**Section 3**

**Cardiovascular Diseases** 

38 Oxidative Stress and Diseases

Zhu, M., Qin, Z.-J., Hu, D., Munishkina, L.A. & Fink, A.L. (2006). α-Synuclein can function

Zinkham, W.H., Lenhard, R.E., Jr. & Childs, B. (1958). A deficiency of glucose-6-phosphate

*Biochemistry*, Vol. 45, pp. 8135-8142.

*Johns Hopkins Hospital*, Vol. 102, No. 4, pp. 169-175.

as an antioxidant preventing oxidation of unsaturated lipid in vesicles,

dehydrogenase activity in erythrocytes from patients with favism, *Bulletin of the* 

**3** 

*Brazil* 

**Reactive Oxygen Species** 

Celso Ferreira2 and Paulo H. N. Saldiva1 *1Faculdade de Medicina da Universidade de São Paulo,* 

*2Faculdade de Medicina do ABC,* 

**and Cardiovascular Diseases** 

Vitor Engrácia Valenti1,2, Luiz Carlos de Abreu2,

Reduction or oxidation caused by addition or loss of any electron is responsible for alterations in functional and structural profile of molecules, hence, changing signaling mechanism. Reactive free radicals play a crucial role in different physiological mechanisms ranging from the immune defense to cell signaling and inflammation (Elahi & Matata, 2006). There is increasing evidence that irregular production of free radicals lead to enhanced stress on cellular structures and causes changes in molecular pathways that underpins the pathogenesis of several relevant human disorders, such as cancer, heart diseases, the process of physiological ageing and neurological diseases (Pacher & Szabo, 2008; Lushchak, 2011a; Lushchak, 2011b). Comprehending the involvement of free radical stress in the pathogenesis of disease will allow us to investigate the development of oxidative stress; a condition that occurs due to an imbalance between cellular production of oxidant molecules and the availability of appropriate antioxidants species that defend against them. It is hoped that this knowledge will subsequently lead to the development of effective therapeutic

The main molecules that are involved in redox signaling are called as reactive oxygen species (ROS), in which we may include hydrogen peroxide (H2O2), nitric oxide (NO), hydroxyl radical, superoxide (O2•−) and peroxynitrite. Current redox signaling investigations indicate that all the vascular constituents, including vascular smooth muscle cells (VSMCs), endothelial and adventitial cells and macrophages, produce ROS (Papaharalambus & Griendling, 2007). ROS are involved in signal transduction which is related to relaxation and contraction of blood vessels, migration, growth and death of

It is known that vascular diseases such as peripheral vascular disease, coronary artery disease and cerebrovascular diseases are the largest cause of morbidity and mortality in industrialized countries. Some common risk factors for vascular disease, including diabetes and hypertension are still prevalent in Western and other populations, indicating that vascular disease will possible continue to impose a substantial burden on health care resources throughout the next generation. The earliest detectable changes in vascular

**1. Introduction** 

interventions against oxidative stress.

vascular cells, and also extracellular matrix (ECM) alterations.

### **Reactive Oxygen Species and Cardiovascular Diseases**

Vitor Engrácia Valenti1,2, Luiz Carlos de Abreu2, Celso Ferreira2 and Paulo H. N. Saldiva1 *1Faculdade de Medicina da Universidade de São Paulo, 2Faculdade de Medicina do ABC, Brazil* 

#### **1. Introduction**

Reduction or oxidation caused by addition or loss of any electron is responsible for alterations in functional and structural profile of molecules, hence, changing signaling mechanism. Reactive free radicals play a crucial role in different physiological mechanisms ranging from the immune defense to cell signaling and inflammation (Elahi & Matata, 2006). There is increasing evidence that irregular production of free radicals lead to enhanced stress on cellular structures and causes changes in molecular pathways that underpins the pathogenesis of several relevant human disorders, such as cancer, heart diseases, the process of physiological ageing and neurological diseases (Pacher & Szabo, 2008; Lushchak, 2011a; Lushchak, 2011b). Comprehending the involvement of free radical stress in the pathogenesis of disease will allow us to investigate the development of oxidative stress; a condition that occurs due to an imbalance between cellular production of oxidant molecules and the availability of appropriate antioxidants species that defend against them. It is hoped that this knowledge will subsequently lead to the development of effective therapeutic interventions against oxidative stress.

The main molecules that are involved in redox signaling are called as reactive oxygen species (ROS), in which we may include hydrogen peroxide (H2O2), nitric oxide (NO), hydroxyl radical, superoxide (O2•−) and peroxynitrite. Current redox signaling investigations indicate that all the vascular constituents, including vascular smooth muscle cells (VSMCs), endothelial and adventitial cells and macrophages, produce ROS (Papaharalambus & Griendling, 2007). ROS are involved in signal transduction which is related to relaxation and contraction of blood vessels, migration, growth and death of vascular cells, and also extracellular matrix (ECM) alterations.

It is known that vascular diseases such as peripheral vascular disease, coronary artery disease and cerebrovascular diseases are the largest cause of morbidity and mortality in industrialized countries. Some common risk factors for vascular disease, including diabetes and hypertension are still prevalent in Western and other populations, indicating that vascular disease will possible continue to impose a substantial burden on health care resources throughout the next generation. The earliest detectable changes in vascular

Reactive Oxygen Species and Cardiovascular Diseases 43

high-salt diet and the role of enhanced ROS in contributing to salt-induced changes in vascular function and hypertension are not completely understood (Cai & Harrison, 2000). Hypertension is a result of enhanced ROS, however, data regarding the most potent cause of ROS-induced hypertension is controversial. In fact, oxidative stress does not elucidate the cause of every kind of hypertension, which develops through many processes. A small number of clinical investigations indicated the protective property of antioxidants (Ceriello et al, 1991; Galley et al, 1997). Nevertheless, it is known that not all animal models of hypertension are related to ROS (Rajagopalan et al, 1996). Additionally, clinical studies demonstrated negative correlation between arterial pressure and oxidative stress markers in subjects with mild to moderate hypertension (Cracowski et al, 2003). It is hard to find a cause–effect association between hypertension and oxidative stress in clinical studies, however, some studies indicated that increased ROS is a risk factor for human hypertension

Animal models are important to support the link between hypertension and oxidative stress. Some procedures performed in animal models helped to comprehend the mechanisms involved in ROS-induced hypertension (Rajagopalan et al, 1996; Puzserova et

The group of Zucker and coworkers from Nebraska have used a model based on heart failure in rabbits (Mousa et al, 2008). According to their method, a platinum wire pacing electrode is sutured to the epicardium of the left ventricle in the rabbits. A ground electrode is secured to the left atrium. All wires are tunneled beneath the skin and exited in the midscapular area. The chest is closed and evacuated; in the same setting, a radiotelemetry unit is implanted into the right femoral artery with the tip of its catheter in the descending aorta to monitor blood pressure and heart rate in the conscious state. Rabbits were allowed to recover from surgery for two weeks before they were used in the study. They developed a rapid pacing model of chronic heart failure. After recovery from surgery, animals are paced at a rate of 360–380 beats/min with the use of a small, light-weight pacing unit of their own design. The pacing rate is adjusted and monitored by frequent echocardiograms. In general, each rabbit is paced at 360 beats/minute for the first week to determine whether it would tolerate this protocol. After the first week, the pacing rate is enhanced to 380 beats/minute and continued at this rate for the remainder of the protocol. The rabbits are continuously paced for 3 weeks. Cardiac dimensions (left ventricular end-diastolic diameter, left ventricular end-systolic diameter, fractional shortening, and ejection fraction) and other hemodynamic parameters are monitored on a weekly basis. Additionally, to left ventricular dimension changes, clinical signs of chronic heart failure such as ascites, pulmonary congestion, and cachexia are appreciated as symptoms of this chronic heart failure model. This model is well accepted in the literature and it was observed enhanced production of ROS in the heart and in the brainstem. Furthermore, they measured two of the three major SOD isoforms (Cu,Zn-SOD and Mn-SOD) and the catalytic subunit of NAD(P)H oxidase, gp91phox into the brainstem. They reported that protein expression of both CuZn SOD and Mn SOD was significantly downregulated in the chronic heart failure condition. The gp91phox protein was significantly enhanced in chronic heart failure rabbits (Gao et al, 2007). The more affected area was the rostroventrolateral area of the medulla oblongata.

(Adbilla et al, 2007).

al, 2010; Valenti et al, 2011a).

disease states are irregularities of the endothelium, resulting in loss of the endothelium's normal homeostatic functions that normally act to inhibit disease-related mechanisms such as thrombosis and inflammation. Particularly, it was previously demonstrated that nitric oxide (NO) produced by NO synthase (eNOS) in the vascular endothelium modulates blood flow and pressure and presents important antiatherogenic effects on platelets, vascular smooth muscle and endothelial cells (Umans and Levi, 1995).

Many previous studies have already demonstrated the effects of oxidative stress on the cardiovascular system. Superoxide dismutase (SOD), an enzyme that catalyze the dismutation of O2•− into oxygen and H2O2, injected into brainstem areas involved in cardiovascular regulation decreased sympathetic nerve activity and decreased blood pressure in swine (Zanzinger and Czachurski, 2009). According to Campese et al. (2004) the lack of low-density lipoprotein (LDL) receptor-enhanced cholesterol blood levels enhanced ROS and impared baroceptor reflex function. Monahan et al (2004) indicated that oxidative stress collaborates to age-associated decreases in cardiovagal baroreflex sensitivity in healthy subjects. Conversely, it was indicated in male smokers that circulating antioxidants had no effect on baroceptor reflex, and minor effects on the cardiovascular system were seen following acute fat and vitamin ingestion (Wright et al, 2009). Overall, comprehending the process which redox signaling modulates cardiovascular system will provide further precise ROS regulation as a therapy for cardiovascular disorders. In this chapter we summarize concepts regarding oxidative stress related to cardiovascular disorders.

#### **2. Models of ROS-induced cardiovascular diseases**

Basic science applied in animal is indispensable to comprehend the pathogenesis, mechanisms involved in therapeutic agents, molecular process, and environmental or genetic factors that increases the risks of disease development. The species of animal studied are influenced by numerous aspects. Usually, animals with small size are preferred because they are more manageable and experiments are less expensive. According to the guide of the principles of animal research, we should use the lowest possible animal and, nowadays, permission would not be granted for using larger animals unless a similar experiment could not be performed on rodent. Nevertheless, a major criticism of using rodents is that they may not adequately correspond to the human situation and this fact occasionally justifies the use of larger animals such as pigs and monkeys (Rees & Alcolado, 2005). In this topic we described the main animal models used in the literature to investigate the mechanisms involved in ROS-induced cardiovascular disorders.

The relationship between enhanced ROS and hypertension is well established in many studies involving diet or endocrine-induced and surgically-induced hypertensive animals (Banday et al, 2007). A variety of evidence suggests that ROS collaborate to impaired endothelial function in several forms of hypertension and that there is enhanced ROS in the microvessels of spontaneously hypertensive rats (SHR) and Dahl salt-sensitive hypertensive rats (Manning et al, 2003). An interesting study by Lenda et al (2000) suggested that ROS can also collaborate to a decreased endothelium-dependent dilation in normotensive rats under an enhanced salt diet. Although the latent level of ROS in contributing to damaged endothelium-dependent vasodilation and decreased NO production during increased dietary salt intake, the nature and mechanisms of the impaired vascular relaxation with the

disease states are irregularities of the endothelium, resulting in loss of the endothelium's normal homeostatic functions that normally act to inhibit disease-related mechanisms such as thrombosis and inflammation. Particularly, it was previously demonstrated that nitric oxide (NO) produced by NO synthase (eNOS) in the vascular endothelium modulates blood flow and pressure and presents important antiatherogenic effects on platelets, vascular

Many previous studies have already demonstrated the effects of oxidative stress on the cardiovascular system. Superoxide dismutase (SOD), an enzyme that catalyze the dismutation of O2•− into oxygen and H2O2, injected into brainstem areas involved in cardiovascular regulation decreased sympathetic nerve activity and decreased blood pressure in swine (Zanzinger and Czachurski, 2009). According to Campese et al. (2004) the lack of low-density lipoprotein (LDL) receptor-enhanced cholesterol blood levels enhanced ROS and impared baroceptor reflex function. Monahan et al (2004) indicated that oxidative stress collaborates to age-associated decreases in cardiovagal baroreflex sensitivity in healthy subjects. Conversely, it was indicated in male smokers that circulating antioxidants had no effect on baroceptor reflex, and minor effects on the cardiovascular system were seen following acute fat and vitamin ingestion (Wright et al, 2009). Overall, comprehending the process which redox signaling modulates cardiovascular system will provide further precise ROS regulation as a therapy for cardiovascular disorders. In this chapter we summarize

Basic science applied in animal is indispensable to comprehend the pathogenesis, mechanisms involved in therapeutic agents, molecular process, and environmental or genetic factors that increases the risks of disease development. The species of animal studied are influenced by numerous aspects. Usually, animals with small size are preferred because they are more manageable and experiments are less expensive. According to the guide of the principles of animal research, we should use the lowest possible animal and, nowadays, permission would not be granted for using larger animals unless a similar experiment could not be performed on rodent. Nevertheless, a major criticism of using rodents is that they may not adequately correspond to the human situation and this fact occasionally justifies the use of larger animals such as pigs and monkeys (Rees & Alcolado, 2005). In this topic we described the main animal models used in the literature to investigate the mechanisms

The relationship between enhanced ROS and hypertension is well established in many studies involving diet or endocrine-induced and surgically-induced hypertensive animals (Banday et al, 2007). A variety of evidence suggests that ROS collaborate to impaired endothelial function in several forms of hypertension and that there is enhanced ROS in the microvessels of spontaneously hypertensive rats (SHR) and Dahl salt-sensitive hypertensive rats (Manning et al, 2003). An interesting study by Lenda et al (2000) suggested that ROS can also collaborate to a decreased endothelium-dependent dilation in normotensive rats under an enhanced salt diet. Although the latent level of ROS in contributing to damaged endothelium-dependent vasodilation and decreased NO production during increased dietary salt intake, the nature and mechanisms of the impaired vascular relaxation with the

smooth muscle and endothelial cells (Umans and Levi, 1995).

concepts regarding oxidative stress related to cardiovascular disorders.

**2. Models of ROS-induced cardiovascular diseases** 

involved in ROS-induced cardiovascular disorders.

high-salt diet and the role of enhanced ROS in contributing to salt-induced changes in vascular function and hypertension are not completely understood (Cai & Harrison, 2000).

Hypertension is a result of enhanced ROS, however, data regarding the most potent cause of ROS-induced hypertension is controversial. In fact, oxidative stress does not elucidate the cause of every kind of hypertension, which develops through many processes. A small number of clinical investigations indicated the protective property of antioxidants (Ceriello et al, 1991; Galley et al, 1997). Nevertheless, it is known that not all animal models of hypertension are related to ROS (Rajagopalan et al, 1996). Additionally, clinical studies demonstrated negative correlation between arterial pressure and oxidative stress markers in subjects with mild to moderate hypertension (Cracowski et al, 2003). It is hard to find a cause–effect association between hypertension and oxidative stress in clinical studies, however, some studies indicated that increased ROS is a risk factor for human hypertension (Adbilla et al, 2007).

Animal models are important to support the link between hypertension and oxidative stress. Some procedures performed in animal models helped to comprehend the mechanisms involved in ROS-induced hypertension (Rajagopalan et al, 1996; Puzserova et al, 2010; Valenti et al, 2011a).

The group of Zucker and coworkers from Nebraska have used a model based on heart failure in rabbits (Mousa et al, 2008). According to their method, a platinum wire pacing electrode is sutured to the epicardium of the left ventricle in the rabbits. A ground electrode is secured to the left atrium. All wires are tunneled beneath the skin and exited in the midscapular area. The chest is closed and evacuated; in the same setting, a radiotelemetry unit is implanted into the right femoral artery with the tip of its catheter in the descending aorta to monitor blood pressure and heart rate in the conscious state. Rabbits were allowed to recover from surgery for two weeks before they were used in the study. They developed a rapid pacing model of chronic heart failure. After recovery from surgery, animals are paced at a rate of 360–380 beats/min with the use of a small, light-weight pacing unit of their own design. The pacing rate is adjusted and monitored by frequent echocardiograms. In general, each rabbit is paced at 360 beats/minute for the first week to determine whether it would tolerate this protocol. After the first week, the pacing rate is enhanced to 380 beats/minute and continued at this rate for the remainder of the protocol. The rabbits are continuously paced for 3 weeks. Cardiac dimensions (left ventricular end-diastolic diameter, left ventricular end-systolic diameter, fractional shortening, and ejection fraction) and other hemodynamic parameters are monitored on a weekly basis. Additionally, to left ventricular dimension changes, clinical signs of chronic heart failure such as ascites, pulmonary congestion, and cachexia are appreciated as symptoms of this chronic heart failure model. This model is well accepted in the literature and it was observed enhanced production of ROS in the heart and in the brainstem. Furthermore, they measured two of the three major SOD isoforms (Cu,Zn-SOD and Mn-SOD) and the catalytic subunit of NAD(P)H oxidase, gp91phox into the brainstem. They reported that protein expression of both CuZn SOD and Mn SOD was significantly downregulated in the chronic heart failure condition. The gp91phox protein was significantly enhanced in chronic heart failure rabbits (Gao et al, 2007). The more affected area was the rostroventrolateral area of the medulla oblongata.

Reactive Oxygen Species and Cardiovascular Diseases 45

In hypertensive renovascular rats, there is a significant increase in systemic ROS, estimated by the thiobarbituric acid reactive substance (TBARS) level in plasma, compared with control rats. Administration of tempol or Vitamin C systemically decreased blood pressure and RSNA only in renovascular hypertensive rats, indicating that the depressor effect in response to the anti-oxidant administration is mediated by a reduction in sympathetic

Some studies evaluated the effects of ROS on vascular properties in rat aorta (Toba et al, 2010; Olukman et al, 2010). Others tried to reveal the mechanisms involved in ROS-induced cardiovascular disease inside the brainstem (Zanzinger et al, 2009; Valenti et al, 2011a;

The SHR is a model which has been well investigated (He et al, 2011). SHR and stroke-prone SHR (SPSHR), genetic models that develop hypertension spontaneously, exhibit enhanced NAD(P)H driven O2•− generation in resistance (mesenteric) and conduit (aortic) vessels (Rodriguez-Iturbe et al, 2003). This is associated with NAD(P)H oxidase subunit overexpression and enhanced oxidase activity (Kishi et al, 2004). Several polymorphisms in the promoter region of the p22phox gene have been identified in SHR (Zalba et al, 2001). This has clinical relevance because an association between a p22phox gene polymorphism and NAD(P)H oxidase–mediated O2•− production in the vascular wall of patients with

Enhanced expression of p47phox has been reported in the renal vasculature, macula densa, and distal nephron from young SHR, suggesting that renal NAD(P)H oxidase upregulation precedes development of hypertension (Kishi et al, 2004). Diminished nitric oxide bioavailability as a consequence of enhanced vascular O2•− generation and downregulation of the thioredoxin system may also collaborate to oxidative stress in SHR and SPSHR (Touyz 2003). Treatment with antioxidant vitamins, NAD(P)H oxidase inhibitors, SOD mimetics, and BH4 and Ang II type-1 (AT1) receptor blockers decrease vascular O2•− production and attenuate development of hypertension in these models (Rodriguez-Iturbe et al, 2003; Shokoji et al, 2003). Taken together, these findings suggest that oxidative stress in genetic hypertension involves enhanced NAD(P)H oxidase activity and dysfunctional endothelial nitric oxide synthase (uncoupled NOS) and is regulated, in part, by AT1 receptors. Figure 1 presents a surgical procedure to record mean arterial pressure and heart rate, while Figure 2 shows recordings from one normotensive Wistar Kyoto and one SHR rat illustrating reflex bradycardia (top) in response to blood pressure increases. In this Figure we may observe the

In animal models of diabetes, several functional and structural alterations of the heart or in cardiac muscle have been documented (Russel et al, 2006). In most studies of type 1 diabetes mellitus, diabetes are induced after administration of the pancreatic beta-cell toxin streptozotocin, and most studies of type 2 diabetes mellitus have been performed in genetic models of obesity and insulin resistance such as the Zucker fatty rat or db/db mice, both of which have mutations that impair leptin receptor signaling, or ob/ob mice, which lacks leptin. Furthermore, because diabetes mellitus develops at varying tempos in these models, it is important to bear in mind that studies performed in animals before the onset of diabetes may reflect changes that are secondary to the underlying obesity and insulin resistance, and

hypertension and atherosclerosis has been described (Moreno et al, 2003).

enhanced mean arterial pressure of the SHR compared to the control animal.

vasomotor activity (Oliveira-Sales et al, 2008).

Campos et al, 2011).

The renovascular model of hypertension is a model which presented enhanced level of ROS (Campos et al, 2011). In this model, rats are anesthetized with ketamine and xylazine (40 mg and 10 mg/kg, respectively, ip); the left renal artery is exposed through an abdominal incision, and the renal artery and renal vein are dissected free from the adherent tissues. The left renal artery is partially obstructed with a silver clip of 0.2-mm width. No clip obstruction is applied to the sham-operated group (n = 15). Animals are submitted to the final experimental procedures 3 or 6 weeks after the surgical procedure. Systolic blood pressure is measured in conscious rats using a pneumatic tail-cuff method.

The role of ROS has been shown in this model of ROS-induced cardiovascular injury, in which the chronic administration of a SOD mimetic, tempol, to reduce ROS was shown to reduce blood pressure (Welch et al, 2003). Moreover, tempol was more effective than AT1 antagonist (candesartan) in reducing blood pressure and in improving renal function in renovascular hypertensive rats, suggesting that ROS plays an important role in mediating of renovascular hypertension (Palm et al, 2010). However, besides the actions of ROS on many tissues, the brain is one of the Ang II targets most affected by ROS. Even when an increase in plasma renin activity is modest after moderate renal artery stenosis, ROS remains increased and collaborates to hypertension (Lerman et al, 1991).Therefore, the involvement of Ang II is believed to decline, whereas ROS increases, during the progression of the 2K1C model. Our hypothesis is that even with a modest increase in circulating Ang II, this peptide acting in the CNS through AT1 receptors might collaborate to NADPH activation, which leads to an increase in local ROS production, causing sympathoexcitation and arterial hypertension.

The central regulation of the sympathetic nervous system (SNS) involved in cardiovascular regulation is complex, involving multiple reflex pathways and neural connections with a large number of neurotransmitters and neuromodulators acting in specific groups of neurons in the CNS involved in the tonic and reflex control of the cardiovascular system. In the CNS, Ang II is able to increase sympathetic vasomotor tone and blood pressure, and is involved in the pathogenesis of many experimental models of hypertension (Campos, 2009). Therefore, the close functional association between NADPH oxidase and the Ang II is of particular relevance in linking oxidative stress in the brain to sympathoexcitation and hypertension. For instance, intracerebroventricular infusion of NADPH oxidase inhibitor antagonizes the pressor response induced by centrally mediated Ang II actions (Gao et al, 2007). In the brain, the overexpression of SOD, an enzyme responsible for O2•− breakdown, also abolishes the central pressor effect of the octapeptide, suggesting that in the CNS there is a positive correlation between the increase in ROS and the central pressor response mediated by Ang II (Zimmerman et al, 2004). Considering that the paraventricular nucleus of the hypothalamus (PVN) and the rostroventrolateral medulla (RVLM) contain critically important neurons involved in the control of sympathetic vasomotor tone and arterial pressure (Valenti et al, 2011a), in the studies reviewed in this article it was examined an increase in AT1 receptor expression and oxidative stress markers within these two nuclei in renovascular hypertension. NAD(P)H oxidase subunits (p47phox and gp91phox) and antioxidant enzyme CuZnSOD mRNA expression were quantified in the RVLM and PVN of renovascular hypertensive rats. It was hypothesized that the overactivity of NADPH oxidase-derived ROS associated with a reduction in the activity of CuZnSOD within the RVLM and PVN could collaborate to renovascular hypertension, particularly in the renindependent phase of hypertension.

The renovascular model of hypertension is a model which presented enhanced level of ROS (Campos et al, 2011). In this model, rats are anesthetized with ketamine and xylazine (40 mg and 10 mg/kg, respectively, ip); the left renal artery is exposed through an abdominal incision, and the renal artery and renal vein are dissected free from the adherent tissues. The left renal artery is partially obstructed with a silver clip of 0.2-mm width. No clip obstruction is applied to the sham-operated group (n = 15). Animals are submitted to the final experimental procedures 3 or 6 weeks after the surgical procedure. Systolic blood

The role of ROS has been shown in this model of ROS-induced cardiovascular injury, in which the chronic administration of a SOD mimetic, tempol, to reduce ROS was shown to reduce blood pressure (Welch et al, 2003). Moreover, tempol was more effective than AT1 antagonist (candesartan) in reducing blood pressure and in improving renal function in renovascular hypertensive rats, suggesting that ROS plays an important role in mediating of renovascular hypertension (Palm et al, 2010). However, besides the actions of ROS on many tissues, the brain is one of the Ang II targets most affected by ROS. Even when an increase in plasma renin activity is modest after moderate renal artery stenosis, ROS remains increased and collaborates to hypertension (Lerman et al, 1991).Therefore, the involvement of Ang II is believed to decline, whereas ROS increases, during the progression of the 2K1C model. Our hypothesis is that even with a modest increase in circulating Ang II, this peptide acting in the CNS through AT1 receptors might collaborate to NADPH activation, which leads to an increase in local ROS production, causing sympathoexcitation and arterial hypertension.

The central regulation of the sympathetic nervous system (SNS) involved in cardiovascular regulation is complex, involving multiple reflex pathways and neural connections with a large number of neurotransmitters and neuromodulators acting in specific groups of neurons in the CNS involved in the tonic and reflex control of the cardiovascular system. In the CNS, Ang II is able to increase sympathetic vasomotor tone and blood pressure, and is involved in the pathogenesis of many experimental models of hypertension (Campos, 2009). Therefore, the close functional association between NADPH oxidase and the Ang II is of particular relevance in linking oxidative stress in the brain to sympathoexcitation and hypertension. For instance, intracerebroventricular infusion of NADPH oxidase inhibitor antagonizes the pressor response induced by centrally mediated Ang II actions (Gao et al, 2007). In the brain, the overexpression of SOD, an enzyme responsible for O2•− breakdown, also abolishes the central pressor effect of the octapeptide, suggesting that in the CNS there is a positive correlation between the increase in ROS and the central pressor response mediated by Ang II (Zimmerman et al, 2004). Considering that the paraventricular nucleus of the hypothalamus (PVN) and the rostroventrolateral medulla (RVLM) contain critically important neurons involved in the control of sympathetic vasomotor tone and arterial pressure (Valenti et al, 2011a), in the studies reviewed in this article it was examined an increase in AT1 receptor expression and oxidative stress markers within these two nuclei in renovascular hypertension. NAD(P)H oxidase subunits (p47phox and gp91phox) and antioxidant enzyme CuZnSOD mRNA expression were quantified in the RVLM and PVN of renovascular hypertensive rats. It was hypothesized that the overactivity of NADPH oxidase-derived ROS associated with a reduction in the activity of CuZnSOD within the RVLM and PVN could collaborate to renovascular hypertension, particularly in the renin-

pressure is measured in conscious rats using a pneumatic tail-cuff method.

dependent phase of hypertension.

In hypertensive renovascular rats, there is a significant increase in systemic ROS, estimated by the thiobarbituric acid reactive substance (TBARS) level in plasma, compared with control rats. Administration of tempol or Vitamin C systemically decreased blood pressure and RSNA only in renovascular hypertensive rats, indicating that the depressor effect in response to the anti-oxidant administration is mediated by a reduction in sympathetic vasomotor activity (Oliveira-Sales et al, 2008).

Some studies evaluated the effects of ROS on vascular properties in rat aorta (Toba et al, 2010; Olukman et al, 2010). Others tried to reveal the mechanisms involved in ROS-induced cardiovascular disease inside the brainstem (Zanzinger et al, 2009; Valenti et al, 2011a; Campos et al, 2011).

The SHR is a model which has been well investigated (He et al, 2011). SHR and stroke-prone SHR (SPSHR), genetic models that develop hypertension spontaneously, exhibit enhanced NAD(P)H driven O2•− generation in resistance (mesenteric) and conduit (aortic) vessels (Rodriguez-Iturbe et al, 2003). This is associated with NAD(P)H oxidase subunit overexpression and enhanced oxidase activity (Kishi et al, 2004). Several polymorphisms in the promoter region of the p22phox gene have been identified in SHR (Zalba et al, 2001). This has clinical relevance because an association between a p22phox gene polymorphism and NAD(P)H oxidase–mediated O2•− production in the vascular wall of patients with hypertension and atherosclerosis has been described (Moreno et al, 2003).

Enhanced expression of p47phox has been reported in the renal vasculature, macula densa, and distal nephron from young SHR, suggesting that renal NAD(P)H oxidase upregulation precedes development of hypertension (Kishi et al, 2004). Diminished nitric oxide bioavailability as a consequence of enhanced vascular O2•− generation and downregulation of the thioredoxin system may also collaborate to oxidative stress in SHR and SPSHR (Touyz 2003). Treatment with antioxidant vitamins, NAD(P)H oxidase inhibitors, SOD mimetics, and BH4 and Ang II type-1 (AT1) receptor blockers decrease vascular O2•− production and attenuate development of hypertension in these models (Rodriguez-Iturbe et al, 2003; Shokoji et al, 2003). Taken together, these findings suggest that oxidative stress in genetic hypertension involves enhanced NAD(P)H oxidase activity and dysfunctional endothelial nitric oxide synthase (uncoupled NOS) and is regulated, in part, by AT1 receptors. Figure 1 presents a surgical procedure to record mean arterial pressure and heart rate, while Figure 2 shows recordings from one normotensive Wistar Kyoto and one SHR rat illustrating reflex bradycardia (top) in response to blood pressure increases. In this Figure we may observe the enhanced mean arterial pressure of the SHR compared to the control animal.

In animal models of diabetes, several functional and structural alterations of the heart or in cardiac muscle have been documented (Russel et al, 2006). In most studies of type 1 diabetes mellitus, diabetes are induced after administration of the pancreatic beta-cell toxin streptozotocin, and most studies of type 2 diabetes mellitus have been performed in genetic models of obesity and insulin resistance such as the Zucker fatty rat or db/db mice, both of which have mutations that impair leptin receptor signaling, or ob/ob mice, which lacks leptin. Furthermore, because diabetes mellitus develops at varying tempos in these models, it is important to bear in mind that studies performed in animals before the onset of diabetes may reflect changes that are secondary to the underlying obesity and insulin resistance, and

Reactive Oxygen Species and Cardiovascular Diseases 47

Fig. 2. Recordings from one Wistar Kyoto control rat and one spontaneously hypertensive rat illustrating reflex bradycardia (top) in response to blood pressure increases. Infusions were given in bolus. MAP: mean arterial pressure; PAP: pulsatile arterial pressure; HR:

Enhanced ROS production in the diabetic heart is a contributing matter to the progression and the development of diabetic cardiomyopathy (Cai et al, 2006). Cumulative superoxidemediated damage or cellular dysfunction results when an imbalance exists in ROS generation and ROS-degrading pathways. Enhanced ROS generation and impaired antioxidant defenses could both collaborate to oxidative stress in diabetic hearts. Several groups have shown that ROS is overproduced in both type 1 and type 2 diabetes (Cai et al, 2006). Under physiological states, most of the ROS generated within cells arises from mitochondria. Whereas enhanced mitochondrial ROS generation has been shown in various tissues such as endothelial cells that are exposed to hyperglycemia (Brownlee, 1995), relatively few studies to date have directly measured mitochondrial ROS production in mitochondria obtained from diabetic hearts. However, overexpression of mitochondrial superoxide dismutase (SOD2) in the heart of a mouse model of type 1 diabetes mellitus reversed altered mitochondrial morphology and function and maintained cardiomyocyte function (Shen et al, 2006). Evidence also exists for enhanced production of ROS from non mitochondrial sources such as NADPH oxidase or decreased neuronal nitric oxide synthase (NOS1) activity coupled with enhanced activation of xanthine oxidoreductase (Saraiva et al, 2006). Whereas evidence for enhanced ROS production in diabetes mellitus is reasonably

heart rate; PHE: phenylephrine.

Fig. 1. Surgical procedure to record basal mean arterial pressure and heart rate in one spontaneously hypertensive rat.

studies performed after the onset of diabetes may reflect the added effects of hyperglycemia of various durations. Most studies have been performed in isolated perfused hearts and reveal depressed cardiac function (Aasum et al, 2002; Aasum et al, 2003). In vivo studies in these rodent models have provided evidence for systolic and diastolic dysfunction by echocardiography (Christoffersen et al, 2003) but in some studies using invasive left ventricle catheterization in mouse models of obesity and diabetes mellitus, left ventricle contractility as determined by developed pressure/developed tension was initially enhanced and may reflect the impact of the enhanced plasma volume and perhaps sympathetic activation associated in part with the underlying obesity (Buchanan et al, 2005). These first observations were additionally clarified later (Van den Bergh et al, 2006). It was assessed the hemodynamic changes in db/db mouse hearts in vivo using a pressure– volume instrument. It was reported decreased contractility using load-independent variables such as preload recruitable stroke work, but steady-state measurements of cardiac output and other load-dependent parameters were increased in db/db mice compared with control mice because of favorable loading conditions, specifically enhanced preload and decreased afterload.

Fig. 1. Surgical procedure to record basal mean arterial pressure and heart rate in one

studies performed after the onset of diabetes may reflect the added effects of hyperglycemia of various durations. Most studies have been performed in isolated perfused hearts and reveal depressed cardiac function (Aasum et al, 2002; Aasum et al, 2003). In vivo studies in these rodent models have provided evidence for systolic and diastolic dysfunction by echocardiography (Christoffersen et al, 2003) but in some studies using invasive left ventricle catheterization in mouse models of obesity and diabetes mellitus, left ventricle contractility as determined by developed pressure/developed tension was initially enhanced and may reflect the impact of the enhanced plasma volume and perhaps sympathetic activation associated in part with the underlying obesity (Buchanan et al, 2005). These first observations were additionally clarified later (Van den Bergh et al, 2006). It was assessed the hemodynamic changes in db/db mouse hearts in vivo using a pressure– volume instrument. It was reported decreased contractility using load-independent variables such as preload recruitable stroke work, but steady-state measurements of cardiac output and other load-dependent parameters were increased in db/db mice compared with control mice because of favorable loading conditions, specifically enhanced preload and

spontaneously hypertensive rat.

decreased afterload.

Fig. 2. Recordings from one Wistar Kyoto control rat and one spontaneously hypertensive rat illustrating reflex bradycardia (top) in response to blood pressure increases. Infusions were given in bolus. MAP: mean arterial pressure; PAP: pulsatile arterial pressure; HR: heart rate; PHE: phenylephrine.

Enhanced ROS production in the diabetic heart is a contributing matter to the progression and the development of diabetic cardiomyopathy (Cai et al, 2006). Cumulative superoxidemediated damage or cellular dysfunction results when an imbalance exists in ROS generation and ROS-degrading pathways. Enhanced ROS generation and impaired antioxidant defenses could both collaborate to oxidative stress in diabetic hearts. Several groups have shown that ROS is overproduced in both type 1 and type 2 diabetes (Cai et al, 2006). Under physiological states, most of the ROS generated within cells arises from mitochondria. Whereas enhanced mitochondrial ROS generation has been shown in various tissues such as endothelial cells that are exposed to hyperglycemia (Brownlee, 1995), relatively few studies to date have directly measured mitochondrial ROS production in mitochondria obtained from diabetic hearts. However, overexpression of mitochondrial superoxide dismutase (SOD2) in the heart of a mouse model of type 1 diabetes mellitus reversed altered mitochondrial morphology and function and maintained cardiomyocyte function (Shen et al, 2006). Evidence also exists for enhanced production of ROS from non mitochondrial sources such as NADPH oxidase or decreased neuronal nitric oxide synthase (NOS1) activity coupled with enhanced activation of xanthine oxidoreductase (Saraiva et al, 2006). Whereas evidence for enhanced ROS production in diabetes mellitus is reasonably

Reactive Oxygen Species and Cardiovascular Diseases 49

Others mechanisms involved in ROS production are better described in the chapters from

Based on the literature, the NOX family includes seven members, which are NOX1-5 and DUOX1-2. NOX2 NADPH oxidase is the predominant source of ROS production in humans (Nauseef, 2004). The main sources of ROS are phagocytic cells—neutrophils and macrophages. NOX2-NADPH oxidase is formed by functional transmembrane heterodimers, gp91 phox and p22 phox (also known collectively as the cytochrome b558), and four regulatory cytosolic subunits—p40 phox , p47 phox , p67 phox , and the small GTPase, Rac2. In the dormant state, cytochrome b558 resides in intracellular vesicles, while cytosolic Rac2 remains inactive in the guanosine diphosphate (GDP) bound state via interaction with RhoGDI (Ando et al, 1992). Upon the initiation of phagocytosis, GDP-Rac2 is converted to GTP-Rac2 through the activity of a Rac guanine nucleotide exchange factor. This allows for Rac2 translocation to the plasma or phagosomal membrane, thereby allowing the subsequent transit of cytochrome b558 from the vesicle to the membrane (Diebold and Bokoch, 2001). Concurrently, p47 phox is phosphorylated and undergoes a conformational change that now exposes two SRC-homology 3 regions to interact with the proline rich motif on p22 phox (Dusi et al, 1993). Furthermore, Phox homology domains on p47 phox allow for binding to phosphatidylinositol 3-phosphate (PI(3)P) and PI(3,4)P2, transient phosphoinositides that are generated only at the plasma membrane upon phagocytosis, thus, further stabilizing p47 phox localization to cytochrome b558 (Nauseef,

Among ROS sources, the NADPH oxidases are considered unique because from those components it is generated ROS in a highly regulated mode whereas ROS are generated as a by-product of enzymatic activity for all the other sources (Cave et al, 2006). Moreover, NADPH oxidases can stimulate further ROS production from one or more of the above enzymes, thereby being able to act as initiating sources of ROS. O2•−radical is the first moiety that is generated by NADPH oxidases (or most of the other sources) and can be rapidly dismutated to H2O2. The biological effects of ROS are expected to depend on the specific moiety generated, its localization and the relative balance between levels generated and the activity of antioxidant mechanisms; most signaling effects of ROS are considered to

XO is another potential source for ROS in vasculature. XO is a form of xanthine oxidoreductase that occurs in two different forms. The predominant form, xanthine dehydrogenase (XDH), can be converted into XO reversibly by direct oxidation of critical cysteine residues or irreversibly by proteolysis (Harris et al, 1999). XO is expressed mainly in the endothelium, and its expression and activity are enhanced by Ang II or oscillatory

Mitochondrial electron transport generates SO2•− as a side product of electron transport during oxidative phosphorylation. Most superoxide never escapes the highly reducing state of the mitochondrial matrix. If the SO2•− generation is excessive, however, superoxide can escape to the intermembraneous space and cytosol via anion channels (Aon et al, 2004).

In summary, the investigation of the sources of reactive oxygen species is essential to describe new pathways involved in the pathogenesis of cardiovascular diseases as well as to

be mediated by H2O2 which is more stable and diffusible than O2•−.

shear in a NADPH oxidase-dependent manner (Landmesser et al, 2007).

develop new therapies to treat those disorders.

this book. Thus, we will only briefly describe the main sources in this topic.

2004).

strong, the effect of diabetes on antioxidant defenses in the heart is controversial. Thus, the activities/expression levels of glutathione peroxidase, copper/zinc SOD, or catalase were either enhanced (Li et al, 2006) or decreased (Matkovics et al, 1997). Enhanced ROS generation may activate maladaptive signaling pathways, which may lead to cell death, which could collaborate to the pathogenesis of diabetic cardiomyopathy. Enhanced ROS production was associated with enhanced apoptosis, as evidenced by enhanced in situ nick end-labeling (TUNEL) staining and caspase 3 activation in ob/ob and db/db hearts (Barouch et al, 2003). In the same study, enhanced ROS was also associated with enhanced DNA impairment and loss of activity of DNA repair pathways that declined more rapidly with age in diabetic versus control animals.

Therefore, enhanced ROS-modulated cell death is able to promote irregular cardiac remodeling, which ultimately may collaborate to the morphological characteristic and functional abnormalities that are associated with diabetic cardiomyopathy. In addition to causing cellular injury, enhanced ROS production might lead to cardiac dysfunction via other mechanisms. For instance, enhanced ROS has been proposed to amplify hyperglycemia-induced activation of protein kinase C isoforms, enhanced formation of glucose-derived advanced glycation end products, and enhanced glucose flux through the aldose reductase pathways (Brownlee et al, 1995), which may all collaborate to various ways to the development of cardiac complications in diabetes mellitus. Enhanced ROS also might collaborate to mitochondrial uncoupling, which could impair myocardial energetic metabolism in diabetes.

Strategies that enhance mitochondrial ROS scavenging systems have been demonstrate to be effective in decreasing diabetes-induced cardiac dysfunction. Overexpression of metallothionein, catalase, and manganese SOD (Shen et al, 2006) in the heart reversed diabetic cardiomyopathy in animal models of both type 1 and type 2 diabetes. Therefore, strategies that either reduce ROS or augment myocardial antioxidant defense mechanisms might have therapeutic efficacy in improving myocardial function in diabetes mellitus.

In summary, experimental protocols applied in animals are necessary to understand the pathological events involved in cardiovascular diseases development. However, there are differences between human and animals like rat and mouse. Thus, we should be careful when interpreting data aiming to apply in humans regarding processes related to therapeutic agents, molecular process, and environmental or genetic factors that enhances the risks for cardiovascular disorders development.

#### **3. Sources of ROS in cells**

The literature indicated that vascular cells, as well as cardiomyocytes and neurons, produce ROS, contributing to the development of disorders related to the cardiovascular system. Although several enzyme systems produce ROS, many of them are prevalent in pathologic processes. Among the main ROS generators we may include cytochrome P450, the mitochondrial respiratory chain, xanthine oxidase (XO), uncoupled endothelial nitric oxide synthase (eNOS), heme oxygenase, myeloperoxidase, lipoxygenase, cyclooxygenase and NADPH oxidases. Some of these systems have been proven to be relevant to hypertension (Lee & Griendling, 2008).

strong, the effect of diabetes on antioxidant defenses in the heart is controversial. Thus, the activities/expression levels of glutathione peroxidase, copper/zinc SOD, or catalase were either enhanced (Li et al, 2006) or decreased (Matkovics et al, 1997). Enhanced ROS generation may activate maladaptive signaling pathways, which may lead to cell death, which could collaborate to the pathogenesis of diabetic cardiomyopathy. Enhanced ROS production was associated with enhanced apoptosis, as evidenced by enhanced in situ nick end-labeling (TUNEL) staining and caspase 3 activation in ob/ob and db/db hearts (Barouch et al, 2003). In the same study, enhanced ROS was also associated with enhanced DNA impairment and loss of activity of DNA repair pathways that declined more rapidly

Therefore, enhanced ROS-modulated cell death is able to promote irregular cardiac remodeling, which ultimately may collaborate to the morphological characteristic and functional abnormalities that are associated with diabetic cardiomyopathy. In addition to causing cellular injury, enhanced ROS production might lead to cardiac dysfunction via other mechanisms. For instance, enhanced ROS has been proposed to amplify hyperglycemia-induced activation of protein kinase C isoforms, enhanced formation of glucose-derived advanced glycation end products, and enhanced glucose flux through the aldose reductase pathways (Brownlee et al, 1995), which may all collaborate to various ways to the development of cardiac complications in diabetes mellitus. Enhanced ROS also might collaborate to mitochondrial uncoupling, which could impair myocardial energetic

Strategies that enhance mitochondrial ROS scavenging systems have been demonstrate to be effective in decreasing diabetes-induced cardiac dysfunction. Overexpression of metallothionein, catalase, and manganese SOD (Shen et al, 2006) in the heart reversed diabetic cardiomyopathy in animal models of both type 1 and type 2 diabetes. Therefore, strategies that either reduce ROS or augment myocardial antioxidant defense mechanisms might have therapeutic efficacy in improving myocardial function in diabetes mellitus.

In summary, experimental protocols applied in animals are necessary to understand the pathological events involved in cardiovascular diseases development. However, there are differences between human and animals like rat and mouse. Thus, we should be careful when interpreting data aiming to apply in humans regarding processes related to therapeutic agents, molecular process, and environmental or genetic factors that enhances

The literature indicated that vascular cells, as well as cardiomyocytes and neurons, produce ROS, contributing to the development of disorders related to the cardiovascular system. Although several enzyme systems produce ROS, many of them are prevalent in pathologic processes. Among the main ROS generators we may include cytochrome P450, the mitochondrial respiratory chain, xanthine oxidase (XO), uncoupled endothelial nitric oxide synthase (eNOS), heme oxygenase, myeloperoxidase, lipoxygenase, cyclooxygenase and NADPH oxidases. Some of these systems have been proven to be relevant to hypertension

with age in diabetic versus control animals.

the risks for cardiovascular disorders development.

**3. Sources of ROS in cells** 

(Lee & Griendling, 2008).

metabolism in diabetes.

Others mechanisms involved in ROS production are better described in the chapters from this book. Thus, we will only briefly describe the main sources in this topic.

Based on the literature, the NOX family includes seven members, which are NOX1-5 and DUOX1-2. NOX2 NADPH oxidase is the predominant source of ROS production in humans (Nauseef, 2004). The main sources of ROS are phagocytic cells—neutrophils and macrophages. NOX2-NADPH oxidase is formed by functional transmembrane heterodimers, gp91 phox and p22 phox (also known collectively as the cytochrome b558), and four regulatory cytosolic subunits—p40 phox , p47 phox , p67 phox , and the small GTPase, Rac2. In the dormant state, cytochrome b558 resides in intracellular vesicles, while cytosolic Rac2 remains inactive in the guanosine diphosphate (GDP) bound state via interaction with RhoGDI (Ando et al, 1992). Upon the initiation of phagocytosis, GDP-Rac2 is converted to GTP-Rac2 through the activity of a Rac guanine nucleotide exchange factor. This allows for Rac2 translocation to the plasma or phagosomal membrane, thereby allowing the subsequent transit of cytochrome b558 from the vesicle to the membrane (Diebold and Bokoch, 2001). Concurrently, p47 phox is phosphorylated and undergoes a conformational change that now exposes two SRC-homology 3 regions to interact with the proline rich motif on p22 phox (Dusi et al, 1993). Furthermore, Phox homology domains on p47 phox allow for binding to phosphatidylinositol 3-phosphate (PI(3)P) and PI(3,4)P2, transient phosphoinositides that are generated only at the plasma membrane upon phagocytosis, thus, further stabilizing p47 phox localization to cytochrome b558 (Nauseef, 2004).

Among ROS sources, the NADPH oxidases are considered unique because from those components it is generated ROS in a highly regulated mode whereas ROS are generated as a by-product of enzymatic activity for all the other sources (Cave et al, 2006). Moreover, NADPH oxidases can stimulate further ROS production from one or more of the above enzymes, thereby being able to act as initiating sources of ROS. O2•−radical is the first moiety that is generated by NADPH oxidases (or most of the other sources) and can be rapidly dismutated to H2O2. The biological effects of ROS are expected to depend on the specific moiety generated, its localization and the relative balance between levels generated and the activity of antioxidant mechanisms; most signaling effects of ROS are considered to be mediated by H2O2 which is more stable and diffusible than O2•−.

XO is another potential source for ROS in vasculature. XO is a form of xanthine oxidoreductase that occurs in two different forms. The predominant form, xanthine dehydrogenase (XDH), can be converted into XO reversibly by direct oxidation of critical cysteine residues or irreversibly by proteolysis (Harris et al, 1999). XO is expressed mainly in the endothelium, and its expression and activity are enhanced by Ang II or oscillatory shear in a NADPH oxidase-dependent manner (Landmesser et al, 2007).

Mitochondrial electron transport generates SO2•− as a side product of electron transport during oxidative phosphorylation. Most superoxide never escapes the highly reducing state of the mitochondrial matrix. If the SO2•− generation is excessive, however, superoxide can escape to the intermembraneous space and cytosol via anion channels (Aon et al, 2004).

In summary, the investigation of the sources of reactive oxygen species is essential to describe new pathways involved in the pathogenesis of cardiovascular diseases as well as to develop new therapies to treat those disorders.

Reactive Oxygen Species and Cardiovascular Diseases 51

changes and allows the phox homology (PX) domain and the SH3 domain in p47phox to interact with phosphoinositides and p22phox in the membrane, respectively (Ago et al, 2004). As p67phox and p40phox interact with p47phox, this process leads to membrane translocation of p67phox and p40phox. Rac1 translocates to the membrane independently of p47phox and p67phox, where they form a functional complex with the Nox2-p22phox heterodimer, followed by a transfer of electrons to molecular oxygen (Quinn et al, 1993). Therefore, the activity of Nox2 is subjected to regulation through multiple mechanisms.

In relation to myocardial damage and NADPH oxidase, the loss of cardiomyocytes through apoptosis or necrosis causes impairment in cardiac function in the heart submitted to chronic myocardial infarction (Wencker et al, 2003). Oxidative stress is involved in the pathogenesis of apoptosis through various pathways, in which it is included activation of enzymes involved in pro-apoptotic signaling, for example, JNK, p38, ASK-1, and CaMKII (Matsuzawa and Ichijo, 2005), effects on the cellular anti-apoptotic signaling and direct

Although excessive production of ROS by Noxs is detrimental, local and modest production of H2O2 and O2•− by Noxs allows those component to function as signaling molecules, thereby mediating physiological responses. For instance, since Noxs are functional at low pO2, Noxs may function as a sensor, and ROS generated by Noxs as a transducer, for hypoxia (Shiose et al, 2001). Erythropoietin (EPO) synthesis occurs in the renal tubular cells, where Nox4 is abundantly expressed (Lacombe et al., 1988). Since DPI, an antioxidant drug, not only blocks oxygen sensing but also inhibits Nox4 in renal tubular cells, it has been proposed that Nox4 is an O2 sensor in the kidney and may regulate EPO production. The causative role of Nox4 in mediating EPO synthesis through its function as an O2 sensor remains to be shown. Recently, a role of Nox4 in mediating angiogenesis during cardiac hypertrophy was reported. Pathological hypertrophy induces upregulation/activation of Nox4, which in turn causes stabilization of HIF-1α, upregulation of VEGF, and increases in angiogenesis (Zhang et al, 2006). It appears that the protective effect of Nox4 prevails under the authors' experimental conditions. It remains unknown, however, whether such a mechanism is sufficient to overcome increases in cell death and mitochondrial dysfunction directly caused by

In addition, the regulation of Ca2+ level in cardiac myocytes is centrally important not only in excitation–contraction coupling but also in many other processes such as the regulation of gene expression and cellular energetics. ROS are recognized to be capable of influencing cellular Ca2+ regulation at several levels, notably via redox alterations of key amino acid residues involved in the function and gating properties of intracellular and plasma membrane ion channels and transporters — e.g., L-type channels, the Na+/Ca2+ exchanger, the sarcoplasmic reticulum (SR) ATPase (SERCA) and the ryanodine receptor (Hool and Corry, 2007). Recent studies have started to address the role of NADPH oxidase-derived

It has been reported that ryanodine receptor-mediated Ca2+-induced Ca2+ release in rat cardiac myocytes is inhibited by an endogenous NADH oxidase activity in the SR, although the molecular nature of this oxidase was not established (Cherednichenko et al, 2004). In contrast to this study, Sanchez et al. (2008) reported the presence of Nox2 NADPH oxidase activity in canine cardiac SR and showed that oxidase activation enhanced S-

effects of ROS on mitochondria, leading to cytochrome-c release.

upregulation of Nox4 in response to hypertrophic stimuli (Ago et al, 2003).

ROS in these effects.

#### **4. ROS in the heart**

It is already known in the literature some mechanisms regarding the role of antioxidants during oxidative stress caused by ROS in the heart tissue. Among the enzymatic and nonenzymatic antioxidants involved in ROS-induced heart tissue injury we may include catalase, glutathione, SOD, ascorbid acid, melatonin, Vitamin C and E, among others. The antioxidant system in SHR cardiomyopathic hearts is induced, possibly due to events of enhanced ROS. This conditioning of the antioxidant system may help to overcome acute stress situations caused by ROS in the failing myocardium (Takimoto & Kass, 2007).

There are several potential sources of ROS in the heart with chronic heart failure (CHF). Excessive ROS derived from mitochondria have been shown in cardiomyocytes from experimental models of myocardial infarction and rapid pacing‐induced heart failure (Ide et al, 2001). The enzyme xanthine oxidase produces O2•− as a byproduct of the terminal steps of purine catabolism and recent studies suggest that it collaborates to oxidative stress in CHF. Xanthine oxidase expression and activity are enhanced in experimental models of CHF as well as in human end‐stage CHF.

Nitric oxide synthase enzymes normally generate nitric oxide, but may instead generate O2•− if this molecule becomes "uncoupled", a state that is mainly observed likely in the setting of lack of the BH4, which is a NOS cofactor or the NOS substrate l‐arginine. NOS uncoupling and subsequent O2•− production are implicated in the genesis of vascular endothelial dysfunction in patients with heart failure (Dixon et al, 2003).

In this context, infiltrating inflammatory cells are also an important source of ROS, mainly in conditions such as myocarditis and in the early stages after myocardial infarction. Recent evidence suggests that complex enzymes called NADPH oxidases are mainly important with regard to redox signalling in CHF and its antecedent conditions (Li et al, 2002). These enzymes catalyse electron transfer from NADPH to molecular oxygen, resulting in the formation of O2•−. NADPH oxidase activity has been found to be enhanced in experimental models of left ventricle hypertrophy and CHF as well as in end‐stage failing human myocardium (Li et al, 2002).

Interestingly, ROS produced by NADPH oxidases can promote ROS generation by other sources, thereby increasing total levels of ROS. For instance, O2•− from NADPH oxidase may oxidize and degrade BH4, thereby leading to NOS uncoupling, and this mechanism has been shown in diabetes and experimental hypertension (Verhaar et al, 2004). Similarly, NADPH oxidase‐derived ROS may also activate xanthine oxidase (Li and Shah, 2004).

Previous studies have already investigated the relationship between the regulation of myocardial growth and death by NADPH oxidase. The classical phagocyte oxidase (gp91phox or Nox2) is also expressed in non-phagocytic cells in the heart, such as cardiomyocytes and fibroblasts (Bendall et al, 2002; Zhang et al, 2006). Activation of Nox2 requires stimulus-induced membrane translocation of cytosolic regulatory subunits, including p47phox, p67phox, p40phox, and Rac1, a small GTPase (Uhlinger et al, 1994). In resting cells, p47phox, p67phox, and p40phox form a ternary complex in the cytoplasm, whereas Rac associates with Rho-GDP dissociation inhibitor. When cells are stimulated with agonists for G protein-coupled receptors, such as angiotensin II (Ang II) type 1 receptors, p47phox is phosphorylated by protein kinase C, which in turn undergoes conformational

It is already known in the literature some mechanisms regarding the role of antioxidants during oxidative stress caused by ROS in the heart tissue. Among the enzymatic and nonenzymatic antioxidants involved in ROS-induced heart tissue injury we may include catalase, glutathione, SOD, ascorbid acid, melatonin, Vitamin C and E, among others. The antioxidant system in SHR cardiomyopathic hearts is induced, possibly due to events of enhanced ROS. This conditioning of the antioxidant system may help to overcome acute

There are several potential sources of ROS in the heart with chronic heart failure (CHF). Excessive ROS derived from mitochondria have been shown in cardiomyocytes from experimental models of myocardial infarction and rapid pacing‐induced heart failure (Ide et al, 2001). The enzyme xanthine oxidase produces O2•− as a byproduct of the terminal steps of purine catabolism and recent studies suggest that it collaborates to oxidative stress in CHF. Xanthine oxidase expression and activity are enhanced in experimental models of

Nitric oxide synthase enzymes normally generate nitric oxide, but may instead generate O2•− if this molecule becomes "uncoupled", a state that is mainly observed likely in the setting of lack of the BH4, which is a NOS cofactor or the NOS substrate l‐arginine. NOS uncoupling and subsequent O2•− production are implicated in the genesis of vascular

In this context, infiltrating inflammatory cells are also an important source of ROS, mainly in conditions such as myocarditis and in the early stages after myocardial infarction. Recent evidence suggests that complex enzymes called NADPH oxidases are mainly important with regard to redox signalling in CHF and its antecedent conditions (Li et al, 2002). These enzymes catalyse electron transfer from NADPH to molecular oxygen, resulting in the formation of O2•−. NADPH oxidase activity has been found to be enhanced in experimental models of left ventricle hypertrophy and CHF as well as in end‐stage failing human

Interestingly, ROS produced by NADPH oxidases can promote ROS generation by other sources, thereby increasing total levels of ROS. For instance, O2•− from NADPH oxidase may oxidize and degrade BH4, thereby leading to NOS uncoupling, and this mechanism has been shown in diabetes and experimental hypertension (Verhaar et al, 2004). Similarly, NADPH oxidase‐derived ROS may also activate xanthine oxidase (Li and Shah, 2004).

Previous studies have already investigated the relationship between the regulation of myocardial growth and death by NADPH oxidase. The classical phagocyte oxidase (gp91phox or Nox2) is also expressed in non-phagocytic cells in the heart, such as cardiomyocytes and fibroblasts (Bendall et al, 2002; Zhang et al, 2006). Activation of Nox2 requires stimulus-induced membrane translocation of cytosolic regulatory subunits, including p47phox, p67phox, p40phox, and Rac1, a small GTPase (Uhlinger et al, 1994). In resting cells, p47phox, p67phox, and p40phox form a ternary complex in the cytoplasm, whereas Rac associates with Rho-GDP dissociation inhibitor. When cells are stimulated with agonists for G protein-coupled receptors, such as angiotensin II (Ang II) type 1 receptors, p47phox is phosphorylated by protein kinase C, which in turn undergoes conformational

endothelial dysfunction in patients with heart failure (Dixon et al, 2003).

stress situations caused by ROS in the failing myocardium (Takimoto & Kass, 2007).

**4. ROS in the heart** 

CHF as well as in human end‐stage CHF.

myocardium (Li et al, 2002).

changes and allows the phox homology (PX) domain and the SH3 domain in p47phox to interact with phosphoinositides and p22phox in the membrane, respectively (Ago et al, 2004). As p67phox and p40phox interact with p47phox, this process leads to membrane translocation of p67phox and p40phox. Rac1 translocates to the membrane independently of p47phox and p67phox, where they form a functional complex with the Nox2-p22phox heterodimer, followed by a transfer of electrons to molecular oxygen (Quinn et al, 1993). Therefore, the activity of Nox2 is subjected to regulation through multiple mechanisms.

In relation to myocardial damage and NADPH oxidase, the loss of cardiomyocytes through apoptosis or necrosis causes impairment in cardiac function in the heart submitted to chronic myocardial infarction (Wencker et al, 2003). Oxidative stress is involved in the pathogenesis of apoptosis through various pathways, in which it is included activation of enzymes involved in pro-apoptotic signaling, for example, JNK, p38, ASK-1, and CaMKII (Matsuzawa and Ichijo, 2005), effects on the cellular anti-apoptotic signaling and direct effects of ROS on mitochondria, leading to cytochrome-c release.

Although excessive production of ROS by Noxs is detrimental, local and modest production of H2O2 and O2•− by Noxs allows those component to function as signaling molecules, thereby mediating physiological responses. For instance, since Noxs are functional at low pO2, Noxs may function as a sensor, and ROS generated by Noxs as a transducer, for hypoxia (Shiose et al, 2001). Erythropoietin (EPO) synthesis occurs in the renal tubular cells, where Nox4 is abundantly expressed (Lacombe et al., 1988). Since DPI, an antioxidant drug, not only blocks oxygen sensing but also inhibits Nox4 in renal tubular cells, it has been proposed that Nox4 is an O2 sensor in the kidney and may regulate EPO production. The causative role of Nox4 in mediating EPO synthesis through its function as an O2 sensor remains to be shown. Recently, a role of Nox4 in mediating angiogenesis during cardiac hypertrophy was reported. Pathological hypertrophy induces upregulation/activation of Nox4, which in turn causes stabilization of HIF-1α, upregulation of VEGF, and increases in angiogenesis (Zhang et al, 2006). It appears that the protective effect of Nox4 prevails under the authors' experimental conditions. It remains unknown, however, whether such a mechanism is sufficient to overcome increases in cell death and mitochondrial dysfunction directly caused by upregulation of Nox4 in response to hypertrophic stimuli (Ago et al, 2003).

In addition, the regulation of Ca2+ level in cardiac myocytes is centrally important not only in excitation–contraction coupling but also in many other processes such as the regulation of gene expression and cellular energetics. ROS are recognized to be capable of influencing cellular Ca2+ regulation at several levels, notably via redox alterations of key amino acid residues involved in the function and gating properties of intracellular and plasma membrane ion channels and transporters — e.g., L-type channels, the Na+/Ca2+ exchanger, the sarcoplasmic reticulum (SR) ATPase (SERCA) and the ryanodine receptor (Hool and Corry, 2007). Recent studies have started to address the role of NADPH oxidase-derived ROS in these effects.

It has been reported that ryanodine receptor-mediated Ca2+-induced Ca2+ release in rat cardiac myocytes is inhibited by an endogenous NADH oxidase activity in the SR, although the molecular nature of this oxidase was not established (Cherednichenko et al, 2004). In contrast to this study, Sanchez et al. (2008) reported the presence of Nox2 NADPH oxidase activity in canine cardiac SR and showed that oxidase activation enhanced S-

Reactive Oxygen Species and Cardiovascular Diseases 53

vascular diseases are the largest cause of mortality and morbidity in industrialized countries. Many common risk factors for vascular disease, such as hypertension and diabetes, remain prevalent in Western and other populations, suggesting that vascular disease will continue to impose a substantial burden on health care resources throughout the next generation. The earliest detectable changes in vascular disease states are irregularities of the endothelium, resulting in loss of the endothelium normal homeostatic functions that normally act to inhibit disease-related processes such as inflammation and thrombosis. In particular, nitric oxide (NO) produced by NO synthase (eNOS) in the vascular endothelium modulates blood flow and pressure (Umans & Levi, 1995) and has important antiatherogenic effects on platelets, vascular smooth muscle and endothelial cells. It is known that ROS causes vascular tone increase, because it influences endothelium regulatory role and also due to its effects on vascular smooth muscle contractility. By influencing phenotype regulation of vascular smooth muscle cells, death of vascular cells, cell migration, atypical growth, and extracellular matrix (ECM) reorganization, ROS

The fact that nitric oxide (NO) is scavenged by superoxide suggests that superoxide production may in part underlie endothelial dysfunction in human atherosclerosis, as it

In vitro (Lambeth et al, 2000) and in vivo (Vita et al, 1990) studies indicate that AChmediated vasorelaxations in human vessels are inversely related to the number of atherosclerotic risks factors present. Nonetheless, functional studies of human vascular superoxide production have been more limited (Lambeth et al, 2000). It was found large variability in both NO mediated vascular relaxations and basal superoxide production in internal mammary arteries (Huraux et al. (1999), however, there was no consistent associations between these two parameters or with clinical risk factors (Lambeth et al, 2000). It was investigated superoxide production by NAD(P)H oxidase in human vessels and the relationships between superoxide production, atherosclerotic risk factor profile and endothelial dysfunction. It was reported the expected inverse correlation between risk factor profile and NO-mediated endothelium-dependent relaxations in vessel ring isometric tension studies. However, it was also found that superoxide production by NAD(P)H oxidases progressively enhanced with increasing risk factor profile (Guzik et al, 2000). Furthermore, NAD(P)H oxidase-mediated superoxide production was inversely correlated with NO-mediated vasorelaxations in individual patients, such that patients with the

highest superoxide production had the most deficient endothelial function.

The association between enhanced vascular NAD(P)H oxidase activity and impaired endothelial vasorelaxations may be due to direct scavenging of NO by superoxide, as has been demonstrated in animal model systems. However, the both could result independently from increasing exposure of endothelium, media and adventitia to factors acting through different signaling pathways. Alternatively, superoxide may directly modulate NOmediated vascular signaling, for instance by peroxynitrite-induced nitration of G proteins or other membrane components (Feron et al, 1999). Previous data suggest that G proteincoupled receptor function is deficient in atherosclerosis (Liao & Clark, 1995). Previous observation that vasorelaxations to ACh were significantly lower than maximal relaxations

collaborate to vascular remodeling (Lee & Griendling, 2008).

does in some experimental models of vascular disease.

glutathionylation of ryanodine receptors and hence SR Ca2+ release — effects which were abrogated by apocynin (a purported Nox inhibitor, but which may act as a non-selective antioxidant). The same group also showed that oxidase activity and the effects on SR Ca2+ release were augmented by tachycardia (Sanchez et al, 2008). O2•−radical production by NADPH oxidase on the SR of bovine coronary artery smooth muscle cells has also been shown to regulate calcium-induced calcium release (Yi et al, 2006). In isolated cardiac myocytes, plasma L-type Ca2+ channel open-state probability was reportedly enhanced by endothelin-1 together with enhanced NADPH oxidase activity, effects which were abolished in cardiomyocytes pre-treated with a specific NADPH oxidase inhibitor, gp91ds-tat (Zeng et al, 2008). These studies suggest that NADPH oxidases may acutely regulate at least two channels directly involved in intracellular Ca2+ homeostasis, i.e. the L-type Ca2+ channel and the ryanodine receptor.

In summary, Figure 3 presents the main mechanisms involved in ROS-induced cardiovascular disorders. Many studies implicate ROS-generating NADPH oxidases in redox signaling in cardiovascular cells and involvement in pathological processes such as cardiac hypertrophy, fibrosis, apoptosis and ventricular remodeling.

Fig. 3. Main mechanisms involved in the potential effects of NADPH oxidase-derived ROS in the cardiac myocyte. SR: sarcoplasmic reticulum; MMPs: matrix metalloproteinases; CICR: calcium-induced calcium release.

#### **5. ROS-Induced vascular damage**

ROS are involved in pathological and physiological processes in the vasculature. Enhanced arterial pressure is partially caused by enhanced total peripheral vascular resistance, which is due to disorders of structural remodeling of blood vessels and vasomotor function. Vascular diseases including coronary artery disease, cerebrovascular and peripheral

glutathionylation of ryanodine receptors and hence SR Ca2+ release — effects which were abrogated by apocynin (a purported Nox inhibitor, but which may act as a non-selective antioxidant). The same group also showed that oxidase activity and the effects on SR Ca2+ release were augmented by tachycardia (Sanchez et al, 2008). O2•−radical production by NADPH oxidase on the SR of bovine coronary artery smooth muscle cells has also been shown to regulate calcium-induced calcium release (Yi et al, 2006). In isolated cardiac myocytes, plasma L-type Ca2+ channel open-state probability was reportedly enhanced by endothelin-1 together with enhanced NADPH oxidase activity, effects which were abolished in cardiomyocytes pre-treated with a specific NADPH oxidase inhibitor, gp91ds-tat (Zeng et al, 2008). These studies suggest that NADPH oxidases may acutely regulate at least two channels directly involved in intracellular Ca2+ homeostasis, i.e. the L-type Ca2+ channel and

In summary, Figure 3 presents the main mechanisms involved in ROS-induced cardiovascular disorders. Many studies implicate ROS-generating NADPH oxidases in redox signaling in cardiovascular cells and involvement in pathological processes such as

Fig. 3. Main mechanisms involved in the potential effects of NADPH oxidase-derived ROS in the cardiac myocyte. SR: sarcoplasmic reticulum; MMPs: matrix metalloproteinases;

ROS are involved in pathological and physiological processes in the vasculature. Enhanced arterial pressure is partially caused by enhanced total peripheral vascular resistance, which is due to disorders of structural remodeling of blood vessels and vasomotor function. Vascular diseases including coronary artery disease, cerebrovascular and peripheral

cardiac hypertrophy, fibrosis, apoptosis and ventricular remodeling.

the ryanodine receptor.

CICR: calcium-induced calcium release.

**5. ROS-Induced vascular damage** 

vascular diseases are the largest cause of mortality and morbidity in industrialized countries. Many common risk factors for vascular disease, such as hypertension and diabetes, remain prevalent in Western and other populations, suggesting that vascular disease will continue to impose a substantial burden on health care resources throughout the next generation. The earliest detectable changes in vascular disease states are irregularities of the endothelium, resulting in loss of the endothelium normal homeostatic functions that normally act to inhibit disease-related processes such as inflammation and thrombosis. In particular, nitric oxide (NO) produced by NO synthase (eNOS) in the vascular endothelium modulates blood flow and pressure (Umans & Levi, 1995) and has important antiatherogenic effects on platelets, vascular smooth muscle and endothelial cells.

It is known that ROS causes vascular tone increase, because it influences endothelium regulatory role and also due to its effects on vascular smooth muscle contractility. By influencing phenotype regulation of vascular smooth muscle cells, death of vascular cells, cell migration, atypical growth, and extracellular matrix (ECM) reorganization, ROS collaborate to vascular remodeling (Lee & Griendling, 2008).

The fact that nitric oxide (NO) is scavenged by superoxide suggests that superoxide production may in part underlie endothelial dysfunction in human atherosclerosis, as it does in some experimental models of vascular disease.

In vitro (Lambeth et al, 2000) and in vivo (Vita et al, 1990) studies indicate that AChmediated vasorelaxations in human vessels are inversely related to the number of atherosclerotic risks factors present. Nonetheless, functional studies of human vascular superoxide production have been more limited (Lambeth et al, 2000). It was found large variability in both NO mediated vascular relaxations and basal superoxide production in internal mammary arteries (Huraux et al. (1999), however, there was no consistent associations between these two parameters or with clinical risk factors (Lambeth et al, 2000).

It was investigated superoxide production by NAD(P)H oxidase in human vessels and the relationships between superoxide production, atherosclerotic risk factor profile and endothelial dysfunction. It was reported the expected inverse correlation between risk factor profile and NO-mediated endothelium-dependent relaxations in vessel ring isometric tension studies. However, it was also found that superoxide production by NAD(P)H oxidases progressively enhanced with increasing risk factor profile (Guzik et al, 2000). Furthermore, NAD(P)H oxidase-mediated superoxide production was inversely correlated with NO-mediated vasorelaxations in individual patients, such that patients with the highest superoxide production had the most deficient endothelial function.

The association between enhanced vascular NAD(P)H oxidase activity and impaired endothelial vasorelaxations may be due to direct scavenging of NO by superoxide, as has been demonstrated in animal model systems. However, the both could result independently from increasing exposure of endothelium, media and adventitia to factors acting through different signaling pathways. Alternatively, superoxide may directly modulate NOmediated vascular signaling, for instance by peroxynitrite-induced nitration of G proteins or other membrane components (Feron et al, 1999). Previous data suggest that G proteincoupled receptor function is deficient in atherosclerosis (Liao & Clark, 1995). Previous observation that vasorelaxations to ACh were significantly lower than maximal relaxations

Reactive Oxygen Species and Cardiovascular Diseases 55

have demonstrated that peroxynitrite oxidizes BH4 to the (non-protonated) BH3 (trihydrobiopterin) radical, and then to BH2, with a rate constant estimated to be 6 × 103 M−1 s−1, several-fold higher than reactions between peroxynitrite and ascorbate, glutathione or thiol groups (Gao et al, 2009). Oxidation not only directly reduces BH4 bioavailability, but the oxidation products themselves (such as BH2), which have no cofactor activity, may compete with BH4 for binding to endothelial NOS (eNOS) (Crabtree et al,

ROS are also involved in vascular remodeling. Vascular remodeling is defined as alteration of structure leading to alteration in wall thickness and lumen diameter. It can be induced through passive adaptation to chronic changes in hemodynamics and/or through neurohumoral factors including Ang II and ROS. The progression of hypertension involves two different types of vascular remodeling: inward eutrophic remodeling and hypertrophic remodeling (Schiffrin, 2004). Eutrophic remodeling is characterized by decreased lumen size, thickening of the media, enhanced media:lumen ratio and, usually, little change in medial cross-sectional area. In this case, the change in vascular smooth muscle (VSMC) size is negligible (Korsgaard et al, 1993), and medial growth toward the lumen is mainly mediated by reorganization of cellular and non cellular material of the existing vascular wall, accompanied by enhanced apoptosis in the periphery of the blood vessel (166). This is common in small resistance arteries of essential hypertensive patients and SHR (Korsgaard

On the other hand, hypertrophic remodeling characterized by an increase in wall crosssectional area predominates in conduit arteries of secondary hypertension, such as those of renovascular hypertensive patients or Ang II-infused hypertensive rats. An increase in cell size and enhanced accumulation of ECM proteins such as collagen and fibronectin are specific features of hypertrophic remodeling (Rizzoni et al, 2000). Hence, VSMC hypertrophy and ECM synthesis are required for hypertrophic remodeling. Both mechanical wall stress and humoral mediators such as Ang II collaborate to hypertrophic remodeling

The both forms of remodeling frequently coexist in different vascular beds and at different stages of hypertension, which occurs even in the same subject. Even though the determinants of each type of remodeling have not been clearly described, the reorganization of media mediated by phenotype modulation of VSMCs, migration, cellular growth, apoptosis, and ECM production and rearrangement is thought to be common to both

Thus, it is not easy to distinguish contributions of each component in vivo. Vascular remodeling is improved by treatment with tempol, antioxidant vitamins (Chen et al, 2011), Ang II receptor antagonists, or NADPH oxidase inhibitors in animal experimental models,

Recent studies with improved forms of ROS scavenging enzymes, specific inhibitors for different ROS generating enzymes, and redox signaling pathway blocking agents allow subtle modulation of redox signaling and may overcome the redundancy of general antioxidant treatments. Therefore, the spatial and temporal aspects of redox signaling in the vasculature are of much importance to understand the etiological role of ROS and to

processes. These events occur cooperatively and simultaneously.

develop better strategies to treat hypertension.

as well as in clinical trials (Zhou et al, 2005), emphasizing the role of ROS.

2008).

et al, 1993).

(Rizzoni et al, 2000).

to the calcium ionophore A23187 is consistent with this hypothesis, and with observations in human internal mammary arteries (Hurax et al, 1999). Nevertheless, the significant correlation between ACh and A23187 - induced relaxations, and the association of NADHdependent superoxide production with both ACh and A231287- stimulated vasorelaxations suggest that a change in G protein-coupled receptor signaling is unlikely to be the sole mechanism underlying decreased NO-mediated vasorelaxations, as A23187 activates endothelial NO synthase independently of any receptor mediated pathway. Alternatively, superoxide may impair endothelial function by direct effects on endothelial NO synthase activity, (Peterson et al, 1999), possibly mediated through oxidation of the NOS cofactor, tetrahydrobiopterin (BH4).

Many studies have focused on the potential role of BH4 oxidation different oxidized biopterin species in reducing BH4 bioavailability for eNOS (Figure 4).

Fig. 4. Schematic representation of nitric oxide synthase (NO synthase) reaction leading to lcitrulline and nitric oxide (NO) from l-arginine and oxygen (O2) without (A) and with BH4 (B). It is important to note that this reaction without BH4 increases ROS.

Although superoxide can indeed react directly with BH4, the rate constant of this reaction is many orders of magnitude lower than that for NO with superoxide (Vasquez-Vivar et al, 2002). A more likely mechanism for BH4 oxidation is the interaction with peroxynitrite (generated from the interaction between NO and superoxide). It was indicated that peroxynitrite can oxidize BH4 within minutes at physiologically relevant concentrations (Crabtree et al, 2011). EPR (electron paramagnetic resonance) spectroscopy experiments

to the calcium ionophore A23187 is consistent with this hypothesis, and with observations in human internal mammary arteries (Hurax et al, 1999). Nevertheless, the significant correlation between ACh and A23187 - induced relaxations, and the association of NADHdependent superoxide production with both ACh and A231287- stimulated vasorelaxations suggest that a change in G protein-coupled receptor signaling is unlikely to be the sole mechanism underlying decreased NO-mediated vasorelaxations, as A23187 activates endothelial NO synthase independently of any receptor mediated pathway. Alternatively, superoxide may impair endothelial function by direct effects on endothelial NO synthase activity, (Peterson et al, 1999), possibly mediated through oxidation of the NOS cofactor,

Many studies have focused on the potential role of BH4 oxidation different oxidized

Fig. 4. Schematic representation of nitric oxide synthase (NO synthase) reaction leading to lcitrulline and nitric oxide (NO) from l-arginine and oxygen (O2) without (A) and with BH4

Although superoxide can indeed react directly with BH4, the rate constant of this reaction is many orders of magnitude lower than that for NO with superoxide (Vasquez-Vivar et al, 2002). A more likely mechanism for BH4 oxidation is the interaction with peroxynitrite (generated from the interaction between NO and superoxide). It was indicated that peroxynitrite can oxidize BH4 within minutes at physiologically relevant concentrations (Crabtree et al, 2011). EPR (electron paramagnetic resonance) spectroscopy experiments

(B). It is important to note that this reaction without BH4 increases ROS.

biopterin species in reducing BH4 bioavailability for eNOS (Figure 4).

tetrahydrobiopterin (BH4).

have demonstrated that peroxynitrite oxidizes BH4 to the (non-protonated) BH3 (trihydrobiopterin) radical, and then to BH2, with a rate constant estimated to be 6 × 103 M−1 s−1, several-fold higher than reactions between peroxynitrite and ascorbate, glutathione or thiol groups (Gao et al, 2009). Oxidation not only directly reduces BH4 bioavailability, but the oxidation products themselves (such as BH2), which have no cofactor activity, may compete with BH4 for binding to endothelial NOS (eNOS) (Crabtree et al, 2008).

ROS are also involved in vascular remodeling. Vascular remodeling is defined as alteration of structure leading to alteration in wall thickness and lumen diameter. It can be induced through passive adaptation to chronic changes in hemodynamics and/or through neurohumoral factors including Ang II and ROS. The progression of hypertension involves two different types of vascular remodeling: inward eutrophic remodeling and hypertrophic remodeling (Schiffrin, 2004). Eutrophic remodeling is characterized by decreased lumen size, thickening of the media, enhanced media:lumen ratio and, usually, little change in medial cross-sectional area. In this case, the change in vascular smooth muscle (VSMC) size is negligible (Korsgaard et al, 1993), and medial growth toward the lumen is mainly mediated by reorganization of cellular and non cellular material of the existing vascular wall, accompanied by enhanced apoptosis in the periphery of the blood vessel (166). This is common in small resistance arteries of essential hypertensive patients and SHR (Korsgaard et al, 1993).

On the other hand, hypertrophic remodeling characterized by an increase in wall crosssectional area predominates in conduit arteries of secondary hypertension, such as those of renovascular hypertensive patients or Ang II-infused hypertensive rats. An increase in cell size and enhanced accumulation of ECM proteins such as collagen and fibronectin are specific features of hypertrophic remodeling (Rizzoni et al, 2000). Hence, VSMC hypertrophy and ECM synthesis are required for hypertrophic remodeling. Both mechanical wall stress and humoral mediators such as Ang II collaborate to hypertrophic remodeling (Rizzoni et al, 2000).

The both forms of remodeling frequently coexist in different vascular beds and at different stages of hypertension, which occurs even in the same subject. Even though the determinants of each type of remodeling have not been clearly described, the reorganization of media mediated by phenotype modulation of VSMCs, migration, cellular growth, apoptosis, and ECM production and rearrangement is thought to be common to both processes. These events occur cooperatively and simultaneously.

Thus, it is not easy to distinguish contributions of each component in vivo. Vascular remodeling is improved by treatment with tempol, antioxidant vitamins (Chen et al, 2011), Ang II receptor antagonists, or NADPH oxidase inhibitors in animal experimental models, as well as in clinical trials (Zhou et al, 2005), emphasizing the role of ROS.

Recent studies with improved forms of ROS scavenging enzymes, specific inhibitors for different ROS generating enzymes, and redox signaling pathway blocking agents allow subtle modulation of redox signaling and may overcome the redundancy of general antioxidant treatments. Therefore, the spatial and temporal aspects of redox signaling in the vasculature are of much importance to understand the etiological role of ROS and to develop better strategies to treat hypertension.

Reactive Oxygen Species and Cardiovascular Diseases 57

produce superoxide when BH4 is oxidized to BH4, leading to an increase in ROS and a reduction in NO (Figure 4). This could set up a vicious cycle that accentuates NO dysfunction and tends to perpetuate oxidative stress. These alterations may constitute an important mechanism of dysregulation that produces hypertension and renal dysfunction (see later). An example of this alteration is the SHR in which blood pressure can be

Researches performed in the renovascular model of hypertension in rats have shown relevant data regarding the relationship between oxidative stress and hypertension. The

involved in cardiovascular regulation is complex, involving multiple reflex pathways and neural connections with a large number of neurotransmitters and neuromodulators acting in specific groups of neurons in the central nervous sysem (CNS) involved in the tonic and reflex control of the cardiovascular system. In the CNS, Ang II is able to increase sympathetic vasomotor tone and blood pressure, and is involved in the pathogenesis of many experimental models of hypertension. Thus, the close functional association between NADPH oxidase and the Ang II is of particular relevance in linking oxidative stress in the brain to sympathoexcitation and hypertension (Campos, 2009). For instance, intracerebroventricular infusion of NADPH oxidase inhibitor antagonizes the pressor

In the brain, the overexpression of SOD, an enzyme responsible for O2 breakdown, also abolishes the central pressor effect of the octapeptide (Zimmerman et al, 2004) suggesting that in the CNS there is a positive correlation between the increase in ROS and the central pressor response mediated by Ang II. Considering that the paraventricular nucleus of the hypothalamus (PVN) and the rostroventrolateral medulla (RVLM) contain critically important neurons involved in the control of sympathetic vasomotor tone and arterial

Previous studies reviewed and examined whether there was an increase in AT1 receptor expression and oxidative stress markers within these two nuclei in 2K1C hypertension. NAD(P)H oxidase subunits (p47phox and gp91phox) and antioxidant enzyme CuZnSOD mRNA expression were quantified in the RVLM and PVN of 2K1C hypertensive rats. It was hypothesized that the overactivity of NADPH oxidase-derived ROS associated with a reduction in the activity of CuZnSOD within the RVLM and PVN could collaborate to 2K1C

In summary, the recent studies support the idea that an increase in ROS in the kidney is involved in the development of cardiovascular disorders, playing a major role in maintaining high arterial pressure and sympathetic drive under conditions of renovascular hypertension.

**7. The involvement of the nervous system in ROS-induced cardiovascular** 

Neurons in the brain present increased density of polyunsaturated fatty acids in its cell membranes. Fatty acids are targets of free radicals. An indirect marker of ROS, TBARS is enhanced in the brainstem of SPSHR compared to age-matched control (Hirooka, 2008). Others reported enhanced ROS in the brainstem of rabbits with heart failure (Gao et al, 2007).

normalized by the administration of BH4 (McIntyre et al, 1997).

central regulation of the sympathetic nervous system (SNS)

pressure (Colombari et al, 2001).

**disease** 

response induced by centrally mediated Ang II actions (Gao et al, 2004).

hypertension, particularly in the renin-dependent phase of hypertension.

#### **6. Oxidative stress in the kidney**

Renal artery obstruction can cause arterial hypertension, which is followed by impaired renal function and renal atrophy. Cardiovascular disorders caused by kidney injury are in part regulated by renin release from the stenotic kidney, with a subsequent increase in angiotensin II (Ang II) synthesis (Trinquart et al, 2010). Ang II results in the activation of O2•− generation through nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, a multi-subunit enzyme, which is one of the enzymatic sources of O2•<sup>−</sup> (Chabrashvili et al, 2002).

The mechanism(s) by which ANG II produces superoxide is not entirely elucidated yet. However, Mollnau et al. (2002) found that ANG II infusion during seven days enhanced expression of nox 1, gp91(phox), and p22(phox) subunits of NADPH oxidase via a PKC system. The mechanism which involves ANG II-mediated increase in the release of superoxide locates into activity a series of events that may play relevant functions in increased blood pressure.

Previous studies were conducted in attempt to clarify the specific components of ROS that are involved in the development of ANG II-induced hypertension. Haas et al. (1999) are among the first to demonstrate that the slow hypertensive response to ANG II was accompanied by a significant elevation of ROS as estimated indirectly by increases in plasma F2-isoprostanes, an oxidative metabolite of arachidonic acid (Morrow et al, 1990). Nishiyama et al. (2002) also demonstrated that a prolonged infusion of ANG II in rats stimulates ROS production. In this study, the administration of tempol, a SOD mimetic, reversed the vasoconstriction and produced vasodilation via an NO-dependent mechanism.

Ortiz et al. (2011) found in rats that the development of slow pressor responses to ANG II could be inhibited by the administration by antioxidants such as tempol and vitamin E. As a result of antioxidant treatment, there was a fall in renal blood flow and glomerular flow rate, whereas the indexes of oxidative stress, TBARS, and isoprostanes were found to be decreased in peripheral circulation as well as the renal vein.

Some investigations suggest that the decrease in the NO concentration due to interaction with superoxide anon radical constitutes a major component in the development of the observed vasoconstriction. Supporting the assumption that inhibition of NO synthesis enhances the vasoconstrictor effect of ANG II are the studies of Kitamoto et al. (2000). These investigators found that the continuous administration of l-NAME (a NOS inhibitor) to Sprague-Dawley rats for seven days induced ROS production, which was dependent on ANG II, because the effect was blocked by the administration of ANG II receptor blockers. Relevant to these findings are the studies of Usui et al. (1999), who also found an increase in ROS produced byl-NAME, which was blocked by the administration of antioxidants. In this study, l-NAME blockade was associated with an increase in angiotensin converting enzyme activity in the aorta. The studies of Kitamoto et al. (2000) and Usui et al. (199) reveal an interesting aspect of ANG II, NO, and oxidative stress. They suggest that a simple decrease in NO synthesis leaves unbalanced ANG II, which induces ROS release. This situation will be further stimulated by the increase in converting enzyme activity, which can accelerate the production of ANG II causing a positive feedback for oxidative stress.

The question of whether NO inhibition alone can cause ROS production without the participation of ANG II should be further explored. As mentioned previously, NOS can

Renal artery obstruction can cause arterial hypertension, which is followed by impaired renal function and renal atrophy. Cardiovascular disorders caused by kidney injury are in part regulated by renin release from the stenotic kidney, with a subsequent increase in angiotensin II (Ang II) synthesis (Trinquart et al, 2010). Ang II results in the activation of O2•− generation through nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, a multi-subunit

The mechanism(s) by which ANG II produces superoxide is not entirely elucidated yet. However, Mollnau et al. (2002) found that ANG II infusion during seven days enhanced expression of nox 1, gp91(phox), and p22(phox) subunits of NADPH oxidase via a PKC system. The mechanism which involves ANG II-mediated increase in the release of superoxide locates into activity a series of events that may play relevant functions in

Previous studies were conducted in attempt to clarify the specific components of ROS that are involved in the development of ANG II-induced hypertension. Haas et al. (1999) are among the first to demonstrate that the slow hypertensive response to ANG II was accompanied by a significant elevation of ROS as estimated indirectly by increases in plasma F2-isoprostanes, an oxidative metabolite of arachidonic acid (Morrow et al, 1990). Nishiyama et al. (2002) also demonstrated that a prolonged infusion of ANG II in rats stimulates ROS production. In this study, the administration of tempol, a SOD mimetic, reversed the vasoconstriction and produced vasodilation via an NO-dependent mechanism. Ortiz et al. (2011) found in rats that the development of slow pressor responses to ANG II could be inhibited by the administration by antioxidants such as tempol and vitamin E. As a result of antioxidant treatment, there was a fall in renal blood flow and glomerular flow rate, whereas the indexes of oxidative stress, TBARS, and isoprostanes were found to be

Some investigations suggest that the decrease in the NO concentration due to interaction with superoxide anon radical constitutes a major component in the development of the observed vasoconstriction. Supporting the assumption that inhibition of NO synthesis enhances the vasoconstrictor effect of ANG II are the studies of Kitamoto et al. (2000). These investigators found that the continuous administration of l-NAME (a NOS inhibitor) to Sprague-Dawley rats for seven days induced ROS production, which was dependent on ANG II, because the effect was blocked by the administration of ANG II receptor blockers. Relevant to these findings are the studies of Usui et al. (1999), who also found an increase in ROS produced byl-NAME, which was blocked by the administration of antioxidants. In this study, l-NAME blockade was associated with an increase in angiotensin converting enzyme activity in the aorta. The studies of Kitamoto et al. (2000) and Usui et al. (199) reveal an interesting aspect of ANG II, NO, and oxidative stress. They suggest that a simple decrease in NO synthesis leaves unbalanced ANG II, which induces ROS release. This situation will be further stimulated by the increase in converting enzyme activity, which can accelerate the

The question of whether NO inhibition alone can cause ROS production without the participation of ANG II should be further explored. As mentioned previously, NOS can

enzyme, which is one of the enzymatic sources of O2•<sup>−</sup> (Chabrashvili et al, 2002).

decreased in peripheral circulation as well as the renal vein.

production of ANG II causing a positive feedback for oxidative stress.

**6. Oxidative stress in the kidney** 

increased blood pressure.

produce superoxide when BH4 is oxidized to BH4, leading to an increase in ROS and a reduction in NO (Figure 4). This could set up a vicious cycle that accentuates NO dysfunction and tends to perpetuate oxidative stress. These alterations may constitute an important mechanism of dysregulation that produces hypertension and renal dysfunction (see later). An example of this alteration is the SHR in which blood pressure can be normalized by the administration of BH4 (McIntyre et al, 1997).

Researches performed in the renovascular model of hypertension in rats have shown relevant data regarding the relationship between oxidative stress and hypertension. The central regulation of the sympathetic nervous system (SNS)

involved in cardiovascular regulation is complex, involving multiple reflex pathways and neural connections with a large number of neurotransmitters and neuromodulators acting in specific groups of neurons in the central nervous sysem (CNS) involved in the tonic and reflex control of the cardiovascular system. In the CNS, Ang II is able to increase sympathetic vasomotor tone and blood pressure, and is involved in the pathogenesis of many experimental models of hypertension. Thus, the close functional association between NADPH oxidase and the Ang II is of particular relevance in linking oxidative stress in the brain to sympathoexcitation and hypertension (Campos, 2009). For instance, intracerebroventricular infusion of NADPH oxidase inhibitor antagonizes the pressor response induced by centrally mediated Ang II actions (Gao et al, 2004).

In the brain, the overexpression of SOD, an enzyme responsible for O2 breakdown, also abolishes the central pressor effect of the octapeptide (Zimmerman et al, 2004) suggesting that in the CNS there is a positive correlation between the increase in ROS and the central pressor response mediated by Ang II. Considering that the paraventricular nucleus of the hypothalamus (PVN) and the rostroventrolateral medulla (RVLM) contain critically important neurons involved in the control of sympathetic vasomotor tone and arterial pressure (Colombari et al, 2001).

Previous studies reviewed and examined whether there was an increase in AT1 receptor expression and oxidative stress markers within these two nuclei in 2K1C hypertension. NAD(P)H oxidase subunits (p47phox and gp91phox) and antioxidant enzyme CuZnSOD mRNA expression were quantified in the RVLM and PVN of 2K1C hypertensive rats. It was hypothesized that the overactivity of NADPH oxidase-derived ROS associated with a reduction in the activity of CuZnSOD within the RVLM and PVN could collaborate to 2K1C hypertension, particularly in the renin-dependent phase of hypertension.

In summary, the recent studies support the idea that an increase in ROS in the kidney is involved in the development of cardiovascular disorders, playing a major role in maintaining high arterial pressure and sympathetic drive under conditions of renovascular hypertension.

#### **7. The involvement of the nervous system in ROS-induced cardiovascular disease**

Neurons in the brain present increased density of polyunsaturated fatty acids in its cell membranes. Fatty acids are targets of free radicals. An indirect marker of ROS, TBARS is enhanced in the brainstem of SPSHR compared to age-matched control (Hirooka, 2008). Others reported enhanced ROS in the brainstem of rabbits with heart failure (Gao et al, 2007).

Reactive Oxygen Species and Cardiovascular Diseases 59

sympathetic outflow in both normal and chronic heart failure rabbits (Gao et al, 2007). Taken together, those data suggest that antioxidant enzymes, i.e., SOD and catalase, into the brainstem are involved in baroceptor reflex regulation, since baroreflex is modulated by

Recent studies from our laboratory have investigated the effects of ROS into the fourth

In one study (Valenti et al, 2011a) it was evaluated the effects of 3-amino-1,2,4-triazole (ATZ), a catalase inhibitor, into the 4th V on baroreflex components in conscious rats. It was revealed that this drug significantly attenuated bradycardic and tachycardic reflex, bradycardic peak and it also decreased heart rate range 30 minutes after its injection. While in Wistar rats treated with vehicle (saline 0.9%) there were no significant changes regarding baseline mean arterial pressure (MAP) and heart rate (HR) and baroreflex components. Considering that the tachycardia (tachycardic reflex) in response to SNP is mediated by both sympathetic and parasympathetic activity (Stornetta et al, 1987) and that we reported reduction in the maximal parasympathetic responses to elevation in mean arterial pressure, while there were no changes in tachycardic peak response to decrease in mean arterial pressure (highest sympathetic response), we suggest that ATZ into the 4th V is acutely involved with parasympathetic activity but is not involved in baroreflex changes. The lack of any change in the vehicles groups is consistent with this assumption. In view of the anatomical scope of the 4th V, an action on an only one neuronal cluster is not an easy accomplishment. However, prior researches indicated a preference for parasympathetic system which modulates HR, such as the dorsal motor nucleus of the vagus and nucleus ambiguous, which receive glutamathergic projections from the nucleus of the solitary tract

In another study (Valenti et al, 2011b), it was evaluated the effects of catalase inhibition into the 4th V on cardiopulmonary reflex in conscious Wistar rats. In this method, we used male Wistar rats, which were implanted with a stainless steel guide cannula in the 4th V. The femoral artery and vein were cannulated for MAP and HR measurement and for drug infusion, respectively. After basal mean arterial pressure and heart rate recordings, the cardiopulmonary reflex was tested with a dose of phenylbiguanide (PBG, 8 μg/kg, bolus). Cardiopulmonary reflex was evaluated before and 15 minutes after 1 μl of ATZ (0.01 g/100 μl) injection into the 4th V. Vehicle treatment did not change cardiopulmonary reflex responses. ATZ injected into the 4th V significantly enhanced hypotensive responses without influencing the bradycardic reflex. Taken together, those data suggested that ATZ injected into the 4th V increases sympathetic inhibition but does not change the

parasympathetic component of the cardiopulmonary reflex in conscious Wistar rats.

minutes after its injection in conscious WKY rats.

Nevertheless, opposite findings were found in SHR. Another study (Valenti et al, 2011c) was undertaken to evaluate the acute effects of central n-acetylcysteine, an antioxidant drug, on baroreflex in juvenile SHR and age-matched Wistar Kyoto (WKY) rats. It was observed that n-acetylcysteine injection into the 4th V did not significantly change baroreflex gain, bradycardic and tachycardic reflex, bradycardic and tachycardic peak in SHR and WKY rats. Interestingly, n-acetylcysteine caused slight but significant increase in basal heart rate 15

sympathetic and parasympathetic activity (Valenti et al, 2009a).

cerebral ventricle (4th V) on cardiovascular responses.

(Colombari et al, 2001).

The activity of sympathetic and parasympathetic systems, which are both involved in cardiopulmonary reflex, as well as the cardiovascular regulation, is under the control of a medullary circuitry comprising the nucleus of the solitary tract (NTS), rostral (RVLM) and caudal ventrolateral medulla (CVLM) and the nucleus ambiguous. Drugs injection into the fourth cerebral ventricle (4th V) may easily reach structures surrounding the ventricular system like the area postrema and the dorsal motor nucleus of the vagus (Colombari et al, 2001) (Figure 5). Those areas are also involved in cardiovascular reflex responses, in which we may include baroreflex (Valenti et al, 2009a; Valenti et al, 2009b; Cisternas et al, 2010).

Fig. 5. Schematic sagittal view of the medulla oblongata showing brain pathways implicated in neurogenic hypertension. Premotor neurons from the RVLM send excitatory synapses to preganglionic neurons situated in the intermediolateral cell column (IML), providing sympathetic efference to target organs. The RVLM is the group of neurons that receive excitatory afference from the commissural nucleus of the solitary tract (NTS) and area postrema. It also receives inhibitory afferences from the caudoventrolateral medulla (CVLM). Adapted from Valenti et al, 2007.

A previous investigation suggested that brain ROS is associated with enhanced sympathetic activity (Gao et al, 2007) and systemic ROS is also related to impaired baroreflex (Bertagnolli et al, 2006). In addition, it was reported increase of NAD(P)H oxidase activity and expression into the RVLM, the primary central site for the maintenance of sympathetic nerve activity, in CHF rabbits. In the same sequence of procedures, the same authors observed that a reduction of brain O2•− by tempol, a SOD mimetic, decreased the sympathetic outflow in chronic heart failure rabbits. Conversely, an increase of central O2•<sup>−</sup> due to administration of the SOD inhibitor diethyldithiocarbamic acid enhanced the

The activity of sympathetic and parasympathetic systems, which are both involved in cardiopulmonary reflex, as well as the cardiovascular regulation, is under the control of a medullary circuitry comprising the nucleus of the solitary tract (NTS), rostral (RVLM) and caudal ventrolateral medulla (CVLM) and the nucleus ambiguous. Drugs injection into the fourth cerebral ventricle (4th V) may easily reach structures surrounding the ventricular system like the area postrema and the dorsal motor nucleus of the vagus (Colombari et al, 2001) (Figure 5). Those areas are also involved in cardiovascular reflex responses, in which we may include baroreflex (Valenti et al, 2009a; Valenti et al, 2009b; Cisternas et al, 2010).

Fig. 5. Schematic sagittal view of the medulla oblongata showing brain pathways implicated in neurogenic hypertension. Premotor neurons from the RVLM send excitatory synapses to preganglionic neurons situated in the intermediolateral cell column (IML), providing sympathetic efference to target organs. The RVLM is the group of neurons that receive excitatory afference from the commissural nucleus of the solitary tract (NTS) and area postrema. It also receives inhibitory afferences from the caudoventrolateral medulla

A previous investigation suggested that brain ROS is associated with enhanced sympathetic activity (Gao et al, 2007) and systemic ROS is also related to impaired baroreflex (Bertagnolli et al, 2006). In addition, it was reported increase of NAD(P)H oxidase activity and expression into the RVLM, the primary central site for the maintenance of sympathetic nerve activity, in CHF rabbits. In the same sequence of procedures, the same authors observed that a reduction of brain O2•− by tempol, a SOD mimetic, decreased the sympathetic outflow in chronic heart failure rabbits. Conversely, an increase of central O2•<sup>−</sup> due to administration of the SOD inhibitor diethyldithiocarbamic acid enhanced the

(CVLM). Adapted from Valenti et al, 2007.

sympathetic outflow in both normal and chronic heart failure rabbits (Gao et al, 2007). Taken together, those data suggest that antioxidant enzymes, i.e., SOD and catalase, into the brainstem are involved in baroceptor reflex regulation, since baroreflex is modulated by sympathetic and parasympathetic activity (Valenti et al, 2009a).

Recent studies from our laboratory have investigated the effects of ROS into the fourth cerebral ventricle (4th V) on cardiovascular responses.

In one study (Valenti et al, 2011a) it was evaluated the effects of 3-amino-1,2,4-triazole (ATZ), a catalase inhibitor, into the 4th V on baroreflex components in conscious rats. It was revealed that this drug significantly attenuated bradycardic and tachycardic reflex, bradycardic peak and it also decreased heart rate range 30 minutes after its injection. While in Wistar rats treated with vehicle (saline 0.9%) there were no significant changes regarding baseline mean arterial pressure (MAP) and heart rate (HR) and baroreflex components. Considering that the tachycardia (tachycardic reflex) in response to SNP is mediated by both sympathetic and parasympathetic activity (Stornetta et al, 1987) and that we reported reduction in the maximal parasympathetic responses to elevation in mean arterial pressure, while there were no changes in tachycardic peak response to decrease in mean arterial pressure (highest sympathetic response), we suggest that ATZ into the 4th V is acutely involved with parasympathetic activity but is not involved in baroreflex changes. The lack of any change in the vehicles groups is consistent with this assumption. In view of the anatomical scope of the 4th V, an action on an only one neuronal cluster is not an easy accomplishment. However, prior researches indicated a preference for parasympathetic system which modulates HR, such as the dorsal motor nucleus of the vagus and nucleus ambiguous, which receive glutamathergic projections from the nucleus of the solitary tract (Colombari et al, 2001).

In another study (Valenti et al, 2011b), it was evaluated the effects of catalase inhibition into the 4th V on cardiopulmonary reflex in conscious Wistar rats. In this method, we used male Wistar rats, which were implanted with a stainless steel guide cannula in the 4th V. The femoral artery and vein were cannulated for MAP and HR measurement and for drug infusion, respectively. After basal mean arterial pressure and heart rate recordings, the cardiopulmonary reflex was tested with a dose of phenylbiguanide (PBG, 8 μg/kg, bolus). Cardiopulmonary reflex was evaluated before and 15 minutes after 1 μl of ATZ (0.01 g/100 μl) injection into the 4th V. Vehicle treatment did not change cardiopulmonary reflex responses. ATZ injected into the 4th V significantly enhanced hypotensive responses without influencing the bradycardic reflex. Taken together, those data suggested that ATZ injected into the 4th V increases sympathetic inhibition but does not change the parasympathetic component of the cardiopulmonary reflex in conscious Wistar rats.

Nevertheless, opposite findings were found in SHR. Another study (Valenti et al, 2011c) was undertaken to evaluate the acute effects of central n-acetylcysteine, an antioxidant drug, on baroreflex in juvenile SHR and age-matched Wistar Kyoto (WKY) rats. It was observed that n-acetylcysteine injection into the 4th V did not significantly change baroreflex gain, bradycardic and tachycardic reflex, bradycardic and tachycardic peak in SHR and WKY rats. Interestingly, n-acetylcysteine caused slight but significant increase in basal heart rate 15 minutes after its injection in conscious WKY rats.

Reactive Oxygen Species and Cardiovascular Diseases 61

the mechanism involved in antioxidant species applied in clinical therapies. Therefore, the presentation of data regarding the systems different from the cardiovascular system implicated in ROS-induced cardiovascular diseases is relevant to the integrative physiology and more particularly to control physiology and clinical therapies that aim to prevent

Aasum, E., Belke, D.D., Severson, D.L., Riemersma, R.A., Cooper, M., Andreassen, M.,

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cardiovascular disorders such as hypertension and heart failure.

**10. References** 

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Many previous studies have already demonstrated the effects of oxidative stress in cardiovascular reflex. Zanzinger and

Czachurski (2009) demonstrated that SOD injected into the RVLM decreased sympathetic nerve activity in swine. Several groups have now shown that ROSs stimulate sympathetic outflow (Campese et al, 2004). Campos et al (2011) evidenced that the lack of LDL receptor enhanced cholesterol blood levels, enhanced ROS and impaired baroreflex sentivity. Monahan et al (2007) supported the hypothesis that oxidative stress collaborates to ageassociated decreases in cardiovagal baroreflex sensitivity in healthy men. On the other hand, Wright et al (2009) indicated that in male smokers, circulating antioxidants had no effect on baroceptor reflex function and minor effects on the cardiovascular system were seen following acute fat and vitamin ingestion.

In the central nervous system, the complex regulating blood pressure is contained within topographically selective networks characterized at all levels of the neuraxis. Adjustments in this modulation network may lead to labile changes in autonomic function. The development of neurogenic hypertension may involve improper alterations in synaptic function within these networks. Thus, investigations regarding the relationship between the nervous system and the cardiovascular system and its importance to regulation of the physiologic homeostasis are always welcome in the basic and clinical research.

#### **8. Perspectives**

At the moment, relevant milestones were achieved with the availability of more overt facts that demonstrates that cardiovascular disorders mechanisms are linked to ROS increase and dysregulation of oxidant-antioxidants systems. The oxidation and nitration of cellular lipids, proteins and nucleic acids, and formation of aggregates of oxidized molecules underlie the loss of cellular function, cellular ageing and the inability of cells to withstand physiological stresses. Moreover, ROS regulate energy metabolism and signal transduction mechanisms in response to situations of nitrosativeor or oxidative stress. Sources of ROS, physiological and pathophysiological conditions, and cellular oxidant targets determine the profile nature of a disease process and resultant outcomes.

In summary, the data presented in this chapter is significant to the literature, because progress in redox signaling provides insight into the function of ROS in the pathological and physiological mechanisms involved in cardiovascular disorders. Nonetheless, the literature raises more questions. Regarding hypertension treatment, there are a few points to be underscored. ROS act as signaling molecules associated with diverse physiological mechanism which are indispensable for normal function of the brainstem. Inappropriate modulation of ROS impairs redox signaling, which is assumed to stimulate pathologic situations, in which we may include hypertension. Moreover, our study reinforces the importance to investigate the integrative neuroscience.

#### **9. Concluding remarks**

Advances in ROS signaling provide insight into the role of ROS in the pathological and physiological mechanisms related to cardiovascular disorders. The comprehension of how redox state regulates the cardiovascular system is a relevant step for a best explanation for the mechanism involved in antioxidant species applied in clinical therapies. Therefore, the presentation of data regarding the systems different from the cardiovascular system implicated in ROS-induced cardiovascular diseases is relevant to the integrative physiology and more particularly to control physiology and clinical therapies that aim to prevent cardiovascular disorders such as hypertension and heart failure.

#### **10. References**

60 Oxidative Stress and Diseases

Many previous studies have already demonstrated the effects of oxidative stress in

Czachurski (2009) demonstrated that SOD injected into the RVLM decreased sympathetic nerve activity in swine. Several groups have now shown that ROSs stimulate sympathetic outflow (Campese et al, 2004). Campos et al (2011) evidenced that the lack of LDL receptor enhanced cholesterol blood levels, enhanced ROS and impaired baroreflex sentivity. Monahan et al (2007) supported the hypothesis that oxidative stress collaborates to ageassociated decreases in cardiovagal baroreflex sensitivity in healthy men. On the other hand, Wright et al (2009) indicated that in male smokers, circulating antioxidants had no effect on baroceptor reflex function and minor effects on the cardiovascular system were seen

In the central nervous system, the complex regulating blood pressure is contained within topographically selective networks characterized at all levels of the neuraxis. Adjustments in this modulation network may lead to labile changes in autonomic function. The development of neurogenic hypertension may involve improper alterations in synaptic function within these networks. Thus, investigations regarding the relationship between the nervous system and the cardiovascular system and its importance to regulation of the

At the moment, relevant milestones were achieved with the availability of more overt facts that demonstrates that cardiovascular disorders mechanisms are linked to ROS increase and dysregulation of oxidant-antioxidants systems. The oxidation and nitration of cellular lipids, proteins and nucleic acids, and formation of aggregates of oxidized molecules underlie the loss of cellular function, cellular ageing and the inability of cells to withstand physiological stresses. Moreover, ROS regulate energy metabolism and signal transduction mechanisms in response to situations of nitrosativeor or oxidative stress. Sources of ROS, physiological and pathophysiological conditions, and cellular oxidant targets determine the profile nature of a

In summary, the data presented in this chapter is significant to the literature, because progress in redox signaling provides insight into the function of ROS in the pathological and physiological mechanisms involved in cardiovascular disorders. Nonetheless, the literature raises more questions. Regarding hypertension treatment, there are a few points to be underscored. ROS act as signaling molecules associated with diverse physiological mechanism which are indispensable for normal function of the brainstem. Inappropriate modulation of ROS impairs redox signaling, which is assumed to stimulate pathologic situations, in which we may include hypertension. Moreover, our study reinforces the

Advances in ROS signaling provide insight into the role of ROS in the pathological and physiological mechanisms related to cardiovascular disorders. The comprehension of how redox state regulates the cardiovascular system is a relevant step for a best explanation for

physiologic homeostasis are always welcome in the basic and clinical research.

cardiovascular reflex. Zanzinger and

following acute fat and vitamin ingestion.

disease process and resultant outcomes.

**9. Concluding remarks** 

importance to investigate the integrative neuroscience.

**8. Perspectives** 


Reactive Oxygen Species and Cardiovascular Diseases 63

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**4** 

*Chile* 

**Oxidative Stress in the Carotid Body: Implications for the Cardioventilatory** 

The obstructive sleep apnea (OSA) syndrome is recognized as an independent risk factor for systemic hypertension. The OSA syndrome is characterized by cyclic episodes of oxygen desaturation due to the partial or complete obstruction of the air flow during sleep. Among the disturbances produced by OSA, the chronic intermittent hypoxia is considered the main factor for developing hypertension. Oxidative stress, inflammation, and sympathetic hyperactivity have been proposed as pathogenic mechanisms involved in the hypertension. However, evidence for a single mechanism has been difficult to establish in OSA patients, because of concomitant comorbidities. Since OSA patients show augmented reflex sympathetic, cardiovascular and ventilatory responses to acute hypoxia, it has been proposed that an enhance carotid body responsiveness to hypoxia is involved in the pathological alterations induced by OSA. This proposal has received further support, since studies performed in animals have shown that intermittent hypoxia selectively enhances the carotid body chemosensory and ventilatory responses to acute hypoxia, producing longterm potentiation of the motor ventilatory and sympathetic discharges. The mechanisms underlying the enhanced carotid body chemosensory reactivity to hypoxia induced by intermittent hypoxia are not completely known. Nevertheless, the available evidence indicates that the repeated episodes of hypoxia-reoxygenation produce local oxidative stress in the carotid body due to the accumulation of reactive oxygen species. In this chapter, we will review and discuss the new evidence supporting the essential role played by the carotid body chemoreceptors, and the contribution of the oxidative stress, endothelin-1 and proinflammatory cytokines to the progression of the cardioventilatory alterations induced by

Most of the mammalian cells respond to hypoxia modifying the expression of genes and proteins, which induce a physiological response to recover the tissue oxygen levels (i.e.

**1. Introduction** 

chronic intermittent hypoxia.

**2. The carotid body chemoreceptors** 

**Alterations Induced by** 

**Obstructive Sleep Apnea** 

Rodrigo Iturriaga and Rodrigo Del Rio

*P. Universidad Católica de Chile, Santiago,* 

*Facultad de Ciencias Biológicas,* 

*Laboratorio de Neurobiología, Departamento de Fisiología,* 

smooth muscle. *American Journal of Physiology Heart and Circulatory Physiology,* Vol. 290, No. 3 (March), pp. H1136–H1144, ISSN 0363-6135


### **Oxidative Stress in the Carotid Body: Implications for the Cardioventilatory Alterations Induced by Obstructive Sleep Apnea**

Rodrigo Iturriaga and Rodrigo Del Rio

*Laboratorio de Neurobiología, Departamento de Fisiología, Facultad de Ciencias Biológicas, P. Universidad Católica de Chile, Santiago, Chile* 

#### **1. Introduction**

70 Oxidative Stress and Diseases

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with superoxide production in angiotensin II hypertension. *Journal of Hypertension,*

caused by angiotensin II infusion involves enhanced superoxide production in the central nervous system. *Circulation Research,* Vol. 95 No. 2 (July) pp. 210–6, ISSN The obstructive sleep apnea (OSA) syndrome is recognized as an independent risk factor for systemic hypertension. The OSA syndrome is characterized by cyclic episodes of oxygen desaturation due to the partial or complete obstruction of the air flow during sleep. Among the disturbances produced by OSA, the chronic intermittent hypoxia is considered the main factor for developing hypertension. Oxidative stress, inflammation, and sympathetic hyperactivity have been proposed as pathogenic mechanisms involved in the hypertension. However, evidence for a single mechanism has been difficult to establish in OSA patients, because of concomitant comorbidities. Since OSA patients show augmented reflex sympathetic, cardiovascular and ventilatory responses to acute hypoxia, it has been proposed that an enhance carotid body responsiveness to hypoxia is involved in the pathological alterations induced by OSA. This proposal has received further support, since studies performed in animals have shown that intermittent hypoxia selectively enhances the carotid body chemosensory and ventilatory responses to acute hypoxia, producing longterm potentiation of the motor ventilatory and sympathetic discharges. The mechanisms underlying the enhanced carotid body chemosensory reactivity to hypoxia induced by intermittent hypoxia are not completely known. Nevertheless, the available evidence indicates that the repeated episodes of hypoxia-reoxygenation produce local oxidative stress in the carotid body due to the accumulation of reactive oxygen species. In this chapter, we will review and discuss the new evidence supporting the essential role played by the carotid body chemoreceptors, and the contribution of the oxidative stress, endothelin-1 and proinflammatory cytokines to the progression of the cardioventilatory alterations induced by chronic intermittent hypoxia.

#### **2. The carotid body chemoreceptors**

Most of the mammalian cells respond to hypoxia modifying the expression of genes and proteins, which induce a physiological response to recover the tissue oxygen levels (i.e.

Oxidative Stress in the Carotid Body:

nucleus (Lin et al., 2007, Yan et al., 2008).

Implications for the Cardioventilatory Alterations Induced by Obstructive Sleep Apnea 73

and arousal. During the airway occlusion, hypoxia and hypercapnia stimulate the carotid body chemoreceptors increasing the respiratory muscle effort, the vascular sympathetic tone and the arterial blood pressure. Finally, the stimulation of the carotid body chemoreceptors and probably the pulmonary mechanoreceptors elicit arousal and restores the ventilation. Among the disturbances produced by OSA, the exposure to intermittent hypoxia is considered the main factor for developing hypertension. Oxidative stress, inflammation and sympathetic overflow have been proposed as potential pathogenic mechanisms involved in the onset of the hypertension and cardiovascular diseases (Arnardottir et al., 2009, Garvey at al., 2009, Lavie 2003, Somers et al., 2008). However, conclusions from studies performed in humans are conflictive, because of concomitant effects of comorbidities (obesity, cardiovascular diseases, diabetes, etc) associated with OSA (Gozal & Kheirandish-Gozal, 2008, Somer et al., 2008). Thus, animal models of chronic intermittent hypoxia (CIH), which simulate the hypoxic-reoxygenation cycles observed in OSA patients, reproduce several pathologic cardiovascular features of OSA (Fletcher et al., 1992, Pack 2009; Schulz et al., 2008). The hypoxic-reoxygenation episodes in OSA patients enhance the cardiorespiratory and sympathetic responses to acute hypoxia (Carlson et al., 1993; Narkiewicz et al., 1998a, 1998b, 1999); impair the autonomic regulation of the heart rate and the arterial blood pressure (Narkiewicz et al., 1999b, Shiomi et al., 1996) and exacerbate the renin-angiotensin system (Fletcher et al., 2002, Moller et al., 2003). Similarly, animals exposed to intermittent hypoxia show potentiated sympathetic discharges and vascular responses to hypoxia, and develop systemic hypertension (Dick et al., 2007; Fletcher et al., 1992, Greenberg et al., 1999, Zoccal et al., 2008). The autonomic hyperactivity is associated with a reduction of the efficiency of the baroreflex control of heart rate and alterations of heart rate variability in OSA patients (Narkiewicz et al., 1998b, Shiomi et al., 1996) and animals exposed to intermittent hypoxia (Lai et al., 2006; Lin et al., 2007; Rey et al., 2004, 2008). Thus, it is likely that the enhanced sympathetic activity along with the reduction of the baroreflex efficiency would impair the regulation of heart rate and the vasomotor tone of blood vessels eliciting hypertension. Besides that, it has been found that intermittent hypoxia produces parasympathetic withdrawal, attributed in part to neuronal loss in the vagal ambiguous

**4. Contribution of the carotid body to the cardiorespiratory alterations in obstructive sleep apnea and animal exposed to intermittent hypoxia** 

Patients with recently diagnosed OSA show enhanced ventilatory, sympathetic and vasopressor responses to acute hypoxia, attributed to a potentiated hypoxic chemoreflex (Cistulli & Sullivan, 1994). Narkiewicz et al., (1999*)* studied the ventilatory, tachycardic and hypertensive responses to acute hypoxia in untreated normotensive OSA patients, and found that the hypoxic stimulation evokes higher ventilatory, tachycardic, and blood pressor responses in OSA patients than control subjects, but the ventilatory and blood pressor responses induced by hypercapnia and by the cold pressor tested in OSA patients were not different from control subjects. Loredo et al., (2001) reported that OSA hypertensive patients present higher basal tidal volumes, suggesting an enhanced carotid body chemosensory drive. Leuenberger et al., (2007) measured changes in sympathetic discharges recorded from the peroneal nerve of normal humans in response to acute

angiogenesis). However, gene expression induction is not fast enough to counteract a rapid drop in systemic oxygen levels. Only the peripheral chemoreceptors located in the carotid and aortic bodies are capable to evoke fast systemic adjustments to overcome a hypoxic episode. The carotid body located in the bifurcation of the carotid arteries is the main arterial chemoreceptor in terms of its contribution to the reflex ventilatory responses to hypoxia (Gonzalez et al., 1994). In humans and mammals, the carotid body initiates the hyperventilatory response induced by hypoxia and activates the sympathetic nervous system. The carotid body is a complex chemoreceptor organ with a high blood flow formed by different types of cells. The glomus cells are considered the oxygen sensors in the carotid body. Glomus cells establish synaptic contacts with the nerve terminals of the primary sensory neurons, whose soma are located in the petrosal ganglion (Gonzalez et al., 1994, Iturriaga & Alcayaga, 2004, Iturriaga et al., 2007). The current model for oxygen chemoreception in the carotid body states that low oxygen induced the inhibition of a voltage-independent potassium TASK-like current, leading to the depolarization of the glomus cells, followed by the entry of Ca2+ through L-type Ca2+ channels and the subsequent release of one or more excitatory transmitters, which in turn increase the discharges of action potentials in the nerve endings of the chemosensory neurons (Iturriaga & Alcayaga 2004, Iturriaga et al., 2007). The glomus cells contain several molecules proposed as putative excitatory transmitters, such as dopamine, acetylcholine, adenosine nucleotides and peptides. Among these molecules present in glomus cells, acetylcholine and adenosine triphosphate fulfill most of the criteria to be considered as the excitatory transmitters between the glomus cells and petrosal nerve ending (Iturriaga et al., 2007). However, other molecules such as dopamine, histamine, nitric oxide and endothelin-1 acts as modulators of the chemosensory process, acting on the glomus cells or controlling the vasomotor tone of the blood vessel (Iturriaga et al., 2007). More recently, it has been proposed that proinflammatory cytokines, such as tumor necrosis factor-α (TNF-α), interleukin 6 (IL-6) and interleukin 1β (IL-1β) are excitatory modulators of the chemoreception process in the rat carotid body (Lam et al., 2008, Liu et al., 2009, Shu et al., 2007).

#### **3. Cardiovascular alterations in patients with obstructive sleep apnea and animals exposed to intermittent hypoxia**

The OSA syndrome, a highly prevalent sleep-breathing disorder is now recognized as an independent risk factor for systemic hypertension. Approximately 50% of the OSA patients develop systemic diurnal hypertension and 30% of the hypertensive patients have OSA. The OSA syndrome is also associated with stroke, pulmonary hypertension, coronary artery disease and atrial fibrillation (Garvey et al., 2009, Parati et al., 2007, Somers et al., 2008). The OSA syndrome affect up to 5% worldwide adult population, but according to the report of the American Heart Association in collaboration with the National Heart, Lung, and Blood Institute National Center on Sleep Disorders Research (Somers et al., 2008) "85% of patients with clinically significant and treatable OSA have never been diagnosed, and referral populations of OSA patients represent only the *tip of the iceberg* of OSA prevalence". OSA is characterized by recurrent episodes of partial or complete obstruction of the air flow during sleep produced by the collapse of the pharyngeal airway. The interruption of the air flow produces hypoxia and hypercapnia, negative intrathoraxic pressure, sleep fragmentation

angiogenesis). However, gene expression induction is not fast enough to counteract a rapid drop in systemic oxygen levels. Only the peripheral chemoreceptors located in the carotid and aortic bodies are capable to evoke fast systemic adjustments to overcome a hypoxic episode. The carotid body located in the bifurcation of the carotid arteries is the main arterial chemoreceptor in terms of its contribution to the reflex ventilatory responses to hypoxia (Gonzalez et al., 1994). In humans and mammals, the carotid body initiates the hyperventilatory response induced by hypoxia and activates the sympathetic nervous system. The carotid body is a complex chemoreceptor organ with a high blood flow formed by different types of cells. The glomus cells are considered the oxygen sensors in the carotid body. Glomus cells establish synaptic contacts with the nerve terminals of the primary sensory neurons, whose soma are located in the petrosal ganglion (Gonzalez et al., 1994, Iturriaga & Alcayaga, 2004, Iturriaga et al., 2007). The current model for oxygen chemoreception in the carotid body states that low oxygen induced the inhibition of a voltage-independent potassium TASK-like current, leading to the depolarization of the glomus cells, followed by the entry of Ca2+ through L-type Ca2+ channels and the subsequent release of one or more excitatory transmitters, which in turn increase the discharges of action potentials in the nerve endings of the chemosensory neurons (Iturriaga & Alcayaga 2004, Iturriaga et al., 2007). The glomus cells contain several molecules proposed as putative excitatory transmitters, such as dopamine, acetylcholine, adenosine nucleotides and peptides. Among these molecules present in glomus cells, acetylcholine and adenosine triphosphate fulfill most of the criteria to be considered as the excitatory transmitters between the glomus cells and petrosal nerve ending (Iturriaga et al., 2007). However, other molecules such as dopamine, histamine, nitric oxide and endothelin-1 acts as modulators of the chemosensory process, acting on the glomus cells or controlling the vasomotor tone of the blood vessel (Iturriaga et al., 2007). More recently, it has been proposed that proinflammatory cytokines, such as tumor necrosis factor-α (TNF-α), interleukin 6 (IL-6) and interleukin 1β (IL-1β) are excitatory modulators of the chemoreception process in the rat

carotid body (Lam et al., 2008, Liu et al., 2009, Shu et al., 2007).

**animals exposed to intermittent hypoxia** 

**3. Cardiovascular alterations in patients with obstructive sleep apnea and** 

The OSA syndrome, a highly prevalent sleep-breathing disorder is now recognized as an independent risk factor for systemic hypertension. Approximately 50% of the OSA patients develop systemic diurnal hypertension and 30% of the hypertensive patients have OSA. The OSA syndrome is also associated with stroke, pulmonary hypertension, coronary artery disease and atrial fibrillation (Garvey et al., 2009, Parati et al., 2007, Somers et al., 2008). The OSA syndrome affect up to 5% worldwide adult population, but according to the report of the American Heart Association in collaboration with the National Heart, Lung, and Blood Institute National Center on Sleep Disorders Research (Somers et al., 2008) "85% of patients with clinically significant and treatable OSA have never been diagnosed, and referral populations of OSA patients represent only the *tip of the iceberg* of OSA prevalence". OSA is characterized by recurrent episodes of partial or complete obstruction of the air flow during sleep produced by the collapse of the pharyngeal airway. The interruption of the air flow produces hypoxia and hypercapnia, negative intrathoraxic pressure, sleep fragmentation and arousal. During the airway occlusion, hypoxia and hypercapnia stimulate the carotid body chemoreceptors increasing the respiratory muscle effort, the vascular sympathetic tone and the arterial blood pressure. Finally, the stimulation of the carotid body chemoreceptors and probably the pulmonary mechanoreceptors elicit arousal and restores the ventilation. Among the disturbances produced by OSA, the exposure to intermittent hypoxia is considered the main factor for developing hypertension. Oxidative stress, inflammation and sympathetic overflow have been proposed as potential pathogenic mechanisms involved in the onset of the hypertension and cardiovascular diseases (Arnardottir et al., 2009, Garvey at al., 2009, Lavie 2003, Somers et al., 2008). However, conclusions from studies performed in humans are conflictive, because of concomitant effects of comorbidities (obesity, cardiovascular diseases, diabetes, etc) associated with OSA (Gozal & Kheirandish-Gozal, 2008, Somer et al., 2008). Thus, animal models of chronic intermittent hypoxia (CIH), which simulate the hypoxic-reoxygenation cycles observed in OSA patients, reproduce several pathologic cardiovascular features of OSA (Fletcher et al., 1992, Pack 2009; Schulz et al., 2008). The hypoxic-reoxygenation episodes in OSA patients enhance the cardiorespiratory and sympathetic responses to acute hypoxia (Carlson et al., 1993; Narkiewicz et al., 1998a, 1998b, 1999); impair the autonomic regulation of the heart rate and the arterial blood pressure (Narkiewicz et al., 1999b, Shiomi et al., 1996) and exacerbate the renin-angiotensin system (Fletcher et al., 2002, Moller et al., 2003). Similarly, animals exposed to intermittent hypoxia show potentiated sympathetic discharges and vascular responses to hypoxia, and develop systemic hypertension (Dick et al., 2007; Fletcher et al., 1992, Greenberg et al., 1999, Zoccal et al., 2008). The autonomic hyperactivity is associated with a reduction of the efficiency of the baroreflex control of heart rate and alterations of heart rate variability in OSA patients (Narkiewicz et al., 1998b, Shiomi et al., 1996) and animals exposed to intermittent hypoxia (Lai et al., 2006; Lin et al., 2007; Rey et al., 2004, 2008). Thus, it is likely that the enhanced sympathetic activity along with the reduction of the baroreflex efficiency would impair the regulation of heart rate and the vasomotor tone of blood vessels eliciting hypertension. Besides that, it has been found that intermittent hypoxia produces parasympathetic withdrawal, attributed in part to neuronal loss in the vagal ambiguous nucleus (Lin et al., 2007, Yan et al., 2008).

#### **4. Contribution of the carotid body to the cardiorespiratory alterations in obstructive sleep apnea and animal exposed to intermittent hypoxia**

Patients with recently diagnosed OSA show enhanced ventilatory, sympathetic and vasopressor responses to acute hypoxia, attributed to a potentiated hypoxic chemoreflex (Cistulli & Sullivan, 1994). Narkiewicz et al., (1999*)* studied the ventilatory, tachycardic and hypertensive responses to acute hypoxia in untreated normotensive OSA patients, and found that the hypoxic stimulation evokes higher ventilatory, tachycardic, and blood pressor responses in OSA patients than control subjects, but the ventilatory and blood pressor responses induced by hypercapnia and by the cold pressor tested in OSA patients were not different from control subjects. Loredo et al., (2001) reported that OSA hypertensive patients present higher basal tidal volumes, suggesting an enhanced carotid body chemosensory drive. Leuenberger et al., (2007) measured changes in sympathetic discharges recorded from the peroneal nerve of normal humans in response to acute

Oxidative Stress in the Carotid Body:

chemosensory discharges, expressed in Hz.

hypoxia (See for review Kumar, 2011).

Implications for the Cardioventilatory Alterations Induced by Obstructive Sleep Apnea 75

Fig. 1. Carotid body chemosensory potentiation induced by intermittent hypoxia in the rat. The carotid chemosensory discharges in response to various levels of inspired O2 (FiO2 ~15 to 5%) were measured from the carotid sinus nerve of a sham rat exposed to air to air cycles and a rat exposed to chronic intermittent hypoxia (CIH) for 21 days. ƒx, frequency of carotid

**6. Mechanisms underlying the potentiation of carotid body chemosensory** 

The enhance carotid chemosensory responses to hypoxia has been associated to increased levels of reactive oxygen species (Peng et al., 2003, Iturriaga et al., 2009, Del Rio et al., 2010) and endothelin-1 within the CB (Rey et al., 2006, Pawar et al., 2009), but it is possible that pro-inflammatory cytokines, which increased in the plasma of OSA patients (Lavie 2003, Jelic et al., 2008) may also contributes to the enhanced carotid body chemosensory responses to acute hypoxia (Iturriaga et al., 2009; Del Rio et al., 2011). Although some studies addressed the effects of intermittent hypoxia on transmitter production and release in the carotid body, very little is known on the functional significance of the role played by the neurotransmitters in the carotid body chemosensory potentiation induced by intermittent

**responses to hypoxia induced by chronic intermittent hypoxia** 

hypoxic stimulation before and after the exposure to 30 episodes of apnea. The episodes of apnea do not only increased sympathetic discharges and produced mild increases in arterial blood pressure, but also enhanced the sympathetic neural response to acute hypoxia, indicating that short-term intermittent hypoxia produces a facilitation of the hypoxic chemoreflex in normal humans. Thus, the available evidence supports the proposal that the enhanced oxygen chemoreflex response in OSA patients is produced by the intermittent hypoxia. Similarly, rats and cats exposed to chronic intermittent hypoxia show enhanced hypoxic ventilatory responses to acute hypoxia (Iturriaga et al., 2009, Rey et al., 2004; Reeves et al., 2003) and long-term facilitation of respiratory motor responses (McGuire et al., 2003, Dick et al., 2007, Prahbakar et al., 2005). The long-term potentiated ventilatory responses to acute hypoxia observed in animals exposed to intermittent hypoxia has been attributed to a central facilitation of the serotonin-mediated motor ventilatory output (McGuire et al., 2003). Although, Narkiewicz et al., (1998a, 1998b) found that sympathetic, pressor and ventilatory responses to acute hypoxia were enhanced in OSA patients, and Fletcher et al., (1992) reported that the bilateral carotid body denervation prevents the hypertension in rats exposed to intermittent hypoxia, the idea that carotid body chemoreceptors are involved in the progression of the hypertension did not receive much attention. However, new evidence obtained in the last decade have shown that an abnormal potentiated carotid chemosensory reactivity to hypoxia is crucial to potentiate the sympathetic activity (Iturriaga et al., 2005, 2009, Feng et al., 2008, Garvey at al., 2009; Prabhakar et al., 2005, Rey et al., 2004; Smith & Pacchia, 2007).

#### **5. Intermittent hypoxia enhanced the carotid body chemosensory responses to acute hypoxia**

Recording of chemosensory discharges from the carotid sinus nerve have shown that chronic intermittent hypoxia produces long-term potentiation of the carotid body chemosensory responses to acute hypoxia. Indeed, exposure of cats and rats to intermittent hypoxia for 4 to 10 days increases the basal carotid body chemosensory discharges measured in normoxia and enhances the chemosensory responses to acute hypoxia (Peng et al., 2003, Rey et al., 2004, Del Rio et al., 2010). Peng et al., (2003) found that the baseline carotid discharge and the chemosensory responses to acute hypoxia were higher in rats exposed to short cyclic hypoxic episodes followed by normoxia, applied during 8 hrs for 10 days. Similarly, we found that cats exposed to intermittent hypoxia during 8 hrs for 4 days showed enhanced CB chemosensory and ventilatory responses to acute hypoxia (Rey et al., 2004). In rats, we found that intermittent hypoxia for 7 days potentiates the carotid chemosensory responses to acute hypoxia, effect that persisted until 21 days of intermittent hypoxia when animals developed hypertension (Del Rio et al., 2011). Figure 1 illustrates representative recordings of carotid chemosensory responses induced by short hypoxic challenges in a sham rat and in one carotid body from a rat exposed to 5% O2, 12 times/hr during 8 hrs for 21 days. As is shown in fig. 1, chronic intermittent hypoxia increased the baseline carotid chemosensory discharges measured in normoxia and induced a potentiation of chemosensory responses to acute hypoxia. Since these alterations in the carotid chemosensory function occurred without significant elevation of the arterial blood pressure until 21 days of intermittent hypoxia, the hypertension was preceded by an early potentiation of the carotid body chemosensory and ventilatory responses to hypoxia.

hypoxic stimulation before and after the exposure to 30 episodes of apnea. The episodes of apnea do not only increased sympathetic discharges and produced mild increases in arterial blood pressure, but also enhanced the sympathetic neural response to acute hypoxia, indicating that short-term intermittent hypoxia produces a facilitation of the hypoxic chemoreflex in normal humans. Thus, the available evidence supports the proposal that the enhanced oxygen chemoreflex response in OSA patients is produced by the intermittent hypoxia. Similarly, rats and cats exposed to chronic intermittent hypoxia show enhanced hypoxic ventilatory responses to acute hypoxia (Iturriaga et al., 2009, Rey et al., 2004; Reeves et al., 2003) and long-term facilitation of respiratory motor responses (McGuire et al., 2003, Dick et al., 2007, Prahbakar et al., 2005). The long-term potentiated ventilatory responses to acute hypoxia observed in animals exposed to intermittent hypoxia has been attributed to a central facilitation of the serotonin-mediated motor ventilatory output (McGuire et al., 2003). Although, Narkiewicz et al., (1998a, 1998b) found that sympathetic, pressor and ventilatory responses to acute hypoxia were enhanced in OSA patients, and Fletcher et al., (1992) reported that the bilateral carotid body denervation prevents the hypertension in rats exposed to intermittent hypoxia, the idea that carotid body chemoreceptors are involved in the progression of the hypertension did not receive much attention. However, new evidence obtained in the last decade have shown that an abnormal potentiated carotid chemosensory reactivity to hypoxia is crucial to potentiate the sympathetic activity (Iturriaga et al., 2005, 2009, Feng et al., 2008, Garvey at al., 2009; Prabhakar et al., 2005, Rey et al., 2004; Smith &

**5. Intermittent hypoxia enhanced the carotid body chemosensory responses** 

Recording of chemosensory discharges from the carotid sinus nerve have shown that chronic intermittent hypoxia produces long-term potentiation of the carotid body chemosensory responses to acute hypoxia. Indeed, exposure of cats and rats to intermittent hypoxia for 4 to 10 days increases the basal carotid body chemosensory discharges measured in normoxia and enhances the chemosensory responses to acute hypoxia (Peng et al., 2003, Rey et al., 2004, Del Rio et al., 2010). Peng et al., (2003) found that the baseline carotid discharge and the chemosensory responses to acute hypoxia were higher in rats exposed to short cyclic hypoxic episodes followed by normoxia, applied during 8 hrs for 10 days. Similarly, we found that cats exposed to intermittent hypoxia during 8 hrs for 4 days showed enhanced CB chemosensory and ventilatory responses to acute hypoxia (Rey et al., 2004). In rats, we found that intermittent hypoxia for 7 days potentiates the carotid chemosensory responses to acute hypoxia, effect that persisted until 21 days of intermittent hypoxia when animals developed hypertension (Del Rio et al., 2011). Figure 1 illustrates representative recordings of carotid chemosensory responses induced by short hypoxic challenges in a sham rat and in one carotid body from a rat exposed to 5% O2, 12 times/hr during 8 hrs for 21 days. As is shown in fig. 1, chronic intermittent hypoxia increased the baseline carotid chemosensory discharges measured in normoxia and induced a potentiation of chemosensory responses to acute hypoxia. Since these alterations in the carotid chemosensory function occurred without significant elevation of the arterial blood pressure until 21 days of intermittent hypoxia, the hypertension was preceded by an early

potentiation of the carotid body chemosensory and ventilatory responses to hypoxia.

Pacchia, 2007).

**to acute hypoxia** 

Fig. 1. Carotid body chemosensory potentiation induced by intermittent hypoxia in the rat. The carotid chemosensory discharges in response to various levels of inspired O2 (FiO2 ~15 to 5%) were measured from the carotid sinus nerve of a sham rat exposed to air to air cycles and a rat exposed to chronic intermittent hypoxia (CIH) for 21 days. ƒx, frequency of carotid chemosensory discharges, expressed in Hz.

#### **6. Mechanisms underlying the potentiation of carotid body chemosensory responses to hypoxia induced by chronic intermittent hypoxia**

The enhance carotid chemosensory responses to hypoxia has been associated to increased levels of reactive oxygen species (Peng et al., 2003, Iturriaga et al., 2009, Del Rio et al., 2010) and endothelin-1 within the CB (Rey et al., 2006, Pawar et al., 2009), but it is possible that pro-inflammatory cytokines, which increased in the plasma of OSA patients (Lavie 2003, Jelic et al., 2008) may also contributes to the enhanced carotid body chemosensory responses to acute hypoxia (Iturriaga et al., 2009; Del Rio et al., 2011). Although some studies addressed the effects of intermittent hypoxia on transmitter production and release in the carotid body, very little is known on the functional significance of the role played by the neurotransmitters in the carotid body chemosensory potentiation induced by intermittent hypoxia (See for review Kumar, 2011).

Oxidative Stress in the Carotid Body:

Implications for the Cardioventilatory Alterations Induced by Obstructive Sleep Apnea 77

Fig. 2. Effect of ascorbic acid on the potentiated carotid body chemosensory responses to hypoxia induced by intermittent hypoxia. CIH, rat exposed to intermittent hypoxia. CIH-AA, rat exposed to intermittent hypoxia and treated with ascorbic acid. Note that ascorbic acid reduced both the baseline and the chemosensory response to 10% O2. ƒx, frequency of

Although the current information suggests that an increased local oxidative stress contributes to the carotid body chemosensory potentiation induced by intermittent hypoxia, the direct participation of ROS on the oxygen chemotransduction process is matter of debate, because no chemosensory excitatory effects of ROS have been observed (Gonzalez et al., 2007). A possible explanation is that an increased level of the superoxide radical in the carotid body may reacts with nitric oxide generating peroxynitrite, a powerful oxidizing agent that nitrates tyrosine residues forming 3-nitrotyrosine. We already found the excessive formation of 3-nitrotyrosine in glomus cells and blood vessels from carotid bodies harvested from rats exposed to intermittent hypoxia (Del Rio et al., 2010, 2011), as is shown in fig. 3. The increased formation of 3-nitrotyrosine indicates that the carotid body tissue is continuously exposed to oxidative stress during the intermittent hypoxic exposure. In addition, we found that a correlation between the marked increase of 3-nitrotyrosine immunoreactivity in the carotid body exposed to intermittent hypoxia and the enhanced carotid chemosensory responses to acute hypoxia (Del Rio et al., 2011), supporting and extending the idea that oxidative-nitrosative stress plays a critical role in the CB chemosensory potentiation (Iturriaga et al., 2009, Peng & Prabhakar, 2003). In OSA patients, Jelic et al., (2008) found that the expression of 3-nitrotyrosine in endothelial cells was greater than controls subjects, indicating that the oxidative stress contributes to the endothelial dysfunction caused by the intermittent hypoxia. In addition to the formation of nitrotyrosine residues, peroxynitrites may also modify iron sulfur clusters, zinc thiolates and other residues. Moreover, peroxynitrites may react with inorganic molecules such as CO2

producing other free radicals that may modify DNA, lipids or proteins.

carotid chemosensory discharges, expressed in Hz.

#### **6.1 Reactive oxygen and nitrogen species**

Reactive oxygen species (ROS) and reactive nitrogen species (RNS) have been proposed as mediators of the cardiovascular and cognitive morbidities in several diseases including the OSA syndrome (Christou et al., 2003; Gozal & Kheirandish-Gozal, 2008, Lavie 2003) and in pathological consequences of intermittent hypoxia in animal models (Chen et al., 2005, Del Rio et al., 2010, Peng et al., 2003, Peng et al., 2009). Studies performed in OSA patients and animals exposed to chronic intermittent hypoxia have shown that the cyclical episodes of hypoxia-reoxygenation produces systemic oxidative stress due to the accumulation of ROS and RNS, which are well known potential sources of cellular damage. Peng et al., (2003) found evidence that the superoxide radical participates in the potentiation of the rat carotid chemosensory responses to hypoxia induced by intermittent hypoxia. They found that pretreatment of rats for 10 days before the exposure to intermittent hypoxia with the superoxide dismutase mimetic, manganese (III) tetrakis (1-methyl-4-pyridyl) porphyrin pentachloride (MnTMPyP) prevents the potentiation of the carotid body chemosensory response to hypoxia. In addition, they found that intermittent hypoxia decreases the activity of the ROS sensitive enzyme aconitase in the whole carotid body, as well as the activity of the complex I of the mitochondrial electron transport chain, suggesting that the mitochondria is one of the sources of ROS (Peng & Prabhakar, 2003). More recently, Peng et al., (2009) tested the hypothesis that ROS generated by NADPH oxidase (NOX) mediate the intermittent hypoxia-induced carotid body potentiation. They found that acute hypoxia produced a larger increase in NOX activity in carotid body of rats exposed to intermittent hypoxia for 10 days than that of control carotid bodies. The carotid body chemosensory potentiation was prevented by NOX inhibitors and was not observed in NOX2 deficient mice. On the other hand, MacFarlane and Mitchell (2008) found that application of MnTMPyP into the intrathecal space of the cervical spinal cord abolished the phrenic longterm potentiation induced by acute intermittent hypoxia in rats, suggesting that ROS production is needed for enhancing the phrenic nerve ventilatory discharge. Consequently, ROS formation seems to be necessary for respiratory plasticity induced by intermittent hypoxia, at the level of the carotid body and respiratory motor output. Recently, we tested the hypothesis that oxidative stress contributes to the carotid chemosensory potentiation and the progression of the hypertension in rats exposed to intermittent hypoxia (Del Rio et al., 2010). We hypothesized that oral supplementation of the common antioxidant ascorbic acid (vitamin C) may prevent the carotid chemosensory potentiation and the cardioventilatory alterations including the hypertension induced by the intermittent hypoxic exposure. Accordingly, we studied the effects of ascorbic acid supplementation in the drinking water (1.25 g/l) on plasma lipid peroxidation, arterial blood pressure, and carotid chemosensory responses to acute hypoxia in rats exposed to short hypoxic episodes (5% O2, 12 times/hr for 8 hrs) for 21 days (Del Rio et al., 2010). We found that exposure of the rats to intermittent hypoxia increased the plasma lipid peroxidation and the formation of 3-nitrotyrosine in the carotid body, the arterial blood pressure and enhanced the carotid chemosensory and ventilatory responses to hypoxia. Ascorbic acid treatment reduced the increased plasma lipid peroxidation and the formation of 3-nitrotyrosine in the carotid body, the potentiation of carotid body chemosensory responses (See Fig. 2), the ventilatory responses to acute hypoxia, as well as the hypertension.

Reactive oxygen species (ROS) and reactive nitrogen species (RNS) have been proposed as mediators of the cardiovascular and cognitive morbidities in several diseases including the OSA syndrome (Christou et al., 2003; Gozal & Kheirandish-Gozal, 2008, Lavie 2003) and in pathological consequences of intermittent hypoxia in animal models (Chen et al., 2005, Del Rio et al., 2010, Peng et al., 2003, Peng et al., 2009). Studies performed in OSA patients and animals exposed to chronic intermittent hypoxia have shown that the cyclical episodes of hypoxia-reoxygenation produces systemic oxidative stress due to the accumulation of ROS and RNS, which are well known potential sources of cellular damage. Peng et al., (2003) found evidence that the superoxide radical participates in the potentiation of the rat carotid chemosensory responses to hypoxia induced by intermittent hypoxia. They found that pretreatment of rats for 10 days before the exposure to intermittent hypoxia with the superoxide dismutase mimetic, manganese (III) tetrakis (1-methyl-4-pyridyl) porphyrin pentachloride (MnTMPyP) prevents the potentiation of the carotid body chemosensory response to hypoxia. In addition, they found that intermittent hypoxia decreases the activity of the ROS sensitive enzyme aconitase in the whole carotid body, as well as the activity of the complex I of the mitochondrial electron transport chain, suggesting that the mitochondria is one of the sources of ROS (Peng & Prabhakar, 2003). More recently, Peng et al., (2009) tested the hypothesis that ROS generated by NADPH oxidase (NOX) mediate the intermittent hypoxia-induced carotid body potentiation. They found that acute hypoxia produced a larger increase in NOX activity in carotid body of rats exposed to intermittent hypoxia for 10 days than that of control carotid bodies. The carotid body chemosensory potentiation was prevented by NOX inhibitors and was not observed in NOX2 deficient mice. On the other hand, MacFarlane and Mitchell (2008) found that application of MnTMPyP into the intrathecal space of the cervical spinal cord abolished the phrenic longterm potentiation induced by acute intermittent hypoxia in rats, suggesting that ROS production is needed for enhancing the phrenic nerve ventilatory discharge. Consequently, ROS formation seems to be necessary for respiratory plasticity induced by intermittent hypoxia, at the level of the carotid body and respiratory motor output. Recently, we tested the hypothesis that oxidative stress contributes to the carotid chemosensory potentiation and the progression of the hypertension in rats exposed to intermittent hypoxia (Del Rio et al., 2010). We hypothesized that oral supplementation of the common antioxidant ascorbic acid (vitamin C) may prevent the carotid chemosensory potentiation and the cardioventilatory alterations including the hypertension induced by the intermittent hypoxic exposure. Accordingly, we studied the effects of ascorbic acid supplementation in the drinking water (1.25 g/l) on plasma lipid peroxidation, arterial blood pressure, and carotid chemosensory responses to acute hypoxia in rats exposed to short hypoxic episodes (5% O2, 12 times/hr for 8 hrs) for 21 days (Del Rio et al., 2010). We found that exposure of the rats to intermittent hypoxia increased the plasma lipid peroxidation and the formation of 3-nitrotyrosine in the carotid body, the arterial blood pressure and enhanced the carotid chemosensory and ventilatory responses to hypoxia. Ascorbic acid treatment reduced the increased plasma lipid peroxidation and the formation of 3-nitrotyrosine in the carotid body, the potentiation of carotid body chemosensory responses (See Fig. 2), the ventilatory

**6.1 Reactive oxygen and nitrogen species** 

responses to acute hypoxia, as well as the hypertension.

Fig. 2. Effect of ascorbic acid on the potentiated carotid body chemosensory responses to hypoxia induced by intermittent hypoxia. CIH, rat exposed to intermittent hypoxia. CIH-AA, rat exposed to intermittent hypoxia and treated with ascorbic acid. Note that ascorbic acid reduced both the baseline and the chemosensory response to 10% O2. ƒx, frequency of carotid chemosensory discharges, expressed in Hz.

Although the current information suggests that an increased local oxidative stress contributes to the carotid body chemosensory potentiation induced by intermittent hypoxia, the direct participation of ROS on the oxygen chemotransduction process is matter of debate, because no chemosensory excitatory effects of ROS have been observed (Gonzalez et al., 2007). A possible explanation is that an increased level of the superoxide radical in the carotid body may reacts with nitric oxide generating peroxynitrite, a powerful oxidizing agent that nitrates tyrosine residues forming 3-nitrotyrosine. We already found the excessive formation of 3-nitrotyrosine in glomus cells and blood vessels from carotid bodies harvested from rats exposed to intermittent hypoxia (Del Rio et al., 2010, 2011), as is shown in fig. 3. The increased formation of 3-nitrotyrosine indicates that the carotid body tissue is continuously exposed to oxidative stress during the intermittent hypoxic exposure. In addition, we found that a correlation between the marked increase of 3-nitrotyrosine immunoreactivity in the carotid body exposed to intermittent hypoxia and the enhanced carotid chemosensory responses to acute hypoxia (Del Rio et al., 2011), supporting and extending the idea that oxidative-nitrosative stress plays a critical role in the CB chemosensory potentiation (Iturriaga et al., 2009, Peng & Prabhakar, 2003). In OSA patients, Jelic et al., (2008) found that the expression of 3-nitrotyrosine in endothelial cells was greater than controls subjects, indicating that the oxidative stress contributes to the endothelial dysfunction caused by the intermittent hypoxia. In addition to the formation of nitrotyrosine residues, peroxynitrites may also modify iron sulfur clusters, zinc thiolates and other residues. Moreover, peroxynitrites may react with inorganic molecules such as CO2 producing other free radicals that may modify DNA, lipids or proteins.

Oxidative Stress in the Carotid Body:

**6.3 Pro-inflammatory cytokines**

intermittent hypoxia.

Implications for the Cardioventilatory Alterations Induced by Obstructive Sleep Apnea 79

Rio et al., 2011), suggesting that ET-1 may contribute to the enhanced carotid body responsiveness to hypoxia in the early phase of the intermittent hypoxic exposures. In addition to the transient changes in ET-1 expression, we found a significant decrease in the eNOS expression in the rat carotid body at 7 days of intermittent hypoxia (Del Rio et al., 2011), suggesting that chronic intermittent hypoxia may decrease the nitric oxide (NO) levels within the carotid body. Since NO at low concentration is an inhibitory modulator of the carotid chemosensory activity (Iturriaga et al., 2000), a reduced NO level may contribute to enhance the carotid body chemosensitivity, as well as to amplify the vasoconstrictor effect of ET-1. This interpretation is supported by the finding that intermittent hypoxia decreases the expression of the neuronal NO synthase in the rat carotid body (Marcus et al., 2010), suggesting that the removal of the inhibitory NO effects may also contribute to enhance the carotid chemosensory responses to hypoxia. Our results also showed that carotid body iNOS immunorreactive levels increased after 21 days of intermittent hypoxic exposure. Since iNOS produce higher amounts of NO, it is plausible that the NO levels in the carotid body will increase during long-term intermittent hypoxic exposure. It is worth noting that NO has a dual effect on carotid chemosensory discharges. Indeed, Iturriaga et al., (2000) found that at low levels NO is predominantly an inhibitor of the chemosensory discharges, whereas at high concentration NO increases carotid body chemosensory discharges. Thus, it is plausible that high NO levels in the carotid body following long-term intermittent hypoxia may

partially contribute to maintain the carotid body chemosensory potentiation.

Endothelial dysfunction has been related to the progression of hypertension in OSA patients and animals exposed to intermittent hypoxia due to the increased plasmatic levels of proinflammatory cytokines (Biltagi et al., 2008, Jelic et al., 2008, Jun et al., 2008, Tam et al., 2007, Williams & Scharf, 2007). It is likely that an increased production of ROS induced by the hypoxia-reoxygention cycles may evoke the expression of genes and the synthesis of proinflammatory cytokines, mediated by the activation of transcription factors such as the nuclear factor kappa B (NF-κB), the activator protein 1 and HIF-1α (Semenza & Prahbakar, 2007). In response to oxidative stress, HIF-1α induces the expression of several genes including ET-1 and iNOS, but ROS also produces the translocation of NF-κB to the nucleus, increasing the expression of several inflammatory genes such as IL-1β, IL-6, TNF-α, adhesion molecules, iNOS and ET-1 (Janseen-Heininger et al., 2000). Recently, we found that intermittent hypoxia increased the levels of TNF-α and IL-1β in the rat carotid body after 21 days of exposure (Del Rio et al, 2011). We found that glomus cells constitutively expresses TNF-α and IL-1β in the cell bodies and that chronic intermittent hypoxia upregulates the expression of both TNF-α and IL-1β, without inducing carotid body tissue infiltration with macrophages or changes in TNF-α and IL-1β plasmatic levels (Del Rio et al., 2011). Our results showed that exposure to intermittent hypoxia enhances the rat carotid chemosensory responses to acute hypoxia, and progressively increase the immunorreactive TNF-α and IL-1β expression in the carotid body, suggesting a potential role for this cytokines in modulating the enhanced carotid body chemosensory activity after exposure to

Fig. 3. Exposure to CIH increased 3-nitrotyrosine formation in the glomus cells (white arrows) and endothelial cells (black arrows) from rat carotid bodies. Scale bar, 20 µm.

#### **6.2 Vasoactive molecules**

An interesting molecule, which may mediate the carotid body chemosensory potentiation induced by intermittent hypoxia, is endothelin-1 (ET-1). It is known that the plasmatic ET-1 level increases in rats exposed to intermittent hypoxia (Kanagy et al., 2001) and OSA patients (Phillips et al., 1999). This potent vasoconstrictor peptide is expressed in the endothelium, blood vessels and glomus cells of the carotid body (Rey et al., 2007). The application of ET-1 produces chemosensory excitation in both *in situ* and *in vitro* carotid body perfused preparations, but not in the superfused preparation devoid of vascular effects (Rey & Iturriaga, 2004). We found that ET-1 was increased locally in the carotid body of cats exposed to 4 days to intermittent hypoxia by ~10-fold, while ET-1 plasma levels remains unchanged (Rey et al., 2006). The enhanced carotid body chemosensory responses to hypoxia were reduced by the ET receptor blocker bosentan in the intermittent hypoxic treated cats, but have no effects on the carotid body chemosensory activity in control animals (Rey et al., 2006), indicating that a local increase of ET-1 contributes to enhance the carotid chemosensory responses. Pawar et al., (2009) tested the hypothesis that ET-1 induced by ROS plays a role in intermittent hypoxia induced chemosensory potentiation in the rat neonatal carotid body. They found that intermittent hypoxia enhanced the release of ET-1 and the expression of the ET-A receptor in response to intermittent hypoxia. Systemic administration of MnTMPyP, which prevent the elevation of ROS, reduced the increased basal release of ET-1, the overexpression of ET-A receptor mRNA and the enhanced carotid body chemosensory response to acute hypoxia. These results support the idea that a ROSinduced increase of ET-1 release is involved in the potentiation of carotid body chemosensory response elicited by intermittent hypoxia. Increased plasmatic levels of ROS and ET-1 have been also implicated in the hypertension induced by intermittent hypoxia. Troncoso-Brindeiro et al., (2007) reported that the concurrent treatments of rats exposed to intermittent hypoxia with the SOD mimetic, 4-hydroxy-2,2,6,6-tetramethylpiperidine-*N*-oxyl (TEMPOL), prevents the increased ROS plasmatic level and the hypertension. However, it is worth noting that intermittent hypoxia increases the expression of ET-1 in the rat CB during the first week of hypoxia, and later the ET-1 levels returned back to the control levels (Del Rio et al., 2011), suggesting that ET-1 may contribute to the enhanced carotid body responsiveness to hypoxia in the early phase of the intermittent hypoxic exposures. In addition to the transient changes in ET-1 expression, we found a significant decrease in the eNOS expression in the rat carotid body at 7 days of intermittent hypoxia (Del Rio et al., 2011), suggesting that chronic intermittent hypoxia may decrease the nitric oxide (NO) levels within the carotid body. Since NO at low concentration is an inhibitory modulator of the carotid chemosensory activity (Iturriaga et al., 2000), a reduced NO level may contribute to enhance the carotid body chemosensitivity, as well as to amplify the vasoconstrictor effect of ET-1. This interpretation is supported by the finding that intermittent hypoxia decreases the expression of the neuronal NO synthase in the rat carotid body (Marcus et al., 2010), suggesting that the removal of the inhibitory NO effects may also contribute to enhance the carotid chemosensory responses to hypoxia. Our results also showed that carotid body iNOS immunorreactive levels increased after 21 days of intermittent hypoxic exposure. Since iNOS produce higher amounts of NO, it is plausible that the NO levels in the carotid body will increase during long-term intermittent hypoxic exposure. It is worth noting that NO has a dual effect on carotid chemosensory discharges. Indeed, Iturriaga et al., (2000) found that at low levels NO is predominantly an inhibitor of the chemosensory discharges, whereas at high concentration NO increases carotid body chemosensory discharges. Thus, it is plausible

that high NO levels in the carotid body following long-term intermittent hypoxia may

partially contribute to maintain the carotid body chemosensory potentiation.

#### **6.3 Pro-inflammatory cytokines**

78 Oxidative Stress and Diseases

Fig. 3. Exposure to CIH increased 3-nitrotyrosine formation in the glomus cells (white arrows) and endothelial cells (black arrows) from rat carotid bodies. Scale bar, 20 µm.

An interesting molecule, which may mediate the carotid body chemosensory potentiation induced by intermittent hypoxia, is endothelin-1 (ET-1). It is known that the plasmatic ET-1 level increases in rats exposed to intermittent hypoxia (Kanagy et al., 2001) and OSA patients (Phillips et al., 1999). This potent vasoconstrictor peptide is expressed in the endothelium, blood vessels and glomus cells of the carotid body (Rey et al., 2007). The application of ET-1 produces chemosensory excitation in both *in situ* and *in vitro* carotid body perfused preparations, but not in the superfused preparation devoid of vascular effects (Rey & Iturriaga, 2004). We found that ET-1 was increased locally in the carotid body of cats exposed to 4 days to intermittent hypoxia by ~10-fold, while ET-1 plasma levels remains unchanged (Rey et al., 2006). The enhanced carotid body chemosensory responses to hypoxia were reduced by the ET receptor blocker bosentan in the intermittent hypoxic treated cats, but have no effects on the carotid body chemosensory activity in control animals (Rey et al., 2006), indicating that a local increase of ET-1 contributes to enhance the carotid chemosensory responses. Pawar et al., (2009) tested the hypothesis that ET-1 induced by ROS plays a role in intermittent hypoxia induced chemosensory potentiation in the rat neonatal carotid body. They found that intermittent hypoxia enhanced the release of ET-1 and the expression of the ET-A receptor in response to intermittent hypoxia. Systemic administration of MnTMPyP, which prevent the elevation of ROS, reduced the increased basal release of ET-1, the overexpression of ET-A receptor mRNA and the enhanced carotid body chemosensory response to acute hypoxia. These results support the idea that a ROSinduced increase of ET-1 release is involved in the potentiation of carotid body chemosensory response elicited by intermittent hypoxia. Increased plasmatic levels of ROS and ET-1 have been also implicated in the hypertension induced by intermittent hypoxia. Troncoso-Brindeiro et al., (2007) reported that the concurrent treatments of rats exposed to intermittent hypoxia with the SOD mimetic, 4-hydroxy-2,2,6,6-tetramethylpiperidine-*N*-oxyl (TEMPOL), prevents the increased ROS plasmatic level and the hypertension. However, it is worth noting that intermittent hypoxia increases the expression of ET-1 in the rat CB during the first week of hypoxia, and later the ET-1 levels returned back to the control levels (Del

**6.2 Vasoactive molecules** 

Endothelial dysfunction has been related to the progression of hypertension in OSA patients and animals exposed to intermittent hypoxia due to the increased plasmatic levels of proinflammatory cytokines (Biltagi et al., 2008, Jelic et al., 2008, Jun et al., 2008, Tam et al., 2007, Williams & Scharf, 2007). It is likely that an increased production of ROS induced by the hypoxia-reoxygention cycles may evoke the expression of genes and the synthesis of proinflammatory cytokines, mediated by the activation of transcription factors such as the nuclear factor kappa B (NF-κB), the activator protein 1 and HIF-1α (Semenza & Prahbakar, 2007). In response to oxidative stress, HIF-1α induces the expression of several genes including ET-1 and iNOS, but ROS also produces the translocation of NF-κB to the nucleus, increasing the expression of several inflammatory genes such as IL-1β, IL-6, TNF-α, adhesion molecules, iNOS and ET-1 (Janseen-Heininger et al., 2000). Recently, we found that intermittent hypoxia increased the levels of TNF-α and IL-1β in the rat carotid body after 21 days of exposure (Del Rio et al, 2011). We found that glomus cells constitutively expresses TNF-α and IL-1β in the cell bodies and that chronic intermittent hypoxia upregulates the expression of both TNF-α and IL-1β, without inducing carotid body tissue infiltration with macrophages or changes in TNF-α and IL-1β plasmatic levels (Del Rio et al., 2011). Our results showed that exposure to intermittent hypoxia enhances the rat carotid chemosensory responses to acute hypoxia, and progressively increase the immunorreactive TNF-α and IL-1β expression in the carotid body, suggesting a potential role for this cytokines in modulating the enhanced carotid body chemosensory activity after exposure to intermittent hypoxia.

Oxidative Stress in the Carotid Body:

the hypertension.

the systemic oxidative stress.

**9. Conclusion** 

Implications for the Cardioventilatory Alterations Induced by Obstructive Sleep Apnea 81

finally contributes to the development of the hypertension. We postulate that cyclic episodes of hypoxia-reoxygenation enhance the carotid body chemosensitivity to hypoxia, which in turn contributes to elicit a persistent facilitation of the sympathetic neural output. The enhanced sympathetic activity along with a reduction of the baroreflex efficiency should impair the regulation of the heart rate variability and the vasomotor tone of blood vessels, resulting in an elevation of arterial blood pressure. On the other hand, systemic oxidative stress and the inflammation *per se* may contribute to the endothelial dysfunction, leading to

Fig. 5. Proposed mechanisms involved in the hypertension induced by the potentiation of the carotid body chemosensory responses to hypoxia induced by intermittent hypoxia, and

disfunction

Autonomic dysfunction has been associated to exposure to chronic intermittent hypoxia in animal models, and is thought to be involved in the increased risk of hypertension and cardiovascular mortality in OSA patients. The cyclic hypoxic episodes in OSA patients potentiate cardiovascular and sympathetic responses induced by hypoxic stimulation of peripheral chemoreceptors, and impair the regulation of arterial blood pressure and the renin–angiotensin system. Intermittent hypoxia enhances the ventilatory and cardiovascular responses to acute hypoxia, suggesting a major role of the carotid body in the pathological

#### **7. Proposed targets of the effects of ROS in the carotid body**

The available evidence indicates that oxidative stress mediated the potentiation of the carotid body chemosensory responses to acute hypoxia, induced by the exposure to intermittent hypoxia. However, the nature of the molecular mechanism by which ROS induced chemosensory potentiation is not known. Based on the presented evidences, we hypothesized that chronic intermittent hypoxia may increase the expression of proinflammatory cytokines and other chemosensory modulators, such as ET-1 and NO, which may potentially contribute to enhance the carotid body chemosensory responses to hypoxia. Figure 4 summarized the possible targets of the effects of ROS on oxygen chemoreception in the carotid body. It is plausible that excessive amounts of free radicals may modify the O2 sensitive K+ channels, increasing the intracellular Ca2+ levels, which in turn evokes the release of excitatory transmitters, but a direct participation of ROS on the O2 chemotransduction process in the carotid body is not clear (Gonzalez et al., 2007). ROS or other molecules, produced downstream of the ROS signal, which act upon the mitochondria, membrane channels or the gene expression machinery may modify the oxygen sensing in the carotid body. Further studies are required to determine which protein or enzyme complexes involved in the carotid body chemosensory process are affected by ROS or ROS-dependent molecules induced by intermittent hypoxia.

Fig. 4. Proposed targets of the effects of ROS on the potentiation of the carotid body induced by intermittent hypoxia.

#### **8. Integrative model**

Figure 5 shown a diagram of the proposed mechanisms involved in the potentiation of the carotid body chemosensory responses to hypoxia induced by intermittent hypoxia, which finally contributes to the development of the hypertension. We postulate that cyclic episodes of hypoxia-reoxygenation enhance the carotid body chemosensitivity to hypoxia, which in turn contributes to elicit a persistent facilitation of the sympathetic neural output. The enhanced sympathetic activity along with a reduction of the baroreflex efficiency should impair the regulation of the heart rate variability and the vasomotor tone of blood vessels, resulting in an elevation of arterial blood pressure. On the other hand, systemic oxidative stress and the inflammation *per se* may contribute to the endothelial dysfunction, leading to the hypertension.

Fig. 5. Proposed mechanisms involved in the hypertension induced by the potentiation of the carotid body chemosensory responses to hypoxia induced by intermittent hypoxia, and the systemic oxidative stress.

#### **9. Conclusion**

80 Oxidative Stress and Diseases

The available evidence indicates that oxidative stress mediated the potentiation of the carotid body chemosensory responses to acute hypoxia, induced by the exposure to intermittent hypoxia. However, the nature of the molecular mechanism by which ROS induced chemosensory potentiation is not known. Based on the presented evidences, we hypothesized that chronic intermittent hypoxia may increase the expression of proinflammatory cytokines and other chemosensory modulators, such as ET-1 and NO, which may potentially contribute to enhance the carotid body chemosensory responses to hypoxia. Figure 4 summarized the possible targets of the effects of ROS on oxygen chemoreception in the carotid body. It is plausible that excessive amounts of free radicals may modify the O2 sensitive K+ channels, increasing the intracellular Ca2+ levels, which in turn evokes the release of excitatory transmitters, but a direct participation of ROS on the O2 chemotransduction process in the carotid body is not clear (Gonzalez et al., 2007). ROS or other molecules, produced downstream of the ROS signal, which act upon the mitochondria, membrane channels or the gene expression machinery may modify the oxygen sensing in the carotid body. Further studies are required to determine which protein or enzyme complexes involved in the carotid body chemosensory process are affected by

Fig. 4. Proposed targets of the effects of ROS on the potentiation of the carotid body induced

Figure 5 shown a diagram of the proposed mechanisms involved in the potentiation of the carotid body chemosensory responses to hypoxia induced by intermittent hypoxia, which

by intermittent hypoxia.

**8. Integrative model** 

**7. Proposed targets of the effects of ROS in the carotid body** 

ROS or ROS-dependent molecules induced by intermittent hypoxia.

Autonomic dysfunction has been associated to exposure to chronic intermittent hypoxia in animal models, and is thought to be involved in the increased risk of hypertension and cardiovascular mortality in OSA patients. The cyclic hypoxic episodes in OSA patients potentiate cardiovascular and sympathetic responses induced by hypoxic stimulation of peripheral chemoreceptors, and impair the regulation of arterial blood pressure and the renin–angiotensin system. Intermittent hypoxia enhances the ventilatory and cardiovascular responses to acute hypoxia, suggesting a major role of the carotid body in the pathological

Oxidative Stress in the Carotid Body:

Implications for the Cardioventilatory Alterations Induced by Obstructive Sleep Apnea 83

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#### **10. Acknowledgements**

Present work was supported by grant 1100405 from the National Fund for Scientific and Technological Development of Chile (FONDECYT). Rodrigo Del Rio was supported by a CONICYT AT-24091043 fellowship.

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**5** 

*México* 

**Adipocytokines, Oxidative Stress and** 

Ana Bertha Zavalza Gómez1, María Cristina Islas Carbajal2

*West National Medical Center, Mexican Institute of Social Security,* 

In spite of the considerable progress in their diagnosis, prevention and treatment, cardiovascular diseases remain the number one cause of death worldwide. This is partially due to the rapidly growing incidence of obesity, which is a well-known independent risk factor for insulin resistance, diabetes, dyslipidaemia, high blood pressure and thrombosis

The metabolic complications of obesity, often referred to as the metabolic syndrome, characterized by a heterogenic complex of symptoms and consist of glucose intolerance, central obesity, dyslipidemia (hypertriglyceridemia, elevated nonesterified fatty acids (NEFAs), and decreased high-density lipoprotein (HDL) cholesterol), and hypertension. These, often culminating in -cell failure, impaired glucose tolerance and type 2 diabetes (T2D). In addition, dyslipidaemia, coronary heart disease (CHD), systemic hypertension and premature heart failure are pathologies related (Hubert et al., 1983). Abdominal obesity, ectopic lipid accumulation, hepatic steatosis, and sleep apnea can also be included in the

On the other hand, obesity leads to an alteration in the profile of hormones secreted by adipose tissue (adipokines). Secretion of adipocytokines has been shown particularly for visceral fat (Dusserre et al., 2000; Fontana et al., 2007; Yang & Smith, 2007). It is evident that many of these adipokines have the ability to influence other tissues such as the liver, muscle and brain, e.g. the adipokine leptin affects appetite regulation, others have an important impact on the consequences of adipose tissue inflammation (e.g. interleukin 6 (IL), PAI-1, monocyte chemoattractant protein 1 [MCP-1]) and vascular biology (e.g. serum amyloid A [SAA]) (Bastard et al., 2002; Mutch et al., 2001; Sartipy et al., 2003; Stofkova, 2009; Yang et al., 2006). In addition, increased tumor necrosis factor (TNF) and IL-6 expression and

**1. Introduction**

(Lopaschuck et al., 2007).

metabolic complications of obesity (Parati et al., 2007).

**Impaired Cardiovascular Functions** 

and Ana Rosa Rincón Sánchez3

*1Specialties Hospital, Medical Unit of High Specialty,* 

*University of Guadalajara, Guadalajara, Jalisco,* 

*2Cardiovascular Research Unit, Physiology Department, Health Science University Center, University of Guadalajara, 3Physiology Department, Health Science University Center,* 

chronic intermittent hypoxia. Am J Physiol Regul Integr Comp Physiol, Vol. 295. pp. 28-37.


### **Adipocytokines, Oxidative Stress and Impaired Cardiovascular Functions**

Ana Bertha Zavalza Gómez1, María Cristina Islas Carbajal2 and Ana Rosa Rincón Sánchez3 *1Specialties Hospital, Medical Unit of High Specialty,* 

*West National Medical Center, Mexican Institute of Social Security, 2Cardiovascular Research Unit, Physiology Department, Health Science University Center, University of Guadalajara, 3Physiology Department, Health Science University Center, University of Guadalajara, Guadalajara, Jalisco, México* 

#### **1. Introduction**

86 Oxidative Stress and Diseases

Semenza, G,L. & Prabhakar, N.R. (2007). HIF-1-dependent respiratory, cardiovascular, and

Shiomi, T.; Guilleminault, C.; Sasanabe, R.; Hirota, I.; Maekawa, M. & Kobayashi, T. (1996).

Shu, H.F.; Wang, B.R.; Wang, S.R.; Yao, W.; Huang, H.P.; Zhou, Z.; Wang, X.; Fan, J.; Wang,

Schulz, R.; Eisele, H.J.; Murzabekova, G. & Weissmann N. (2008). Sleep apnea and

Smith, M,L. & Pacchia Ch,F. (2007). Sleep apnoea and hypertension: Role of chemoreflexes

Somers, V.K.; White, D,P.; Amin, R.; Abraham, W.T.; Costa, F.; Culebras, A.; Daniels, S.;

Troncoso-Brindeiro, C.M.; Da Silva, A.Q.; Allahdadi, K,J.; Youngblood, V. & Kanagy, N.L.

Williams, A. & Scharf S.M. (2007). Obstructive sleep apnea, cardiovascular disease, and inflammation: is NF-kappaB the key? Sleep Breath, Vol. 11. pp. 69-76. Yan, B.; Soukhova-O'Hare, G.K.; Li, L.; Lin, Y.; Gozal, D.; Wead, W.B.; Wurster, R.D. &

Zoccal, D.B.; Simms, A.E.; Bonagamba, L.G.; Braga, V.A.; Pickering, A.E.; Paton, J.F. &

on Sleep Disorders Research. J Am Coll Cardiol, Vol. 52. pp. 686-717. Tam, C.S.; Wong, M.; Tam, K.; Aouad, L. & Waters K.A. (2007). The effect of acute

rats. Am J Physiol Heart Circ Physiol, Vol. 293. pp. 2971-2976.

obstructive sleep apnea. Sleep, Vol. 119. pp. 370-377.

in humans. Exp Physiol, Vol. 92. pp. 45-50.

piglets. Sleep, Vol. 30. pp. 723-727.

344 rats. Neurocience, Vol. 153. pp. 709-720.

Physiol. Vol. 586, 3253-3265.

pp. 28-37.

1391-1396.

25. pp. 3638-3647.

22.

chronic intermittent hypoxia. Am J Physiol Regul Integr Comp Physiol, Vol. 295.

redox responses to chronic intermittent hypoxia. Antioxid Redox Signal, Vol. 9. pp.

Augmented very low frequency component of heart rate variability during

T. & Ju, G. (2007). IL-1βeta inhibits IK and increases [Ca2+]i in the carotid body glomus cells and increases carotid sinus nerve firings in the rat. Eur J Neurosci, Vol.

cardiovascular disease. Results from animal studies. Pneumologie, Vol. 62. pp.18-

Floras, J.S.; Hunt, C,E.; Olson, L.J.; Pickering, T.G.; Russell, R.; Woo, M. & Young, T. (2008). Sleep apnea and cardiovascular disease: An American Heart Association. In collaboration with the National Heart, Lung, and Blood Institute National Center

intermittent hypercapnic hypoxia treatment on IL-6, TNF-alpha, and CRP levels in

(2007). Reactive oxygen species contribute to sleep apnea-induced hypertension in

Cheng Z.J. (2008). Attenuation of heart rate control and neural degeneration in nucleus ambiguus following chronic intermittent hypoxia in young adult Fischer

Machado, B.H. (2008). Increased sympathetic outflow in juvenile rats submitted to chronic intermittent hypoxia correlates with enhanced expiratory activity. J In spite of the considerable progress in their diagnosis, prevention and treatment, cardiovascular diseases remain the number one cause of death worldwide. This is partially due to the rapidly growing incidence of obesity, which is a well-known independent risk factor for insulin resistance, diabetes, dyslipidaemia, high blood pressure and thrombosis (Lopaschuck et al., 2007).

The metabolic complications of obesity, often referred to as the metabolic syndrome, characterized by a heterogenic complex of symptoms and consist of glucose intolerance, central obesity, dyslipidemia (hypertriglyceridemia, elevated nonesterified fatty acids (NEFAs), and decreased high-density lipoprotein (HDL) cholesterol), and hypertension. These, often culminating in -cell failure, impaired glucose tolerance and type 2 diabetes (T2D). In addition, dyslipidaemia, coronary heart disease (CHD), systemic hypertension and premature heart failure are pathologies related (Hubert et al., 1983). Abdominal obesity, ectopic lipid accumulation, hepatic steatosis, and sleep apnea can also be included in the metabolic complications of obesity (Parati et al., 2007).

On the other hand, obesity leads to an alteration in the profile of hormones secreted by adipose tissue (adipokines). Secretion of adipocytokines has been shown particularly for visceral fat (Dusserre et al., 2000; Fontana et al., 2007; Yang & Smith, 2007). It is evident that many of these adipokines have the ability to influence other tissues such as the liver, muscle and brain, e.g. the adipokine leptin affects appetite regulation, others have an important impact on the consequences of adipose tissue inflammation (e.g. interleukin 6 (IL), PAI-1, monocyte chemoattractant protein 1 [MCP-1]) and vascular biology (e.g. serum amyloid A [SAA]) (Bastard et al., 2002; Mutch et al., 2001; Sartipy et al., 2003; Stofkova, 2009; Yang et al., 2006). In addition, increased tumor necrosis factor (TNF) and IL-6 expression and

Adipocytokines, Oxidative Stress and Impaired Cardiovascular Functions 89

diabetes). Older Americans over 50 years of age without MetS regardless of diabetes status had the lowest CHD prevalence (8.7% without diabetes, 7.5% with diabetes). Those with MetS without diabetes had higher CHD prevalence (13.9%) and, those with both MetS and diabetes had the highest prevalence of CHD (19.2%) compared with those with neither

The Systematic Coronary Risk Evaluation (SCORE) data set comprises data from 12 European cohort studies. The SCORE population was also divided into gender and age strata: under 40, 40–49, 50–59, and over 60. The rate of CVD mortality in each body mass index (BMI) category was calculated, each 5-unit increase in BMI was associated with an increase in CVD mortality of 34% in men and 29% in women. This increases the public health importance of BMI as both a simple indicator and mediator of CVD risk (Dudina et

Impaired myocardial diastolic relaxation (e.g., diastolic dysfunction) is the earliest myocardial contractility observed in metabolic conditions such as obesity, insulin resistance, and hypertension. Diastolic dysfunction manifests as a reduction in velocity of myocardial relaxation, as well as decreasing myocardial compliance. Mechanisms that contribute to this selective cardiac dysfunction include decreases in energy production due to reductions in mitochondrial respiration, increased oxidative stress, and defective contractile and intracellular "Ca2+" regulatory proteins. Abnormalities in "Ca2+" signaling/flux and myofilament function contribute to the cardiomyopathic alterations observed in the metabolic syndrome (Ren et al., 2010). Reductions in the oxidative capacity of the mitochondrial electron transport chain are manifested in obese, insulin-resistant persons as well as diabetic patients. Mitochondria in endothelial cells are thought to play an important role in cellular signaling as sensors for local oxygen concentration and regulations of nitric oxide (NO) production. Renin-angiotensin-aldosterone system (RAAS)-mediated increases in nicotinamide-adenine dinucleotide phosphate (NADPH) oxidase activity and generation of reactive oxygen species (ROS) may result in mitochondrial damage and associated decreases in oxidative phosphorylation, Adenosine triphosphate (ATP) production and

The mechanisms underlying ventricular dysfunction are dysfunction of cardiac myocytes and longstanding pressure or volume overload. As myocardial contractility decreases, the stroke volume drops and the end-diastolic volume and pressure increase. If sustained in the long-term, this volume increase leads to what is termed cardiac remodelling. This involves myocardial hypertrophy, chamber enlargement and an increase in ventricular wall stress, and increases oxygen demand. An increase in ventricular stiffness also occurs due to increased collagen deposition in the heart, which impairs filling and exacerbates the

The new paradigm of atherosclerosis links oxidative stress, inammation, thrombosis, and endothelial dysfunction. Growing evidence indicates that chronic and acute overproduction of ROS under pathophysiologic conditions is integral in the development of CVD (Madamanchi et al., 2005). Coronary artery disease (CAD) is one of the most frequent causes of death and disabling symptoms worldwide. Epidemiological studies have indicated the

**2.3 Pathologies associated to cardiovascular morbidity** 

(Alexander et al., 2003).

bioavailable NO (Ren et al., 2010).

situation (Kotzé & Howell, 2008).

al., 2011).

secretion from adipose tissue are involved in both whole-body and local insulin resistance at different tissue sites.

The principal purpose of this chapter is to describe how the adipocytokines and oxidative stress interact with insulin signaling in the context of low-grade inflammation related to obesity in order to promote cardiovascular complications.

#### **2. Pathophysiology of cardiovascular morbidity**

#### **2.1 Introduction**

The pathophysiology of cardiovascular morbidity is complex and multifactorial. Oxidative stress is an important contributory factor to the etiology of many cardiovascular diseases, including atherosclerosis, coronary heart disease (heart attack), cerebrovascular disease (stroke), cardiomyopathies, peripheral vascular disease, diabetes, heart failure, and hypertension (Dusting & Triggle, 2005). Ischemic heart disease and hypertension are the two most important causes of heart failure in the Western world. Other common causes include valvular heart disease (especially aortic stenosis and mitral regurgitation).

Arterial hypertension is the most prevalent cardiovascular risk factor and the leading cause of morbidity and mortality from cardiovascular disease (CVD) worldwide (Gómez-Marcos et al., 2009). Heart failure (HF) is a complex clinical syndrome caused by impaired ventricular performance. It is the final common pathway for a variety of cardiovascular disease processes, leading to potentially disabling symptoms and shortened life expectancy. Currently, 1% of the population aged 50–59 yr, and 10% of those over 80 yr, have HF; is the only major cardiovascular condition that is increasing in prevalence, because of an ageing population and improved survival from other CVD (Kotzé & Howell, 2008). Understanding these profound mechanisms of disease can help clinicians identify and treat CVD, as well as help patients prevent these potentially devastating complications.

#### **2.2 Epidemiology**

Cardiovascular diseases are the world's largest killers, claiming 17.1 million lives a year, CVD contributed to a third of global deaths. An estimated 79 400 000 American adults (1 in 3) have 1 or more types of CVD. Of these, 37 500 000 are estimated to be age 65 or older (Rosamond et al., 2007).

Extensive epidemiological research has established diabetes, hyperlipidemia, hypertension, and cigarette smoking, as independent risk factors for CHD. The risk increases 2–3 folds with tobacco smoking, with age and is greater for women than for men. In contrast, cardiac events fall 50% in people who stop smoking and the risk of CVDs, also decreases significantly over the first two years after stopping smoking (Khot, et al., 2003).

The health interview part of the National Health and Nutrition Examination Survey (NHANES) III was used to categorize adults over 50 years of age by presence of metabolic syndrome (National Cholesterol Education Program [NCEP] definition) with or without diabetes. The prevalence of CHD for each group was then determined. Metabolic syndrome (MetS) is very common, with ∼44% of the U.S. population over 50 years of age meeting the NCEP criteria. In contrast, diabetes without MetS is uncommon (13% of those with

secretion from adipose tissue are involved in both whole-body and local insulin resistance at

The principal purpose of this chapter is to describe how the adipocytokines and oxidative stress interact with insulin signaling in the context of low-grade inflammation related to

The pathophysiology of cardiovascular morbidity is complex and multifactorial. Oxidative stress is an important contributory factor to the etiology of many cardiovascular diseases, including atherosclerosis, coronary heart disease (heart attack), cerebrovascular disease (stroke), cardiomyopathies, peripheral vascular disease, diabetes, heart failure, and hypertension (Dusting & Triggle, 2005). Ischemic heart disease and hypertension are the two most important causes of heart failure in the Western world. Other common causes include

Arterial hypertension is the most prevalent cardiovascular risk factor and the leading cause of morbidity and mortality from cardiovascular disease (CVD) worldwide (Gómez-Marcos et al., 2009). Heart failure (HF) is a complex clinical syndrome caused by impaired ventricular performance. It is the final common pathway for a variety of cardiovascular disease processes, leading to potentially disabling symptoms and shortened life expectancy. Currently, 1% of the population aged 50–59 yr, and 10% of those over 80 yr, have HF; is the only major cardiovascular condition that is increasing in prevalence, because of an ageing population and improved survival from other CVD (Kotzé & Howell, 2008). Understanding these profound mechanisms of disease can help clinicians identify and treat CVD, as well as

Cardiovascular diseases are the world's largest killers, claiming 17.1 million lives a year, CVD contributed to a third of global deaths. An estimated 79 400 000 American adults (1 in 3) have 1 or more types of CVD. Of these, 37 500 000 are estimated to be age 65 or older

Extensive epidemiological research has established diabetes, hyperlipidemia, hypertension, and cigarette smoking, as independent risk factors for CHD. The risk increases 2–3 folds with tobacco smoking, with age and is greater for women than for men. In contrast, cardiac events fall 50% in people who stop smoking and the risk of CVDs, also decreases

The health interview part of the National Health and Nutrition Examination Survey (NHANES) III was used to categorize adults over 50 years of age by presence of metabolic syndrome (National Cholesterol Education Program [NCEP] definition) with or without diabetes. The prevalence of CHD for each group was then determined. Metabolic syndrome (MetS) is very common, with ∼44% of the U.S. population over 50 years of age meeting the NCEP criteria. In contrast, diabetes without MetS is uncommon (13% of those with

significantly over the first two years after stopping smoking (Khot, et al., 2003).

valvular heart disease (especially aortic stenosis and mitral regurgitation).

help patients prevent these potentially devastating complications.

obesity in order to promote cardiovascular complications.

**2. Pathophysiology of cardiovascular morbidity** 

different tissue sites.

**2.1 Introduction** 

**2.2 Epidemiology** 

(Rosamond et al., 2007).

diabetes). Older Americans over 50 years of age without MetS regardless of diabetes status had the lowest CHD prevalence (8.7% without diabetes, 7.5% with diabetes). Those with MetS without diabetes had higher CHD prevalence (13.9%) and, those with both MetS and diabetes had the highest prevalence of CHD (19.2%) compared with those with neither (Alexander et al., 2003).

The Systematic Coronary Risk Evaluation (SCORE) data set comprises data from 12 European cohort studies. The SCORE population was also divided into gender and age strata: under 40, 40–49, 50–59, and over 60. The rate of CVD mortality in each body mass index (BMI) category was calculated, each 5-unit increase in BMI was associated with an increase in CVD mortality of 34% in men and 29% in women. This increases the public health importance of BMI as both a simple indicator and mediator of CVD risk (Dudina et al., 2011).

#### **2.3 Pathologies associated to cardiovascular morbidity**

Impaired myocardial diastolic relaxation (e.g., diastolic dysfunction) is the earliest myocardial contractility observed in metabolic conditions such as obesity, insulin resistance, and hypertension. Diastolic dysfunction manifests as a reduction in velocity of myocardial relaxation, as well as decreasing myocardial compliance. Mechanisms that contribute to this selective cardiac dysfunction include decreases in energy production due to reductions in mitochondrial respiration, increased oxidative stress, and defective contractile and intracellular "Ca2+" regulatory proteins. Abnormalities in "Ca2+" signaling/flux and myofilament function contribute to the cardiomyopathic alterations observed in the metabolic syndrome (Ren et al., 2010). Reductions in the oxidative capacity of the mitochondrial electron transport chain are manifested in obese, insulin-resistant persons as well as diabetic patients. Mitochondria in endothelial cells are thought to play an important role in cellular signaling as sensors for local oxygen concentration and regulations of nitric oxide (NO) production. Renin-angiotensin-aldosterone system (RAAS)-mediated increases in nicotinamide-adenine dinucleotide phosphate (NADPH) oxidase activity and generation of reactive oxygen species (ROS) may result in mitochondrial damage and associated decreases in oxidative phosphorylation, Adenosine triphosphate (ATP) production and bioavailable NO (Ren et al., 2010).

The mechanisms underlying ventricular dysfunction are dysfunction of cardiac myocytes and longstanding pressure or volume overload. As myocardial contractility decreases, the stroke volume drops and the end-diastolic volume and pressure increase. If sustained in the long-term, this volume increase leads to what is termed cardiac remodelling. This involves myocardial hypertrophy, chamber enlargement and an increase in ventricular wall stress, and increases oxygen demand. An increase in ventricular stiffness also occurs due to increased collagen deposition in the heart, which impairs filling and exacerbates the situation (Kotzé & Howell, 2008).

The new paradigm of atherosclerosis links oxidative stress, inammation, thrombosis, and endothelial dysfunction. Growing evidence indicates that chronic and acute overproduction of ROS under pathophysiologic conditions is integral in the development of CVD (Madamanchi et al., 2005). Coronary artery disease (CAD) is one of the most frequent causes of death and disabling symptoms worldwide. Epidemiological studies have indicated the

Adipocytokines, Oxidative Stress and Impaired Cardiovascular Functions 91

Obesity has been increasing in epidemic proportions in both adults and children. In adults, overweight is dened as a BMI 25 to 29.9 Kg/m2 and obesity as BMI ≥30 Kg/m2. Other indexes that have been used less commonly but possibly with more predictive power include body fatness, waist circumference (WC), waist-to-hip ratio (WHR), and weight-toheight ratio. A recent study of nearly 360,000 participants from 9 European countries showed that both general obesity and abdominal adiposity are associated with risk of death and support the importance of WC or WHR in addition to BMI for assessing mortality risk. Obesity has many adverse effects on hemodynamics and CV structure and function (Lavie

Elevated BMI predisposes to congestive heart failure (CHF) by promoting increased blood pressure, diabetes, and CHD. Factors related to obesity and hypertension, include: endothelial dysfunction, insulin resistance, sympathetic nervous system, substances released

The role of obesity in the initiation and acceleration of tissue inflammation has been well studied. Excess adipose tissue can contribute to inflammation in two ways: (a) ectopic fat storage induces lipotoxicity, promoting an intracellular inflammatory response and (b) altered adipokine production in obesity contributes to the inflammatory response. It is now recognized that adiponectin has a role in both of these processes. Related to demonstrated association between hypo-adiponectinaemia and metabolic dysfunction (Cnop et al., 2003) the proposal provides that a replacement of adiponectin may function as pharmacological

Healthy endothelium regulates blood vessel tone, platelet activation, leukocyte adhesion, thrombogenesis, and inflammation. The net effect of healthy endothelium is vasodilatory, anti-atherogenic, and anti-inflammatory (Dokken, 2008). Endothelial dysfunction has been observed in patients with established coronary artery disease or coronary risk factors, both

As shown in figure 1, endothelial dysfunction a key factor in atherogenesis, is associated with an increased risk of cardiovascular events and highest risk for vascular morbidity and mortality. A major risk for atherosclerotic plaque rupture is aging. One possible mechanism is that aging is associated with endothelial cell senescence, which is a risk factor for endothelial apoptosis and endothelial denudation, rendering the atherosclerotic plaque

A primary event in atherogenesis is the inltration of activated inammatory cells into the arterial wall. ROS can be produced from both endogenous and exogenous substances. Potential endogenous sources include mitochondria, cytochrome P450 metabolism, peroxisomes, and inflammatory cell activation. In the vascular wall, ROS are generated by several mechanisms, including NADPH oxidases, xanthine oxidase, the mitochondrial respiratory chain, lipoxygenases, and nitric oxide synthases. ROS formation can be stimulated by mechanical forces (e.g., stretch, pressure, shear stress), environmental factors (such as hypoxia), secreted factors coupled to tyrosine kinase receptors (e.g., platelet derived

from adipocytes (IL-6, TNF-, etc.), and sleep apnea (Poirier et al., 2006).

in the coronary and peripheral vasculature (Heitzer et al., 2001).

**2.3.3 Obesity** 

et al., 2009).

therapy (Chandran et al., 2003).

**2.3.4 Endothelial dysfunction** 

prone to rupture (Hulsmans et al., 2011).

rising prevalence of atherosclerosis globally (Tedgui & Mallat, 2006). Formation of atheromatous plaques in the arteries obstructs the supply of oxygen and nutrients to the myocardium, resulting in CHD (Woods et al., 2000).

#### **2.3.1 Diabetes**

Diabetes is a prime risk factor for CVD, the link between diabetes and CVD is complex and multifactorial. The presence of insulin resistance, impaired glucose tolerance, and overt diabetes, are associated with an increased risk of CVD, these conditions are also accompanied by the presence of oxidative stress (Woods et al., 2000). Vascular disorders include retinopathy and nephropathy, peripheral vascular disease (PVD), stroke, and CAD. Diabetes also affects the heart muscle, causing both systolic and diastolic heart failure.

The etiology of this excess cardiovascular morbidity and mortality is not completely clear (Dokken, 2008). Evidence suggests that although hyperglycemia, the hallmark of diabetes, contributes to myocardial damage after ischemic events, it is clearly not the only factor, because both pre-diabetes and the presence of the MetS, even in normoglycemic patients, increase the risk of most types of CVD (Alexander, 2003; Dokken, 2008). In diabetes, where CVD is of particular concern, there are multiple sources of ROS including the auto-oxidation of glucose, increased substrate flux, and decreased levels of NADPH through the polyol pathway. Formation of advanced glycation end products (AGEs) and their interaction with cellular targets, such as endothelial cells, may lead to oxidative stress and promote formation of oxidized LDL (ox-LDL) (Ceriello & Motz, 2004).

The recent explosion of the worldwide epidemic of MetS combining disturbances in glucose and insulin metabolism, excess predominantly abdominally distributed weight, mild dyslipidemia, and hypertension, with the subsequent development of obesity, T2D and CVD, compromises progress made in reducing the morbidity and mortality of CVD in recent years. Cardiovascular risk increases in parallel to insulin resistance (as estimated by the homeostasis model assessment index (HOMA) both patients with diabetes and nondiabetic (Saely et al., 2005).

#### **2.3.2 Metabolic syndrome**

The incidence of CVD, coronary heart disease, and T2D has not been well defined in persons with the MetS. Conclusions were that MetS is common and is associated with an increased risk for CVD and T2D in both sexes, according to metabolic syndrome traits. MetS accounts for up to one third of CVD in men and approximately half of new T2D over 8 years of follow-up (Wilson et al., 2005).

A large family study of T2D in Finland and Sweden (the Botnia study) were included in the analysis of cardiovascular risk associated with the MetS. The aim of the study was to assess the prevalence of cardiovascular morbidity and mortality associated with the MetS by applying the WHO denition. In women and men, respectively, the MetS was seen in 10 and 15% of subjects with normal glucose tolerance, 42 and 64% of those with impaired fasting glucose (IFG)/impaired glucose tolerance (IGT), and 78 and 84% of those with T2D. Cardiovascular mortality was markedly increased in subjects with the MetS (12.0 vs. 2.2%, *p< 0.001)*. Of the individual components of the MetS, microalbuminuria conferred the strongest risk of cardiovascular death (RR 2.80; *p< 0.002)* (Isomaa et al., 2001).

#### **2.3.3 Obesity**

90 Oxidative Stress and Diseases

rising prevalence of atherosclerosis globally (Tedgui & Mallat, 2006). Formation of atheromatous plaques in the arteries obstructs the supply of oxygen and nutrients to the

Diabetes is a prime risk factor for CVD, the link between diabetes and CVD is complex and multifactorial. The presence of insulin resistance, impaired glucose tolerance, and overt diabetes, are associated with an increased risk of CVD, these conditions are also accompanied by the presence of oxidative stress (Woods et al., 2000). Vascular disorders include retinopathy and nephropathy, peripheral vascular disease (PVD), stroke, and CAD. Diabetes also affects the heart muscle, causing both systolic and diastolic heart failure.

The etiology of this excess cardiovascular morbidity and mortality is not completely clear (Dokken, 2008). Evidence suggests that although hyperglycemia, the hallmark of diabetes, contributes to myocardial damage after ischemic events, it is clearly not the only factor, because both pre-diabetes and the presence of the MetS, even in normoglycemic patients, increase the risk of most types of CVD (Alexander, 2003; Dokken, 2008). In diabetes, where CVD is of particular concern, there are multiple sources of ROS including the auto-oxidation of glucose, increased substrate flux, and decreased levels of NADPH through the polyol pathway. Formation of advanced glycation end products (AGEs) and their interaction with cellular targets, such as endothelial cells, may lead to oxidative stress and promote

The recent explosion of the worldwide epidemic of MetS combining disturbances in glucose and insulin metabolism, excess predominantly abdominally distributed weight, mild dyslipidemia, and hypertension, with the subsequent development of obesity, T2D and CVD, compromises progress made in reducing the morbidity and mortality of CVD in recent years. Cardiovascular risk increases in parallel to insulin resistance (as estimated by the homeostasis model assessment index (HOMA) both patients with diabetes and

The incidence of CVD, coronary heart disease, and T2D has not been well defined in persons with the MetS. Conclusions were that MetS is common and is associated with an increased risk for CVD and T2D in both sexes, according to metabolic syndrome traits. MetS accounts for up to one third of CVD in men and approximately half of new T2D over 8 years of

A large family study of T2D in Finland and Sweden (the Botnia study) were included in the analysis of cardiovascular risk associated with the MetS. The aim of the study was to assess the prevalence of cardiovascular morbidity and mortality associated with the MetS by applying the WHO denition. In women and men, respectively, the MetS was seen in 10 and 15% of subjects with normal glucose tolerance, 42 and 64% of those with impaired fasting glucose (IFG)/impaired glucose tolerance (IGT), and 78 and 84% of those with T2D. Cardiovascular mortality was markedly increased in subjects with the MetS (12.0 vs. 2.2%, *p< 0.001)*. Of the individual components of the MetS, microalbuminuria conferred the

strongest risk of cardiovascular death (RR 2.80; *p< 0.002)* (Isomaa et al., 2001).

myocardium, resulting in CHD (Woods et al., 2000).

formation of oxidized LDL (ox-LDL) (Ceriello & Motz, 2004).

nondiabetic (Saely et al., 2005).

follow-up (Wilson et al., 2005).

**2.3.2 Metabolic syndrome** 

**2.3.1 Diabetes** 

Obesity has been increasing in epidemic proportions in both adults and children. In adults, overweight is dened as a BMI 25 to 29.9 Kg/m2 and obesity as BMI ≥30 Kg/m2. Other indexes that have been used less commonly but possibly with more predictive power include body fatness, waist circumference (WC), waist-to-hip ratio (WHR), and weight-toheight ratio. A recent study of nearly 360,000 participants from 9 European countries showed that both general obesity and abdominal adiposity are associated with risk of death and support the importance of WC or WHR in addition to BMI for assessing mortality risk. Obesity has many adverse effects on hemodynamics and CV structure and function (Lavie et al., 2009).

Elevated BMI predisposes to congestive heart failure (CHF) by promoting increased blood pressure, diabetes, and CHD. Factors related to obesity and hypertension, include: endothelial dysfunction, insulin resistance, sympathetic nervous system, substances released from adipocytes (IL-6, TNF-, etc.), and sleep apnea (Poirier et al., 2006).

The role of obesity in the initiation and acceleration of tissue inflammation has been well studied. Excess adipose tissue can contribute to inflammation in two ways: (a) ectopic fat storage induces lipotoxicity, promoting an intracellular inflammatory response and (b) altered adipokine production in obesity contributes to the inflammatory response. It is now recognized that adiponectin has a role in both of these processes. Related to demonstrated association between hypo-adiponectinaemia and metabolic dysfunction (Cnop et al., 2003) the proposal provides that a replacement of adiponectin may function as pharmacological therapy (Chandran et al., 2003).

#### **2.3.4 Endothelial dysfunction**

Healthy endothelium regulates blood vessel tone, platelet activation, leukocyte adhesion, thrombogenesis, and inflammation. The net effect of healthy endothelium is vasodilatory, anti-atherogenic, and anti-inflammatory (Dokken, 2008). Endothelial dysfunction has been observed in patients with established coronary artery disease or coronary risk factors, both in the coronary and peripheral vasculature (Heitzer et al., 2001).

As shown in figure 1, endothelial dysfunction a key factor in atherogenesis, is associated with an increased risk of cardiovascular events and highest risk for vascular morbidity and mortality. A major risk for atherosclerotic plaque rupture is aging. One possible mechanism is that aging is associated with endothelial cell senescence, which is a risk factor for endothelial apoptosis and endothelial denudation, rendering the atherosclerotic plaque prone to rupture (Hulsmans et al., 2011).

A primary event in atherogenesis is the inltration of activated inammatory cells into the arterial wall. ROS can be produced from both endogenous and exogenous substances. Potential endogenous sources include mitochondria, cytochrome P450 metabolism, peroxisomes, and inflammatory cell activation. In the vascular wall, ROS are generated by several mechanisms, including NADPH oxidases, xanthine oxidase, the mitochondrial respiratory chain, lipoxygenases, and nitric oxide synthases. ROS formation can be stimulated by mechanical forces (e.g., stretch, pressure, shear stress), environmental factors (such as hypoxia), secreted factors coupled to tyrosine kinase receptors (e.g., platelet derived

Adipocytokines, Oxidative Stress and Impaired Cardiovascular Functions 93

Hypertriglyceridemia can lead to increased production of the small, dense form of LDL and to decrease HDL transport of cholesterol back to the liver (Poirier et al., 2006). In addition to the characteristic pattern of increased triglycerides and decreased HDL cholesterol found in the plasma of patients with diabetes, abnormalities are seen in the structure of the lipoprotein particles, where the predominant form of LDL cholesterol is the small, dense form. Small LDL particles are more atherogenic than large LDL particles because they can more easily penetrate and form stronger attachments to the arterial wall, and they are more susceptible to oxidation (Stocker & Keaney, 2004). In diabetic patients, LDL particles can also become glycated, in a process similar to the glycation of hemoglobin. Glycation of LDL lengthens its half-life and therefore increases the ability of the LDL to promote atherogenesis

Atherosclerosis is no longer considered a pure lipid disorder. It has become increasingly clear that inflammation is at the root of atherosclerosis and its complications. In addition to playing a causal role in lesion formation, inflammation can yield predictive and prognostic information of considerable clinical utility. In addition to serving as biomarkers of atherosclerotic events, inflammatory mediators directly participate in lesion formation, propagation, and eventual rupture and in this fashion may represent a powerful tool to assess endothelial cell activation. Clearly, understanding the mechanisms and mediators of endothelial dysregulation and inflammation may yield new targets to predict, prevent, and

Many common conditions predisposing to atherosclerosis, such as hypercholesterolemia, hypertension, diabetes, and smoking, are associated with a reduced vascular availability of NO, a free radical that not only produces vasodilation but also has potent antiatherogenic properties, such as inhibition of platelet aggregation, prevention of smooth muscle cell proliferation, reduction of lipid peroxidation, and inhibition of adhesion molecule expression (Landmesser & Harrison, 2001). Impaired endothelium-dependent vasodilation, a surrogate for NO bioavailability, may predict cardiovascular events. Thus, the loss of NO not only alters vascular tone but also may explaining in part why these conditions are risk

**3. Reactive oxygen species and oxidative stress in cardiovascular diseases**  Oxidative stress (OS) is an imbalance between production and degradation of ROS in cells, leading eventually to enhanced oxidative modification of biomolecules. Therefore, is a phenomenon associated with pathogenetic mechanisms of several diseases including atherosclerosis, cancer, diabetes mellitus, heart failure, hypertension, inflammatory diseases, as well as psychological diseases or aging processes (Naito et al., 2010). An increase in ROS and/or a weakening in the antioxidant defense mechanisms can cause OS. Accumulating evidence suggests that OS increases with age, and that therapeutic and life style approaches that reduce oxidative stress likely slow the development of atherosclerotic cardiovascular disease. Increased cellular ROS is an important contributor to the pathophysiology of vascular diseases, including atherosclerosis, restenosis, myocardial infarction and stroke. Additionally, some ROS act as intracellular messengers, and ROS accumulation activates

(Dokken, 2008).

**2.3.6 Atherosclerosis** 

factors for atherosclerosis.

treat cardiovascular disease (Szmitko, 2003).

growth factor, PDGF), and secreted factors coupled to G protein-coupled receptors such as angiotensin II (Lehoux et al., 2006; Dokken, 2008; Hulsmans et al., 2011).

The general process of lipid peroxidation consists of three stages: initiation, propagation, and termination (Catalá, 2006). The initiation phase of lipid peroxidation includes hydrogen atom abstraction. Several species can abstract the first hydrogen atom and include the radicals: hydroxyl (−OH), alkoxyl (RO−), peroxyl (ROO−), and possibly HO2− but not H2O2 or O2−. The membrane lipids, mainly phospholipids, containing polyunsaturated fatty acids are predominantly susceptible to peroxidation because abstraction from a methylene (CH2-) group of a hydrogen atom, which contains only one electron, leaves at the back an unpaired electron on the carbon, CH-. The presence of a double bond in the fatty acid weakens the C–H bonds on the carbon atom nearby to the double bond and thus facilitates H- subtraction. The initial reaction of -OH with polyunsaturated fatty acids produces a lipid radical (L-), which in turn reacts with molecular oxygen to form a lipid peroxyl radical (LOO−).There they secrete ROS and oxidize lipoproteins, inducing foam cell formation and endothelial cell apoptosis, which in turn lead to plaque growth, erosion, and rupture (Hulsmans et al., 2011).

It is now widely recognized that chronic low-grade inammation and oxidative stress play a key role in the initiation, propagation, and development of metabolic disorders. The aim of Hulsmans et al., (2011), was to review the functional roles of various microRNAs (miRs) in regulating oxidative stress and inammation in adipose and vascular tissues leading to obesity and atherosclerosis, in order to analyze how these processes can be linked through communication between cells even at a remarkable distance, thus highlighting the communication between inammatory and endothelial cells. The work of Targonski et al., was performed to evaluate the magnitude of the association between coronary endothelial dysfunction (CED) and cerebrovascular events. Kaplan-Meier analysis indicated that patients with CED had a significantly higher cumulative cerebrovascular event rate than those without CED (*P*=0.04). Presence of CED in patients without obstructive CAD is independently associated with an increased risk of cerebrovascular events (Targonski et al., 2003).

#### **2.3.5 Dyslipidemia**

The major threat to the macrovasculature for patients with and without diabetes is atherosclerosis, and dyslipidemia is highly correlated with atherosclerosis, up to 97% of patients with diabetes are dyslipidemic (Dokken, 2008). Insulin deficiency and insulin resistance promote dyslipidemia accompanied by increased oxidation, glycosylation, and triglyceride enrichment of lipoproteins.

Nonenzymatic glycosylation of HDL shortens its half-life and renders it less protective against atherosclerosis (Duell, 1991). The study of Marsuki et al., was undertaken to evaluate the effect, on macrophage cholesterol efflux, of functional modification of HDL by its glycation. They also investigated the effects of the glycation-inhibitors, metformin (MF) and aminoguanidine (AG), on glycated HDL-mediated cholesterol efflux. The conclusion was that glycated HDL particles are ineffective as acceptors of ATP-binding cassette transporter (ABCG1) mediated cholesterol efflux; and this may explain, at least in part, accelerated atherosclerosis in diabetic patients. Metformin serves as a possible candidate to restore impaired cholesterol efflux and reverse cholesterol transport (Matsuki et al., 2009).

Hypertriglyceridemia can lead to increased production of the small, dense form of LDL and to decrease HDL transport of cholesterol back to the liver (Poirier et al., 2006). In addition to the characteristic pattern of increased triglycerides and decreased HDL cholesterol found in the plasma of patients with diabetes, abnormalities are seen in the structure of the lipoprotein particles, where the predominant form of LDL cholesterol is the small, dense form. Small LDL particles are more atherogenic than large LDL particles because they can more easily penetrate and form stronger attachments to the arterial wall, and they are more susceptible to oxidation (Stocker & Keaney, 2004). In diabetic patients, LDL particles can also become glycated, in a process similar to the glycation of hemoglobin. Glycation of LDL lengthens its half-life and therefore increases the ability of the LDL to promote atherogenesis (Dokken, 2008).

#### **2.3.6 Atherosclerosis**

92 Oxidative Stress and Diseases

growth factor, PDGF), and secreted factors coupled to G protein-coupled receptors such as

The general process of lipid peroxidation consists of three stages: initiation, propagation, and termination (Catalá, 2006). The initiation phase of lipid peroxidation includes hydrogen atom abstraction. Several species can abstract the first hydrogen atom and include the radicals: hydroxyl (−OH), alkoxyl (RO−), peroxyl (ROO−), and possibly HO2− but not H2O2 or O2−. The membrane lipids, mainly phospholipids, containing polyunsaturated fatty acids are predominantly susceptible to peroxidation because abstraction from a methylene (CH2-) group of a hydrogen atom, which contains only one electron, leaves at the back an unpaired electron on the carbon, CH-. The presence of a double bond in the fatty acid weakens the C–H bonds on the carbon atom nearby to the double bond and thus facilitates H- subtraction. The initial reaction of -OH with polyunsaturated fatty acids produces a lipid radical (L-), which in turn reacts with molecular oxygen to form a lipid peroxyl radical (LOO−).There they secrete ROS and oxidize lipoproteins, inducing foam cell formation and endothelial cell apoptosis,

angiotensin II (Lehoux et al., 2006; Dokken, 2008; Hulsmans et al., 2011).

which in turn lead to plaque growth, erosion, and rupture (Hulsmans et al., 2011).

associated with an increased risk of cerebrovascular events (Targonski et al., 2003).

The major threat to the macrovasculature for patients with and without diabetes is atherosclerosis, and dyslipidemia is highly correlated with atherosclerosis, up to 97% of patients with diabetes are dyslipidemic (Dokken, 2008). Insulin deficiency and insulin resistance promote dyslipidemia accompanied by increased oxidation, glycosylation, and

Nonenzymatic glycosylation of HDL shortens its half-life and renders it less protective against atherosclerosis (Duell, 1991). The study of Marsuki et al., was undertaken to evaluate the effect, on macrophage cholesterol efflux, of functional modification of HDL by its glycation. They also investigated the effects of the glycation-inhibitors, metformin (MF) and aminoguanidine (AG), on glycated HDL-mediated cholesterol efflux. The conclusion was that glycated HDL particles are ineffective as acceptors of ATP-binding cassette transporter (ABCG1) mediated cholesterol efflux; and this may explain, at least in part, accelerated atherosclerosis in diabetic patients. Metformin serves as a possible candidate to restore

impaired cholesterol efflux and reverse cholesterol transport (Matsuki et al., 2009).

**2.3.5 Dyslipidemia** 

triglyceride enrichment of lipoproteins.

It is now widely recognized that chronic low-grade inammation and oxidative stress play a key role in the initiation, propagation, and development of metabolic disorders. The aim of Hulsmans et al., (2011), was to review the functional roles of various microRNAs (miRs) in regulating oxidative stress and inammation in adipose and vascular tissues leading to obesity and atherosclerosis, in order to analyze how these processes can be linked through communication between cells even at a remarkable distance, thus highlighting the communication between inammatory and endothelial cells. The work of Targonski et al., was performed to evaluate the magnitude of the association between coronary endothelial dysfunction (CED) and cerebrovascular events. Kaplan-Meier analysis indicated that patients with CED had a significantly higher cumulative cerebrovascular event rate than those without CED (*P*=0.04). Presence of CED in patients without obstructive CAD is independently Atherosclerosis is no longer considered a pure lipid disorder. It has become increasingly clear that inflammation is at the root of atherosclerosis and its complications. In addition to playing a causal role in lesion formation, inflammation can yield predictive and prognostic information of considerable clinical utility. In addition to serving as biomarkers of atherosclerotic events, inflammatory mediators directly participate in lesion formation, propagation, and eventual rupture and in this fashion may represent a powerful tool to assess endothelial cell activation. Clearly, understanding the mechanisms and mediators of endothelial dysregulation and inflammation may yield new targets to predict, prevent, and treat cardiovascular disease (Szmitko, 2003).

Many common conditions predisposing to atherosclerosis, such as hypercholesterolemia, hypertension, diabetes, and smoking, are associated with a reduced vascular availability of NO, a free radical that not only produces vasodilation but also has potent antiatherogenic properties, such as inhibition of platelet aggregation, prevention of smooth muscle cell proliferation, reduction of lipid peroxidation, and inhibition of adhesion molecule expression (Landmesser & Harrison, 2001). Impaired endothelium-dependent vasodilation, a surrogate for NO bioavailability, may predict cardiovascular events. Thus, the loss of NO not only alters vascular tone but also may explaining in part why these conditions are risk factors for atherosclerosis.

#### **3. Reactive oxygen species and oxidative stress in cardiovascular diseases**

Oxidative stress (OS) is an imbalance between production and degradation of ROS in cells, leading eventually to enhanced oxidative modification of biomolecules. Therefore, is a phenomenon associated with pathogenetic mechanisms of several diseases including atherosclerosis, cancer, diabetes mellitus, heart failure, hypertension, inflammatory diseases, as well as psychological diseases or aging processes (Naito et al., 2010). An increase in ROS and/or a weakening in the antioxidant defense mechanisms can cause OS. Accumulating evidence suggests that OS increases with age, and that therapeutic and life style approaches that reduce oxidative stress likely slow the development of atherosclerotic cardiovascular disease. Increased cellular ROS is an important contributor to the pathophysiology of vascular diseases, including atherosclerosis, restenosis, myocardial infarction and stroke. Additionally, some ROS act as intracellular messengers, and ROS accumulation activates

Adipocytokines, Oxidative Stress and Impaired Cardiovascular Functions 95

oxidation of glucose, increased substrate flux, and decreased levels of NADPH through the polyol pathway. Formation of AGEs products and their interaction with cellular targets, such as endothelial cells, may lead to oxidative stress and promote formation of oxidized

Increased production of oxygen-derived free radicals such as the superoxide anion has been linked to impaired endothelial vasomotor function in experimental models of atherosclerosis. Accordingly, treatment with antioxidants has been shown to improve coronary and peripheral endothelial function in patients with CAD or coronary risk factors (Heitzer et al., 2001). Mechanisms that contribute to this selective cardiac dysfunction include decreases in energy production due to reductions in mitochondrial respiration, increased oxidative stress, and defective contractile and intracellular "Ca2+" regulatory proteins. Changes in mitochondrial biogenesis and function have been documented in the metabolic syndrome and diabetes. Alterations in mitochondrial biogenesis as well as mitochondrial content and function provoke a heterogeneous group of CVD risk factors that constitute the metabolic syndrome. (Ren et al., 2010). It is increasingly recognized that important aspects of mitochondrial dysfunction that contribute to CVDs are induction of apoptosis and changes in mitochondrial morphology under the influence of oxidative stress. Finally, inefficient mitochondrial oxidative phosphorylation/biogenesis and increases in oxidative stress appear to be overarching abnormalities contributing to cardiac diastolic

function, the hallmark of metabolic cardiomyopathy (Ren et al., 2010).

**4. Adipocytokines and the metabolic complications of obesity** 

lipid and glucose metabolism, inflammation, and atherosclerosis (see figure 1).

IL1R1 (IL-1R; production of which is proportional to body weight).

During positive caloric balance there are two factors important for the development of metabolic disease. First one is a type of the fat accumulation, i.e., due to increase in size (hypertrophy) or in number (hyperplasia) of fat cells. The next factor is a place of fat storage, i.e. subcutaneous (SC) or visceral (Vis) fat (Wajchenberg, 2000; Bays et al., 2008). In humans, white adipose tissue (WAT) produces over 50 'adipokines', including TNF- which contributes to the low-grade inflammation found in obesity, leptin which has effects on food intake, and a host of other agents with a variety of effects (Lago et al., 2007). In parallel with these proinflammatory events, WAT also produces anti-inflammatory cytokines such as adiponectin (which, paradoxically, tends to be lower in obese individuals) and IL-10 and

**4.1 Fat depots, adipocitokines and their relation to the human metabolic syndrome**  Adipose tissue is composed of adipocytes embedded in a loose connective tissue meshwork containing adipocyte precursors, fibroblasts, immune cells, and various other cell types. Adipose tissue was traditionally considered an energy storage depot with few interesting attributes. However, adipocytes express and secrete a variety of products known as 'adipokines', including leptin, adiponectin, resistin and visfatin, as well as cytokines and chemokines such as TNF-, IL-6 and monocyte chemoattractant protein (Antuna-Puente et al., 2008) and due to the dramatic rise in obesity and its metabolic sequelae during the past decades, adipose tissue gained tremendous scientific interest. It is now regarded as an active endocrine organ that, in addition to regulating fat mass and nutrient homeostasis, releases a large number of bioactive mediators (adipokines) modulating hemostasis, blood pressure,

LDL (Ceriello & Motz, 2004).

proinflammatory signaling pathways with an increased propensity for the formation of atherosclerotic lesions within the vessel wall (Runge et al., 2010).

The antioxidant enzymes superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx) serve as primary line of defense in destroying free radicals. However, there are several human antioxidant genes, classified according to genes whose products are defined as "antioxidant enzymes" the first two groups, and genes whose products are not enzymes, but also deal directly with reactive species. Also, are subclassified into 3 functional groups: peroxidases: catalase, ceruloplasmin (ferroxidase), glutathione peroxidase 1-7, lactoperoxidase, myeloperoxidase, peroxiredoxin 1-6; superoxide dismutases: copper chaperone for superoxide dismutase, superoxide dismutase 1, soluble (amyotrophic lateral sclerosis 1 (adult), superoxide dismutase 2, mitochondrial, superoxide dismutase 3, extracellular and thiol redox proteins: glutaredoxin (thioltransferase), glutaredoxin 2,3,5, glutathione reductase,methionine sulfoxide reductase A, metallothionein 1A, 1B, 1E,1F,1G,1H,1M,1X,2A, protein disulfide isomerase family A, member 6, selenoprotein P, plasma, 1, sulfiredoxin 1 homolog (S. cerevisiae), thioredoxin, thioredoxin 2, thioredoxin domain containing 1,2,3,4,5,6,8,9,10,11,12,13,14,17, thioredoxin interacting protein, thioredoxin-like 1, 4A, 4B, thioredoxin reductase 1,2,3 (Dusting & Triggle, 2005).

An elevation of ROS may cause CVD due the overproduction of superoxide anion (O2•-). This overproduction is detrimental, because of the rapid interaction of O2•- with NO, which leads to the loss of NO bioavailability and increase in the production of peroxynitrite (ONOO−). A subsequent reduction in the vascular effects of NO, as well as a reduction in the antiatherogenic effects of NO, as a consequence will compromise cardiovascular function. An elevation of O2•- will also lead to the oxidation of the important co-factor in the regulation of nitric oxide synthase, tetrahydrobiopterin (BH4), and this will lead to an "uncoupled eNOS", which will then synthesize O2•- rather than NO (Dusting & Triggle, 2005).

The term ROS refers to a subset of molecules called "free radicals", however there are some ROS which are not free radicals, such as hydrogen peroxide. This term refers to any molecule that contains an unpaired electron in the outer orbital. This unpaired electron makes the molecule highly reactive that leads to the formation of bonds between the ROS and other compounds (Dokken, 2008). Unpaired electron makes the molecule highly reactive, seeking to either donate an electron to another compound or take up protons from another compound to obtain a stable electron pair. These free radicals include superoxide anion (O2•-), hydroxyl radical (•OH), and the free radical form of nitric oxide (•NO). Other members of the ROS family include hydrogen peroxide (H2O2) and peroxynitrite (ONOO−) (Dusting & Triggle, 2005). On the other hand, several enzyme systems are known to be sources of ROS including the mitochondrial respiratory chain, xanthine oxidase, NADPH oxidase, cyclooxygenase, cytochrome P450, and uncoupled eNOS. Mitochondria are the source of ROS. There is also growing evidence that NADPH-oxidase is a major source of vascular superoxide production.

The high reactivity of free radicals leads to the formation of bonds between the ROS and other compounds, altering the structure and function of the tissue. Because of the reactive propensity of these molecules, ROS can directly damage a number of cell components, such as plasma membranes and organelles (Dokken, 2008). In diabetes, where cardiovascular disease is of particular concern, there are multiple sources of ROS including the auto-

proinflammatory signaling pathways with an increased propensity for the formation of

The antioxidant enzymes superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx) serve as primary line of defense in destroying free radicals. However, there are several human antioxidant genes, classified according to genes whose products are defined as "antioxidant enzymes" the first two groups, and genes whose products are not enzymes, but also deal directly with reactive species. Also, are subclassified into 3 functional groups: peroxidases: catalase, ceruloplasmin (ferroxidase), glutathione peroxidase 1-7, lactoperoxidase, myeloperoxidase, peroxiredoxin 1-6; superoxide dismutases: copper chaperone for superoxide dismutase, superoxide dismutase 1, soluble (amyotrophic lateral sclerosis 1 (adult), superoxide dismutase 2, mitochondrial, superoxide dismutase 3, extracellular and thiol redox proteins: glutaredoxin (thioltransferase), glutaredoxin 2,3,5, glutathione reductase,methionine sulfoxide reductase A, metallothionein 1A, 1B, 1E,1F,1G,1H,1M,1X,2A, protein disulfide isomerase family A, member 6, selenoprotein P, plasma, 1, sulfiredoxin 1 homolog (S. cerevisiae), thioredoxin, thioredoxin 2, thioredoxin domain containing 1,2,3,4,5,6,8,9,10,11,12,13,14,17, thioredoxin interacting protein,

thioredoxin-like 1, 4A, 4B, thioredoxin reductase 1,2,3 (Dusting & Triggle, 2005).

which will then synthesize O2•- rather than NO (Dusting & Triggle, 2005).

vascular superoxide production.

An elevation of ROS may cause CVD due the overproduction of superoxide anion (O2•-). This overproduction is detrimental, because of the rapid interaction of O2•- with NO, which leads to the loss of NO bioavailability and increase in the production of peroxynitrite (ONOO−). A subsequent reduction in the vascular effects of NO, as well as a reduction in the antiatherogenic effects of NO, as a consequence will compromise cardiovascular function. An elevation of O2•- will also lead to the oxidation of the important co-factor in the regulation of nitric oxide synthase, tetrahydrobiopterin (BH4), and this will lead to an "uncoupled eNOS",

The term ROS refers to a subset of molecules called "free radicals", however there are some ROS which are not free radicals, such as hydrogen peroxide. This term refers to any molecule that contains an unpaired electron in the outer orbital. This unpaired electron makes the molecule highly reactive that leads to the formation of bonds between the ROS and other compounds (Dokken, 2008). Unpaired electron makes the molecule highly reactive, seeking to either donate an electron to another compound or take up protons from another compound to obtain a stable electron pair. These free radicals include superoxide anion (O2•-), hydroxyl radical (•OH), and the free radical form of nitric oxide (•NO). Other members of the ROS family include hydrogen peroxide (H2O2) and peroxynitrite (ONOO−) (Dusting & Triggle, 2005). On the other hand, several enzyme systems are known to be sources of ROS including the mitochondrial respiratory chain, xanthine oxidase, NADPH oxidase, cyclooxygenase, cytochrome P450, and uncoupled eNOS. Mitochondria are the source of ROS. There is also growing evidence that NADPH-oxidase is a major source of

The high reactivity of free radicals leads to the formation of bonds between the ROS and other compounds, altering the structure and function of the tissue. Because of the reactive propensity of these molecules, ROS can directly damage a number of cell components, such as plasma membranes and organelles (Dokken, 2008). In diabetes, where cardiovascular disease is of particular concern, there are multiple sources of ROS including the auto-

atherosclerotic lesions within the vessel wall (Runge et al., 2010).

oxidation of glucose, increased substrate flux, and decreased levels of NADPH through the polyol pathway. Formation of AGEs products and their interaction with cellular targets, such as endothelial cells, may lead to oxidative stress and promote formation of oxidized LDL (Ceriello & Motz, 2004).

Increased production of oxygen-derived free radicals such as the superoxide anion has been linked to impaired endothelial vasomotor function in experimental models of atherosclerosis. Accordingly, treatment with antioxidants has been shown to improve coronary and peripheral endothelial function in patients with CAD or coronary risk factors (Heitzer et al., 2001). Mechanisms that contribute to this selective cardiac dysfunction include decreases in energy production due to reductions in mitochondrial respiration, increased oxidative stress, and defective contractile and intracellular "Ca2+" regulatory proteins. Changes in mitochondrial biogenesis and function have been documented in the metabolic syndrome and diabetes. Alterations in mitochondrial biogenesis as well as mitochondrial content and function provoke a heterogeneous group of CVD risk factors that constitute the metabolic syndrome. (Ren et al., 2010). It is increasingly recognized that important aspects of mitochondrial dysfunction that contribute to CVDs are induction of apoptosis and changes in mitochondrial morphology under the influence of oxidative stress. Finally, inefficient mitochondrial oxidative phosphorylation/biogenesis and increases in oxidative stress appear to be overarching abnormalities contributing to cardiac diastolic function, the hallmark of metabolic cardiomyopathy (Ren et al., 2010).

#### **4. Adipocytokines and the metabolic complications of obesity**

#### **4.1 Fat depots, adipocitokines and their relation to the human metabolic syndrome**

Adipose tissue is composed of adipocytes embedded in a loose connective tissue meshwork containing adipocyte precursors, fibroblasts, immune cells, and various other cell types. Adipose tissue was traditionally considered an energy storage depot with few interesting attributes. However, adipocytes express and secrete a variety of products known as 'adipokines', including leptin, adiponectin, resistin and visfatin, as well as cytokines and chemokines such as TNF-, IL-6 and monocyte chemoattractant protein (Antuna-Puente et al., 2008) and due to the dramatic rise in obesity and its metabolic sequelae during the past decades, adipose tissue gained tremendous scientific interest. It is now regarded as an active endocrine organ that, in addition to regulating fat mass and nutrient homeostasis, releases a large number of bioactive mediators (adipokines) modulating hemostasis, blood pressure, lipid and glucose metabolism, inflammation, and atherosclerosis (see figure 1).

During positive caloric balance there are two factors important for the development of metabolic disease. First one is a type of the fat accumulation, i.e., due to increase in size (hypertrophy) or in number (hyperplasia) of fat cells. The next factor is a place of fat storage, i.e. subcutaneous (SC) or visceral (Vis) fat (Wajchenberg, 2000; Bays et al., 2008). In humans, white adipose tissue (WAT) produces over 50 'adipokines', including TNF- which contributes to the low-grade inflammation found in obesity, leptin which has effects on food intake, and a host of other agents with a variety of effects (Lago et al., 2007). In parallel with these proinflammatory events, WAT also produces anti-inflammatory cytokines such as adiponectin (which, paradoxically, tends to be lower in obese individuals) and IL-10 and IL1R1 (IL-1R; production of which is proportional to body weight).

Adipocytokines, Oxidative Stress and Impaired Cardiovascular Functions 97

Several studies have linked hypoadiponectinemia to diabetes (Kern et al., 2003), hypertension (Kim et al., 2007), atherosclerosis, and endothelial dysfunction (Chow et al., 2007). More recent studies have shown that the high-molecular weight (HMW) oligomer is inversely associated with the risk for diabetes independent of total adiponectin (Kadowaki et al., 2007), and the HMW oligomer is responsible for the association of adiponectin with traits of metabolic syndrome (Heidemann et al., 2008; Lara-Castro et al., 2006). On the other hand, adiponectin improves insulin sensitivity by increasing energy expenditure and fatty acid oxidation through activation of AMPK, and by increasing the expression of PPAR target genes such as CD36, acyl-coenzyme oxidase, and uncoupling protein-2 (Kadowaki et al., 2007). Alternatively, adiponectin may lead to an improved metabolic profile by the expansion of SC adipose tissue with decreased levels of macrophage infiltration (Nawrocki et al., 2006), similar to the actions of peroxisome proliferator-activated receptor (PPAR)-γ agonists; reduction of lipotoxicity and inflammation associated with obesity (Wang et al., 2007), and adiponectin has also had vasculoprotective effects mediated via an increase in endothelial nitric oxide production, or modulation of expression of adhesion molecules and

In addition, work in experimental models has shown that adiponectin mediates beneficial actions in cardiovascular and metabolic-associated diseases (Sam & Walsh, 2010). For example, in mouse models, adiponectin modulates hypertrophic signals in the heart and exhibits direct anti-hypertrophic properties; in addition to improving vascular function and pathological remodeling (Antuna-Puente et al., 2008); the hypoadiponectinemia might be observed in subjects with hypertension and other cardiovascular diseases and could be a useful pharmacologic tool to improve membrane microviscosity in hypertension, via the NO

It has been demonstrated that plasma adiponectin levels increased during weight reduction or blockade of the rennin angiotensin system indicating that adiponectin might be benecial for preventing the development of atherosclerotic changes. The results of Kurata et al. indicate that blockade of Angiotensin II receptor ameliorates adipocytokine dysregulation and that such action is mediated, at least in part, by targeting oxidative stress in obese adipose tissue.

Resistin is a 12-kDa peptide that was originally discovered as a result of examining differential gene expression of mouse adipose tissue after thiazolidinediones (TZD) treatment (Steppan et al., 2001). The thiazolidinediones a class of drugs that work through PPARγ agonism, are insulin sensitizers and have been shown to improve cardiac risk factors and decrease cardiovascular events; may potentially correct the inflammatory disarray, endothelial dysfunction, dyslipidemia, and plaque vulnerability associated with diabetic cardiovascular disease through their effects on insulin resistance and fat metabolism. Resistin was decreased by TZD treatment of mice and was increased in insulin-resistant mice. Furthermore, treatment with antiresistin antibody improved insulin sensitivity and glucose transport in mice and mouse adipocytes, respectively (Steppan et al., 2001). Additional studies in mice suggest that an important site of action of resistin is on hepatic glucose production (Rajala et al., 2003). Therefore, resistin is clearly an important adipokine that likely plays a role in the development of insulin resistance; however, it appears to be

scavenger receptors (Chow et al., 2007; Zhu et al., 2008).

quantitatively less important in humans than other adipokines.

dependent mechanisms (Tsuda, 2011).

**4.4 Resistin** 

#### **4.2 Leptin**

Hyperleptinaemia is common in obesity and reflects increased adiposity and leptin resistance. Nevertheless, leptin resistance may not be complete as several actions of leptin, such as cardiovascular sympatho-activation, might be preserved in obese subjects known to be resistant to the metabolic effects of leptin (i.e. selective leptin resistance). Notably, the renal and sympathetic actions of leptin may play an important role in the pathogenesis of hypertension related to obesity and metabolic syndrome. Furthermore, the lipotoxic effect of leptin resistance may cause insulin resistance and β cell dysfunction, increasing the risk of T2D. Leptin has also been shown to possess proliferative, pro-inflammatory, prothrombotic, and pro-oxidative actions (Buettner et al., 2006).

#### **4.3 Adiponectin**

Adiponectin, referred to as adipocyte complement-related protein of 30 kDa (ACRP30), is a protein secreted from adipocytes (Correia & Rahmouni, 2006), that is abundantly present in plasma (Scherer et al., 1995; Berg et al., 2001). It is now well established that adiponectin has potent salutary actions on peripheral insulin sensitivity, and circulating adiponectin levels are reduced in obesity, insulin resistance and T2D (Scherer et al., 1995; Kern et al., 2003). Mice lacking adiponectin have reduced insulin sensitivity (Weyer et al., 2001; Kubota et al., 2002; Maeda et al., 2002); in contrast, adiponectin overexpression in ob/ob mice, confers dramatic metabolic improvements, e.g., in various mouse models, Holland et al. (2011) show that the insulin-sensitizing and antiapoptotic actions of adiponectin are partly related to its effects on sphingolipid metabolism, providing a new unifying mechanism for the pleiotropic beneficial actions of adiponectin. Adiponectin stimulates the cellular activity of ceramidase, which removes the fatty acyl chain from ceramides. This liberates sphingosine, which can subsequently be phosphorylated by sphingosine kinases to generate the antiapoptotic metabolite sphingosine-1-phosphate (S1P). Furthermore, liver-specific overexpression of the adiponectin receptors, AdipoR1 and AdipoR2, increased hepatic ceramidase activity and, concomitantly, reduced hepatic ceramide content. These *in vivo*  models of varying adiponectin expression and AdipoR1 and R2 overexpression demonstrate a strong association between adiponectin levels, hepatic ceramide content and insulin sensitivity (Holland et al., 2011).

In the beta cell model of apoptosis, adiponectin protected against the development of hyperglycemia-a key feature of pancreatic insufficiency-by partially preserving beta cell mass and insulin content. Using mouse primary cardiomyocytes and a pancreatic beta cell line, Holland et al. (2011) showed that adiponectin prevents cell death induced by the saturated fatty acid palmitate and a short chain ceramide analog, C2-ceramide. Mechanistically, the insulin-sensitizing actions of adiponectin, which include enhanced glucose use and fatty acid oxidation (Yamauchi et al., 2003), inhibition of serine kinases that antagonize insulin signaling and enhanced mitochondrial biogenesis, are believed to occur via receptor-dependent activation of the 5′-AMP–activated protein kinase (AMPK). Intriguingly, it is known that adiponectin also exerts potent antiapoptotic effects and prevents myocardial apoptosis in response to ischemia-reperfusion injury (Shibata et al., 2005) and lipid-induced pancreatic beta cell apoptosis (Rakatzi et al., 2004).

Several studies have linked hypoadiponectinemia to diabetes (Kern et al., 2003), hypertension (Kim et al., 2007), atherosclerosis, and endothelial dysfunction (Chow et al., 2007). More recent studies have shown that the high-molecular weight (HMW) oligomer is inversely associated with the risk for diabetes independent of total adiponectin (Kadowaki et al., 2007), and the HMW oligomer is responsible for the association of adiponectin with traits of metabolic syndrome (Heidemann et al., 2008; Lara-Castro et al., 2006). On the other hand, adiponectin improves insulin sensitivity by increasing energy expenditure and fatty acid oxidation through activation of AMPK, and by increasing the expression of PPAR target genes such as CD36, acyl-coenzyme oxidase, and uncoupling protein-2 (Kadowaki et al., 2007). Alternatively, adiponectin may lead to an improved metabolic profile by the expansion of SC adipose tissue with decreased levels of macrophage infiltration (Nawrocki et al., 2006), similar to the actions of peroxisome proliferator-activated receptor (PPAR)-γ agonists; reduction of lipotoxicity and inflammation associated with obesity (Wang et al., 2007), and adiponectin has also had vasculoprotective effects mediated via an increase in endothelial nitric oxide production, or modulation of expression of adhesion molecules and scavenger receptors (Chow et al., 2007; Zhu et al., 2008).

In addition, work in experimental models has shown that adiponectin mediates beneficial actions in cardiovascular and metabolic-associated diseases (Sam & Walsh, 2010). For example, in mouse models, adiponectin modulates hypertrophic signals in the heart and exhibits direct anti-hypertrophic properties; in addition to improving vascular function and pathological remodeling (Antuna-Puente et al., 2008); the hypoadiponectinemia might be observed in subjects with hypertension and other cardiovascular diseases and could be a useful pharmacologic tool to improve membrane microviscosity in hypertension, via the NO dependent mechanisms (Tsuda, 2011).

It has been demonstrated that plasma adiponectin levels increased during weight reduction or blockade of the rennin angiotensin system indicating that adiponectin might be benecial for preventing the development of atherosclerotic changes. The results of Kurata et al. indicate that blockade of Angiotensin II receptor ameliorates adipocytokine dysregulation and that such action is mediated, at least in part, by targeting oxidative stress in obese adipose tissue.

#### **4.4 Resistin**

96 Oxidative Stress and Diseases

Hyperleptinaemia is common in obesity and reflects increased adiposity and leptin resistance. Nevertheless, leptin resistance may not be complete as several actions of leptin, such as cardiovascular sympatho-activation, might be preserved in obese subjects known to be resistant to the metabolic effects of leptin (i.e. selective leptin resistance). Notably, the renal and sympathetic actions of leptin may play an important role in the pathogenesis of hypertension related to obesity and metabolic syndrome. Furthermore, the lipotoxic effect of leptin resistance may cause insulin resistance and β cell dysfunction, increasing the risk of T2D. Leptin has also been shown to possess proliferative, pro-inflammatory, pro-

Adiponectin, referred to as adipocyte complement-related protein of 30 kDa (ACRP30), is a protein secreted from adipocytes (Correia & Rahmouni, 2006), that is abundantly present in plasma (Scherer et al., 1995; Berg et al., 2001). It is now well established that adiponectin has potent salutary actions on peripheral insulin sensitivity, and circulating adiponectin levels are reduced in obesity, insulin resistance and T2D (Scherer et al., 1995; Kern et al., 2003). Mice lacking adiponectin have reduced insulin sensitivity (Weyer et al., 2001; Kubota et al., 2002; Maeda et al., 2002); in contrast, adiponectin overexpression in ob/ob mice, confers dramatic metabolic improvements, e.g., in various mouse models, Holland et al. (2011) show that the insulin-sensitizing and antiapoptotic actions of adiponectin are partly related to its effects on sphingolipid metabolism, providing a new unifying mechanism for the pleiotropic beneficial actions of adiponectin. Adiponectin stimulates the cellular activity of ceramidase, which removes the fatty acyl chain from ceramides. This liberates sphingosine, which can subsequently be phosphorylated by sphingosine kinases to generate the antiapoptotic metabolite sphingosine-1-phosphate (S1P). Furthermore, liver-specific overexpression of the adiponectin receptors, AdipoR1 and AdipoR2, increased hepatic ceramidase activity and, concomitantly, reduced hepatic ceramide content. These *in vivo*  models of varying adiponectin expression and AdipoR1 and R2 overexpression demonstrate a strong association between adiponectin levels, hepatic ceramide content and insulin

In the beta cell model of apoptosis, adiponectin protected against the development of hyperglycemia-a key feature of pancreatic insufficiency-by partially preserving beta cell mass and insulin content. Using mouse primary cardiomyocytes and a pancreatic beta cell line, Holland et al. (2011) showed that adiponectin prevents cell death induced by the saturated fatty acid palmitate and a short chain ceramide analog, C2-ceramide. Mechanistically, the insulin-sensitizing actions of adiponectin, which include enhanced glucose use and fatty acid oxidation (Yamauchi et al., 2003), inhibition of serine kinases that antagonize insulin signaling and enhanced mitochondrial biogenesis, are believed to occur via receptor-dependent activation of the 5′-AMP–activated protein kinase (AMPK). Intriguingly, it is known that adiponectin also exerts potent antiapoptotic effects and prevents myocardial apoptosis in response to ischemia-reperfusion injury (Shibata et al.,

2005) and lipid-induced pancreatic beta cell apoptosis (Rakatzi et al., 2004).

thrombotic, and pro-oxidative actions (Buettner et al., 2006).

**4.2 Leptin** 

**4.3 Adiponectin** 

sensitivity (Holland et al., 2011).

Resistin is a 12-kDa peptide that was originally discovered as a result of examining differential gene expression of mouse adipose tissue after thiazolidinediones (TZD) treatment (Steppan et al., 2001). The thiazolidinediones a class of drugs that work through PPARγ agonism, are insulin sensitizers and have been shown to improve cardiac risk factors and decrease cardiovascular events; may potentially correct the inflammatory disarray, endothelial dysfunction, dyslipidemia, and plaque vulnerability associated with diabetic cardiovascular disease through their effects on insulin resistance and fat metabolism. Resistin was decreased by TZD treatment of mice and was increased in insulin-resistant mice. Furthermore, treatment with antiresistin antibody improved insulin sensitivity and glucose transport in mice and mouse adipocytes, respectively (Steppan et al., 2001). Additional studies in mice suggest that an important site of action of resistin is on hepatic glucose production (Rajala et al., 2003). Therefore, resistin is clearly an important adipokine that likely plays a role in the development of insulin resistance; however, it appears to be quantitatively less important in humans than other adipokines.

Adipocytokines, Oxidative Stress and Impaired Cardiovascular Functions 99

The vascular endothelium, located at the interface of blood and tissue, is able to sense changes in hemodynamic forces and blood borne signals and react by synthesizing and releasing vasoactive substances. Vascular homeostasis is maintained by a balance between endothelium-derived relaxing and contracting factors. With disruption of this balance, mediated by inflammatory and traditional cardiovascular risk factors, the vasculature becomes susceptible to atheroma formation. Inflammatory mediators appear to play a fundamental role in the initiation, progression, and eventual rupture of atherosclerotic

Endothelial dysfunction implies diminished production or availability of NO and/or an imbalance in the relative contribution of endothelium-derived relaxing and contracting factors, those included endothelin-1 (ET-1), angiotensin, and several oxidants. However, endothelial dysfunction, as assessed in terms of vasomotor dysfunction, can occur well before the structural manifestation of atherosclerosis and thus can serve as an independent

Hypercholesterolemia, traditional cardiovascular risk factor, promotes attachment of blood leukocytes to the endothelium. Oxidized low-density lipoprotein causes endothelial activation and changes its biological characteristics in part by reducing the intracellular

On the other hand, angiotensin II can induce the production of ROS, increase the expression of the proinflammatory cytokines as IL-6 and monocyte chemoattractant protein-1 (MCP-1), and upregulate VCAM-1 on ECs. High levels of CRP can also promote endothelial dysfunction by quenching the production of NO and diminishing its bioactivity (Verma, 2002). These endothelial modifications promote inflammation within the vessel wall, setting

Recent research has focused on the origin of the inflammatory markers in obesity and the extent to which adipose tissue has a direct effect. The production of adipokines by visceral adipose tissue is of particular interest since their local secretion by visceral fat depots may provide a novel mechanistic link between obesity and the associated vascular complications. Under conditions of inflammation associated with cardiovascular disease, as well as an increase in mobilization of fatty acids from adipose tissue, there is increased secretion of

The cardiometabolic benefits of adiponectin may be driven largely through improvements in vascular homeostasis, especially through improving endothelial function. Some studies have demonstrated impaired endothelial function in adiponectin-deficient mice (Teoh et al., 2008) demonstrated that adiponectin plays an important role to limit endothelial activation

predictor of future cardiovascular events (Behrendt & Ganz, 2002).

the stage for the initiation and progression of an atherosclerotic lesion.

pro-atherogenic, pro-inflammatory adipocytokines and chemokines.

**5. Inflammatory pathway activation and interactions with endothelial cells**

**5.1 Endothelial cells** 

**5.1.1 Endothelial dysfunction** 

concentration of NO (Cominacini, 2001).

**5.2 Adiponectin and inflammatory activation** 

plaques.

#### **4.5 Visfatin**

Visfatin is expressed in many cells and tissues, and was previously identified as a protein involved in B-cell maturation (pre-B colony enhancing factor) (Kitani et al., 2003; Samal et al., 1994). More recently, visfatin was described to be a highly expressed protein with insulin-like functions, and was predominantly found in visceral adipose tissue, from which the name visfatin was derived (Fukuhara et al., 2005). Injection of visfatin in mice lowered blood glucose, and mice with a mutation in visfatin, and nicotinamide adenine dinucleotide (NAD) biosynthetic activity ionotropy, which is essential for -cell function (Revollo et al., 2007). In human studies, a positive correlation between visceral adipose tissue visfatin gene expression and BMI was noted, along with a negative correlation between BMI and SC fat visfatin (Berndt et al., 2005; Varma et al., 2007), suggesting that visfatin regulation in these different depots is different, and adipose depot ratios are highly dependent on the obesity of the subjects. Variable results were obtained regarding the relationship between visfatin and diabetes or insulin resistance (Varma et al., 2007; Chen et al., 2006; Hammarstedt et al., 2005; Haider et al., 2006). Therefore, there are a number of inconsistencies among the different studies of visfatin, and the role of this adipokine in obesity and insulin resistance is not clear.

#### **4.6 Apelin**

Apelin is another short peptide released from adipocytes upon stimulation by e.g. insulin and the endogenous ligand of the human orphan G-protein-coupled APJ receptor. In line with this, plasma apelin levels are increased in obesity associated with insulin resistance and hyperinsulinemia (Beltowski, 2006). In the cardiovascular system, apelin elicits endothelium-dependent, nitric oxide-mediated vasorelaxation and in rodents, apelin also increases cardiac contractility *in vivo* (Ashley et al., 2005; Atluri et al., 2007) and causes a rapid fall in both arterial blood pressure and systemic venous tone (Tatemoto et al., 2001; Lee, 2005) with corresponding reductions in left ventricular afterload and preload (Ashley et al., 2005; Tatemoto et al., 2001).

Apelin-APJ system, expressed in the central nervous system and in a variety of peripheral tissues, is involved in the regulation of the immune response, brain signaling, hemodynamic homeostasis, vasodilatation, inotropy, angiogenesis and glucose metabolism (Sorli et al., 2006; Zhang et al., 2009). In the cardiovascular system, high expression of APJ mRNA has been observed in the heart (Zhang et al., 2009). Apelin expression is restricted to endothelial cells and negligible in cardiomyocytes in normal myocardium, but detectable in failing hearts (Földes et al., 2003). Of all the active fragments identified to date, apelin-13 may represent the most potent biological ligand (Kawamata et al., 2001). Current studies suggest that apelin expression is at least maintained and possibly augmented in mild, compensated chronic heart failure but declines in severe disease (Japp & Newby, 2008). Exogenous apelin administration during myocardial injury can preserve cardiac function (Chandrasekaran, 2008). Some researchers suggested that apelin reduces infarct size and protects myocardial cells against ischemia-reperfusion (I/R) injury by activating the reperfusion injury salvage kinase (RISK) pathway. The RISK pathway incorporates phosphatidylinositol 3-OH kinase (PI3K)/Akt, p44/42 mitogen-activated protein kinase (MAPK) and extracellular signalregulated MAPK (ERK1/2) (Simpkin, 2007).

#### **5. Inflammatory pathway activation and interactions with endothelial cells**

#### **5.1 Endothelial cells**

98 Oxidative Stress and Diseases

Visfatin is expressed in many cells and tissues, and was previously identified as a protein involved in B-cell maturation (pre-B colony enhancing factor) (Kitani et al., 2003; Samal et al., 1994). More recently, visfatin was described to be a highly expressed protein with insulin-like functions, and was predominantly found in visceral adipose tissue, from which the name visfatin was derived (Fukuhara et al., 2005). Injection of visfatin in mice lowered blood glucose, and mice with a mutation in visfatin, and nicotinamide adenine dinucleotide (NAD) biosynthetic activity ionotropy, which is essential for -cell function (Revollo et al., 2007). In human studies, a positive correlation between visceral adipose tissue visfatin gene expression and BMI was noted, along with a negative correlation between BMI and SC fat visfatin (Berndt et al., 2005; Varma et al., 2007), suggesting that visfatin regulation in these different depots is different, and adipose depot ratios are highly dependent on the obesity of the subjects. Variable results were obtained regarding the relationship between visfatin and diabetes or insulin resistance (Varma et al., 2007; Chen et al., 2006; Hammarstedt et al., 2005; Haider et al., 2006). Therefore, there are a number of inconsistencies among the different studies of visfatin,

Apelin is another short peptide released from adipocytes upon stimulation by e.g. insulin and the endogenous ligand of the human orphan G-protein-coupled APJ receptor. In line with this, plasma apelin levels are increased in obesity associated with insulin resistance and hyperinsulinemia (Beltowski, 2006). In the cardiovascular system, apelin elicits endothelium-dependent, nitric oxide-mediated vasorelaxation and in rodents, apelin also increases cardiac contractility *in vivo* (Ashley et al., 2005; Atluri et al., 2007) and causes a rapid fall in both arterial blood pressure and systemic venous tone (Tatemoto et al., 2001; Lee, 2005) with corresponding reductions in left ventricular afterload and preload (Ashley et

Apelin-APJ system, expressed in the central nervous system and in a variety of peripheral tissues, is involved in the regulation of the immune response, brain signaling, hemodynamic homeostasis, vasodilatation, inotropy, angiogenesis and glucose metabolism (Sorli et al., 2006; Zhang et al., 2009). In the cardiovascular system, high expression of APJ mRNA has been observed in the heart (Zhang et al., 2009). Apelin expression is restricted to endothelial cells and negligible in cardiomyocytes in normal myocardium, but detectable in failing hearts (Földes et al., 2003). Of all the active fragments identified to date, apelin-13 may represent the most potent biological ligand (Kawamata et al., 2001). Current studies suggest that apelin expression is at least maintained and possibly augmented in mild, compensated chronic heart failure but declines in severe disease (Japp & Newby, 2008). Exogenous apelin administration during myocardial injury can preserve cardiac function (Chandrasekaran, 2008). Some researchers suggested that apelin reduces infarct size and protects myocardial cells against ischemia-reperfusion (I/R) injury by activating the reperfusion injury salvage kinase (RISK) pathway. The RISK pathway incorporates phosphatidylinositol 3-OH kinase (PI3K)/Akt, p44/42 mitogen-activated protein kinase (MAPK) and extracellular signal-

and the role of this adipokine in obesity and insulin resistance is not clear.

**4.5 Visfatin** 

**4.6 Apelin** 

al., 2005; Tatemoto et al., 2001).

regulated MAPK (ERK1/2) (Simpkin, 2007).

The vascular endothelium, located at the interface of blood and tissue, is able to sense changes in hemodynamic forces and blood borne signals and react by synthesizing and releasing vasoactive substances. Vascular homeostasis is maintained by a balance between endothelium-derived relaxing and contracting factors. With disruption of this balance, mediated by inflammatory and traditional cardiovascular risk factors, the vasculature becomes susceptible to atheroma formation. Inflammatory mediators appear to play a fundamental role in the initiation, progression, and eventual rupture of atherosclerotic plaques.

#### **5.1.1 Endothelial dysfunction**

Endothelial dysfunction implies diminished production or availability of NO and/or an imbalance in the relative contribution of endothelium-derived relaxing and contracting factors, those included endothelin-1 (ET-1), angiotensin, and several oxidants. However, endothelial dysfunction, as assessed in terms of vasomotor dysfunction, can occur well before the structural manifestation of atherosclerosis and thus can serve as an independent predictor of future cardiovascular events (Behrendt & Ganz, 2002).

Hypercholesterolemia, traditional cardiovascular risk factor, promotes attachment of blood leukocytes to the endothelium. Oxidized low-density lipoprotein causes endothelial activation and changes its biological characteristics in part by reducing the intracellular concentration of NO (Cominacini, 2001).

On the other hand, angiotensin II can induce the production of ROS, increase the expression of the proinflammatory cytokines as IL-6 and monocyte chemoattractant protein-1 (MCP-1), and upregulate VCAM-1 on ECs. High levels of CRP can also promote endothelial dysfunction by quenching the production of NO and diminishing its bioactivity (Verma, 2002). These endothelial modifications promote inflammation within the vessel wall, setting the stage for the initiation and progression of an atherosclerotic lesion.

#### **5.2 Adiponectin and inflammatory activation**

Recent research has focused on the origin of the inflammatory markers in obesity and the extent to which adipose tissue has a direct effect. The production of adipokines by visceral adipose tissue is of particular interest since their local secretion by visceral fat depots may provide a novel mechanistic link between obesity and the associated vascular complications. Under conditions of inflammation associated with cardiovascular disease, as well as an increase in mobilization of fatty acids from adipose tissue, there is increased secretion of pro-atherogenic, pro-inflammatory adipocytokines and chemokines.

The cardiometabolic benefits of adiponectin may be driven largely through improvements in vascular homeostasis, especially through improving endothelial function. Some studies have demonstrated impaired endothelial function in adiponectin-deficient mice (Teoh et al., 2008) demonstrated that adiponectin plays an important role to limit endothelial activation

Adipocytokines, Oxidative Stress and Impaired Cardiovascular Functions 101

formation and angiogenesis, insulin resistance, adipogenesis and T-lymphocyte activation) and is deregulated in human diseases such as cancer and T2D (Laplante & Sabatini, 2009). *In vivo* stimulators of adipogenesis have not been clearly identified, but may include insulin, IGF-1, as well as certain fatty acids and/or their metabolites. Insulin/IGF-1 acts on cell surface receptors, activating key intracellular signaling proteins. One of these signaling pathways, mTOR that is binds to, and inhibited by, rapamycin, an immunosuppressant that blocks T cell proliferation. The mTOR protein is a 289-kDa serine-threonine kinase that belongs to the phosphoinositide 3-kinase (PI3K)-related kinase family and is conserved throughout evolution (Laplante & Sabatini, 2009). The role of mTORC1 in regulating lipid synthesis, which is required for cell growth and proliferation, is beginning to be appreciated. It has been demonstrated that mTORC1 positively regulates the activity of sterol regulatory element binding protein 1 (SREBP1) (Porstmann et al., 2008) and of PPAR- (Kim & Chen, 2004), two transcription factors that control the expression of genes encoding

The binding of insulin to its cell-surface receptor promotes the tyrosine kinase activity of the insulin receptor, the recruitment of insulin receptor substrate 1 (IRS1), the production of phosphatidylinositol (3,4,5)-triphosphate [PtdIns(3,4,5)*P*3] through the activation of PI3K, and the recruitment and activation of AKT at the plasma membrane. In many cell types, activation of mTORC1 strongly represses the PI3K-AKT axis upstream of PI3K. Activation of S6 kinase 1 (S6K1) by mTORC1 promotes the phosphorylation of insulin receptor substrate 1 (IRS-1) and reduces its stability (Harrington et al., 2005). This auto-regulatory pathway, characterized as the S6K1-dependent negative feedback loop, has been shown to have profound implications for both metabolic diseases and tumorigenesis (Manning, 2004) and pro-inflammatory cytokines, such as TNF, activate IkB kinase-(IKK), which physically interacts with and inactivates tuberous sclerosis complex 1 (TSC1), leading to mTORC1 activation (Lee et al., 2007). This positive relationship between inflammation and mTORC1 activation is thought to be important in tumor angiogenesis and in the

Adiponectin exerts potent anti-inflammatory effects, as documented in experimental studies where authors demonstrate that reduces TNF-α production in response to various stresses in plasma, adipose tissue, vascular wall, heart, and liver (Kojima et al., 2003; Ujiie et al., 2006). In addition, antagonizes several of the inflammatory effects of TNF-α (Ouchi et al., 2003); can facilitate the removal of early apoptotic cells by macrophages and modulate the processes of inflammation and autoimmunity. This activity was mediated by calreticulin expressed on the phagocytic cell surface and not by any of the previously identified adiponectin receptors. Because the accumulation of cell corpses can cause inflammation and immune system dysfunction, these authors suggest a mechanism by which hypoadiponectinemia can contribute to the development of diabetes, atherosclerosis, and other complex diseases in which chronic inflammation is a contributing factor (Takemura et al., 2007). Thus, while AdipoR1 and AdipoR2 may mediate the metabolic properties of adiponectin (Yamauchi et al., 2003), calreticulin controls aspects of adiponectin's antiinflammatory actions (Hug et al., 2004). *In vitro* studies demonstrate that adiponectin adheres to injured vascular endothelium

proteins involved in lipid and cholesterol homeostasis.

development of insulin resistance.

**5.6 Adiponectin as anti-inflammatory action** 

and inflammation in experimental sepsis. On the contrary, in adiponectin-deficient mice exhibit profound reduction in survival following cecal ligation and puncture.

#### **5.3 Mediators of inflammation**

The inflammatory processes are mediated by several factors secreted by adipocytes collectively called adipocytokines (adiponectin, leptin, ghrelin, visfatin and resistin) some of which seem to play an important role in obesity-associated insulin resistance and cardiovascular complications. Tissue levels of TNF-α, IL-6, leptin and visfatin were significantly higher in patients with CAD relative to control subjects. Significantly higher tissue levels of these four cytokines from abdominal fat depots were found compared to those from epicardial fat in CAD patients.

IL-6 is secreted by a wide variety of cells such as endothelial cells, adipocytes, β pancreatic cells, monocytes, and macrophages. This cytokine is essential in reducing the inflammatory process by promoting the synthesis of anti-inflammatory cytokines and by negatively regulating inflammatory targets. In humans, higher circulating IL-6 levels have been associated with obesity and visceral fat deposition, increased risk of impaired glucose tolerance, T2D and high blood pressure. IL-6 is a central mediator of the acute-phase response and a primary determinant of hepatic production of CRP. Visceral adipose tissue secretes about two to three times more IL-6 than subcutaneous tissue, secreting also other molecules that stimulate further IL-6 expression (Curti, 2011).

In obesity, the pro-inflammatory effects of cytokines through intracellular signaling pathways involve the NF-κB and JNK systems. Thus, it can be considered that obesity corresponds to a sub-clinical inflammatory condition that promotes the production of proinflammatory factors involved in the pathogenesis of insulin resistance (Bastard et al., 2002).

#### **5.4 Vasculature as part of the immune system**

Blood vessels are integral components of the immune system; they are important part in lymphocyte circulation and act as portals between tissue and blood compartments. Endothelial cells express toll-like receptors, (Kunjathoor, 2002) whose ligation induces expression of leukocyte adhesion molecules, inducible NO synthase 2, endothelin, IL-1, and other inflammatory molecules. These cells also express the scavenger receptors CD36 and LOX-1, and can internalize ligands such as modified LDL particles. ECs are located at the interface of blood and tissues, and play a pivotal role in the inflammatory response. Their activation causes leukocyte recruitment, increased permeability, edema, and other characteristic features of inflammation. Furthermore, ECs can activate adaptive immunity by presenting foreign antigens to specific T cells.

#### **5.5 Mammalian Target Of Rapamycin (mTOR) signaling pathway**

The mammalian target of rapamycin (mTOR) signaling pathway integrates both intracellular and extracellular signals and serves as a central regulator of cell metabolism, growth, proliferation and survival. Discoveries that have been made over the last decade show that the mTOR pathway is activated during various cellular processes (e.g. tumor

and inflammation in experimental sepsis. On the contrary, in adiponectin-deficient mice

The inflammatory processes are mediated by several factors secreted by adipocytes collectively called adipocytokines (adiponectin, leptin, ghrelin, visfatin and resistin) some of which seem to play an important role in obesity-associated insulin resistance and cardiovascular complications. Tissue levels of TNF-α, IL-6, leptin and visfatin were significantly higher in patients with CAD relative to control subjects. Significantly higher tissue levels of these four cytokines from abdominal fat depots were found compared to

IL-6 is secreted by a wide variety of cells such as endothelial cells, adipocytes, β pancreatic cells, monocytes, and macrophages. This cytokine is essential in reducing the inflammatory process by promoting the synthesis of anti-inflammatory cytokines and by negatively regulating inflammatory targets. In humans, higher circulating IL-6 levels have been associated with obesity and visceral fat deposition, increased risk of impaired glucose tolerance, T2D and high blood pressure. IL-6 is a central mediator of the acute-phase response and a primary determinant of hepatic production of CRP. Visceral adipose tissue secretes about two to three times more IL-6 than subcutaneous tissue, secreting also other

In obesity, the pro-inflammatory effects of cytokines through intracellular signaling pathways involve the NF-κB and JNK systems. Thus, it can be considered that obesity corresponds to a sub-clinical inflammatory condition that promotes the production of proinflammatory factors involved in the pathogenesis of insulin resistance (Bastard et al., 2002).

Blood vessels are integral components of the immune system; they are important part in lymphocyte circulation and act as portals between tissue and blood compartments. Endothelial cells express toll-like receptors, (Kunjathoor, 2002) whose ligation induces expression of leukocyte adhesion molecules, inducible NO synthase 2, endothelin, IL-1, and other inflammatory molecules. These cells also express the scavenger receptors CD36 and LOX-1, and can internalize ligands such as modified LDL particles. ECs are located at the interface of blood and tissues, and play a pivotal role in the inflammatory response. Their activation causes leukocyte recruitment, increased permeability, edema, and other characteristic features of inflammation. Furthermore, ECs can activate adaptive immunity

The mammalian target of rapamycin (mTOR) signaling pathway integrates both intracellular and extracellular signals and serves as a central regulator of cell metabolism, growth, proliferation and survival. Discoveries that have been made over the last decade show that the mTOR pathway is activated during various cellular processes (e.g. tumor

exhibit profound reduction in survival following cecal ligation and puncture.

**5.3 Mediators of inflammation** 

those from epicardial fat in CAD patients.

molecules that stimulate further IL-6 expression (Curti, 2011).

**5.4 Vasculature as part of the immune system** 

by presenting foreign antigens to specific T cells.

**5.5 Mammalian Target Of Rapamycin (mTOR) signaling pathway** 

formation and angiogenesis, insulin resistance, adipogenesis and T-lymphocyte activation) and is deregulated in human diseases such as cancer and T2D (Laplante & Sabatini, 2009). *In vivo* stimulators of adipogenesis have not been clearly identified, but may include insulin, IGF-1, as well as certain fatty acids and/or their metabolites. Insulin/IGF-1 acts on cell surface receptors, activating key intracellular signaling proteins. One of these signaling pathways, mTOR that is binds to, and inhibited by, rapamycin, an immunosuppressant that blocks T cell proliferation. The mTOR protein is a 289-kDa serine-threonine kinase that belongs to the phosphoinositide 3-kinase (PI3K)-related kinase family and is conserved throughout evolution (Laplante & Sabatini, 2009). The role of mTORC1 in regulating lipid synthesis, which is required for cell growth and proliferation, is beginning to be appreciated. It has been demonstrated that mTORC1 positively regulates the activity of sterol regulatory element binding protein 1 (SREBP1) (Porstmann et al., 2008) and of PPAR- (Kim & Chen, 2004), two transcription factors that control the expression of genes encoding proteins involved in lipid and cholesterol homeostasis.

The binding of insulin to its cell-surface receptor promotes the tyrosine kinase activity of the insulin receptor, the recruitment of insulin receptor substrate 1 (IRS1), the production of phosphatidylinositol (3,4,5)-triphosphate [PtdIns(3,4,5)*P*3] through the activation of PI3K, and the recruitment and activation of AKT at the plasma membrane. In many cell types, activation of mTORC1 strongly represses the PI3K-AKT axis upstream of PI3K. Activation of S6 kinase 1 (S6K1) by mTORC1 promotes the phosphorylation of insulin receptor substrate 1 (IRS-1) and reduces its stability (Harrington et al., 2005). This auto-regulatory pathway, characterized as the S6K1-dependent negative feedback loop, has been shown to have profound implications for both metabolic diseases and tumorigenesis (Manning, 2004) and pro-inflammatory cytokines, such as TNF, activate IkB kinase-(IKK), which physically interacts with and inactivates tuberous sclerosis complex 1 (TSC1), leading to mTORC1 activation (Lee et al., 2007). This positive relationship between inflammation and mTORC1 activation is thought to be important in tumor angiogenesis and in the development of insulin resistance.

#### **5.6 Adiponectin as anti-inflammatory action**

Adiponectin exerts potent anti-inflammatory effects, as documented in experimental studies where authors demonstrate that reduces TNF-α production in response to various stresses in plasma, adipose tissue, vascular wall, heart, and liver (Kojima et al., 2003; Ujiie et al., 2006). In addition, antagonizes several of the inflammatory effects of TNF-α (Ouchi et al., 2003); can facilitate the removal of early apoptotic cells by macrophages and modulate the processes of inflammation and autoimmunity. This activity was mediated by calreticulin expressed on the phagocytic cell surface and not by any of the previously identified adiponectin receptors. Because the accumulation of cell corpses can cause inflammation and immune system dysfunction, these authors suggest a mechanism by which hypoadiponectinemia can contribute to the development of diabetes, atherosclerosis, and other complex diseases in which chronic inflammation is a contributing factor (Takemura et al., 2007). Thus, while AdipoR1 and AdipoR2 may mediate the metabolic properties of adiponectin (Yamauchi et al., 2003), calreticulin controls aspects of adiponectin's antiinflammatory actions (Hug et al., 2004). *In vitro* studies demonstrate that adiponectin adheres to injured vascular endothelium

Adipocytokines, Oxidative Stress and Impaired Cardiovascular Functions 103

A member of the lipocalin family, lipocalin-2, also known as neutrophil gelatinase– associated lipocalin, modulates inflammation and is another adipokine that is elevated in the adipose tissue of obese mouse models and in the plasma of obese and insulin-resistant humans. *In vitro* studies suggest that lipocalin-2 induces insulin resistance in adipocytes and hepatocytes. The plasma level of another member of the lipocalin family, lipocalin-type prostaglandin D synthase, serves as a biomarker of coronary atherosclerosis (Yan, 2007).

**6. Oxidative Stress in conditions and comorbidities that aggregate with** 

Obesity, is associated with inflammation and ROS production, while advanced glycation end-products (AGEs), through their receptor (AGER or RAGE), play an important role on these processes. This is a multiligand receptor of the immunoglobulin superfamily that binds advanced glycation end-products. Thus, increased epicardial, pericardial (EAT), or subcutaneous adipose tissue (SAT) is associated with the presence and severity of coronary artery calcium. The AGE–RAGE engagement is widely related with CVD and ROS generation, mainly mediated by NADPH-oxidase. This enzyme consists in two membrane-bound subunits, the gp91-PHOX protein (NOX2) or some of its homologs (named NOX from 1 to 5) and p22-PHOX protein. Once activated, NADPH-oxidase produces superoxide anions from oxygen and NADPH or NADH. Enhanced ROS production is an important factor associated with some CVD such as CAD. Furthermore, Rodino-Janeiro et al. (2010, 2011) have previously observed that EAT may undergone higher oxidative stress than SAT in patients with CAD because of lower expression of some antioxidant enzymes, like catalase, and higher expression of RAGE in EAT than in SAT. Oxidation of phospholipids in LDL, which inltrates into the injured vessel wall, results in the formation and accumulation of ox-LDL. These, is pro-atherogenic, produces several abnormal biological responses, such as attracting leukocytes to the intimal of the vessel, improving the ability of the leukocytes to ingest lipids and differentiate into foam cells, and stimulating the proliferation of leukocytes, endothelial cells, and smooth muscle cells, all of which are steps in the formation of atherosclerotic plaque. Furthermore, activated macrophages express scavenger receptors that internalize ox-LDL. However, unregulated uptake of ox-LDL leads to production of lipid-loaded foam cells (Hulsmans

Glucotoxicity, lipotoxicity, and glucolipotoxicity are secondary phenomena that are proposed to play a role in all forms of T2D. They are implicated in the pathogenesis of cell dysfunction (Poitout & Robertson, 2008). Hyperglycemia and hyperlipidemia follow the primary pathogenesis of diabetes and exert additional toxic effects on -cells. The concept of toxicity derived because physiologically, it presents a continuous overstimulation of the βcell by glucose could eventually lead to depletion of insulin stores, worsening of hyperglycemia, and finally deterioration of β-cell function. So, a prolonged *in vitro* exposure of isolated islets or insulin-secreting cells to elevated levels of fatty acids is associated with

**5.7.3 Lipocalin-2** 

**cardiovascular disease** 

et al., 2011).

**6.1 Oxidative stress and the beta-cell** 

(Okamoto et al., 2000) and inhibits TNF--induced monocyte adhesion to endothelial cells. It also decreases the expression of endothelial cell adhesion molecules (Ouchi et al., 1999) and TNF--induced NFkB activation (Ouchi N et al., 2000).

Adiponectin has been shown to have a role in hepatic inflammation and steatosis. Hypoadiponectinaemia is associated with nonalcoholic steatohepatitis (Targher et al., 2004) and adiponectin has been shown to have beneficial anti-inflammatory effects in liver, reducing steatosis, hepatomegaly and inflammation in mouse models of alcoholic and non-alcoholic fatty liver disease (Xu et al., 2003).

#### **5.7 New mediators of inflammation and endothelial cell activation**

#### **5.7.1 Oxidized low-density lipoprotein receptor-1 and LOX-1**

Oxidatively modied Ox-LDL and lectin-like oxidized LDL receptor-1 (LOX-1) are contributing factors of endothelial dysfunction, an early cellular event during atherogenesis. The primary receptor for Ox-LDL in endothelial cells is LOX-1. Under physiological conditions, LOX-1 may play a role in host defense (is expressed at low levels), whereas pathological states such as atherosclerosis, diabetes, dyslipedemia, hypertension dramatically and disease states that promote vascular injury, LOX-1 is highly expressed in blood vessels increase (Mattaliano et al., 2010), and may be involved in binding proatherogenic materials, such as ox-LDL, that activate the endothelium. With its ability to bind products that induce inflammation and endothelial activation, elevated LOX-1 expression was observed in both initial and advanced atherosclerotic lesions (Li et al., 2002). Induction of LOX-1 expression is mediated by angiotensin II and endothelin-1, both antagonists of NO (Chen et al., 2006). LOX-1 is a type II transmembrane glycoprotein that is known to recognize a wide array of structurally distinct ligands besides Ox-LDL. These include activated platelets, AGEs, apoptotic bodies, bacteria, and CRP. LOX-1 plays a critical role in the development of atherosclerosis. This may suggest that increased LOX-1 transcriptional promoter activity may equal increased LOX-1 gene expression and elevated risk of atherosclerosis. Accordingly, decreased LOX-1 promoter activation may reduce the incidence of atherosclerosis and related diseases (Chen et al., 2006).

#### **5.7.2 Protease-Activated Receptors (PARs)**

Protease-Activated Receptors (PARs) are a family of 7-transmembrane–domain, G-protein– coupled receptors that function to link tissue injury to appropriate cellular responses, such as inflammation and tissue repair, which may contribute to disease. Under the influence of the traditional cardiovascular risk factors, the endogenous defenses of the vascular endothelium begin to break down, resulting in endothelial dysfunction and injury (see figure 1). PAR activation is also linked to the secretion of IL–6, the cytokine that promotes CRP synthesis, which itself triggers many of the steps in the inflammatory process. Overall, PAR activation appears to promote the inflammatory response within the intimal tissue, enhancing the initiation and progression of atherosclerotic plaques. Rosiglitazone, a selective PPARγ agonist, exerts anti-inflammatory effects in both obese and T2D individuals by decreasing plasma concentrations of CRP, serum amyloid-A, and matrix metalloproteinase (Stienstra, 2007).

#### **5.7.3 Lipocalin-2**

102 Oxidative Stress and Diseases

(Okamoto et al., 2000) and inhibits TNF--induced monocyte adhesion to endothelial cells. It also decreases the expression of endothelial cell adhesion molecules (Ouchi et al., 1999) and

Adiponectin has been shown to have a role in hepatic inflammation and steatosis. Hypoadiponectinaemia is associated with nonalcoholic steatohepatitis (Targher et al., 2004) and adiponectin has been shown to have beneficial anti-inflammatory effects in liver, reducing steatosis, hepatomegaly and inflammation in mouse models of alcoholic and non-alcoholic

Oxidatively modied Ox-LDL and lectin-like oxidized LDL receptor-1 (LOX-1) are contributing factors of endothelial dysfunction, an early cellular event during atherogenesis. The primary receptor for Ox-LDL in endothelial cells is LOX-1. Under physiological conditions, LOX-1 may play a role in host defense (is expressed at low levels), whereas pathological states such as atherosclerosis, diabetes, dyslipedemia, hypertension dramatically and disease states that promote vascular injury, LOX-1 is highly expressed in blood vessels increase (Mattaliano et al., 2010), and may be involved in binding proatherogenic materials, such as ox-LDL, that activate the endothelium. With its ability to bind products that induce inflammation and endothelial activation, elevated LOX-1 expression was observed in both initial and advanced atherosclerotic lesions (Li et al., 2002). Induction of LOX-1 expression is mediated by angiotensin II and endothelin-1, both antagonists of NO (Chen et al., 2006). LOX-1 is a type II transmembrane glycoprotein that is known to recognize a wide array of structurally distinct ligands besides Ox-LDL. These include activated platelets, AGEs, apoptotic bodies, bacteria, and CRP. LOX-1 plays a critical role in the development of atherosclerosis. This may suggest that increased LOX-1 transcriptional promoter activity may equal increased LOX-1 gene expression and elevated risk of atherosclerosis. Accordingly, decreased LOX-1 promoter activation may reduce the

Protease-Activated Receptors (PARs) are a family of 7-transmembrane–domain, G-protein– coupled receptors that function to link tissue injury to appropriate cellular responses, such as inflammation and tissue repair, which may contribute to disease. Under the influence of the traditional cardiovascular risk factors, the endogenous defenses of the vascular endothelium begin to break down, resulting in endothelial dysfunction and injury (see figure 1). PAR activation is also linked to the secretion of IL–6, the cytokine that promotes CRP synthesis, which itself triggers many of the steps in the inflammatory process. Overall, PAR activation appears to promote the inflammatory response within the intimal tissue, enhancing the initiation and progression of atherosclerotic plaques. Rosiglitazone, a selective PPARγ agonist, exerts anti-inflammatory effects in both obese and T2D individuals by decreasing plasma concentrations of CRP, serum amyloid-A, and matrix

TNF--induced NFkB activation (Ouchi N et al., 2000).

**5.7 New mediators of inflammation and endothelial cell activation** 

**5.7.1 Oxidized low-density lipoprotein receptor-1 and LOX-1** 

incidence of atherosclerosis and related diseases (Chen et al., 2006).

**5.7.2 Protease-Activated Receptors (PARs)** 

metalloproteinase (Stienstra, 2007).

fatty liver disease (Xu et al., 2003).

A member of the lipocalin family, lipocalin-2, also known as neutrophil gelatinase– associated lipocalin, modulates inflammation and is another adipokine that is elevated in the adipose tissue of obese mouse models and in the plasma of obese and insulin-resistant humans. *In vitro* studies suggest that lipocalin-2 induces insulin resistance in adipocytes and hepatocytes. The plasma level of another member of the lipocalin family, lipocalin-type prostaglandin D synthase, serves as a biomarker of coronary atherosclerosis (Yan, 2007).

#### **6. Oxidative Stress in conditions and comorbidities that aggregate with cardiovascular disease**

Obesity, is associated with inflammation and ROS production, while advanced glycation end-products (AGEs), through their receptor (AGER or RAGE), play an important role on these processes. This is a multiligand receptor of the immunoglobulin superfamily that binds advanced glycation end-products. Thus, increased epicardial, pericardial (EAT), or subcutaneous adipose tissue (SAT) is associated with the presence and severity of coronary artery calcium. The AGE–RAGE engagement is widely related with CVD and ROS generation, mainly mediated by NADPH-oxidase. This enzyme consists in two membrane-bound subunits, the gp91-PHOX protein (NOX2) or some of its homologs (named NOX from 1 to 5) and p22-PHOX protein. Once activated, NADPH-oxidase produces superoxide anions from oxygen and NADPH or NADH. Enhanced ROS production is an important factor associated with some CVD such as CAD. Furthermore, Rodino-Janeiro et al. (2010, 2011) have previously observed that EAT may undergone higher oxidative stress than SAT in patients with CAD because of lower expression of some antioxidant enzymes, like catalase, and higher expression of RAGE in EAT than in SAT. Oxidation of phospholipids in LDL, which inltrates into the injured vessel wall, results in the formation and accumulation of ox-LDL. These, is pro-atherogenic, produces several abnormal biological responses, such as attracting leukocytes to the intimal of the vessel, improving the ability of the leukocytes to ingest lipids and differentiate into foam cells, and stimulating the proliferation of leukocytes, endothelial cells, and smooth muscle cells, all of which are steps in the formation of atherosclerotic plaque. Furthermore, activated macrophages express scavenger receptors that internalize ox-LDL. However, unregulated uptake of ox-LDL leads to production of lipid-loaded foam cells (Hulsmans et al., 2011).

#### **6.1 Oxidative stress and the beta-cell**

Glucotoxicity, lipotoxicity, and glucolipotoxicity are secondary phenomena that are proposed to play a role in all forms of T2D. They are implicated in the pathogenesis of cell dysfunction (Poitout & Robertson, 2008). Hyperglycemia and hyperlipidemia follow the primary pathogenesis of diabetes and exert additional toxic effects on -cells. The concept of toxicity derived because physiologically, it presents a continuous overstimulation of the βcell by glucose could eventually lead to depletion of insulin stores, worsening of hyperglycemia, and finally deterioration of β-cell function. So, a prolonged *in vitro* exposure of isolated islets or insulin-secreting cells to elevated levels of fatty acids is associated with

Adipocytokines, Oxidative Stress and Impaired Cardiovascular Functions 105

deleterious to the endothelial function of T2D patients (Ceriello et al., 2008). Overall, these data outline the importance of steady glucose control and the potential involvement of oxidative and nitrosative stress in the pathogenesis of complications due to poorly controlled diabetes. Diabetic subjects have reduced antioxidant capacity which could favor oxidative stress. A decline in important cellular antioxidant defense mechanisms, including the glutathione redox system and vitamin C-vitamin E cycle, significantly increases the susceptibility to oxidative stress. Thus, attempts have been made to reduce oxidative stressdependent cellular changes in patients with diabetes by supplementation with naturally occurring antioxidants, especially vitamins E and C, lipoic acid levels are reduced in diabetic

It has now been established that measurement of F2-isoprostanes is the most reliable approach to assess oxidative stress status *in vivo*, providing an important tool to explore the role of oxidative stress in the pathogenesis of human disease. In addition, products of the isoprostane (IsoP) pathway have been found to exert potent biological actions and therefore may be pathophysiologic mediators of disease. IsoPs, 8-iso-PGF2α and 8-iso-PGE2 possess potent biological effects in various systems and they also serve as mediators of oxidant stress through their vasoconstrictive and inflammatory properties (Kaviarasan et al., 2009). There exists a significant correlation between blood glucose and urinary IsoPs levels, suggesting that peroxidation is related to glycemic control. In vascular smooth muscle cells,

F2-IsoPs formation was found to be induced *in vitro* by high glucose concentrations.

The diverse responses of the microvasculature to CVD risk factors include oxidative stress, enhanced leukocyte and platelet-endothelial cell adhesion, impaired endothelial barrier function, altered capillary proliferation, enhanced thrombosis, and vasomotor dysfunction

As shown in figure 1, an imbalance between the production and detoxification of ROS in vascular endothelial cells can result in the oxidative modification of cell components, impair cell function and/or can enhance cell death via apoptosis or necrosis. The oxidative activation of enzymes (phospholipase A2) and transcription factors (nuclear factor kB, NFkB) that accompanies excess ROS production can also result in an enhanced biosynthesis of lipids (platelet activating factor, leukotrienes) and proteins (adhesion molecules, cytokines) that promote inflammation. Superoxide, by virtue of its ability to inactivate nitric oxide (an anti-inflammatory molecule), is another link between oxidative stress and the induction of a pro-inflammatory phenotype in the vasculature. This oxidative stress in the vessel wall is often accompanied by an increased production of superoxide anion by circulating immune cells, and there is evidence for a causal link between these two sources

Different enzymatic sources have been implicated in the enhanced ROS production, including NADPH oxidase, xanthine oxidase, mitochondrial enzymes, and uncoupled nitric oxide synthase. It remains to clarify whether the pro-hypertensive effects of the superoxide anion relate to its ability to inactivate NO or to indirectly promote the production of endogenous vasoconstrictors, such as endothelin. NADPH-oxidase has received the most

patients.

**6.3 Cardiovascular disease** 

of ROS: circulating cells and vessel wall.

(Granger et al., 2010).

inhibited glucose-induced insulin secretion, impaired insulin gene expression, and induction of cell death by apoptosis.

#### **6.2 Oxidative stress and diabetic vascular complications**

Cardiovascular risk factors promote the production of ROS, excessive generation of ROS and has expression of eNOS been implicated in a variety of pathological events such as diabetes, hypertension, atherosclerosis, ischemia-reperfusion injury, CVD and neurodegenerative disease (Halliwell & Gutteridge, 2007). Results from several studies showed that the increase in ROS levels precedes the hyperglycemia and insulin resistance, suggesting a causal role of ROS in the disease process. Atherosclerosis is considered as the underlying pathology of cardiovascular diseases such as peripheral vascular disease, stroke, and coronary heart disease. The pathology of atherosclerosis is complex and involves structural elements of the arterial wall, platelets, leukocytes, and inflammatory cells such as monocytes and macrophages (Libby et al., 2002; Weber et al., 2008). The endothelium is a dynamic interface between the arterial wall and the circulating cells. Therefore, endothelial dysfunction accounts for one of the primary causes of atherosclerosis. Since the endothelium is the major source of NO in the vasculature, loss of normal cellular function can result in altered NO synthesis. The endothelium provides a constitutive supply of NO from eNOS, and under certain conditions (e.g. inflammation) it can produce excessive NO from the inducible isoform of NOS (iNOS). Therefore, regulation of NOS is central in the development and progression of atherosclerosis.

In particular, increased glucose leads to increased mitochondrial formation of ROS. Superoxide is a ROS that produces peroxynitrite when reacting with NO. Peroxynitrite induces cellular damage through depletion of the co-factor of the eNOS, tetrahydrobiopterin (BH4). Also, it activates the denominated classic pathways of diabetic complications, including: a) the polyol pathway, b) the AGE pathway, c) the protein kinase C (PKC) pathway, and d) the hexosamine pathway. Several studies suggested that intermittent low and high glucose conditions are even more deleterious to endothelial cell function than a steady, constant increase of glucose. These conditions also induce endothelial cells to enter into a proinflammatory state, and this state is associated with the upregulation of various adhesion molecules and proinflammatory cytokines (Piconi et al., 2004).

iNOS is very relevant to diabetic pathophysiology. Recent reports reveal that decreased expression of eNOS accompanies increased expression of iNOS and nitrotyrosine during the progression of diabetes in rats (Nagareddy et al., 2005). This finding suggests that induction of iNOS in cardiovascular tissues is dependent on the duration of diabetes and contributes significantly to depressed responses to vasoactive agents. *In vivo* studies revealed that oxidative stress due to hyperglycemia, occurring before late complications, become clinically evident (Pitocco et al., 2009). This finding suggests that oxidative stress plays a crucial role in the pathogenesis of late diabetic complications. It has also been described in human studies that endothelial cells in diabetes fail to produce sufficient amount of NO and fail to relax in response to endothelium-dependent vasorelaxants e.g. acetylcholine, bradykinin, shear stress, etc (Avogaro et al., 2006).

Further clinical data have demonstrated that rapid glycemic swings are associated with an exacerbated degree of oxidant production in human diabetes (Monnier et al., 2006), and are deleterious to the endothelial function of T2D patients (Ceriello et al., 2008). Overall, these data outline the importance of steady glucose control and the potential involvement of oxidative and nitrosative stress in the pathogenesis of complications due to poorly controlled diabetes. Diabetic subjects have reduced antioxidant capacity which could favor oxidative stress. A decline in important cellular antioxidant defense mechanisms, including the glutathione redox system and vitamin C-vitamin E cycle, significantly increases the susceptibility to oxidative stress. Thus, attempts have been made to reduce oxidative stressdependent cellular changes in patients with diabetes by supplementation with naturally occurring antioxidants, especially vitamins E and C, lipoic acid levels are reduced in diabetic patients.

It has now been established that measurement of F2-isoprostanes is the most reliable approach to assess oxidative stress status *in vivo*, providing an important tool to explore the role of oxidative stress in the pathogenesis of human disease. In addition, products of the isoprostane (IsoP) pathway have been found to exert potent biological actions and therefore may be pathophysiologic mediators of disease. IsoPs, 8-iso-PGF2α and 8-iso-PGE2 possess potent biological effects in various systems and they also serve as mediators of oxidant stress through their vasoconstrictive and inflammatory properties (Kaviarasan et al., 2009). There exists a significant correlation between blood glucose and urinary IsoPs levels, suggesting that peroxidation is related to glycemic control. In vascular smooth muscle cells, F2-IsoPs formation was found to be induced *in vitro* by high glucose concentrations.

#### **6.3 Cardiovascular disease**

104 Oxidative Stress and Diseases

inhibited glucose-induced insulin secretion, impaired insulin gene expression, and

Cardiovascular risk factors promote the production of ROS, excessive generation of ROS and has expression of eNOS been implicated in a variety of pathological events such as diabetes, hypertension, atherosclerosis, ischemia-reperfusion injury, CVD and neurodegenerative disease (Halliwell & Gutteridge, 2007). Results from several studies showed that the increase in ROS levels precedes the hyperglycemia and insulin resistance, suggesting a causal role of ROS in the disease process. Atherosclerosis is considered as the underlying pathology of cardiovascular diseases such as peripheral vascular disease, stroke, and coronary heart disease. The pathology of atherosclerosis is complex and involves structural elements of the arterial wall, platelets, leukocytes, and inflammatory cells such as monocytes and macrophages (Libby et al., 2002; Weber et al., 2008). The endothelium is a dynamic interface between the arterial wall and the circulating cells. Therefore, endothelial dysfunction accounts for one of the primary causes of atherosclerosis. Since the endothelium is the major source of NO in the vasculature, loss of normal cellular function can result in altered NO synthesis. The endothelium provides a constitutive supply of NO from eNOS, and under certain conditions (e.g. inflammation) it can produce excessive NO from the inducible isoform of NOS (iNOS). Therefore, regulation of NOS is central in the

In particular, increased glucose leads to increased mitochondrial formation of ROS. Superoxide is a ROS that produces peroxynitrite when reacting with NO. Peroxynitrite induces cellular damage through depletion of the co-factor of the eNOS, tetrahydrobiopterin (BH4). Also, it activates the denominated classic pathways of diabetic complications, including: a) the polyol pathway, b) the AGE pathway, c) the protein kinase C (PKC) pathway, and d) the hexosamine pathway. Several studies suggested that intermittent low and high glucose conditions are even more deleterious to endothelial cell function than a steady, constant increase of glucose. These conditions also induce endothelial cells to enter into a proinflammatory state, and this state is associated with the upregulation of various

iNOS is very relevant to diabetic pathophysiology. Recent reports reveal that decreased expression of eNOS accompanies increased expression of iNOS and nitrotyrosine during the progression of diabetes in rats (Nagareddy et al., 2005). This finding suggests that induction of iNOS in cardiovascular tissues is dependent on the duration of diabetes and contributes significantly to depressed responses to vasoactive agents. *In vivo* studies revealed that oxidative stress due to hyperglycemia, occurring before late complications, become clinically evident (Pitocco et al., 2009). This finding suggests that oxidative stress plays a crucial role in the pathogenesis of late diabetic complications. It has also been described in human studies that endothelial cells in diabetes fail to produce sufficient amount of NO and fail to relax in response to endothelium-dependent vasorelaxants e.g. acetylcholine, bradykinin,

Further clinical data have demonstrated that rapid glycemic swings are associated with an exacerbated degree of oxidant production in human diabetes (Monnier et al., 2006), and are

adhesion molecules and proinflammatory cytokines (Piconi et al., 2004).

induction of cell death by apoptosis.

**6.2 Oxidative stress and diabetic vascular complications** 

development and progression of atherosclerosis.

shear stress, etc (Avogaro et al., 2006).

The diverse responses of the microvasculature to CVD risk factors include oxidative stress, enhanced leukocyte and platelet-endothelial cell adhesion, impaired endothelial barrier function, altered capillary proliferation, enhanced thrombosis, and vasomotor dysfunction (Granger et al., 2010).

As shown in figure 1, an imbalance between the production and detoxification of ROS in vascular endothelial cells can result in the oxidative modification of cell components, impair cell function and/or can enhance cell death via apoptosis or necrosis. The oxidative activation of enzymes (phospholipase A2) and transcription factors (nuclear factor kB, NFkB) that accompanies excess ROS production can also result in an enhanced biosynthesis of lipids (platelet activating factor, leukotrienes) and proteins (adhesion molecules, cytokines) that promote inflammation. Superoxide, by virtue of its ability to inactivate nitric oxide (an anti-inflammatory molecule), is another link between oxidative stress and the induction of a pro-inflammatory phenotype in the vasculature. This oxidative stress in the vessel wall is often accompanied by an increased production of superoxide anion by circulating immune cells, and there is evidence for a causal link between these two sources of ROS: circulating cells and vessel wall.

Different enzymatic sources have been implicated in the enhanced ROS production, including NADPH oxidase, xanthine oxidase, mitochondrial enzymes, and uncoupled nitric oxide synthase. It remains to clarify whether the pro-hypertensive effects of the superoxide anion relate to its ability to inactivate NO or to indirectly promote the production of endogenous vasoconstrictors, such as endothelin. NADPH-oxidase has received the most

Adipocytokines, Oxidative Stress and Impaired Cardiovascular Functions 107

mechanisms underlying both the initiation and perpetuation of AF are not well established but are thought to involve inflammation and oxidative stress. Furthermore, a number of studies have shown that concentrations of inflammatory mediators or markers, such as IL-6 and high-sensitivity CRP (hs-CRP), are increased in patients with AF. One mechanism that may mediate the effects of inflammation in AF is oxidative stress. Elevated inflammatory biomarkers are strongly associated with AF. Inflammation has important prognostic implications in AF; large prospective studies have shown that elevated hs-CRP levels correlated with risk factors for stroke and overall prognosis. The positive correlation between elevated levels of TNF-α and N-terminal pro-brain natriuretic peptide (NTpBNP) and severity of AF suggests that these biomarkers could be prognostic markers for AF in

A large number of studies have evidenced the pivotal role of oxidative stress in insulin resistance states such as metabolic syndrome, obesity, and T2D (Atabek et al., 2004; Block et al., 2002). Decreased antioxidant capacity, increased production of ROS with oxidation products of lipids, DNA, and proteins have been reported in plasma, urine, and various tissues, suggesting systemic and organ-specific oxidative stress. Recent evidence for systemic oxidative stress includes the detection of increased circulating and urinary levels of the lipid peroxidation product F2-isoprostane (8-epi-prostaglandin F2α) in both T1D and T2D patients (Davi et al., 2003). As described above, ROS and reactive nitrogen species (RNS) are able to directly modify the expression of adiponectin. Secreted almost exclusively from adipocytes, it is inversely correlated with fat mass in obesity and with its associated cardiovascular risk. It should be considered that, plasma and urinary lipid peroxidation markers indicative of systemic OS correlated with lower circulating adiponectin levels.

Figure 1 summarizes much of the content of this chapter, because it shows most of the cellular elements and signaling pathways in which highlights the participation of adipocytokines involved in the immune response and oxidative stress on the vascular endothelium. These alterations lead to development of atherosclerosis. And finally this endothelial damage, together with the increase in free radicals can cause multiorgan

In conclusion, abnormal adipocytokine expression with consequent inflammation, oxidative stress itself may result from the inflammatory changes that occur in obesity. Therefore, a vicious cycle that provokes increased oxidative stress in obesity may exist. Reactive oxygen species that lead to increased oxidative stress can be generated in adipocytes and in other cell types such as leukocytes, all of which can be a source of increased oxidative stress in obese humans. Increased oxidative stress is independently associated with obesity measures including body mass index and waist-hip ratio. It is also associated with several CVD risk factors including smoking, blood glucose, and hyperlipidemia. Oxidative stress and increased adipocytokines may also promote endothelial dysfunction, atherogenesis, and

coronary heart disease independent of traditional risk factors.

clinical practice (Li et al., 2010).

**6.6 Insulin resistance** 

**7. Conclusion** 

damage.

attention as a potential source of ROS in hypertension (HTN), followed by xanthine oxidase. Both endothelial cell- and leukocyte-associated NADPH-oxidase have been implicated in HTN-induced superoxide production, and there is evidence linking both cellular sources of the enzyme to activation of the angiotensin II type 1 receptor (AT1r) and to cytokines (TNF- ) derived from circulating immune cells (Crimi et al., 2007; Harrison & Gongora, 2009).

While some adipokines as leptin and adiponectin have been shown to promote the expression of endothelial cell adhesion molecules (CAMs) and leukocyte-endothelial cell adhesion (LECA) are known to exert an inhibitory effect on these responses. The absence of LECA in the microcirculation of obese mice under basal conditions suggests either that the pro- and anti-adhesive adipokines are in balance or that the systemic plasma levels achieved by these mediators do not cause overt inflammation in tissues distant from their source (adipose tissue). The latter possibility is supported by evidence of an increased sensitivity (priming) of endothelial cells and leukocytes in obese animals to inflammatory stimuli.

However, within the microvasculature of adipose tissue, a robust inflammatory response is noted under basal conditions, as reflected by an increased expression of the endothelial cell adhesion molecules ICAM-1 and E- and P-select in, with an accompanying recruitment of rolling and firmly adherent leukocytes, and the formation of platelet-leukocyte aggregates. The reduced LECA may be linked to adiponectin deficiency since the adipokine is a potent inhibitor of LECA and its production/release is diminished during adipogenesis (Singer & Granger, 2007).

#### **6.4 Oxidative stress in aortic valves**

Superoxide levels also are increased in stenotic aortic valves from humans. Heistad et al., (2009) found, in stenotic valves removed during surgical replacement of the aortic valve, that superoxide is increased greatly near calcified regions of the valve. Others authors (Miller et al., 2008) also found, in valves obtained at surgery or autopsy, that oxidative stress is increased in stenotic aortic valves. Thus, in calcified stenotic aortic valves as well as in atherosclerotic lesions, oxidative stress is increased. But, there are important differences in mechanisms that account for oxidative stress in aortic valves and in atherosclerotic arteries. In calcific aortic stenosis, increased production of superoxide may be mediated by "uncoupling" of NOS, as NOS primarily produces superoxide instead of nitric oxide NAD(P)H expression and activity do not appear to be increased in aortic valves (Miller et al., 2008). In striking contrast, increased expression and activity of NADPH-oxidase appears to be a major mechanism for oxidative stress in atherosclerotic lesions. Oxidative stress, in addition to contributing to fibrosis, may activate matrix metalloproteinases (MMPs) in the aortic valve and arteries. In the valve, MMPs may play a permissive role in expansion of calcification of the valve, and degraded fragments of collagen and elastin also may increase pro-calcific signaling in valvular interstitial cells. Activation of MMPs in arteries probably is harmful in a different way, by contributing to plaque rupture.

#### **6.5 Atrial fibrillation**

Atrial fibrillation (AF) is the most common sustained cardiac arrhythmia in clinical practice and contributes to impaired quality of life, and increased morbidity and mortality. The mechanisms underlying both the initiation and perpetuation of AF are not well established but are thought to involve inflammation and oxidative stress. Furthermore, a number of studies have shown that concentrations of inflammatory mediators or markers, such as IL-6 and high-sensitivity CRP (hs-CRP), are increased in patients with AF. One mechanism that may mediate the effects of inflammation in AF is oxidative stress. Elevated inflammatory biomarkers are strongly associated with AF. Inflammation has important prognostic implications in AF; large prospective studies have shown that elevated hs-CRP levels correlated with risk factors for stroke and overall prognosis. The positive correlation between elevated levels of TNF-α and N-terminal pro-brain natriuretic peptide (NTpBNP) and severity of AF suggests that these biomarkers could be prognostic markers for AF in clinical practice (Li et al., 2010).

#### **6.6 Insulin resistance**

106 Oxidative Stress and Diseases

attention as a potential source of ROS in hypertension (HTN), followed by xanthine oxidase. Both endothelial cell- and leukocyte-associated NADPH-oxidase have been implicated in HTN-induced superoxide production, and there is evidence linking both cellular sources of the enzyme to activation of the angiotensin II type 1 receptor (AT1r) and to cytokines (TNF- ) derived from circulating immune cells (Crimi et al., 2007; Harrison & Gongora, 2009).

While some adipokines as leptin and adiponectin have been shown to promote the expression of endothelial cell adhesion molecules (CAMs) and leukocyte-endothelial cell adhesion (LECA) are known to exert an inhibitory effect on these responses. The absence of LECA in the microcirculation of obese mice under basal conditions suggests either that the pro- and anti-adhesive adipokines are in balance or that the systemic plasma levels achieved by these mediators do not cause overt inflammation in tissues distant from their source (adipose tissue). The latter possibility is supported by evidence of an increased sensitivity (priming) of endothelial cells and leukocytes in obese animals to inflammatory stimuli.

However, within the microvasculature of adipose tissue, a robust inflammatory response is noted under basal conditions, as reflected by an increased expression of the endothelial cell adhesion molecules ICAM-1 and E- and P-select in, with an accompanying recruitment of rolling and firmly adherent leukocytes, and the formation of platelet-leukocyte aggregates. The reduced LECA may be linked to adiponectin deficiency since the adipokine is a potent inhibitor of LECA and its production/release is diminished during adipogenesis (Singer &

Superoxide levels also are increased in stenotic aortic valves from humans. Heistad et al., (2009) found, in stenotic valves removed during surgical replacement of the aortic valve, that superoxide is increased greatly near calcified regions of the valve. Others authors (Miller et al., 2008) also found, in valves obtained at surgery or autopsy, that oxidative stress is increased in stenotic aortic valves. Thus, in calcified stenotic aortic valves as well as in atherosclerotic lesions, oxidative stress is increased. But, there are important differences in mechanisms that account for oxidative stress in aortic valves and in atherosclerotic arteries. In calcific aortic stenosis, increased production of superoxide may be mediated by "uncoupling" of NOS, as NOS primarily produces superoxide instead of nitric oxide NAD(P)H expression and activity do not appear to be increased in aortic valves (Miller et al., 2008). In striking contrast, increased expression and activity of NADPH-oxidase appears to be a major mechanism for oxidative stress in atherosclerotic lesions. Oxidative stress, in addition to contributing to fibrosis, may activate matrix metalloproteinases (MMPs) in the aortic valve and arteries. In the valve, MMPs may play a permissive role in expansion of calcification of the valve, and degraded fragments of collagen and elastin also may increase pro-calcific signaling in valvular interstitial cells. Activation of MMPs in arteries probably is

Atrial fibrillation (AF) is the most common sustained cardiac arrhythmia in clinical practice and contributes to impaired quality of life, and increased morbidity and mortality. The

Granger, 2007).

**6.5 Atrial fibrillation** 

**6.4 Oxidative stress in aortic valves** 

harmful in a different way, by contributing to plaque rupture.

A large number of studies have evidenced the pivotal role of oxidative stress in insulin resistance states such as metabolic syndrome, obesity, and T2D (Atabek et al., 2004; Block et al., 2002). Decreased antioxidant capacity, increased production of ROS with oxidation products of lipids, DNA, and proteins have been reported in plasma, urine, and various tissues, suggesting systemic and organ-specific oxidative stress. Recent evidence for systemic oxidative stress includes the detection of increased circulating and urinary levels of the lipid peroxidation product F2-isoprostane (8-epi-prostaglandin F2α) in both T1D and T2D patients (Davi et al., 2003). As described above, ROS and reactive nitrogen species (RNS) are able to directly modify the expression of adiponectin. Secreted almost exclusively from adipocytes, it is inversely correlated with fat mass in obesity and with its associated cardiovascular risk. It should be considered that, plasma and urinary lipid peroxidation markers indicative of systemic OS correlated with lower circulating adiponectin levels.

#### **7. Conclusion**

Figure 1 summarizes much of the content of this chapter, because it shows most of the cellular elements and signaling pathways in which highlights the participation of adipocytokines involved in the immune response and oxidative stress on the vascular endothelium. These alterations lead to development of atherosclerosis. And finally this endothelial damage, together with the increase in free radicals can cause multiorgan damage.

In conclusion, abnormal adipocytokine expression with consequent inflammation, oxidative stress itself may result from the inflammatory changes that occur in obesity. Therefore, a vicious cycle that provokes increased oxidative stress in obesity may exist. Reactive oxygen species that lead to increased oxidative stress can be generated in adipocytes and in other cell types such as leukocytes, all of which can be a source of increased oxidative stress in obese humans. Increased oxidative stress is independently associated with obesity measures including body mass index and waist-hip ratio. It is also associated with several CVD risk factors including smoking, blood glucose, and hyperlipidemia. Oxidative stress and increased adipocytokines may also promote endothelial dysfunction, atherogenesis, and coronary heart disease independent of traditional risk factors.

Adipocytokines, Oxidative Stress and Impaired Cardiovascular Functions 109

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Fig. 1. Impaired cardiovascular functions and adipocytokines actions on oxidative stress. This figure shows the majority of cellular elements and signaling pathways in which highlights the participation of adipocytokines (framed) involved in the immune response and oxidative stress on the vascular endothelium. These alterations lead to development of atherosclerosis. And finally, this endothelial dysfunction can generate harmful free radicals and cause tissue and multiorgan damage.

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Fig. 1. Impaired cardiovascular functions and adipocytokines actions on oxidative stress. This figure shows the majority of cellular elements and signaling pathways in which highlights the participation of adipocytokines (framed) involved in the immune response and oxidative stress on the vascular endothelium. These alterations lead to development of atherosclerosis. And finally, this endothelial dysfunction can generate harmful free radicals

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**6** 

 *USA* 

Mahdi Garelnabi1,

*2Department of Surgery,* 

*School of Health and Environment,* 

 *University of Massachusetts Lowell, MA,* 

*The Ohio State University, Columbus, OH,* 

**Role of Oxidized Lipids in Atherosclerosis** 

The role of oxidized lipids in cardiovascular diseases (CVD) has been investigated over the last three decades extensively. A number of studies have been carried out on the mechanisms, and pathways leading to the arterial atherosclerosis. These studies originated from the oxidation hypothesis of the atherosclerosis which was originally proposed more than 25 years ago (Steinberg et al., 1989), and since then experiments were performed by many investigators to further examine and explore the contribution of oxidation and oxidized lipids to cardiovascular diseases. Oxidized fatty acids in the ester and free forms, their decomposition products, cholesterol and its oxidized products, proteins with oxidized amino acid residues and cross-links, and polypeptides with varying extents of covalent modification with lipid oxidation products, and many others substances derived from oxidation have been the subject of detailed studies by many investigators. These products originated *in vivo* from oxidized lipoproteins and lipid membranes were linked to initiation and propagation of atherosclerosis (Zhang & Salomon, 2005; Mitra et al., 2011; Hulsmans et al., 2010). The effect of dietary oxidized fat as a contributor to the oxidative stress was also investigated by several groups including our group (Catapano et al., 2000; Drüeke et al., 2001; Garelnabi et al., 2008; Mitra et al., 2011). While there is a consensus in understanding of initial oxidative steps in the generation of early fatty streak lesions as well as the role of products of peroxidized lipid decomposition such as aldehydes in atherosclerosis, the role of further oxidation into neutral carboxylic acids is still obscure. In this chapter we will review the background of the oxidation theory of lipoproteins and the current state of the knowledge. We will review and summarizes data leading to the current understanding of the role of oxidized lipids in atherosclerosis and some pathways involved in this process. We will also discuss recent studies that elucidate factors leading to oxidative stress including chemical, physical and biological factors. In addition, we will explain the current knowledge of the use of antioxidants; and explain their benefits if any to inhibit oxidation of LDL. This part

**1. Introduction** 

will discuss in brief some selected clinical data.

Srikanth Kakumanu1 and Dmitry Litvinov2

*1Department of Clinical Laboratory and Nutritional Sciences,* 

reactive protein attenuates nitric oxide production and inhibits angiogenesis. Circulation, Vol. 106, pp. 913-919


### **Role of Oxidized Lipids in Atherosclerosis**

#### Mahdi Garelnabi1,

Srikanth Kakumanu1 and Dmitry Litvinov2 *1Department of Clinical Laboratory and Nutritional Sciences, School of Health and Environment, University of Massachusetts Lowell, MA, 2Department of Surgery, The Ohio State University, Columbus, OH, USA* 

#### **1. Introduction**

118 Oxidative Stress and Diseases

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Shaul, PW. & Mineo, C. (2008). The scavenger receptor class B type I adaptor protein PDZK1 maintains endothelial monolayer integrity. Circ Res, Vol. 102, No. The role of oxidized lipids in cardiovascular diseases (CVD) has been investigated over the last three decades extensively. A number of studies have been carried out on the mechanisms, and pathways leading to the arterial atherosclerosis. These studies originated from the oxidation hypothesis of the atherosclerosis which was originally proposed more than 25 years ago (Steinberg et al., 1989), and since then experiments were performed by many investigators to further examine and explore the contribution of oxidation and oxidized lipids to cardiovascular diseases. Oxidized fatty acids in the ester and free forms, their decomposition products, cholesterol and its oxidized products, proteins with oxidized amino acid residues and cross-links, and polypeptides with varying extents of covalent modification with lipid oxidation products, and many others substances derived from oxidation have been the subject of detailed studies by many investigators. These products originated *in vivo* from oxidized lipoproteins and lipid membranes were linked to initiation and propagation of atherosclerosis (Zhang & Salomon, 2005; Mitra et al., 2011; Hulsmans et al., 2010). The effect of dietary oxidized fat as a contributor to the oxidative stress was also investigated by several groups including our group (Catapano et al., 2000; Drüeke et al., 2001; Garelnabi et al., 2008; Mitra et al., 2011). While there is a consensus in understanding of initial oxidative steps in the generation of early fatty streak lesions as well as the role of products of peroxidized lipid decomposition such as aldehydes in atherosclerosis, the role of further oxidation into neutral carboxylic acids is still obscure. In this chapter we will review the background of the oxidation theory of lipoproteins and the current state of the knowledge. We will review and summarizes data leading to the current understanding of the role of oxidized lipids in atherosclerosis and some pathways involved in this process. We will also discuss recent studies that elucidate factors leading to oxidative stress including chemical, physical and biological factors. In addition, we will explain the current knowledge of the use of antioxidants; and explain their benefits if any to inhibit oxidation of LDL. This part will discuss in brief some selected clinical data.

Role of Oxidized Lipids in Atherosclerosis 121

When platelets interact with or adhere to sub-endothelial connective tissue, they are stimulated to release their granule contents. Endothelial cells normally prevent platelet adherence because of the non-thrombogenic character of their surface and their capacity to form antithrombotic substances (e.g., prostacyclin and heparin). When endothelium is injured, platelets are promoted to adhere to its surface and thus, the release of platelet constituents, although it is not clear that platelet adherence to modified endothelium is a common event (Ross, 1986). Several investigators have demonstrated that if platelets are absent from the site of endothelial injury, or if are prevented from the injury sites pharmacologically as in experimental models, then the intimal proliferative lesions that usually accompany such injury will not occur (Friedman et al., 1977; Haker et al., 1983). Oxidized low density lipoproteins (OxLDLs) have been shown to play a key role in the pathogenesis of atherosclerosis, since they are present in atherosclerotic lesions. Indeed, oxidized LDLs inhibit endothelium-dependent relaxation of the rabbit aorta in response to acetylcholine, as well as of porcine coronary artery in response to serotonin and platelets

The major constituents of plaques are lipid-laden foam cells are formed and their remains. Foam cells form when macrophages or other cells uptake an excessive amount of LDL, and die. An oxidative hypothesis of atherosclerosis was proposed in 1989 and suggested modification of LDL as a primary reason of foam cell formation and development of atherosclerosis (Steinberget al, 1989; Parthasarathy et al., 2010). A massive amount of confirming data was collected since then. It is well accepted now that oxidative processes

LDL is a microparticle consisting of one ApoB protein molecule and a mixture of triacylglycerol, cholesterol and its esters, phospholidpids, and vitamin E. Oxidation of LDL is a gradual process starting with oxidation of vitamin E and polyunsaturated fatty acids. Peroxides, the primary oxidation products, undergo further transformations with generation of aldehydes among other products. Aldehydes modify amino acid residues of ApoB, primarily lysine, resulting in malondialdehyde modified ApoB (MDA-ApoB) and 4 hydroxy-2-nonenal modified ApoB (4-HNE-ApoB). Biological effect of oxidized LDL varies greatly depending on the grade of oxidation. There are several terms for oxidized LDL that indicate the level of oxidation, such as MM-LDL (minimally modified LDL), fully oxidized LDL, and MDA-LDL (malondialdehyde-modified LDL). It is difficult to determine the level of oxidation in many cases. The term OxLDL (oxidized LDL) is used for any oxidized LDL

Development of atherosclerotic lesion starts with accumulation of OxLDL in intima, the innermost part of vessel, consisting of single layer of endothelial cells that rest on basement membrane. Intimal basement membrane separates endothelial cells and smooth muscle cells in arterial blood vessels. It consists of extracellular matrix, mostly collagen and

There is detectable level of OxLDL in circulating blood, and OxLDL is observed in vascular wall. Immunoglobulin M (IgM) is essential for noninflamatory clearance of OxLDL by macrophages. IgM co-localizes with CD68-positive macrophages in lesions. Double

proteoglycans, with sparse immune cells and smooth muscle cells (SMC) in it.

and oxidized lipids play pivotal role in initiation and progression of the disease.

(Tanner et al., 1990).

**2. Oxidation of LDL** 

regardless of the extent of oxidation.

#### **1.1 Atherosclerosis**

Atherosclerosis is the principal contributor to the pathogenesis of myocardial and cerebral infarction, gangrene, and loss of function in the extremities. The process, which under normal circumstances is a protective response to insults against the endothelium and smooth muscle cells of arterial walls, consists of the formation of fibrofatty and fibrous lesions, and is preceded and accompanied by inflammation. The advanced lesions of atherosclerosis become pathologic, and may cause occlusion of the affected artery, result from an excessive inflammatory-fibroproliferative response to numerous different forms of insult (Ross, 1986).

The earliest recognizable lesion of atherosclerosis is the so-called 'fatty streak', an aggregation of lipid-rich macrophages and T lymphocytes within the innermost layer of the arterial wall, the intima. The ubiquity of the atherosclerotic process is attested by the finding of fatty streaks in the coronary arteries of half of the autopsy specimens from children aged 10 to 14 years (WHO, 1985). Animal observations have shown that fatty streaks precede the development of intermediate lesions, which are composed of layers of macrophages and smooth muscle cells and, in turn, develop into the more advanced, complex, occlusive lesions called fibrous plaques (Fig 1). The fibrous plaques increase in size and, by projecting into the arterial lumen, may impede the flow of blood. They are covered by a dense cap of connective tissue with embedded smooth muscle cells that usually overly a core of lipid and necrotic debris (Garelnabi, 2010).

Most of the sudden deaths from myocardial infarcts are due to ruptures or fissures, particularly in the margins of the fibrous cap where there are more macrophages, resulting in hemorrhage into the plaque, thrombosis, and occlusion of the artery (Ross, 1993). As the process continues, migrating cells reach further beneath the arterial surface, where the monocytes become macrophages, accumulate lipid, become foam cells, and together with the accompanying lymphocytes, become the fatty streak. These often form at sites of preexisting collections of intimal smooth muscle. Thereafter, continued cell influx and proliferation lead to the more advanced lesions, distinguished by their fibrous character, and ultimately to the fibrous plaque (Ross, 1993).

Studies on animals with artificially induced hypercholesterolemia have confirmed that three processes are involved in the formation of atherosclerotic lesions : (1) The proliferation of smooth muscle cells, macrophages, and possibly lymphocytes; (2) the formation of a connective tissue matrix by smooth muscle cells comprised of elastic fiber proteins, collagen, and proteoglycans; and (3) the accumulation of lipid and mostly free esterified cholesterol in the surrounding matrix and the associate cells (Daley et al., 1994).

There are numerous signals, biochemical in nature, which underlie smooth muscle proliferation. Platelet derived growth factor (PDGF), the first postulated growth factor in atherogenesis is produced by many of the cells involved in the process (i.e., platelets, macrophages, endothelial cells and smooth muscle cells). Activated macrophages can also synthesize fibroblast growth factor (FGF), endothelial derived growth factor (EDGF), and transforming growth factor beta (-TGF). The combination of these growth factors has been shown to be extremely potent in stimulating the migration and proliferation of fibroblasts and smooth muscle cells, as well as the formation of connective tissue element.

Atherosclerosis is the principal contributor to the pathogenesis of myocardial and cerebral infarction, gangrene, and loss of function in the extremities. The process, which under normal circumstances is a protective response to insults against the endothelium and smooth muscle cells of arterial walls, consists of the formation of fibrofatty and fibrous lesions, and is preceded and accompanied by inflammation. The advanced lesions of atherosclerosis become pathologic, and may cause occlusion of the affected artery, result from an excessive inflammatory-fibroproliferative response to numerous different forms of

The earliest recognizable lesion of atherosclerosis is the so-called 'fatty streak', an aggregation of lipid-rich macrophages and T lymphocytes within the innermost layer of the arterial wall, the intima. The ubiquity of the atherosclerotic process is attested by the finding of fatty streaks in the coronary arteries of half of the autopsy specimens from children aged 10 to 14 years (WHO, 1985). Animal observations have shown that fatty streaks precede the development of intermediate lesions, which are composed of layers of macrophages and smooth muscle cells and, in turn, develop into the more advanced, complex, occlusive lesions called fibrous plaques (Fig 1). The fibrous plaques increase in size and, by projecting into the arterial lumen, may impede the flow of blood. They are covered by a dense cap of connective tissue with embedded smooth muscle cells that usually overly a core of lipid and

Most of the sudden deaths from myocardial infarcts are due to ruptures or fissures, particularly in the margins of the fibrous cap where there are more macrophages, resulting in hemorrhage into the plaque, thrombosis, and occlusion of the artery (Ross, 1993). As the process continues, migrating cells reach further beneath the arterial surface, where the monocytes become macrophages, accumulate lipid, become foam cells, and together with the accompanying lymphocytes, become the fatty streak. These often form at sites of preexisting collections of intimal smooth muscle. Thereafter, continued cell influx and proliferation lead to the more advanced lesions, distinguished by their fibrous character,

Studies on animals with artificially induced hypercholesterolemia have confirmed that three processes are involved in the formation of atherosclerotic lesions : (1) The proliferation of smooth muscle cells, macrophages, and possibly lymphocytes; (2) the formation of a connective tissue matrix by smooth muscle cells comprised of elastic fiber proteins, collagen, and proteoglycans; and (3) the accumulation of lipid and mostly free esterified cholesterol in

There are numerous signals, biochemical in nature, which underlie smooth muscle proliferation. Platelet derived growth factor (PDGF), the first postulated growth factor in atherogenesis is produced by many of the cells involved in the process (i.e., platelets, macrophages, endothelial cells and smooth muscle cells). Activated macrophages can also synthesize fibroblast growth factor (FGF), endothelial derived growth factor (EDGF), and transforming growth factor beta (-TGF). The combination of these growth factors has been shown to be extremely potent in stimulating the migration and proliferation of fibroblasts

and smooth muscle cells, as well as the formation of connective tissue element.

**1.1 Atherosclerosis** 

insult (Ross, 1986).

necrotic debris (Garelnabi, 2010).

and ultimately to the fibrous plaque (Ross, 1993).

the surrounding matrix and the associate cells (Daley et al., 1994).

When platelets interact with or adhere to sub-endothelial connective tissue, they are stimulated to release their granule contents. Endothelial cells normally prevent platelet adherence because of the non-thrombogenic character of their surface and their capacity to form antithrombotic substances (e.g., prostacyclin and heparin). When endothelium is injured, platelets are promoted to adhere to its surface and thus, the release of platelet constituents, although it is not clear that platelet adherence to modified endothelium is a common event (Ross, 1986). Several investigators have demonstrated that if platelets are absent from the site of endothelial injury, or if are prevented from the injury sites pharmacologically as in experimental models, then the intimal proliferative lesions that usually accompany such injury will not occur (Friedman et al., 1977; Haker et al., 1983). Oxidized low density lipoproteins (OxLDLs) have been shown to play a key role in the pathogenesis of atherosclerosis, since they are present in atherosclerotic lesions. Indeed, oxidized LDLs inhibit endothelium-dependent relaxation of the rabbit aorta in response to acetylcholine, as well as of porcine coronary artery in response to serotonin and platelets (Tanner et al., 1990).

#### **2. Oxidation of LDL**

The major constituents of plaques are lipid-laden foam cells are formed and their remains. Foam cells form when macrophages or other cells uptake an excessive amount of LDL, and die. An oxidative hypothesis of atherosclerosis was proposed in 1989 and suggested modification of LDL as a primary reason of foam cell formation and development of atherosclerosis (Steinberget al, 1989; Parthasarathy et al., 2010). A massive amount of confirming data was collected since then. It is well accepted now that oxidative processes and oxidized lipids play pivotal role in initiation and progression of the disease.

LDL is a microparticle consisting of one ApoB protein molecule and a mixture of triacylglycerol, cholesterol and its esters, phospholidpids, and vitamin E. Oxidation of LDL is a gradual process starting with oxidation of vitamin E and polyunsaturated fatty acids. Peroxides, the primary oxidation products, undergo further transformations with generation of aldehydes among other products. Aldehydes modify amino acid residues of ApoB, primarily lysine, resulting in malondialdehyde modified ApoB (MDA-ApoB) and 4 hydroxy-2-nonenal modified ApoB (4-HNE-ApoB). Biological effect of oxidized LDL varies greatly depending on the grade of oxidation. There are several terms for oxidized LDL that indicate the level of oxidation, such as MM-LDL (minimally modified LDL), fully oxidized LDL, and MDA-LDL (malondialdehyde-modified LDL). It is difficult to determine the level of oxidation in many cases. The term OxLDL (oxidized LDL) is used for any oxidized LDL regardless of the extent of oxidation.

Development of atherosclerotic lesion starts with accumulation of OxLDL in intima, the innermost part of vessel, consisting of single layer of endothelial cells that rest on basement membrane. Intimal basement membrane separates endothelial cells and smooth muscle cells in arterial blood vessels. It consists of extracellular matrix, mostly collagen and proteoglycans, with sparse immune cells and smooth muscle cells (SMC) in it.

There is detectable level of OxLDL in circulating blood, and OxLDL is observed in vascular wall. Immunoglobulin M (IgM) is essential for noninflamatory clearance of OxLDL by macrophages. IgM co-localizes with CD68-positive macrophages in lesions. Double

Role of Oxidized Lipids in Atherosclerosis 123

intima and becomes absorbed by macrophages through scavenger receptors. There are many scavenger receptors that vary in the substrate specificity, expression in different tissues, and biological roles. Some of them play essential role in atherosclerosis (Table 1). Excessive loading of macrophages by OxLDL convert them to dysfunctional "foam" cells. OxLDL itself or products of spontaneous or enzyme-assisted decomposition act as pro-

OxLDL are cytotoxic for all spectra of atherosclerosis-related cells: T-cells (Alcouffe et al., 1999), macrophages, endothelial cells, smooth muscle cells. OxLDL cytotoxicity in human fibroblasts is mediated through OxLDL-derived lipid peroxides and hydroperoxides, but

High load of OxLDL induces two separate lethal processes in macrophages. The first process is activation of caspases-3 in Fas-independent manner. Other caspases, caspase-6, caspase-8, caspase-9, are likely involved as well. It ultimately leads to apoptosis with characteristic DNA fragmentation. The second process is OxLDL-induced plasma membrane lysis (necrosis) mediated by reactive oxygen species (ROS). Both processes occur concurrently, however lysis

Caspase activation might contribute to macrophage death, however some experiments demonstrate that the extent of the activation is not enough for OxLDL cytotoxicity, since a higher level of caspase-3 activity through activation of Fas is not lethal for macrophages. At the same time inhibitors of caspase-3 do not suppress macrophage lysis by OxLDL, while peroxyl radical scavengers Trolox, and N,N'-diphenyl-1,4-phenylene diamine (DPPD) inhibit cytotoxicity of OxLDL. Generation of peroxyl radical as primary reactive oxygen species (ROS) in OxLDL-activated macrophages was confirmed with several specific ROS-sensitive fluorescent dyes. So, OxLDL cytotoxicity is mediated by peroxyl radicals, but not superoxide. ROS-mediated lysis and caspase activation are independent processes since inhibitors of caspase-3 do not suppress macrophage lysis by OxLDL, and Trolox does not inhibit caspase

activation when it inhibits OxLDL-induced macrophage lysis (Asmis & Begley, 2003).

In response to OxLDL, macrophages start to generate intracellularly an increased amount of ROS. Excessive load with OxLDL and ROS generation leads to necrosis of foam cells. There are several NADPH oxidases expressed in macrophages. Nox2 (Gp91phox), a hemecontaining subunit of NADPH oxidase, is the major source of ROS during phagocytosis. Nox2 likely does not contribute to atherosclerosis, since Nox2 knockout mouse does not

Nox4 is another NADPH oxidase. Protein expression of Nox4 and its binding partner p22phox in macrophages is increased by OxLDL but not by native LDL through MEK1/2 pathway. Inhibition of MEK1/2 or siRNA knockdown of Nox4 suppresses ROS production

NF-κB is a family of transcription factors and their precursors sharing Rel homology domain. They function as homo or heterodimers, such as RelA/p50. In resting cells, NF-κB

and macrophage death assessed by membrane integrity (Lee et al., 2010).

inflammatory, chemotactic, growth-promoting factors (Fig 1).

of plasma membrane is likely the actual reason for macrophages death.

**2.1 Induction of oxidative stress by OxLDL** 

slow development of lesions (Kirk et al., 2000).

**2.2 NF-κB response to OxLDL and atherosclerosis** 

not superoxide (Coffey et al., 1995).

knockout Ldlr-/- and soluble IgM-/- mice develop lesions seven time bigger than Ldlr-/ control. C1qa is a complement participating in IgM-mediated clearance. There is a pronounced increase in the size of aortic root lesion in double knockout Ldlr-/-, C1qa-/ mouse as compared to Ldlr-/- mouse/- (Lewis et al., 2009).

Immunization of atherosclerosis-prone Ldlr-/- mice with MDA-LDL or native LDL before feeding with cholesterol-rich atherogenic diet resulted in smaller lesion areas without significant reduction of plasma cholesterol (Freigang et al., 1998). Both type of immunization generated antibodies that recognize a wide pattern of modified and oxidized LDL likely because of some oxidation of LDL during immunization. Binding of OxLDL with antibodies demonstrated antiatherogenic effect, whether it limits the influx of OxLDL into artery wall or helps to clear retained OxLDL. Similar results were obtained in rabbit (Ameli et al., 1996).

While immunization with MDA-LDL prior or at initial stages of atherosclerosis suppresses growth of lesions in mouse and rabbit, there is a controversy in whether higher titer of antibodies to OxLDL in blood correlates with higher or lower grade of atherosclerosis (Palinski et al., 1995; Tsimikas et al., 2007, reviewed in Shoenfeld et al., 2004).

Fig. 1. **OxLDL effects and fate in healthy and atherosclerotic artery wall.** LDL (green circles) enter vessel wall and become gradually oxidized (depicted by changing circle color from green to red). In healthy artery tissue lymphocytes, primarily macrophages (Mɸ), uptake OxLDL, and egress the vessel to lymphatic system. The removal of OxLDL is impaired in atherosclerotic artery. Macrophages get overloaded with OxLDL and die generating foam cells. Overloaded macrophages release inflammatory signals that affect endothelial cells and patrolling leukocytes on the vessel surface (depicted with red arrows). Endothelial cells respond to accumulating OxLDL by inflammation as well.

Currently, the general consensus is that oxidation of LDL occurs mostly within vascular wall. Both native LDL and OxLDL are able to pass through endothelial layer passively through interendothelial junctions, or by endothelial transcytosis, an active transport process ( von Eckardstein & Rohrer, 2009). LDL and OxLDL are retained in intima through interaction of the LDL protein ApoB-100 and proteoglycans. LDL undergoes oxidation in

knockout Ldlr-/- and soluble IgM-/- mice develop lesions seven time bigger than Ldlr-/ control. C1qa is a complement participating in IgM-mediated clearance. There is a pronounced increase in the size of aortic root lesion in double knockout Ldlr-/-, C1qa-/-

Immunization of atherosclerosis-prone Ldlr-/- mice with MDA-LDL or native LDL before feeding with cholesterol-rich atherogenic diet resulted in smaller lesion areas without significant reduction of plasma cholesterol (Freigang et al., 1998). Both type of immunization generated antibodies that recognize a wide pattern of modified and oxidized LDL likely because of some oxidation of LDL during immunization. Binding of OxLDL with antibodies demonstrated antiatherogenic effect, whether it limits the influx of OxLDL into artery wall or helps to clear retained OxLDL. Similar results were obtained in rabbit (Ameli et al., 1996). While immunization with MDA-LDL prior or at initial stages of atherosclerosis suppresses growth of lesions in mouse and rabbit, there is a controversy in whether higher titer of antibodies to OxLDL in blood correlates with higher or lower grade of atherosclerosis

(Palinski et al., 1995; Tsimikas et al., 2007, reviewed in Shoenfeld et al., 2004).

Fig. 1. **OxLDL effects and fate in healthy and atherosclerotic artery wall.** LDL (green circles) enter vessel wall and become gradually oxidized (depicted by changing circle color from green to red). In healthy artery tissue lymphocytes, primarily macrophages (Mɸ), uptake OxLDL, and egress the vessel to lymphatic system. The removal of OxLDL is impaired in atherosclerotic artery. Macrophages get overloaded with OxLDL and die generating foam cells. Overloaded macrophages release inflammatory signals that affect endothelial cells and patrolling leukocytes on the vessel surface (depicted with red arrows).

Currently, the general consensus is that oxidation of LDL occurs mostly within vascular wall. Both native LDL and OxLDL are able to pass through endothelial layer passively through interendothelial junctions, or by endothelial transcytosis, an active transport process ( von Eckardstein & Rohrer, 2009). LDL and OxLDL are retained in intima through interaction of the LDL protein ApoB-100 and proteoglycans. LDL undergoes oxidation in

Endothelial cells respond to accumulating OxLDL by inflammation as well.

mouse as compared to Ldlr-/- mouse/- (Lewis et al., 2009).

intima and becomes absorbed by macrophages through scavenger receptors. There are many scavenger receptors that vary in the substrate specificity, expression in different tissues, and biological roles. Some of them play essential role in atherosclerosis (Table 1). Excessive loading of macrophages by OxLDL convert them to dysfunctional "foam" cells. OxLDL itself or products of spontaneous or enzyme-assisted decomposition act as proinflammatory, chemotactic, growth-promoting factors (Fig 1).

#### **2.1 Induction of oxidative stress by OxLDL**

OxLDL are cytotoxic for all spectra of atherosclerosis-related cells: T-cells (Alcouffe et al., 1999), macrophages, endothelial cells, smooth muscle cells. OxLDL cytotoxicity in human fibroblasts is mediated through OxLDL-derived lipid peroxides and hydroperoxides, but not superoxide (Coffey et al., 1995).

High load of OxLDL induces two separate lethal processes in macrophages. The first process is activation of caspases-3 in Fas-independent manner. Other caspases, caspase-6, caspase-8, caspase-9, are likely involved as well. It ultimately leads to apoptosis with characteristic DNA fragmentation. The second process is OxLDL-induced plasma membrane lysis (necrosis) mediated by reactive oxygen species (ROS). Both processes occur concurrently, however lysis of plasma membrane is likely the actual reason for macrophages death.

Caspase activation might contribute to macrophage death, however some experiments demonstrate that the extent of the activation is not enough for OxLDL cytotoxicity, since a higher level of caspase-3 activity through activation of Fas is not lethal for macrophages. At the same time inhibitors of caspase-3 do not suppress macrophage lysis by OxLDL, while peroxyl radical scavengers Trolox, and N,N'-diphenyl-1,4-phenylene diamine (DPPD) inhibit cytotoxicity of OxLDL. Generation of peroxyl radical as primary reactive oxygen species (ROS) in OxLDL-activated macrophages was confirmed with several specific ROS-sensitive fluorescent dyes. So, OxLDL cytotoxicity is mediated by peroxyl radicals, but not superoxide. ROS-mediated lysis and caspase activation are independent processes since inhibitors of caspase-3 do not suppress macrophage lysis by OxLDL, and Trolox does not inhibit caspase activation when it inhibits OxLDL-induced macrophage lysis (Asmis & Begley, 2003).

In response to OxLDL, macrophages start to generate intracellularly an increased amount of ROS. Excessive load with OxLDL and ROS generation leads to necrosis of foam cells. There are several NADPH oxidases expressed in macrophages. Nox2 (Gp91phox), a hemecontaining subunit of NADPH oxidase, is the major source of ROS during phagocytosis. Nox2 likely does not contribute to atherosclerosis, since Nox2 knockout mouse does not slow development of lesions (Kirk et al., 2000).

Nox4 is another NADPH oxidase. Protein expression of Nox4 and its binding partner p22phox in macrophages is increased by OxLDL but not by native LDL through MEK1/2 pathway. Inhibition of MEK1/2 or siRNA knockdown of Nox4 suppresses ROS production and macrophage death assessed by membrane integrity (Lee et al., 2010).

#### **2.2 NF-κB response to OxLDL and atherosclerosis**

NF-κB is a family of transcription factors and their precursors sharing Rel homology domain. They function as homo or heterodimers, such as RelA/p50. In resting cells, NF-κB

Role of Oxidized Lipids in Atherosclerosis 125

participates in tight adhesion of monocytes. VCAM-1 knockout is lethal for mouse; however a study of a transgenic mouse with suppressed expression of VCAM-1(D4D) demonstrated

While NF-κB pathway responds to OxLDL, activation of NF-κB stimulates expression of Lox-1 and OxLDL uptake. A study of transgenic ApoE-/-, SIRT1+/- mouse with decreased SIRT1 function revealed that NF-κB inhibition decreases expression of Lox-1 and Ox-LDL uptake. SIRT1, a NAD-dependent class III deacetylases, is known to inhibit NF-κB activity by deacetylating RelA/p65. Indeed transgenic ApoE-/-, SIRT1+/- mouse has decreased SIRT1 activity an increased level of Lox-1 in aorta, and develops atherosclerosis faster compared to ApoE-/-, SIRT1+/+ mouse. Experiments with bone marrow transplantation revealed that pro-atherogenic effect of decreased SIRT1 function is mostly associated with leukocytes. ApoE-/-, SIRT1+/- peritoneal thioglycolate-elicited macrophages uptake

It is believed that lipid peroxidation is involved in the oxidative modification of low density lipoprotein (LDL) and the formation of the potent oxidant peroxynitrite (ONOO) (Roger et al., 1994). Despite intensive research into this key step, the identity of the radical is still a mystery, especially for the in vivo situation. It may result from preformed or lipoxygenasederived lipid hydroperoxides or hydrogen peroxide, which decompose in the presence of metal ions to lipid alkoxyl radicals and lipid peroxyl radicals and to hydroxyl radical, respectively. Once formed, the carbon-centred PUFA radical reacts very quickly with molecular oxygen yielding a lipid peroxyl radical which in turn abstracts a hydrogen atom from an adjacent PUFA, yielding a lipid hydroperoxide and a new PUFA radical. It is the latter reaction that carries the lipid peroxidation chain. If no chain termination took place, a single initiating event could convert all LDL. The precise length of the chain, i.e., the number of PUFAs oxidized per one initiating radical depends on many factors especially on the antioxidants. The antioxidants of LDL compete with chain propagation by very efficiently

Lipid peroxidation can be measured in a laboratory setting by a variety of methods. Oxidized lipid extracts is measurable in spectrophotometer technique. Recent methods of analysis includes the free oxygen radicals monitor (FORM) system (Garelnabi et al, 2008), Electron Spin Resonance Spin Trapping Techniques (ESRT), and several other traditional techniques. Peroxidation of fatty acids containing three or more double bonds will produce malondialdehyde (MDA). Malondialdehyde produced by peroxidation can cause crosslinking and polymerization of membrane components (Nielsen, 1981). This can alter intrinsic membrane properties such as deformability, ion transport, enzyme activity, and the aggregation state of cell surface determinants. Because MDA is diffusible, it will also react with nitrogenous bases of DNA (Bruce & James 1982). Increased formation of MDA has been associated with arachidonic acid metabolism and platelet aggregation (Marie, 1979; Macfarlane et al., 1977; Garelnabi et al. 2008; Garelnabi et al. 2010). Experimental studies have shown that free radicals promote platelet aggregation and thrombosis and chain breaking antioxidants, such as vitamin E, inhibit or delay arterial thrombogenesis (Ikeda et

reduced lesion development (Cybulsky et al., 2001).

showed increased uptake of OxLDL (Stein et al., 2010).

**3. Lipid peroxidation: NO Implication** 

scavenging lipid peroxyl radicals.

al., 1994; Jourdan et al., 1995).

dimer is associated with IκB, an inhibitory subunit of NF-κB. There are several members in IκB family. The canonical pathway of NF-κB activation is IκB phosphorylation by activated IκB kinase complex consisting of IKKα and IKKβ subunits and regulatory protein NEMO. Phosphorylated IκB becomes ubiquitinated and undergoes degradation. Degradation of inhibitory subunit releases NF-κB dimer, which translocates from cytoplasm to nucleus and initiates transcription of target genes. Various signals activate IKK complex including tumor necrosis factor (TNF) and interleukin-1 (IL-1). In an alternative pathway, activated NF-κB inducing kinase (NIK) phosphorylates precursor protein p100 that results in ubiquitination and proteasomal processing of a precursor protein p100 into mature p52 subunit. The subunit binds with RelB, and RelB/p52 dimer is an active transcription factor. B-cell– activating factor and other stimuli can activate NIK and thus initiate the alternative pathway. Factors such as lipopolysaccharide (LPS), CD40 ligand can activate both pathways, canonical and alternative. However, there is no data yet on regulation of NF-κB via alternative pathway in smooth muscle cells, macrophages, and endothelial cells (de Winther et al., 2005). OxLDL initiates inflammatory response in endothelial cells and leukocytes. Inflamed cells induce factors that attract leukocytes. Activation of NF-κB is one of the pathways that are involved in atherosclerosis. Activation of this pathway is observed in lesions in endothelial cells, macrophages and SMC (Brand et al., 1996).

OxLDL exerts dual effect on NF-κB activation in monocytes and macrophages. It activates NF-κB in short term, and suppresses it in long term (Brand et al., 1997; Eligini et al., 2002). Activation of NF-κB by OxLDL in atherosclerotic endothelial cells is more stable. An essential mechanism of NF-κB activation is mediated through scavenger receptor LOX-1 (lectin-like oxidized low-density lipoprotein receptor 1). Binding of OxLDL to LOX-1 induces superoxide and hydrogen peroxide generation, and NF-κB activation trough activation of p38 MAP kinase, PI3K, ERK1/2 pathway (Cominacini et al., 2000; Tanigawa et al., 2006). Knockdown of LOX-1 gene suppresses endothelial cell injury measured as LDH release, abates expression of MCP-1 and decreases monocyte adhesion to endothelial cells (Li & Mehta, 2000). Knockout of LOX-1 in Ldlr-/- mouse suppresses activation of p38 MAPK, decreases NF-κB p65 protein level, and inhibits development of atherosclerosis (Mehta et al., 2007).

The importance of NF-κB in endothelial cells in progression of atherosclerosis is demonstrated in ApoE-/- mouse. NF-κB pathway was disrupted by ablation of NEMO/IKKγ or expression of dominant-negative IκBα in endothelial cells. In both cases the lesions developed slower than in control ApoE-/- mouse (Gareus et al., 2008).

Inflammation is central process in development of atherosclerosis. Presentation of P-, E-, Lselectins by endothelial cells initiates vascular recruitment of circulating monocytes through selectin ligands that are expressed on surface of leukocytes, such as PSGL-1 (Yang et al., 1999; Sperandio et al., 2003). Inhibition of leukocyte recruitment slows development of atherosclerosis. Indeed, P-selectin knockout mice have smaller lesions than control animals (Dong et al., 2000). NF-κB regulates expression of P-selectin and other inflammation-related genes including E-selectin, ICAM-1, VCAM-1, and MCP-1 (Cominacini et al., 1997).

MCP-1 is another cytokine essential for development of atherosclerosis: Ldlr-/- Mcp1-/ mouse has smaller lesions compare to Ldlr-/- (Gu et al., 1998). VCAM-1 on endothelial cells

dimer is associated with IκB, an inhibitory subunit of NF-κB. There are several members in IκB family. The canonical pathway of NF-κB activation is IκB phosphorylation by activated IκB kinase complex consisting of IKKα and IKKβ subunits and regulatory protein NEMO. Phosphorylated IκB becomes ubiquitinated and undergoes degradation. Degradation of inhibitory subunit releases NF-κB dimer, which translocates from cytoplasm to nucleus and initiates transcription of target genes. Various signals activate IKK complex including tumor necrosis factor (TNF) and interleukin-1 (IL-1). In an alternative pathway, activated NF-κB inducing kinase (NIK) phosphorylates precursor protein p100 that results in ubiquitination and proteasomal processing of a precursor protein p100 into mature p52 subunit. The subunit binds with RelB, and RelB/p52 dimer is an active transcription factor. B-cell– activating factor and other stimuli can activate NIK and thus initiate the alternative pathway. Factors such as lipopolysaccharide (LPS), CD40 ligand can activate both pathways, canonical and alternative. However, there is no data yet on regulation of NF-κB via alternative pathway in smooth muscle cells, macrophages, and endothelial cells (de Winther et al., 2005). OxLDL initiates inflammatory response in endothelial cells and leukocytes. Inflamed cells induce factors that attract leukocytes. Activation of NF-κB is one of the pathways that are involved in atherosclerosis. Activation of this pathway is observed

in lesions in endothelial cells, macrophages and SMC (Brand et al., 1996).

(Mehta et al., 2007).

OxLDL exerts dual effect on NF-κB activation in monocytes and macrophages. It activates NF-κB in short term, and suppresses it in long term (Brand et al., 1997; Eligini et al., 2002). Activation of NF-κB by OxLDL in atherosclerotic endothelial cells is more stable. An essential mechanism of NF-κB activation is mediated through scavenger receptor LOX-1 (lectin-like oxidized low-density lipoprotein receptor 1). Binding of OxLDL to LOX-1 induces superoxide and hydrogen peroxide generation, and NF-κB activation trough activation of p38 MAP kinase, PI3K, ERK1/2 pathway (Cominacini et al., 2000; Tanigawa et al., 2006). Knockdown of LOX-1 gene suppresses endothelial cell injury measured as LDH release, abates expression of MCP-1 and decreases monocyte adhesion to endothelial cells (Li & Mehta, 2000). Knockout of LOX-1 in Ldlr-/- mouse suppresses activation of p38 MAPK, decreases NF-κB p65 protein level, and inhibits development of atherosclerosis

The importance of NF-κB in endothelial cells in progression of atherosclerosis is demonstrated in ApoE-/- mouse. NF-κB pathway was disrupted by ablation of NEMO/IKKγ or expression of dominant-negative IκBα in endothelial cells. In both cases the

Inflammation is central process in development of atherosclerosis. Presentation of P-, E-, Lselectins by endothelial cells initiates vascular recruitment of circulating monocytes through selectin ligands that are expressed on surface of leukocytes, such as PSGL-1 (Yang et al., 1999; Sperandio et al., 2003). Inhibition of leukocyte recruitment slows development of atherosclerosis. Indeed, P-selectin knockout mice have smaller lesions than control animals (Dong et al., 2000). NF-κB regulates expression of P-selectin and other inflammation-related

MCP-1 is another cytokine essential for development of atherosclerosis: Ldlr-/- Mcp1-/ mouse has smaller lesions compare to Ldlr-/- (Gu et al., 1998). VCAM-1 on endothelial cells

lesions developed slower than in control ApoE-/- mouse (Gareus et al., 2008).

genes including E-selectin, ICAM-1, VCAM-1, and MCP-1 (Cominacini et al., 1997).

participates in tight adhesion of monocytes. VCAM-1 knockout is lethal for mouse; however a study of a transgenic mouse with suppressed expression of VCAM-1(D4D) demonstrated reduced lesion development (Cybulsky et al., 2001).

While NF-κB pathway responds to OxLDL, activation of NF-κB stimulates expression of Lox-1 and OxLDL uptake. A study of transgenic ApoE-/-, SIRT1+/- mouse with decreased SIRT1 function revealed that NF-κB inhibition decreases expression of Lox-1 and Ox-LDL uptake. SIRT1, a NAD-dependent class III deacetylases, is known to inhibit NF-κB activity by deacetylating RelA/p65. Indeed transgenic ApoE-/-, SIRT1+/- mouse has decreased SIRT1 activity an increased level of Lox-1 in aorta, and develops atherosclerosis faster compared to ApoE-/-, SIRT1+/+ mouse. Experiments with bone marrow transplantation revealed that pro-atherogenic effect of decreased SIRT1 function is mostly associated with leukocytes. ApoE-/-, SIRT1+/- peritoneal thioglycolate-elicited macrophages uptake showed increased uptake of OxLDL (Stein et al., 2010).

#### **3. Lipid peroxidation: NO Implication**

It is believed that lipid peroxidation is involved in the oxidative modification of low density lipoprotein (LDL) and the formation of the potent oxidant peroxynitrite (ONOO) (Roger et al., 1994). Despite intensive research into this key step, the identity of the radical is still a mystery, especially for the in vivo situation. It may result from preformed or lipoxygenasederived lipid hydroperoxides or hydrogen peroxide, which decompose in the presence of metal ions to lipid alkoxyl radicals and lipid peroxyl radicals and to hydroxyl radical, respectively. Once formed, the carbon-centred PUFA radical reacts very quickly with molecular oxygen yielding a lipid peroxyl radical which in turn abstracts a hydrogen atom from an adjacent PUFA, yielding a lipid hydroperoxide and a new PUFA radical. It is the latter reaction that carries the lipid peroxidation chain. If no chain termination took place, a single initiating event could convert all LDL. The precise length of the chain, i.e., the number of PUFAs oxidized per one initiating radical depends on many factors especially on the antioxidants. The antioxidants of LDL compete with chain propagation by very efficiently scavenging lipid peroxyl radicals.

Lipid peroxidation can be measured in a laboratory setting by a variety of methods. Oxidized lipid extracts is measurable in spectrophotometer technique. Recent methods of analysis includes the free oxygen radicals monitor (FORM) system (Garelnabi et al, 2008), Electron Spin Resonance Spin Trapping Techniques (ESRT), and several other traditional techniques. Peroxidation of fatty acids containing three or more double bonds will produce malondialdehyde (MDA). Malondialdehyde produced by peroxidation can cause crosslinking and polymerization of membrane components (Nielsen, 1981). This can alter intrinsic membrane properties such as deformability, ion transport, enzyme activity, and the aggregation state of cell surface determinants. Because MDA is diffusible, it will also react with nitrogenous bases of DNA (Bruce & James 1982). Increased formation of MDA has been associated with arachidonic acid metabolism and platelet aggregation (Marie, 1979; Macfarlane et al., 1977; Garelnabi et al. 2008; Garelnabi et al. 2010). Experimental studies have shown that free radicals promote platelet aggregation and thrombosis and chain breaking antioxidants, such as vitamin E, inhibit or delay arterial thrombogenesis (Ikeda et al., 1994; Jourdan et al., 1995).

Role of Oxidized Lipids in Atherosclerosis 127

The autoxidation of polyunsaturated lipids is an irreversible destructive process; and in tissues it may be associated with accelerated cell aging and premature cell death. Because such biological autoxidation is essentially slow process, the quantitative measurement of susceptibility to oxidation requires standard experimental stress conduction (Dildar et al.,

The biochemical defenses that protect organism from the ROS include both small molecules (low molecular weight compounds such as antioxidants and free radical scavengers) and complex enzyme systems. These defenses serve to lower concentrations of free radical

the cell, ROS will cause excessive damage to cell components. ROS scavengers have also been used to characterize the production, nature, and toxicity of free radical species in *in* 

A variety of molecules that preferentially partition into membranes function by reducing lipophilic free radical species to less toxic forms. Vitamin E (a series of isomers of

peroxy radicals, and other radical species. Ascorbate is proposed to have similar properties and may serve to maintain tocopherols in the reduced active form. Ascorbate serves as a water-soluble reductant and radical scavenger (Bruce & James 1982). The ascorbateglutathione pathway represent an avenue through which ascorbate consumed in H2O2 reduction get recycled at the expense of NADPH. In the first step of this pathway, H2O2 is reduced to water by ascorbate peroxidase (APX) using ascorbate as the electron donor. The oxidized ascorbate (monodehydroascorbate) is regenerated by monodehydroascorbate; a radical and if not rapidly reduced it disproportionates into ascorbate and dehydroascorbate. Dehydroascorbate is reduced to ascorbate by dehydroascorbate reductase at the expense of GSH, yielding oxidized glutathione GSSG which is reduced by glutathione reductase (GR) using NADPH as electron donor (Fig 2), (Blokhina and Fagerstedt KV, 2010; Palma et. al, 2009; Halliwell, 2009). Enzymatic ROS scavengers: Catalase and peroxidases lower the steady state concentration of H2O2 which is a precursor of potent radical species. Thus, the cytotoxic potential of H2O2 is in large part a function of intracellular catalase and peroxidase activities that scavenge H2O2, and concentration of free ions of transition metals that

isozymes are known, cellular GPx, extracellular GPx, and phospholipid hydroperoxide GPx, and each contains a selenocysteine in its catalytic center. Cellular GPx; the most characterized form, can react with hydrogen peroxide and organic peroxides but not lipid hydroperoxide (Michio et al., 1995). Platelet GPx has been shown to influence the platelet arachidonic acid metabolism by stimulating lipoxygenase and inhibiting cyclooxygenase, since oxidative stress enhances the arachidonic acid metabolism and thereby creates greater demands on the regulatory systems (Malmgren et al., 1990). Phospholipid hydroperoxide

), hydroxyl radical (

NO) hydroxyl radical (

OH from H2O2. Three glutathione peroxidase (GPx; EC1.11.1.9)

). If ROS generation exceeds defense capacity of

), and strong oxidants and precursors of free radicals such as hydrogen

OH), lipid peroxyl

OH), singlet oxygen (1O2), lipid

), nitric oxide (

1998).

**4. Cellular defenses against ROS** 

peroxide (H2O2) and peroxynitrite (ONOO

species such as superoxide (O2

*vitro* and *in vivo* systems.

promote generation of

**4.1 Lipid soluble scavengers** 

tocopherol) will reduce superoxide (O2

radicals (L-OO


Less studied scavenger receptors such as MARCO, SRCL (Class A), CD68 (Class D), SREC-1 (Class F), SR-PSOX/CXCL16 (Class G) are not included in the table. The table is based on review (Moore & Freeman, 2006)

Table 1. Scavenger receptors involved in atherosclerosis

**Other substrates Effect of** 

Apoptotic cells, beta-amyloid peptide, anionic phospholipids, advanced glycation end-products, Gram-negative and Gram-positive pathogen-related molecules

apoptotic cells, beta-amyloid, anionic

phospholipids, advanced glycation end-products, amyloid

Native LDL, HDL, apoptotic cells, beta-amyloid, anionic

phospholipids, advanced glycation end-products, thrombospondin-1, collagen, fatty acids, protozoan and bacterial peptides and lipopeptides

OxLDL Lox1 knockout

OxLDL Native LDL, HDL,

**knockout in mouse** 

Controversial results on atherosclerosis development in knockout of both SR-AI and SR-AII genes (Msr- /-) in Apo-/- or Ldlr-/- mice

Srb1 knockout in Apoe-/- or Ldlr- /- mouse promotes atherosclerosis

Knockout of Cd36 in Apoe-/ mouse partly protects from atherosclerosis

inhibits

2007)

atherosclerosis in Ldlr-/- mouse (Mehta et al,.

**Expression LDL-related** 

**substrates** 

Moderately oxidized LDL, POV-PC (1 palmytoyl-2-(5 oxovaleryl) snglycero-3 phosphocholine) ; does not bind acetylated LDL or extensively oxidized LDL

Less studied scavenger receptors such as MARCO, SRCL (Class A), CD68 (Class D), SREC-1 (Class F), SR-PSOX/CXCL16 (Class G) are not included in the table. The table is based on review (Moore &

Acetylated LDL, lower affinity for OxLDL; recognize modified ApoB

**Scavenger receptor** 

Class A: SR-AI, SR-AII

Class B: SR-B1 (and another minor splice variant of the same gene SR-B2)

Class B: CD36

Class E: LOX-1

Freeman, 2006)

Tissue macrophages, arterial endothelial cells, smooth muscle cells

Liver,

macrophages; adrenal glands, ovaries, and testes - reverse cholesterol transport

Macrophages, dendritic cells, endothelial cells

Endothelial cells,

SMC

macrophages,

Table 1. Scavenger receptors involved in atherosclerosis

The autoxidation of polyunsaturated lipids is an irreversible destructive process; and in tissues it may be associated with accelerated cell aging and premature cell death. Because such biological autoxidation is essentially slow process, the quantitative measurement of susceptibility to oxidation requires standard experimental stress conduction (Dildar et al., 1998).

#### **4. Cellular defenses against ROS**

The biochemical defenses that protect organism from the ROS include both small molecules (low molecular weight compounds such as antioxidants and free radical scavengers) and complex enzyme systems. These defenses serve to lower concentrations of free radical species such as superoxide (O2 ), nitric oxide ( NO) hydroxyl radical ( OH), lipid peroxyl radicals (L-OO ), and strong oxidants and precursors of free radicals such as hydrogen peroxide (H2O2) and peroxynitrite (ONOO ). If ROS generation exceeds defense capacity of the cell, ROS will cause excessive damage to cell components. ROS scavengers have also been used to characterize the production, nature, and toxicity of free radical species in *in vitro* and *in vivo* systems.

#### **4.1 Lipid soluble scavengers**

A variety of molecules that preferentially partition into membranes function by reducing lipophilic free radical species to less toxic forms. Vitamin E (a series of isomers of tocopherol) will reduce superoxide (O2 ), hydroxyl radical ( OH), singlet oxygen (1O2), lipid peroxy radicals, and other radical species. Ascorbate is proposed to have similar properties and may serve to maintain tocopherols in the reduced active form. Ascorbate serves as a water-soluble reductant and radical scavenger (Bruce & James 1982). The ascorbateglutathione pathway represent an avenue through which ascorbate consumed in H2O2 reduction get recycled at the expense of NADPH. In the first step of this pathway, H2O2 is reduced to water by ascorbate peroxidase (APX) using ascorbate as the electron donor. The oxidized ascorbate (monodehydroascorbate) is regenerated by monodehydroascorbate; a radical and if not rapidly reduced it disproportionates into ascorbate and dehydroascorbate. Dehydroascorbate is reduced to ascorbate by dehydroascorbate reductase at the expense of GSH, yielding oxidized glutathione GSSG which is reduced by glutathione reductase (GR) using NADPH as electron donor (Fig 2), (Blokhina and Fagerstedt KV, 2010; Palma et. al, 2009; Halliwell, 2009). Enzymatic ROS scavengers: Catalase and peroxidases lower the steady state concentration of H2O2 which is a precursor of potent radical species. Thus, the cytotoxic potential of H2O2 is in large part a function of intracellular catalase and peroxidase activities that scavenge H2O2, and concentration of free ions of transition metals that promote generation of OH from H2O2. Three glutathione peroxidase (GPx; EC1.11.1.9) isozymes are known, cellular GPx, extracellular GPx, and phospholipid hydroperoxide GPx, and each contains a selenocysteine in its catalytic center. Cellular GPx; the most characterized form, can react with hydrogen peroxide and organic peroxides but not lipid hydroperoxide (Michio et al., 1995). Platelet GPx has been shown to influence the platelet arachidonic acid metabolism by stimulating lipoxygenase and inhibiting cyclooxygenase, since oxidative stress enhances the arachidonic acid metabolism and thereby creates greater demands on the regulatory systems (Malmgren et al., 1990). Phospholipid hydroperoxide

Role of Oxidized Lipids in Atherosclerosis 129

the release of free calcium ions (Ca2+) from the mitochondrial matrix into the cell cytosol. Nitric oxide also reacts with lipophilic peroxyl radicals, important propagating species in biological chain reaction of lipid peroxidation, to generate alkyl peroxynitrites (LOONO).

and phagocyte adhesion to the endothelium. However, in atherosclerotic lesions excess

for irreversibly oxidation of thiols to higher oxidation states, but nitrosothiols can also form,

molecule such as nitrosothiol (Liu et al., 1994). Repeated exposure to ONOO results in a

There are a vast number of studies on the role of anti-oxidants particularly in the area of atherosclerosis and CVD. These studies are controversial, and do not provide clear evidences on the benefits of antioxidants for prevention or treatment of the diseases. Supplementation of antioxidant vitamins such as α-tocopherol, ascorbic acid and β-carotene used alone or in combination had long been considered to be cardio protective. However, controlled clinical trials using antioxidant vitamin supplements to prevent CVD have yielded conflicting results (Raghavamenon et al., 2009). While some secondary prevention interventions have been shown with α-tocopherol supplementation alone or in combination with ascorbic acid is reported to reduce CVD risk, other studies have shown no effect of α-

Vitamin E (α-tocopherol) is found in plant oils (Honarbakshsh & Schachter, 2009). This vitamin is extensively studied as a possible antioxidant agent against oxidation-induced cardiovascular diseases. Administration of 1000 IU/day α-tocopherol has been shown to reduce LDL oxidation (Princen et al., 1992). A human study shown that α-tocopherol supplementation of 150 IU/day to 1200 IU/day increases it level in plasma and in LDL in concentration-dependent manner. *In vitro* oxidation of LDL was partly inhibited in LDL with higher tocopherol content (Dieber-Rotheneder et al., 1991). α-Tocopherol is reported to reduce plasma OxLDL levels at 25 IU/day in both men and women, and the effect rises with increased supplementation until 800 IU/day (Princen et al., 1995). Tocopherol accumulation in monocytes decreases stress-induced adhesion of monocytes to endothelial cells (Islam et al., 1998; Devraj et al., 1996; Faruqi et al., 1994; Zapolska-Downar et al., 2000), which in turn inhibit the formation of atherosclerotic lesions. Overall, a number of *in vitro* studies demonstrate anti-atherogenic effect of vitamin E by decreasing the production of ROS, lipid oxidation, monocyte endothelial cell adhesion and cytokines secretion. However clinical

may cause loss of the modulatory action of

vasorelaxation occurs by a mechanism characteristic of release of

NO to stop lipid peroxidation.

NO donors. Indeed, when isolated vascular tissues are exposed to

which is pro-aggregatory and so could commit platelets in this environment

NO may lead to

NO with peroxyl radicals is

NO and at the same time

, a molecule responsible

NO from a carrier

NO inhibits platelet

. If LOONO derivatives can be metabolised

of FeS centres in enzymes. Persistent blockade of cytochrome c oxidase by

without the release of toxic free radicals then the reaction of

Protective mechanism: Several antioxidants can scavenge ONOO

progressive decrease in the efficiency of the vasorelaxing effect.

tocopherol supplementation in both primary and secondary prevention.

studies have not revealed anti-atherogenic effect in human (Yusuf et al., 2000).

**4.2 Benefits of antioxidants against lipid peroxidation** 

These appear far more stable than ONOO

potentially beneficial because it allows

to thrombus formation (Roger et al., 1994).

production of O2

and later may act as

yield ONOO

ONOO

glutathione peroxidase (PHGPx) is an intracellular antioxidant selenoenzyme which interacts directly with peroxidized phospholipids and cholesterol and cholesteryl esters (Imai and Nakagawa 2003) . Selenium (Se) is an essential micronutrient for animals and humans that exerts its biological functions through selenoproteins. These proteins contain Se in the form of selenocysteine (Sec), Phospholipid hydroperoxide glutathione peroxidase (PHGPx or GPx4, E.C. 1.11.1.12) is characterized by the presence of selenocysteine at the active site, and belongs to the important family of glutathione peroxidases (GPx). Since the discovery of PHGPx, a number of studies have demonstrated that this seleno-enzyme is essential to organisms. However on the other hand glutaghione-S-transferase possessing glutathione peroxidase activity toward lipid peroxides, but not having selenocysteine in its active site (Ursini et al. 1982; Yagi et. al 1996)

Fig. 2. The glutathione-ascorbate cycle.

Superoxide dismutases (SOD; EC 1.15.1.1) are metalloproteins that catalyze dismutation of superoxide anion radical to H2O2. Several types of SOD have been discovered. Mn-SOD (MW 85,000) has been found in mitochondria matrices and CuZn-SOD (MW 33,000) is contained in cellular cytosol. However, Mn-SOD and CuZn-SOD have been found also in extracellular fluids (Wesiger & Fridovich, 1973; Marklund et al., 1982). The superoxide radical has been reported as being produced from stimulated platelets (Levine et al., 1981) but its biological value in platelet function is not clearly understood (Violi et al., 1985). A decrease in cytosolic SOD the main defense against superoxide, could lead to increased cellular peroxides. Role of diet in the activity of Cu,Zn-SOD in platelets was studied and found to be influenced by the availability of Cu in diet (Catherine et al., 1993). Furthermore insufficiency in dietary copper was found to increase platelet thromboxane production, which in turn significantly correlated with endogenous lipid hydroperoxides. Evidence obtained from *in vitro* experiments indicates that superoxide dismutase may also inhibit platelet aggregation. That is, SOD given as adjuvant therapy with thrombolysis may both blunt free radicals mediated reperfusion injury and limit the incidence of spontaneous reocclusion after restoration of blood flow (Karin & Robert, 1993). Superoxide dismutase may protect endogenous NO from inactivation by scavenging superoxide anion. *In vitro* the inhibitory action of NO on platelet aggregation as well as their adhesion to endothelium induced by thrombin is potentiated by SOD consistent with its preventing inactivation of endothelium-derived NO (Meng et al., 1995).

Nitric oxide derived reactive nitrogen species (RNS) such as nitrogen dioxide ( NO2) and peroxynitrite (ONOO ) are indicated in the mediation of oxidative damage.Nitric oxide reacts very rapidly with oxygen radicals. Thus NO reacting with O2 generates peroxynitrite (IUPAC–recommended name is oxoperoxonitrate O=NOO ). The peroxynitrite anion (ONOO ) is relatively stable but its acid form (ONOOH) decays to nitrite with a half life of at most 1 sec at physiological pH and temperature (Ducrocq et al., 1999). Peroxynitrite mediates several of the cytotoxic effects of NO such as the destruction

glutathione peroxidase (PHGPx) is an intracellular antioxidant selenoenzyme which interacts directly with peroxidized phospholipids and cholesterol and cholesteryl esters (Imai and Nakagawa 2003) . Selenium (Se) is an essential micronutrient for animals and humans that exerts its biological functions through selenoproteins. These proteins contain Se in the form of selenocysteine (Sec), Phospholipid hydroperoxide glutathione peroxidase (PHGPx or GPx4, E.C. 1.11.1.12) is characterized by the presence of selenocysteine at the active site, and belongs to the important family of glutathione peroxidases (GPx). Since the discovery of PHGPx, a number of studies have demonstrated that this seleno-enzyme is essential to organisms. However on the other hand glutaghione-S-transferase possessing glutathione peroxidase activity toward lipid peroxides, but not having selenocysteine in its

Superoxide dismutases (SOD; EC 1.15.1.1) are metalloproteins that catalyze dismutation of superoxide anion radical to H2O2. Several types of SOD have been discovered. Mn-SOD (MW 85,000) has been found in mitochondria matrices and CuZn-SOD (MW 33,000) is contained in cellular cytosol. However, Mn-SOD and CuZn-SOD have been found also in extracellular fluids (Wesiger & Fridovich, 1973; Marklund et al., 1982). The superoxide radical has been reported as being produced from stimulated platelets (Levine et al., 1981) but its biological value in platelet function is not clearly understood (Violi et al., 1985). A decrease in cytosolic SOD the main defense against superoxide, could lead to increased cellular peroxides. Role of diet in the activity of Cu,Zn-SOD in platelets was studied and found to be influenced by the availability of Cu in diet (Catherine et al., 1993). Furthermore insufficiency in dietary copper was found to increase platelet thromboxane production, which in turn significantly correlated with endogenous lipid hydroperoxides. Evidence obtained from *in vitro* experiments indicates that superoxide dismutase may also inhibit platelet aggregation. That is, SOD given as adjuvant therapy with thrombolysis may both blunt free radicals mediated reperfusion injury and limit the incidence of spontaneous reocclusion after restoration of blood flow (Karin & Robert, 1993). Superoxide dismutase

induced by thrombin is potentiated by SOD consistent with its preventing inactivation of

nitrite with a half life of at most 1 sec at physiological pH and temperature (Ducrocq et al.,

Nitric oxide derived reactive nitrogen species (RNS) such as nitrogen dioxide (

peroxynitrite (IUPAC–recommended name is oxoperoxonitrate O=NOO

NO (Meng et al., 1995).

1999). Peroxynitrite mediates several of the cytotoxic effects of

reacts very rapidly with oxygen radicals. Thus

NO from inactivation by scavenging superoxide anion. *In vitro* the

NO2) and

). The

generates

NO such as the destruction

NO on platelet aggregation as well as their adhesion to endothelium

) are indicated in the mediation of oxidative damage.Nitric oxide

) is relatively stable but its acid form (ONOOH) decays to

NO reacting with O2

active site (Ursini et al. 1982; Yagi et. al 1996)

Fig. 2. The glutathione-ascorbate cycle.

may protect endogenous

inhibitory action of

endothelium-derived

peroxynitrite (ONOO

peroxynitrite anion (ONOO

of FeS centres in enzymes. Persistent blockade of cytochrome c oxidase by NO may lead to the release of free calcium ions (Ca2+) from the mitochondrial matrix into the cell cytosol. Nitric oxide also reacts with lipophilic peroxyl radicals, important propagating species in biological chain reaction of lipid peroxidation, to generate alkyl peroxynitrites (LOONO). These appear far more stable than ONOO . If LOONO derivatives can be metabolised without the release of toxic free radicals then the reaction of NO with peroxyl radicals is potentially beneficial because it allows NO to stop lipid peroxidation. NO inhibits platelet and phagocyte adhesion to the endothelium. However, in atherosclerotic lesions excess production of O2 may cause loss of the modulatory action of NO and at the same time yield ONOO which is pro-aggregatory and so could commit platelets in this environment to thrombus formation (Roger et al., 1994).

Protective mechanism: Several antioxidants can scavenge ONOO , a molecule responsible for irreversibly oxidation of thiols to higher oxidation states, but nitrosothiols can also form, and later may act as NO donors. Indeed, when isolated vascular tissues are exposed to ONOO vasorelaxation occurs by a mechanism characteristic of release of NO from a carrier molecule such as nitrosothiol (Liu et al., 1994). Repeated exposure to ONOO results in a progressive decrease in the efficiency of the vasorelaxing effect.

#### **4.2 Benefits of antioxidants against lipid peroxidation**

There are a vast number of studies on the role of anti-oxidants particularly in the area of atherosclerosis and CVD. These studies are controversial, and do not provide clear evidences on the benefits of antioxidants for prevention or treatment of the diseases. Supplementation of antioxidant vitamins such as α-tocopherol, ascorbic acid and β-carotene used alone or in combination had long been considered to be cardio protective. However, controlled clinical trials using antioxidant vitamin supplements to prevent CVD have yielded conflicting results (Raghavamenon et al., 2009). While some secondary prevention interventions have been shown with α-tocopherol supplementation alone or in combination with ascorbic acid is reported to reduce CVD risk, other studies have shown no effect of αtocopherol supplementation in both primary and secondary prevention.

Vitamin E (α-tocopherol) is found in plant oils (Honarbakshsh & Schachter, 2009). This vitamin is extensively studied as a possible antioxidant agent against oxidation-induced cardiovascular diseases. Administration of 1000 IU/day α-tocopherol has been shown to reduce LDL oxidation (Princen et al., 1992). A human study shown that α-tocopherol supplementation of 150 IU/day to 1200 IU/day increases it level in plasma and in LDL in concentration-dependent manner. *In vitro* oxidation of LDL was partly inhibited in LDL with higher tocopherol content (Dieber-Rotheneder et al., 1991). α-Tocopherol is reported to reduce plasma OxLDL levels at 25 IU/day in both men and women, and the effect rises with increased supplementation until 800 IU/day (Princen et al., 1995). Tocopherol accumulation in monocytes decreases stress-induced adhesion of monocytes to endothelial cells (Islam et al., 1998; Devraj et al., 1996; Faruqi et al., 1994; Zapolska-Downar et al., 2000), which in turn inhibit the formation of atherosclerotic lesions. Overall, a number of *in vitro* studies demonstrate anti-atherogenic effect of vitamin E by decreasing the production of ROS, lipid oxidation, monocyte endothelial cell adhesion and cytokines secretion. However clinical studies have not revealed anti-atherogenic effect in human (Yusuf et al., 2000).

Role of Oxidized Lipids in Atherosclerosis 131

while antioxidant supplementation trials have been found to be largely ineffective in preventing cardiovascular outcomes, other interventions including aerobic exercise training and pharmacological treatment with lipid and blood pressure-lowering medications may have significant antioxidant effects that are related to reductions in CVD risk. Another study have shown that oxidized lipoprotein(a) is significantly correlated with blood glucose level among healthy young women, suggesting that lipoprotein(a) may be oxidized with increased glucose

There is some controversy on the role of antioxidants on development of atherosclerosis. A number of clinical studies have demonstrated an anti-atherosclerotic effect of antioxidants while a group of other studies do not see any appreciable benefit of the use of antioxidants. The following are examples of these studies that have suggested an inhibiting effect of antioxidants on lesion development. Gey & Puska (1989) have reported that vitamin E and A concentrations in the plasma were inversely proportional to cardiovascular risks. A study of 667 cases of atherosclerosis-induced coronary disease developed in originally healthy (not diagnosed with coronary heart disease, diabetes, or hypercholesterolemia) 39,910 US men have shown a protective effect of vitamin E but not vitamin C. Carotene appeared to be protective in non-smoking men, however increased the risk of coronary disease among smokers (Rimm et al., 1993). A protective effect of vitamin E was observed in similar study of 87,245 women developed 552 cases of major coronary disease in eight years (Stampher et

However a large the Heart Outcomes Prevention Evaluation (HOPE) study did not show any anti-atherogenic effect of vitamin E (Yusuf et al., 2000). Subjects who were taking vitamin E and placebo developed atherosclerosis-related diseases such as myocardial infarction, stroke, unstable angina, congestive heart failure at the same rate. Potential explanation for the failure of antioxidants in clinical studies may include the type of dose, duration, time of introduction, i.e. stages of the disease at which the treatment/supplementation were introduced and the selection of an optimal doses of antioxidants. Also, most of the studies did not measure the oxidative stress markers in the

Research has provided strong evidence that LDL oxidation plays an important role in the pathogenesis of atherosclerosis and cardiovascular diseases. The involvement of lipid peroxidation in the propagation of the disease is well supported by clinical and scientific research using cell culture and animal models; these studies clearly point that modification of the LDL and the accompanied oxidative damage trigger an inflammation response that mediate the development of the atherosclerosis. One may assume that antioxidants should inhibit the oxidative damage and slow the inflammation processes that lead to CVD and associated with metabolic disorders. However despite of some positive findings, antioxidant compounds did not consistently prove to be potent protective agents against atherosclerosis. In animal atherosclerosis, which is studied in the short term, the emphasis is on establishing the lesions. Thus, antioxidants, such as α-tocopherol, might affect predominantly the initial formation and progression of the lesion. In humans, particularly in those who already have clinically significant events, the early steps might have already occurred. In such cases, αtocopherol and similar antioxidants could affect the conversion of aldehydes into carboxylic

concentration even within the normal glucose level (Kotani et al., 2010).

plasma to take it into account (Parthasarathy et al., 2001).

al., 1993).

Vitamin C (ascorbic acid) is principally found in citrus fruits, broccoli, red pepper, and cauliflowers, etc. Ascorbate acts in combination with vitamin E and beta–carotene to protect them from excretion and recycle them for further use. It is also reported to inhibit OxLDL formation indirectly by protecting vitamin E and beta-carotene (Jialal & Grundy, 1991; Kagan et al., 1992). Apart from this vitamin C is reported to inhibit endothelial apoptosis initiated by inflammatory cytokines *in vitro*, and reduces circulating apoptotic microparticles in human (Rössig et al., 2001). Adhesion proteins such as ICAM-1 can be involved in atherosclerosis. Ascorbate supplementation of subjects with low baseline level of this vitamin suppresses mRNA and protein expression of ICAM-1 in monocytes (Rayment et al, 2003). While these and other studies suggest that vitamin C might have antiatherogenic effect, there is no conclusive clinical evidence of such effect.

β-Carotene is indicated in preventing oxidation of lipids which might decrease atherosclerotic lesions formation. β-Carotene is proposed to be efficient scavenger of singlet oxygen and it attenuates oxidative stress, however it does not directly inhibit lipid peroxidation (Briviba et al., 2004).

Polyphenols are another group of antioxidants which are abundant in vegetables and fruits and are found to reduce the risk of CVD (Naderi et al., 2003). They contain both hydrophilic and hydrophobic moieties (Woodman & Chan, 2004). Polyphenols are suggested to inhibit lipid peroxidation (Madrau et al, 2009). It has also been reported that flavonoids chelates copper and iron ions, rendering them inactive to participate in free radical generating reactions (Fernandez et al., 2002). Polyphenols are also known to inhibit enzymes responsible for generation of ROS such as NADPH oxidase, lipoxygenase, phospholipase A2, and xanthine oxidase (Rice-Evans et al., 1997). Indirectly inhibiting the formation of OxLDL, the benefits of flavonoids goes beyond the protection against LDL oxidation to protect the HDL-associated paraoxonase activity (Patel et al., 2007). The antiatherogenic effect of mulberry leaf extracts (MLE) and the polyphenolic extracts (MLPE), which contain polyphenols including quercetin (11.70%), naringenin (9.01%) and gallocatechin gallate (10.02%) was studied by Yang et al. 2011. Both MLE and MLPE inhibited the oxidation and lipid peroxidation of LDL, while MLPE was shown to be more potent.

#### **5. Clinical studies: OxLDL and antioxidants**

A number of studies have demonstrated an association of circulating OxLDL with atherosclerosis disease (Itabe & Ueda, 2007; Hulthe & Fagerberg, 2002). The size of LDL particles might have an effect on LDL oxidation. Smaller LDL was associated with higher level of OxLDL. However the association was observed in diabetic subjects, but not in nondiabetic subjects (Scheffer et al., 2003).

OxLDL level normalized to LDL or ApoB protein levels was increased in diabetic subject with macrovascular diseases compared to diabetic subjects without such diseases. Increased OxLDL normalized level was associated with TT genotype of 108C/T polymorphism in PON1 promoter with lower level of expression of the gene (Tsuzura et al., 2004; Brinkley et al., 2009) have demonstrated for the first time that plasma OxLDL levels are related to arterial stiffness in elderly men and women; suggesting that the oxidative modification of LDL may be associated with changes in the elastic properties of blood vessels. Their findings suggest that

Vitamin C (ascorbic acid) is principally found in citrus fruits, broccoli, red pepper, and cauliflowers, etc. Ascorbate acts in combination with vitamin E and beta–carotene to protect them from excretion and recycle them for further use. It is also reported to inhibit OxLDL formation indirectly by protecting vitamin E and beta-carotene (Jialal & Grundy, 1991; Kagan et al., 1992). Apart from this vitamin C is reported to inhibit endothelial apoptosis initiated by inflammatory cytokines *in vitro*, and reduces circulating apoptotic microparticles in human (Rössig et al., 2001). Adhesion proteins such as ICAM-1 can be involved in atherosclerosis. Ascorbate supplementation of subjects with low baseline level of this vitamin suppresses mRNA and protein expression of ICAM-1 in monocytes (Rayment et al, 2003). While these and other studies suggest that vitamin C might have anti-

β-Carotene is indicated in preventing oxidation of lipids which might decrease atherosclerotic lesions formation. β-Carotene is proposed to be efficient scavenger of singlet oxygen and it attenuates oxidative stress, however it does not directly inhibit lipid

Polyphenols are another group of antioxidants which are abundant in vegetables and fruits and are found to reduce the risk of CVD (Naderi et al., 2003). They contain both hydrophilic and hydrophobic moieties (Woodman & Chan, 2004). Polyphenols are suggested to inhibit lipid peroxidation (Madrau et al, 2009). It has also been reported that flavonoids chelates copper and iron ions, rendering them inactive to participate in free radical generating reactions (Fernandez et al., 2002). Polyphenols are also known to inhibit enzymes responsible for generation of ROS such as NADPH oxidase, lipoxygenase, phospholipase A2, and xanthine oxidase (Rice-Evans et al., 1997). Indirectly inhibiting the formation of OxLDL, the benefits of flavonoids goes beyond the protection against LDL oxidation to protect the HDL-associated paraoxonase activity (Patel et al., 2007). The antiatherogenic effect of mulberry leaf extracts (MLE) and the polyphenolic extracts (MLPE), which contain polyphenols including quercetin (11.70%), naringenin (9.01%) and gallocatechin gallate (10.02%) was studied by Yang et al. 2011. Both MLE and MLPE inhibited the oxidation and

A number of studies have demonstrated an association of circulating OxLDL with atherosclerosis disease (Itabe & Ueda, 2007; Hulthe & Fagerberg, 2002). The size of LDL particles might have an effect on LDL oxidation. Smaller LDL was associated with higher level of OxLDL. However the association was observed in diabetic subjects, but not in non-

OxLDL level normalized to LDL or ApoB protein levels was increased in diabetic subject with macrovascular diseases compared to diabetic subjects without such diseases. Increased OxLDL normalized level was associated with TT genotype of 108C/T polymorphism in PON1 promoter with lower level of expression of the gene (Tsuzura et al., 2004; Brinkley et al., 2009) have demonstrated for the first time that plasma OxLDL levels are related to arterial stiffness in elderly men and women; suggesting that the oxidative modification of LDL may be associated with changes in the elastic properties of blood vessels. Their findings suggest that

atherogenic effect, there is no conclusive clinical evidence of such effect.

lipid peroxidation of LDL, while MLPE was shown to be more potent.

**5. Clinical studies: OxLDL and antioxidants** 

diabetic subjects (Scheffer et al., 2003).

peroxidation (Briviba et al., 2004).

while antioxidant supplementation trials have been found to be largely ineffective in preventing cardiovascular outcomes, other interventions including aerobic exercise training and pharmacological treatment with lipid and blood pressure-lowering medications may have significant antioxidant effects that are related to reductions in CVD risk. Another study have shown that oxidized lipoprotein(a) is significantly correlated with blood glucose level among healthy young women, suggesting that lipoprotein(a) may be oxidized with increased glucose concentration even within the normal glucose level (Kotani et al., 2010).

There is some controversy on the role of antioxidants on development of atherosclerosis. A number of clinical studies have demonstrated an anti-atherosclerotic effect of antioxidants while a group of other studies do not see any appreciable benefit of the use of antioxidants. The following are examples of these studies that have suggested an inhibiting effect of antioxidants on lesion development. Gey & Puska (1989) have reported that vitamin E and A concentrations in the plasma were inversely proportional to cardiovascular risks. A study of 667 cases of atherosclerosis-induced coronary disease developed in originally healthy (not diagnosed with coronary heart disease, diabetes, or hypercholesterolemia) 39,910 US men have shown a protective effect of vitamin E but not vitamin C. Carotene appeared to be protective in non-smoking men, however increased the risk of coronary disease among smokers (Rimm et al., 1993). A protective effect of vitamin E was observed in similar study of 87,245 women developed 552 cases of major coronary disease in eight years (Stampher et al., 1993).

However a large the Heart Outcomes Prevention Evaluation (HOPE) study did not show any anti-atherogenic effect of vitamin E (Yusuf et al., 2000). Subjects who were taking vitamin E and placebo developed atherosclerosis-related diseases such as myocardial infarction, stroke, unstable angina, congestive heart failure at the same rate. Potential explanation for the failure of antioxidants in clinical studies may include the type of dose, duration, time of introduction, i.e. stages of the disease at which the treatment/supplementation were introduced and the selection of an optimal doses of antioxidants. Also, most of the studies did not measure the oxidative stress markers in the plasma to take it into account (Parthasarathy et al., 2001).

Research has provided strong evidence that LDL oxidation plays an important role in the pathogenesis of atherosclerosis and cardiovascular diseases. The involvement of lipid peroxidation in the propagation of the disease is well supported by clinical and scientific research using cell culture and animal models; these studies clearly point that modification of the LDL and the accompanied oxidative damage trigger an inflammation response that mediate the development of the atherosclerosis. One may assume that antioxidants should inhibit the oxidative damage and slow the inflammation processes that lead to CVD and associated with metabolic disorders. However despite of some positive findings, antioxidant compounds did not consistently prove to be potent protective agents against atherosclerosis. In animal atherosclerosis, which is studied in the short term, the emphasis is on establishing the lesions. Thus, antioxidants, such as α-tocopherol, might affect predominantly the initial formation and progression of the lesion. In humans, particularly in those who already have clinically significant events, the early steps might have already occurred. In such cases, αtocopherol and similar antioxidants could affect the conversion of aldehydes into carboxylic

Role of Oxidized Lipids in Atherosclerosis 133

The low density lipoprotein oxidation hypothesis is pivotal to the explanation of the formation of fatty streak lesions. A wide range of atherogenic processes has been reported to be influenced by OxLDL and its components. The presence of OxLDL in lesions and plasma of patients with various forms of coronary artery diseases and other related metabolic disorder confirms the role of oxidized lipids in atherosclerosis. This conclusion led to numerous studies on the role of antioxidants in the prevention or treatment of atherosclerosis. However they did not yield uniformed outcome on the role of antioxidants in suppressing of the atherosclerotic process. Possible reasons might include discrepancies in experimental models, study designs, and schemes of treatment. Results shown in cell culture or animal models do not necessarily translate to similar results in human due to the major difference between the atherosclerosis development and stages in the animal models and human. Another factor that has not been tested yet is a possible inhibition of oxidation of OxLDL-released aldehydes by antioxidants. If oxidation of aldehydes is inhibited, they modify proteins and cause wide spectra of biological effects that exaggerate atherosclerotic processes. The future studies on the role of antioxidants in atherosclerosis should take in

Alcouffe J, Caspar-Bauguil S, Garcia V, Salvayre R, Thomsen M, Benoist H. (1999). Oxidized

mononuclear cells and in the Jurkat T-cell line. *J Lipid Res*.;40(7):1200-10. Ameli S, Hultgårdh-Nilsson A, Regnström J, Calara F, Yano J, Cercek B, Shah PK, Nilsson J.

low density lipoproteins induce apoptosis in PHA-activated peripheral blood

(1996). Effect of immunization with homologous LDL and oxidized LDL on early

155 Tsuzura et al., 2004

391 Hulthe & Fagerberg, 2002

1889 Holvoet et al., 2008

116 Scheffer et al., 2003

9541 Yusuf et al., 2000

Clinical study Findings No of patients References

OxLDL increased in subjects with PON1 genotype that lead to decreased expression of PON1

syndrome and in abdominal obesity, hyperglycemia and hypertriglyceridemia

higher level of OxLDL

development of CVD

Table summarizes some clinical studies measured OxLDL in plasma

Smaller LDL are associated with

protein

CARDIA study OxLDL indication metabolic

HOPE Study No effect of vitamin E on

Table 3. Clinical studies on OxLDL

consideration these factors.

**7. References** 

**6. Conclusions and perspectives** 

AIR study OxLDL role in atherosclerosis and inflammation

Department of Internal Medicine, Kochi Medical School, Kochi, Japan.

Metabolic Laboratory, Department of Clinical

Chemistry study, Netherlands

acids. The latter, are presumed to be nonatherogenic and are easily degraded *via* fatty-acid degradation pathways (Raghavamenon et al., 2009). Based on these arguments it may be necessary for the scientific community to revisit the topic and investigate in well structured studies the type, dose, duration of the anitoxidants on a well defined population of subjects with various stages of CVD and its associated metabolic disorders such as diabetes, obesity and hyperlipidemia.


The table describes the currently investigated antioxidants and their relation to markers of CVD.

Table 2. Role of Antioxidants in Cardiovascular Disease

acids. The latter, are presumed to be nonatherogenic and are easily degraded *via* fatty-acid degradation pathways (Raghavamenon et al., 2009). Based on these arguments it may be necessary for the scientific community to revisit the topic and investigate in well structured studies the type, dose, duration of the anitoxidants on a well defined population of subjects with various stages of CVD and its associated metabolic disorders such as diabetes, obesity

> trapper of singlet oxygen, acts against LDL oxidation

other anti-oxidants such as Vitamin E; inhibit formation of OxLDL, Il-1β secretion or chemokines and monocyte – endothelial cell adhesion and β-carotene which are anti-

inhibit formation of OxLDL, Il-1β secretion or chemokines and monocyte –endothelial

peroxidase. Has antioxidant

dismutase. Protects cells from

Activation of NF-κB, which is involved in development of

oxidative damage by reactive

The table describes the currently investigated antioxidants and their relation to markers of CVD.

ions, scavenging of ROS, inhibiting lipid peroxidation Protects anti-oxidant

oxidative damage.

and inhibition ROS.

atherosclerosis.

lipid peroxidation

nitrogen species.

Honarbakshsh & Schachter, 2009.

Honarbakshsh & Schachter, 2009.

Honarbakshsh & Schachter, 2009.

Zapolska-Downar et al., 2000

Wongcharoen & Phrommintikul,

Princen et al., 1992

Dunstan et al., 2007

Jialal & Grundy, 1991. Kagan et al, 1992. Rössig et al, 2001. Gokce et al., 1999, Rayment et al., 2003. Heller et al., 1999

Dunstan et al., 2007

Princen et al., 1992. Islam et al., 1998. Devraj et al., 1996. Faruqi et al, 1994.

Michiels et al., 1994

Michiels et.al., 1994

Cho et al., 2003

Martin, 2010

Ramprasath & Jones, 2010

2009

Anti-oxidants Mechanism References

atherogenic

cell adhesion

capacity.

enzymes.

β-carotene Scavenging ROS and excellent

Vitamin C Scavenging ROS, reactivating

Vitamin E Scavenging ROS, reported to

Selenium Cofactor for glutathione

Zinc Cofactor for superoxide

Curcumin Chelating of iron and copper

Quercetin Scavenging of metals ions,

Resevetrol Inhibits ROS production and

Ergothioneine Protects endothelial cells from

Table 2. Role of Antioxidants in Cardiovascular Disease

and hyperlipidemia.


Table summarizes some clinical studies measured OxLDL in plasma

Table 3. Clinical studies on OxLDL

#### **6. Conclusions and perspectives**

The low density lipoprotein oxidation hypothesis is pivotal to the explanation of the formation of fatty streak lesions. A wide range of atherogenic processes has been reported to be influenced by OxLDL and its components. The presence of OxLDL in lesions and plasma of patients with various forms of coronary artery diseases and other related metabolic disorder confirms the role of oxidized lipids in atherosclerosis. This conclusion led to numerous studies on the role of antioxidants in the prevention or treatment of atherosclerosis. However they did not yield uniformed outcome on the role of antioxidants in suppressing of the atherosclerotic process. Possible reasons might include discrepancies in experimental models, study designs, and schemes of treatment. Results shown in cell culture or animal models do not necessarily translate to similar results in human due to the major difference between the atherosclerosis development and stages in the animal models and human. Another factor that has not been tested yet is a possible inhibition of oxidation of OxLDL-released aldehydes by antioxidants. If oxidation of aldehydes is inhibited, they modify proteins and cause wide spectra of biological effects that exaggerate atherosclerotic processes. The future studies on the role of antioxidants in atherosclerosis should take in consideration these factors.

#### **7. References**


Role of Oxidized Lipids in Atherosclerosis 135

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**7** 

**Oxidative Damage in Cardiac** 

Juliana C. Fantinelli\*, Claudia Caldiz\*,

*Centro de Investigaciones Cardiovasculares,* 

 **Effect of Ageing** 

*Facultad de Ciencias Médicas, Universidad Nacional de La Plata* 

 *Argentina* 

 **Tissue from Normotensive and** 

María Cecilia Álvarez, Carolina D. Garciarena,

**Spontaneously Hypertensive Rats:** 

Gladys E. Chiappe de Cingolani and Susana M. Mosca

The spontaneously hypertensive rat (SHR) is a laboratory model of naturally developing hypertension and heart failure that appears to be similar in many aspects to essential hypertension in humans (Trippodo & Frohlich, 1981). Systolic blood pressure in SHR rapidly increases during 5 to 10 weeks of age and develops cardiac hypertrophy between 9 and 12 weeks of age (Shimamoto et al., 1982). Increasing evidence from different experimental models supports the concept that oxidative stress contributes to the pathogenesis of myocardial hypertrophy and in the process of myocardial remodeling

The oxidative stress is the result of an increase of reactive oxygen species (ROS) and/or inadequate antioxidant defense mechanisms. It has been shown that an increase in the activity and expression of myocardial NAD(P)H oxidase (NOX) is the main source of ROS in cardiac hypertrophy (Bendall et al., 2002; Griendling et al., 2000; Xiao et al et al., 2002). However, existing data about the antioxidant status in hypertension are inconsistent. Some studies have shown that the activities of one or more antioxidant enzymes are lower (Ito et al, 1995; Newaz & Nawal, 1999), higher (Czonka et al., 2000) or without changes (Gómez-Amores et al., 2006; Girard et al, 2005) compared with normotensive controls. Although the underlying causes of these discrepancies are unknown, it may be possibly due to the use of different hypertension models, animals at different hypertensive stages and/or different

On the other hand, ROS are thought to be a key mechanism in the aging process (Beckman & Ames, 1998; Colavitti & Finkel, 2004; Harman, 1988) and there are arguments that NOX-

leading to heart failure (Yücel et al., 1998; Lasségue & Griendling, 2004).

**1. Introduction** 

experimental preparations.

These authors contributed equally to the present work

 \*


### **Oxidative Damage in Cardiac Tissue from Normotensive and Spontaneously Hypertensive Rats: Effect of Ageing**

Juliana C. Fantinelli\*, Claudia Caldiz\*, María Cecilia Álvarez, Carolina D. Garciarena, Gladys E. Chiappe de Cingolani and Susana M. Mosca *Centro de Investigaciones Cardiovasculares, Facultad de Ciencias Médicas, Universidad Nacional de La Plata Argentina* 

#### **1. Introduction**

140 Oxidative Stress and Diseases

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protein which protects liposomes and biomembranes from peroxidative degradation and exhibits glutathione peroxidase activity on phosphatidylcholine

of endothelial permeability and integrity by lipoproteins. *Curr Opin* 

The spontaneously hypertensive rat (SHR) is a laboratory model of naturally developing hypertension and heart failure that appears to be similar in many aspects to essential hypertension in humans (Trippodo & Frohlich, 1981). Systolic blood pressure in SHR rapidly increases during 5 to 10 weeks of age and develops cardiac hypertrophy between 9 and 12 weeks of age (Shimamoto et al., 1982). Increasing evidence from different experimental models supports the concept that oxidative stress contributes to the pathogenesis of myocardial hypertrophy and in the process of myocardial remodeling leading to heart failure (Yücel et al., 1998; Lasségue & Griendling, 2004).

The oxidative stress is the result of an increase of reactive oxygen species (ROS) and/or inadequate antioxidant defense mechanisms. It has been shown that an increase in the activity and expression of myocardial NAD(P)H oxidase (NOX) is the main source of ROS in cardiac hypertrophy (Bendall et al., 2002; Griendling et al., 2000; Xiao et al et al., 2002). However, existing data about the antioxidant status in hypertension are inconsistent. Some studies have shown that the activities of one or more antioxidant enzymes are lower (Ito et al, 1995; Newaz & Nawal, 1999), higher (Czonka et al., 2000) or without changes (Gómez-Amores et al., 2006; Girard et al, 2005) compared with normotensive controls. Although the underlying causes of these discrepancies are unknown, it may be possibly due to the use of different hypertension models, animals at different hypertensive stages and/or different experimental preparations.

On the other hand, ROS are thought to be a key mechanism in the aging process (Beckman & Ames, 1998; Colavitti & Finkel, 2004; Harman, 1988) and there are arguments that NOX-

<sup>\*</sup> These authors contributed equally to the present work

Oxidative Damage in Cardiac Tissue from

densitometric analysis (Scion Image).

**2.4 Measurement of superoxide (***O2*

**2.5 SOD, CAT and GPx activities assays** 

**2.3 Determination of NAD(P)H oxidase (NOX) activity**

Normotensive and Spontaneously Hypertensive Rats: Effect of Ageing 143

denatured and equal amounts of protein subjected to PAGE and electrotransferred to PVDF membranes. Membranes were incubated with an anti-nitrotyrosine polyclonal antibody (Cayman Chemical). A peroxidase-conjugated, anti-rabbit IgG (Santa Cruz Biotechnology) was used as secondary antibody, and finally bands were visualized with ECL-Plus chemiluminescence detection system (Amersham). Autoradiograms were analyzed by

Left ventricular slices (LVS, 1 x 5 mm, 3 – 3.5 mg dry weight) were incubated for 5 min at 37 °C in Krebs-Hepes buffer (in mmol/l: 99 ClNa, 4.69 ClK, 1.87 Cl2Ca, 1.2 SO4Mg, 1.03 K2PO4, 25 CO3HNa, 20 Hepes, 11.1 glucose) bubbled with 95% O2 - 5% CO2 to maintain pH 7.4 and then transferred to glass scintillation vials containing the same buffer with 5 M lucigenin. Chemiluminiscence was assessed at 37°C over 15 minutes in a Scintillation counter (Packard 1900 TR) at 1-minute intervals. Vials containing all components without tissue were previously counted and the values were substracted from the chemiluminiscence signals obtained in the presence of LVS. NOX activity was measured in the presence of 100 mM

*–.* **) production** Superoxide production was measured in LVS with lucigenin-enhanced chemiluminiscence in Krebs-Hepes buffer with 5 M lucigenin (Khan et al., 2004). The chemiluminiscence in arbitrary units (AU) was recorded with a luminometer (Chameleon, Hidex) during 30 seconds each with 4.5 min interval during 30 minutes. O2–. production was expressed as AU per mg dry weight per minute. To determine the involvement of NOX in O2–. production,

SOD activity was determined by inhibition of formazan production (produced by nitroblue tetrazolium (NBT) reduction by superoxide anion) at pH 10.2 and 25º C. The reaction mixture consists in: 100 M xanthine, 100 M EDTA, 25 M NBT, 50 mM CO3Na2, pH 10.2. The reaction was started by the addition of xanthine oxidase, reading the absorbance at 560 nm each 30 sec for 5 min (Beauchamp & Fridovich, 1971). One unit of SOD assay was defined as the amount of enzymatic protein required to inhibit 50 % of NBT reduction.

CAT activity was determined by the procedure of Aebi (1984). Decrease in absorbance at 240 nm by the addition of 30 mM H2O2 was monitored each 15 sec and for 30 sec. One unit of CAT assay was defined as the amount of the enzyme that decomposed 1 mol of H2O2.

The GPx activity was measured according to Lawrence and Burk method (1976). The assay reaction comprised 50 mM K2HPO4 buffer, 1 mM EDTA, 1 mM NaN3, 1 mM reduced glutathione, 0.2 mM NADPH, 0.25 mM H2O2 and 1 U/ml glutathione reductase. Gpx activity was assayed by following NADPH oxidation at 340 nm, measuring the absorbance each 15 sec for 5 min. The activity was calculated using a molar extinction coefficient for NADPH of 6.22 103 M−1 x cm−1 at 340 nm. One unit of the enzyme was represented the

NAD(P)H and expressed as cpm/mg dry weight of LVS (Souza et al., 2002).

the slices were pretreated during 30 min with 300 M apocynin.

decrease of 1 mol of NADPH/min under assay conditions.

derived ROS may lead to cellular senescence (Ago et al., 2010a; Ago et al., 2010b; Imanishi et al., 2005). Thus, lipid peroxidation and oxidative modification of proteins by ROS like peroxynitrite-the product of combination of superoxide (O2 –.) and nitric oxide (NO)- are implicated in the pathogenesis of hypertrophy (Nadruz et al., 2004) and in cardiac normal aging (Beal, 2002).

The aim of this study was to assess the oxidative stress in hearts from young and old SHR compared to age-matched Wistar rats.

#### **2. Methods**

Experiments were conducted with 40 days and 4-, 11- and 19-month-old male SHR and agematched Wistar rats. All animals were identically housed under controlled lighting (12 hs) and temperature (20 °C) conditions with free access to standard rat chow and tap water. The experiments were conducted in accordance with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised in 1996). Systolic blood pressure (SBP) was recorded by the tail-cuff method (Camilión de Hurtado et al., 2002). Left ventricular hypertrophy (LVH) was evaluated by the ratio between heart weight (HW) and tibia length (TL) as previously described (Yin et al., 1982). Wistar strain was used as normotensive control rat. For the biochemical determinations SHR and Wistar rats of 4- and 19 months-old were used. The animals were decapitated and hearts were quickly removed and perfused with ice-cold saline solution (0.9% NaCl) to remove the blood. Left ventricle (LV) samples were taken to assay NOX activity, superoxide production and protein nitration. The rest of the heart was homogenized in 5 volume of 25 mM PO4KH2 - 140 mM ClK at pH = 7.4 containing protease inhibitors cocktail (Complete Mini Roche) with a Polytron homogenizer. An aliquot of heart homogenate was used to assess lipid peroxidation. The remaining homogenate was centrifuged at 12000 x g for 5 min at 4º C and the supernatant stored at -70 ºC until superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx) activities were assayed. Protein concentration was evaluated by Bradford method (Bradford, 1976) using bovine serum albumin as a standard.

#### **2.1 Assessment of lipid peroxidation**

Lipid peroxidation was determined by measuring the level of thiobarbituric acid reactive substances (TBARS), expressed as nmol/mg protein. Heart homogenates were centrifuged at 2000 x g for 10 min. Supernatants (0.5 ml) were mixed with 1.5 ml trichloroacetic acid (30 % w/v), 1 ml thiobarbituric acid (0.7% w/v) and 0.5 ml water followed by boiling during 15 min. After cooling, absorbance was determined spectrophotometrically at 535 nm, using a ε value of 1.56 x 105 M-1 cm−1 (Buege & Aust, 1978).

#### **2.2 Assessment of protein nitration**

The interaction of peroxynitrite leads to nitrotyrosine formation actually considered as an indirect marker of oxidative /nitrosative stress (Halliwell, 1997). Thus, we assessed nitrotyrosine level by Western blot analysis. A sample of left ventricle was homogenized in lysis buffer (300 mM sucrose; 1 mM DTT; 4 mM EGTA, protease inhibitors cocktail: 1 tablet/15 ml of buffer; 20 mM Tris-HCl, pH 7.4). After a brief centrifugation proteins were

derived ROS may lead to cellular senescence (Ago et al., 2010a; Ago et al., 2010b; Imanishi et al., 2005). Thus, lipid peroxidation and oxidative modification of proteins by ROS like peroxynitrite-the product of combination of superoxide (O2–.) and nitric oxide (NO)- are implicated in the pathogenesis of hypertrophy (Nadruz et al., 2004) and in cardiac normal

The aim of this study was to assess the oxidative stress in hearts from young and old SHR

Experiments were conducted with 40 days and 4-, 11- and 19-month-old male SHR and agematched Wistar rats. All animals were identically housed under controlled lighting (12 hs) and temperature (20 °C) conditions with free access to standard rat chow and tap water. The experiments were conducted in accordance with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised in 1996). Systolic blood pressure (SBP) was recorded by the tail-cuff method (Camilión de Hurtado et al., 2002). Left ventricular hypertrophy (LVH) was evaluated by the ratio between heart weight (HW) and tibia length (TL) as previously described (Yin et al., 1982). Wistar strain was used as normotensive control rat. For the biochemical determinations SHR and Wistar rats of 4- and 19 months-old were used. The animals were decapitated and hearts were quickly removed and perfused with ice-cold saline solution (0.9% NaCl) to remove the blood. Left ventricle (LV) samples were taken to assay NOX activity, superoxide production and protein nitration. The rest of the heart was homogenized in 5 volume of 25 mM PO4KH2 - 140 mM ClK at pH = 7.4 containing protease inhibitors cocktail (Complete Mini Roche) with a Polytron homogenizer. An aliquot of heart homogenate was used to assess lipid peroxidation. The remaining homogenate was centrifuged at 12000 x g for 5 min at 4º C and the supernatant stored at -70 ºC until superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx) activities were assayed. Protein concentration was evaluated by Bradford method (Bradford, 1976)

Lipid peroxidation was determined by measuring the level of thiobarbituric acid reactive substances (TBARS), expressed as nmol/mg protein. Heart homogenates were centrifuged at 2000 x g for 10 min. Supernatants (0.5 ml) were mixed with 1.5 ml trichloroacetic acid (30 % w/v), 1 ml thiobarbituric acid (0.7% w/v) and 0.5 ml water followed by boiling during 15 min. After cooling, absorbance was determined spectrophotometrically at 535 nm, using a ε

The interaction of peroxynitrite leads to nitrotyrosine formation actually considered as an indirect marker of oxidative /nitrosative stress (Halliwell, 1997). Thus, we assessed nitrotyrosine level by Western blot analysis. A sample of left ventricle was homogenized in lysis buffer (300 mM sucrose; 1 mM DTT; 4 mM EGTA, protease inhibitors cocktail: 1 tablet/15 ml of buffer; 20 mM Tris-HCl, pH 7.4). After a brief centrifugation proteins were

aging (Beal, 2002).

**2. Methods** 

compared to age-matched Wistar rats.

using bovine serum albumin as a standard.

value of 1.56 x 105 M-1 cm−1 (Buege & Aust, 1978).

**2.1 Assessment of lipid peroxidation** 

**2.2 Assessment of protein nitration** 

denatured and equal amounts of protein subjected to PAGE and electrotransferred to PVDF membranes. Membranes were incubated with an anti-nitrotyrosine polyclonal antibody (Cayman Chemical). A peroxidase-conjugated, anti-rabbit IgG (Santa Cruz Biotechnology) was used as secondary antibody, and finally bands were visualized with ECL-Plus chemiluminescence detection system (Amersham). Autoradiograms were analyzed by densitometric analysis (Scion Image).

#### **2.3 Determination of NAD(P)H oxidase (NOX) activity**

Left ventricular slices (LVS, 1 x 5 mm, 3 – 3.5 mg dry weight) were incubated for 5 min at 37 °C in Krebs-Hepes buffer (in mmol/l: 99 ClNa, 4.69 ClK, 1.87 Cl2Ca, 1.2 SO4Mg, 1.03 K2PO4, 25 CO3HNa, 20 Hepes, 11.1 glucose) bubbled with 95% O2 - 5% CO2 to maintain pH 7.4 and then transferred to glass scintillation vials containing the same buffer with 5 M lucigenin. Chemiluminiscence was assessed at 37°C over 15 minutes in a Scintillation counter (Packard 1900 TR) at 1-minute intervals. Vials containing all components without tissue were previously counted and the values were substracted from the chemiluminiscence signals obtained in the presence of LVS. NOX activity was measured in the presence of 100 mM NAD(P)H and expressed as cpm/mg dry weight of LVS (Souza et al., 2002).

#### **2.4 Measurement of superoxide (***O2 –.* **) production**

Superoxide production was measured in LVS with lucigenin-enhanced chemiluminiscence in Krebs-Hepes buffer with 5 M lucigenin (Khan et al., 2004). The chemiluminiscence in arbitrary units (AU) was recorded with a luminometer (Chameleon, Hidex) during 30 seconds each with 4.5 min interval during 30 minutes. O2–. production was expressed as AU per mg dry weight per minute. To determine the involvement of NOX in O2–. production, the slices were pretreated during 30 min with 300 M apocynin.

#### **2.5 SOD, CAT and GPx activities assays**

SOD activity was determined by inhibition of formazan production (produced by nitroblue tetrazolium (NBT) reduction by superoxide anion) at pH 10.2 and 25º C. The reaction mixture consists in: 100 M xanthine, 100 M EDTA, 25 M NBT, 50 mM CO3Na2, pH 10.2. The reaction was started by the addition of xanthine oxidase, reading the absorbance at 560 nm each 30 sec for 5 min (Beauchamp & Fridovich, 1971). One unit of SOD assay was defined as the amount of enzymatic protein required to inhibit 50 % of NBT reduction.

CAT activity was determined by the procedure of Aebi (1984). Decrease in absorbance at 240 nm by the addition of 30 mM H2O2 was monitored each 15 sec and for 30 sec. One unit of CAT assay was defined as the amount of the enzyme that decomposed 1 mol of H2O2.

The GPx activity was measured according to Lawrence and Burk method (1976). The assay reaction comprised 50 mM K2HPO4 buffer, 1 mM EDTA, 1 mM NaN3, 1 mM reduced glutathione, 0.2 mM NADPH, 0.25 mM H2O2 and 1 U/ml glutathione reductase. Gpx activity was assayed by following NADPH oxidation at 340 nm, measuring the absorbance each 15 sec for 5 min. The activity was calculated using a molar extinction coefficient for NADPH of 6.22 103 M−1 x cm−1 at 340 nm. One unit of the enzyme was represented the decrease of 1 mol of NADPH/min under assay conditions.

Oxidative Damage in Cardiac Tissue from

months-old SHR.
