate intensity activity on a daily basis, maintaining long-term adherence.

Weight reduction among overweight and obese persons is recommended to reduce blood pressure and risk of stroke.

Bianchi C et al. stipulates the basics of food diet for prevention and treatment of DMS:


The medical care is represented by "a multi-drug treatment", in order to reduce morbidity and prevent DMS complications; the treatment must be with metabolic and glucidic neutrality, with respect to the accompanying disturbances of the DMS":


Framingham risk scoring (or other risk scores, although they include different components) will be used to estimate "10-year atherosclerotic disease risk" and may guide the use of

The initial management of metabolic syndrome involves lifestyle modifications (changes in

The choice of drug and dose should be individualized to the patient and titrated to achieve

Diets that promote the consumption of fruits, vegetables, and low-fat dairy products ("DASH-style diet") help lower blood pressure and may lower risk of stroke. The

Increasing physical activity is associated with a reduction in the risk of stroke, at least 30 minutes of moderate intensity activity on a daily basis, maintaining long-term adherence. Weight reduction among overweight and obese persons is recommended to reduce blood




The medical care is represented by "a multi-drug treatment", in order to reduce morbidity and prevent DMS complications; the treatment must be with metabolic and glucidic





Bianchi C et al. stipulates the basics of food diet for prevention and treatment of DMS:

further lowered to < 8%, if serum LDL-cholesterol level is increased;

cereals: the most appropriate sources of carbohydrates;

hypertension, T2DM, and obesity (Bianchi et al., 2006).

neutrality, with respect to the accompanying disturbances of the DMS":


Rimonabant (the first inhibitor of CB1 receptors);

medication therapy (Spellman & Chemitiganti, 2010; Nicholls et al., 2007).

consumption of products with high-glycemic-index will be avoided.

**6.3 Medical care** 

diet and exercise habits).

guideline-recommended goals.

pressure and risk of stroke.

diabetes mellitus ";

action;




In dyslipidemia, primary aims for therapy are:


Fibrates (PPAR alpha agonists) improve all components of atherogenic dyslipidaemia (elevated triglyceride and low HDL-C levels), especially in overweight patients, and reduce the risk for cardiovascular disease in DMS,

For patients with elevated triglyceride levels, the addition of omega-3 fatty acids is likely to produce added benefit (according to clinical trials).

Statins are administered in order to reduce all apoB-containing lipoproteins and to achieve ATP III goals for LDL-cholesterol, as well as for non-HDL-Cholesterol; the multiple benefits are confirmed through many clinical trials "at all indicated ranges", the pleiotropic and metabolic effects being an "undisputed reality".

Management of reduced high-density lipoprotein cholesterol (HDL-C) remains controversial, not yet having a specific treatment; some measures have proved a positive influence: dietary changes, sustained physical efort, some statins (ex. Rosuvatatin), etc.

The latest guidelines insist on LDL-cholesterol being "the primary target of treatment by adequate use of statins"; although high levels of LDL-cholesterol are not necessarily associated with DMS, they will be properly quantified and treated, for reducing the cardiovascular risk.

Fibric-acid derivatives, bile acid sequestrants, and ezetimibe may be useful in patients who have not achieved target LDL with statin therapy or cannot tolerate statins (Eckel et al., 2005; Alberti et al., 2009; Robins et al., 2003; Heart Protection Study Collaborative Group, 2003; The Task Force for the management of dyslipidaemias of the European Society of Cardiology (ESC) and the European Atherosclerosis Society 2011).

Hypertension remains a clinical target according to present guidelines; regular blood pressure screening, lifestyle modification, and drug therapy are recommended. The focus must be on clinical situations associated with target organ damage, diabetes, or renal complications. Hypertension and diabetes, both components of metabolic syndrome, are known to be associated with renal dysfunction; oxidative stress and inflammation mediated by renin-angiotensin system activation are the most frequently involved mechanisms. Other mechanisms acting singly or in combination, linking obesity to chronic kidney disease, have been proposed: renal adaptation to increased body mass, with an increased excretory load, sodium retention, insulin resistance, renal lipotoxicity, etc. (Praga, 2002).

Microalbuminuria, however, was more common in subjects who had a constellation of all 3 components of metabolic syndrome than in those without. Presently, microalbuminuria is an early marker of renal dysfunction due to hypertension, predicts the onset of kidney

Dysmetabolic Syndrome 241

Moreover, it is required to follow up the effect/effects of the prescribed treatment, especially in cases of hypertension, DLP and DM, with a periodic assessment of patient adherence to treatment; attention will be directed to prescribe optimal combination

Of course that the management of DMS is impossible to be wholly presented; it is a subject of great practical interest, but its defining elements are found in specialized literature and

With all these questions, different opinions and debates, "the metabolic syndrome serves as a call to action for practitioners to focus more carefully on risk prevention above and beyond

Jean Pierre Després (Institut Universitaire de Cardiologie et de Pneumologie de Québec) presents the DMS as "a work in progress" and adds :« I think it should be redefined as a constellation of metabolic abnormalities associated with visceral fat and insulin resistance;

DMS was and remains an attractive subject in many ways, theoretical and practical, as demonstrated by all of the research so far; it remains a concept with great practical use, a heterogeneous entity based on a metabolic imbalance that has not yet found the best definition, the optimal nosological framing, widely accepted, even if it will reach soon 100

We propose the terminology of CARDIOVASCULAR DISMETABOLIC SYNDROME (CV DMS) which express a metabolic disorder, multifactorial entity that require the participation of several medical specialties (even over 10 specialities) in order to quantify and consolidate the defining elements of cardiometabolic risk and defining appropriate management in real time. In order to reduce the confusion in the medical community, universal agreement on the definition and clinical tools to assess the DMS would be very helpful and eforts for additional international consensus activities have been made; all this research will determine all the "preventive and screening strategies for the dismetabolic syndrome".

Theoreticians and practitioners, laboratory doctors, lipidologists, diabetologists, nutritionists, or hypertensiologists, cardiologists, nephrologists, neurologists, endocrinologists, but also pediatricians, geneticists, family doctors and so on all participate in this complex process, because only teamwork can, indeed, to define actively, to monitor and improve multiple abnormal components of this entity called CARDIOVASCULAR

[1] Adamczak, M,; Wiecek, A. & Funahashi, T. (2003). *Decreased plasma adiponectin concentration in patients with essential hypertension*. Am J Hypertens;16:72-5 [2] Alberti, KG.; Zimmet, P. & Shaw, J. (2006). *Metabolic syndrome-a new world-wide definition.* 

*A Consensus Statement from the International Diabetes Federation*. Diabet Med,

this would simply things and clarify a lot of confusion over this." (Després, 2008)

regimens, respectively those recognized in reducing the CV risk.

guides.

**7. Conclusions** 

years of existence.

**8. References** 

23(5):469-80.

DISMETABOLIC SYNDROME (CV DMS).

traditional …". (Smith, 2006).

disease in diabetic and nondiabetic subjects, and reflects widespread endothelial dysfunction, microvascular damage, and possibly inflammation. (Gerstein et al., 2001; Festa et al., 2000). The precise pathogenetic basis of microalbuminuria in metabolic syndrome is not known; it is, however, possible that microalbuminuria in metabolic syndrome reflects renin-angiotensin system activation and resultant oxidant stress, inflammation, and endothelial injury.

Dzau VJ, Safar ME, amongst many other elite scientists, have shown that hypertension, dyslipidemia, and insulin resistance are associated with renin-angiotensin system activation and generation of large amounts of angiotensin II (Dzau & Safar, 1988; Shinozaki et al., 2004).

Angiotensin converting enzyme inhibitors and angiotensin receptor blockers are useful antihypertensive drugs; some clinical trials suggest that they have advantages over other drugs, in patients with diabetes, and that the risk reduction associated with antihypertensive drugs is "the result of blood pressure lowering per se, and not due to a particular type of drug" (Chobanian et al., 2003).

Diabetes mellitus, recognized as "a true cardiovascular disease", raises particular problems of follow-up and treatment; the periodic screening with the assessment of end-organ complications is required even from the beginning of its evolution;


Medical treatment of hyperglycemia in DMS begins with an insulin-sensitizing agent (ex. metformin), which proved that it can reverse the complications DMS, especially together with fibrates and thiazolidinediones.

Multiple research have shown the possibility that drugs that reduce insulin resistance will delay the onset of type 2 diabetes and will reduce CVD risk, when DMS is present (ex. "Diabetes Prevention Program" (DPP) with metformin, thiazolidinedione in delaying or preventing type 2 diabetes in impaired glucose tolerance (IGT) and insulin resistance) (www.idf.org).

Of course, we should not avoid prescribing aspirin for its well-deserved preventive actions, especially in patients with a high CV risk given by DMS, unless contraindicated.

Many other recommendations are necessary:


disease in diabetic and nondiabetic subjects, and reflects widespread endothelial dysfunction, microvascular damage, and possibly inflammation. (Gerstein et al., 2001; Festa et al., 2000). The precise pathogenetic basis of microalbuminuria in metabolic syndrome is not known; it is, however, possible that microalbuminuria in metabolic syndrome reflects renin-angiotensin system activation and resultant oxidant stress, inflammation, and

Dzau VJ, Safar ME, amongst many other elite scientists, have shown that hypertension, dyslipidemia, and insulin resistance are associated with renin-angiotensin system activation and generation of large amounts of angiotensin II (Dzau & Safar, 1988; Shinozaki et al., 2004). Angiotensin converting enzyme inhibitors and angiotensin receptor blockers are useful antihypertensive drugs; some clinical trials suggest that they have advantages over other drugs, in patients with diabetes, and that the risk reduction associated with antihypertensive drugs is "the result of blood pressure lowering per se, and not due to a

Diabetes mellitus, recognized as "a true cardiovascular disease", raises particular problems of follow-up and treatment; the periodic screening with the assessment of end-organ


Medical treatment of hyperglycemia in DMS begins with an insulin-sensitizing agent (ex. metformin), which proved that it can reverse the complications DMS, especially together

Multiple research have shown the possibility that drugs that reduce insulin resistance will delay the onset of type 2 diabetes and will reduce CVD risk, when DMS is present (ex. "Diabetes Prevention Program" (DPP) with metformin, thiazolidinedione in delaying or preventing type 2 diabetes in impaired glucose tolerance (IGT) and insulin resistance)

Of course, we should not avoid prescribing aspirin for its well-deserved preventive actions,





especially in patients with a high CV risk given by DMS, unless contraindicated.

methods of weight loss have failed, etc., for more than 5 years;

endothelial injury.

particular type of drug" (Chobanian et al., 2003).

with fibrates and thiazolidinediones.

Many other recommendations are necessary:

cardiometabolic risk parameters.

(www.idf.org).

endocrinologist;

prevention of CVD;

complications is required even from the beginning of its evolution;

each of "these pursue matters" (ex. JNC 7, update 2009).

Moreover, it is required to follow up the effect/effects of the prescribed treatment, especially in cases of hypertension, DLP and DM, with a periodic assessment of patient adherence to treatment; attention will be directed to prescribe optimal combination regimens, respectively those recognized in reducing the CV risk.

Of course that the management of DMS is impossible to be wholly presented; it is a subject of great practical interest, but its defining elements are found in specialized literature and guides.

#### **7. Conclusions**

With all these questions, different opinions and debates, "the metabolic syndrome serves as a call to action for practitioners to focus more carefully on risk prevention above and beyond traditional …". (Smith, 2006).

Jean Pierre Després (Institut Universitaire de Cardiologie et de Pneumologie de Québec) presents the DMS as "a work in progress" and adds :« I think it should be redefined as a constellation of metabolic abnormalities associated with visceral fat and insulin resistance; this would simply things and clarify a lot of confusion over this." (Després, 2008)

DMS was and remains an attractive subject in many ways, theoretical and practical, as demonstrated by all of the research so far; it remains a concept with great practical use, a heterogeneous entity based on a metabolic imbalance that has not yet found the best definition, the optimal nosological framing, widely accepted, even if it will reach soon 100 years of existence.

We propose the terminology of CARDIOVASCULAR DISMETABOLIC SYNDROME (CV DMS) which express a metabolic disorder, multifactorial entity that require the participation of several medical specialties (even over 10 specialities) in order to quantify and consolidate the defining elements of cardiometabolic risk and defining appropriate management in real time.

In order to reduce the confusion in the medical community, universal agreement on the definition and clinical tools to assess the DMS would be very helpful and eforts for additional international consensus activities have been made; all this research will determine all the "preventive and screening strategies for the dismetabolic syndrome".

Theoreticians and practitioners, laboratory doctors, lipidologists, diabetologists, nutritionists, or hypertensiologists, cardiologists, nephrologists, neurologists, endocrinologists, but also pediatricians, geneticists, family doctors and so on all participate in this complex process, because only teamwork can, indeed, to define actively, to monitor and improve multiple abnormal components of this entity called CARDIOVASCULAR DISMETABOLIC SYNDROME (CV DMS).

#### **8. References**


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**12** 

*China* 

**The Relationship Between AST/ALT** 

In previous studies, the relationship between aspartate aminotransferase to alanine aminotransferase ratio(AST/ALT ratio) and liver disease has been evaluated. In viral hepatitis, alcoholic liver disease and primary biliary cirrhosis, AST/ALT ratio has been proven to be an indicator of liver cirrhosis.1-3 AST/ALT ratio was a potential value in differentiating nonalcoholic steatohepatitis from alcoholic liver disease.The value < 1 suggestted nonalcoholic steatohepatitis, a ratio of 2 was strongly suggestive of alcoholic

Nonalcoholic fatty liver disease(NAFLD) is the most common cause of elevated liver enzymes and also one of the most common forms of liver disease in the world.5 The AST/ALT ratio of the ultrasound-diagnosed NAFLD patients was lower than controls.6 AST/ALT ratio < 1 was common NAFLD-related feature.7 NAFLD is now considered the hepatic manifestation of the metabolic syndrome. When compared with individuals without NAFLD, individuals with NAFLD had significantly higher fasting glucose, insulin, lowdensity lipoprotein cholesterol, triglycerides, systolic blood pressure and diastolic blood pressure.8 Recently, NAFLD marker the AST/ALT ratio have attracted great interest as

Very little information was available about the association of AST/ALT ratio with metabolic syndrome in Han young adults. Thus, this study evaluated the relationship between

**1. Introduction** 

liver disease.4

potential novel marker of metabolic syndrome.9

AST/ALT ratio and metabolic syndrome in Han young adults.

**Ratio and Metabolic Syndrome in** 

**Han Young Adults – AST/ALT** 

Qiang Lu1, Xiaoli Liu1,

Shuhua Liu2, Changshun Xie3, Yali Liu4 and Chunming Ma1 *1Department of Endocrinology, 2Department of Cardiology, 3Department of Gastroenterology, 4Medical Examination Center, The First Hospital of Qinhuangdao, Qinhuangdao, Hebei Province,* 

**Ratio and Metabolic Syndrome** 


### **The Relationship Between AST/ALT Ratio and Metabolic Syndrome in Han Young Adults – AST/ALT Ratio and Metabolic Syndrome**

Qiang Lu1, Xiaoli Liu1, Shuhua Liu2, Changshun Xie3, Yali Liu4 and Chunming Ma1 *1Department of Endocrinology, 2Department of Cardiology, 3Department of Gastroenterology, 4Medical Examination Center, The First Hospital of Qinhuangdao, Qinhuangdao, Hebei Province, China* 

#### **1. Introduction**

246 Recent Advances in Cardiovascular Risk Factors

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In previous studies, the relationship between aspartate aminotransferase to alanine aminotransferase ratio(AST/ALT ratio) and liver disease has been evaluated. In viral hepatitis, alcoholic liver disease and primary biliary cirrhosis, AST/ALT ratio has been proven to be an indicator of liver cirrhosis.1-3 AST/ALT ratio was a potential value in differentiating nonalcoholic steatohepatitis from alcoholic liver disease.The value < 1 suggestted nonalcoholic steatohepatitis, a ratio of 2 was strongly suggestive of alcoholic liver disease.4

Nonalcoholic fatty liver disease(NAFLD) is the most common cause of elevated liver enzymes and also one of the most common forms of liver disease in the world.5 The AST/ALT ratio of the ultrasound-diagnosed NAFLD patients was lower than controls.6 AST/ALT ratio < 1 was common NAFLD-related feature.7 NAFLD is now considered the hepatic manifestation of the metabolic syndrome. When compared with individuals without NAFLD, individuals with NAFLD had significantly higher fasting glucose, insulin, lowdensity lipoprotein cholesterol, triglycerides, systolic blood pressure and diastolic blood pressure.8 Recently, NAFLD marker the AST/ALT ratio have attracted great interest as potential novel marker of metabolic syndrome.9

Very little information was available about the association of AST/ALT ratio with metabolic syndrome in Han young adults. Thus, this study evaluated the relationship between AST/ALT ratio and metabolic syndrome in Han young adults.

The Relationship Between AST/ALT Ratio and Metabolic

used adjustment for age and sex. The *-*

significance was established at *P*<0.05.

**3.1 Clinical and laboratory characteristics** 

**3.2 AST/ALT ratio and metabolic syndrome** 

**3. Results** 

1 (*P*>0.05).(Table 1)

(*P*<0.05). (Table 2)

**4. Discussion** 

*vs* 1.9%,*-*

Syndrome in Han Young Adults – AST/ALT Ratio and Metabolic Syndrome 249

analysis. Continuous variables were analyzed with covariance adjustment for age and sex. To measure the strength of association between 2 variables, partial correlation analysis was

To examine the association between AST/ALT ratio and metabolic syndrome, multivariate logistic regression was tested. The results of the logistic regression analysis were expressed as odds ratios with 95% confidence intervals(CI). Analyses were performed with the computer software SPSS version 11.5 software(SPSS Inc., Chicago, IL, U.S.A.). Statistical

In this sample, AST/ALT ratio < 1 was detected in 146 subjects (34.4%). Males had a significantly higher frequency of AST/ALT ratio < 1 than females(48.6% *vs* 19.6%,*-*

39.595,*P*=0.000). The age was similar in two groups(20.3±0.8 *vs* 20.3±0.8, *t*=0.057,*P*=0.955). Table 1 showed clinical and laboratory characteristics in the study subjects. After adjustment for age and sex, young adults with AST/ALT ratio < 1 had higher BMI, WC, SBP, DBP, TG, TI and HOMA-IR than subjects with AST/ALT ratio 1. Subjects with AST/ALT ratio < 1 also had significantly lower HDL-C than subjects with AST/ALT ratio 1 (*P*<0.05). The level of FPG was similar between subjects with AST/ALT ratio < 1 and

Associations of AST/ALT ratio with anthropometric and metabolic variables were presented in Table 2. After adjustment for age and sex, AST/ALT ratio showed positive correlations with HDL-C and negative correlations with BMI, WC, TGTI and HOMA-IR

The prevalence of metabolic syndrome was 2.1% and was similar in males and females(2.3%

frequency of abdominal obesity, high triglycerides, elevated blood pressure and metabolic syndrome(*P*<0.05). (Table 3) After adjustment for age, sex and BMI, the frequency of metabolic syndrome among young adults with AST/ALT ratio < 1 was 6.975(95%CI: 1.430

Using Adult Treatment Panel III criteria's definition, we estimated that approximately 2.1% of Han young adults have the metabolic syndrome. The prevalence of metabolic syndrome in this study was lower than previously report from Chinese adults and was similar to Chinese adolescents.11,12 It was because of a positive effect of age on the prevalence of metabolic syndrome.13,14 Abdominal obesity, elevated blood pressure and low HDL-C were common components of the metabolic syndrome in this sample. In the insulin resistance atherosclerosis study, NAFLD marker the AST/ALT ratio predict metabolic syndrome independently of potential confounding variables.15 In this study, we found that the

to 34.019, *P*=0.016) times compared with young adults with AST/ALT ratio 1.

2 = 0.082,*P*= 0.774). Subjects with AST/ALT ratio < 1 had a significantly higher

2 test was used to test for differences in proportions.

2 =

### **2. Materials and methods**

#### **2.1 Subjects**

After obtaining the informed consent from all subjects, a cross-sectional, population-based study was conducted. The study population was determined according to 2-stage cluster sampling. In the first stage, a random sample of universities in Qinhuangdao, Hebei Province, China, were obtained; in the second stage, young adults aged 19 to 24 years, randomly selected from these schools, were invited to participate. In the end, 425 Han young adults(males/females 216/209) participated in 2009. Subjects with evidence of alcohol intake, hepatitis B (hepatitis B surface antigen), hepatitis C (hepatitis C antibody), autoimmune hepatitis (antinuclear antibody and anti-smooth muscle antibody) and drug toxicity were excluded. The study protocol was approved and supervised by the ethical committee of the First Hospital of Qinhuangdao.

#### **2.2 Measurements**

Anthropometric measurements, including height, weight and waist circumference(WC) were performed when subjects were without shoes and in light clothing. Height and weight were measured to the nearest 0.1 cm and 0.1 kg, respectively. Standing height without shoes was measured three times with a stadiometer, and the three measurements were averaged for analysis. Body mass index (BMI) was defined as weight (kg) divided by height (m) squared. Blood pressure was measured three times with a mercury sphygmomanometer while the subjects were seated after 10 min of rest, and the three measurements were averaged for analysis.

After an overnight fast of 10–12h, blood samples were drawn from an antecubital vein in each subjects into vacutainer tubes. Fasting plasma glucose(FPG) concentration was measured using the glucose oxidase method, serum lipids and alanine aminotransferase (ALT) and aspartate alanine aminotransferase (AST) were measured using enzymatic procedures with an autoanalyzer (Hitachi, Tokyo, Japan). Serum true insulin(TI) was measured using enzyme linked immunosorbent assay(ELISA) with model 680 microplate reader(BIO-RAD, America).The ELISA kits were purchased from USCNLIFE company, America. The following equation for homeostasis model assessment of insulin resistance(HOMA-IR) was used: fasting insulin level (/U/ml) x fasting glucose level (mmol/l)/22.5.

#### **2.3 Definition for metabolic syndrome**

Metabolic syndrome was defined as having 3 of the 5 factors with the following cut points: abdominal obesity (waist circumference 90cm in males and 80 cm in females); high triglycerides(1.70mmol/L [150mg/dL]); low high density lipoprotein cholesterol (HDL-C) (<1.03 mmol/L[40mg/dL] for males and <1.30 mmol/L [50 mg/dL] for females); elevated blood pressure (systolic blood pressure135mmHg and/or diastolic blood pressure85mmHg) and impaired fasting glucose(5.6mmol/L[100mg/dL]).10

#### **2.4 Statistical analysis**

Data are expressed as mean ± standard deviation(SD) or medians with interquartile ranges(IQR).When data was not normally distributed, they were ln transformed for analysis. Continuous variables were analyzed with covariance adjustment for age and sex. To measure the strength of association between 2 variables, partial correlation analysis was used adjustment for age and sex. The *-*2 test was used to test for differences in proportions. To examine the association between AST/ALT ratio and metabolic syndrome, multivariate logistic regression was tested. The results of the logistic regression analysis were expressed as odds ratios with 95% confidence intervals(CI). Analyses were performed with the computer software SPSS version 11.5 software(SPSS Inc., Chicago, IL, U.S.A.). Statistical significance was established at *P*<0.05.

#### **3. Results**

248 Recent Advances in Cardiovascular Risk Factors

After obtaining the informed consent from all subjects, a cross-sectional, population-based study was conducted. The study population was determined according to 2-stage cluster sampling. In the first stage, a random sample of universities in Qinhuangdao, Hebei Province, China, were obtained; in the second stage, young adults aged 19 to 24 years, randomly selected from these schools, were invited to participate. In the end, 425 Han young adults(males/females 216/209) participated in 2009. Subjects with evidence of alcohol intake, hepatitis B (hepatitis B surface antigen), hepatitis C (hepatitis C antibody), autoimmune hepatitis (antinuclear antibody and anti-smooth muscle antibody) and drug toxicity were excluded. The study protocol was approved and supervised by the ethical

Anthropometric measurements, including height, weight and waist circumference(WC) were performed when subjects were without shoes and in light clothing. Height and weight were measured to the nearest 0.1 cm and 0.1 kg, respectively. Standing height without shoes was measured three times with a stadiometer, and the three measurements were averaged for analysis. Body mass index (BMI) was defined as weight (kg) divided by height (m) squared. Blood pressure was measured three times with a mercury sphygmomanometer while the subjects were seated after 10 min of rest, and the three measurements were

After an overnight fast of 10–12h, blood samples were drawn from an antecubital vein in each subjects into vacutainer tubes. Fasting plasma glucose(FPG) concentration was measured using the glucose oxidase method, serum lipids and alanine aminotransferase (ALT) and aspartate alanine aminotransferase (AST) were measured using enzymatic procedures with an autoanalyzer (Hitachi, Tokyo, Japan). Serum true insulin(TI) was measured using enzyme linked immunosorbent assay(ELISA) with model 680 microplate reader(BIO-RAD, America).The ELISA kits were purchased from USCNLIFE company, America. The following equation for homeostasis model assessment of insulin resistance(HOMA-IR) was used: fasting

Metabolic syndrome was defined as having 3 of the 5 factors with the following cut points: abdominal obesity (waist circumference 90cm in males and 80 cm in females); high triglycerides(1.70mmol/L [150mg/dL]); low high density lipoprotein cholesterol (HDL-C) (<1.03 mmol/L[40mg/dL] for males and <1.30 mmol/L [50 mg/dL] for females); elevated blood pressure (systolic blood pressure135mmHg and/or diastolic blood

Data are expressed as mean ± standard deviation(SD) or medians with interquartile ranges(IQR).When data was not normally distributed, they were ln transformed for

pressure85mmHg) and impaired fasting glucose(5.6mmol/L[100mg/dL]).10

**2. Materials and methods** 

committee of the First Hospital of Qinhuangdao.

insulin level (/U/ml) x fasting glucose level (mmol/l)/22.5.

**2.3 Definition for metabolic syndrome** 

**2.1 Subjects** 

**2.2 Measurements** 

averaged for analysis.

**2.4 Statistical analysis** 

#### **3.1 Clinical and laboratory characteristics**

In this sample, AST/ALT ratio < 1 was detected in 146 subjects (34.4%). Males had a significantly higher frequency of AST/ALT ratio < 1 than females(48.6% *vs* 19.6%,*-*2 = 39.595,*P*=0.000). The age was similar in two groups(20.3±0.8 *vs* 20.3±0.8, *t*=0.057,*P*=0.955). Table 1 showed clinical and laboratory characteristics in the study subjects. After adjustment for age and sex, young adults with AST/ALT ratio < 1 had higher BMI, WC, SBP, DBP, TG, TI and HOMA-IR than subjects with AST/ALT ratio 1. Subjects with AST/ALT ratio < 1 also had significantly lower HDL-C than subjects with AST/ALT ratio 1 (*P*<0.05). The level of FPG was similar between subjects with AST/ALT ratio < 1 and 1 (*P*>0.05).(Table 1)

Associations of AST/ALT ratio with anthropometric and metabolic variables were presented in Table 2. After adjustment for age and sex, AST/ALT ratio showed positive correlations with HDL-C and negative correlations with BMI, WC, TGTI and HOMA-IR (*P*<0.05). (Table 2)

#### **3.2 AST/ALT ratio and metabolic syndrome**

The prevalence of metabolic syndrome was 2.1% and was similar in males and females(2.3% *vs* 1.9%,*-*2 = 0.082,*P*= 0.774). Subjects with AST/ALT ratio < 1 had a significantly higher frequency of abdominal obesity, high triglycerides, elevated blood pressure and metabolic syndrome(*P*<0.05). (Table 3) After adjustment for age, sex and BMI, the frequency of metabolic syndrome among young adults with AST/ALT ratio < 1 was 6.975(95%CI: 1.430 to 34.019, *P*=0.016) times compared with young adults with AST/ALT ratio 1.

#### **4. Discussion**

Using Adult Treatment Panel III criteria's definition, we estimated that approximately 2.1% of Han young adults have the metabolic syndrome. The prevalence of metabolic syndrome in this study was lower than previously report from Chinese adults and was similar to Chinese adolescents.11,12 It was because of a positive effect of age on the prevalence of metabolic syndrome.13,14 Abdominal obesity, elevated blood pressure and low HDL-C were common components of the metabolic syndrome in this sample. In the insulin resistance atherosclerosis study, NAFLD marker the AST/ALT ratio predict metabolic syndrome independently of potential confounding variables.15 In this study, we found that the

The Relationship Between AST/ALT Ratio and Metabolic

of insulin resistance.

in Han young aduts.

The *-*

low high density lipoprotein

aspartate aminotransferase.

Syndrome in Han Young Adults – AST/ALT Ratio and Metabolic Syndrome 251

variable *r P\* r P\*\**

BMI(kg/m2) -0.296 0.000 -0.281 0.000

WC(cm) -0.310 0.000 -0.264 0.000

SBP(mmHg) -0.141 0.004 -0.072 0.134

DBP(mmHg) -0.102 0.036 -0.048 0.321

FPG(mmol/L) -0.051 0.295 -0.039 0.413

TG(mmol/L) -0.134 0.006 -0.134 0.006

HDL-C(mmol/L) 0.174 0.000 0.125 0.010

TI( uU/ml) -0.154 0.001 -0.118 0.015

HOMA-IR -0.158 0.001 -0.121 0.012

\*Pearson correlation.*\*\**Partial correlation analysis, adjustment for age and sex. ALT: alanine

Factor AST/ALT level ratio<sup>1</sup>

aminotransferase; AST: aspartate aminotransferase; BMI: body mass index; WC: waist circumference; SBP: systolic blood press; DBP: diastolic blood press; FPG: fasting plasma glucose; TG: triglycerides; HDL-C: high density lipoprotein cholesterol; TI: true insulin; HOMA-IR: homeostasis model assessment

Table 2 Correlation analysis of AST/ALT ratio with anthropometric and metabolic variables

(n=279)

abdominal obesity(%) 9.7 32.2 0.000

high triglycerides(%) 0.0 3.4 0.008

elevated blood pressure(%) 12.9 25.3 0.001

impaired fasting glucose(%) 0.7 0.0 0.548

2 test was used to test for differences in proportions. ALT: alanine aminotransferase; AST:

Table 3 Prevalence of metabolic syndrome in different levels of AST/ALT ratio(%).

metabolic syndrome(%) 0.7 4.8 0.016

cholesterol(%) 15.1 21.2 0.109

AST/ALT level ratio< 1

(n=146) *<sup>P</sup>*

metabolic syndrome was related with a lower AST/ALT ratio in Han young adults. Subjects with AST/ALT ratio < 1 had a significantly higher frequency of abdominal obesity, high triglycerides, elevated blood pressure and metabolic syndrome. The association was not modified by age, sex and BMI.


Data are expressed as mean ± standard deviation(SD) or medians with interquartile ranges(IQR). Continuous variables were analyzed with covariance adjustment for age and sex. When data was not normally distributed, they were ln transformed for analysis. ALT: alanine aminotransferase; AST: aspartate aminotransferase; BMI: body mass index; WC: waist circumference; SBP: systolic blood press; DBP: diastolic blood press; FPG: fasting plasma glucose; TG: triglycerides; HDL-C: high density lipoprotein cholesterol; TI: true insulin; HOMA-IR: homeostasis model assessment of insulin resistance; SD: standard deviation; IQR:indicates interquartile range.

Table 1. Clinical and laboratory characteristics of the subjects in different levels of AST/ALT ratio.


metabolic syndrome was related with a lower AST/ALT ratio in Han young adults. Subjects with AST/ALT ratio < 1 had a significantly higher frequency of abdominal obesity, high triglycerides, elevated blood pressure and metabolic syndrome. The association was not

(n=279)

Age(years) mean(SD) 20.3(0.8) 20.3(0.8) 0.955

Sex(male/female) 111/168 105/41 0.000

BMI(kg/m2) mean(SD) 22.9(3.8) 26.3(4.0) 0.000

WC(cm) mean(SD) 70.8(9.3) 81.4(12.3) 0.000

SBP(mmHg) mean(SD) 109.3(10.7) 115.2(11.5) 0.004

DBP(mmHg) mean (SD) 73.1(8.2) 76.7(9.9) 0.030

FPG(mmol/L) mean (SD) 4.30(0.44) 4.38(0.40) 0.101

TG(mmol/L) mean (SD) 0.69(0.24) 0.83(0.46) 0.000

HDL-C(mmol/L) mean (SD) 1.42(0.22) 1.30(0.24) 0.001

TI( uU/ml) median (IQR) 6.58(3.88) 7.75(4.86) 0.014

HOMA-IR median(IQR) 1.25(0.76) 1.53(1.08) 0.006

ALT(U/L) mean(SD) 10.6(5.0) 30.6(17.3) 0.000

AST(U/L) mean(SD) 17.9(6.0) 20.6(8.5) 0.223

Data are expressed as mean ± standard deviation(SD) or medians with interquartile ranges(IQR). Continuous variables were analyzed with covariance adjustment for age and sex. When data was not normally distributed, they were ln transformed for analysis. ALT: alanine aminotransferase; AST: aspartate aminotransferase; BMI: body mass index; WC: waist circumference; SBP: systolic blood press; DBP: diastolic blood press; FPG: fasting plasma glucose; TG: triglycerides; HDL-C: high density lipoprotein cholesterol; TI: true insulin; HOMA-IR: homeostasis model assessment of insulin resistance;

Table 1. Clinical and laboratory characteristics of the subjects in different levels of AST/ALT

SD: standard deviation; IQR:indicates interquartile range.

ratio.

AST/ALT level ratio< 1

(n=146) *<sup>P</sup>*

variable AST/ALT level ratio<sup>1</sup>

modified by age, sex and BMI.


\*Pearson correlation.*\*\**Partial correlation analysis, adjustment for age and sex. ALT: alanine aminotransferase; AST: aspartate aminotransferase; BMI: body mass index; WC: waist circumference; SBP: systolic blood press; DBP: diastolic blood press; FPG: fasting plasma glucose; TG: triglycerides; HDL-C: high density lipoprotein cholesterol; TI: true insulin; HOMA-IR: homeostasis model assessment of insulin resistance.

Table 2 Correlation analysis of AST/ALT ratio with anthropometric and metabolic variables in Han young aduts.


The *-*2 test was used to test for differences in proportions. ALT: alanine aminotransferase; AST: aspartate aminotransferase.

Table 3 Prevalence of metabolic syndrome in different levels of AST/ALT ratio(%).

The Relationship Between AST/ALT Ratio and Metabolic

Circulation. 2008;118:277-283.

JAMA. 2002;287:356-359.

2009;2009:136501.

study. Nutr Metab (Lond). 2009;6:30.

among adults in China. Lancet. 2005;365:1398-1405.

atherosclerosis study. Diabetes. 2005;54:3140-3147.

2006;41:566-572.

2497.

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Syndrome in Han Young Adults – AST/ALT Ratio and Metabolic Syndrome 253

[5] Kim CH, Younossi ZM. Nonalcoholic fatty liver disease: a manifestation of the metabolic

[6] Lee S, Jin Kim Y, Yong Jeon T, Hoi Kim H, Woo Oh S, Park Y, Soo Kim S.Obesity is the

[7] Khurram M, Ashraf MM. A clinical and biochemical profile of biopsy-proven nonalcoholic Fatty liver disease subjects. J Coll Physicians Surg Pak. 2007;17:531-534. [8] Schwimmer JB, Pardee PE, Lavine JE, Blumkin AK, Cook S. Cardiovascular risk factors

[9] Tzima N, Pitsavos C, Panagiotakos DB, Chrysohoou C, Polychronopoulos E, Skoumas J,

[10] Expert Panel on Detection, Evaluation, and Treatment of High Blood Cholesterol in

[11] Gu D, Reynolds K, Wu X, Chen J, Duan X, Reynolds RF, Whelton PK, He J; InterASIA

[12] Li Y, Yang X, Zhai F, Kok FJ, Zhao W, Piao J, Zhang J, Cui Z, Ma G. Prevalence of the metabolic syndrome in Chinese adolescents. Br J Nutr. 2008;99:565-570. [13] Ford ES, Giles WH, Dietz WH. Prevalence of the metabolic syndrome among US

[14] Sharifi F, Mousavinasab SN, Saeini M, Dinmohammadi M. Prevalence of metabolic

[15] Hanley AJ, Williams K, Festa A, Wagenknecht LE, D'Agostino RB Jr, Haffner SM. Liver

[16] Hanley AJ, Wagenknecht LE, Festa A, D'Agostino RB Jr, Haffner SM. Alanine

[18] Yan HM, Gao X, Liu M, Gu Q, Zhang B, Li X. Relationship of liver fat content to insulin resistance and metabolic syndrome.Zhonghua Yi Xue Za Zhi 2008;88:1255-1258.

United States: impact of ethnicity. Hepatology. 2004;40:1387-1395.

only independent factor associated with ultrasound-diagnosed non-alcoholic fatty liver disease: a cross-sectional case-control study. Scand J Gastroenterol.

and the metabolic syndrome in pediatric nonalcoholic fatty liver disease.

Stefanadis C. Adherence to the Mediterranean diet moderates the association of aminotransferases with the prevalence of the metabolic syndrome; the ATTICA

Adults. Executive Summary of the Third Report of the National Cholesterol Education Program(NCEP) expert panel on detection, evaluation, and treatment of high blood cholesterol in adults(Adult Treatment Panel III) .JAMA. 2001;285:2486-

Collaborative Group. Prevalence of the metabolic syndrome and overweight

adults: findings from the third National Health and Nutrition Examination Survey.

syndrome in an adult urban population of the west of Iran. Exp Diabetes Res.

markers and development of the metabolic syndrome: the insulin resistance

aminotransferase and directly measured insulin sensitivity in a multiethnic cohort: the Insulin Resistance Atherosclerosis Study. Diabetes Care. 2007;30:1819-1827. [17] Browning JD, Szczepaniak LS, Dobbins R, Nuremberg P, Horton JD, Cohen JC, Grundy

SM, Hobbs HH. Prevalence of hepatic steatosis in an urban population in the

In our study, AST/ALT ratio showed negative correlation with HOMA-IR. Insulin resistance is a central feature in the pathogenesis of metabolic syndrome,while insulin resistance is now considered the main link between metabolic disturbances and liver enzymes. Hanley AJ et al.16 reported that AST/ALT ratio was associated with directly measured insulin sensitivity when conventionally availables were adjusted. This result can be explained by the following possible mechanism. Liver markers were known to be significantly correlated with increased hepatic fat content.17 Liver fat content was a significant risk factor of HOMA-IR while BMI and waist circumference were not.18

In our study, we found that young adults with AST/ALT ratio < 1 had higher TI than subjects with AST/ALT ratio 1. But the level of FPG was similar between subjects with AST/ALT ratio < 1 and 1. The liver plays an important role in maintaining normal glucose concentrations during fasting as well as postprandially. The loss of a direct effect of insulin to suppress hepatic glucose production and glycogenolysis in the liver causes an increase in hepatic glucose production.19 Increased insulin levels with increasing insulin resistance in subjects with AST/ALT ratio < 1 suggested that elevated insulin concentrations reflected increased beta cell output to the elevated insulin concentrations. The hyperinsulinemia can thus be seen as a compensatory mechanism for the preexisting insulin resistance, which represents a mechanism for protection against the development of impaired fasting glucose and diabetes. T2DM will result when there is insufficient insulin secretion to counter preexisting insulin resistance.20

Diet habit modifies the relationship of the liver enzymes ratio with metabolic syndrome. For example, Mediterranean diet moderates the association of the liver enzymes ratio with the prevalence of the metabolic syndrome.9 In China, people's eating habits are quite different in different area. Regrettably, diet habit was not evaluated in our study. This is a limitation of our study.

In conclusion, the prevalence of metabolic syndrome was low in Han young adults. AST/ALT ratio was related with metabolic syndrome in Han young adults.

#### **5. References**


In our study, AST/ALT ratio showed negative correlation with HOMA-IR. Insulin resistance is a central feature in the pathogenesis of metabolic syndrome,while insulin resistance is now considered the main link between metabolic disturbances and liver enzymes. Hanley AJ et al.16 reported that AST/ALT ratio was associated with directly measured insulin sensitivity when conventionally availables were adjusted. This result can be explained by the following possible mechanism. Liver markers were known to be significantly correlated with increased hepatic fat content.17 Liver fat content was a

In our study, we found that young adults with AST/ALT ratio < 1 had higher TI than subjects with AST/ALT ratio 1. But the level of FPG was similar between subjects with AST/ALT ratio < 1 and 1. The liver plays an important role in maintaining normal glucose concentrations during fasting as well as postprandially. The loss of a direct effect of insulin to suppress hepatic glucose production and glycogenolysis in the liver causes an increase in hepatic glucose production.19 Increased insulin levels with increasing insulin resistance in subjects with AST/ALT ratio < 1 suggested that elevated insulin concentrations reflected increased beta cell output to the elevated insulin concentrations. The hyperinsulinemia can thus be seen as a compensatory mechanism for the preexisting insulin resistance, which represents a mechanism for protection against the development of impaired fasting glucose and diabetes. T2DM will result when there is insufficient insulin secretion to counter

Diet habit modifies the relationship of the liver enzymes ratio with metabolic syndrome. For example, Mediterranean diet moderates the association of the liver enzymes ratio with the prevalence of the metabolic syndrome.9 In China, people's eating habits are quite different in different area. Regrettably, diet habit was not evaluated in our study. This is a limitation of

In conclusion, the prevalence of metabolic syndrome was low in Han young adults.

[1] Nyblom H, Björnsson E, Simrén M, Aldenborg F, Almer S, Olsson R. The AST/ALT ratio as an indicator of cirrhosis in patients with PBC. Liver Int. 2006;26:840-845. [2] Giannini E, Risso D, Botta F, Chiarbonello B, Fasoli A, Malfatti F, Romagnoli P, Testa E,

[3] Nyblom H, Berggren U, Balldin J, Olsson R. High AST/ALT ratio may indicate

[4] Sorbi D, Boynton J, Lindor KD. The ratio of aspartate aminotransferase to alanine

from alcoholic liver disease. Am J Gastroenterol. 1999;94:1018-1022.

Ceppa P, Testa R. Validity and clinical utility of the aspartate aminotransferasealanine aminotransferase ratio in assessing disease severity and prognosis in patients with hepatitis C virus-related chronic liver disease. Arch Intern Med.

advanced alcoholic liver disease rather than heavy drinking. Alcohol Alcohol.

aminotransferase: potential value in differentiating nonalcoholic steatohepatitis

AST/ALT ratio was related with metabolic syndrome in Han young adults.

significant risk factor of HOMA-IR while BMI and waist circumference were not.18

preexisting insulin resistance.20

2003;163:218-224.

2004;39:336-339.

our study.

**5. References** 


**13** 

*USA*

**On the Mechanism of Action of** 

B. Shariat-Madar1, M. Taherian2 and Z. Shariat-Madar3

*2Department of Anesthesia, Massachusetts General Hospital,* 

*3School of Pharmacy, Department of Pharmacology,* 

*1College of Literature, Science, and the Arts, University of Michigan, Ann Arber, MI,* 

Many mechanisms are involved in the regulation of blood pressure. Among the molecules, hormones, and factors known to be involved in blood pressure regulation, PRCP can generate the most potent inflammatory vasodilator molecules, including nitric oxide (Zhao et al., 2001c), prostaglandins (Kolte et al., 2011), bradykinin (BK)-(Chajkowski et al., 2010) and angiotensin 1-7 (Ang1-7)-(Mallela et al., 2008) dependent pathways in the endothelium and astrocytes. Experimental evidence suggest that PRCP appears to metabolize its main substrates, des-Arg9-bradykinin (des-Arg9-BK, BK1-8)(Chajkowski et al., 2010) and alphamelanocyte stimulating hormone 1-13 (-MSH1-13)(Wallingford et al., 2009), to prevent the production of prostaglandins and NO, indicating that the enzyme may promote antiinflammatory effects in cardiovascular and cerebrovascular systems. Evidence shows that *Prcp* gene expression is elevated in the developing murine brain vasculature during the intermediate phase of chick chorio-allantoic membrane (CAM)(Javerzat et al., 2009), suggesting that PRCP has a role during the active phase of vascular remodeling. Interestingly, *Prcp* gene is overexpressed in glioblastoma and it is suggested that PRCP also has a role in tumor angiogenesis(Javerzat et al., 2009). Thus, PRCP plays important roles in response to stress and may be an endothelial gate-keeper regulating blood flow, which tightly regulates endothelial barrier function. The historical perspective of knowledge of PRCP and its roles in physiological and cardiovascular diseases such as inflammation,

The cardiovascular system is composed of the heart, blood, and a complex network of blood vessels. Humans have a closed cardiovascular system in which the system allows a complete separation of function and blood loss due to having a relatively low blood volume compared to species with open cardiovascular systems. In a closed cardiovascular system, blood flows rapidly from arteries to capillaries and returns through the venous system. The

heart sustains the high pressure necessary for the blood to reach the body's tissues.

hypertension, thrombosis, and obesity is tabulated in Table 1.

**2. Cardiovascular system function**

**1. Introduction** 

**Prolylcarboxypeptidase** 

*Harvard Medical School, Boston, MA,* 

*University of Mississippi, University, MS,* 


### **On the Mechanism of Action of Prolylcarboxypeptidase**

B. Shariat-Madar1, M. Taherian2 and Z. Shariat-Madar3

*1College of Literature, Science, and the Arts, University of Michigan, Ann Arber, MI, 2Department of Anesthesia, Massachusetts General Hospital, Harvard Medical School, Boston, MA, 3School of Pharmacy, Department of Pharmacology, University of Mississippi, University, MS,* 

*USA*

#### **1. Introduction**

254 Recent Advances in Cardiovascular Risk Factors

[19] Michael MD, Kulkarni RN, Postic C, Previs SF, Shulman GI, Magnuson MA, Kahn CR.

[20] Ten S, Maclaren N. Insulin resistance syndrome in children. J Clin Endocrinol Metab

progressive hepatic dysfunction. Mol Cell 2000,6:87–97.

2004;89:2526-2539.

Loss of insulin signaling in hepatocytes leads to severe insulin resistance and

Many mechanisms are involved in the regulation of blood pressure. Among the molecules, hormones, and factors known to be involved in blood pressure regulation, PRCP can generate the most potent inflammatory vasodilator molecules, including nitric oxide (Zhao et al., 2001c), prostaglandins (Kolte et al., 2011), bradykinin (BK)-(Chajkowski et al., 2010) and angiotensin 1-7 (Ang1-7)-(Mallela et al., 2008) dependent pathways in the endothelium and astrocytes. Experimental evidence suggest that PRCP appears to metabolize its main substrates, des-Arg9-bradykinin (des-Arg9-BK, BK1-8)(Chajkowski et al., 2010) and alphamelanocyte stimulating hormone 1-13 (-MSH1-13)(Wallingford et al., 2009), to prevent the production of prostaglandins and NO, indicating that the enzyme may promote antiinflammatory effects in cardiovascular and cerebrovascular systems. Evidence shows that *Prcp* gene expression is elevated in the developing murine brain vasculature during the intermediate phase of chick chorio-allantoic membrane (CAM)(Javerzat et al., 2009), suggesting that PRCP has a role during the active phase of vascular remodeling. Interestingly, *Prcp* gene is overexpressed in glioblastoma and it is suggested that PRCP also has a role in tumor angiogenesis(Javerzat et al., 2009). Thus, PRCP plays important roles in response to stress and may be an endothelial gate-keeper regulating blood flow, which tightly regulates endothelial barrier function. The historical perspective of knowledge of PRCP and its roles in physiological and cardiovascular diseases such as inflammation, hypertension, thrombosis, and obesity is tabulated in Table 1.

#### **2. Cardiovascular system function**

The cardiovascular system is composed of the heart, blood, and a complex network of blood vessels. Humans have a closed cardiovascular system in which the system allows a complete separation of function and blood loss due to having a relatively low blood volume compared to species with open cardiovascular systems. In a closed cardiovascular system, blood flows rapidly from arteries to capillaries and returns through the venous system. The heart sustains the high pressure necessary for the blood to reach the body's tissues.

On the Mechanism of Action of Prolylcarboxypeptidase 257

To deal with proper delivery of blood to the tissues as well as many types of insults, including chemical and physical damage as well as infection, the cardiovascular system has evolved an intricate multilayer of specialized biochemical pathways. Multiple feedback mechanisms controlling blood flow with contrasting effects regulate normal vascular function, which have either stimulatory or inhibitory effects. These features enable the cardiovascular system to perform its tasks effectively. Of these regulatory feedback machinery, a few have multiple and redundant control mechanisms that tightly uphold cardiovascular functions. Among these regulatory feedback mechanisms within the mammalian cardiovascular system, we review the renin-angiotensin(Savinetskaia et al., 2008; Hayashi et al., 2010), kallikrein-kinin(Bryant and Shariat-Madar, 2009; Saxena et al., 2011), and pro-opiomelanocortin (POMC) (Wang et al., 2008) systems that communicate with each other. Although within each of these systems are surely a network of molecular interactions with other pathways, their interactions are not either compared or reviewed.

The systemic RAS is a complex, but fairly well understood pathway(Perret-Guillaume et al., 2009; Molteni, 1982; Brown et al., 1983; Oishi et al., 1996; Renshaw et al., 2001; Hettinger et al., 2002; Volzke et al., 2005; Clerk et al., 2005). Although the physiological importance of the RAS for regulation of blood pressure and water homeostasis has been established(Fukuchi et al., 1973; Bing and Nielsen, 1973), emerging evidence suggests that the components of RAS are important players for maintenance and regulation of many physiological and pathophysiological processes, such as angiogenesis (Abali et al., 2002), inflammation (Akishita et al., 2001), tumorigenesis(Ager et al., 2008), thrombosis (Asselbergs et al., 2008; Asselbergs et al., 2007), vascular hypertrophy (Brede et al., 2001), vascular development (Lasaitiene et al., 2006), and remodeling (Akishita et al., 2000; Alcazar et al., 2009). The molecular mechanisms by which the components of RAS exert their pleiotropic effects are redundant in order to ensure an appropriate balance between the stimulatory and inhibitory signals favoring normal blood delivery. More interestingly, there is also a balance between RAS function and its counter-regulatory hormones, in particular plasma kinins, the main effectors of the plasma Kallikrein-Kinin System (KKS) (Shariat-Madar et al., 2002a). The active metabolites of these two systems regulate each other in an antagonistic manner. In view of the opposing effects of these two systems(Bader, 2001), the complex interplay of RAS and KKS with the local microenvironment of endothelial cells lining blood vessels is responsible for delivering nutrients. The RAS and KKS signaling pathways are also responsible for sustaining the high pressure necessary for the blood to reach all of the tissues

The RAS encompasses numerous peptides that act in concert in a cascade triggered by low blood volume or a drop in blood pressure cascades or water-fall(Taquini, Jr. and Taquini, 1961; Nicholls and Robertson, 2000; Shariat-Madar and Schmaier, 2004; Ritz, 2005; Oudart, 2005). These peptides exert their actions, which can be autocrine (Chan and Wong, 1996), paracrine (Haulica et al., 2005), or endocrine(Gohlke et al., 1992), acting through specific cell surface receptors on their target cells to control blood pressure and body fluid homeostasis. In a stepwise enzymatic process, the aspartyl protease renin (a rate-limiting enzyme) cleaves circulating angiotensinogen to the inactive angiotensin I (Ang I) (Helmer, 1965). Subsequently,

However, special attention is given to the role of PRCP in health and disease.

of the body while maintaining blood pressure within a narrow range.

**2.1 Renin-angiotensin system (RAS)** 


Table 1. Historical Perspective

(Sorrells and Erdos, 1971) (Odya et al., 1978)

(Skidgel et al., 1981) (Kumamoto et al., 1981)

(Deddish et al., 1990)

(Tamaoki et al., 1994)

(Suzawa et al., 1995) (Suga et al., 1995)

(Jackman et al., 1995) (Watson, Jr. et al., 1997) (Shariat-Madar et al., 2002) (Moreira et al., 2002)

(Wang et al., 2006)

(Hooley et al., 2007)

(Soisson et al., 2010)

(Javerzat et al., 2009)

(Zhao et al., 2010) (Zhu et al., 2010)

(Duan et al., 2011)

(Adams et al., 2011)

(Chajkowski et al., 2010)

(ZHANG Yan et al., 2009)

(Wallingford et al., 2009)

(Shariat-Madar et al., 2005)

(Tan et al., 1993)

**Year References** 

PRCP was purified from human kidney and its potential substrates was reported. The two substrates were angiotensin II, and

PRCP was found in both inflammatory exudates and in cells that

Cultured Madin-Darby canine kidney (MDCK) distal tubular cells

Human PRCP was sequenced and cloned. It was noted that PRCP might have both exopeptidase and endopeptidase activities PRCP was found to have a modulatory role in the angiotensin II-

PRCP was purified from cell free extracts of Xanthomonas maltophilia, a bacteria species which is normally found in clinical

Overexpression of PRCP on Chinese hamster ovary cells was

Wang and associates described the functional significance of PRCP

Hooley and associates described a C-terminal extension in the PK

Wallingford and associates described that *Prcp*-null mice had elevated levels of alpha-MSH1-13 in the hypothalamus and were leaner and shorter than the wild-type controls on a regular chow diet. *Prcp* null mice were also resistant to high-fat diet-induced

Soisson and associates described the crystal structure of human

E112D polymorphism in the prolylcarboxypeptidase gene was found to be associated with blood pressure response to benazepril

Javerzat and associates described overexpression of PRCP during the intermediate phase of the chick chorio-allantoic membrane Recombinant PRCP (rPRCP) metabolized BK1-8 to BK1-7, whereas

PRCP is a 4OHTAM resistance factor in estrogen receptor-positive

PRCPgt/gt mice were found to be hypertensive and prothrombotic

PRCP was found to be active in human cerebrospinal fluid Overexpression of angiotensin type 2 (AT2) receptor in mouse coronary artery endothelial cells was found to increase expression

serine protease domain as a potential substrate for PRCP.

Human PRCP (EC 3.4.16.2) was found in biological fluids

A novel and rapid radioassay for PRCP was developed

PRCP was found to be highly active in macrophages Watson and associates described localization of *Prcp* gene PRCP was found to be an endothelial cell prekallikrein activator Moreira and associates described the extracellular PRCP that

1971 1978

1981 1981 angiotensin III

specimens

reported

obesity

PRCP

activates prekallikrein

in the etiology of preeclampsia

in Chinese hypertensive patients

breast cancer cells

Table 1. Historical Perspective

rPRCP was ineffective in metabolizing BK 1-9

of PRCP, which may contribute to kinin release

appear at sites of inflammation

induced pulmonary vasoconstriction PRCP was found in monkey kidney

was found to contain PRCP

1990

1993

1994

1995 1995

2005

2006

2007

2009

2009

2009

2009

2010

2010 2010

2011

2011

To deal with proper delivery of blood to the tissues as well as many types of insults, including chemical and physical damage as well as infection, the cardiovascular system has evolved an intricate multilayer of specialized biochemical pathways. Multiple feedback mechanisms controlling blood flow with contrasting effects regulate normal vascular function, which have either stimulatory or inhibitory effects. These features enable the cardiovascular system to perform its tasks effectively. Of these regulatory feedback machinery, a few have multiple and redundant control mechanisms that tightly uphold cardiovascular functions. Among these regulatory feedback mechanisms within the mammalian cardiovascular system, we review the renin-angiotensin(Savinetskaia et al., 2008; Hayashi et al., 2010), kallikrein-kinin(Bryant and Shariat-Madar, 2009; Saxena et al., 2011), and pro-opiomelanocortin (POMC) (Wang et al., 2008) systems that communicate with each other. Although within each of these systems are surely a network of molecular interactions with other pathways, their interactions are not either compared or reviewed. However, special attention is given to the role of PRCP in health and disease.

#### **2.1 Renin-angiotensin system (RAS)**

The systemic RAS is a complex, but fairly well understood pathway(Perret-Guillaume et al., 2009; Molteni, 1982; Brown et al., 1983; Oishi et al., 1996; Renshaw et al., 2001; Hettinger et al., 2002; Volzke et al., 2005; Clerk et al., 2005). Although the physiological importance of the RAS for regulation of blood pressure and water homeostasis has been established(Fukuchi et al., 1973; Bing and Nielsen, 1973), emerging evidence suggests that the components of RAS are important players for maintenance and regulation of many physiological and pathophysiological processes, such as angiogenesis (Abali et al., 2002), inflammation (Akishita et al., 2001), tumorigenesis(Ager et al., 2008), thrombosis (Asselbergs et al., 2008; Asselbergs et al., 2007), vascular hypertrophy (Brede et al., 2001), vascular development (Lasaitiene et al., 2006), and remodeling (Akishita et al., 2000; Alcazar et al., 2009). The molecular mechanisms by which the components of RAS exert their pleiotropic effects are redundant in order to ensure an appropriate balance between the stimulatory and inhibitory signals favoring normal blood delivery. More interestingly, there is also a balance between RAS function and its counter-regulatory hormones, in particular plasma kinins, the main effectors of the plasma Kallikrein-Kinin System (KKS) (Shariat-Madar et al., 2002a). The active metabolites of these two systems regulate each other in an antagonistic manner. In view of the opposing effects of these two systems(Bader, 2001), the complex interplay of RAS and KKS with the local microenvironment of endothelial cells lining blood vessels is responsible for delivering nutrients. The RAS and KKS signaling pathways are also responsible for sustaining the high pressure necessary for the blood to reach all of the tissues of the body while maintaining blood pressure within a narrow range.

The RAS encompasses numerous peptides that act in concert in a cascade triggered by low blood volume or a drop in blood pressure cascades or water-fall(Taquini, Jr. and Taquini, 1961; Nicholls and Robertson, 2000; Shariat-Madar and Schmaier, 2004; Ritz, 2005; Oudart, 2005). These peptides exert their actions, which can be autocrine (Chan and Wong, 1996), paracrine (Haulica et al., 2005), or endocrine(Gohlke et al., 1992), acting through specific cell surface receptors on their target cells to control blood pressure and body fluid homeostasis. In a stepwise enzymatic process, the aspartyl protease renin (a rate-limiting enzyme) cleaves circulating angiotensinogen to the inactive angiotensin I (Ang I) (Helmer, 1965). Subsequently,

On the Mechanism of Action of Prolylcarboxypeptidase 259

Although AT2 appears to be cell-type specific with a low expression pattern in adult tissues, it is the dominate receptor in fetal tissues(Akishita et al., 1999; Guan et al., 2008). Experimental evidence in support of AT2-independent G-protein signaling pathway has been demonstrated (Bottari et al., 1991). AT2 receptors are essential for triggering vasodilation and it can also mediate cell death (Gwathmey et al., 2009). Ang II-induced AT2 receptor-dependent vasodilation is mediated by activation of nitric oxide/cyclic guanosine 3',5'-monophosphate (cGMP) signaling cascades. On the other hand, AT2 activation by Ang II may lead to the activation of proapoptotic second messengers, MAPK phosphatase 1 (MKP-1)(Horiuchi et al., 1997), SH2 domain-containing phosphatase 1 (SHP-1)(Feng et al., 2002), and protein phosphatase 2A (PP2A)(Huang et al., 1996) and subsequent inactivation of MAPKs(Bedecs et al., 1997; Horiuchi et al., 1997), and ERK1/2(Horiuchi et al., 1997). While identification and characterization of the molecular switch that controls differential activation of distinct downstream signaling pathways by Ang II are poorly understood, our

In the next step of the RAS cascades, angiotensin converting enzyme 2 (ACE2) and PRCP metabolize Ang II (a prothrombotic risk factor) to angiotensin 1-7 (a vasodilator, antithrombotic factor)(Mallela et al., 2008), highlighting the functional significance of these two enzymes. Notably, ACE2 (Gurley et al., 2006) and PRCP (Adams et al., 2011)null mice do not exhibit excessive elevated blood pressure compared to control littermates, suggesting that these enzymes play a critical role in the maintenance of normal vascular physiology, Figure 1. PRCP also metabolizes angiotensin III (Ang III, Ang2-8) to angiotensin 2-7 (Ang 2- 7)(Mallela et al., 2008). Ang III, like Ang II, is also a pressor agent whose response is mediated by AT1 receptors (Campbell et al., 1979). Ang III is implicated in cell proliferation and matrix accumulation(Ruiz-Ortega et al., 1998). Ang II- and Ang III-induced AT1 receptor stimulation mediate vasoconstriction, mitogenic and hypertrophic effects and inflammatory responses (Ishanov et al., 1998; Schluter and Wenzel, 2008; Marshall et al., 2000). Hydrolysis of Ang III by PRCP leads to a decrease in Ang III-mediated effects, further

pointing out the importance of PRCP in contributing to regulation of blood pressure.

Ang II is a proangiogenic hormone and AT1 mediates its proangiogenic actions(Wilop et al., 2009). The proangiogenic effect of Ang II leads to upregulation of EGF (Friedlander and Terzi, 2006; Lautrette et al., 2005), FGF (Liu et al., 2009), PDGF(Blaschke et al., 2002; Li et al., 2005), IGF-1 (Kajstura et al., 2001; Song et al., 2005(Jia et al., 2011), hepatocytes growth factor(Bataller et al., 2003), and NO(Abassi et al., 1997). Ang II-induced AT1 mediated signaling pathway cause an activation of mitogen-activated protein kinases (MAPK) (Guo et

The MAPK pathway induces cellular protein synthesis, volume regulation, gene expression, and growth (Morrell et al., 1999). In contrast, activation of AT1-independent G-protein signaling pathway by Ang II can also cause endothelial cell proliferation through non-receptor tyrosine kinase Src-mediated increase of NADP oxidase activation, phosphorylation of extracellular signal-regulated kinases 1 and 2 (ERK1/2), P38 MAP kinase, and a decrease in phosphorylation of Src homology 2 (SH2)-containing inositol phosphatase 2 (SHIP2)(Ali et al., 1997b; Feng et al., 2002; Bedecs et al., 1997). These experimental observations suggest that Ang

**2.2 RAS and angiogenesis** 

al., 2006; Morrell et al., 1999; Taniyama et al., 2004).

understanding of the downstream events is exponentially mounting.

angiotensin converting enzyme (ACE) cleaves the two C-terminal amino acid residues of Ang I to generate the key effector of RAS, angiotensin II (Ang II, Ang 1-8) under physiological conditions (Figure 1)(Peart, 1975). However, the tonin-, cathepsin G-, tissue plasminogen activator- or chymase-induced Ang II generation is apparently important under pathophysiological conditions (Doggrell and Wanstall, 2004; Doggrell and Wanstall, 2003; Leckie, 2005; Belova, 2000). Recent evidence suggests that Ang I can block a downstream protease, PRCP *in vitro* (Mallela et al., 2008). Up to now, the physiological significant role of Ang I in blocking PRCP is uncertain. Ang II primarily exerts its physiological effects by activating angiotensin type 1 (AT1) receptors (Figure 1) (Chassagne et al., 2000). However, angiotensin type 2 (AT2) receptors also appear to mediate Ang II effects (Figure 1)(Anavekar and Solomon, 2005). Activation of this signaling pathway leads to cardioprotection. Both AT1 and AT2 belong to the G-protein couple receptor (GPCR) superfamily(Ali et al., 1997a). While AT1 can couple to two members of the G family (Gq/11 and Gi) and to G complexes (Okuda et al., 1996), AT2 can couple to Gi and Gs subunits (Kai et al., 1996).

Fig. 1. Metabolites of the renin-angiotensin system influence cardiovascular system. **Ang I**; Angiotensin I, **Ang II**; Angiotensin II, **Ang III**; Angiotensin III, **Ang IV**; Angiotensin IV, Ang1-9; Angiotensin 1-9, Ang1-7; Angiotensin 1-7, **ACE;** Angiotensin converting enzyme, **ACE2;** Angiotensin converting enzyme 2, **AT1**; angiotensin type 1, **AT2**; angiotensin type 2, **AT4**; angiotensin type 4, **PAI-1**; plasminogen activator 1, **TF**; tissue activator, **Mas**; Ang1-7 Mas receptor, **NO**; nitric oxide, **PG**; prostaglandin.

Ang II-induced AT1 receptor-dependent vasoconstriction is mediated by activation of Gq/11, Gi, and G complexes(Kai et al., 1996). The activation of these heterotrimeric G proteins triggers the activation of phospholipase C (PLC)(Griendling et al., 1997) and phospholipase D (PLD)(Griendling et al., 1997; Ushio-Fukai et al., 1999), leading to an increase in intracellular calcium, actin-myosin interactions, and release of leukotrienes.

angiotensin converting enzyme (ACE) cleaves the two C-terminal amino acid residues of Ang I to generate the key effector of RAS, angiotensin II (Ang II, Ang 1-8) under physiological conditions (Figure 1)(Peart, 1975). However, the tonin-, cathepsin G-, tissue plasminogen activator- or chymase-induced Ang II generation is apparently important under pathophysiological conditions (Doggrell and Wanstall, 2004; Doggrell and Wanstall, 2003; Leckie, 2005; Belova, 2000). Recent evidence suggests that Ang I can block a downstream protease, PRCP *in vitro* (Mallela et al., 2008). Up to now, the physiological significant role of Ang I in blocking PRCP is uncertain. Ang II primarily exerts its physiological effects by activating angiotensin type 1 (AT1) receptors (Figure 1) (Chassagne et al., 2000). However, angiotensin type 2 (AT2) receptors also appear to mediate Ang II effects (Figure 1)(Anavekar and Solomon, 2005). Activation of this signaling pathway leads to cardioprotection. Both AT1 and AT2 belong to the G-protein couple receptor (GPCR) superfamily(Ali et al., 1997a). While AT1 can couple to two members of the G family (Gq/11 and Gi) and to G complexes

(Okuda et al., 1996), AT2 can couple to Gi and Gs subunits (Kai et al., 1996).

ACE2

**PRCP** ACE 2

**PRCP**

Angiotensinogen

Ang I

Ang IV

APN

Ang II

APA

ACE

AT2 Ang III

Ang 1-9

ACE 2

Ang1-7

Mas

NO, PG

• Vasodilation • Hypotension • Thromboprevention

Fig. 1. Metabolites of the renin-angiotensin system influence cardiovascular system. **Ang I**; Angiotensin I, **Ang II**; Angiotensin II, **Ang III**; Angiotensin III, **Ang IV**; Angiotensin IV, Ang1-9; Angiotensin 1-9, Ang1-7; Angiotensin 1-7, **ACE;** Angiotensin converting enzyme, **ACE2;** Angiotensin converting enzyme 2, **AT1**; angiotensin type 1, **AT2**; angiotensin type 2, **AT4**; angiotensin type 4, **PAI-1**; plasminogen activator 1, **TF**; tissue activator, **Mas**; Ang1-7

Ang II-induced AT1 receptor-dependent vasoconstriction is mediated by activation of Gq/11, Gi, and G complexes(Kai et al., 1996). The activation of these heterotrimeric G proteins triggers the activation of phospholipase C (PLC)(Griendling et al., 1997) and phospholipase D (PLD)(Griendling et al., 1997; Ushio-Fukai et al., 1999), leading to an increase in intracellular calcium, actin-myosin interactions, and release of leukotrienes.

Mas receptor, **NO**; nitric oxide, **PG**; prostaglandin.

AT4

• Vasoconstriction • Blood pressure • Thrombosis

AT1

TF, PAI-1

Although AT2 appears to be cell-type specific with a low expression pattern in adult tissues, it is the dominate receptor in fetal tissues(Akishita et al., 1999; Guan et al., 2008). Experimental evidence in support of AT2-independent G-protein signaling pathway has been demonstrated (Bottari et al., 1991). AT2 receptors are essential for triggering vasodilation and it can also mediate cell death (Gwathmey et al., 2009). Ang II-induced AT2 receptor-dependent vasodilation is mediated by activation of nitric oxide/cyclic guanosine 3',5'-monophosphate (cGMP) signaling cascades. On the other hand, AT2 activation by Ang II may lead to the activation of proapoptotic second messengers, MAPK phosphatase 1 (MKP-1)(Horiuchi et al., 1997), SH2 domain-containing phosphatase 1 (SHP-1)(Feng et al., 2002), and protein phosphatase 2A (PP2A)(Huang et al., 1996) and subsequent inactivation of MAPKs(Bedecs et al., 1997; Horiuchi et al., 1997), and ERK1/2(Horiuchi et al., 1997). While identification and characterization of the molecular switch that controls differential activation of distinct downstream signaling pathways by Ang II are poorly understood, our understanding of the downstream events is exponentially mounting.

In the next step of the RAS cascades, angiotensin converting enzyme 2 (ACE2) and PRCP metabolize Ang II (a prothrombotic risk factor) to angiotensin 1-7 (a vasodilator, antithrombotic factor)(Mallela et al., 2008), highlighting the functional significance of these two enzymes. Notably, ACE2 (Gurley et al., 2006) and PRCP (Adams et al., 2011)null mice do not exhibit excessive elevated blood pressure compared to control littermates, suggesting that these enzymes play a critical role in the maintenance of normal vascular physiology, Figure 1. PRCP also metabolizes angiotensin III (Ang III, Ang2-8) to angiotensin 2-7 (Ang 2- 7)(Mallela et al., 2008). Ang III, like Ang II, is also a pressor agent whose response is mediated by AT1 receptors (Campbell et al., 1979). Ang III is implicated in cell proliferation and matrix accumulation(Ruiz-Ortega et al., 1998). Ang II- and Ang III-induced AT1 receptor stimulation mediate vasoconstriction, mitogenic and hypertrophic effects and inflammatory responses (Ishanov et al., 1998; Schluter and Wenzel, 2008; Marshall et al., 2000). Hydrolysis of Ang III by PRCP leads to a decrease in Ang III-mediated effects, further pointing out the importance of PRCP in contributing to regulation of blood pressure.

#### **2.2 RAS and angiogenesis**

Ang II is a proangiogenic hormone and AT1 mediates its proangiogenic actions(Wilop et al., 2009). The proangiogenic effect of Ang II leads to upregulation of EGF (Friedlander and Terzi, 2006; Lautrette et al., 2005), FGF (Liu et al., 2009), PDGF(Blaschke et al., 2002; Li et al., 2005), IGF-1 (Kajstura et al., 2001; Song et al., 2005(Jia et al., 2011), hepatocytes growth factor(Bataller et al., 2003), and NO(Abassi et al., 1997). Ang II-induced AT1 mediated signaling pathway cause an activation of mitogen-activated protein kinases (MAPK) (Guo et al., 2006; Morrell et al., 1999; Taniyama et al., 2004).

The MAPK pathway induces cellular protein synthesis, volume regulation, gene expression, and growth (Morrell et al., 1999). In contrast, activation of AT1-independent G-protein signaling pathway by Ang II can also cause endothelial cell proliferation through non-receptor tyrosine kinase Src-mediated increase of NADP oxidase activation, phosphorylation of extracellular signal-regulated kinases 1 and 2 (ERK1/2), P38 MAP kinase, and a decrease in phosphorylation of Src homology 2 (SH2)-containing inositol phosphatase 2 (SHIP2)(Ali et al., 1997b; Feng et al., 2002; Bedecs et al., 1997). These experimental observations suggest that Ang

On the Mechanism of Action of Prolylcarboxypeptidase 261

birth, FXI levels are reached to normal adult plasma levels by six months of age. FXI circulates in the plasma as a disulfide bond-linked dimer complexed with HK. It is important for propagation of blood coagulation(Walsh and Griffin, 1981). FXI can be activated to activated FXI (FXIa) by FXIIa-mediated activation on the negatively charged surfaces or thrombinmediated activation on the platelet surface(Baglia et al., 2004), Figure 2. FXIa augments thrombin generation via interaction with a coagulation factor of the extrinsic pathway, factor IX(Baglia and Walsh, 1998; Taketomi et al., 2008). It appears that FXI along with activated platelet are a prerequisite for optimum thrombin generation in which the procoagulant activity

Plasma KKS activation has been demonstrated under physiological conditions and in numerous inflammatory human diseases. Under pathological conditions, conversion of plasma factor XII (FXII) to its active form (FXIIa) is concomitant with the appearance of hematologic responses including coagulation, fibrinolytic, and inflammation. FXIIa initiates propagation of the intrinsic pathway (IP) of blood coagulation via factor XI activation, Fig. **2** (1; Pathway 1). In the presence of HK, FXIIa converts plasma prekallikrein (PK) to kallikrein, Fig. **2** (2; Pathway 2). In a reciprocal feedback mechanism, kallikrein accelerates FXII activation. Kallikrein causes the release of tissue plasminogen activator (tPA) via a

Fig. 2. A schematic representation of factor XII (FXII) activation during pathological plasma proteolytic states. **FXIIa;** activated factor XII, 1; pathway 1, **FXI;** factor XI, **FXIa**; activated factor XI, 2; pathway 2, **HK;** high molecular weight kininogen, **PK;** prekallikrein, **BK;** bradykinin, **tPA;** tissue plasminogen activator, **FDP**; Fibrin degradation Products.

of KKS is so just below the threshold point (Keularts et al., 2001).

bradykinin-dependent pathway.

II is proangiogenic. However, evidence suggests that Ang II may inhibit angiogenesis under certain conditions by activating AT2-receptors, suggesting that AT1 mediates effects that counteract those mediated by AT2 receptors (Chung et al., 1996).

#### **2.3 RAS in cardiovascular disease**

Since Ang II, Ang III, Ang IV, and Ang 1-7 can act systemically and locally to control blood pressure and body fluid homeostasis, components of RAS have become therapeutic targets for diseases such as hypertension(Farmer, 2000), cardiac hypertrophy(Brasier et al., 2000; Billet et al., 2008), congestive heart failure(Volpe and De, 2000; Yang et al., 1998), ischemic heart disease(Burrell et al., 2005), coronary heart disease(Farmer and Torre-Amione, 2001), and renal diseases.

#### **3. Plasma kallikrein kinin system (KKS)**

The primary functions of plasma KKS are to 1) assist hemostasis and limit blood loss, 2) increase capillary permeability and dilate arterioles, 3) promote antiatherogenic and antithrombogenic properties of endothelium by maintaining or increasing antiatherogenic agents including nitric oxide, prostaglandins, and tissue plasminogen activator, and 4) induce acute vascular pain. In addition to its hemodynamic actions, antifibrotic and renoprotective effects of kallikrein have been reported (Pawluczyk et al., 2008). Although the expression and activity of components of plasma KKS are known to be age-dependent (Gordon et al., 1980) for three decades, the mechanism underlying this phenomenon is yet to be unravelled.

The components of KKS, also called the contact activation pathway, consist of three serine zymogens (prekallikrein, factor XII, and factor XI) as well as a kinin and antiangiogenic precursor (high molecular weight kininogen)(Figure 2)(Ratnoff and Saito, 1979). Plasma kallikrein, a serine protease, is synthesized in the liver. It is predominantly secreted by hepatocytes as an inactive molecule called prekallikrein (PK) that circulates in plasma as a heterodimer complex bound to high molecular weight kininogen (HK) with 1:1 molar stoichiometry. PK is a single chain +-globulin that is present in the plasma of humans and of other animal species. About 80-90% of PK is normally in complexed with HK.

HK is a single-chained glycoprotein synthesised by liver and secreted into the circulation (Sueyoshi et al., 1987). HK consists of three domains: an amino acid-terminal heavy chain, a carboxyl-terminal light chain, and bradykinin moiety (Figure 3)(Kleniewski et al., 1988). The heavy chain linked to the light chain by a single disulfide bond (Kleniewski et al., 1988). Domain 6 of HK has a PK and FXI-binding site (Reddigari and Kaplan, 1989). HK binding to cell surface proteins or artificial surfaces is vital for activation of the plasma KKS(Pixley et al., 2011; Joseph et al., 2001; Zhao et al., 2001). The procoagulant activity of HK is dependent on whether it is in complexed with PK or FXI (Gailani and Broze, Jr., 1991). The proteolytic cleavage of HK by kallikrein results in the production of BK and cleaved HK (HKa). HK could be metabolized by activated factor XI into three peptide fragments, yet its physiological significance has been unclear. HK is involved in angiogenesis and in cell adhesion and the matrix of various endothelial cells (Motta et al., 2001).

Unlike PK, factor XI (FXI) is involved in normal hemostasis *in vivo*. FXI synthesis is primarily in the liver. Factor XI activity is age-dependent (Andrew et al., 1987). While its activity is low at

II is proangiogenic. However, evidence suggests that Ang II may inhibit angiogenesis under certain conditions by activating AT2-receptors, suggesting that AT1 mediates effects that

Since Ang II, Ang III, Ang IV, and Ang 1-7 can act systemically and locally to control blood pressure and body fluid homeostasis, components of RAS have become therapeutic targets for diseases such as hypertension(Farmer, 2000), cardiac hypertrophy(Brasier et al., 2000; Billet et al., 2008), congestive heart failure(Volpe and De, 2000; Yang et al., 1998), ischemic heart disease(Burrell et al., 2005), coronary heart disease(Farmer and Torre-Amione, 2001),

The primary functions of plasma KKS are to 1) assist hemostasis and limit blood loss, 2) increase capillary permeability and dilate arterioles, 3) promote antiatherogenic and antithrombogenic properties of endothelium by maintaining or increasing antiatherogenic agents including nitric oxide, prostaglandins, and tissue plasminogen activator, and 4) induce acute vascular pain. In addition to its hemodynamic actions, antifibrotic and renoprotective effects of kallikrein have been reported (Pawluczyk et al., 2008). Although the expression and activity of components of plasma KKS are known to be age-dependent (Gordon et al., 1980) for

The components of KKS, also called the contact activation pathway, consist of three serine zymogens (prekallikrein, factor XII, and factor XI) as well as a kinin and antiangiogenic precursor (high molecular weight kininogen)(Figure 2)(Ratnoff and Saito, 1979). Plasma kallikrein, a serine protease, is synthesized in the liver. It is predominantly secreted by hepatocytes as an inactive molecule called prekallikrein (PK) that circulates in plasma as a heterodimer complex bound to high molecular weight kininogen (HK) with 1:1 molar stoichiometry. PK is a single chain +-globulin that is present in the plasma of humans and of

HK is a single-chained glycoprotein synthesised by liver and secreted into the circulation (Sueyoshi et al., 1987). HK consists of three domains: an amino acid-terminal heavy chain, a carboxyl-terminal light chain, and bradykinin moiety (Figure 3)(Kleniewski et al., 1988). The heavy chain linked to the light chain by a single disulfide bond (Kleniewski et al., 1988). Domain 6 of HK has a PK and FXI-binding site (Reddigari and Kaplan, 1989). HK binding to cell surface proteins or artificial surfaces is vital for activation of the plasma KKS(Pixley et al., 2011; Joseph et al., 2001; Zhao et al., 2001). The procoagulant activity of HK is dependent on whether it is in complexed with PK or FXI (Gailani and Broze, Jr., 1991). The proteolytic cleavage of HK by kallikrein results in the production of BK and cleaved HK (HKa). HK could be metabolized by activated factor XI into three peptide fragments, yet its physiological significance has been unclear. HK is involved in angiogenesis and in cell

Unlike PK, factor XI (FXI) is involved in normal hemostasis *in vivo*. FXI synthesis is primarily in the liver. Factor XI activity is age-dependent (Andrew et al., 1987). While its activity is low at

three decades, the mechanism underlying this phenomenon is yet to be unravelled.

other animal species. About 80-90% of PK is normally in complexed with HK.

adhesion and the matrix of various endothelial cells (Motta et al., 2001).

counteract those mediated by AT2 receptors (Chung et al., 1996).

**2.3 RAS in cardiovascular disease** 

**3. Plasma kallikrein kinin system (KKS)** 

and renal diseases.

birth, FXI levels are reached to normal adult plasma levels by six months of age. FXI circulates in the plasma as a disulfide bond-linked dimer complexed with HK. It is important for propagation of blood coagulation(Walsh and Griffin, 1981). FXI can be activated to activated FXI (FXIa) by FXIIa-mediated activation on the negatively charged surfaces or thrombinmediated activation on the platelet surface(Baglia et al., 2004), Figure 2. FXIa augments thrombin generation via interaction with a coagulation factor of the extrinsic pathway, factor IX(Baglia and Walsh, 1998; Taketomi et al., 2008). It appears that FXI along with activated platelet are a prerequisite for optimum thrombin generation in which the procoagulant activity of KKS is so just below the threshold point (Keularts et al., 2001).

Plasma KKS activation has been demonstrated under physiological conditions and in numerous inflammatory human diseases. Under pathological conditions, conversion of plasma factor XII (FXII) to its active form (FXIIa) is concomitant with the appearance of hematologic responses including coagulation, fibrinolytic, and inflammation. FXIIa initiates propagation of the intrinsic pathway (IP) of blood coagulation via factor XI activation, Fig. **2** (1; Pathway 1). In the presence of HK, FXIIa converts plasma prekallikrein (PK) to kallikrein, Fig. **2** (2; Pathway 2). In a reciprocal feedback mechanism, kallikrein accelerates FXII activation. Kallikrein causes the release of tissue plasminogen activator (tPA) via a bradykinin-dependent pathway.

Fig. 2. A schematic representation of factor XII (FXII) activation during pathological plasma proteolytic states. **FXIIa;** activated factor XII, 1; pathway 1, **FXI;** factor XI, **FXIa**; activated factor XI, 2; pathway 2, **HK;** high molecular weight kininogen, **PK;** prekallikrein, **BK;** bradykinin, **tPA;** tissue plasminogen activator, **FDP**; Fibrin degradation Products.

On the Mechanism of Action of Prolylcarboxypeptidase 263

Under physiological conditions, PK bound to HK on endothelial cells can lead to PK activation through a FXIIa-independent mechanism, which in turn leads to BK generation. While assembly and activation of the plasma KKS is regulated by PRCP on several cultured cell lines, heat shock protein 90 (HSP90) is the main activator of the plasma PK in mesothelium-derived cell lines (Varano, V et al., 2011). Thus, the plasma KKS participates in both surface-dependent activation of blood clotting and inflammatory process. Further, PRCP participates in cell surface – dependent activation of PK and its dependent pathways. The question remains how PRCP-dependent PK signaling pathway is assembled, activated, and regulated in response to plasma levels of Ang II and/or Ang III. However, in an elegant study, the authors find evidence for a cross-talk among the kinin, PRCP, and angiotensin

Whereas the normally functioning KKS can successfully counteract blood pressure, promote smooth blood flow, modulates neovascularisation and eliminates pathogens by promoting the recruitment of neutrophils to the site of injury, inappropriate responses can lead to extensive tissue damage. KKS has been implicated in the pathogenesis of inflammation, hypertension, endotoxemia, and coagulopathy. Increased BK levels is a hallmark in all of these cases. In some cases, the persistent production of BK due to the deficiency of the blood protein C1-inhibitor, which controls kallikrein and activated FXII, is detrimental to the survival of the patients with hereditary angioedema (HAE). In others, the inability of angiotensin converting enzyme (ACE) to degrade BK leads to elevated BK levels and edema

Various cancers exhibit upregulation of either epidermal growth factor or urokinase receptor or both, which leads to poor prognosis. On studies done on human prostate cancer cells (DU145) the cleaved fragment of HK, HKa and also domain 5 prevented colocalization of uPAR and EGFR, decreased downstream extracellular signal-regulated kinase (ERK) and AKT-mediated signal transduction at EGFR(Wuepper and Cochrane, 1972). This indicates therapeutic potential of HKa and D5 to inhibit metastasis involving migration and invasion

Both HK and LK are proangiogenic as demonstrated by induction of vascularization on chorioallantoic membrane. Monoclonal antibody directed against HK has been shown to inhibit HK-induced neovascularization on chorioallatoic membrane as well as HK-induced fibrosarcoma formation. Domain 5 of HK, also called kininostatin, is antiangiogenic(Colman et al., 2000). There are reports that peptides derived from domain 3 and domain 5 inhibit angiogenesis in Zn2+ independent and dependent manner (Zhang et al., 2002). Antibody

Like HK, BK also is proangiogenic and a reduction in its levels may be associated with impaired neovascularization(Krankel et al., 2008). But HKa is antiangiogenic as it prevents VEGF and FGF2 induced vascularization. HKa through its D5 domain has been shown to inhibit sphingosine 1-phosphate (S1P) and vascular endothelial growth factor (VEGF) mediated endothelial cell migration by modifying PI3 kinase-Akt signaling (Katkade et al.,

directed against domain 5 also prevents neovascularization(Song et al., 2004).

signaling pathways(Zhu et al., 2010).

in patients on ACE inhibitors.

of human prostate cancer cells.

**3.1 The plasma KKS and cardiovascular diseases** 

**3.2 The importance of KKS for angiogenesis** 

Fibrinolysis is the process by which plasmin digests fibrin, Fig. **2**. This aids in thrombus (clot) dissolution and limits the promotion of coagulation (clot formation). tPA converts plasma plasminogen to plasmin, which in turn remodels the thrombus. Plasmin breaks down the clot fibrin to fibrin degradation products (FDP). Consequently, FDP blocks thrombin-induced fibrin (clot) formation, Figure 2. Notably, plasmin triggers a positive feedback amplification by accelerating conversion of PK to kallikrein.

Kallikrein acts on HK to generate HKa (cleaved HK) and bradykinin (BK), Figure 3. BKinduced activation of the B2 (B2) receptor leads to the formation of tissue plasminogen activator (tPA, a potent fibrinolytic molecule)(Czokalo-Plichta et al., 2001), Fig. **3**. BK upon activation of its constitutive B2 receptors on endothelial cells leads to an increase in intracellular Ca2+ levels and subsequent production of nitric oxide (NO, a vasodilator) and prostacyclin (PGI2, a platelet activation inhibitor), ultimately leading to vasodilation, increased vascular permeability and edema (Zhao et al., 2001b; Hong, 1980; Palmer et al., 1987). Whereas B2 receptors are constitutively expressed, the expression of bradykinin B 1 (B1) receptors is induced during inflammation (Marceau, 1995). Metabolism of BK by carboxypeptidase N (CPN) in plasma or carboxypeptidase M (CPM) on endothelial cells yields des-Arg9-BK which interacts with B1 receptors to potentiate and/or perpetuate the inflammatory response (Figure 3)(Marceau et al., 1981).

Fig. 3. A schematic representation of PRCP-dependent prekallikrein activation. **HK**; high molecular weight kininogen**, PK;** prekallikrein, **PRCP**; prolylcarboxypeptidase, **K**; kallikrein, **BK1-9**; bradykinin, **HKa**; cleaved HK, **BK**; bradykinin, **NO**; nitric oxide, **PGI2**; prostacyclin, **tPA**; tissue plasminogen activator, **CPN**; carbox peptidase N, **CPM**; Carboxypeptidase M, **BK1-8**; bradykinin 1-8 (des-Arg9-bradykinin), **BK1-7**; bradykinin 1-7.

Fibrinolysis is the process by which plasmin digests fibrin, Fig. **2**. This aids in thrombus (clot) dissolution and limits the promotion of coagulation (clot formation). tPA converts plasma plasminogen to plasmin, which in turn remodels the thrombus. Plasmin breaks down the clot fibrin to fibrin degradation products (FDP). Consequently, FDP blocks thrombin-induced fibrin (clot) formation, Figure 2. Notably, plasmin triggers a positive

Kallikrein acts on HK to generate HKa (cleaved HK) and bradykinin (BK), Figure 3. BKinduced activation of the B2 (B2) receptor leads to the formation of tissue plasminogen activator (tPA, a potent fibrinolytic molecule)(Czokalo-Plichta et al., 2001), Fig. **3**. BK upon activation of its constitutive B2 receptors on endothelial cells leads to an increase in intracellular Ca2+ levels and subsequent production of nitric oxide (NO, a vasodilator) and prostacyclin (PGI2, a platelet activation inhibitor), ultimately leading to vasodilation, increased vascular permeability and edema (Zhao et al., 2001b; Hong, 1980; Palmer et al., 1987). Whereas B2 receptors are constitutively expressed, the expression of bradykinin B 1 (B1) receptors is induced during inflammation (Marceau, 1995). Metabolism of BK by carboxypeptidase N (CPN) in plasma or carboxypeptidase M (CPM) on endothelial cells yields des-Arg9-BK which interacts with B1 receptors to potentiate and/or perpetuate the

Fig. 3. A schematic representation of PRCP-dependent prekallikrein activation. **HK**; high molecular weight kininogen**, PK;** prekallikrein, **PRCP**; prolylcarboxypeptidase, **K**; kallikrein, **BK1-9**; bradykinin, **HKa**; cleaved HK, **BK**; bradykinin, **NO**; nitric oxide, **PGI2**; prostacyclin, **tPA**; tissue plasminogen activator, **CPN**; carbox peptidase N, **CPM**;

Carboxypeptidase M, **BK1-8**; bradykinin 1-8 (des-Arg9-bradykinin), **BK1-7**; bradykinin 1-7.

feedback amplification by accelerating conversion of PK to kallikrein.

inflammatory response (Figure 3)(Marceau et al., 1981).

Under physiological conditions, PK bound to HK on endothelial cells can lead to PK activation through a FXIIa-independent mechanism, which in turn leads to BK generation. While assembly and activation of the plasma KKS is regulated by PRCP on several cultured cell lines, heat shock protein 90 (HSP90) is the main activator of the plasma PK in mesothelium-derived cell lines (Varano, V et al., 2011). Thus, the plasma KKS participates in both surface-dependent activation of blood clotting and inflammatory process. Further, PRCP participates in cell surface – dependent activation of PK and its dependent pathways. The question remains how PRCP-dependent PK signaling pathway is assembled, activated, and regulated in response to plasma levels of Ang II and/or Ang III. However, in an elegant study, the authors find evidence for a cross-talk among the kinin, PRCP, and angiotensin signaling pathways(Zhu et al., 2010).

#### **3.1 The plasma KKS and cardiovascular diseases**

Whereas the normally functioning KKS can successfully counteract blood pressure, promote smooth blood flow, modulates neovascularisation and eliminates pathogens by promoting the recruitment of neutrophils to the site of injury, inappropriate responses can lead to extensive tissue damage. KKS has been implicated in the pathogenesis of inflammation, hypertension, endotoxemia, and coagulopathy. Increased BK levels is a hallmark in all of these cases. In some cases, the persistent production of BK due to the deficiency of the blood protein C1-inhibitor, which controls kallikrein and activated FXII, is detrimental to the survival of the patients with hereditary angioedema (HAE). In others, the inability of angiotensin converting enzyme (ACE) to degrade BK leads to elevated BK levels and edema in patients on ACE inhibitors.

#### **3.2 The importance of KKS for angiogenesis**

Various cancers exhibit upregulation of either epidermal growth factor or urokinase receptor or both, which leads to poor prognosis. On studies done on human prostate cancer cells (DU145) the cleaved fragment of HK, HKa and also domain 5 prevented colocalization of uPAR and EGFR, decreased downstream extracellular signal-regulated kinase (ERK) and AKT-mediated signal transduction at EGFR(Wuepper and Cochrane, 1972). This indicates therapeutic potential of HKa and D5 to inhibit metastasis involving migration and invasion of human prostate cancer cells.

Both HK and LK are proangiogenic as demonstrated by induction of vascularization on chorioallantoic membrane. Monoclonal antibody directed against HK has been shown to inhibit HK-induced neovascularization on chorioallatoic membrane as well as HK-induced fibrosarcoma formation. Domain 5 of HK, also called kininostatin, is antiangiogenic(Colman et al., 2000). There are reports that peptides derived from domain 3 and domain 5 inhibit angiogenesis in Zn2+ independent and dependent manner (Zhang et al., 2002). Antibody directed against domain 5 also prevents neovascularization(Song et al., 2004).

Like HK, BK also is proangiogenic and a reduction in its levels may be associated with impaired neovascularization(Krankel et al., 2008). But HKa is antiangiogenic as it prevents VEGF and FGF2 induced vascularization. HKa through its D5 domain has been shown to inhibit sphingosine 1-phosphate (S1P) and vascular endothelial growth factor (VEGF) mediated endothelial cell migration by modifying PI3 kinase-Akt signaling (Katkade et al.,

On the Mechanism of Action of Prolylcarboxypeptidase 265

also evidence that kinin or B2-receptor inhibitor administration increases renin secretion or decrease plasma renin levels in anesthetized rabbits, respectively(Chiu and Reid, 1997). This data plus the location of B2 receptors in different parts of the kidney and vascular endothelium suggests a role for the plasma KKS in the cardiovascular system (Schricker et

There have been numerous suggestions that kallikrein converts prorenin to renin and a few results suggest that RAS and KKS are interdependent and KKS regulates RAS activity (Sealey et al., 1979). In this report, the plasma prorenin was activated to renin *in vitro* by kallikrein when plasma acidity increased (Sealey et al., 1979). Later, Purdon and colleagues evaluated the effect of kallikrein converting prorenin to renin in plasma deficient in C1 inhibitor (hereditary angioedema) at neutral pH(Purdon et al., 1985). Although PK was completely activated to kallikrein and despite normal prorenin concentration, prorenin was not converted to an active enzyme. Regardless of kallikrein metabolic capability in converting prorenin to renin, studies suggest the presence of a crosstalk between the

In summary, the increased activity of RAS could be responsible both for arterial wall constriction and KKS activation and subsequent BK production to relief vasoconstriction. This explains the previous finding when the pressure is raised in the perfused heart muscle and brain, the flow can be held constant independently of any external neurogenic control(Mosher et al., 1964). The regulation of tissue flow as perfusion pressure changes can be affected by the rate of delivery or removal of BK and Ang II to the perfused region, influencing the state of contraction of the smooth muscle cells of the blocked vessels directly. This signifies the importance of the feedback mechanism between KKS and RAS. These systems may serve as a "on-off" biological switch impacting the risk of thrombosis/bleeding

PRCP has a subtle role in preserving the blood flow through the endothelial lining of blood vessels so that the tissues can maintain cellular function. PRCP exerts this effect through regulation of local levels of autocrine-paracrine hormones such as angiotensin II (Ang II), angiotensin III (Ang III), and bradykinin generation via kallikrein-kinin pathway. Subsequently, the modification of these vasoactive substances influences the secretion or expression of a diverse group of endothelium mediators that are involved in the contraction and relaxation of smooth muscles, regulating blood flow, blood clotting, inhibition of platelet aggregation, and electrolyte homeostasis. It is well-established that dysfunction of

PRCP is a serine peptidase that cleaves peptides at C-terminal amino acids linked to a proline residue. The first evidence that PRCP might play a role in the cardiovascular system was provided by catalytic activity analysis, which showed the metabolism of angiotensin II (Ang II) to angiotensin 1-7 (Ang 1-7)(Odya et al., 1978b). The mere inactivation of Ang II results in two events that broaden the functional spectrum of PRCP. First, it is a negative regulator of the pressor actions of the renin-angiotensin system (RAS). Second, PRCP

vasoconstricting RAS and the vasodilating KKS(Yokosawa et al., 1979).

endothelium significantly contributes to the development of thrombosis.

and those of low/high blood pressure.

**6.1 Physiology** 

**6. Prolylcarboxypeptidase or angiotensinase C** 

al., 1995).

2005). HKa acting through uPAR can also bring about apoptosis in endothelial cells, which is due to inhibition of uPA binding to uPAR thus preventing subsequent uPAR internalization and regeneration to the cell surface (Cao et al., 2004). This results in interference with all aspects of angiogenesis such as cell migration, proliferation, and survival.

Histidine proline rich glycoprotein (HPRG) is present in plasma abundantly and might be the predecessor of HK(Koide and Odani, 1987). HPRG is similar in sequence and function to HKa and is shown to bind to tropomyosin and inhibit angiogenesis through apoptosis (Donate et al., 2004).

### **4. Pro-opiomelanocortin (POMC)**

POMC is expressed in numerous mammalian tissues (Tatro et al., 1992; Tatro and Reichlin, 1987). It undergoes extensive posttranslational modification by a family of serine proteases and the prohormone convertases (PCs). The metabolism of POMC results in the generation of alpha-melanocyte stimulating hormone (+-MSH, -MSH1-13) along with -MSH, -MSH, and -endorphin (Shariat-Madar et al., 2010). Although all four of these hormones are important, only -MSH1-13 will be discussed here. The synthesis of +-MSH1-13 from POMC involves numerous specific enzymes in addition to PC1 and PC2. It appears that PRCP plays an important role in regulating -MSH.

#### **5. The interactions among KKS, RAS, and POMC**

The participation of the components of the plasma kallikrein-kinin system in some pathological processes like hypertension and cardiovascular diseases is still a matter of controversy. The availability of transgenic and knock-out mice for B2 and B1 receptors and angiotensinogen and angiotensin II receptors have advanced our understanding about the roles of KKS and specifically these receptors in cardiovascular regulation and inflammatory processes.

Bradykinin (BK) is the natural hypotensive agent that causes vasodilatation by stimulation of endothelial B2 receptors of arteries and arterioles, and the subsequent endothelial release of nitric oxide and prostaglandins(Sharma, 2009). BK influences the kidneys to produce diuresis and natriuresis (Katori and Majima, 2008; Katori and Majima, 2003). The diuretic effect of the exogenous BK administered by the renal artery is by B2 receptors (Boric et al., 1998; Croxatto et al., 1999), whereas the BK–induced natriuresis and increase in renal blood flow are due to both B1 and B2, Figure 3 (Lortie et al., 1992). Tissue injury such as myocardial ischemia and inflammation can cause the expression of B1 receptors on their surfaces (Foucart et al., 1997b; Foucart et al., 1997a). In addition, elevated concentrations of BK have been observed in ischemic myocardium(Burch and DePasquale, 1962; Burch and DePasquale, 1963). Occlusion of the proximal left anterior coronary artery causes a change in BK(Hashimoto et al., 1979; Kimura et al., 1973; Hashimoto et al., 1977). Exposure of the epicardium of anesthetized dogs to BK leads to stimulation of sympathetic afferent fibers protruding into the left ventricle(Uchida and Murao, 1974). Inhibition of the B2 receptor inhibitor resulted in increased kallikrein activity, but not kallikrein mRNA levels, in kidney of adult rats. There is a direct correlation between increased renal interstitial BK levels in dogs in low-sodium balance and the presence of B2 receptors(Siragy et al., 1997). There is

2005). HKa acting through uPAR can also bring about apoptosis in endothelial cells, which is due to inhibition of uPA binding to uPAR thus preventing subsequent uPAR internalization and regeneration to the cell surface (Cao et al., 2004). This results in interference with all aspects of angiogenesis such as cell migration, proliferation, and

Histidine proline rich glycoprotein (HPRG) is present in plasma abundantly and might be the predecessor of HK(Koide and Odani, 1987). HPRG is similar in sequence and function to HKa and is shown to bind to tropomyosin and inhibit angiogenesis through apoptosis

POMC is expressed in numerous mammalian tissues (Tatro et al., 1992; Tatro and Reichlin, 1987). It undergoes extensive posttranslational modification by a family of serine proteases and the prohormone convertases (PCs). The metabolism of POMC results in the generation of alpha-melanocyte stimulating hormone (+-MSH, -MSH1-13) along with -MSH, -MSH, and -endorphin (Shariat-Madar et al., 2010). Although all four of these hormones are important, only -MSH1-13 will be discussed here. The synthesis of +-MSH1-13 from POMC involves numerous specific enzymes in addition to PC1 and PC2. It appears that PRCP plays

The participation of the components of the plasma kallikrein-kinin system in some pathological processes like hypertension and cardiovascular diseases is still a matter of controversy. The availability of transgenic and knock-out mice for B2 and B1 receptors and angiotensinogen and angiotensin II receptors have advanced our understanding about the roles of KKS and specifically these receptors in cardiovascular regulation and inflammatory

Bradykinin (BK) is the natural hypotensive agent that causes vasodilatation by stimulation of endothelial B2 receptors of arteries and arterioles, and the subsequent endothelial release of nitric oxide and prostaglandins(Sharma, 2009). BK influences the kidneys to produce diuresis and natriuresis (Katori and Majima, 2008; Katori and Majima, 2003). The diuretic effect of the exogenous BK administered by the renal artery is by B2 receptors (Boric et al., 1998; Croxatto et al., 1999), whereas the BK–induced natriuresis and increase in renal blood flow are due to both B1 and B2, Figure 3 (Lortie et al., 1992). Tissue injury such as myocardial ischemia and inflammation can cause the expression of B1 receptors on their surfaces (Foucart et al., 1997b; Foucart et al., 1997a). In addition, elevated concentrations of BK have been observed in ischemic myocardium(Burch and DePasquale, 1962; Burch and DePasquale, 1963). Occlusion of the proximal left anterior coronary artery causes a change in BK(Hashimoto et al., 1979; Kimura et al., 1973; Hashimoto et al., 1977). Exposure of the epicardium of anesthetized dogs to BK leads to stimulation of sympathetic afferent fibers protruding into the left ventricle(Uchida and Murao, 1974). Inhibition of the B2 receptor inhibitor resulted in increased kallikrein activity, but not kallikrein mRNA levels, in kidney of adult rats. There is a direct correlation between increased renal interstitial BK levels in dogs in low-sodium balance and the presence of B2 receptors(Siragy et al., 1997). There is

survival.

processes.

(Donate et al., 2004).

**4. Pro-opiomelanocortin (POMC)** 

an important role in regulating -MSH.

**5. The interactions among KKS, RAS, and POMC** 

also evidence that kinin or B2-receptor inhibitor administration increases renin secretion or decrease plasma renin levels in anesthetized rabbits, respectively(Chiu and Reid, 1997). This data plus the location of B2 receptors in different parts of the kidney and vascular endothelium suggests a role for the plasma KKS in the cardiovascular system (Schricker et al., 1995).

There have been numerous suggestions that kallikrein converts prorenin to renin and a few results suggest that RAS and KKS are interdependent and KKS regulates RAS activity (Sealey et al., 1979). In this report, the plasma prorenin was activated to renin *in vitro* by kallikrein when plasma acidity increased (Sealey et al., 1979). Later, Purdon and colleagues evaluated the effect of kallikrein converting prorenin to renin in plasma deficient in C1 inhibitor (hereditary angioedema) at neutral pH(Purdon et al., 1985). Although PK was completely activated to kallikrein and despite normal prorenin concentration, prorenin was not converted to an active enzyme. Regardless of kallikrein metabolic capability in converting prorenin to renin, studies suggest the presence of a crosstalk between the vasoconstricting RAS and the vasodilating KKS(Yokosawa et al., 1979).

In summary, the increased activity of RAS could be responsible both for arterial wall constriction and KKS activation and subsequent BK production to relief vasoconstriction. This explains the previous finding when the pressure is raised in the perfused heart muscle and brain, the flow can be held constant independently of any external neurogenic control(Mosher et al., 1964). The regulation of tissue flow as perfusion pressure changes can be affected by the rate of delivery or removal of BK and Ang II to the perfused region, influencing the state of contraction of the smooth muscle cells of the blocked vessels directly. This signifies the importance of the feedback mechanism between KKS and RAS. These systems may serve as a "on-off" biological switch impacting the risk of thrombosis/bleeding and those of low/high blood pressure.

#### **6. Prolylcarboxypeptidase or angiotensinase C**

PRCP has a subtle role in preserving the blood flow through the endothelial lining of blood vessels so that the tissues can maintain cellular function. PRCP exerts this effect through regulation of local levels of autocrine-paracrine hormones such as angiotensin II (Ang II), angiotensin III (Ang III), and bradykinin generation via kallikrein-kinin pathway. Subsequently, the modification of these vasoactive substances influences the secretion or expression of a diverse group of endothelium mediators that are involved in the contraction and relaxation of smooth muscles, regulating blood flow, blood clotting, inhibition of platelet aggregation, and electrolyte homeostasis. It is well-established that dysfunction of endothelium significantly contributes to the development of thrombosis.

#### **6.1 Physiology**

PRCP is a serine peptidase that cleaves peptides at C-terminal amino acids linked to a proline residue. The first evidence that PRCP might play a role in the cardiovascular system was provided by catalytic activity analysis, which showed the metabolism of angiotensin II (Ang II) to angiotensin 1-7 (Ang 1-7)(Odya et al., 1978b). The mere inactivation of Ang II results in two events that broaden the functional spectrum of PRCP. First, it is a negative regulator of the pressor actions of the renin-angiotensin system (RAS). Second, PRCP

On the Mechanism of Action of Prolylcarboxypeptidase 267

Over thirty years ago, Odya et al (Odya et al., 1978d) showed that PRCP can metabolize Ang III to Ang 2-7. Evidence indicates that Ang 2-7 excites some neurons in the paraventricular nuclease (Ambuhl et al., 1992). Differential regulation of Ang 2-7 during ACE inhibitor treatment suggests a cardiovascular role for this peptide (Campbell et al., 1991). Because of the significance of Ang 2-7, AngIII regulation and metabolism are important. Results from several studies show that Ang III is a pressor agent. It exerts its hypertensive effects via AT1 receptors (Felix et al., 1991b; Gammelgaard et al., 2006a). Ang III can enhance renal disease through the overproduction of aldosterone and subsequent development of arterial hypertension and/or to atrial fibrillation (Al-Aloul et al., 2006). Aldosterone which is produced by adrenal cortex maintains blood volume, pressure, and electrolyte balance. It is tightly regulated by renin, an enzyme produced in the juxtaglomerular apparatus of the kidneys. Renin production is elevated by low blood pressure, decreased blood flow to the kidneys or sodium deficiency. In theory, inactivation of Ang III by PRCP might lead to a decrease in blood pressure. However, further studies are needed before claims can be made

It is well known that cells produce and secrete a large variety of proteases either as proenzymes or activated proteases. Although PRCP can be viewed as a proenzyme based on its primary structure, its post-translational regulation has not been characterized. PRCP is distributed throughout endothelial cells, including lysosome, cytoplasm, and plasma membrane. The membrane bound PRCP activates plasma PK to kallikrein in endothelial cells. The activation of PK is characterized by elevated kallikrein expression along with increased local BK production. Kallikrein-induced BK production causes endothelial cells to produce proinflammatory prostaglandins and vasodilatory nitric oxide (Ngo et al., 2009). Thus, the PRCP-dependent PK activation pathway might be considered as an additional

Local skeletal muscle ischemia and acidosis are shown to increase the generation of BK and prostaglandins, the two circulating end-products of the PRCP-dependent pathways, Figure 3 (Piepoli et al., 2001)**.** The increased acidotic response during exercise and inflammatory mediators such as BK and prostacyclin have been shown to cause the abnormal exerciserelated symptoms and autonomic responses in congestive heart failure syndrome (Scott et al., 2003). Emerging evidence suggest that the long-term elevated concentrations of NO and prostacyclin may be detrimental and eventually responsible for cardiovascular diseases such as congestive heart disease (Kai C.Wollert and Helmut Drexler, 2004). Needless to say, the reduction of endogenous PGI2 synthesis is associated with the occurrence of cyclic reduction of coronary flow(Tada et al., 1984). Thus, PRCP-dependent Ang1-7 and PRCPdependent BK pathways have important biological activities. Altered PRCP expression

PK is markedly elevated during pregnancy(Maki and Soga, 1981). Among proteases that metabolize neuropeptide Y 3-36 (NPY3-36, anorexigenic hormone), plasma kallikrein metabolizes NPY3-36 to NPY3-35(Abid et al., 2009). In addition, specific inhibitor of plasma kallikrein prevents the production of NPY3-35. We find these studies intriguing because elevation of kallikrein should provoke anorectic effect via accelerated inactivation of NPY3- 36. Recent evidence suggests that there is a link between severe prekallikrein deficiency and pregnancy loss (Dasanu and Alexandrescu, 2009). As mentioned earlier, PRCP activates PK to kallikrein. If PRCP is inhibited/down-regulated, kallikrein levels are lower. Thus,

mechanism to counteract the hypertensive activities of both Ang II and Ang III.

levels mediates pathophysiological changes in the cardiovascular system.

of a beneficial effect of PRCP on hypertension.

activity provides a measure of endothelium relaxation due to its ability to initiate nitric oxide generation through Mas receptor activation (Silva et al., 2007). PRCP metabolizes angiotensin III (Ang III) to angiotensin 2-7 (Ang 2-7)(Odya et al., 1978c). Ang III is a pressor agent whose response, like that of Ang II, is mediated by AT1 receptors(Felix et al., 1991a; Gammelgaard et al., 2006b). We described that when the complex of high molecular weight kininogen (HK) and prekallikrein (PK, Fletcher factor) binds to endothelium membrane, PK is rapidly converted to kallikrein by PRCP, Figure 3(Shariat-Madar et al., 2002b). The formed kallikrein then cleaves HK to liberate bradykinin (BK), which leads to vasodilation and subsequent nitric oxide and PGI2 formation by activating constitutive bradykinin B2 and inducible bradykinin B1 receptors (Zhao et al., 2001a; Colman and Schmaier, 1997). BK also causes release of tissue plasminogen activator (tPA, a profibrinolytic factor) to convert plasminogen to plasmin, which inhibits platelet aggregation, thrombus formation and promotes fibrinolysis, Figure 2(Pawluczyk et al., 2006). Thus, PRCP locally regulates nitric oxide-dependent vasodilation and indirectly modulates fibrinolysis within the vascular wall.

#### **6.2 Protein structure and molecular biology**

The human *Prcp* gene has been mapped to 11q14 and the gene is more than 147 kb in length and has 9 exons (Watson, Jr. et al., 1997). More recently, a new *Prcp*-like gene (*Prcp*2) has been discovered. The human Prcp gene family contains at least 2 genes. Both genes share important similarities including significant homology at the nucleotide level. Both genes encode for putative serine proteases. However, the functional role of *Prcp2* gene has not been characterized.

PRCP belongs to the single chain serine peptidase S28 family. It is derived from a larger precursor, prepro-PRCP, whose primary nucleotide structure was first described in a human cDNA clone by Tan et al., 1993. It appears that the precursor is homodimer polypeptides containing a secretory signal sequence. PRCP regulates the activity of bradykinin, Ang II, Ang III, prekallikrein, and -MSH(Odya et al., 1978a; Shariat-Madar et al., 2002c; Moreira et al., 2002; Wallingford et al., 2009), suggesting that PRCP has versatile roles in influencing cardiovascular system. The enzyme is ubiquitously present in all major areas of the vascular bed as well as in the kidney, heart, placenta, and hypothalamus (Jackman et al., 1995b; Skidgel et al., 1981; Rosenblum and Kozarich, 2003; Schmaier, 2003; Shariat-Madar et al., 2010). However, PRCP is highly produced in endothelial cells, which is one cell type involved in the release of vasodilators and antithrombotic mediators.

#### **6.3 PRCP in cardiovascular diseases**

PRCP and angiotensin converting enzyme 2 (ACE2) are exopeptidases, which convert Ang II to Ang 1-7. ACE2 metabolizes AngII at a much faster rate than PRCP (Rice et al., 2004), suggesting that Ang II is a poor substrate for PRCP. Clinical studies have provided reliable evidence that ACE2 is an essential regulator of angiotensin I (Ang I), Ang II and angiotensin-induced cardiac hypertrophy (Huentelman et al., 2005). Increased myocardial levels of Ang II concomitant with a significant decrease in Ang 1-7 in ACE2 deficient hearts has been reported (Kassiri et al., 2009). These data suggest that PRCP may indeed be important in metabolizing Ang II in the absence of ACE2. These observations suggest that PRCP is a redundant catalyst contributing to alternate pathways for Ang II metabolism.

activity provides a measure of endothelium relaxation due to its ability to initiate nitric oxide generation through Mas receptor activation (Silva et al., 2007). PRCP metabolizes angiotensin III (Ang III) to angiotensin 2-7 (Ang 2-7)(Odya et al., 1978c). Ang III is a pressor agent whose response, like that of Ang II, is mediated by AT1 receptors(Felix et al., 1991a; Gammelgaard et al., 2006b). We described that when the complex of high molecular weight kininogen (HK) and prekallikrein (PK, Fletcher factor) binds to endothelium membrane, PK is rapidly converted to kallikrein by PRCP, Figure 3(Shariat-Madar et al., 2002b). The formed kallikrein then cleaves HK to liberate bradykinin (BK), which leads to vasodilation and subsequent nitric oxide and PGI2 formation by activating constitutive bradykinin B2 and inducible bradykinin B1 receptors (Zhao et al., 2001a; Colman and Schmaier, 1997). BK also causes release of tissue plasminogen activator (tPA, a profibrinolytic factor) to convert plasminogen to plasmin, which inhibits platelet aggregation, thrombus formation and promotes fibrinolysis, Figure 2(Pawluczyk et al., 2006). Thus, PRCP locally regulates nitric oxide-dependent vasodilation and indirectly modulates fibrinolysis within the vascular

The human *Prcp* gene has been mapped to 11q14 and the gene is more than 147 kb in length and has 9 exons (Watson, Jr. et al., 1997). More recently, a new *Prcp*-like gene (*Prcp*2) has been discovered. The human Prcp gene family contains at least 2 genes. Both genes share important similarities including significant homology at the nucleotide level. Both genes encode for putative serine proteases. However, the functional role of *Prcp2* gene has not

PRCP belongs to the single chain serine peptidase S28 family. It is derived from a larger precursor, prepro-PRCP, whose primary nucleotide structure was first described in a human cDNA clone by Tan et al., 1993. It appears that the precursor is homodimer polypeptides containing a secretory signal sequence. PRCP regulates the activity of bradykinin, Ang II, Ang III, prekallikrein, and -MSH(Odya et al., 1978a; Shariat-Madar et al., 2002c; Moreira et al., 2002; Wallingford et al., 2009), suggesting that PRCP has versatile roles in influencing cardiovascular system. The enzyme is ubiquitously present in all major areas of the vascular bed as well as in the kidney, heart, placenta, and hypothalamus (Jackman et al., 1995b; Skidgel et al., 1981; Rosenblum and Kozarich, 2003; Schmaier, 2003; Shariat-Madar et al., 2010). However, PRCP is highly produced in endothelial cells, which is one cell type

PRCP and angiotensin converting enzyme 2 (ACE2) are exopeptidases, which convert Ang II to Ang 1-7. ACE2 metabolizes AngII at a much faster rate than PRCP (Rice et al., 2004), suggesting that Ang II is a poor substrate for PRCP. Clinical studies have provided reliable evidence that ACE2 is an essential regulator of angiotensin I (Ang I), Ang II and angiotensin-induced cardiac hypertrophy (Huentelman et al., 2005). Increased myocardial levels of Ang II concomitant with a significant decrease in Ang 1-7 in ACE2 deficient hearts has been reported (Kassiri et al., 2009). These data suggest that PRCP may indeed be important in metabolizing Ang II in the absence of ACE2. These observations suggest that PRCP is a redundant catalyst contributing to alternate pathways for Ang II metabolism.

involved in the release of vasodilators and antithrombotic mediators.

wall.

been characterized.

**6.2 Protein structure and molecular biology** 

**6.3 PRCP in cardiovascular diseases** 

Over thirty years ago, Odya et al (Odya et al., 1978d) showed that PRCP can metabolize Ang III to Ang 2-7. Evidence indicates that Ang 2-7 excites some neurons in the paraventricular nuclease (Ambuhl et al., 1992). Differential regulation of Ang 2-7 during ACE inhibitor treatment suggests a cardiovascular role for this peptide (Campbell et al., 1991). Because of the significance of Ang 2-7, AngIII regulation and metabolism are important. Results from several studies show that Ang III is a pressor agent. It exerts its hypertensive effects via AT1 receptors (Felix et al., 1991b; Gammelgaard et al., 2006a). Ang III can enhance renal disease through the overproduction of aldosterone and subsequent development of arterial hypertension and/or to atrial fibrillation (Al-Aloul et al., 2006). Aldosterone which is produced by adrenal cortex maintains blood volume, pressure, and electrolyte balance. It is tightly regulated by renin, an enzyme produced in the juxtaglomerular apparatus of the kidneys. Renin production is elevated by low blood pressure, decreased blood flow to the kidneys or sodium deficiency. In theory, inactivation of Ang III by PRCP might lead to a decrease in blood pressure. However, further studies are needed before claims can be made of a beneficial effect of PRCP on hypertension.

It is well known that cells produce and secrete a large variety of proteases either as proenzymes or activated proteases. Although PRCP can be viewed as a proenzyme based on its primary structure, its post-translational regulation has not been characterized. PRCP is distributed throughout endothelial cells, including lysosome, cytoplasm, and plasma membrane. The membrane bound PRCP activates plasma PK to kallikrein in endothelial cells. The activation of PK is characterized by elevated kallikrein expression along with increased local BK production. Kallikrein-induced BK production causes endothelial cells to produce proinflammatory prostaglandins and vasodilatory nitric oxide (Ngo et al., 2009). Thus, the PRCP-dependent PK activation pathway might be considered as an additional mechanism to counteract the hypertensive activities of both Ang II and Ang III.

Local skeletal muscle ischemia and acidosis are shown to increase the generation of BK and prostaglandins, the two circulating end-products of the PRCP-dependent pathways, Figure 3 (Piepoli et al., 2001)**.** The increased acidotic response during exercise and inflammatory mediators such as BK and prostacyclin have been shown to cause the abnormal exerciserelated symptoms and autonomic responses in congestive heart failure syndrome (Scott et al., 2003). Emerging evidence suggest that the long-term elevated concentrations of NO and prostacyclin may be detrimental and eventually responsible for cardiovascular diseases such as congestive heart disease (Kai C.Wollert and Helmut Drexler, 2004). Needless to say, the reduction of endogenous PGI2 synthesis is associated with the occurrence of cyclic reduction of coronary flow(Tada et al., 1984). Thus, PRCP-dependent Ang1-7 and PRCPdependent BK pathways have important biological activities. Altered PRCP expression levels mediates pathophysiological changes in the cardiovascular system.

PK is markedly elevated during pregnancy(Maki and Soga, 1981). Among proteases that metabolize neuropeptide Y 3-36 (NPY3-36, anorexigenic hormone), plasma kallikrein metabolizes NPY3-36 to NPY3-35(Abid et al., 2009). In addition, specific inhibitor of plasma kallikrein prevents the production of NPY3-35. We find these studies intriguing because elevation of kallikrein should provoke anorectic effect via accelerated inactivation of NPY3- 36. Recent evidence suggests that there is a link between severe prekallikrein deficiency and pregnancy loss (Dasanu and Alexandrescu, 2009). As mentioned earlier, PRCP activates PK to kallikrein. If PRCP is inhibited/down-regulated, kallikrein levels are lower. Thus,

On the Mechanism of Action of Prolylcarboxypeptidase 269

Baglia, F.A. and Walsh, P.N. (1998). Prothrombin is a cofactor for the binding of factor XI to

Billet, S., Aguilar, F., Baudry, C., and Clauser, E. (2008). Role of angiotensin II AT1 receptor

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disruption of PRCP-dependent PK activation may be a risk factor for pregnancy losses. The inhibitors of both kallikrein and PRCP may be contraindicated during pregnancy. However, further investigations are required to define the safety of these inhibitors.

The activation of POMC is characterized by elevated -MSH1-13 production. -MSH1-13 regulates inflammation and food intake, suggesting that it may contribute to tissue damage production. Recently, experimental evidence indicated that PRCP metabolizes -MSH1-13 to -MSH1-12 (Wallingford et al., 2009). Mice deficient for PRCP present high blood pressure (Adams G.N et al., 2009), a sustained long-term reduction in food intake, and increased energy expenditure (Wallingford et al., 2009). In humans, hypertension is considered to be a multifactorial disorder. *Prcp* gene and its variants contribute to hypertension (Wang et al., 2006c; Zhang et al., 2009; Zhu et al., 2010). Rutaecarpine stimulates expression of PRCP in the circulation and small arteries in renovascular hypertensive rats (Qin et al., 2009). Since PRCP variant promotes disease progression (Zhang et al., 2009) in Chinese hypertensive patients and risk of preeclampsia in black and non-black women (Wang et al., 2006b), it is a candidate for pharmacological intervention.

#### **7. Conclusions and future directions**

In conclusion, PRCP is one of the conserved proteases throughout evolution in vertebrates. In-vitro and in-vivo studies of PRCP, this enzyme serves to generate nitric oxide and prostaglandins. Notably, bradykinin and bradykinin 1-8 are important mediators of pain (Argent et al., 1954) and endotoxin-induced vascular permeability {Ueno, 1995 783 /id}. BK production is increased in ischemic myocardial tissue (Dell'Italia and Oparil, 1999). They are involved in the control of local blood flow. Although PRCP is less efficient than ACE2 induced NO and prostaglandin generation under pathophysiological conditions, this redundant mechanism has presumably evolved for ischemic remodeling and smooth blood flow (Figure 1 and Figure 3). Overactivation of this mechanism may contribute to myocardial tissue damage or endothelial dysfunction. It inactivates BK1-8, suggesting that PRCP may also reduce marked inflammatory response. It appears that PRCP is fine-tuned to discriminate between physiological and pathophysiological changes. Since cardiomyocytes are unable to proliferate (Lagrand et al., 2000), inactivation of BK1-8 by PRCP may contribute to prevention of tissue damage. Further studies are warranted to evaluate the role of PRCP in cardiovascular system, in particular the heart.

#### **8. Acknowledgment**

This work was supported by National Institute of Health [NCRR/NIH P20RR021929] to ZSM.

#### **9. References**


disruption of PRCP-dependent PK activation may be a risk factor for pregnancy losses. The inhibitors of both kallikrein and PRCP may be contraindicated during pregnancy. However,

The activation of POMC is characterized by elevated -MSH1-13 production. -MSH1-13 regulates inflammation and food intake, suggesting that it may contribute to tissue damage production. Recently, experimental evidence indicated that PRCP metabolizes -MSH1-13 to -MSH1-12 (Wallingford et al., 2009). Mice deficient for PRCP present high blood pressure (Adams G.N et al., 2009), a sustained long-term reduction in food intake, and increased energy expenditure (Wallingford et al., 2009). In humans, hypertension is considered to be a multifactorial disorder. *Prcp* gene and its variants contribute to hypertension (Wang et al., 2006c; Zhang et al., 2009; Zhu et al., 2010). Rutaecarpine stimulates expression of PRCP in the circulation and small arteries in renovascular hypertensive rats (Qin et al., 2009). Since PRCP variant promotes disease progression (Zhang et al., 2009) in Chinese hypertensive patients and risk of preeclampsia in black and non-black women (Wang et al., 2006b), it is a

In conclusion, PRCP is one of the conserved proteases throughout evolution in vertebrates. In-vitro and in-vivo studies of PRCP, this enzyme serves to generate nitric oxide and prostaglandins. Notably, bradykinin and bradykinin 1-8 are important mediators of pain (Argent et al., 1954) and endotoxin-induced vascular permeability {Ueno, 1995 783 /id}. BK production is increased in ischemic myocardial tissue (Dell'Italia and Oparil, 1999). They are involved in the control of local blood flow. Although PRCP is less efficient than ACE2 induced NO and prostaglandin generation under pathophysiological conditions, this redundant mechanism has presumably evolved for ischemic remodeling and smooth blood flow (Figure 1 and Figure 3). Overactivation of this mechanism may contribute to myocardial tissue damage or endothelial dysfunction. It inactivates BK1-8, suggesting that PRCP may also reduce marked inflammatory response. It appears that PRCP is fine-tuned to discriminate between physiological and pathophysiological changes. Since cardiomyocytes are unable to proliferate (Lagrand et al., 2000), inactivation of BK1-8 by PRCP may contribute to prevention of tissue damage. Further studies are warranted to evaluate the role of PRCP

This work was supported by National Institute of Health [NCRR/NIH P20RR021929] to ZSM.

Adams, G.N., LaRusch, G.A., Stavrou, E., Zhou, Y., Nieman, M.T., Jacobs, G.H., Cui, Y., Lu,

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Y., Jain, M.K., Mahdi, F., Shariat-Madar, Z., Okada, Y., D'Alecy, L.G., and Schmaier, A.H. (2011). Murine prolylcarboxypeptidase depletion induces vascular dysfunction with hypertension and faster arterial thrombosis. Blood *117*, 3929-3937.

Walsh, P.N. (2004). Factor XI interacts with the leucine-rich repeats of glycoprotein

further investigations are required to define the safety of these inhibitors.

candidate for pharmacological intervention.

**7. Conclusions and future directions** 

in cardiovascular system, in particular the heart.

**8. Acknowledgment**

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**14** 

*Poland* 

**Adolescent Obesity Predicts** 

Jarosaw Derejczyk1, Barbara Kapci\$ska2, Ewa Sadowska-Kr#pa2, Olga St#pie\$-Wyrobiec1, El'bieta Kimsa2 and Katarzyna Kempa2

Rapid increase in the prevalence of obesity among children and adolescents has become a major worldwide health issue. Several large epidemiological studies have demonstrated that childhood and adolescent obesity is a significant independent predictor of metabolic disorders, such as hypertension, dyslipidemia and insulin resistance, which have a major impact on the premature development of atherosclerosis and cardiovascular morbidity and mortality in adulthood (Bibbins-Domingo et al., 2007; Dietz & Robinson, 2005; Franks et al., 2010). The cluster of metabolic disorders including abdominal obesity, insulin resistance, dyslipidemia and elevated blood pressure has been defined as metabolic syndrome (Alberti et al., 2006; Aggoun, 2007, Han & Lean, 2006). It is now generally accepted that overweight or obese children and adolescents are at increased risk for some or all of the metabolic syndrome (MS) features (Carnethon et al., 2004; Franks et al., 2010; Magnussen et al., 2010; Zimmet et al., 2007). The data from the Third National Health and Nutrition Examination Survey (NHANESIII, 1988-1994) have demonstrated that about 4 % of the whole population and almost 30% of the overweight or obese 12 to 19-y-old adolescents met the criteria of MS (Cook et al., 2003). The early occurrence of the MS in childhood and at the pubertal age was also found to have a major impact on the development of atherosclerosis, a life-time risk of cardiovascular disease (Aggoun, 2007), and an increased rate of premature death (Franks et al., 2010; Nieto, 1992). Excessive body weight in childhood and adolescence is considered a strong predictor of adult obesity (Wang et al. 2008) and obesity-related health consequences including diabetes and heart disease (Carnethon et al., 2004; Must et al., 1992; Morrison et al. 2007) regardless of whether the parents were obese. Notably, parental obesity was found to double the risk of

being obese in adulthood (Shengxu et al., 2003; Whitaker et al., 1997).

would help to implement prevention programs targeted at risk groups.

The main reason for undertaking the present study was to evaluate the prevalence of obesity among 15-y-old adolescents and to estimate the risk for adverse health outcomes in this population sample. Another goal was to find out relatively simple biochemical markers that could be suitable for the early diagnosis of metabolic disorders and cardiovascular risk, and

**1. Introduction** 

**Cardiovascular Risk** 

*1John Paul II Geriatric Hospital in Katowice, 2Department of Physiological and Medical Sciences,* 

*Academy of Physical Education in Katowice* 


### **Adolescent Obesity Predicts Cardiovascular Risk**

Jarosaw Derejczyk1, Barbara Kapci\$ska2, Ewa Sadowska-Kr#pa2,

Olga St#pie\$-Wyrobiec1, El'bieta Kimsa2 and Katarzyna Kempa2 *1John Paul II Geriatric Hospital in Katowice, 2Department of Physiological and Medical Sciences, Academy of Physical Education in Katowice Poland* 

#### **1. Introduction**

274 Recent Advances in Cardiovascular Risk Factors

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Wang, S.X., Fan, Z.C., and Tao, Y.X. (2008). Functions of acidic transmembrane residues in

Watson, B., Jr., Nowak, N.J., Myracle, A.D., Shows, T.B., and Warnock, D.G. (1997). The

Wilop, S., von, H.S., Crysandt, M., Esser, A., Osieka, R., and Jost, E. (2009). Impact of

Yang, B., Li, D., Phillips, M.I., Mehta, P., and Mehta, J.L. (1998). Myocardial angiotensin II receptor expression and ischemia-reperfusion injury. Vasc. Med. *3*, 121-130. Yokosawa, N., Takahashi, N., Inagami, T., and Page, D.L. (1979). Isolation of completely

Zhang Yan, Hong Xiu-mei, Hou-xun, Li Jian-ping, Huo Yong, and Xu Xi-ping (2009). E112D

Zhao, X., Southwick, K., Cardasis, H.L., Du, Y., Lassman, M.E., Xie, D., El-Sherbeini, M.,

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Zhu, L., Carretero, O.A., Liao, T.D., Harding, P., Li, H., Sumners, C., and Yang, X.P. (2010).

a candidate gene for essential hypertension. Genomics *44*, 365-367.

between renin and kallikrein. Biochim. Biophys. Acta *569*, 211-219.

Rettig, R. (2005). Renin-angiotensin system and haemostasis gene polymorphisms and outcome after coronary artery bypass graft surgery. Int. J. Cardiol. *98*, 133-139.

Diament, A., Haus, K.A., Shariat-Madar, Z., Mahdi, F., Wardlaw, S.L., Schmaier, A.H., Warden, C.H., and Diano, S. (2009). Prolylcarboxypeptidase regulates food

X. (2006). Prolylcarboxypeptidase gene, chronic hypertension, and risk of

human melanocortin-3 receptor binding and activation. Biochem. Pharmacol. *76*,

human angiotensinase C gene (HUMPCP) maps to 11q14 within 700 kb of D11S901:

angiotensin I converting enzyme inhibitors and angiotensin II type 1 receptor blockers on survival in patients with advanced non-small-cell lung cancer undergoing first-line platinum-based chemotherapy. J. Cancer Res. Clin. Oncol. *135*,

inactive plasma prorenin and its activation by kallikreins. A possible new link

polymorphism in the prolylcarboxypeptidase gene is associated with blood pressure response to benazepril in Chinese hypertensive patients. Chinese Medical

Geissler, W.M., Pryor, K.D., Verras, A., Garcia-Calvo, M., Shen, D.M., Yates, N.A., Pinto, S., and Hendrickon, R.C. (2010). Peptidomic profiling of human cerebrospinal fluid identifies YPRPIHPA as a novel substrate for

Assembly and activation of HK-PK complex on endothelial cells results in bradykinin liberation and NO formation. Am. J. Physiol Heart Circ. Physiol *280*,

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Rapid increase in the prevalence of obesity among children and adolescents has become a major worldwide health issue. Several large epidemiological studies have demonstrated that childhood and adolescent obesity is a significant independent predictor of metabolic disorders, such as hypertension, dyslipidemia and insulin resistance, which have a major impact on the premature development of atherosclerosis and cardiovascular morbidity and mortality in adulthood (Bibbins-Domingo et al., 2007; Dietz & Robinson, 2005; Franks et al., 2010). The cluster of metabolic disorders including abdominal obesity, insulin resistance, dyslipidemia and elevated blood pressure has been defined as metabolic syndrome (Alberti et al., 2006; Aggoun, 2007, Han & Lean, 2006). It is now generally accepted that overweight or obese children and adolescents are at increased risk for some or all of the metabolic syndrome (MS) features (Carnethon et al., 2004; Franks et al., 2010; Magnussen et al., 2010; Zimmet et al., 2007). The data from the Third National Health and Nutrition Examination Survey (NHANESIII, 1988-1994) have demonstrated that about 4 % of the whole population and almost 30% of the overweight or obese 12 to 19-y-old adolescents met the criteria of MS (Cook et al., 2003). The early occurrence of the MS in childhood and at the pubertal age was also found to have a major impact on the development of atherosclerosis, a life-time risk of cardiovascular disease (Aggoun, 2007), and an increased rate of premature death (Franks et al., 2010; Nieto, 1992). Excessive body weight in childhood and adolescence is considered a strong predictor of adult obesity (Wang et al. 2008) and obesity-related health consequences including diabetes and heart disease (Carnethon et al., 2004; Must et al., 1992; Morrison et al. 2007) regardless of whether the parents were obese. Notably, parental obesity was found to double the risk of being obese in adulthood (Shengxu et al., 2003; Whitaker et al., 1997).

The main reason for undertaking the present study was to evaluate the prevalence of obesity among 15-y-old adolescents and to estimate the risk for adverse health outcomes in this population sample. Another goal was to find out relatively simple biochemical markers that could be suitable for the early diagnosis of metabolic disorders and cardiovascular risk, and would help to implement prevention programs targeted at risk groups.

Adolescent Obesity Predicts Cardiovascular Risk 277

Concentrations of serum glucose, total cholesterol (TC), HDL-cholesterol (HDL-C), triglycerides (TG) were assessed by the enzymatic methods using commercially available diagnostic kits (BioMaxima cat.no. 1-033-0400, 1-023-0400, 1-029-0200, 1-053-0400, respectively). Concentrations of low-density lipoprotein cholesterol (LDL-C) were calculated from TC, HDL-C, and TG using the Friedewald formula ( Friedewald et al., 1972). Serum insulin was measured by the immunoradiometric method (Insulin IRMA IM3210, Immunotech SA, France), sensitivity was 2.0 mIU/ml, and autoantibodies against oxidized LDL (oLAb) in serum samples were evaluated using ELISA assay (oLAb-ELISA, BI-20032;

To evaluate risk for vascular disease , the lipid ratios (TC/HDL-C, LDL-C/HDL-C, TG/HDL-C) and atherogenic index of plasma [AIP= log10(TG/HDL-C) with TG and HDL-C expressed in molar concentrations] (Dobiášová & Frohlich, 2001, 2004, 2011) were calculated. The homeostatic model assessment (HOMA) was used to estimate insulin resistance according to the method described by Matthews (1998). Insulin resistance score (HOMA-IR) was computed using the following formula: HOMA-IR=glucose (mmol/L)\*insulin (mIU/L)/22.5, with the cut-off point for adolescents less than or equal to 3.16 , as suggested by Keskin et al. (2005). Moreover, in 231 individuals (121 boys and 110 girls) serum adiponectin was measured by an immunoenzymatic method (Human Adiponectin ELISA, BioVendor, cat. no. RD195023100), and serum insulin-like growth factor (IGF-1) was evaluated in 80 adolescents (40 boys and 40 girls) using a commercially available diagnostic kit (IGF-I ELISA, Immunodiagnostic Systems GmbH, sensitivity 3.1

All statistical analyses were performed with STATISTICA 7.1 software (StatSoft, Tulsa, OK, USA). The data reported as means ± SD were tested for homogeneity of variances by using the Levene test, then two-way ANOVA was performed to analyze the effect of gender and the degree of overweight/obesity on the studied variables, followed where appropriate, by the Tukey post-hoc comparisons. Spearman's rank order correlation analysis was performed to assess relationships between selected variables. Additionally, Pearson's Chi-square test of independence was used to evaluate the impact of the degree of overweight/obesity on SBP, serum triglycerides and surrogate markers of insulin resistance (HOMA-IR, TG/HDL-C). To predict the relative contribution of selected metabolic variables to prevalence of over normal SBP, insulin resistance (as assessed by HOMA-IR) and atherogenic potential of plasma (as assessed by AIP) a stepwise multiple regression analysis was applied. The level of

Physical characteristics of individual groups of the participants are presented in Table 1. In the evaluation of between-group differences, the data obtained in 113 adolescents (55 boys and 58 girls) with underweight, in 100 individuals (54 boys and 46 girls) with a tendency to overweight or overweight, and in 113 obese subjects (57 boys and 56 girls) were compared to the data obtained in 179 adolescents (98 boys and 81 girls) with normal weight. Mean

Biomedica GmbH, Wien, Austria) according to Tatzber & Esterbauer (1995).

/g/L, reference range for 12 to 15 y old subjects 142-525 /g/L).

significance of P<0.05 was chosen for all statistical comparisons.

**2.2 Analytical procedures** 

**2.3 Statistics** 

**3. Results** 

### **2. Methods**

#### **2.1 Population sample**

The data were obtained from 505 adolescents, middle school pupils (264 boys and 241 girls) aged 15 years, who were enrolled into a cross- sectional health screening study undertaken by the municipal health care authorities of Katowice (Poland) - in collaboration with our group. In the morning, after an overnight fast, the participants reported to the hospital to undergo a brief general medical examination, including assessment of anthropometric parameters, measurement of blood pressure, and collection of venous blood samples for biochemical analyses. The participants, boys and girls separately, were classified into one of the four groups, according to their Body Mass Index (BMI) score, using the age and gender-specific cutoff points of the BMI percentiles established for 15-years old Polish youth by Palczewska & Nied&wiecka (2002): group A of the underweight (<25 c), group B of the normal weight (25- 75c), group C of the overweight (75-97 c), and group D of the obese (>97 c) individuals (Table 1). Criterion for diagnosis of obesity at BMI 97th percentile was consistent with the WHO references for children 5 to 19 years old (available at http://www.who.growthref). Informed consent was obtained from all adolescents and their parents prior to participation in the study, the protocol of which was approved by the local Ethics Committee.


Table 1. Anthropometric characteristics, and systolic (SBP) and diastolic (DBP) blood pressure of 15-year-old Polish adolescents. Significance of differences *vs.* normal weight individuals: \*p<0.05, \*\*\* p<0.001

#### **2.2 Analytical procedures**

276 Recent Advances in Cardiovascular Risk Factors

The data were obtained from 505 adolescents, middle school pupils (264 boys and 241 girls) aged 15 years, who were enrolled into a cross- sectional health screening study undertaken by the municipal health care authorities of Katowice (Poland) - in collaboration with our group. In the morning, after an overnight fast, the participants reported to the hospital to undergo a brief general medical examination, including assessment of anthropometric parameters, measurement of blood pressure, and collection of venous blood samples for biochemical analyses. The participants, boys and girls separately, were classified into one of the four groups, according to their Body Mass Index (BMI) score, using the age and gender-specific cutoff points of the BMI percentiles established for 15-years old Polish youth by Palczewska & Nied&wiecka (2002): group A of the underweight (<25 c), group B of the normal weight (25- 75c), group C of the overweight (75-97 c), and group D of the obese (>97 c) individuals (Table 1). Criterion for diagnosis of obesity at BMI 97th percentile was consistent with the WHO references for children 5 to 19 years old (available at http://www.who.growthref). Informed consent was obtained from all adolescents and their parents prior to participation in the study,

the protocol of which was approved by the local Ethics Committee.

Underweight (<25 c) X±SD

Variable Percentile ranges (c)

Normal weight (25-75 c) X±SD

No. of cases N=55 N=98 N=54 N=57 Body height, m 1.65\*±0.08 1.71±0.07 1.69±0.09 1.70±0.08 Body mass, kg 45.9\*\*\*±5.2 57.7±6.0 70.3\*\*\*±7.93 82.7\*\*\*±11.6

No. of cases N=58 N=81 N=46 N=56 Body height, m 1.65±0.08 1.66±0.08 1.65±0.09 1.64±0.08 Body mass, kg 46.1\*\*\*±5.1 54.1±6.5 67.2\*\*\*±7.8 79.4\*\*\*±11.8

Table 1. Anthropometric characteristics, and systolic (SBP) and diastolic (DBP) blood pressure of 15-year-old Polish adolescents. Significance of differences *vs.* normal weight

BMI 16.7\*\*\*±0.9 19.7±1.1 24.6\*\*\*±0.9 28.6\*\*\*±3.4 SBP, mm Hg 118.8±10.6 124.9±14.3 132.8\*±14.8 135.7\*\*±15.2 DBP, mm Hg 71.3±8.5 71.6±11.1 73.4±9.2 73.7±10.2

BMI 17,0\*\*\*±0.9 19.6±1.2 24.7\*\*\*±0.8 29.3\*\*\*±3.2 SBP, mm Hg 118.7±12.9 124.8±12.4 124.7±15.7 132.5\*±15.3 DBP, mm Hg 69.4±0.08 74.4±9.2 72.9±11.1 74.8±11.1

Marginally overweight (75-90 c) and overweight (90-97 c) X±SD

Obese (>97 c) X±SD

**2. Methods**

Gender

Boys

Girls

individuals: \*p<0.05, \*\*\* p<0.001

**2.1 Population sample** 

Concentrations of serum glucose, total cholesterol (TC), HDL-cholesterol (HDL-C), triglycerides (TG) were assessed by the enzymatic methods using commercially available diagnostic kits (BioMaxima cat.no. 1-033-0400, 1-023-0400, 1-029-0200, 1-053-0400, respectively). Concentrations of low-density lipoprotein cholesterol (LDL-C) were calculated from TC, HDL-C, and TG using the Friedewald formula ( Friedewald et al., 1972). Serum insulin was measured by the immunoradiometric method (Insulin IRMA IM3210, Immunotech SA, France), sensitivity was 2.0 mIU/ml, and autoantibodies against oxidized LDL (oLAb) in serum samples were evaluated using ELISA assay (oLAb-ELISA, BI-20032; Biomedica GmbH, Wien, Austria) according to Tatzber & Esterbauer (1995).

To evaluate risk for vascular disease , the lipid ratios (TC/HDL-C, LDL-C/HDL-C, TG/HDL-C) and atherogenic index of plasma [AIP= log10(TG/HDL-C) with TG and HDL-C expressed in molar concentrations] (Dobiášová & Frohlich, 2001, 2004, 2011) were calculated. The homeostatic model assessment (HOMA) was used to estimate insulin resistance according to the method described by Matthews (1998). Insulin resistance score (HOMA-IR) was computed using the following formula: HOMA-IR=glucose (mmol/L)\*insulin (mIU/L)/22.5, with the cut-off point for adolescents less than or equal to 3.16 , as suggested by Keskin et al. (2005). Moreover, in 231 individuals (121 boys and 110 girls) serum adiponectin was measured by an immunoenzymatic method (Human Adiponectin ELISA, BioVendor, cat. no. RD195023100), and serum insulin-like growth factor (IGF-1) was evaluated in 80 adolescents (40 boys and 40 girls) using a commercially available diagnostic kit (IGF-I ELISA, Immunodiagnostic Systems GmbH, sensitivity 3.1 /g/L, reference range for 12 to 15 y old subjects 142-525 /g/L).

#### **2.3 Statistics**

All statistical analyses were performed with STATISTICA 7.1 software (StatSoft, Tulsa, OK, USA). The data reported as means ± SD were tested for homogeneity of variances by using the Levene test, then two-way ANOVA was performed to analyze the effect of gender and the degree of overweight/obesity on the studied variables, followed where appropriate, by the Tukey post-hoc comparisons. Spearman's rank order correlation analysis was performed to assess relationships between selected variables. Additionally, Pearson's Chi-square test of independence was used to evaluate the impact of the degree of overweight/obesity on SBP, serum triglycerides and surrogate markers of insulin resistance (HOMA-IR, TG/HDL-C). To predict the relative contribution of selected metabolic variables to prevalence of over normal SBP, insulin resistance (as assessed by HOMA-IR) and atherogenic potential of plasma (as assessed by AIP) a stepwise multiple regression analysis was applied. The level of significance of P<0.05 was chosen for all statistical comparisons.

#### **3. Results**

Physical characteristics of individual groups of the participants are presented in Table 1. In the evaluation of between-group differences, the data obtained in 113 adolescents (55 boys and 58 girls) with underweight, in 100 individuals (54 boys and 46 girls) with a tendency to overweight or overweight, and in 113 obese subjects (57 boys and 56 girls) were compared to the data obtained in 179 adolescents (98 boys and 81 girls) with normal weight. Mean

Adolescent Obesity Predicts Cardiovascular Risk 279

No. of cases N=55 N=98 N=54 N=57 N=264

mg/dL 169.4±25.3 167.2±26.0 170.5±25.1 174.1±24.3 P<0.05 HDL-C, mg/ dL 54.5±5.3 53.5±5.3 54.8±6.1 54.9±4.8 NS LDL-C, mg/ dL 93.3±22.9 92.1±22.0 93.2±22.8 93.0±24.6 NS oLAb, mU/mL 921.6±817.8 947.6±726.9 845.3±849.8 821.1±672.4 NS TG, mg/ dL 108.0±27.3 108.0±22.9 112.3±29.0 130.7\*\*±50.0 P<0.005 TC/HDL-C 3.12±0.44 3.13±0.41 3.12±0.40 3.18±0.42 NS LDL-C/HDL-C 1.72±0.41 1.72±0.38 1.71±0.40 1.70±0.44 NS TG/HDL-C 2.00±0.55 2.02±0.42 2.07±0.59 2.41\*±0.98 P<0.005 AIP -0.074±0.114 -0.063±0.090 -0.059±0.111 -0.007\*±0.117 P<0.01 Glucose, mg/ dL 84.2±11.9 82.2±11.8 83.7±11.8 81.5±10.2 NS Insulin, mIU/L 9.4±7.8 11.6±8.0 13.7±12.3 16.1\*±9.5 P<0.005 HOMA-IR 1.97±1.61 2.38±1.80 2.89±2.82 3.24±1.94 P<0.05

ng/mL (N=121) 14.3\*\*±19.9 9.5±7.7 9.9±6.4 9.4±5.3 P<0.005

(N=40) 287.8±118.6 500.3±261.4 432.8±127.6 420.8±123.3 NS

mg/dL 173.7±23.2 167.0±26.1 175.5±23.9 171.4±22.5 NS HDL-C, mg/dL 56.7±6.1 55.03±5.486.3 55.23±5.14 55.65±5.91 NS LDL-C, mg/dL 95.8±20.9 89.1±23.4 96.8±20.0 89.5±20.7 NS oLAb, mU/mL 877.3±800.0 790.5±688.7 980.5±800.8 787.5±705.2 NS TG, mg/dL 106.0±25.2 114.7±29.0 117.3±25.4 131.6\*±51.6 P<0.05 TC/HDL-C 3.08±0.44 3.05±0.46 3.18±0.34 3.10±0.44 NS LDL-C/HDL-C 1.71±0.39 1.63±0.43 1.75±0.32 1.62±0.39 NS TG/HDL-C 1.89±0.51 2.10±0.54 2.13±0.44 2.41±1.07 P<0.005 AIP -0.097±0.110 -0.051±0.105 -0.041±0.089 -0.009±0.151 P<0.01 Glucose, mg/ dL 81.2±9.7 80.8±10.4 83.7±12.6 83.3±10.8 NS Insulin, mIU/L 8.4±4.2 12.6±12.9 15.5±13.3 18.3±15.1P=0.06 P<0.005 HOMA-IR 1.69±0.90 2.65±2.86 3.29±3.08 3.74±3.11 P<0.05

(N=40) 385.0±76.5 387.5±170.5 398.0±98.5 388.3±60.8 NS Table 2. Serum biochemical parameters in 15-year-old Polish adolescents. Significance of differences *vs.* normal weight individuals: \*p<0.05, \*\* p<0.005, NS-non significant

14.2\*\*±10.1 8.3±4.7 9.0±4.5 11.3±5.5 P<0.01

No. of cases N=58 N=81 N=46 N=56 N=241

Normal weight (25-75 c) X±SD

Percentile ranges (c)

Marginally overweight (75-90 c) and overweight (90-97 c) X±SD

Obese (>97 c) X±SD

Main effect: degree of overweight P value

Gender

Boys

Girls

Variables

Cholesterol (TC),

Adiponectin,

IGF-1, ng/mL

Cholesterol (TC),

Adiponectin, ng/mL (N=110)

IGF-1, ng/mL

Underweight (<25 c) X±SD

values and percentiles of BMI were similar among the groups of boys and girls with various degree of overweight, while the mean values of BMI in both groups of normal-weight teens were very close to the 50 percentile of BMI distribution for 15 year old boys and girls (Palczewska & Nied&wiecka, 2001; WHO Growth reference data for 5 to 19 years, available at http://www.who.growthref).

The readings of systolic and diastolic blood pressure (SBP and DBP) in all groups of adolescents, stratified according to gender and degree of overweight, were compared with the data of the updated blood pressure charts for children and adolescents according to the child's age and height percentile. For the purposes of this study, the following criteria were adopted. The adolescents were classified as normotensive if their systolic BP was <90th percentile, i.e. <130 mm Hg. The measured SBP >90th percentile, which could indicate prehypertension or hypertension, was classified as "over normal". These criteria were conform to the recommendations of the National High Blood Pressure Education Program The Fourth Report on the Diagnosis, Evaluation and Treatment of High Blood Pressure in Children and Adolescents, (2004), and to the guidelines of the IDF 2007 Consensus Statement (Zimmet et al., 2007; Alberti et al.,2006, 2007). There were numerous cases, in particular in overweight or obese boys and in obese girls, when SBP exceeded 136 mm Hg, the level suggesting occurrence of significant hypertension according to blood pressure criteria established by the Task Force report (Blood pressure charts for children and adolescents by age and height percentile*,* 2005; Update on the 1987 Task Force Report on High Blood Pressure in Children and Adolescents. A Working Group report from the National High Blood Pressure Education Program, 1996). Notably, more than a half of obese teens, especially girls, had over normal systolic BP.

Results of biochemical tests obtained in all groups stratified by gender and the degree of overweight are presented in Table 2. Except for a very limited number of cases, fasting blood glucose level, in all groups of boys and girls, did not exceed the upper limit of the normal range (100 mg/dL), while fasting insulin concentration tended toward higher values with growing degree of overweight to reach, in several cases, levels exceeding the upper limit of the reference range of the method used (2,6 – 22 µU/mL) (Table 2, Fig.1). As expected, the prevalence of insulin resistance, as assessed by the HOMA-IR index, increased significantly with the degree of overweight as measured by BMI. In the prevailing number of cases, the fasting serum levels of total cholesterol, HDL-C, LDL-C and calculated lipid ratios (TC /HDL-C, LDL-C / HDL-C) were within the specific reference ranges, while the concentration of TG and the TG/HDL-C ratio were found to reach the markedly higher values only in both groups of obese individuals (Table 2, Fig.1). Except for serum HDL-C, which was higher (p<0.05) in girls, there were no significant inter-gender differences for the remaining tested variables. There was a clear tendency toward higher levels of SBP, insulin, HOMA-IR , triglycerides, and TG/HDL-C ratio with increased degree of overweight ( Fig.1) in adolescent boys and girls, and most of these measures were affected significantly by the degree of overweight (Table 2). It is also worth to note that the degree of overweight/obesity significantly affected the atherogenic index of plasma (AIP), which adopted the lowest (most favorable) levels in the underweight groups, and then increased progressively with rising body mass index (Table 2, Fig.1). No between-group differences were found in titers of autoantibodies against oxidized LDL (oLAb), while individual oLAb titers were very variable, reaching over normal levels (>600 mU/mL) (Pincemail et al., 2000) in several subjects from each group. Similarly, no between-group differences were observed

values and percentiles of BMI were similar among the groups of boys and girls with various degree of overweight, while the mean values of BMI in both groups of normal-weight teens were very close to the 50 percentile of BMI distribution for 15 year old boys and girls (Palczewska & Nied&wiecka, 2001; WHO Growth reference data for 5 to 19 years, available

The readings of systolic and diastolic blood pressure (SBP and DBP) in all groups of adolescents, stratified according to gender and degree of overweight, were compared with the data of the updated blood pressure charts for children and adolescents according to the child's age and height percentile. For the purposes of this study, the following criteria were adopted. The adolescents were classified as normotensive if their systolic BP was <90th percentile, i.e. <130 mm Hg. The measured SBP >90th percentile, which could indicate prehypertension or hypertension, was classified as "over normal". These criteria were conform to the recommendations of the National High Blood Pressure Education Program The Fourth Report on the Diagnosis, Evaluation and Treatment of High Blood Pressure in Children and Adolescents, (2004), and to the guidelines of the IDF 2007 Consensus Statement (Zimmet et al., 2007; Alberti et al.,2006, 2007). There were numerous cases, in particular in overweight or obese boys and in obese girls, when SBP exceeded 136 mm Hg, the level suggesting occurrence of significant hypertension according to blood pressure criteria established by the Task Force report (Blood pressure charts for children and adolescents by age and height percentile*,* 2005; Update on the 1987 Task Force Report on High Blood Pressure in Children and Adolescents. A Working Group report from the National High Blood Pressure Education Program, 1996). Notably, more than a half of obese

Results of biochemical tests obtained in all groups stratified by gender and the degree of overweight are presented in Table 2. Except for a very limited number of cases, fasting blood glucose level, in all groups of boys and girls, did not exceed the upper limit of the normal range (100 mg/dL), while fasting insulin concentration tended toward higher values with growing degree of overweight to reach, in several cases, levels exceeding the upper limit of the reference range of the method used (2,6 – 22 µU/mL) (Table 2, Fig.1). As expected, the prevalence of insulin resistance, as assessed by the HOMA-IR index, increased significantly with the degree of overweight as measured by BMI. In the prevailing number of cases, the fasting serum levels of total cholesterol, HDL-C, LDL-C and calculated lipid ratios (TC /HDL-C, LDL-C / HDL-C) were within the specific reference ranges, while the concentration of TG and the TG/HDL-C ratio were found to reach the markedly higher values only in both groups of obese individuals (Table 2, Fig.1). Except for serum HDL-C, which was higher (p<0.05) in girls, there were no significant inter-gender differences for the remaining tested variables. There was a clear tendency toward higher levels of SBP, insulin, HOMA-IR , triglycerides, and TG/HDL-C ratio with increased degree of overweight ( Fig.1) in adolescent boys and girls, and most of these measures were affected significantly by the degree of overweight (Table 2). It is also worth to note that the degree of overweight/obesity significantly affected the atherogenic index of plasma (AIP), which adopted the lowest (most favorable) levels in the underweight groups, and then increased progressively with rising body mass index (Table 2, Fig.1). No between-group differences were found in titers of autoantibodies against oxidized LDL (oLAb), while individual oLAb titers were very variable, reaching over normal levels (>600 mU/mL) (Pincemail et al., 2000) in several subjects from each group. Similarly, no between-group differences were observed

at http://www.who.growthref).

teens, especially girls, had over normal systolic BP.


Table 2. Serum biochemical parameters in 15-year-old Polish adolescents. Significance of differences *vs.* normal weight individuals: \*p<0.05, \*\* p<0.005, NS-non significant

Adolescent Obesity Predicts Cardiovascular Risk 281

The analysis carried out to assess the prevalence ( %) of adolescents with over normal values of SBP (>130 mm Hg), serum triglycerides (> 150 mg/dL) (Zimmet et al., 2007), HOMA-IR (>3.16) (Keskin et al., 2005), and TG/HDL-C ratio (>3.0) (McLaughlin et al., 2003; Hannon et al., 2006) revealed a rising number of individuals fulfilling these criteria with an increasing degree of overweight (Table 3). Noteworthy, Pearson's chi-square test showed significant increases in the prevalence (%) of individuals with over normal levels of these parameters

> Normal weight (25-75 c)

B+G 34.5 13.4 31.1 40.0 56.9 48.5 <10-5 B 36.2 13.0 32.3 50.0 52.8 24.1 <10-4 G 32.8 13.8 29.6 28.3 60.7 30.1 <10-5

B+G 22.5 8.0 19.6 25.5 39.3 33.2 <10-5 B 21.7 10.9 20.1 22.6 33.3 8.4 <0.05 G 23.4 5.2 18.5 28.9 45.5 27.5 <10-5

B+G 10.7 6.2 6.7 7.0 24.8 30.3 <10-5 B 9.5 7.3 3.1 7.4 24.6 20.4 <0.0005 G 12.0 5.2 11.1 6.5 25.0 12.9 <0.005

B+G 6.3 4.4 3.4 5.0 14.2 15.3 <0.005 B 7.2 5.5 2.1 7.4 17.5 13.3 <0.005 G 5.4 3.5 5.0 2.2 10.7 4.5 NS

Table 3. Prevalence (%) of 15 year-old boys (B) and girls (G) with over normal values of

In order to assess the impact of the degree of overweight on selected biochemical parameters and biomarkers of atherogenic risk, the Spearman rank correlation coefficients were computed, and selected statistically significant associations are summarized in Tables 4 and 5. As expected, the BMI and serum triglycerides correlated positively with systolic and diastolic blood pressure, insulin, and HOMA-IR. Serum adiponectin correlated negatively with lipid ratios (TG/HDL-C, TC/HDL-C, LDL-C/HDL-C), while it was positively associated with HDL-C. The oLAb titers correlated positively with serum LDL-C

The important finding was that the atherogenic index of plasma (AIP) was positively correlated with the BMI, systolic and diastolic blood pressure, TG, total cholesterol, common lipid ratios (LDL-C/HDL-C, TC/HDL-C), insulin, HOMA-IR, but negatively associated with HDL-C and adiponectin (Table 5). Although these relationships do not infer casual

systolic blood pressure (SBP), HOMA-IR, triglycerides (TG), and TG/HDL-C.

Marginally overweight (75-90 c) and overweight (90-97 c)

Obese (>97c)

Pearson's Chi-square (df=3)

\*2 P value

Prevalence (%) of individuals with over normal values of SBP, HOMA-IR, TG, and TG/HDL-C ratio

> Underweight (<25 c)

with raising BMI.

SBP >130 mmHg

TG >150 mg/dL

C > 3.0

HOMA-IR >3,16

TG/HDL-

and LDL-C/HDL-C ratio (Table 4).

Group: Boys (B) and/or girls (G)

in serum IGF-1, there was only a slight tendency toward higher IGF-1 levels in boys and the lowest values in underweight individuals, although both effects did not reach statistical significance. Serum adiponectin in both groups of overweight or obese individuals was close to the levels recorded in normal weight teens, but the highest concentrations of this adipokine, significantly (p<0.05) different from the values recorded in normal weight individuals, were found in the underweight groups (Table 2, Fig1).

Fig. 1. The impact of the degree of overweight/obesity on systolic blood pressure (SBP), HOMA-IR, serum concentration of triglycerides (TG), TG/HDL ratio, insulin and adiponectin levels, and atherogenic index of plasma (AIP) in 15-year-old Polish adolescents with underweight (A), normal weight (B), overweight (C) and obesity (D). Significance of differences *vs.* individuals with normal weight: \*p<0.05,\*\* p<0.01, \*\*\*p<0.001

in serum IGF-1, there was only a slight tendency toward higher IGF-1 levels in boys and the lowest values in underweight individuals, although both effects did not reach statistical significance. Serum adiponectin in both groups of overweight or obese individuals was close to the levels recorded in normal weight teens, but the highest concentrations of this adipokine, significantly (p<0.05) different from the values recorded in normal weight

Fig. 1. The impact of the degree of overweight/obesity on systolic blood pressure (SBP), HOMA-IR, serum concentration of triglycerides (TG), TG/HDL ratio, insulin and

differences *vs.* individuals with normal weight: \*p<0.05,\*\* p<0.01, \*\*\*p<0.001

8,9 9,5 10,2

**ABCD**

14,2

\*

1,83

16,9 19,7

\*\*\*

**HOMA**

**BMI, kg/m2**

**Adiponectin, ng/mL**

**1 1,5 2 2,5 3 3,5 4**

**TG / HDL**

adiponectin levels, and atherogenic index of plasma (AIP) in 15-year-old Polish adolescents with underweight (A), normal weight (B), overweight (C) and obesity (D). Significance of

**-0,2**

**-0,15**

**-0,1**

**-0,05**

**AIP**

**0**

**Insulin, mU/L**

2,41

\*

**TG, mg/dl**

**SBP, mm Hg**

\*

\*

29

\*\*\*

**-0,086**

**-0,058 -0,051**

8,9 12,1 14,5 17,2

**ABCD**

106,9 <sup>111</sup> 114,6

**ABCD**

118,7 126,8 129,1 134,0

**ABCD**

**ABCD**

**-0,008**

131,1

\*\*

\*\*\*

\*\*

individuals, were found in the underweight groups (Table 2, Fig1).

24,6

\*\*\*

2,49 3,08 3,49

**ABCD**

1,95 2,06 2,1

**ABCD**

**ABCD**

The analysis carried out to assess the prevalence ( %) of adolescents with over normal values of SBP (>130 mm Hg), serum triglycerides (> 150 mg/dL) (Zimmet et al., 2007), HOMA-IR (>3.16) (Keskin et al., 2005), and TG/HDL-C ratio (>3.0) (McLaughlin et al., 2003; Hannon et al., 2006) revealed a rising number of individuals fulfilling these criteria with an increasing degree of overweight (Table 3). Noteworthy, Pearson's chi-square test showed significant increases in the prevalence (%) of individuals with over normal levels of these parameters with raising BMI.


Table 3. Prevalence (%) of 15 year-old boys (B) and girls (G) with over normal values of systolic blood pressure (SBP), HOMA-IR, triglycerides (TG), and TG/HDL-C.

In order to assess the impact of the degree of overweight on selected biochemical parameters and biomarkers of atherogenic risk, the Spearman rank correlation coefficients were computed, and selected statistically significant associations are summarized in Tables 4 and 5. As expected, the BMI and serum triglycerides correlated positively with systolic and diastolic blood pressure, insulin, and HOMA-IR. Serum adiponectin correlated negatively with lipid ratios (TG/HDL-C, TC/HDL-C, LDL-C/HDL-C), while it was positively associated with HDL-C. The oLAb titers correlated positively with serum LDL-C and LDL-C/HDL-C ratio (Table 4).

The important finding was that the atherogenic index of plasma (AIP) was positively correlated with the BMI, systolic and diastolic blood pressure, TG, total cholesterol, common lipid ratios (LDL-C/HDL-C, TC/HDL-C), insulin, HOMA-IR, but negatively associated with HDL-C and adiponectin (Table 5). Although these relationships do not infer casual

Adolescent Obesity Predicts Cardiovascular Risk 283

Multiple studies (Aggoun, 2007; Carnethon et al., 2004; Davis et al., 2001; Dietz, 1998; Dietz & Robinson, 2005; Franks et al., 2010; Han & Lean, 2006; Magnussen et al., 2010; Morrison et al., 2007; Must et al., 1992; Shengxu et al., 2003; Wang et al., 2008; Whitaker et al., 1997) have demonstrated that obesity at the developmental age poses an important risk factor for the development of cardiovascular diseases in youth and adulthood. The main objective of this cross-sectional study was screening for cardiovascular risk factors in a locally living urban population of 15-year old adolescents that could be easily applied in the early diagnosis of metabolic disorders in overweight and obese youth. The following independent risk factors were taken into consideration : degree of overweight/obesity, over normal blood pressure, insulin resistance, dyslipidemia, and adiponectin level (Aggoun, 2007; McLaughlin et al., 2003; Hannon et al., 2006; Kaelber & Pickett, 2009; McNiece et al., 2007;The Fourth report on the Diagnosis, Evaluation, and Treatment of High Blood Pressure in Children and

The important finding of this study is that over normal SBP and DBP values were strongly associated with body mass index (BMI). Namely, the prevalence of over normal SBP (i.e.>130 mmHg) was twice as high in the obese, compared with normal-weight teens, and four times higher in the obese than in the underweight groups, but there was no significant difference in the prevalence of over normal SBP between the two sexes. The marked increase of prevalence of over normal SBP with the degree of overweight in the participants of our study strongly supports the view that obesity is becoming a significant health issue in the young Polish population. Our observations fully support previous studies among various ethnic and racial groups that have shown the higher prevalence of abnormally high blood pressure in obese, compared with non-obese children and adolescents (Mc Niece et al. ,

Obesity-related elevated blood pressure in adolescence, is known to increase the risk of hypertension in adulthood (Cook et al., 2003; The Fourth Report on the Diagnosis, Evaluation and Treatment of High Blood Pressure in Children and Adolescents, 2004; Williams et al., 2002*)*. Increased blood pressure is also considered the independent predictor of atherosclerosis, hyperplasia and hypertrophy of vascular smooth muscle, the factors

There is a large body of evidence that obesity is associated with metabolic pathology such as impaired glucose tolerance, insulin resistance, and dyslipidemia. Studies using specimens collected at autopsy (McGill, 1997; Berenson et al., 1998), or using non-invasive assessment of carotid intima-media thickness (IMT) by ultrasonography (Berenson, 2002; Davis et al., 2001; Raitakari et al., 2003; Shengxu et al., 2003) demonstrated that the atherosclerotic process begins in childhood and adolescence and may track into adulthood. In this regard, the occurrence of highly statistically significant positive correlations between body mass index (BMI) and serum levels of triglycerides and insulin, or surrogate markers of insulin resistance (HOMA-IR and TG/HDL-C) fully confirmed the view that obesity occurring during developmental age raises the risk of developing type 2 diabetes and cardiovascular

In the present study, the assessment of risk for CVD and insulin resistance in 15-year old subjects was based on the analysis of serum biomarkers, such as total cholesterol (TC) and

2007*;* Sorof &Daniels, 2002; Verma et al., 1994; Macedo et al., 1997*)*.

affecting arterial stiffness (Aggoun, 2007).

disease in the young .

**4. Discussion** 

Adolescents, 2004).

dependence between the variables studied, they may suggest that there is a cluster of metabolically linked risk factors that, along with the hypertension and degree of obesity, determines the risk of atherosclerotic vascular disease. This hypothesis was fully supported by our results of a stepwise multivariate regression analyses that have shown that the main predictors of insulin resistance, as assessed by HOMA-IR, were concentrations of insulin and TG or TG/HDL-C ratio (R2=0.96, p<0.0001), while concentrations of TG, LDL-C and of total cholesterol (TC) accounted for 95% (R2=0.95, p<0.0001) of atherogenic potential of plasma (AIP) variance, and the most important predictors of systolic blood pressure (SBP) were BMI, TG and LDL/HDL ratio (R2=0.16, p<0.001).


Table 4. Spearman's rank order correlation coefficients between selected anthropometric and biochemical variables in 15-year- old Polish adolescents.


Table 5. Spearman's rank order correlation coefficients between atherogenic index of plasma (AIP) and selected physical and biochemical variables in 15-year- old Polish adolescents.

#### **4. Discussion**

282 Recent Advances in Cardiovascular Risk Factors

dependence between the variables studied, they may suggest that there is a cluster of metabolically linked risk factors that, along with the hypertension and degree of obesity, determines the risk of atherosclerotic vascular disease. This hypothesis was fully supported by our results of a stepwise multivariate regression analyses that have shown that the main predictors of insulin resistance, as assessed by HOMA-IR, were concentrations of insulin and TG or TG/HDL-C ratio (R2=0.96, p<0.0001), while concentrations of TG, LDL-C and of total cholesterol (TC) accounted for 95% (R2=0.95, p<0.0001) of atherogenic potential of plasma (AIP) variance, and the most important predictors of systolic blood pressure (SBP)

Variables N R p BMI & SBP 498 0.332 P<10-6 BMI & DBP 498 0.128 P<0.005 BMI & insulin 502 0.377 P<10-6 BMI & HOMA-IR 502 0.360 P<10-6 BMI & TG 505 0.189 P<10-4 BMI & TG/HDL-C 505 0.174 P<10-4 TG & SBP 498 0.184 P<10-4 TG & DBP 498 0.170 p<0.0005 TG & insulin 502 0.272 P<10-6 TG & cholesterol 505 0.343 P<10-6 TG & HOMA-IR 502 0.266 P<10-6 TG/HDL-C & HOMA-IR 502 0.262 P<10-6 Adiponectin & HDL-C 231 0.308 P<10-4 Adiponectin & TG/HDL-C 231 -0.194 P<0.005 Adiponeectin & TC/HDL-C 231 -0.208 P<0.005 Adiponectin &LDL/HDL-C 231 -0.198 P<0.005 Adiponectin & oLAb 231 -0.169 P<0.05 oLAb & LDL-C 438 0.106 P<0.05 oLAb & LDL-C/HDL-C 438 0.129 P<0.01 Table 4. Spearman's rank order correlation coefficients between selected anthropometric and

Variables N R p AIP & BMI 505 0.174 P<0.0001 AIP & SBP 498 0.139 P<0.005 AIP & DBP 498 0.104 p<0.05 AIP & TG 505 0.907 P<10-6 AIP & HDL-C 505 -0.297 P<10-6 AIP & INS 502 0.272 P<10-6 AIP & HOMA-IR 502 0.262 P<10-6 AIP & adiponectin 231 -0.194 P<0.005 Table 5. Spearman's rank order correlation coefficients between atherogenic index of plasma (AIP) and selected physical and biochemical variables in 15-year- old Polish adolescents.

were BMI, TG and LDL/HDL ratio (R2=0.16, p<0.001).

biochemical variables in 15-year- old Polish adolescents.

Multiple studies (Aggoun, 2007; Carnethon et al., 2004; Davis et al., 2001; Dietz, 1998; Dietz & Robinson, 2005; Franks et al., 2010; Han & Lean, 2006; Magnussen et al., 2010; Morrison et al., 2007; Must et al., 1992; Shengxu et al., 2003; Wang et al., 2008; Whitaker et al., 1997) have demonstrated that obesity at the developmental age poses an important risk factor for the development of cardiovascular diseases in youth and adulthood. The main objective of this cross-sectional study was screening for cardiovascular risk factors in a locally living urban population of 15-year old adolescents that could be easily applied in the early diagnosis of metabolic disorders in overweight and obese youth. The following independent risk factors were taken into consideration : degree of overweight/obesity, over normal blood pressure, insulin resistance, dyslipidemia, and adiponectin level (Aggoun, 2007; McLaughlin et al., 2003; Hannon et al., 2006; Kaelber & Pickett, 2009; McNiece et al., 2007;The Fourth report on the Diagnosis, Evaluation, and Treatment of High Blood Pressure in Children and Adolescents, 2004).

The important finding of this study is that over normal SBP and DBP values were strongly associated with body mass index (BMI). Namely, the prevalence of over normal SBP (i.e.>130 mmHg) was twice as high in the obese, compared with normal-weight teens, and four times higher in the obese than in the underweight groups, but there was no significant difference in the prevalence of over normal SBP between the two sexes. The marked increase of prevalence of over normal SBP with the degree of overweight in the participants of our study strongly supports the view that obesity is becoming a significant health issue in the young Polish population. Our observations fully support previous studies among various ethnic and racial groups that have shown the higher prevalence of abnormally high blood pressure in obese, compared with non-obese children and adolescents (Mc Niece et al. , 2007*;* Sorof &Daniels, 2002; Verma et al., 1994; Macedo et al., 1997*)*.

Obesity-related elevated blood pressure in adolescence, is known to increase the risk of hypertension in adulthood (Cook et al., 2003; The Fourth Report on the Diagnosis, Evaluation and Treatment of High Blood Pressure in Children and Adolescents, 2004; Williams et al., 2002*)*. Increased blood pressure is also considered the independent predictor of atherosclerosis, hyperplasia and hypertrophy of vascular smooth muscle, the factors affecting arterial stiffness (Aggoun, 2007).

There is a large body of evidence that obesity is associated with metabolic pathology such as impaired glucose tolerance, insulin resistance, and dyslipidemia. Studies using specimens collected at autopsy (McGill, 1997; Berenson et al., 1998), or using non-invasive assessment of carotid intima-media thickness (IMT) by ultrasonography (Berenson, 2002; Davis et al., 2001; Raitakari et al., 2003; Shengxu et al., 2003) demonstrated that the atherosclerotic process begins in childhood and adolescence and may track into adulthood. In this regard, the occurrence of highly statistically significant positive correlations between body mass index (BMI) and serum levels of triglycerides and insulin, or surrogate markers of insulin resistance (HOMA-IR and TG/HDL-C) fully confirmed the view that obesity occurring during developmental age raises the risk of developing type 2 diabetes and cardiovascular disease in the young .

In the present study, the assessment of risk for CVD and insulin resistance in 15-year old subjects was based on the analysis of serum biomarkers, such as total cholesterol (TC) and

Adolescent Obesity Predicts Cardiovascular Risk 285

It has been suggested that accelerated atherosclerosis in diabetes may be due to an enhanced oxidative modification of LDL, and of sdLDL in particular ( Uusitupa et al., 1996). It was also suggested that LDL modified by glycation, as it may occur in diabetic patients, may be more susceptible to oxidation ( Jenkins et al., 2004). Oxidatively modified LDL (oxLDL) that are strongly atherogenic may induce the immune response associated with enhanced production of specific autoantibodies against oxLDL (oLAb) (Steinerová et al., 2001; Shoenfeld et al., 2004). In the present study, the oLAb titers, the mean values of which were within the reference range set by Pincemail et al. (2000) for the method used in our study*,*  appeared to be independent of the degree of obesity and HOMA-IR estimated insulin resistance but, as could be predicted, they were correlated with serum LDL-C and the LDL-C/HDL-C ratio. These results are fully consistent with those reported by Uusitupa et al (1996) and Jenkins et al. (2004) who found that autoantibodies against oxLDL indicate the presence of oxidatively modified LDL *in vivo*, but their titers do not seem to predict

Research into the mechanisms and mediators of obesity-related pathologies also called attention to the involvement of the adipose tissue in the regulation of energy balance by a number of adipose tissue –derived peptide hormones (adipocytokines), such as leptin, adiponectin and resistin, all seem to be involved in insulin resistance associated with obesity. We focused our attention on adiponectin, and we found that the highest circulating levels of this adipocytokine were found in the underweight groups of teens. This observation is consistent with the previously described effects of weight-loss on increases in serum adiponectin (Elloumi et al., 2009). Interestingly, high levels of serum adiponectin have been reported in anorectic patients (Nedvidkova et al., 2005; Pannacciulli et al., 2003). Adiponectin is recognized for its beneficial antiatherogenic, antidiabetogenic, and anti inflammatory action, mainly due to its ability to improve insulin sensitivity (Körner et al., 2007; Cruz et al., 2004; Nedvidková et al., 2005), to inhibit TNF-α mediated adherence of monocytes, to reduce their phagocytic activity, to suppress the accumulation of modified lipoproteins in the vascular wall , and to stimulate endothelial NO production (Ekmekci & Elmekci, 2006). Statistically significant positive correlation between serum adiponectin and HDL-cholesterol, and the negative associations with common lipid ratios (TC/HDL-C and LDL-C/HDL-C) and AIP seem to confirm the beneficial antiatherogenic effect of this hormone, and suggest low risk of dyslipidemia in adolescents with low body weight. Of note, the associations of adiponectin with HOMA-IR, LDL-cholesterol, triglycerides and insulin were also negative, but they did not reach statistical significance, most likely due to

In the present study we have also made a preliminary attempt to investigate the association between serum IGF-1 and obesity-related insulin resistance in adolescent subjects. The data from the literature suggest that IGF-1 may have beneficial effect on glucose homeostasis, due to its glucose lowering and insulin sensitizing actions (Kabir et al., 2010). Adult studies suggest that lower IGF-1 level in childhood predict increased risk for developing insulin resistance and type 2 diabetes (Dunger et al., 2003). Moreover, previous studies reported that circulating fasting free IGF-1 were higher in obese subjects compared with normal weight controls, whereas total IGF-1 were not significantly different between the groups (Nam et al., 1997). Our results on obesity- related changes in serum total IGF-1, that found neither between group differences nor significant associations between total IGF-1 and

selected indices of insulin resistance, are consistent with the latter findings.

cardiovascular morbidity or carotid IMT.

the limited number (N=231) of adiponectin data.

its lipoprotein fractions (LDL-C and HDL-C), triglycerides (TG), insulin, and glucose. Interestingly, no between group differences in serum concentrations of total cholesterol, LDL- and HDL cholesterol, lipid ratios (TC/HDL-C and LDL-C/HDL-C) and glucose were found. The mean values of these variables did not exceed the cut-off points recommended by the American Heart Association (Kavey et al., 2003) set at 170 mg/dL for TC, 110 mg/dL for LDL-C and 35 mg/dL for HDL-C. In this respect, the results of the present study are consistent with those previously reported by Lee at al. (2009), who found that the BMI percentiles do not provide effective discrimination for distinguishing children with abnormal TC and LDL-C levels. On the contrary, compared with the control groups of normal weight teens, the mean concentration of serum TG was significantly higher in the obese boys and girls, although it did not exceed the borderline level (150 mg/dL) as defined by the IDF guidelines (Alberti et al., 2006, 2007; Zimmet et al. , 2007). A similar trend was observed for TG/HDL-C ratio, considered a simple metabolic marker for identification of overweight individuals who are insulin resistant (McLaughlin et al., 2003; Hannon et al., 2006; Brehm et al., 2004).

The usefulness of the TG/HDL-C ratio as a marker of insulin resistance was additionally supported by its significant correlation with HOMA-IR. It should be stressed, however, that not all overweight and obese teens could be diagnosed as insulin resistant. Noteworthy, the prevalence of individuals with HOMA-IR exceeding the cut-off point (3.16) for adolescents (Keskin et al., 2005) rose with degree of overweight, and reached the highest levels in the obese groups, but only in about 40% of obese teens it exceeded the borderline level. Noteworthy is that the risk of becoming insulin resistant was higher among obese girls than among obese boys. Less marked increases were observed for TG or TG/HDL-C levels, as only in about 25% or 15 % of obese individuals they exceeded the respective cutoff points. Significantly higher serum levels of TG in obese individuals may indicate increased risk for atherosclerosis and obesity-related insulin resistance. This hypothesis is strongly supported by our finding of significant correlations between TG and atherogenic index of plasma (AIP), total cholesterol (TC), insulin, and HOMA-IR. Existing evidence suggests that serum TG is a strong predictor of CVD (Gotto, 1998), although it was found that TG can regulate lipoprotein interactions, but is not an independent risk factor.

This opinion is supported by the evidence that hypertriglyceridemia is associated with predominance of more atherogenic small dense LDL particles (sdLDL) (Packard, 1996). The increased risk may be related to easier penetration of sdLDL into the sub-endothelial space, their lower binding affinity to the LDL receptor and a higher susceptibility to oxidation. Although various techniques are available for direct measurement of LDL particle size distribution (Superko, 1996) , an indirect (surrogate) method may be used for evaluation of the atherogenicity of plasma lipoproteins based on the assessment of the Atherogenic Index of Plasma (AIP), calculated as log(TG/HDL-C) (with TG and HDL-C expressed in molar concentrations ) (Dobiášová & Frohlich , 2001). It is well documented that changes in AIP may predict LDL particle size, and provide reliable information about the atherogenicity of plasma (Dobiášová & Frohlich, 2001; Dobiášová, 2004; Dobiášová et al., 2011; Onat et al., 2010). One of the most important findings of the present study is that there was a statistically significant trend toward higher values of AIP with raising degree of overweight, which supports the view that obesity is the main predictor of the CVD risk, and that atherogenic index of plasma (AIP) is a suitable tool to evaluate the risk.

its lipoprotein fractions (LDL-C and HDL-C), triglycerides (TG), insulin, and glucose. Interestingly, no between group differences in serum concentrations of total cholesterol, LDL- and HDL cholesterol, lipid ratios (TC/HDL-C and LDL-C/HDL-C) and glucose were found. The mean values of these variables did not exceed the cut-off points recommended by the American Heart Association (Kavey et al., 2003) set at 170 mg/dL for TC, 110 mg/dL for LDL-C and 35 mg/dL for HDL-C. In this respect, the results of the present study are consistent with those previously reported by Lee at al. (2009), who found that the BMI percentiles do not provide effective discrimination for distinguishing children with abnormal TC and LDL-C levels. On the contrary, compared with the control groups of normal weight teens, the mean concentration of serum TG was significantly higher in the obese boys and girls, although it did not exceed the borderline level (150 mg/dL) as defined by the IDF guidelines (Alberti et al., 2006, 2007; Zimmet et al. , 2007). A similar trend was observed for TG/HDL-C ratio, considered a simple metabolic marker for identification of overweight individuals who are insulin resistant (McLaughlin et al., 2003; Hannon et al.,

The usefulness of the TG/HDL-C ratio as a marker of insulin resistance was additionally supported by its significant correlation with HOMA-IR. It should be stressed, however, that not all overweight and obese teens could be diagnosed as insulin resistant. Noteworthy, the prevalence of individuals with HOMA-IR exceeding the cut-off point (3.16) for adolescents (Keskin et al., 2005) rose with degree of overweight, and reached the highest levels in the obese groups, but only in about 40% of obese teens it exceeded the borderline level. Noteworthy is that the risk of becoming insulin resistant was higher among obese girls than among obese boys. Less marked increases were observed for TG or TG/HDL-C levels, as only in about 25% or 15 % of obese individuals they exceeded the respective cutoff points. Significantly higher serum levels of TG in obese individuals may indicate increased risk for atherosclerosis and obesity-related insulin resistance. This hypothesis is strongly supported by our finding of significant correlations between TG and atherogenic index of plasma (AIP), total cholesterol (TC), insulin, and HOMA-IR. Existing evidence suggests that serum TG is a strong predictor of CVD (Gotto, 1998), although it was found that TG can regulate

This opinion is supported by the evidence that hypertriglyceridemia is associated with predominance of more atherogenic small dense LDL particles (sdLDL) (Packard, 1996). The increased risk may be related to easier penetration of sdLDL into the sub-endothelial space, their lower binding affinity to the LDL receptor and a higher susceptibility to oxidation. Although various techniques are available for direct measurement of LDL particle size distribution (Superko, 1996) , an indirect (surrogate) method may be used for evaluation of the atherogenicity of plasma lipoproteins based on the assessment of the Atherogenic Index of Plasma (AIP), calculated as log(TG/HDL-C) (with TG and HDL-C expressed in molar concentrations ) (Dobiášová & Frohlich , 2001). It is well documented that changes in AIP may predict LDL particle size, and provide reliable information about the atherogenicity of plasma (Dobiášová & Frohlich, 2001; Dobiášová, 2004; Dobiášová et al., 2011; Onat et al., 2010). One of the most important findings of the present study is that there was a statistically significant trend toward higher values of AIP with raising degree of overweight, which supports the view that obesity is the main predictor of the CVD risk, and that

lipoprotein interactions, but is not an independent risk factor.

atherogenic index of plasma (AIP) is a suitable tool to evaluate the risk.

2006; Brehm et al., 2004).

It has been suggested that accelerated atherosclerosis in diabetes may be due to an enhanced oxidative modification of LDL, and of sdLDL in particular ( Uusitupa et al., 1996). It was also suggested that LDL modified by glycation, as it may occur in diabetic patients, may be more susceptible to oxidation ( Jenkins et al., 2004). Oxidatively modified LDL (oxLDL) that are strongly atherogenic may induce the immune response associated with enhanced production of specific autoantibodies against oxLDL (oLAb) (Steinerová et al., 2001; Shoenfeld et al., 2004). In the present study, the oLAb titers, the mean values of which were within the reference range set by Pincemail et al. (2000) for the method used in our study*,*  appeared to be independent of the degree of obesity and HOMA-IR estimated insulin resistance but, as could be predicted, they were correlated with serum LDL-C and the LDL-C/HDL-C ratio. These results are fully consistent with those reported by Uusitupa et al (1996) and Jenkins et al. (2004) who found that autoantibodies against oxLDL indicate the presence of oxidatively modified LDL *in vivo*, but their titers do not seem to predict cardiovascular morbidity or carotid IMT.

Research into the mechanisms and mediators of obesity-related pathologies also called attention to the involvement of the adipose tissue in the regulation of energy balance by a number of adipose tissue –derived peptide hormones (adipocytokines), such as leptin, adiponectin and resistin, all seem to be involved in insulin resistance associated with obesity. We focused our attention on adiponectin, and we found that the highest circulating levels of this adipocytokine were found in the underweight groups of teens. This observation is consistent with the previously described effects of weight-loss on increases in serum adiponectin (Elloumi et al., 2009). Interestingly, high levels of serum adiponectin have been reported in anorectic patients (Nedvidkova et al., 2005; Pannacciulli et al., 2003). Adiponectin is recognized for its beneficial antiatherogenic, antidiabetogenic, and anti inflammatory action, mainly due to its ability to improve insulin sensitivity (Körner et al., 2007; Cruz et al., 2004; Nedvidková et al., 2005), to inhibit TNF-α mediated adherence of monocytes, to reduce their phagocytic activity, to suppress the accumulation of modified lipoproteins in the vascular wall , and to stimulate endothelial NO production (Ekmekci & Elmekci, 2006). Statistically significant positive correlation between serum adiponectin and HDL-cholesterol, and the negative associations with common lipid ratios (TC/HDL-C and LDL-C/HDL-C) and AIP seem to confirm the beneficial antiatherogenic effect of this hormone, and suggest low risk of dyslipidemia in adolescents with low body weight. Of note, the associations of adiponectin with HOMA-IR, LDL-cholesterol, triglycerides and insulin were also negative, but they did not reach statistical significance, most likely due to the limited number (N=231) of adiponectin data.

In the present study we have also made a preliminary attempt to investigate the association between serum IGF-1 and obesity-related insulin resistance in adolescent subjects. The data from the literature suggest that IGF-1 may have beneficial effect on glucose homeostasis, due to its glucose lowering and insulin sensitizing actions (Kabir et al., 2010). Adult studies suggest that lower IGF-1 level in childhood predict increased risk for developing insulin resistance and type 2 diabetes (Dunger et al., 2003). Moreover, previous studies reported that circulating fasting free IGF-1 were higher in obese subjects compared with normal weight controls, whereas total IGF-1 were not significantly different between the groups (Nam et al., 1997). Our results on obesity- related changes in serum total IGF-1, that found neither between group differences nor significant associations between total IGF-1 and selected indices of insulin resistance, are consistent with the latter findings.

Adolescent Obesity Predicts Cardiovascular Risk 287

Bibbins-Domingo, K., Coxson, P., Pletcher, M.J., Lightwood, J. & Goldman, L. (2007)

Blood pressure charts for children and adolescents by age and height percentile (in Polish)

Brehm ,A., Pfeiler, G., Pacini, G., Vierhapper, H. & Roden, M. (2004) Relationship between serum lipoprotein ratios and insulin resistance in obesity *Clin Chem* 50:2316-2322 Carnethon, M.R., Loria, C.M., Hill, J.O., Sidney, S., Savage P.J. & Liu, K. (2004) Risk factors

Cook, S., Weitzman,M., Auinger, P., Nguyen, M. & Dietz, W.H. (2003) Prevalence of a

Cruz, M., Garcia-Macedo. R., Garcia-Valerio, Y. , Gutierez,M., Medina-Navarro, R., Duran,

Davis, P.H., Dawson, J.D., Riley,W.A. & Lauer, RM. (2001) Carotid intimal-medial thickness

Dietz W.H. & Robinson T.N. (2005) Overweight children and adolescents. *N Engl J Med*

Dietz W.H. (1998) Health consequences of obesity in youth: childhood predictors of adult

Dobiášová, M. & Frohlich, J. (2001) The plasma parameter log(TG/HDL-C) as an

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#### **4.1 Conclusions**

We seem to be authorized to conclude that:


#### **5. Acknowledgment**

A preliminary report of this work was presented in the poster session of the conference "Nutrition and physical activity in inhibiting the aging process" in Wroclaw (4-6 November, 2010). This study was partially supported by statutory funding from the Academy of Physical Education, Katowice, Poland.

#### **6. References**


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653-659

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**15** 

**Peculiarities of Coronary Artery Disease in Athletes** 

*2Hopital Foch, Suresnes* 

*1,2,4,5France 3Maroc* 

Halna du Fretay Xavier1,2, Akoudad Hafid3, Hamadou Ouceyni2 and Benhamer Hakim2,4,5

*3Centre Hospitalier Universitaire Hassan II, Fez 4Hôpital Européen de Paris la Roseraie, Aubervilliers* 

*5Institut Cardiovasculaire Paris Sud, Massy* 

*1Centre Hospitalier Universitaire Bichat Claude Bernard, Paris,* 

Intense physical activity increases the risk of acute coronary syndrome but regular physical

Sport related myocardial infarctions (SMI) are rare but serious. They present angiographic features that explain some clinical characteristics, the limits of the detection of coronary disease in this population and encourage decision making in order to have a better regulation of sport

However, athletes could be asymptomatic despite tight coronary stenosis as in the three clinical cases presented. The reasons for the asymptomatic nature of these coronary diseases are probably due to cardiovascular adaptation to regular training. These athletes are at risk of major cardiac events and the place of stress testing remains important in this population.

We report a retrospective study of twelve cases of SMI. All patients had myocardial infarction (MI) defined as ischemic symptoms and ST segment elevation on ECG. MI was considered to be related to sport if it occurred during sport with vigorous exertions (METs > or = 6) or within 2 hours afterward according to the definition of Von Klot et al (Von Klot et

A total of twelve patients are studied, all men. The mean age is 47.7 years (24 to 64). Eight of these patients considered themselves as insufficiently trained. Chest pain is the most

training is a cardio vascular protective factor (Shepard & Balady, 1999).

**2. Sport related myocardial infarctions** 

al, 2008). All patients had emergent coronary angiography.

such as rules of good practice and dissemination of defibrillators on sports sites.

**1. Introduction** 

**2.1 Clinical cases 2.1.1 Methods** 

**2.1.2 Results** 


### **Peculiarities of Coronary Artery Disease in Athletes**

Halna du Fretay Xavier1,2, Akoudad Hafid3, Hamadou Ouceyni2 and Benhamer Hakim2,4,5 *1Centre Hospitalier Universitaire Bichat Claude Bernard, Paris, 2Hopital Foch, Suresnes 3Centre Hospitalier Universitaire Hassan II, Fez 4Hôpital Européen de Paris la Roseraie, Aubervilliers 5Institut Cardiovasculaire Paris Sud, Massy 1,2,4,5France 3Maroc* 

#### **1. Introduction**

290 Recent Advances in Cardiovascular Risk Factors

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Intense physical activity increases the risk of acute coronary syndrome but regular physical training is a cardio vascular protective factor (Shepard & Balady, 1999).

Sport related myocardial infarctions (SMI) are rare but serious. They present angiographic features that explain some clinical characteristics, the limits of the detection of coronary disease in this population and encourage decision making in order to have a better regulation of sport such as rules of good practice and dissemination of defibrillators on sports sites.

However, athletes could be asymptomatic despite tight coronary stenosis as in the three clinical cases presented. The reasons for the asymptomatic nature of these coronary diseases are probably due to cardiovascular adaptation to regular training. These athletes are at risk of major cardiac events and the place of stress testing remains important in this population.

#### **2. Sport related myocardial infarctions**

#### **2.1 Clinical cases**

#### **2.1.1 Methods**

We report a retrospective study of twelve cases of SMI. All patients had myocardial infarction (MI) defined as ischemic symptoms and ST segment elevation on ECG. MI was considered to be related to sport if it occurred during sport with vigorous exertions (METs > or = 6) or within 2 hours afterward according to the definition of Von Klot et al (Von Klot et al, 2008). All patients had emergent coronary angiography.

#### **2.1.2 Results**

A total of twelve patients are studied, all men. The mean age is 47.7 years (24 to 64). Eight of these patients considered themselves as insufficiently trained. Chest pain is the most

Peculiarities of Coronary Artery Disease in Athletes 293

Thus the most common angiographic presentation is a single-vessel disease with thrombus

This patient had inferior SMI. Chest pain occurred during tennis training and he arrived in our cath lab three hours after symptom onset. Cardiac catheterization was performed using a 6 French sheath via right radial artery access. Pharmacotherapy included 70 U/Kg unfractionated heparin and 250 mg aspirin intravenous and clopidogrel 600 mg loading dose. The left coronary artery was normal (figure 1). The right was occluded, flow TIMI 0 (Figure 2). PCI was performed with use of a thrombus aspiration catheter. Fresh clots were

Fig. 1. Left coronary artery with collateral flow in the right coronary artery (black arrow)

retrieved from the filtered aspirated blood (Figure 3).

Fig. 2. Right coronary artery with thrombus (white arrow)

as case N°11.

common presentation (11/12) with initial collapse in one case (case 12). SMI occur after exercise in 3 cases and during in 9 cases with unusual very vigorous exertion in 5 cases. One patient returned to sport after a year off (case 12). In this series, the most common sport was running (33%). The clinical characteristics of patients are presented on table 1.

Only two patients had multivessel diseases. Primary coronary intervention (PCI) was performed in nine patients, fibrinolysis in two patients (cases 1 and 4). The last patient (case 7) had normal coronary angiography just after relief of chest pain but elevated markers of myocardial necrosis. Coronary angiographic findings are presented on table 2.


Table 1. M: male; F: Female; S: cigarette smoking; FC: family history of coronary artery disease; D: dyslipidemia; H: hypertension; 0 : no risk factor


Table 2. Ter: territory ; Ant: anterior wall ; Inf: inferior wall, Glob: global; CA: culprit artery; LM: left main; LAD: left anterior descending artery; RCA : right coronary artery; Mg: marginal branch of circumflex artery; AL: associated lesion, \* lesion < 70 %

common presentation (11/12) with initial collapse in one case (case 12). SMI occur after exercise in 3 cases and during in 9 cases with unusual very vigorous exertion in 5 cases. One patient returned to sport after a year off (case 12). In this series, the most common sport was

Only two patients had multivessel diseases. Primary coronary intervention (PCI) was performed in nine patients, fibrinolysis in two patients (cases 1 and 4). The last patient (case 7) had normal coronary angiography just after relief of chest pain but elevated markers of

N° Age (years) Sex Risk factors Sports Symptoms Circumstances 1 40 M S, FC Swimming Chest pain After sport 2 51 M 0 Running Syncope During sport 3 52 F H, FC Running Chest pain During sport 4 45 M 0 Triathlon Chest pain During sport 5 40 M FC Running Chest pain During sport 6 24 M S, FC Football Chest pain After sport 7 39 M 0 Rugby Chest pain During sport 8 63 M 0 Cycling Chest pain During sport 9 48 M S, FC Karate Chest pain During sport 10 64 M H, FC Running Chest Pain After sport 11 50 M D, FC Tennis Chest Pain During sport 12 33 M D Football Chest pain During sport Table 1. M: male; F: Female; S: cigarette smoking; FC: family history of coronary artery

N° Ter CA TIMI flow Collaterals Thrombus AL 1 Ant LAD 3 0 0 0 2 Ant LAD 2 0 0 0 3 Ant LAD 2 0 + 0 4 Ant LAD 3 0 + 0 5 Ant LAD 2 0 + 0 6 Glob LM 2 0 + CD 7 Inf 0 3 0 0 0 8 Inf RCA 1 0 + 0 9 Inf RCA 2 0 + 0 10 Inf RCA 0 0 + LAD 11 Inf RCA 2 0 + 0 12 Inf RCA 0 + + Mg \* Table 2. Ter: territory ; Ant: anterior wall ; Inf: inferior wall, Glob: global; CA: culprit artery;

LM: left main; LAD: left anterior descending artery; RCA : right coronary artery; Mg: marginal branch of circumflex artery; AL: associated lesion, \* lesion < 70 %

running (33%). The clinical characteristics of patients are presented on table 1.

myocardial necrosis. Coronary angiographic findings are presented on table 2.

disease; D: dyslipidemia; H: hypertension; 0 : no risk factor

Thus the most common angiographic presentation is a single-vessel disease with thrombus as case N°11.

This patient had inferior SMI. Chest pain occurred during tennis training and he arrived in our cath lab three hours after symptom onset. Cardiac catheterization was performed using a 6 French sheath via right radial artery access. Pharmacotherapy included 70 U/Kg unfractionated heparin and 250 mg aspirin intravenous and clopidogrel 600 mg loading dose. The left coronary artery was normal (figure 1). The right was occluded, flow TIMI 0 (Figure 2). PCI was performed with use of a thrombus aspiration catheter. Fresh clots were retrieved from the filtered aspirated blood (Figure 3).

Fig. 1. Left coronary artery with collateral flow in the right coronary artery (black arrow)

Fig. 2. Right coronary artery with thrombus (white arrow)

Peculiarities of Coronary Artery Disease in Athletes 295

It is well reported that MI risk is increased during and after vigorous exercise (Gibson et al, 1980; Mittelman et al, 1993; Willich et al, 1993; Von Klot et al 2008). Ciampriccotti and Taverne reported even one case of MI twice during sporting activities (Ciampriccotti and Taverne, 1992). Mittelman et al reported that 4.4 % of MI were related to exercise which would represent more than 5000 MI per year in France. In addition, the majority of sport-

The prevalence of SSD is more often evaluated, in retrospective studies (Maron et al, 1996; Peddoe, 2007), that of SMI. The SSD rates are between 1/50000 to 1/80000 athletes in these studies. SSD are most commonly related to coronary disease in the general population

Chevallier et al reported in a prospective study a yearly SMI incidence of 2.6/100000

So, SMI are rare but occur in a supposedly healthy population and shock the public opinion and the medical profession especially as the clinical presentation can be severe particularly

Running is the most commonly practiced sport in our study, reflecting European trends. Indeed the number of runners who completed the Paris Marathon has increased from 9000 to 30000 from 1990 to 2010. We have previously reported data for European studies about the type of sport during SMI (figure 6). The most commonly practiced sports in Europe are

Fig. 6. Sports practiced during SMI : Tennis : ; Cycling: ; Football : ; Running : ;

22

18.6

19,6

18,618.6

related sudden deaths (SSD) are due to coronary artery disease and mainly MI.

contrary to young competitive athletes (Marijon et al 2011).

persons. (50 SMI, lethal in 3 cases) (Chevallier et al, 2009).

present. The number of runner is low as this is old data.

28,9

Other : . (Halna du Fretay and Gerardin, 2008)

11

**2.2 Discussion and literature review** 

when it comes to a sudden death.

**2.2.2 Which athletes and which sports?** 

**2.2.1 Prevalence of SMI** 

Fig. 3. Fresh clot removed by aspiration

Although after thromboaspiration only a limited stenosis remained (figure 4) a bare metal stent was placed to optimize the result (Figure 5).

Fig. 4. Result after thrombus aspiration

Fig. 5. Final result

#### **2.2 Discussion and literature review**

#### **2.2.1 Prevalence of SMI**

294 Recent Advances in Cardiovascular Risk Factors

Although after thromboaspiration only a limited stenosis remained (figure 4) a bare metal

Fig. 3. Fresh clot removed by aspiration

Fig. 4. Result after thrombus aspiration

Fig. 5. Final result

stent was placed to optimize the result (Figure 5).

It is well reported that MI risk is increased during and after vigorous exercise (Gibson et al, 1980; Mittelman et al, 1993; Willich et al, 1993; Von Klot et al 2008). Ciampriccotti and Taverne reported even one case of MI twice during sporting activities (Ciampriccotti and Taverne, 1992). Mittelman et al reported that 4.4 % of MI were related to exercise which would represent more than 5000 MI per year in France. In addition, the majority of sportrelated sudden deaths (SSD) are due to coronary artery disease and mainly MI.

The prevalence of SSD is more often evaluated, in retrospective studies (Maron et al, 1996; Peddoe, 2007), that of SMI. The SSD rates are between 1/50000 to 1/80000 athletes in these studies. SSD are most commonly related to coronary disease in the general population contrary to young competitive athletes (Marijon et al 2011).

Chevallier et al reported in a prospective study a yearly SMI incidence of 2.6/100000 persons. (50 SMI, lethal in 3 cases) (Chevallier et al, 2009).

So, SMI are rare but occur in a supposedly healthy population and shock the public opinion and the medical profession especially as the clinical presentation can be severe particularly when it comes to a sudden death.

#### **2.2.2 Which athletes and which sports?**

Running is the most commonly practiced sport in our study, reflecting European trends. Indeed the number of runners who completed the Paris Marathon has increased from 9000 to 30000 from 1990 to 2010. We have previously reported data for European studies about the type of sport during SMI (figure 6). The most commonly practiced sports in Europe are present. The number of runner is low as this is old data.

Fig. 6. Sports practiced during SMI : Tennis : ; Cycling: ; Football : ; Running : ; Other : . (Halna du Fretay and Gerardin, 2008)

Peculiarities of Coronary Artery Disease in Athletes 297

occurred during trainings. Mental stress caused by a competitive situation leads to an greater increase in the heart rate during exertion (Lindholm et al, 2006;) and Viru et al reported that competitive conditions increase the cortisol response to exercise, suggesting that sympatho-adrenal system activation occur in such situations (Viru et al, 2010). Such complications are probably explained by high heart oxygen consumption during exertion at the time of SMI, the activation of the sympathoadrenergic system with increase of plasma nor epinephrine and epinephrine (Strobel et al, 1999), the absence of myocardial preconditioning due to the features of coronary disease in SMI wich could promote

In our study, 1 patient had normal coronary angiography, 8 patients had single vessel disease, and 3 multivessel disease. We had previously reported data from European studies

> **normal 1 vessel 2vessels 3 vessels**

Fig. 7. Angiographic characteristics of SMI in European studies, percentage value of 97

These angiographic characteristics are similar to those of MI in the young (Kanitz et al, 1996;

Giri et al reported in their study 50 % of single-vessel disease in case of MI related to exertion and 28 % in case of MI none related to exertion. Three vessels disease was finding respectively in 8 % and 41 % of cases. Nevertheless, in the first group the population was more likely to have risk factors but was also more likely regular exertions with its probable

Most of the studies on SMI, usually with a younger population, show in more than 50 % of cases single vessel disease. Ambrose et al reported that MI frequently develops from previously non severe lesions (Ambrose et al, 1988). It is recognized that plaque rupture is the most common substrate for coronary thrombosis and occurs in case of vulnerable plaques as thin-caps fibroatheroma (Fuster et all, 2005). During exertion several phenomena

malignant ventricular arrhythmias.

**2.2.4 Angiographic characteristics** 

showing the prevalence of single-vessel disease (Figure 7).

patients, Halna du Fretay and Gerardin, 2008.

**0**

**10**

**20**

**30**

**40**

**50**

**60**

beneficial effect (Giri et all 1999).

Bajaj et al, 2011) despite a mean age of 45.7 years in our study.

Recently, Marijon et al reported that cycling (30.6 %), running (21.3 % and football (13 %) were the most common sports related to sudden death (Marijon et al, 2011).

Hiltgen et al reported that SMI occurred most commonly after exertion and that the recovery period was particularly at high risk of SMI (Hiltgen et al 1989). The vagal stimulation may be the cause of coronary spasm, especially if the athlete smokes at this time. On the contrary, among runners, cardiac events occur in one third of cases at the end or after exercise and the rest during exercise (Robert and Maron, 2005; Pedoe, 2007; Gerardin, 2009). Gerardin reported even one case of SSD just at the start of the Paris Marathon.

SMI concern most often men aged over 35 years as in our study (10/12 patients) and is previously well reported (Roberts & Maron, 2005; Chevallier et al, 2009). Giri et al reported that patients with an exertion-related MI were more likely to have risk factors including male sex, hyperlipidemia, hypertension and current cigarette smoking (59 % versus 37 % in non exertion-related MI) but in a population of which had known coronary artery disease and older than that of our study (Giri et al 1999). There were 28 cigarette smokers in a population of 42 patients in one study on unstable angina, SMI and SSD related to sport (Ciampricotti et al, 1990). We find in our study, with a small number of patients, few risk factors in this population but often family history of coronary disease and 4/12 patients without risk factor. This is similar to those who suffer MI at young age (Kanitz et al 1996; Bajaj et al, 2011).

High frequency of exertion is a protection factor against SMI as reported by Giri and Mittelmann. The latter showed that the risk of MI during exertion or within one hour afterward was elevated 107 times for subjects who usually exercise less than one a week and only 2.4 times for those having more than 5 times (Mittelman et al, 1993). So the subject most at risk of SMI is an untrained man over 35 years, current smoker and SMI can occur throughout or after exercise.

#### **2.2.3 Clinical characteristics**

The existence of prodromata before SMI or SSD is variable in studies. Chest pain is most commonly reported in high cardiac risk population or in case of known ischemic heart disease (Opie, 1975; Northcote et al, 1986). Several studies reported the existence of symptoms neglected before SMI (Droniou et al, 1987; François et al, 1989). In the study of Droniou and all, symptoms were present in 45 % of cases particularly after the age of 40 years with angina in 11/20 cases. The prevalence of risk factor was high in this study.

In a population with few or no (23.8 % of patients) coronary risk factor, Ciampriccotti et al reported only 3 patients with prodromata in their study of 42 cases of SSD, SMI or unstable angina related to sport (Ciampriccotti et al, 1990). Pedoe and Gerardin reported very few or no symptom in cases of SSD most commonly due to ischemic heart diseases (Pedoe, 2007; Gerardin, 2009).

Giri and all found ventricular fibrillation in 20 % of exertion-related MI and 11 % in non exertion-related MI (Giri and all, 1999).

Thus SSD appears to be a common mode of SMI or unstable angina related to sport presentation, as reported by Ciampriccotti (28 % of cases), Gerardin (all cases) (Ciampriccotti et al, 1900; Gerardin, 2009). We find in our study just one case of possible ventricular arrhythmia. It may be due to the small number of patients and the fact that SMI

Recently, Marijon et al reported that cycling (30.6 %), running (21.3 % and football (13 %)

Hiltgen et al reported that SMI occurred most commonly after exertion and that the recovery period was particularly at high risk of SMI (Hiltgen et al 1989). The vagal stimulation may be the cause of coronary spasm, especially if the athlete smokes at this time. On the contrary, among runners, cardiac events occur in one third of cases at the end or after exercise and the rest during exercise (Robert and Maron, 2005; Pedoe, 2007; Gerardin, 2009).

SMI concern most often men aged over 35 years as in our study (10/12 patients) and is previously well reported (Roberts & Maron, 2005; Chevallier et al, 2009). Giri et al reported that patients with an exertion-related MI were more likely to have risk factors including male sex, hyperlipidemia, hypertension and current cigarette smoking (59 % versus 37 % in non exertion-related MI) but in a population of which had known coronary artery disease and older than that of our study (Giri et al 1999). There were 28 cigarette smokers in a population of 42 patients in one study on unstable angina, SMI and SSD related to sport (Ciampricotti et al, 1990). We find in our study, with a small number of patients, few risk factors in this population but often family history of coronary disease and 4/12 patients without risk factor.

were the most common sports related to sudden death (Marijon et al, 2011).

Gerardin reported even one case of SSD just at the start of the Paris Marathon.

This is similar to those who suffer MI at young age (Kanitz et al 1996; Bajaj et al, 2011).

throughout or after exercise.

**2.2.3 Clinical characteristics** 

exertion-related MI (Giri and all, 1999).

Gerardin, 2009).

High frequency of exertion is a protection factor against SMI as reported by Giri and Mittelmann. The latter showed that the risk of MI during exertion or within one hour afterward was elevated 107 times for subjects who usually exercise less than one a week and only 2.4 times for those having more than 5 times (Mittelman et al, 1993). So the subject most at risk of SMI is an untrained man over 35 years, current smoker and SMI can occur

The existence of prodromata before SMI or SSD is variable in studies. Chest pain is most commonly reported in high cardiac risk population or in case of known ischemic heart disease (Opie, 1975; Northcote et al, 1986). Several studies reported the existence of symptoms neglected before SMI (Droniou et al, 1987; François et al, 1989). In the study of Droniou and all, symptoms were present in 45 % of cases particularly after the age of 40 years with angina in 11/20 cases. The prevalence of risk factor was high in this study.

In a population with few or no (23.8 % of patients) coronary risk factor, Ciampriccotti et al reported only 3 patients with prodromata in their study of 42 cases of SSD, SMI or unstable angina related to sport (Ciampriccotti et al, 1990). Pedoe and Gerardin reported very few or no symptom in cases of SSD most commonly due to ischemic heart diseases (Pedoe, 2007;

Giri and all found ventricular fibrillation in 20 % of exertion-related MI and 11 % in non

Thus SSD appears to be a common mode of SMI or unstable angina related to sport presentation, as reported by Ciampriccotti (28 % of cases), Gerardin (all cases) (Ciampriccotti et al, 1900; Gerardin, 2009). We find in our study just one case of possible ventricular arrhythmia. It may be due to the small number of patients and the fact that SMI occurred during trainings. Mental stress caused by a competitive situation leads to an greater increase in the heart rate during exertion (Lindholm et al, 2006;) and Viru et al reported that competitive conditions increase the cortisol response to exercise, suggesting that sympatho-adrenal system activation occur in such situations (Viru et al, 2010). Such complications are probably explained by high heart oxygen consumption during exertion at the time of SMI, the activation of the sympathoadrenergic system with increase of plasma nor epinephrine and epinephrine (Strobel et al, 1999), the absence of myocardial preconditioning due to the features of coronary disease in SMI wich could promote malignant ventricular arrhythmias.

#### **2.2.4 Angiographic characteristics**

In our study, 1 patient had normal coronary angiography, 8 patients had single vessel disease, and 3 multivessel disease. We had previously reported data from European studies showing the prevalence of single-vessel disease (Figure 7).

Fig. 7. Angiographic characteristics of SMI in European studies, percentage value of 97 patients, Halna du Fretay and Gerardin, 2008.

These angiographic characteristics are similar to those of MI in the young (Kanitz et al, 1996; Bajaj et al, 2011) despite a mean age of 45.7 years in our study.

Giri et al reported in their study 50 % of single-vessel disease in case of MI related to exertion and 28 % in case of MI none related to exertion. Three vessels disease was finding respectively in 8 % and 41 % of cases. Nevertheless, in the first group the population was more likely to have risk factors but was also more likely regular exertions with its probable beneficial effect (Giri et all 1999).

Most of the studies on SMI, usually with a younger population, show in more than 50 % of cases single vessel disease. Ambrose et al reported that MI frequently develops from previously non severe lesions (Ambrose et al, 1988). It is recognized that plaque rupture is the most common substrate for coronary thrombosis and occurs in case of vulnerable plaques as thin-caps fibroatheroma (Fuster et all, 2005). During exertion several phenomena

Peculiarities of Coronary Artery Disease in Athletes 299

We have previously emphasized the risk of sudden death or serious ventricular arrhythmias in SMI. Roberts and Maron reported nine cardiac events occurring during marathons, with SD (5 cases) and non fatal cardiac arrest (NFCA) (4 cases) from 1976 to 2004. Seven of the nine runners had underlying atherosclerotic coronary disease. The four patients with NFCA had external defibrillation performed promptly within 5 min. These authors observed a decrease in mortality since 1995, which was largely attributable to the expanded access to

> **1995- 2004**

Fig. 8. Cardiac events occurring during marathons from 1976 to 2004, (Roberts and Maron,

Gerardin reported four cardiac arrests related to sport, with proven coronary diseases and no deaths. Sport events were marathons or running races of 20 km with a medical supervision by mobile intensive care units (ambulances). These four athletes had external

 So for these competitions the organization of medical teams including physicians, paramedics and emergency medical technicians trained to use defibrillators is saving-life.

In France 75 % of people aged 40 to 60 years report taking part in sporting competitions or recreational sport. A proportion of these people will have coronary artery disease either known or unknown. These athletes with unknown ischemic heart disease, sometimes

Case N°1: A 43-year-old man presented with discomfort after running to catch his train. His risk factors were smoking and dyslipidemia. He was a well trained athletes, previous professional basketball player, and tennis player at a regional level with training at least three times per week. He was examined in emergency department a few hours later with a normal examination including ECG and troponin. The diagnosis was a vagal discomfort and consultation from a cardiologist was requested. ECG showed negative T waves in leads II,

**NFCA SD**

**2.2.5.3 Management of complications** 

external defibrillators (figure 8).

2005)

**0**

**1976- 1994**

defibrillation for ventricular fibrillation (SMI in 3 cases) (Gerardin, 2009).

**3. Unrecognized coronary disease among athletes** 

asymptomatic, should be screened.

**3.1 Case reports** 

**1**

**2**

**3**

**4**

occur as increased coronary flow, adrenergic activity, blood pressure and heart rate causing a shearing effect of the coronary arteries and favouring plaque rupture, intimal haemorrhage. Platelets are also implicated in the participation of acute coronary syndromes particularly in exercise ( Hilberg et al, 2003) and strenuous exercise is associated with a transient hypercoagulability state (Lippi and Maffulli, 2009). In vivo platelet activation is reported in marathon runners (Kratz et al, 2006). Exertion can be the trigger of a vulnerable plaque rupture and thrombosis promoted by modifications of haemostasis. Another possible pathophysiological mechanism is the occurrence of spam (Hiltgen et al, 1989) and could explain the case N° 7 of our study. Few cases of spontaneous coronary dissection (Kalaga et al, 2007) and congenital coronary artery anomalies (Corrado et al, 1998) are also reported but concern young athletes with a different pathophysiological mechanism.

We can suppose that most of these patients with single-vessel disease had no severe coronary stenosis before SMI and that would explain the limits of stress testing to prevent SMI. Gerardin reported in their study 3 SMI with negative stress testing a few months before in 2 cases (Gerardin, 2009).

The patient of case N° 11 is a typical example of the probable pathophysiological mechanism of SMI.

#### **2.2.5 Which specific treatment?**

There is no specific treatment of SMI but some features should be noticed.

#### **2.2.5.1 Prevention rules**

Smoking is not only a risk factor of coronary disease but has also deleterious effects in the short term as an increased level of carboxyhemoglobin reducing the amount of haemoglobin available to carry oxygen, increased heart rate and blood pressure but with a decrease in coronary blood flow. It follows in an increase in myocardial consumption but a decreased supply. Smoking is also associated with an increased risk of vasospastic angina (Caralis et al, 1992). This can be mediated by increased catecholamines or during the recovery phase by acetylcholine release with vasospastic paradoxical effect in case of endothelial dysfunction. Chevallier et al reported that 73 % of young athletes smoke a cigarette in the last hour or two hours after a strenuous exercising (Chevallier et al, 2005). So educational measures against smoking are a priority.

Because of platelet activation during exercise aspirin use was discussed by some authors (Mittelman et al, 1993, Burtscher et al, 2007) but criticized by other and not recommended in case of exertion in hot weather (Fred, 1981) or with risk of body collision. Its use should be clarified by further studies, especially in athletes at risk of SMI.

#### **2.2.5.2 Reperfusion therapy**

Reperfusion strategies in SMI must be consistent with the recommendations of ESC for the treatment of STEMI but although the use of manual catheter thrombus aspiration during PCI is only class II a in the recent guidelines on myocardial revascularization (The Task Force on Myocardial Revascularization of ESC/EACTS, 2010), the advantage of this technique seems to be especially great in case of SMI.

#### **2.2.5.3 Management of complications**

298 Recent Advances in Cardiovascular Risk Factors

occur as increased coronary flow, adrenergic activity, blood pressure and heart rate causing a shearing effect of the coronary arteries and favouring plaque rupture, intimal haemorrhage. Platelets are also implicated in the participation of acute coronary syndromes particularly in exercise ( Hilberg et al, 2003) and strenuous exercise is associated with a transient hypercoagulability state (Lippi and Maffulli, 2009). In vivo platelet activation is reported in marathon runners (Kratz et al, 2006). Exertion can be the trigger of a vulnerable plaque rupture and thrombosis promoted by modifications of haemostasis. Another possible pathophysiological mechanism is the occurrence of spam (Hiltgen et al, 1989) and could explain the case N° 7 of our study. Few cases of spontaneous coronary dissection (Kalaga et al, 2007) and congenital coronary artery anomalies (Corrado et al, 1998) are also

reported but concern young athletes with a different pathophysiological mechanism.

There is no specific treatment of SMI but some features should be noticed.

clarified by further studies, especially in athletes at risk of SMI.

technique seems to be especially great in case of SMI.

before in 2 cases (Gerardin, 2009).

**2.2.5 Which specific treatment?** 

against smoking are a priority.

**2.2.5.2 Reperfusion therapy** 

**2.2.5.1 Prevention rules** 

of SMI.

We can suppose that most of these patients with single-vessel disease had no severe coronary stenosis before SMI and that would explain the limits of stress testing to prevent SMI. Gerardin reported in their study 3 SMI with negative stress testing a few months

The patient of case N° 11 is a typical example of the probable pathophysiological mechanism

Smoking is not only a risk factor of coronary disease but has also deleterious effects in the short term as an increased level of carboxyhemoglobin reducing the amount of haemoglobin available to carry oxygen, increased heart rate and blood pressure but with a decrease in coronary blood flow. It follows in an increase in myocardial consumption but a decreased supply. Smoking is also associated with an increased risk of vasospastic angina (Caralis et al, 1992). This can be mediated by increased catecholamines or during the recovery phase by acetylcholine release with vasospastic paradoxical effect in case of endothelial dysfunction. Chevallier et al reported that 73 % of young athletes smoke a cigarette in the last hour or two hours after a strenuous exercising (Chevallier et al, 2005). So educational measures

Because of platelet activation during exercise aspirin use was discussed by some authors (Mittelman et al, 1993, Burtscher et al, 2007) but criticized by other and not recommended in case of exertion in hot weather (Fred, 1981) or with risk of body collision. Its use should be

Reperfusion strategies in SMI must be consistent with the recommendations of ESC for the treatment of STEMI but although the use of manual catheter thrombus aspiration during PCI is only class II a in the recent guidelines on myocardial revascularization (The Task Force on Myocardial Revascularization of ESC/EACTS, 2010), the advantage of this We have previously emphasized the risk of sudden death or serious ventricular arrhythmias in SMI. Roberts and Maron reported nine cardiac events occurring during marathons, with SD (5 cases) and non fatal cardiac arrest (NFCA) (4 cases) from 1976 to 2004. Seven of the nine runners had underlying atherosclerotic coronary disease. The four patients with NFCA had external defibrillation performed promptly within 5 min. These authors observed a decrease in mortality since 1995, which was largely attributable to the expanded access to external defibrillators (figure 8).

Fig. 8. Cardiac events occurring during marathons from 1976 to 2004, (Roberts and Maron, 2005)

Gerardin reported four cardiac arrests related to sport, with proven coronary diseases and no deaths. Sport events were marathons or running races of 20 km with a medical supervision by mobile intensive care units (ambulances). These four athletes had external defibrillation for ventricular fibrillation (SMI in 3 cases) (Gerardin, 2009).

 So for these competitions the organization of medical teams including physicians, paramedics and emergency medical technicians trained to use defibrillators is saving-life.

#### **3. Unrecognized coronary disease among athletes**

In France 75 % of people aged 40 to 60 years report taking part in sporting competitions or recreational sport. A proportion of these people will have coronary artery disease either known or unknown. These athletes with unknown ischemic heart disease, sometimes asymptomatic, should be screened.

#### **3.1 Case reports**

Case N°1: A 43-year-old man presented with discomfort after running to catch his train. His risk factors were smoking and dyslipidemia. He was a well trained athletes, previous professional basketball player, and tennis player at a regional level with training at least three times per week. He was examined in emergency department a few hours later with a normal examination including ECG and troponin. The diagnosis was a vagal discomfort and consultation from a cardiologist was requested. ECG showed negative T waves in leads II,

Peculiarities of Coronary Artery Disease in Athletes 301

Case N° 2: A 54-year-old man had myocardial perfusion scintigraphy for the exploration of abnormal routine ECG. He had FC and dyslipidemia as risk factor and had stopped smoking 10 years before. He ran 10 km twice a week and had completed several marathons and a 100 km race. He was asymptomatic but myocardial perfusion sintigraphy showed defect in the anterior wall. Coronary angiography was performed showing 2 vessel disease

Fig. 11. Distal RCA perfused by collaterals from LAD

Fig. 12. Very tight lesion of marginal branch

(figures 12 and 13).

III and aVF. Conventional ECG exercise testing showed late but profound ST segment depression in leads V1- V4, asymptomatic and coronary angiography was performed showing two vessel disease (figures 9, 10 and 11).

Fig. 9. Left coronary artery with short stenosis of LAD

Fig. 10. RCA occlusion

This athlete was able to play tennis in competition despite at least one chronic lesion (occlusion of the RC may be recent and the cause of the discomfort). Coronary artery bypass grafting was performed in this patient who seeks now to return the competitive sport.

III and aVF. Conventional ECG exercise testing showed late but profound ST segment depression in leads V1- V4, asymptomatic and coronary angiography was performed

This athlete was able to play tennis in competition despite at least one chronic lesion (occlusion of the RC may be recent and the cause of the discomfort). Coronary artery bypass grafting was performed in this patient who seeks now to return the competitive sport.

showing two vessel disease (figures 9, 10 and 11).

Fig. 9. Left coronary artery with short stenosis of LAD

Fig. 10. RCA occlusion

Fig. 11. Distal RCA perfused by collaterals from LAD

Case N° 2: A 54-year-old man had myocardial perfusion scintigraphy for the exploration of abnormal routine ECG. He had FC and dyslipidemia as risk factor and had stopped smoking 10 years before. He ran 10 km twice a week and had completed several marathons and a 100 km race. He was asymptomatic but myocardial perfusion sintigraphy showed defect in the anterior wall. Coronary angiography was performed showing 2 vessel disease (figures 12 and 13).

Fig. 12. Very tight lesion of marginal branch

Peculiarities of Coronary Artery Disease in Athletes 303

PCI was performed to the LAD requiring predilatation. Angioplasty of RCA and Mg was

In this case lesions of LAD, Mg and mild RCA seemed to be chronics, were very tight and had not stopped this athlete completing a marathon. The real culprit lesion was probably the distal occlusion of the RCA reperfused previously the LAD below of the tight stenosis.

In these three different cases of patients with chronic coronary lesions, strenuous exercise was possible without symptom even in competition. Cardiovascular adaptation to regular exercise may explain this fact but these patients have high risk of major cardiac events and

Fig. 15. Stenosis on mild RCA and occlusion of distal RCA

performed few days later.

Fig. 16. Stenosis on Cx

should be screened.

Fig. 13. LAD stenosis including septal a diagonal branches

Coronary angioplasty was performed to LAD and Mg with a good result. This athlete returns to recreational sport and remains asymptomatic.

Case N ° 3: A 50-year-old man presented inaugural anterior STMI revealed by sever chest pain at rest. His risk factors were smoking and dyslipidemia. He was a well trained runner and had performed the Paris marathon two weeks before. Pre-hospital treatment included unfractionated heparin and GPIIb/IIIa antagonist. Coronary angiography was performed 90 mn after onset symptoms via right radial artery access showing 3 vessel disease (Figures 14, 15 and 16).

Fig. 14. Stenosis on proximal LAD

Coronary angioplasty was performed to LAD and Mg with a good result. This athlete

Case N ° 3: A 50-year-old man presented inaugural anterior STMI revealed by sever chest pain at rest. His risk factors were smoking and dyslipidemia. He was a well trained runner and had performed the Paris marathon two weeks before. Pre-hospital treatment included unfractionated heparin and GPIIb/IIIa antagonist. Coronary angiography was performed 90 mn after onset symptoms via right radial artery access showing 3 vessel disease (Figures 14,

Fig. 13. LAD stenosis including septal a diagonal branches

returns to recreational sport and remains asymptomatic.

15 and 16).

Fig. 14. Stenosis on proximal LAD

Fig. 15. Stenosis on mild RCA and occlusion of distal RCA

PCI was performed to the LAD requiring predilatation. Angioplasty of RCA and Mg was performed few days later.

Fig. 16. Stenosis on Cx

In this case lesions of LAD, Mg and mild RCA seemed to be chronics, were very tight and had not stopped this athlete completing a marathon. The real culprit lesion was probably the distal occlusion of the RCA reperfused previously the LAD below of the tight stenosis.

In these three different cases of patients with chronic coronary lesions, strenuous exercise was possible without symptom even in competition. Cardiovascular adaptation to regular exercise may explain this fact but these patients have high risk of major cardiac events and should be screened.

Peculiarities of Coronary Artery Disease in Athletes 305

However some athletes have unknown severe coronary disease probably detectable by a stress test and should be screened. So athletes with multiple risk factors could benefit from screening for coronary artery disease, must respect rules of good practices and be advised of

the possibility of SMI despite a negative stress test.

12, N°. 1, (July 1988), pp. 56-62, ISSN 0735-1097

marathon

**5. References** 

ISSN 0009-7322

pp. 621-624, ISSN 0943-917X.

909, ISSN: 0009-7322

Fig. 17. Personal photo in the famous Mont Saint Michel which is held every year a

Ambrose, JA; Tannenbaum, MA; Alexopoulos, D; Hjemdahl-Monsen, CE; Leavy, J; Weiss,

Bajaj, S; Shamoon, F,; Gupta, N; Parikh, R; Debari ,VA; Hamdan, A & Bikkina, M.. (June

risk? *Coron Artery Dis,* Vol.22, N°.4, (June 2011), pp 238-244, ISSN 0954-6928 Belardinelli, R; Georgiou, D; Ginzton, L; Cianci, G & Purcaro, A. (1998). Effects of moderate

Burtscher, M; Pachinger, O; Schocke, MF & Ulmer, H. (2007). Risk factor profile for sudden

Caralis, DG; Deligonul, U; Kern, MJ & and Cohen, JD. (1992). Smoking is a risk factor for

Chevalier, L ; Douard, H ; Laporte, T ; Hajar, M ; Baudot, C ; Genson, F ; Labanere, C ; Merle,

M; Borrico, S; Gorlin, R & Fuster,V. (1988). Angiographic progression of coronary artery disease and the development of myocardial infarction. *Am Coll Cardiol,* Vol.

2011). Acute ST segment élévation myocardial infarction in young adults: who is at

exercise training on thallium uptake and contractile response to low-dose dobutamine of dysfunctional myocardium in patients with ischémie cardiomyopathy. Circulation. 1998 Feb 17;Vol. 97, N°.6, (Februar 1998), pp. 553-561,

cardiac death during mountain hiking. *Int J Sports Med,* Vol. 28, N°.7, ( July 2007),

coronary spasm in young women. *Circulation,* Vol. 85, N°3, (March 1992), pp. 905-

F &Vincent, MP. (2005). Survey of cardiovascular risk assessment and the

#### **3.2 Discussion**

#### **3.2.1 Why a lack of symptom in these three athletes?**

Firstly, the athletes appear casual in their approach to possibily cardiac symptoms (Chevallier et al 2005). Secondly, silent ischemia in chronic ischemic heart disease has been commonly reported but is usually associated with angina (Rogers et al, 1995). At last, exercise training improves clinical symptoms of patients with stable coronary artery disease and increase maximal exercise tolerance (Wannamethee et al, 2000; Hambrecht et al, 2004). Explanation could be that moderate exercise training improve left ventricular function du to a decrease of blood pressure and an increase in myocardial contractile response to beta adrenergic stimulation. It also promotes coronary collateral development (Belardinelli et al, 1998) and improves peripheral endothelial function (Gokce et al, 2002). However, in these three cases the patients made frequent strenuous exertions. Zinden et al reported a direct demonstration of coronary collateral flow by intense physical endurance exercise. The subject was a 46 year old healthy cardiologist who had performed long distance running. This invasive study including coronary angiography with measurement of coronary flow reserve showed increase of collateral flow index after regular training and one ultra marathon run (Zbinden et al 2004).

So the lack of symptoms in these patients could be du to cardiovascular adaptation to regular training and a development of coronary collateral particularly important.

#### **3.2.2 What consequences?**

It is recognized that ischaemia is a factor of poor prognosis even if it is silent (Rogers, 1995). Secondly although the major acute coronary syndromes occur more on moderate coronary stenosis, small luminal area is a predictor of events at lesion site (Stone et al, 2011). It can be assumed that these patients were at risk of serious cardiac events either acute coronary syndrome through occlusion of tight sténosis or plaque rupture but also severe ischemic arrhythmias related to strenuous exercises (6 or more metabolic equivalents as in practice of football, running, cycling, tennis). However increasing levels of habitual physical activity is associated with progressively lower relative risks os SMI.

Finally, these patients should not have been allowed to practice sports in competition (exept low-moderate dynamic and low static sports, I A, B as bowling, golf, table tennis, doubles tennis, volleyball) and could be screened as recommended by ESC because of their risk factors (Pelliccia et al, 2005). Indeed these three athletes are men over 40 years with at least two risk factors.

#### **4. Conclusions**

SMI are rare but can have a severe clinical presentation and are the primary cause of SD in athletes over 35 years. The probable pathophysiological mechanism of SMI is a rupture and thrombosis of a vulnerable plaque often in a patient with single vessel coronary disease. These features may have some therapeutic consequences such as the use manual catheter thrombus aspiration during PCI and may explain the limits of the effectiveness of stress testing for the prevention of these events. This prompts us to determine rules of good practice, including information on the risks of smoking and promote equipment defibrillator on sports sites.

Firstly, the athletes appear casual in their approach to possibily cardiac symptoms (Chevallier et al 2005). Secondly, silent ischemia in chronic ischemic heart disease has been commonly reported but is usually associated with angina (Rogers et al, 1995). At last, exercise training improves clinical symptoms of patients with stable coronary artery disease and increase maximal exercise tolerance (Wannamethee et al, 2000; Hambrecht et al, 2004). Explanation could be that moderate exercise training improve left ventricular function du to a decrease of blood pressure and an increase in myocardial contractile response to beta adrenergic stimulation. It also promotes coronary collateral development (Belardinelli et al, 1998) and improves peripheral endothelial function (Gokce et al, 2002). However, in these three cases the patients made frequent strenuous exertions. Zinden et al reported a direct demonstration of coronary collateral flow by intense physical endurance exercise. The subject was a 46 year old healthy cardiologist who had performed long distance running. This invasive study including coronary angiography with measurement of coronary flow reserve showed increase of collateral flow index after regular training and one ultra marathon run (Zbinden et al 2004).

So the lack of symptoms in these patients could be du to cardiovascular adaptation to

It is recognized that ischaemia is a factor of poor prognosis even if it is silent (Rogers, 1995). Secondly although the major acute coronary syndromes occur more on moderate coronary stenosis, small luminal area is a predictor of events at lesion site (Stone et al, 2011). It can be assumed that these patients were at risk of serious cardiac events either acute coronary syndrome through occlusion of tight sténosis or plaque rupture but also severe ischemic arrhythmias related to strenuous exercises (6 or more metabolic equivalents as in practice of football, running, cycling, tennis). However increasing levels of habitual physical activity is

Finally, these patients should not have been allowed to practice sports in competition (exept low-moderate dynamic and low static sports, I A, B as bowling, golf, table tennis, doubles tennis, volleyball) and could be screened as recommended by ESC because of their risk factors (Pelliccia et al, 2005). Indeed these three athletes are men over 40 years with at least

SMI are rare but can have a severe clinical presentation and are the primary cause of SD in athletes over 35 years. The probable pathophysiological mechanism of SMI is a rupture and thrombosis of a vulnerable plaque often in a patient with single vessel coronary disease. These features may have some therapeutic consequences such as the use manual catheter thrombus aspiration during PCI and may explain the limits of the effectiveness of stress testing for the prevention of these events. This prompts us to determine rules of good practice, including information on the risks of smoking and promote equipment defibrillator

regular training and a development of coronary collateral particularly important.

**3.2.1 Why a lack of symptom in these three athletes?** 

associated with progressively lower relative risks os SMI.

**3.2 Discussion** 

**3.2.2 What consequences?** 

two risk factors.

**4. Conclusions** 

on sports sites.

However some athletes have unknown severe coronary disease probably detectable by a stress test and should be screened. So athletes with multiple risk factors could benefit from screening for coronary artery disease, must respect rules of good practices and be advised of the possibility of SMI despite a negative stress test.

Fig. 17. Personal photo in the famous Mont Saint Michel which is held every year a marathon

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Peculiarities of Coronary Artery Disease in Athletes 307

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**16** 

*Japan* 

**Blood Pressure Regulation During Bathing:** 

Water immersion-induced augmentations of external hydrostatic pressure increase central venous and atrial volume and pressure (Arborelius et al., 1972; Gabrielsen et al., 1993; Onodera et al., 2001). The increased left ventricular preload results in the elevation of stroke volume and cardiac output (Arborelius et al., 1972; Ueno et al., 2005). In Poiseuille's law, an elevated stroke volume should increase blood pressure. Therefore, it is reasonable to predict that blood pressure increases during bathing. However, previous studies have reported contradictory results. Ohnaka et al. (1995) have demonstrated that 10-min bathing (40 ºC) decreased systolic blood pressure (SBP) in young and middle-aged subjects. Additionally, Nagasawa et al. (2001) have reported that SBP decreased during hot-water immersion (40 ºC, 10 min) in young subjects, but increased at the onset of immersion and recovered during the latter phase of immersion in older subjects. Furthermore, Allison & Reger (1998) have reported that SBP in young and middle-aged men showed initial decrease, followed by a gradual increase back toward the baseline during 21-min bathing (40.0 ºC). Taken together, the vasopressor effects of bathing are less likely to be marked. Blood pressure during

Blood pressure is regulated mainly by autonomic nervous, endocrine, and renal body-fluid modulation systems. Particularly, the autonomic nervous system rapidly responds to changes in blood pressure. The cardiovagal baroreflex is one of the autonomic functions that regulate blood pressure. Arterial baroreceptors, stretch receptors in the carotid arterial and aortic bodies, detect any blood pressure-associated strain of arterial walls and send afferent signal to increase or decrease heart rate (HR). In this way, the cardiovagal baroreflex regulates blood pressure. Since the baroreceptor firing rate is proportional to changes in arterial circumference at a physiological blood pressure (Aars, 1969), cardiovagal baroreflex sensitivity (BRS) is considered to be affected by carotid arterial or aortic stiffness (Kingwell et al., 1995; Monahan et al., 2001; Rowe, 1987). Additionally, *in vitro* diameter-pressure curves at different temperatures, illustrated using harvested human common carotid arteries, have suggested that hyperthermia may have softening effects on the carotid artery (Guinea et al., 2005). Therefore, bathing-induced hyperthermia may reduce carotid arterial stiffness and consequently increase cardiovagal BRS. Evidence demonstrating that shortterm heat stress (5 min, 46.0-48.0 ºC) increases skeletal muscle sympathetic BRS (Keller et al., 2006), may support this hypothesis. However, it has remained unclear whether cardiovagal

bathing may be maintained by some facilitated regulatory mechanisms.

**1. Introduction** 

BRS increases during bathing.

**Is There a Cardiovascular Risk?** 

*Ryutsu Keizai University & Hiroshima Bunka Gakuen University,* 

Takeshi Otsuki and Yasuko Okuda

Pericardial Diseases of the European Society of Cardiology. *Eur Heart J,* Vol.26, N°.14, (Jully 2005), pp. 1422-1445, ISSN 1520-765X


### **Blood Pressure Regulation During Bathing: Is There a Cardiovascular Risk?**

Takeshi Otsuki and Yasuko Okuda *Ryutsu Keizai University & Hiroshima Bunka Gakuen University, Japan* 

#### **1. Introduction**

308 Recent Advances in Cardiovascular Risk Factors

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death associated with thé marathon. (2005). *J Am Coll Cardiol,* vol..46, N°. 7,

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coronary collatéral growth by physical endurance exercise in a healthy marathon runner. *Heart,* Vol.90, N°. 11, (November 2004), pp.1350-1351, ISSN 1355-6037

Water immersion-induced augmentations of external hydrostatic pressure increase central venous and atrial volume and pressure (Arborelius et al., 1972; Gabrielsen et al., 1993; Onodera et al., 2001). The increased left ventricular preload results in the elevation of stroke volume and cardiac output (Arborelius et al., 1972; Ueno et al., 2005). In Poiseuille's law, an elevated stroke volume should increase blood pressure. Therefore, it is reasonable to predict that blood pressure increases during bathing. However, previous studies have reported contradictory results. Ohnaka et al. (1995) have demonstrated that 10-min bathing (40 ºC) decreased systolic blood pressure (SBP) in young and middle-aged subjects. Additionally, Nagasawa et al. (2001) have reported that SBP decreased during hot-water immersion (40 ºC, 10 min) in young subjects, but increased at the onset of immersion and recovered during the latter phase of immersion in older subjects. Furthermore, Allison & Reger (1998) have reported that SBP in young and middle-aged men showed initial decrease, followed by a gradual increase back toward the baseline during 21-min bathing (40.0 ºC). Taken together, the vasopressor effects of bathing are less likely to be marked. Blood pressure during bathing may be maintained by some facilitated regulatory mechanisms.

Blood pressure is regulated mainly by autonomic nervous, endocrine, and renal body-fluid modulation systems. Particularly, the autonomic nervous system rapidly responds to changes in blood pressure. The cardiovagal baroreflex is one of the autonomic functions that regulate blood pressure. Arterial baroreceptors, stretch receptors in the carotid arterial and aortic bodies, detect any blood pressure-associated strain of arterial walls and send afferent signal to increase or decrease heart rate (HR). In this way, the cardiovagal baroreflex regulates blood pressure. Since the baroreceptor firing rate is proportional to changes in arterial circumference at a physiological blood pressure (Aars, 1969), cardiovagal baroreflex sensitivity (BRS) is considered to be affected by carotid arterial or aortic stiffness (Kingwell et al., 1995; Monahan et al., 2001; Rowe, 1987). Additionally, *in vitro* diameter-pressure curves at different temperatures, illustrated using harvested human common carotid arteries, have suggested that hyperthermia may have softening effects on the carotid artery (Guinea et al., 2005). Therefore, bathing-induced hyperthermia may reduce carotid arterial stiffness and consequently increase cardiovagal BRS. Evidence demonstrating that shortterm heat stress (5 min, 46.0-48.0 ºC) increases skeletal muscle sympathetic BRS (Keller et al., 2006), may support this hypothesis. However, it has remained unclear whether cardiovagal BRS increases during bathing.

Blood Pressure Regulation During Bathing: Is There a Cardiovascular Risk? 311

beginning of the second heart sound and notch of the carotid artery pulse wave. The distance traveled by the pulse waves was estimated according to the height of subjects using the following equation: 0.2437 x height - 18.999 (Kimoto et al., 2003; Kobayashi et al., 2004).

Cardiovagal BRS was assessed according to previous studies with minor modifications (Bertinieri et al., 1988; Hayashi et al., 2006). Under all recording conditions, an applanation tonometer (BP-508SD, Omron Colin) was placed at the forth intercostal level. The applanation tonometer sampled the radial arterial pressure waveforms and stored data in a computer. HR was calculated from pulse intervals. We identified baroreflex sequences (three or more beats relating to pulse intervals and progressively and spontaneously changing SBP at the same detection points). Next, we determined the slope of the linear relationship between the pulse intervals and SBP at these points. The minimum changes observed were 1 mmHg for SBP and 1 ms for the pulse interval. Linear regressions relating SBP to the pulse interval were plotted for each sequence; only those sequences with linear *r* values > 0.85 were accepted. For the latter 5-min of respective period, the results were

Data are expressed as means±SE. Statistical analysis was carried out using repeatedmeasures two-way ANOVA followed by Scheffe's test for multiple comparisons. Linear correlation analysis was used to examine the relationship between carotid arterial PWV and

Respective temperatures of shower/bath water were 40.6±0.2/40.6±0.1 ºC. Bathroom temperature in showering and bathing sessions was 27.3±0.1 and 27.8±0.1 ºC, and room

Cardiovagal BRS before, during, and after showering/bathing is indicated in Fig. 1. Twoway ANOVA revealed an interaction between session (showering or bathing) and time in cardiovagal BRS. In multiple comparisons, cardiovagal BRS did not change during showering but increased during bathing compared to the baseline. Also, cardiovagal BRS was greater during bathing than during showering. The elevated cardiovagal BRS recovered

Hemodynamics during sessions are summarized in Table 1. We did not identify interactions (session x time) in all measures. Again, there were no main effects of session in all indices. Main effects of time were detected in SBP, mean blood pressure (MBP), HR, and doubleproduct (DP, *i.e.*, product of SBP and HR). In details, SBP was higher during and after showering and bathing than the baseline. MBP increased after showering and bathing compared to at rest. HR was higher during showering and bathing in comparison to before showering and bathing. Increased DP was observed during and after showering and

bathing. Time was not associated with diastolic blood pressure and pulse pressure.

Carotid arterial PWV was calculated as the distance divided by the transit time.

**2.4 Cardiovagal BRS** 

averaged to provide a single data set.

cardiovagal BRS. *P*<0.05 was regarded as significant.

humidity was 66.7±0.9 and 62.3±1.7 %, respectively.

to the baseline after the cessation of bathing.

**2.5 Statistical analysis** 

**3. Results** 

We hypothesized that cardiovagal BRS increases during bathing to attenuate bathinginduced blood pressure changes. To test this hypothesis, cardiovagal BRS and hemodynamics in women were measured before, during, and after bathing. Cardiovagal BRS was evaluated using the sequence method (Bertinieri et al., 1988; Hayashi et al., 2006). As a control experiment, a showering session was performed according to the protocol for the bathing session. Body temperature before and after showering/bathing and carotid arterial pulse wave velocity (PWV; an index of arterial stiffness) at rest were also investigated.

#### **2. Methods**

#### **2.1 Subjects**

Twelve women [age, 35.5±6.6 (19-69) years; height, 157±2 (147-169) cm; weight, 52.4±2.1 (39.3-62.7) kg; and BMI, 21.2±0.7 (16.2-25.4) kg/m2] volunteered to participate in this study. All subjects were free of signs and symptoms of cardiac diseases, diabetes, and hypertension. None of the participants were currently taking any medications. Premenopausal women were tested at least 3 days before or 3 days after menses. All subjects provided written informed consent before inclusion in the study. This study conformed to the principles outlined in the Helsinki Declaration.

#### **2.2 Experimental protocol**

Two testing sessions, showering and bathing sessions, were performed in a randomized order on the same day, at least 2 h apart, in a quiet and temperature-controlled room. First, carotid arterial PWV was measured at supine position after a rest period (formPWV/ABI; Omron Colin, Tokyo, Japan). Second, radial arterial pulse waveforms were continuously recorded for 5 min by applanation tonometry in a seated position (BP-508SD, Omron Colin). At the onset of waveforms recording, brachial arterial pressure was measured by oscillometry (BP-508SD, Omron Colin). Skin temperature was measure at the end of the rest period using an electronic axillary thermometer (CT513, Citizen Systems, Tokyo, Japan). Also, tympanic temperature was measured via an infrared ear thermometer (CT820, Citizen Systems). Third, subjects were showered with hot water (40 ºC, 12.5 L/min) or immersed in hot water (40 ºC) up to the axillae for 10 min in a sitting position. The radial arterial waveforms and brachial blood pressure waveforms were investigated as previously mentioned. Finally, subjects underwent a 10-min recovery period after the cessation of showering/bathing in the same position as in the pre-showering/bathing period. Again, radial pressure waveforms, brachial blood pressure, and body temperatures were measured in this period.

#### **2.3 Carotid arterial PWV**

Carotid arterial PWV was measured using a device to investigate PWV (formPWV/ABI; Omron Colin), as previously described with minor modifications (Kimoto et al., 2003; Kobayashi et al., 2004). Briefly, subjects assumed a supine position with a cardiac sound microphone placed at the right sternal margin at the second intercostal level, with an applanation tonometer on the common carotid artery. The pulse wave transit time between the aortic valve and carotid artery was determined based on the time delay between the beginning of the second heart sound and notch of the carotid artery pulse wave. The distance traveled by the pulse waves was estimated according to the height of subjects using the following equation: 0.2437 x height - 18.999 (Kimoto et al., 2003; Kobayashi et al., 2004). Carotid arterial PWV was calculated as the distance divided by the transit time.

#### **2.4 Cardiovagal BRS**

310 Recent Advances in Cardiovascular Risk Factors

We hypothesized that cardiovagal BRS increases during bathing to attenuate bathinginduced blood pressure changes. To test this hypothesis, cardiovagal BRS and hemodynamics in women were measured before, during, and after bathing. Cardiovagal BRS was evaluated using the sequence method (Bertinieri et al., 1988; Hayashi et al., 2006). As a control experiment, a showering session was performed according to the protocol for the bathing session. Body temperature before and after showering/bathing and carotid arterial pulse wave velocity (PWV; an index of arterial stiffness) at rest were also

Twelve women [age, 35.5±6.6 (19-69) years; height, 157±2 (147-169) cm; weight, 52.4±2.1 (39.3-62.7) kg; and BMI, 21.2±0.7 (16.2-25.4) kg/m2] volunteered to participate in this study. All subjects were free of signs and symptoms of cardiac diseases, diabetes, and hypertension. None of the participants were currently taking any medications. Premenopausal women were tested at least 3 days before or 3 days after menses. All subjects provided written informed consent before inclusion in the study. This study conformed to

Two testing sessions, showering and bathing sessions, were performed in a randomized order on the same day, at least 2 h apart, in a quiet and temperature-controlled room. First, carotid arterial PWV was measured at supine position after a rest period (formPWV/ABI; Omron Colin, Tokyo, Japan). Second, radial arterial pulse waveforms were continuously recorded for 5 min by applanation tonometry in a seated position (BP-508SD, Omron Colin). At the onset of waveforms recording, brachial arterial pressure was measured by oscillometry (BP-508SD, Omron Colin). Skin temperature was measure at the end of the rest period using an electronic axillary thermometer (CT513, Citizen Systems, Tokyo, Japan). Also, tympanic temperature was measured via an infrared ear thermometer (CT820, Citizen Systems). Third, subjects were showered with hot water (40 ºC, 12.5 L/min) or immersed in hot water (40 ºC) up to the axillae for 10 min in a sitting position. The radial arterial waveforms and brachial blood pressure waveforms were investigated as previously mentioned. Finally, subjects underwent a 10-min recovery period after the cessation of showering/bathing in the same position as in the pre-showering/bathing period. Again, radial pressure waveforms, brachial blood pressure, and body temperatures were measured

Carotid arterial PWV was measured using a device to investigate PWV (formPWV/ABI; Omron Colin), as previously described with minor modifications (Kimoto et al., 2003; Kobayashi et al., 2004). Briefly, subjects assumed a supine position with a cardiac sound microphone placed at the right sternal margin at the second intercostal level, with an applanation tonometer on the common carotid artery. The pulse wave transit time between the aortic valve and carotid artery was determined based on the time delay between the

investigated.

**2. Methods 2.1 Subjects** 

in this period.

**2.3 Carotid arterial PWV** 

the principles outlined in the Helsinki Declaration.

**2.2 Experimental protocol** 

Cardiovagal BRS was assessed according to previous studies with minor modifications (Bertinieri et al., 1988; Hayashi et al., 2006). Under all recording conditions, an applanation tonometer (BP-508SD, Omron Colin) was placed at the forth intercostal level. The applanation tonometer sampled the radial arterial pressure waveforms and stored data in a computer. HR was calculated from pulse intervals. We identified baroreflex sequences (three or more beats relating to pulse intervals and progressively and spontaneously changing SBP at the same detection points). Next, we determined the slope of the linear relationship between the pulse intervals and SBP at these points. The minimum changes observed were 1 mmHg for SBP and 1 ms for the pulse interval. Linear regressions relating SBP to the pulse interval were plotted for each sequence; only those sequences with linear *r* values > 0.85 were accepted. For the latter 5-min of respective period, the results were averaged to provide a single data set.

#### **2.5 Statistical analysis**

Data are expressed as means±SE. Statistical analysis was carried out using repeatedmeasures two-way ANOVA followed by Scheffe's test for multiple comparisons. Linear correlation analysis was used to examine the relationship between carotid arterial PWV and cardiovagal BRS. *P*<0.05 was regarded as significant.

#### **3. Results**

Respective temperatures of shower/bath water were 40.6±0.2/40.6±0.1 ºC. Bathroom temperature in showering and bathing sessions was 27.3±0.1 and 27.8±0.1 ºC, and room humidity was 66.7±0.9 and 62.3±1.7 %, respectively.

Cardiovagal BRS before, during, and after showering/bathing is indicated in Fig. 1. Twoway ANOVA revealed an interaction between session (showering or bathing) and time in cardiovagal BRS. In multiple comparisons, cardiovagal BRS did not change during showering but increased during bathing compared to the baseline. Also, cardiovagal BRS was greater during bathing than during showering. The elevated cardiovagal BRS recovered to the baseline after the cessation of bathing.

Hemodynamics during sessions are summarized in Table 1. We did not identify interactions (session x time) in all measures. Again, there were no main effects of session in all indices. Main effects of time were detected in SBP, mean blood pressure (MBP), HR, and doubleproduct (DP, *i.e.*, product of SBP and HR). In details, SBP was higher during and after showering and bathing than the baseline. MBP increased after showering and bathing compared to at rest. HR was higher during showering and bathing in comparison to before showering and bathing. Increased DP was observed during and after showering and bathing. Time was not associated with diastolic blood pressure and pulse pressure.

Blood Pressure Regulation During Bathing: Is There a Cardiovascular Risk? 313

SBP, mmHg Showering 100 ± 2 103 ± 2 105 ± 2 *F*=0.3

MBP, mmHg Showering 67 ± 2 71 ± 2 73 ± 3 *F*=0.7

DBP, mmHg Showering 55 ± 2 55 ± 2 56 ± 2 *F*=1.3

PP, mmHg Showering 45 ± 2 48 ± 2 49 ± 3 *F*=1.4

HR, bpm Showering 68 ± 1 70 ± 2 68 ± 2 *F*=2.2

DP, AU Showering 68.4 ± 1.7 71.5 ± 1.6 71.3 ± 1.4 *F*=1.6

Table 1. Hemodynamics before, during, and after showering/bathing. Values are means±SE. SBP, systolic blood pressure; MBP, mean blood pressure; DBP, diastolic blood pressure; PP,

The relationship between carotid arterial PWV and cardiovagal BRS at rest is demonstrated

Table 2 shows body temperatures before and after showering/bathing. There was no interaction between session and time in skin temperature. However, two-way ANOVA identified the main effect of time; skin temperature increased after showering and bathing. No differences in skin temperature between showering and bathing were present. On the other hand, we observed an interaction (session x time) in tympanic temperature. In multiple comparisons, showering did not change tympanic temperature but bathing

Spontaneous cardiovagal BRS, hemodynamics, and body temperature in women were measured before, during, and after showering/bathing. Carotid arterial PWV, an index of arterial stiffness, was also measured at rest. We demonstrated for the first time that spontaneous cardiovagal BRS increased during bathing compared to the baseline. There were no differences in the changes from the baseline in blood pressure, HR, and DP between showering and bathing sessions. Tympanic temperature did not change after showering but increased after bathing in comparison to the baseline. Cardiovagal BRS was negatively correlated with carotid arterial PWV. We concluded that cardiovagal BRS increases during bathing. Increased cardiovagal BRS may attenuate the blood pressure changes during

pulse pressure; HR, heart rate; DP, double product; AU, arbitrary unit.

in Fig. 2. Carotid arterial PWV was correlated with cardiovagal BRS.

increased it.

**4. Discussion** 

/Bathing

Bathing 99 ± 3 103 ± 2 102 ± 3 *P*=0.73

Bathing 70 ± 2 71 ± 2 72 ± 2 *P*=0.49

Bathing 52 ± 3 56 ± 4 58 ± 3 *P*=0.28

Bathing 46 ± 2 47 ± 4 44 ± 2 *P*=0.27

Bathing 68 ± 2 72 ± 3 71 ± 2 *P*=0.12

Bathing 66.5 ± 2.7 73.7 ± 3.3 72.0 ± 3.0 *P*=0.20

After Interaction

Before Showering

Fig. 1. Cardiovagal baroreflex sensitivity (BRS) before, during, and after showering/bathing. Data are expressed as means±SE. Repeated-measures two-way ANOVA (session x time) revealed an interaction in cardiovagal BRS (*F*=6.4, *P*<0.01). On multiple comparisons, showering was not associated with cardiovagal BRS, but bathing increased it compared to the baseline. The increased BRS during bathing recovered to the baseline after the cessation of bathing. \**P*<0.05 vs. before bathing; †*P*<0.05 vs. showering.


Table 1. Hemodynamics before, during, and after showering/bathing. Values are means±SE. SBP, systolic blood pressure; MBP, mean blood pressure; DBP, diastolic blood pressure; PP, pulse pressure; HR, heart rate; DP, double product; AU, arbitrary unit.

The relationship between carotid arterial PWV and cardiovagal BRS at rest is demonstrated in Fig. 2. Carotid arterial PWV was correlated with cardiovagal BRS.

Table 2 shows body temperatures before and after showering/bathing. There was no interaction between session and time in skin temperature. However, two-way ANOVA identified the main effect of time; skin temperature increased after showering and bathing. No differences in skin temperature between showering and bathing were present. On the other hand, we observed an interaction (session x time) in tympanic temperature. In multiple comparisons, showering did not change tympanic temperature but bathing increased it.

#### **4. Discussion**

312 Recent Advances in Cardiovascular Risk Factors

Fig. 1. Cardiovagal baroreflex sensitivity (BRS) before, during, and after showering/bathing. Data are expressed as means±SE. Repeated-measures two-way ANOVA (session x time) revealed an interaction in cardiovagal BRS (*F*=6.4, *P*<0.01). On multiple comparisons, showering was not associated with cardiovagal BRS, but bathing increased it compared to the baseline. The increased BRS during bathing recovered to the baseline after the cessation

of bathing. \**P*<0.05 vs. before bathing; †*P*<0.05 vs. showering.

Spontaneous cardiovagal BRS, hemodynamics, and body temperature in women were measured before, during, and after showering/bathing. Carotid arterial PWV, an index of arterial stiffness, was also measured at rest. We demonstrated for the first time that spontaneous cardiovagal BRS increased during bathing compared to the baseline. There were no differences in the changes from the baseline in blood pressure, HR, and DP between showering and bathing sessions. Tympanic temperature did not change after showering but increased after bathing in comparison to the baseline. Cardiovagal BRS was negatively correlated with carotid arterial PWV. We concluded that cardiovagal BRS increases during bathing. Increased cardiovagal BRS may attenuate the blood pressure changes during

Blood Pressure Regulation During Bathing: Is There a Cardiovascular Risk? 315

Skin temperature, ºC Showering 36.4 ± 0.1 36.6 ± 0.2 *F*=0.3

Tympanic temperature, ºC Showering 37.0 ± 0.1 36.9 ± 0.1 *F*=7.4

Water immersion increases venous return and ventricular preload (Arborelius et al., 1972; Gabrielsen et al., 1993; Onodera et al., 2001). Consequently, stroke volume and cardiac output increases in the water-immersed subjects (Arborelius et al., 1972; Ueno et al., 2005). Therefore, theoretically, arterial SBP and pulse pressure are speculated to be elevated during bathing. However, the reported effects of bathing on blood pressure are not consistent (Allison & Reger, 1998; Nagasawa et al., 2001; Ohnaka et al., 1995). Also in this study, we did not observe differences in blood pressure changes between showering and bathing periods. It is reasonable to consider that the effects of bathing on blood pressure are not marked. The mechanisms underlying the paradox (*i.e.*, bathing increases blood flow but not arterial blood pressure) have, up to now, remained unclear. This study demonstrated that cardiovagal BRS increases during bathing. This enhanced cardiovagal BRS may participate in the regulation

Cardiovagal BRS is regulated via arterial baroreceptors, stretch receptors in arterial tissue. The baroreceptors are located in carotid arterial and aortic bodies. Baroreceptors sense arterial wall strain caused by blood pressure changes and send afferent signals corresponding to the strain level to the cardiovascular center. The stiffness of baroreceptorcontaining arteries such as the carotid artery has been hypothesized to be one of the key factors determining cardiovagal BRS (Kingwell et al., 1995; Monahan et al., 2001; Rowe, 1987). Indeed, Monahan et al. (2001) have demonstrated that age-related carotid arterial stiffening, evaluated using echo tracking, was closely associated with decreased cardiovagal BRS, investigated using the Oxford technique. Again, we demonstrated a linear relationship between carotid arterial PWV and cardiovagal BRS. It is well-established that arterial stiffness increases with advancing age (Avolio et al., 1985; Otsuki et al., 2006a; Otsuki et al., 2006b; Vaitkevicius et al., 1993). Central arterial stiffness are also associated with daily physical activities (Iemitsu et al., 2006; Sugawara et al., 2006). Although the present subjects did not experience a marked blood pressure increase during bathing [*e.g.*, maximal SBP increase was 16 mmHg (from 98 at rest to 114 mmHg)], humans with markedly stiff arteries

Body temperature was measured before and after showering/bathing at two sites. The skin temperature increased after both showering and bathing. On the other hand, the effects on tympanic temperature were different between showering and bathing. Showering did not change the tympanic temperature, bathing slightly but significantly increased it. The tympanic temperature is closer to the core temperature than skin temperature. It is possible that bathing elevated not only the surface but also internal temperature. At least, the

hyperthermic effects are likely to be greater in bathing compared to showering.

Table 2. Body temperature before and after showering/bathing.

of blood pressure and reduce cardiovascular risks during bathing.

Values are means±SE. \**P*<0.05 vs.

may need to take precautions.

Before After Interaction

Bathing 36.3 ± 0.2 36.6 ± 0.1 *P*=0.57

Bathing 37.0 ± 0.1 37.1 ± 0.1\* *P*=0.01

bathing, resulting in comparable hemodynamics responses between showering and bathing. We would like to propose that it may be unreasonable to withhold bathing by reason of cardiovascular risk. Taking into consideration the slight but significant hyperthermic effects, bathing may be more beneficial to humans in comparison to showering, although caution may be required for humans with stiffened carotid arteries.

Fig. 2. Relationship between carotid arterial pulse wave velocity (PWV), an index of arterial stiffness, and cardiovagal baroreflex sensitivity (BRS) at rest. Carotid arterial PWV was correlated with cardiovagal BRS.


Values are means±SE. \**P*<0.05 vs.

314 Recent Advances in Cardiovascular Risk Factors

bathing, resulting in comparable hemodynamics responses between showering and bathing. We would like to propose that it may be unreasonable to withhold bathing by reason of cardiovascular risk. Taking into consideration the slight but significant hyperthermic effects, bathing may be more beneficial to humans in comparison to showering, although caution

Fig. 2. Relationship between carotid arterial pulse wave velocity (PWV), an index of arterial stiffness, and cardiovagal baroreflex sensitivity (BRS) at rest. Carotid arterial PWV was

correlated with cardiovagal BRS.

may be required for humans with stiffened carotid arteries.

Table 2. Body temperature before and after showering/bathing.

Water immersion increases venous return and ventricular preload (Arborelius et al., 1972; Gabrielsen et al., 1993; Onodera et al., 2001). Consequently, stroke volume and cardiac output increases in the water-immersed subjects (Arborelius et al., 1972; Ueno et al., 2005). Therefore, theoretically, arterial SBP and pulse pressure are speculated to be elevated during bathing. However, the reported effects of bathing on blood pressure are not consistent (Allison & Reger, 1998; Nagasawa et al., 2001; Ohnaka et al., 1995). Also in this study, we did not observe differences in blood pressure changes between showering and bathing periods. It is reasonable to consider that the effects of bathing on blood pressure are not marked. The mechanisms underlying the paradox (*i.e.*, bathing increases blood flow but not arterial blood pressure) have, up to now, remained unclear. This study demonstrated that cardiovagal BRS increases during bathing. This enhanced cardiovagal BRS may participate in the regulation of blood pressure and reduce cardiovascular risks during bathing.

Cardiovagal BRS is regulated via arterial baroreceptors, stretch receptors in arterial tissue. The baroreceptors are located in carotid arterial and aortic bodies. Baroreceptors sense arterial wall strain caused by blood pressure changes and send afferent signals corresponding to the strain level to the cardiovascular center. The stiffness of baroreceptorcontaining arteries such as the carotid artery has been hypothesized to be one of the key factors determining cardiovagal BRS (Kingwell et al., 1995; Monahan et al., 2001; Rowe, 1987). Indeed, Monahan et al. (2001) have demonstrated that age-related carotid arterial stiffening, evaluated using echo tracking, was closely associated with decreased cardiovagal BRS, investigated using the Oxford technique. Again, we demonstrated a linear relationship between carotid arterial PWV and cardiovagal BRS. It is well-established that arterial stiffness increases with advancing age (Avolio et al., 1985; Otsuki et al., 2006a; Otsuki et al., 2006b; Vaitkevicius et al., 1993). Central arterial stiffness are also associated with daily physical activities (Iemitsu et al., 2006; Sugawara et al., 2006). Although the present subjects did not experience a marked blood pressure increase during bathing [*e.g.*, maximal SBP increase was 16 mmHg (from 98 at rest to 114 mmHg)], humans with markedly stiff arteries may need to take precautions.

Body temperature was measured before and after showering/bathing at two sites. The skin temperature increased after both showering and bathing. On the other hand, the effects on tympanic temperature were different between showering and bathing. Showering did not change the tympanic temperature, bathing slightly but significantly increased it. The tympanic temperature is closer to the core temperature than skin temperature. It is possible that bathing elevated not only the surface but also internal temperature. At least, the hyperthermic effects are likely to be greater in bathing compared to showering.

Blood Pressure Regulation During Bathing: Is There a Cardiovascular Risk? 317

Gabrielsen, A., Johansen, L. B. & Norsk, P. (1993). Central cardiovascular pressures during

Guinea, G. V., Atienza, J. M., Elices, M., Aragoncillo, P. & Hayashi, K. (2005).

Hayashi, K., Miyachi, M., Seno, N., Takahashi, K., Yamazaki, K., Sugawara, J., Yokoi, T.,

Iemitsu, M., Maeda, S., Otsuki, T., Sugawara, J., Tanabe, T., Jesmin, S., Kuno, S., Ajisaka, R.,

Keller, D. M., Cui, J., Davis, S. L., Low, D. A. & Crandall, C. G. (2006). Heat stress enhances

Kingwell, B. A., Cameron, J. D., Gillies, K. J., Jennings, G. L. & Dart, A. M. (1995). Arterial

Kobayashi, K., Akishita, M., Yu, W., Hashimoto, M., Ohni, M. & Toba, K. (2004).

Lee, K., Jackson, D. N., Cordero, D. L., Nishiyasu, T., Peters, J. K. & Mack, G. W. (2003).

Monahan, K. D., Tanaka, H., Dinenno, F. A. & Seals, D. R. (2001). Central arterial compliance

Nagasawa, Y., Komori, S., Sato, M., Tsuboi, Y., Umetani, K., Watanabe, Y. & Tamura, K.

Onodera, S., Miyachi, M., Nishimura, M., Yamamoto, K., Yamaguchi, H., Takahashi, K., In, J.

Otsuki, T., Maeda, S., Kesen, Y., Yokoyama, N., Tanabe, T., Sugawara, J., Miyauchi, T.,

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Onodera, S. & Mesaki, N. (2006). Fluctuations in carotid arterial distensibility during the menstrual cycle do not influence cardiovagal baroreflex sensitivity, *Acta* 

Miyauchi, T. & Matsuda, M. (2006). Polymorphism in endothelin-related genes limits exercise-induced decreases in arterial stiffness in older subjects, *Hypertension*

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Nishizawa, Y. (2003). Preferential stiffening of central over peripheral arteries in

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Change in spontaneous baroreflex control of pulse interval during heat stress in

is associated with age- and habitual exercise-related differences in cardiovagal

(2001). Effects of hot bath immersion on autonomic activity and hemodynamics: Comparison of the elderly patient and the healthy young, *Jpn Circ J* 65 (7):587-592. Ohnaka, T., Tochihara, Y., Kubo, M. & Yamaguchi, C. (1995). Physiological and subjective

responses to standing showers, sitting showers, and sink baths, *Appl Human Sci* 14

Y., Amaoka, H., Yoshioka, A., Matsui, T. & Hara, H. (2001). Effects of water depth on abdominal [correction of abdominails] aorta and inferior vena cava during

Kuno, S., Ajisaka, R. & Matsuda, M. (2006a). Age-related reduction of systemic arterial compliance induces excessive myocardial oxygen consumption during sub-

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*Physiol* 268 (1 Pt 2):H411-418.

*Physiol* 186 (2):103-110.

47 (5):928-936.

(5):235-239.

We showed that cardiovagal BRS increased on short-term (*i.e.*, 10 min, 40.6 ºC) bathing. Also, Keller et al. (2006) have demonstrated that skeletal muscle sympathetic BRS is enhanced by short-term heat stress (5 min, 46.0-48.0 ºC). On the other hand, prolonged bathing may be counterproductive. Lee et al. (2003) have reported that hot-water (44.0 ºC) immersion of the lower legs for 30 min reduced spontaneous cardiovagal BRS. Prolonged hot-water bathing should be refrained from.

The mechanisms responsible for bathing-induced increases in cardiovagal BRS remain unclear. One explanation for this response may be hyperthermia-related reduction of arterial stiffness. Guinea et al. (2005) have reported that hyperthermia shifts the carotid arterial diameter-pressure curve to the right, suggesting that hyperthermia reduces carotid arterial stiffness. Carotid arterial baroreceptors are stretch receptors. A compliant carotid artery is easily stretched and would be sensitive to changes in blood pressure. Indeed, the present and previous (Monahan et al., 2001) studies have showed a negative relation between cardiovagal BRS and carotid arterial stiffness. We observed that showering increased only the skin temperature, but bathing increased both the skin and tympanic temperature. Collectively, it may be possible that a hyperthermia-induced reduction of carotid arterial stiffness is associated with increased cardiovagal BRS during bathing. Another possible explanation for the changes in cardiovagal BRS is the direct effect of an elevated core temperature on central baroreflex pathways. Previous findings have demonstrated that an increased temperature enhances the neuronal firing rate of thermosensitive neurons (Boulant, 1998). While these findings were limited to the hypothalamic region of the brain, it is a possibility that neurons involved in the pathways of baroreflex control are also thermosensitive and are responsible for the changes in cardiovagal BRS with bathing.

In conclusion, cardiovagal BRS appears to increase during bathing. It may attenuate the bathing-induced blood pressure changes.

#### **5. References**


We showed that cardiovagal BRS increased on short-term (*i.e.*, 10 min, 40.6 ºC) bathing. Also, Keller et al. (2006) have demonstrated that skeletal muscle sympathetic BRS is enhanced by short-term heat stress (5 min, 46.0-48.0 ºC). On the other hand, prolonged bathing may be counterproductive. Lee et al. (2003) have reported that hot-water (44.0 ºC) immersion of the lower legs for 30 min reduced spontaneous cardiovagal BRS. Prolonged

The mechanisms responsible for bathing-induced increases in cardiovagal BRS remain unclear. One explanation for this response may be hyperthermia-related reduction of arterial stiffness. Guinea et al. (2005) have reported that hyperthermia shifts the carotid arterial diameter-pressure curve to the right, suggesting that hyperthermia reduces carotid arterial stiffness. Carotid arterial baroreceptors are stretch receptors. A compliant carotid artery is easily stretched and would be sensitive to changes in blood pressure. Indeed, the present and previous (Monahan et al., 2001) studies have showed a negative relation between cardiovagal BRS and carotid arterial stiffness. We observed that showering increased only the skin temperature, but bathing increased both the skin and tympanic temperature. Collectively, it may be possible that a hyperthermia-induced reduction of carotid arterial stiffness is associated with increased cardiovagal BRS during bathing. Another possible explanation for the changes in cardiovagal BRS is the direct effect of an elevated core temperature on central baroreflex pathways. Previous findings have demonstrated that an increased temperature enhances the neuronal firing rate of thermosensitive neurons (Boulant, 1998). While these findings were limited to the hypothalamic region of the brain, it is a possibility that neurons involved in the pathways of baroreflex control are also thermosensitive and are responsible for the changes in

In conclusion, cardiovagal BRS appears to increase during bathing. It may attenuate the

Aars, H. (1969). Relationship between aortic diameter and aortic baroreceptor activity in

Allison, T. G. & Reger, W. E. (1998). Comparison of responses of men to immersion in circulating water at 40.0 and 41.5 degrees c, *Aviat Space Environ Med* 69 (9):845-850. Arborelius, M., Jr., Ballidin, U. I., Lilja, B. & Lundgren, C. E. (1972). Hemodynamic changes in man during immersion with the head above water, *Aerosp Med* 43 (6):592-598. Avolio, A. P., Deng, F. Q., Li, W. Q., Luo, Y. F., Huang, Z. D., Xing, L. F. & O'Rourke, M. F.

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Boulant, J. A. (1998). Cellular mechanisms of temperature sensitivity in hypothalamic

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hot-water bathing should be refrained from.

cardiovagal BRS with bathing.

**5. References** 

bathing-induced blood pressure changes.

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cats, *Am J Physiol* 254 (2 Pt 2):H377-383.


**17** 

*Serbia* 

**Sagittal Abdominal Diameter as the** 

Edita Stoki1, Biljana Srdi2, Vladimir Brtka3 and Dragana Tomi-Nagli<sup>1</sup>

Obesity has a profound impact on the cardiovascular disease development, and is associated with a reduced overall survival. There is a strong correlation between the central (abdominal) type of obesity and the cardiovascular and metabolic diseases. Among a variety of anthropometric measurements of the abdominal fat size, sagittal abdominal diameter has been proposed as the valid measurement of the visceral fat mass and cardiometabolic risk level. Many studies have analyzed the relationship between sagittal abdominal diameter (SAD), visceral fat area, and different markers of cardiometabolic disturbances with respect to age, gender and ethnicity. Some of them have offered the cut-off values that could be useful in clinical practice, in identifying individuals who are at higher risk of comorbidities of the obesity. Using the principles of rough set theory, based on producing *If-Then* rules, we have developed a model that allows better applicability of SAD in identifying patients at higher cardiovascular risk. In this chapter, we describe the basic principles of the proposed model. Furthermore, we give a broad overview of the main concerns regarding the significance of SAD and its use in diagnosing the abdominal obesity and predicting the

The prevalence of obesity has increased dramatically worldwide during the past few decades. Obesity is recognized as an independent factor for the development of the cardiovascular

Obesity implies increased body weight due to the enlargement of the adipose tissue to the extent that impairs health. Regional obesity appears to be an important indicator of the risk level. Thus, the diagnosis of obesity depends on three main aspects: relative weight (total

diseases. It also predisposes to the development of other cardiovascular risk factors.

body mass with relation to body height), total body fat, and fat distribution.

**1. Introduction** 

adverse cardiometabolic outcomes.

**2. Obesity as a cardiovascular risk factor** 

<sup>2</sup>*Department of Anatomy, Medical Faculty, Novi Sad, Medical Faculty Novi Sad,* 

**Anthropometric Measure of** 

*Institute of Internal Disease, Clinical Centre Vojvodina, Novi Sad,* 

**Cardiovascular Risk** 

*3Technical Faculty "Mihajlo Pupin" Zrenjanin,* 

*1Department of Endocrinology,* 

