**Meet the editor**

Dr. S. Ashok Kumar is an Assistant Professor at the Centre for Biotechnology, Anna University, Chennai, Tamil Nadu, India. He has done doctoral research on hypercholesterolemia and obtained his PhD from the University of Madras. Later, he joined as a postdoctoral fellow at CancerCare Manitoba, University of Manitoba. At present, as an Assistant Professor at Anna University,

he is actively involved in the research field of hypercholesterolemia and targeting signaling pathways in metabolic disorder. Dr. Kumar's research work has been supported by grants from national funding agencies such as UGC and DST-SERB. His research findings were published in nine international journals and a book chapter. Dr. Kumar is an active Member of the Board of Studies in Biotechnology and Member of the Editorial Board of Scientific Publication.

## Contents

#### **Preface XI**



Safila Naveed

## Preface

Chapter 8 **Hypercholesterolemia — Statin Therapy — Indications, Side**

Lucía Cid-Conde and José López-Castro

Chapter 10 **Resistance of Statin Therapy, and Methods for its**

Chapter 11 **Interaction Studies of ACE Inhibitors with Statins 203**

Chapter 9 **Pleiotropic Effects of Statins 175** Sigrid Mennickent

**Influence 185**

**VI** Contents

Safila Naveed

**Effects, Common Mistakes in Handling, Last Evidence and Recommendations in Current Clinical Practice 159**

Lyudmila Georgieva Vladimirova-Kitova and Spas Ivanov Kitov

Increased dietary intake of fat has received considerable attention in the past few decades be‐ cause of its link with an increased risk of atherosclerosis and subsequent cardiovascular disease. Apart from dietary origin, there are several other factors such as genetic factors and hormonal changes that play a vital role in hypercholesterolemia-induced cardiovascular disease. A num‐ ber of risk factors have been identified for the development of atherosclerosis, among which, hypercholesterolemia is a well-recognized primary risk factor for cardiovascular disease. A strong relationship between hypercholesterolemia and cardiovascular disease has been estab‐ lished through epidemiological, experimental, and clinical trial data. Understanding and tar‐ geting hypercholesterolemia becomes important since it is an earlier stage in the pathogenesis of atherosclerosis and can also be managed with appropriate treatment.

This book describes the basic information on the causes, diagnosis, consequences, and treat‐ ment strategies for hypercholesterolemia. InTech has invited experts in the related field from different countries to take part in this book. The chapters contain more updated and wide information, and indeed, some chapters take a practical approach towards the man‐ agement of hypercholesterolemia. This book is a collection of information on hypercholes‐ terolemia from different authors in order to present a vast and clear knowledge about hypercholesterolemia to readers.

> **Dr. Sekar Ashok Kumar** Assistant Professor Centre for Biotechnology Anna University Chennai Chennai, Tamil Nadu, India

**Hypercholesterolemia- Basic Information, Animal Models, Consequences and Approaches to Treatment**

## **Animal Models of Diet-induced Hypercholesterolemia**

Jeannie Chan, Genesio M. Karere, Laura A. Cox and John L. VandeBerg

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/59610

#### **1. Introduction**

Cholesterol is a component of the cell membrane and metabolites of cholesterol, such as bile acids, steroid hormones and vitamin D, serve important biologic functions in vertebrates. Cholesterol is synthesized primarily in the liver and transported to cells throughout the body by lipoproteins via the blood, even though all nucleated cells in the body are capable of synthesizing cholesterol. Whole-body cholesterol homeostasis is determined by cholesterol absorption, cholesterol synthesis and cholesterol excretion, and losing control of any of these processes leads to an increase in plasma cholesterol. Liver and intestine are the major sites that control cholesterol homeostasis. The liver synthesizes cholesterol for secretion in nascent lipoproteins when blood levels of cholesterol are low, and removes excess cholesterol from the blood by taking up chylomicron remnants, high density lipoprotein (HDL), very low density lipoprotein (VLDL) and low density lipoprotein (LDL) particles. It converts cholesterol into bile acids, and secretes cholesterol and bile acids into bile for elimination from the body. The intestine regulates influx of cholesterol from the lumen and efflux of cholesterol back into the lumen to control the amount of cholesterol that enters the body [1].

Hypercholesterolemia is characterized by LDL cholesterol exceeding 159 mg/dl [2]. Many developed countries have a high prevalence of hypercholesterolemia. According to an estimate based on data from the 2005-2008 National Health and Nutrition Examination Survey, the Centers for Disease Control and Prevention reported that 33.5% of US adults aged 20 or older had high levels of LDL cholesterol [3]. Diets containing high levels of cholesterol and high levels of fat (HCHF) are frequently the culprit in causing hypercholesterolemia. In addition, genetic factors influence susceptibility to diet-induced hypercholesterolemia.

Hypercholesterolemia is a complex disorder often due to multiple genetic defects and rarely due to a single genetic defect as in the case of familial hypercholesterolemia [4]. Because

© 2015 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

hypercholesterolemia is associated with risk of developing atherosclerosis, much research has been devoted to understanding the genetic variants and environmental factors that contribute to elevated blood LDL cholesterol. There are several challenges to investigating gene-diet interactions in humans. It is difficult to control the diet and environment of human subjects for long periods of time, and ethical constraints limit access to tissue samples. These problems can be circumvented by using animals to study the effects of diets on cholesterol homeostasis and atherosclerosis because animals can be fed the same diet and kept under the same laboratory conditions for long periods of time, and because access to tissue samples from animals is less restricted. Numerous animal species have been used as animal models for investigating hypercholesterolemia, including rabbits [5,6], mice [7], guinea pigs [8], minipigs [9], laboratory opossums [10] and nonhuman primates [11-15]. Nonhuman primates are more similar to humans than other animals, but ethical issues, facilities and high cost limit studies with nonhuman primates. Non-primate animal models have other limitations. However, extensive use of the collage of primate and non-primate models has provided considerable insights into the genes and molecular mechanisms that control plasma cholesterol in response to diets.

In this chapter, we discuss mouse, laboratory opossum and nonhuman primate models of hypercholesterolemia. Mice lipoprotein profiles differ from humans and they are resistant to developing hypercholesterolemia and atherosclerosis, but genetic engineering tools have been used effectively to alter their lipoprotein profiles. A commonly used mouse model in which the apolipoprotein E gene (*apoE)* is disrupted exhibits a lipoprotein profile similar to that of humans. In addition, *apoE* knockout mice become hypercholesterolemic and have a propensity to develop atherosclerosis [16,17]. Mice have also been used extensively to elucidate mecha‐ nisms that regulate cholesterol homeostasis. Cholesterol excretion is one of the major processes that can be targeted to reduce hypercholesterolemia. A nonbiliary pathway for disposal of cholesterol termed transintestinal cholesterol excretion (TICE) was first described in mice almost a decade ago [18]. A growing body of evidence for TICE has since been gathered using genetically modified and normal mice [19,20]. The discovery of TICE has opened a new avenue of research into the role of the intestine in cholesterol excretion [21].

The laboratory opossum is a model of diet-induced hypercholesterolemia developed at Texas Biomedical Research Institute that does not require genetic manipulation to knock out or overexpress specific genes to elevate LDL cholesterol [22,23]. Through many generations of inbreeding and selection for plasma cholesterol response to an HCHF diet, high and low responding strains of opossums were produced. All strains have normal levels of plasma cholesterol on a basal diet. However, high responding opossums exhibit an extremely high LDL cholesterol response when fed an HCHF diet compared to low responding opossums. Hypercholesterolemia in high responding opossums is caused by mutations in at least two genes. One of the causative genes has been identified as *ABCB4;* mutations in the *ABCB4* gene impair biliary cholesterol secretion [24]. The *ABCB4* gene has not been shown previously to be associated with hypercholesterolemia. The opossum model provides an opportunity to investigate genes that interact with *ABCB4* to regulate cholesterol homeostasis.

Nonhuman primates are utilized as models of hypercholesterolemia because of their physio‐ logic and genetic similarities with humans [25]. In addition, nonhuman primates naturally develop hypercholesterolemia and atherosclerosis, both of which can be exacerbated by HCHF diets to mimic diet-induced hypercholesterolemia and atherosclerosis in humans [11,15,26]. Because of these characteristics, nonhuman primates including baboon (*Papio hymadryas*), rhesus macaque (*Macaca mulatta*), green monkey (*Chlorocebus aethiops*) and cynomolgus monkey (*Macaca fascicularis*) have been used as animal models for biomedical research aimed at understanding diet-induced hypercholesterolemia in humans. Nonhuman primates exhibit species and individual variations in plasma cholesterol in response to HCHF diets. Studies using pedigreed baboons and high-throughput sequencing technology have identified genetic factors that influence plasma cholesterol response to HCHF diets [13,27].

#### **2. Mouse models**

hypercholesterolemia is associated with risk of developing atherosclerosis, much research has been devoted to understanding the genetic variants and environmental factors that contribute to elevated blood LDL cholesterol. There are several challenges to investigating gene-diet interactions in humans. It is difficult to control the diet and environment of human subjects for long periods of time, and ethical constraints limit access to tissue samples. These problems can be circumvented by using animals to study the effects of diets on cholesterol homeostasis and atherosclerosis because animals can be fed the same diet and kept under the same laboratory conditions for long periods of time, and because access to tissue samples from animals is less restricted. Numerous animal species have been used as animal models for investigating hypercholesterolemia, including rabbits [5,6], mice [7], guinea pigs [8], minipigs [9], laboratory opossums [10] and nonhuman primates [11-15]. Nonhuman primates are more similar to humans than other animals, but ethical issues, facilities and high cost limit studies with nonhuman primates. Non-primate animal models have other limitations. However, extensive use of the collage of primate and non-primate models has provided considerable insights into the genes and molecular mechanisms that control plasma cholesterol in response

In this chapter, we discuss mouse, laboratory opossum and nonhuman primate models of hypercholesterolemia. Mice lipoprotein profiles differ from humans and they are resistant to developing hypercholesterolemia and atherosclerosis, but genetic engineering tools have been used effectively to alter their lipoprotein profiles. A commonly used mouse model in which the apolipoprotein E gene (*apoE)* is disrupted exhibits a lipoprotein profile similar to that of humans. In addition, *apoE* knockout mice become hypercholesterolemic and have a propensity to develop atherosclerosis [16,17]. Mice have also been used extensively to elucidate mecha‐ nisms that regulate cholesterol homeostasis. Cholesterol excretion is one of the major processes that can be targeted to reduce hypercholesterolemia. A nonbiliary pathway for disposal of cholesterol termed transintestinal cholesterol excretion (TICE) was first described in mice almost a decade ago [18]. A growing body of evidence for TICE has since been gathered using genetically modified and normal mice [19,20]. The discovery of TICE has opened a new avenue

The laboratory opossum is a model of diet-induced hypercholesterolemia developed at Texas Biomedical Research Institute that does not require genetic manipulation to knock out or overexpress specific genes to elevate LDL cholesterol [22,23]. Through many generations of inbreeding and selection for plasma cholesterol response to an HCHF diet, high and low responding strains of opossums were produced. All strains have normal levels of plasma cholesterol on a basal diet. However, high responding opossums exhibit an extremely high LDL cholesterol response when fed an HCHF diet compared to low responding opossums. Hypercholesterolemia in high responding opossums is caused by mutations in at least two genes. One of the causative genes has been identified as *ABCB4;* mutations in the *ABCB4* gene impair biliary cholesterol secretion [24]. The *ABCB4* gene has not been shown previously to be associated with hypercholesterolemia. The opossum model provides an opportunity to

of research into the role of the intestine in cholesterol excretion [21].

investigate genes that interact with *ABCB4* to regulate cholesterol homeostasis.

to diets.

4 Hypercholesterolemia

#### **2.1. Mouse models of cholesterol metabolism**

Mouse models are the most widely used animal models because of several advantages such as ease of breeding, large litter size, a short generation time of 9 months and economies of colony maintenance. An additional advantage of mice is the availability of tools to add exogenous genes to the germ line to create transgenic mice, or to disrupt endogenous genes by homologous recombination in murine embryonic stem cells to create knockout mice. Although there are important differences between mice and humans in lipoprotein and cholesterol metabolism, genetic manipulation has provided mouse models that resemble some aspects of hypercholesterolemia in humans.

Non-genetically modified mice have high levels of HDL cholesterol and low levels of LDL cholesterol, whereas humans have high levels of LDL cholesterol and low levels of HDL cholesterol. The difference in lipid profiles between mice and humans is due to absence of the cholesteryl ester transfer protein (CETP) in mice [28,29]. CETP is an enzyme that transfers cholesterol ester from HDL to VLDL and LDL in exchange for triglycerides [30]. In normal mice lacking CETP, more than 80% of plasma cholesterol is carried on HDL, so mice with high levels of HDL cholesterol are resistant to hypercholesterolemia and atherosclerosis. To overcome this problem to using mice as models for research aimed at understanding choles‐ terol metabolism in humans, several genetically engineered strains of mice were generated to alter the distribution of plasma cholesterol from HDL to VLDL and LDL. The genetically modified mice include CETP transgenic, apoE knockout and LDL receptor knockout mice.

*CETP transgenic mice*. Transgenic mice carrying human and cynomolgus monkey versions of the *CETP* gene were studied to investigate the effects of CETP on distribution of plasma lipoprotein cholesterol. Transgenic mice expressing high levels of human CETP showed a small decrease in HDL cholesterol and a small increase in VLDL and LDL cholesterol on the basal (chow) diet [29]. Transgenic mice expressing high levels of cynomolgus monkey CETP showed greater responsiveness than nontransgenic mice when challenged with an HCHF diet. Total plasma cholesterol of *CETP* transgenic mice averaged 250 mg/dl whereas those of nontransgenic mice averaged 163 mg/dl. Furthermore, *CETP* transgenic mice showed that CETP activity was inversely associated with apoA-I, but positively associated with apoB [28]. These observations demonstrated that human and monkey CETP can interact with mouse lipoproteins to mediate its effects in lipoprotein metabolism.

*ApoE deficient mice*. ApoE is a 34 kD glycoprotein produced primarily in the liver and to a lesser extent in other tissues. With the exception of LDL particles, apoE is a structural component of all lipoprotein particles and chylomicrons. It binds to the LDL receptor and to the LDL receptor-related protein to remove VLDL and chylomicron remnants from the plasma [31]. Using gene targeting to disrupt the *apoE* gene, mutant mice developed severe hypercholes‐ terolemia as expected from a defect in lipoprotein clearance from plasma. Total plasma cholesterol levels were highly elevated in homozygous apoE deficient (*apoE*-/-) mice (400-500 mg/dl) compared with normal mice (80 mg/dl) on a chow diet with 0.01% cholesterol and 4.5% fat. A more dramatic increase in plasma cholesterol was observed in *apoE*-/- mice (1800 mg/dl) fed an HCHF diet with 0.15% cholesterol and 20% fat. Plasma cholesterol concentrations in the VLDL and intermediate density lipoprotein (IDL) fractions were increased on both diets. The *apoE*-/- mice were highly susceptible to atherosclerosis, even on chow diet, as a result of the increase in plasma cholesterol concentrations [16,17].

*LDL receptor deficient mice*. The LDL receptor is a cell surface receptor expressed in many cell types. In the liver, the LDL receptor regulates plasma cholesterol by binding to apoB and apoE on the surface of lipoprotein particles and removes these particles from the plasma [32]. Patients with familial hypercholesterolemia [33] and Watanabe-heritable hyperlipidemic rabbits [34] develop elevated levels of LDL cholesterol due to mutations in the LDL receptor. Based on this knowledge, LDL receptor knock out (*LDLR*-/-) mice were generated to increase plasma LDL cholesterol concentrations. The loss of functional LDL receptors elevated total plasma cholesterol in *LDLR*-/- mice, but the effect was more moderate compared to *apoE*-/ mice. The mean total plasma cholesterol on a chow diet was 293 mg/dl and on a diet enriched with cholesterol (0.2%) and fat (19%) was 425 mg/dl. The increase in plasma cholesterol in *LDLR*-/- mice was attributed to increases in IDL and LDL cholesterol [35]. Compared with familial hypercholesterolemia patients (receptor-negative homozygotes) who have plasma cholesterol levels over 700 mg/dl, the effect of LDL receptor deficiency is less severe in mice. This is due to the fact that mice produce VLDL particles containing both apoB-48 and apoB-100, whereas humans only produce VLDL particles containing apoB-100 [36]. In mice, VLDL particles containing apoB-48 can be cleared from the plasma by the chylomicron remnant receptor in addition to the LDL receptor, so fewer VLDL particles are converted to LDL particles. Therefore, LDL receptor deficiency in mice does not increase plasma cholesterol to the same extent as in humans.

#### **2.2. Mouse models of atherosclerosis**

Disruption of the *apoE* gene causes hypercholesterolemic *apoE* knockout mice to develop atherosclerotic lesions spontaneously [16,17]. Foam cell lesions were observed in chow-fed *apoE*-/- mice 10 weeks after birth and the lesions progress to fibrous plaques by 20 weeks of age. An HCHF diet accelerates all stages of lesion formation and increases the size of lesions; thus, formation of atherosclerotic lesions in *apoE-/-* mice is responsive to diet as in humans. Because of the short period of time for lesion formation in apoE-/- mice, they have been used extensively to study dietary and genetic factors affecting atherosclerosis and mechanisms of atherogenesis, as well as to assess efficacy of pharmacologic agents on lesion size. Progression of early lesions to advanced lesions in *apoE-/-* mice is similar to that in humans; lesions often develop at vascular branch points and progress rapidly to foam cells with fibrous plaques and necrotic lipid cores [37]. The major difference from humans is a low incidence of ruptured plaques that leads to thrombosis and arterial occlusion. Plaque rupture is the event that causes a heart attack in humans. A higher incidence of ruptured plaques was obtained by feeding a diet enriched with 0.15% cholesterol and 21% fat to *apoE-/-* mice. After 8 weeks of feeding the HCHF diet to *apoE-/-* mice, acute plaque ruptures were observed in the brachiocephalic arteries of more than half of the animals [38].

#### **2.3. Mouse models of cholesterol excretion**

nontransgenic mice averaged 163 mg/dl. Furthermore, *CETP* transgenic mice showed that CETP activity was inversely associated with apoA-I, but positively associated with apoB [28]. These observations demonstrated that human and monkey CETP can interact with mouse

*ApoE deficient mice*. ApoE is a 34 kD glycoprotein produced primarily in the liver and to a lesser extent in other tissues. With the exception of LDL particles, apoE is a structural component of all lipoprotein particles and chylomicrons. It binds to the LDL receptor and to the LDL receptor-related protein to remove VLDL and chylomicron remnants from the plasma [31]. Using gene targeting to disrupt the *apoE* gene, mutant mice developed severe hypercholes‐ terolemia as expected from a defect in lipoprotein clearance from plasma. Total plasma cholesterol levels were highly elevated in homozygous apoE deficient (*apoE*-/-) mice (400-500 mg/dl) compared with normal mice (80 mg/dl) on a chow diet with 0.01% cholesterol and 4.5% fat. A more dramatic increase in plasma cholesterol was observed in *apoE*-/- mice (1800 mg/dl) fed an HCHF diet with 0.15% cholesterol and 20% fat. Plasma cholesterol concentrations in the VLDL and intermediate density lipoprotein (IDL) fractions were increased on both diets. The *apoE*-/- mice were highly susceptible to atherosclerosis, even on chow diet, as a result of the

*LDL receptor deficient mice*. The LDL receptor is a cell surface receptor expressed in many cell types. In the liver, the LDL receptor regulates plasma cholesterol by binding to apoB and apoE on the surface of lipoprotein particles and removes these particles from the plasma [32]. Patients with familial hypercholesterolemia [33] and Watanabe-heritable hyperlipidemic rabbits [34] develop elevated levels of LDL cholesterol due to mutations in the LDL receptor. Based on this knowledge, LDL receptor knock out (*LDLR*-/-) mice were generated to increase plasma LDL cholesterol concentrations. The loss of functional LDL receptors elevated total plasma cholesterol in *LDLR*-/- mice, but the effect was more moderate compared to *apoE*-/ mice. The mean total plasma cholesterol on a chow diet was 293 mg/dl and on a diet enriched with cholesterol (0.2%) and fat (19%) was 425 mg/dl. The increase in plasma cholesterol in *LDLR*-/- mice was attributed to increases in IDL and LDL cholesterol [35]. Compared with familial hypercholesterolemia patients (receptor-negative homozygotes) who have plasma cholesterol levels over 700 mg/dl, the effect of LDL receptor deficiency is less severe in mice. This is due to the fact that mice produce VLDL particles containing both apoB-48 and apoB-100, whereas humans only produce VLDL particles containing apoB-100 [36]. In mice, VLDL particles containing apoB-48 can be cleared from the plasma by the chylomicron remnant receptor in addition to the LDL receptor, so fewer VLDL particles are converted to LDL particles. Therefore, LDL receptor deficiency in mice does not increase plasma cholesterol to

Disruption of the *apoE* gene causes hypercholesterolemic *apoE* knockout mice to develop atherosclerotic lesions spontaneously [16,17]. Foam cell lesions were observed in chow-fed *apoE*-/- mice 10 weeks after birth and the lesions progress to fibrous plaques by 20 weeks of age. An HCHF diet accelerates all stages of lesion formation and increases the size of lesions;

lipoproteins to mediate its effects in lipoprotein metabolism.

6 Hypercholesterolemia

increase in plasma cholesterol concentrations [16,17].

the same extent as in humans.

**2.2. Mouse models of atherosclerosis**

*Hepatobiliary cholesterol excretion*. The human body cannot degrade cholesterol, but it can convert cholesterol to bile acids in the liver. Fecal excretion is the major route for the body to remove cholesterol as bile acids or neutral sterols (cholesterol and its metabolites formed by bacteria in the intestine). Diet-induced hypercholesterolemia can be mitigated by increasing the loss of cholesterol in feces. Fecal excretion of cholesterol and bile acids depends on transporting these molecules from the liver into bile. The ABCG5/G8 [39] and ABCB11 [40] proteins transport cholesterol and bile acids, respectively. The ABCB4 protein transports phospholipids (mainly phosphatidylcholine), which is essential for secretion of cholesterol into bile [41,42]. ABCB11 plays a central role in hepatobiliary secretion as bile flow is the driving force for biliary secretion of cholesterol and phospholipids [43].

Many studies have been carried out using transgenic and knockout mouse models to further our knowledge of the physiologic pathways and molecular mechanisms that control choles‐ terol excretion. Transgenic mice expressing the human *ABCG5* and *ABCG8* genes directed by their own regulatory DNA sequences (*hG5G8Tg* mice) had higher levels (5-fold) of biliary cholesterol and higher levels (3- to 6-fold) of fecal neutral sterol compared with nontransgenic mice, providing evidence that ABCG5 and ABCG8 function as cholesterol transporters [44]. Moreover, cholesterol absorption in these transgenic mice was reduced by 50% because the proteins encoded by *ABCG5* and *ABCG8* genes are also expressed in the small intestine and they transport cholesterol from the intestine into the lumen [44]. In another study, *Abcg5* and *Abcg8* double knockout (*G5G8*-/-) mice were created to investigate the effects of disrupting these genes on biliary cholesterol secretion. *G5G8*-/- mice were more diet-responsive than normal mice on a 2% cholesterol diet. Plasma cholesterol increased 2-fold in *G5G8*-/- mice, but not in normal mice. Hepatic cholesterol was markedly increased in *G5G8*-/- mice (18-fold) compared with normal mice (3-fold). Accordingly, expression of the cholesterol synthesis genes HMG-CoA synthase and HMG-CoA reductase was lower in *G5G8*-/- mice than normal mice. Fecal neutral sterol was reduced by 36% in *G5G8*-/- mice relative to that in normal mice. The findings using double knockout mice also supported ABCG5 and ABCG8 function in cholesterol transport [45].

Another series of experiments was conducted to investigate the role of phospholipids in secretion of biliary cholesterol. Disruption of the *Abcb4* (also known as *Mdr2*) gene in mice led to a severe liver disease. Phospholipids were undetectable in the bile of homozygous *Abcb4* knockout (*Abcb4-/-*) mice, while the levels in heterozygous *Abcb4* knockout (*Abcb4+/-*) mice were half of those in nontransgenic mice. Interestingly, *Abcb4-/-* mice secreted extremely low levels of cholesterol into bile, but cholesterol levels in flowing bile from *Abcb4+/-* mice were similar to those in nontransgenic mice [41]. Langheim et al. [46] investigated whether overex‐ pression of ABCG5 and ABCG8 could restore biliary cholesterol secretion in *Abcb4-/-* mice by breeding *Abcb4-/-* mice with *hG5G8Tg* mice to generate *Abcb4-/-*;*hG5G8Tg* mice. The *Abcb4-/-*;*hG5G8Tg* mice also secreted very low levels of biliary cholesterol, which were similar to the levels in *Abcb4-/-* mice. Taken together, these results indicate that biliary cholesterol secretion requires a minimal concentration of phospholipids in the bile. Biliary phospholipid secretion in the liver serves two purposes. One is to protect the canalicular membrane of hepatocytes exposed to high concentrations of bile acids from damage by the detergent action of bile acids. Secretion of phospholipids by the liver into the bile reduces bile salt micelles to extract phospholipids from the membranes of hepatocytes. The other is to make phospholipids available for incorporation into pure bile salt micelles to form bile salt mixed micelles. Solubility of cholesterol in bile salt mixed micelles is greater than that in pure bile salt micelles, thus phospholipid secretion prevents formation of gallstones [47].

*Nonbiliary cholesterol excretion*. Hepatobiliary secretion is known to be the main pathway for eliminating cholesterol from the body, but an increasing body of evidence suggests that plasma cholesterol is also eliminated by a nonbiliary pathway in mice. As mentioned above, *G5G8*-/ mice did not show a dramatic reduction in fecal neutral sterol excretion despite they had extremely low levels of biliary cholesterol. Normal or even increased fecal neutral sterol excretion was observed in other mouse models (*Abcb4-/-*, *Npc1l1*-LiverTg) that are severely impaired in hepatobiliary cholesterol secretion [20]. This observation suggests the existence of an alternate route, known as TICE, which does not involve biliary cholesterol secretion. Plasma cholesterol is transported via blood to intestinal cells and eventually secreted into the intestinal lumen for disposal in feces [20]. It should be mentioned that a nonbiliary cholesterol excretion pathway has also been postulated to explain fecal neutral sterol loss in bile-diverted dogs [48, 49] and bile-diverted rats [50].

Intestinal perfusion studies [19] and *in vivo* stable isotope studies [51] in mice that have an intact hepatobiliary secretion and enterohepatic cycling system revealed that TICE accounted for ~30% of fecal cholesterol excretion. The site of action of TICE is the proximal small intestine [19,52]. It is thought that TICE involves plasma lipoproteins to deliver cholesterol to the intestine, then cholesterol is taken up by receptors at the basolateral membrane of enterocytes and traverses the enterocytes to the apical membrane where it is excreted into the intestinal lumen. There are still many gaps in our understanding of the molecular mechanism of TICE. HDL is ruled out as the lipoprotein that delivers cholesterol to the intestine because TICE was not diminished in *Abca*1 knockout mice having extremely low concentrations of plasma HDL [53,54]. Evidence to support VLDL remnants or LDL as the plasma cholesterol carrier came from studies using antisense oligonucleotides (ASO) to knockdown expression of proteins critical for production of VLDL and alter plasma VLDL cholesterol concentrations. ASOmediated knockdown of acyl-CoA:cholesterol acyltransferase activity 2 (ACAT2) in mice fed a high cholesterol diet resulted in an increase in both VLDL cholesterol and fecal neutral sterol excretion [55]. Conversely, ASO-mediated knockdown of microsomal triglyceride transfer protein resulted in a decrease in both VLDL cholesterol and fecal neutral sterol excretion [56]. As for the receptor that takes up cholesterol at the basolateral membrane, it is not likely to be the LDL receptor nor scavenger receptor BI (SR-BI) because neutral sterol excretion was not affected in *LDLR*-/- mice [57] and SR-BI-/- mice [58,59]. It may involve other members of the LDL receptor or a novel receptor. Lastly, Abcg5/Abcg8 and Abcb1 participate in the secretion of cholesterol from enterocytes into the intestinal lumen [57].

A study in patients with complete biliary obstruction revealed they still excreted substantial amounts of neutral sterol into feces [60]. Data from these patients showed that ~20% to 30% of neutral sterols were excreted by TICE, which is similar to that excreted by TICE in normal mice. However, the relevance of TICE for disposal of cholesterol in humans without biliary obstruction has yet to be established. A recent *ex vivo* study showed that human and mouse intestinal (duodenual) explants mounted on Ussing chambers were capable of effluxing cholesterol, providing evidence for the activity of TICE in humans [57].

#### **3. Laboratory opossum model**

Another series of experiments was conducted to investigate the role of phospholipids in secretion of biliary cholesterol. Disruption of the *Abcb4* (also known as *Mdr2*) gene in mice led to a severe liver disease. Phospholipids were undetectable in the bile of homozygous *Abcb4* knockout (*Abcb4-/-*) mice, while the levels in heterozygous *Abcb4* knockout (*Abcb4+/-*) mice were half of those in nontransgenic mice. Interestingly, *Abcb4-/-* mice secreted extremely low levels of cholesterol into bile, but cholesterol levels in flowing bile from *Abcb4+/-* mice were similar to those in nontransgenic mice [41]. Langheim et al. [46] investigated whether overex‐ pression of ABCG5 and ABCG8 could restore biliary cholesterol secretion in *Abcb4-/-* mice by breeding *Abcb4-/-* mice with *hG5G8Tg* mice to generate *Abcb4-/-*;*hG5G8Tg* mice. The *Abcb4-/-*;*hG5G8Tg* mice also secreted very low levels of biliary cholesterol, which were similar to the levels in *Abcb4-/-* mice. Taken together, these results indicate that biliary cholesterol secretion requires a minimal concentration of phospholipids in the bile. Biliary phospholipid secretion in the liver serves two purposes. One is to protect the canalicular membrane of hepatocytes exposed to high concentrations of bile acids from damage by the detergent action of bile acids. Secretion of phospholipids by the liver into the bile reduces bile salt micelles to extract phospholipids from the membranes of hepatocytes. The other is to make phospholipids available for incorporation into pure bile salt micelles to form bile salt mixed micelles. Solubility of cholesterol in bile salt mixed micelles is greater than that in pure bile salt micelles,

*Nonbiliary cholesterol excretion*. Hepatobiliary secretion is known to be the main pathway for eliminating cholesterol from the body, but an increasing body of evidence suggests that plasma cholesterol is also eliminated by a nonbiliary pathway in mice. As mentioned above, *G5G8*-/ mice did not show a dramatic reduction in fecal neutral sterol excretion despite they had extremely low levels of biliary cholesterol. Normal or even increased fecal neutral sterol excretion was observed in other mouse models (*Abcb4-/-*, *Npc1l1*-LiverTg) that are severely impaired in hepatobiliary cholesterol secretion [20]. This observation suggests the existence of an alternate route, known as TICE, which does not involve biliary cholesterol secretion. Plasma cholesterol is transported via blood to intestinal cells and eventually secreted into the intestinal lumen for disposal in feces [20]. It should be mentioned that a nonbiliary cholesterol excretion pathway has also been postulated to explain fecal neutral sterol loss in bile-diverted dogs [48,

Intestinal perfusion studies [19] and *in vivo* stable isotope studies [51] in mice that have an intact hepatobiliary secretion and enterohepatic cycling system revealed that TICE accounted for ~30% of fecal cholesterol excretion. The site of action of TICE is the proximal small intestine [19,52]. It is thought that TICE involves plasma lipoproteins to deliver cholesterol to the intestine, then cholesterol is taken up by receptors at the basolateral membrane of enterocytes and traverses the enterocytes to the apical membrane where it is excreted into the intestinal lumen. There are still many gaps in our understanding of the molecular mechanism of TICE. HDL is ruled out as the lipoprotein that delivers cholesterol to the intestine because TICE was not diminished in *Abca*1 knockout mice having extremely low concentrations of plasma HDL [53,54]. Evidence to support VLDL remnants or LDL as the plasma cholesterol carrier came from studies using antisense oligonucleotides (ASO) to knockdown expression of proteins

thus phospholipid secretion prevents formation of gallstones [47].

49] and bile-diverted rats [50].

8 Hypercholesterolemia

#### **3.1. Characteristics of laboratory opossums**

The gray short-tailed opossum (*Monodelphis domestica*) is a docile, nocturnal marsupial native to Brazil and adjacent countries. It is the only marsupial species that has adapted to breeding in captivity to produce large numbers of animals [61]. In addition to being able to breed in captivity throughout the year, *Monodelphis* have a short generation time of 6 months and produce large litters (typically 6-13). Breeding colonies have been established in the United States, Brazil, Germany, United Kingdom, Japan, and Australia. Opossums in captive colonies are quite different genetically from their wild counterparts due to selection that has undoubt‐ edly taken place while the animals were bred in isolated colonies for many generations; therefore laboratory stocks of this species are referred to as laboratory opossums. Adult laboratory opossums weigh 70-150 g, which is intermediate between mice (20-30 g) and rats (250-300 g). They are maintained in polycarbonate rodent cages, and the standard laboratory diet is commercial pelleted fox food provided *ad libitium*. Owing to its physical characteristics as a laboratory animal and economic production in captivity, *Monodelphis domestica* has become the most widely used marsupial in biomedical research. Furthermore, it was the first marsupial species to have its genome sequenced and analyzed [62]. The availability of genome sequence data has accelerated progress on genetic aspects of research involving *Monodelphis*.

The opossum model has advantageous characteristics for understanding cholesterol homeo‐ stasis. *Monodelphis* is omnivorous like humans, and its natural diet includes cholesterol derived from the consumption of insects and small vertebrates. Therefore, laboratory opossums and humans are likely to have many similarities in lipoprotein and cholesterol metabolism. Unlike genetically modified (transgenic and knockout) mouse models, the opossum model provides an opportunity to identify naturally occurring variants of genes and to study how interactions among gene variants lead to development of hypercholesterolemia. Some partially inbred strains of opossums have inbreeding coefficients in excess of 0.95, and strains with high or low responses to an HCHF diet have inbreeding coefficients in excess of 0.8. Since more than 80% of the genes in each partially inbred strain have alleles that are identical by descent, the work required to identify genes that cause diet-induced hypercholesterolemia in the opossum model is substantially reduced.

#### **3.2. Development of partially inbred strains**

Nine wild-caught animals were imported from Brazil into the United States in 1978 by the National Zoo in Washington, D.C. The founders of the breeding colony of laboratory opossums at Texas Biomedical Research Institute were comprised of 20 first and second generation descendants of those nine founders, together with 17 additional wild-caught opossums from Brazil and two from Bolivia. After several generations of inbreeding, some individuals were fed a high cholesterol (0.6% by weight) and high fat (17% by weight) diet for 6 months, and total blood cholesterol was measured after an overnight fast. Low and high responses were observed among opossums, with few animals exhibiting an intermediate response, i.e. the phenotypes are clustered at the high and low ends of the range. Low responding opossums had blood cholesterol levels ranging from 62-171 mg/dl whereas high responding opossums had levels of 215-932 mg/dl [22]. Furthermore, analysis of lipoprotein particles by gradient gel electrophoresis showed elevated levels of LDL particles in high responding opossums [22]. Subsequently, inbreeding and selection for either low responsiveness or high responsiveness to the HCHF diet led to development of two related low responding partially inbred strains (designated ATHE and ATHL) and a high responding partially inbred strain (designated ATHH) that show extreme difference (>5-fold) in plasma cholesterol concentrations in response to the HCHF diet. These strains are being used to identify genetic variants and molecular mechanisms that cause diet-induced hypercholesterolemia.

#### **3.3. Hypercholesterolemia in high responders**

*Dietary challenge and plasma cholesterol response*. Studies were conducted to compare lipoprotein characteristics of low responders from the ATHE strain and high responders from the ATHH strain. The standard laboratory diet is the basal diet, which contains 0.04% cholesterol and 8.1% fat, by weight. Plasma cholesterol concentrations of low and high responders do not differ on this diet. Most of the plasma cholesterol is carried by HDL, and ~30% of total plasma cholesterol is carried by LDL [10]. After consuming the HCHF diet for at least 4 weeks, total plasma cholesterol increases slightly (< 2-fold) in low responders, but dramatically in high responders (>5-fold). HDL cholesterol levels of low and high responders show small (< 2-fold) but significant increases. The increase in plasma cholesterol in high responders is mainly due to an increase in VLDL and LDL (V+LDL) cholesterol such that the percentage of total plasma cholesterol carried by V+LDL is increased to ~85% in response to diet. The increase in V+LDL cholesterol on the HCHF diet alters the plasma lipid profile of high responding opossums to resemble more closely that of humans. Plasma triglyceride concentrations are relatively low in low and high responders on the basal diet, and they are not responsive to dietary challenge. The major lipoprotein is apoA-I and the minor lipoprotein is apoE in HDL particles from low and high responders on both diets. ApoB is the major lipoprotein in V+LDL particles from low and high responders on the basal diet and low responders on the HCHF diet. The V+LDL particles from high responders on the HCHF diet are more heterogeneous, and they carry apoE in addition to apoB [10].

genetically modified (transgenic and knockout) mouse models, the opossum model provides an opportunity to identify naturally occurring variants of genes and to study how interactions among gene variants lead to development of hypercholesterolemia. Some partially inbred strains of opossums have inbreeding coefficients in excess of 0.95, and strains with high or low responses to an HCHF diet have inbreeding coefficients in excess of 0.8. Since more than 80% of the genes in each partially inbred strain have alleles that are identical by descent, the work required to identify genes that cause diet-induced hypercholesterolemia in the opossum model

Nine wild-caught animals were imported from Brazil into the United States in 1978 by the National Zoo in Washington, D.C. The founders of the breeding colony of laboratory opossums at Texas Biomedical Research Institute were comprised of 20 first and second generation descendants of those nine founders, together with 17 additional wild-caught opossums from Brazil and two from Bolivia. After several generations of inbreeding, some individuals were fed a high cholesterol (0.6% by weight) and high fat (17% by weight) diet for 6 months, and total blood cholesterol was measured after an overnight fast. Low and high responses were observed among opossums, with few animals exhibiting an intermediate response, i.e. the phenotypes are clustered at the high and low ends of the range. Low responding opossums had blood cholesterol levels ranging from 62-171 mg/dl whereas high responding opossums had levels of 215-932 mg/dl [22]. Furthermore, analysis of lipoprotein particles by gradient gel electrophoresis showed elevated levels of LDL particles in high responding opossums [22]. Subsequently, inbreeding and selection for either low responsiveness or high responsiveness to the HCHF diet led to development of two related low responding partially inbred strains (designated ATHE and ATHL) and a high responding partially inbred strain (designated ATHH) that show extreme difference (>5-fold) in plasma cholesterol concentrations in response to the HCHF diet. These strains are being used to identify genetic variants and

*Dietary challenge and plasma cholesterol response*. Studies were conducted to compare lipoprotein characteristics of low responders from the ATHE strain and high responders from the ATHH strain. The standard laboratory diet is the basal diet, which contains 0.04% cholesterol and 8.1% fat, by weight. Plasma cholesterol concentrations of low and high responders do not differ on this diet. Most of the plasma cholesterol is carried by HDL, and ~30% of total plasma cholesterol is carried by LDL [10]. After consuming the HCHF diet for at least 4 weeks, total plasma cholesterol increases slightly (< 2-fold) in low responders, but dramatically in high responders (>5-fold). HDL cholesterol levels of low and high responders show small (< 2-fold) but significant increases. The increase in plasma cholesterol in high responders is mainly due to an increase in VLDL and LDL (V+LDL) cholesterol such that the percentage of total plasma cholesterol carried by V+LDL is increased to ~85% in response to diet. The increase in V+LDL cholesterol on the HCHF diet alters the plasma lipid profile of high responding opossums to

molecular mechanisms that cause diet-induced hypercholesterolemia.

**3.3. Hypercholesterolemia in high responders**

is substantially reduced.

10 Hypercholesterolemia

**3.2. Development of partially inbred strains**

*Cholesterol absorption*. Additional studies were conducted to determine whether low and high responders differ in cholesterol absorption, which is one of the physiologic processes that govern cholesterol homeostasis. Cholesterol absorption was measured by the fecal isotope ratio method. On the basal diet, the percentage of cholesterol absorbed through the intestine was ~60% in low and high responding opossums. On the HCHF diet, low responders reduced the percentage of absorbed cholesterol by 50%, whereas high responders did not [63]. Several genes that play a role in cholesterol absorption were analyzed to determine whether their expression differed between low and high responders on the HCHF diet. Dietary cholesterol increased expression of *ABCG5* and *ABCG8* in the small intestine of low and high responders to limit absorption of cholesterol by transporting cholesterol from enterocytes to the intestinal lumen; the extent of increase was similar in both strains of opossums. The *NPC1L1* gene transports cholesterol from the lumen into enterocytes, and the *ACAT2* and *MTP* genes facilitate chylomicron formation and secretion in the small intestine. These genes were expressed at similar levels in low and high responders. Therefore, the difference in cholesterol absorption between low and high responders is not due to differences in expression of genes that regulate influx and efflux of cholesterol in the small intestine [64].

*Bile acid synthesis*. Liver is the other major site that controls cholesterol homeostasis. In the liver, cholesterol is converted to bile acids, and bile acids and free cholesterol are secreted into bile for disposal via fecal excretion. Bile acids are synthesized by two pathways, the classic pathway and the alternate pathway. The rate-limiting enzyme in the classic pathway is 7α-hydroxylase. The enzyme sterol 27-hydroxylase initiates bile acid synthesis in the alternate pathway and catalyzes several oxidation reactions in the classic and alternate bile acid synthesis pathways [65]. Low and high responders on the HCHF diet had similar 7α-hydroxylase activities but differed in sterol 27-hydroxylase activities. Low responders had higher activity of hepatic sterol 27-hydroxylase (14.1 ± 0.8 pmol/mg protein/min) compared with high responders (10.1 ± 0.8 pmol/mg protein/min; *P*<0.01) [66]. Sterol 27-hydroxylase is encoded by the *CYP27A1* gene. Expression of the *CYP27A1* gene was also higher (2-fold) in low responders fed a high cholesterol diet [64]. Given that sterol 27-hydroxylase catalyzes several reactions in the classic and alternate pathways of bile acid synthesis, a reduction in enzyme activity was consistent with lower concentrations of total bile acids in gall bladder bile of high responders (194 ± 19 mol/ml for high responders versus 244 ± 15 mol/ml for low responders; *P*=0.05) [66].

*Biliary cholesterol and biliary phospholipids*. Bile samples collected from gall bladders were analyzed to determine whether there are any differences in the secretion of cholesterol into bile by low and high responders fed the HCHF diet. The results revealed differences in biliary cholesterol concentration and biliary phospholipid concentration in gall bladder bile. Choles‐ terol concentration in the bile of low responders (5.7 ± 1.3 mg/ml) was higher than that of high responders (1.3 ± 0.7 mg/ml; *P*<0.05). Similarly, phospholipid concentration in the bile of low responders (29.7 ± 3.7 mg/ml) was also higher than that of high responders (6.9 ± 5.5 mg/ml for high responders; *P*<0.05) [24]. Biliary cholesterol secretion is mediated by ABCG5, ABCG8 and NPC1L1. ABCG5 and ABCG8 transport cholesterol from the liver into bile [45] whereas NPC1L1 transports cholesterol from the bile back into the liver [67]. Biliary phospholipid secretion is mediated by ABCB4 [41,42]. The difference in biliary cholesterol and biliary phospholipid secretion prompted an investigation of the expression of these genes in response to dietary challenge in low and high responders. *ABCG5* and *ABCG8* mRNA levels did not differ between low and high responding opossums, whereas *NPC1L1* mRNA levels were down-regulated in high responders [68]. There was no significant difference in *ABCB4* mRNA levels between low and high responders on the two diets [69]. Therefore, expression of cholesterol and phospholipid transporter genes cannot explain differences in biliary lipid concentrations.

*Association of ABCB4 with hypercholesterolemia*. Development of a genetic linkage map of *Monodelphis* [70], coupled with the *Monodelphis* genome sequence [62], facilitated the identifi‐ cation of genes that predispose high responders to develop hypercholesterolemia on the HCHF diet. Genome-wide linkage analyses on data from pedigreed opossums located two quantita‐ tive trait loci (QTL) influencing V+LDL cholesterol levels. The QTL on chromosome 1 influ‐ ences V+LDL cholesterol on the basal diet, and the QTL on chromosome 8 influences V+LDL cholesterol on the HCHF diet [69]. One gene in the chromosome 8 QTL is *ABCB4*. Since ABCB4 plays a role in biliary secretion of phospholipids and cholesterol, lower levels of biliary lipids in high responding opossums could be due to an impairment of ABCB4 function. We tested this hypothesis by sequencing the *ABCB4* gene to identify mutations and found two single nucleotide polymorphisms (SNPs) that cause missense mutations in exons 2 and 7 of the *ABCB4* gene from high responders. In exon 2, Gly at amino acid 29 in allele 1 is substituted by Arg in allele 2. In exon 7, Leu at amino acid 235 in allele 1 is substituted by Ile in allele 2. Allele 1 is predominant in the high responding strain, whereas allele 2 is predominant in the two low responding strains [24,69].

Using a pedigree-based genetic association approach, matings of high responders with low responders were carried out to produce F2 progeny in two different crosses (designated JCX and KUSH6) to determine whether ABCB4 has an effect on response to dietary cholesterol. Animals from both crosses were genotyped for the ABCB4 Ile235Leu polymorphism and subjected to measured genotype analysis using plasma cholesterol data from a basal diet and from a 4-week HCHF diet. The average concentration of plasma total cholesterol and V+LDL cholesterol on the HCHF diet was highest in JCX animals homozygous for the *ABCB4* '1' allele, intermediate in animals with the *ABCB4* '1/2' genotype, and lowest in animals homozygous for the *ABCB4* '2' allele. A similar pattern was observed in animals from the KUSH6 cross. The results showed that genetic variation in *ABCB4* had a significant effect on variation in V+LDL cholesterol levels in response to the HCHF diet, and implicated defects in biliary phospholipid and biliary cholesterol secretion in causing diet-induced hypercholesterolemia in the opossum model. However, it was apparent from the analysis that there is at least one additional gene that influences diet-induced hypercholesterolemia because some opossums that are homozy‐ gous for the missense mutations are not high responders [24,69].

Variations in the *ABCB4* gene have not been shown previously to be associated with variations in plasma LDL cholesterol in response to diet in other experimental animals or humans. ABCB4 mutations affect secretion of phospholipids, and clinical symptoms are due to production of bile with a low phospholipid content which cannot prevent bile salts from damaging the membranes of cells lining the bile ducts. Moreover, the phospholipid deficient bile has a high cholesterol saturation index. In humans, *ABCB4* variants that have a severe effect are associated with progressive familial intrahepatic cholestasis type 3, a liver disease that often develops in the first year of life. *ABCB4* variants that have a moderate effect are associated with a gallstone disease in adults known as low-phospholipid associated cholelithiasis, and a reversible form of cholestasis known as intrahepatic cholestasis of pregnancy that develops in women during the third trimester of pregnancy and resolves after delivery of their babies [71]. *ABCB4* knockout mice lacking phospholipid transport function develop sclerosing cholangitis, which progresses to metastatic liver cancer [72,73]. Mutations in the opossum A*BCB4* gene do not have a severe effect as high responders exhibit no adverse symptoms when the animals are fed the basal diet. The reduction in biliary cholesterol and biliary phospholipids associated with *ABCB4* mutations leads to an increase in plasma V+LDL cholesterol when high responders are challenged with the HCHF diet. However, a gene whose identity is still unknown seems to be able to compensate for the reduction in biliary cholesterol secretion and rescues high responders that are homozygous for the *ABCB4* mutations from developing diet-induced hypercholesterolemia. Identification of this gene will lead to a better understanding of the process involving *ABCB*4 in controlling plasma LDL cholesterol concentration in response to dietary cholesterol.

#### **3.4. Pathologic features of high responders**

terol concentration in the bile of low responders (5.7 ± 1.3 mg/ml) was higher than that of high responders (1.3 ± 0.7 mg/ml; *P*<0.05). Similarly, phospholipid concentration in the bile of low responders (29.7 ± 3.7 mg/ml) was also higher than that of high responders (6.9 ± 5.5 mg/ml for high responders; *P*<0.05) [24]. Biliary cholesterol secretion is mediated by ABCG5, ABCG8 and NPC1L1. ABCG5 and ABCG8 transport cholesterol from the liver into bile [45] whereas NPC1L1 transports cholesterol from the bile back into the liver [67]. Biliary phospholipid secretion is mediated by ABCB4 [41,42]. The difference in biliary cholesterol and biliary phospholipid secretion prompted an investigation of the expression of these genes in response to dietary challenge in low and high responders. *ABCG5* and *ABCG8* mRNA levels did not differ between low and high responding opossums, whereas *NPC1L1* mRNA levels were down-regulated in high responders [68]. There was no significant difference in *ABCB4* mRNA levels between low and high responders on the two diets [69]. Therefore, expression of cholesterol and phospholipid transporter genes cannot explain differences in biliary lipid

*Association of ABCB4 with hypercholesterolemia*. Development of a genetic linkage map of *Monodelphis* [70], coupled with the *Monodelphis* genome sequence [62], facilitated the identifi‐ cation of genes that predispose high responders to develop hypercholesterolemia on the HCHF diet. Genome-wide linkage analyses on data from pedigreed opossums located two quantita‐ tive trait loci (QTL) influencing V+LDL cholesterol levels. The QTL on chromosome 1 influ‐ ences V+LDL cholesterol on the basal diet, and the QTL on chromosome 8 influences V+LDL cholesterol on the HCHF diet [69]. One gene in the chromosome 8 QTL is *ABCB4*. Since ABCB4 plays a role in biliary secretion of phospholipids and cholesterol, lower levels of biliary lipids in high responding opossums could be due to an impairment of ABCB4 function. We tested this hypothesis by sequencing the *ABCB4* gene to identify mutations and found two single nucleotide polymorphisms (SNPs) that cause missense mutations in exons 2 and 7 of the *ABCB4* gene from high responders. In exon 2, Gly at amino acid 29 in allele 1 is substituted by Arg in allele 2. In exon 7, Leu at amino acid 235 in allele 1 is substituted by Ile in allele 2. Allele 1 is predominant in the high responding strain, whereas allele 2 is predominant in the two low

Using a pedigree-based genetic association approach, matings of high responders with low responders were carried out to produce F2 progeny in two different crosses (designated JCX and KUSH6) to determine whether ABCB4 has an effect on response to dietary cholesterol. Animals from both crosses were genotyped for the ABCB4 Ile235Leu polymorphism and subjected to measured genotype analysis using plasma cholesterol data from a basal diet and from a 4-week HCHF diet. The average concentration of plasma total cholesterol and V+LDL cholesterol on the HCHF diet was highest in JCX animals homozygous for the *ABCB4* '1' allele, intermediate in animals with the *ABCB4* '1/2' genotype, and lowest in animals homozygous for the *ABCB4* '2' allele. A similar pattern was observed in animals from the KUSH6 cross. The results showed that genetic variation in *ABCB4* had a significant effect on variation in V+LDL cholesterol levels in response to the HCHF diet, and implicated defects in biliary phospholipid and biliary cholesterol secretion in causing diet-induced hypercholesterolemia in the opossum model. However, it was apparent from the analysis that there is at least one additional gene

concentrations.

12 Hypercholesterolemia

responding strains [24,69].

*Fatty livers*. Dysregulated cholesterol homeostasis causes high responders to develop fatty livers and atherosclerotic lesions. Cholesterol accumulates in the livers of high responders as a result of impaired biliary cholesterol secretion. After 4 weeks of HCHF diet, serum levels of liver enzymes (alanine aminotransferase, aspartate aminotransferase, and γ– glutamyltransferase) and bilirubin were significantly elevated, indicating high responders had liver injury. Histology revealed steatosis, inflammation and ballooned hepatocytes in their livers after 8 weeks of HCHF diet. The pathologic condition in the liver worsened as high responders continued to consume the HCHF diet. In one study in which high and low responders were fed the HCHF diet for 24 weeks, livers of high responders were markedly enlarged compared to those of low responders. The enlarged livers had an increase in free cholesterol (2-fold), esterified cholesterol (11-fold) and triglycerides (2 fold), but no significant increase in free fatty acids. Low responders did not display any significant morphological changes in the liver after 24 weeks on the HCHF diet. Pro‐ longed HCHF dietary challenge caused high responders to develop fibrosis in addition to steatosis, inflammation and ballooned cells. Liver fibrosis is a characteristic feature of the severe form of nonalcoholic fatty liver disease known as nonalcoholic steatohepatitis. Expression of a set of hepatic genes associated with inflammation, oxidative stress and fibrogenesis was up-regulated in high responders, and the gene expression pattern was consistent with the histopathological features in the livers of high responders [74].

*Atherosclerotic lesions*. Similar to humans, hypercholesterolemia leads to development of atherosclerotic lesions in the arteries of high responding opossums. Low responding opossums whose V+LDL cholesterol was below 75 mg/dl did not develop gross or histologically detect‐ able lesions after consuming the HCHF diet for one year. In contrast, high responding opossums whose V+LDL cholesterol was over 500 mg/dl developed gross and histologically detectable lesions after 40 weeks of HCHF diet. The opossum lesions were similar in histologic characteristics to those observed in cholesterol-fed mouse models of atherosclerosis [23].

#### **4. Non-human primate models**

Non-human primate models stand out as the most biologically similar to humans in physio‐ logic and genetic characteristics of hypercholesterolemia [25]. This is because nonhuman primates and humans share similar biochemical, anatomical and physiological characteristics, including lipid synthesis and metabolism. Both humans and primates exhibit spontaneous and diet-induced hypercholesterolemia [15] and develop atherosclerosis [11,75]. Commonly used nonhuman primates include African green monkey (green monkey), rhesus monkey, cyno‐ molgus monkey and baboon. These species not only have a high degree of physiological similarity with humans, but also have many of the same genes underlying relevant pheno‐ types. The size of nonhuman primates by comparison to mice enables the collection of tissue and organ samples of equivalent sizes to humans, including arteries and hearts. It is important to mention that great apes share greater similarities to humans than other nonhuman primates. However, cost and ethical considerations prohibit use of great apes for most human disease studies.

#### **4.1. Nonhuman primate responses to HCHF diet**

Nonhuman primates respond to HCHF diet as do humans. Most nonhuman primates respond to HCHF diet by an increase in average total plasma cholesterol concentration ranging from 200-800 mg/dl with no change in weight [11,15]. Total plasma cholesterol levels positively correlate with LDL cholesterol, VLDL cholesterol and triglyceride levels with no change in HDL cholesterol levels. In addition, triglyceride concentration is positively correlated with VLDL cholesterol and LDL cholesterol concentrations [15,25]. However, there are differences among species and among individuals in response to HCHF diet in nonhuman primates.

*Variation among species.* Nonhuman primate species differ in their response to HCHF diet challenges by exhibiting variation in plasma cholesterol [11,76]. Baboons and green monkeys display moderate response with an average plasma cholesterol concentration of 204 and 275 mg/dl, respectively, while cynomolgus monkeys and rhesus macaque have the highest response, 307 and 467 mg/dl, respectively [11,15,76]. The response of baboons is similar to that of humans [11].

In addition, green monkeys and baboons show unusual increases in HDL cholesterol levels in response to HCHF diet compared to most nonhuman primate species [11,76]. The mechanisms underlying the marked difference in response to HCHF diet for green monkeys and baboons are not well understood. Sorci-Thomas et al. [76] compared the responses of green and cynomolgus monkeys to HCHF diet. Because green monkeys develop modest hypercholes‐ terolemia compared to cynomolgus monkeys when challenged with HCHF diet, green monkeys were fed diet with more cholesterol than cynomolgus monkeys to induce equivalent extent of hypercholesterolemia in both species. Surprisingly green monkeys still had 2-3-fold higher plasma HDL cholesterol and apoA-I concentrations than cynomolgus monkeys, indicating that higher plasma HDL cholesterol in green monkeys was due to factors inde‐ pendent of level of dietary cholesterol. Further investigation indicated that green monkey hepatic apoA-I and mRNA expression levels were respectively 2-fold and 3.7-fold higher, and intestinal *apoA-I* mRNA level was 3.7-folder higher than in cynomolgus monkeys. These observations indicate that factors that regulate mRNA transcription and post-transcription, including microRNA (miRNA) gene regulation, may be determinants of resistance to HCHF diet.

Expression of a set of hepatic genes associated with inflammation, oxidative stress and fibrogenesis was up-regulated in high responders, and the gene expression pattern was

*Atherosclerotic lesions*. Similar to humans, hypercholesterolemia leads to development of atherosclerotic lesions in the arteries of high responding opossums. Low responding opossums whose V+LDL cholesterol was below 75 mg/dl did not develop gross or histologically detect‐ able lesions after consuming the HCHF diet for one year. In contrast, high responding opossums whose V+LDL cholesterol was over 500 mg/dl developed gross and histologically detectable lesions after 40 weeks of HCHF diet. The opossum lesions were similar in histologic characteristics to those observed in cholesterol-fed mouse models of atherosclerosis [23].

Non-human primate models stand out as the most biologically similar to humans in physio‐ logic and genetic characteristics of hypercholesterolemia [25]. This is because nonhuman primates and humans share similar biochemical, anatomical and physiological characteristics, including lipid synthesis and metabolism. Both humans and primates exhibit spontaneous and diet-induced hypercholesterolemia [15] and develop atherosclerosis [11,75]. Commonly used nonhuman primates include African green monkey (green monkey), rhesus monkey, cyno‐ molgus monkey and baboon. These species not only have a high degree of physiological similarity with humans, but also have many of the same genes underlying relevant pheno‐ types. The size of nonhuman primates by comparison to mice enables the collection of tissue and organ samples of equivalent sizes to humans, including arteries and hearts. It is important to mention that great apes share greater similarities to humans than other nonhuman primates. However, cost and ethical considerations prohibit use of great apes for most human disease

Nonhuman primates respond to HCHF diet as do humans. Most nonhuman primates respond to HCHF diet by an increase in average total plasma cholesterol concentration ranging from 200-800 mg/dl with no change in weight [11,15]. Total plasma cholesterol levels positively correlate with LDL cholesterol, VLDL cholesterol and triglyceride levels with no change in HDL cholesterol levels. In addition, triglyceride concentration is positively correlated with VLDL cholesterol and LDL cholesterol concentrations [15,25]. However, there are differences among species and among individuals in response to HCHF diet in nonhuman primates.

*Variation among species.* Nonhuman primate species differ in their response to HCHF diet challenges by exhibiting variation in plasma cholesterol [11,76]. Baboons and green monkeys display moderate response with an average plasma cholesterol concentration of 204 and 275 mg/dl, respectively, while cynomolgus monkeys and rhesus macaque have the highest response, 307 and 467 mg/dl, respectively [11,15,76]. The response of baboons is similar to that

consistent with the histopathological features in the livers of high responders [74].

**4. Non-human primate models**

**4.1. Nonhuman primate responses to HCHF diet**

studies.

14 Hypercholesterolemia

of humans [11].

*Variation among individuals.* Similar to humans, nonhuman primates display variation among individual animals of the same species in response to HCHF diet [77-80]. This variation is one of the important features of nonhuman primate models that enables us to identify genetic variants that predispose individuals to develop hypercholesterolemia.

**a.** *Plasma lipoprotein cholesterol levels.* The response of plasma cholesterol level to HCHF diet differs among individuals. Baboons challenged with HCHF diet for 2 years exhibited an increase in plasma cholesterol levels from 5 to 197 mg/dl [81]. Based on these observations, McGill et al. selectively bred two lines of baboons with extreme plasma cholesterol levels; low responders and high responders to HCHF diet. Subsequent studies have shown that low and high responders differed in LDL cholesterol levels when challenged with HCHF diet. High responders had approximately 2-fold higher plasma cholesterol than low responders [11,80]. In addition, LDL *apoB* concentrations were 2-3-fold higher in high responders compared with low responders [11]. This difference was due to higher production of apoB in high responders. However, apoB mRNA levels did not differ between low and high responders on HCHF diet, suggesting that apoB production is regulated at the post-transcriptional level and is influenced by plasma cholesterol levels, which differ between low and high responders.

McGill et al. [82] examined the effect of cholesterol or saturated fat on plasma cholesterol response in low and high responders. The study revealed that high responders challenged with diet containing high cholesterol (1.7 mg/kcal) displayed a higher percent increase of LDL and VLDL cholesterol levels than low responders, and that there was no difference in HDL cholesterol levels between high and low responders. The type of saturated fat, corn or coconut oil in the diet did not influence plasma cholesterol variation between the two lines of baboons. Genetic analysis revealed that genetic factors explained 57% of the response to dietary cholesterol. These findings indicated that dietary cholesterol, and not saturated fat, drives variation in plasma cholesterol in baboons.


Together, these findings suggest that plasma cholesterol variation in nonhuman primates may be influenced by the level of sterol 27-hydroxylase activity in the liver. A decrease in conversion of cholesterol to bile acids may lead to an increase in plasma cholesterol, which in turn decreases LDL receptor expression. As a consequence, the rate of clearance of plasma VLDL and LDL cholesterol is reduced and circulating levels of LDL cholesterol are elevated.

#### **4.2. Genetic mechanisms that influence individual variation in plasma cholesterol levels**

*Baboon genetic resources to study lipid metabolism.* The baboon is the most commonly used primate model for genetic studies of complex traits and susceptibility to complex diseases [14]. The Southwest National Primate Research Center (SNPRC) at Texas Biomedical Research Institute maintains approximately 1,500 living baboons for biological research. These baboons have been used to develop nonhuman primate genomic resources to study responses to environ‐ mental factors, such as diet, and how these factors interact with genomic factors in causing complex diseases or disorders. In addition, SNPRC maintains an extensive pedigree database consisting of 16,000 baboons across seven generations. This is the largest nonhuman primate pedigree in the world. The pedigreed population includes approximately 384 founders of olive (*P. h. anubis*) and yellow (*P. h. cynocephalus)* baboons, and their hybrid descendants. These resources provided a unique opportunity to map baboon genes, resulting in the first ever nonhuman primate linkage map [84,85]. In addition, tissues and blood samples have been collected from 8,000 baboons, and DNA, serum and buffy coats from 4,000 animals [14].

**b.** *Expression of 27-hydroxylase.* Nonhuman primates exhibit individual variation in the synthesis of bile acids from cholesterol. Sterol 27-hydroxylase is an important enzyme for bile acid synthesis in both the classic and alternate pathways. Kushwaha et al. [83] measured plasma and hepatic 27-hydroxycholesterol levels, hepatic 27-hydroxylase activity and mRNA levels in 12 low and 12 high responding baboons. Low responders displayed higher 27-hydroxycholesterol levels, 27-hydroxylase activities and mRNA levels than high responders when fed the HCHF diet but not when fed the chow diet. These parameters were negatively correlated with LDL and VLDL cholesterol concentra‐ tions in low responders. These findings indicate that sterol 27-hydroxylase is induced by HCHF diet and that the induction is higher in low responding baboons, resulting in higher bile acid synthesis. Thus, the ability to induce sterol 27-hydroxylase influences LDL

**c.** *ApoE levels and LDL receptor expression.* The liver clears excess plasma lipoprotein choles‐ terol through the LDL receptor and the LDL receptor-related protein. ApoE is a compo‐ nent of chylomicrons and most of the lipoproteins, and aids in receptor mediatedclearance of plasma lipoprotein cholesterol [31]. A study in cynomolgus and green monkeys fed an HCHF diet demonstrated that apoE concentrations were positively correlated with total plasma cholesterol concentrations, plasma LDL cholesterol concen‐ trations and LDL particle size [12]. Since apoE is a high-affinity ligand for the LDL receptor and the LDL receptor-related protein, plasma cholesterol clearance by the liver is expected to increase when apoE levels are elevated, but this is not the case. A possible explanation is that LDL receptors are down-regulated in hypercholesterolemic monkeys, and clear‐ ance of VLDL and LDL particles is impeded. ApoE-enriched LDL particles accumulate in the plasma of hypercholesterolemic animals because VLDL particles are metabolized to LDL particles rather than being removed from the circulation by the LDL receptor as in

Together, these findings suggest that plasma cholesterol variation in nonhuman primates may be influenced by the level of sterol 27-hydroxylase activity in the liver. A decrease in conversion of cholesterol to bile acids may lead to an increase in plasma cholesterol, which in turn decreases LDL receptor expression. As a consequence, the rate of clearance of plasma VLDL and LDL cholesterol is reduced and circulating levels of LDL cholesterol are elevated.

**4.2. Genetic mechanisms that influence individual variation in plasma cholesterol levels**

*Baboon genetic resources to study lipid metabolism.* The baboon is the most commonly used primate model for genetic studies of complex traits and susceptibility to complex diseases [14]. The Southwest National Primate Research Center (SNPRC) at Texas Biomedical Research Institute maintains approximately 1,500 living baboons for biological research. These baboons have been used to develop nonhuman primate genomic resources to study responses to environ‐ mental factors, such as diet, and how these factors interact with genomic factors in causing complex diseases or disorders. In addition, SNPRC maintains an extensive pedigree database consisting of 16,000 baboons across seven generations. This is the largest nonhuman primate pedigree in the world. The pedigreed population includes approximately 384 founders of olive

monkeys with normal plasma cholesterol concentrations [12].

cholesterol variation in baboons.

16 Hypercholesterolemia

*Genetic factors for hypercholesterolemia in baboons.* Baboon response to HCHF diet is modest, similar to the response of humans. Because of this similarity as well as other baboon charac‐ teristics that mimic human characteristics, numerous studies have utilized baboon resources available at SNPRC to understand how genetic variation influences lipoprotein cholesterol in response to diet. This initiative started more than three decades ago when scientists at the Texas Biomedical Research Institute observed differential response of baboons to HCHF diet [81]. These observations led to selective breeding and characterization of distinct phenotypes of baboons and revealed that differential response to HCHF diet is heritable [86,87].

Attempts to find the major genes influencing plasma cholesterol in response to dietary challenge revealed that polymorphisms in the LDL receptor gene contributed only 6% of the variation [88], suggesting that lipid response to HCHF diet is multigenic. Hypercholesterole‐ mia is a complex disorder plausibly influenced by complex genetic networks. Therefore, to elucidate the mechanisms that underlie cholesterol variation, a system biological approach is most appropriate. Using available pedigree and genotypic information for more than 2,400 baboons, important lipid/lipoprotein-related QTL have been identified [27,89,90]. In addition, improved Next Gen Sequencing techniques for RNA and DNA sequencing and genetic network analyses have enabled understanding of genes encoding these QTL.

Studies were undertaken to interrogate the QTL to discover gene, genetic variants and functional mechanisms that influence variation in response to diet in baboons. In one study, four novel candidate genes (*TENC1, ACVR1B, ERBB3, DGKA*) were identified that encode a QTL for LDL cholesterol concentration variation. This QTL overlaps multiple other QTL for LDL related traits, including particle size, suggesting that these genes have pleiotropic effects [13]. *TENC1* was downregulated while *ACVR1B, ERBB3* and *DGKA* were upregulated in response to HCHF diet. The protein products of all four genes are central molecules for a single pathway, affirming that multiple genes influence LDL cholesterol variation. Interestingly these genes are associated with cancer in the *AKT/GSK3B* signaling pathway [91]. Several studies have alluded a link between cancer, hypercholesterolemia and atherosclerosis [92-95], but the link is not well understood. One aspect that is clear is that cholesterol is intertwined in the etiology of cancer and atherosclerosis. In addition, tumorigenesis thrives by the ability to alter important biological processes, including regulation of cholesterol levels. For cancer cells to proliferate uncontrollably, essential cell components, such as cholesterol must be available for plasma membrane synthesis. In order to meet the demand for cholesterol, pathways regulating cellular cholesterol homeostasis are altered in cancer cells.

Other studies have investigated the role of miRNA in LDL cholesterol variation in baboons [13,80]. miRNAs were hypothesized to regulate genes encoding variation in LDL cholesterol in response to HCHF diet. Hepatic miRNA expression profiling in low and high LDL choles‐ terol half–sibling baboons by RNA sequencing revealed 226 miRNAs were differentially expressed (160 downregulated and 66 upregulated) between low and high responders in response to HCHF diet. In order to identify molecular mechanisms that may regulate LDL cholesterol variation, these miRNAs were overlaid onto gene networks that differ between low and high baboon responders. Seven miRNAs were inversely expressed with respect to the four candidate genes. Together, these findings demonstrate that hepatic miRNAs are responsive to diet, and that response differs among baboons with different plasma LDL cholesterol levels.

#### **4.3. Nonhuman primates and atherosclerosis, the clinical endpoint of hypercholesterolemia**

Atherosclerosis, a complex progressive disease, is the leading cause of mortality and morbidity in developed countries [96,97]. The clinical end-point of atherosclerosis is cardiovascular disease primarily caused by thickening and/or occlusion of coronary arteries. Atherosclerotic heart disease is the leading cause of death in the world and is projected to remain the single leading cause of death by 2030 [98].

*Atherogenesis is similar in nonhuman primates and humans.* Atherogenesis is a multifactorial process. Initial events during atherogenesis include deposition of modified or oxidized cholesterol (ox-cholesterol) in the artery wall, resulting in endothelial dysfunction; adhesion of circulating monocytes onto the endothelium; entry of ox-cholesterol and monocytes into the intima layer of the artery; engulfment of ox-cholesterol by monocytes and transformation into macrophages and foam cells; production of pro-inflammatory cytokines and connective matrix; conversion and proliferation of smooth muscle cells; cell apoptosis; and intima thickening. During these processes, atherosclerotic lesions, which are grossly and microscop‐ ically heterogeneous, develop on the intimal arterial surface. In nonhuman primates, as in humans, the initial lesions are flat fatty streaks, which are not elevated on the intimal surface, containing predominately foam cells derived from monocytes and smooth muscle cells filled with minimal lipid. These lesions advance to raised fatty streaks that are characterized by lipidfilled foam cells. Raised lesions may progress to advanced fibrous plaques with lipid cores and accumulated connective matrix [26,75,99].

*Lipoprotein cholesterol and atherosclerosis.* Nonhuman primates provide a unique opportunity not only to understand the factors that underlie differential response to HCHF diet but the link between diet response and development of atherosclerosis in humans. In both nonhuman primates and humans, dyslipidemia is associated with atherosclerosis. High levels of non-HDL cholesterol, including LDL cholesterol, VLDL cholesterol and triglycerides, induced by HCHF diet are positively correlated with the extent and severity of atherosclerosis while HDL cholesterol is negatively correlated [75]. This implies that HCHF diets indirectly influence atherogenesis through induction of hypercholesterolemia. Another study with baboons revealed that plasma HDL1 levels are negatively correlated with extent and severity of atherosclerosis [11]. These results are consistent with results from human studies that indicate plasma lipoprotein cholesterol levels and lipoprotein subclasses are indicators of the extent of atherosclerosis [100]. Stevenson et al. [12] observed in cynomolgus monkeys that an increase in apoE correlates with extent of atherosclerosis, suggesting that apoE may represent an atherogenic feature of diet-induced hypercholesterolemia.

*Arterial distribution of atherosclerosis.* Atherosclerosis in nonhuman primates and humans displays a distinctive topographical distribution in the arterial system. The extent and severity of the disease is greater in the abdominal and common iliac arteries than in the thoracic and aortic arch, whereas flat lesions are more abundant in thoracic and aortic arch in nonhuman primates and humans [11,26]. It is hypothesized that the distinct localization of atherosclerotic lesions is a consequence of hemodynamic stress induced by blood flow; and anatomic, cellular, or biochemical variations in the arterial wall, particularly in the endothelium. These hypoth‐ eses are consistent with observations of more abundant lesions in branches and bifurcations of medium-sized arteries, including abdominal and common iliac arteries in nonhuman primates [101]. However, the mechanisms underlying the varied distribution of atherosclerosis in both humans and nonhuman primates are not well understood.

*Variation among species and individuals in development of atherosclerosis.* Nonhuman primate species display variation in susceptibility to developing atherosclerosis. Paralleling the different responses to HCHF diet, rhesus and cynomolgus monkeys are more susceptible to atherosclerosis [102] than green monkeys and baboons, which develop moderate atheroscle‐ rosis [103] as do humans [11]. Individuals within any one species also display differential susceptibility to atherosclerosis. High responders to HCHF diet develop more severe athero‐ sclerosis than low responders, consistent with differential levels of plasma non-HDL choles‐ terol [26]. It is this variation that is critical for identification of genetic factors underlying variation in atherosclerosis development. This variation is heritable [104] and may correspond to genetic variation that underlies observed plasma cholesterol variation in response to HCHF diet.

#### **5. Conclusion**

response to HCHF diet. In order to identify molecular mechanisms that may regulate LDL cholesterol variation, these miRNAs were overlaid onto gene networks that differ between low and high baboon responders. Seven miRNAs were inversely expressed with respect to the four candidate genes. Together, these findings demonstrate that hepatic miRNAs are responsive to diet, and that response differs among baboons with different plasma LDL cholesterol levels.

**4.3. Nonhuman primates and atherosclerosis, the clinical endpoint of hypercholesterolemia** Atherosclerosis, a complex progressive disease, is the leading cause of mortality and morbidity in developed countries [96,97]. The clinical end-point of atherosclerosis is cardiovascular disease primarily caused by thickening and/or occlusion of coronary arteries. Atherosclerotic heart disease is the leading cause of death in the world and is projected to remain the single

*Atherogenesis is similar in nonhuman primates and humans.* Atherogenesis is a multifactorial process. Initial events during atherogenesis include deposition of modified or oxidized cholesterol (ox-cholesterol) in the artery wall, resulting in endothelial dysfunction; adhesion of circulating monocytes onto the endothelium; entry of ox-cholesterol and monocytes into the intima layer of the artery; engulfment of ox-cholesterol by monocytes and transformation into macrophages and foam cells; production of pro-inflammatory cytokines and connective matrix; conversion and proliferation of smooth muscle cells; cell apoptosis; and intima thickening. During these processes, atherosclerotic lesions, which are grossly and microscop‐ ically heterogeneous, develop on the intimal arterial surface. In nonhuman primates, as in humans, the initial lesions are flat fatty streaks, which are not elevated on the intimal surface, containing predominately foam cells derived from monocytes and smooth muscle cells filled with minimal lipid. These lesions advance to raised fatty streaks that are characterized by lipidfilled foam cells. Raised lesions may progress to advanced fibrous plaques with lipid cores and

*Lipoprotein cholesterol and atherosclerosis.* Nonhuman primates provide a unique opportunity not only to understand the factors that underlie differential response to HCHF diet but the link between diet response and development of atherosclerosis in humans. In both nonhuman primates and humans, dyslipidemia is associated with atherosclerosis. High levels of non-HDL cholesterol, including LDL cholesterol, VLDL cholesterol and triglycerides, induced by HCHF diet are positively correlated with the extent and severity of atherosclerosis while HDL cholesterol is negatively correlated [75]. This implies that HCHF diets indirectly influence atherogenesis through induction of hypercholesterolemia. Another study with baboons revealed that plasma HDL1 levels are negatively correlated with extent and severity of atherosclerosis [11]. These results are consistent with results from human studies that indicate plasma lipoprotein cholesterol levels and lipoprotein subclasses are indicators of the extent of atherosclerosis [100]. Stevenson et al. [12] observed in cynomolgus monkeys that an increase in apoE correlates with extent of atherosclerosis, suggesting that apoE may represent an

*Arterial distribution of atherosclerosis.* Atherosclerosis in nonhuman primates and humans displays a distinctive topographical distribution in the arterial system. The extent and severity of the disease is greater in the abdominal and common iliac arteries than in the thoracic and

leading cause of death by 2030 [98].

18 Hypercholesterolemia

accumulated connective matrix [26,75,99].

atherogenic feature of diet-induced hypercholesterolemia.

ApoE deficient mice, generated by gene targeting, have a lipoprotein profile similar to humans in that most of the plasma cholesterol is carried on VLDL and IDL particles rather than HDL particles as in non-genetically modified mice. *ApoE*-/- mice have elevated levels of plasma cholesterol even on a chow diet and develop atherosclerotic lesions spontaneously. Advanced lesions with plaque rupture that resemble those in humans are frequently observed in *apoE*-/ mice fed an HCHF diet, and these mice are used for developing drugs to reduce atherosclerosis.

A nonbiliary pathway for cholesterol excretion in humans was suggested more than five decades ago based on measurement of intestinal cholesterol secretion from patients with bile duct obstruction. This finding was largely ignored because hepatobiliary secretion is believed to be the only route to dispose of cholesterol in feces. Observations from studies using several genetically modified mice (*G5G8*-/-, *Abcb4-/-* and *Npc1l1*-LiverTg) that have severe defects in biliary cholesterol secretion, but normal or even increased fecal neutral sterol excretion, prompted several groups of researchers to investigate the TICE pathway. They reported that TICE accounts for 20%-30% of fecal neutral sterol excretion. However, mechanistic details of TICE still remain unknown. Because cholesterol excretion is an important process to eliminate cholesterol from the body, stimulation of TICE by pharmacological agents may be a novel therapeutic strategy to limit atherogenesis.

Studies using genetically modified mice have shown that they are indispensable for advancing our knowledge of the genes and pathways that govern cholesterol homeostasis, as well as for developing pharmacological agents to treat atherosclerosis. Traditional gene targeting using embryonic stem cells is a complex and time-consuming procedure to produce mutant mice and is limited to targeting one gene at a time. Mice carrying mutations in multiple genes are produced either by sequential gene targeting or intercrossing mice with a single mutation. The CRISPR (clustered regularly interspaced short palindromic repeat)-Cas9 (CRISPR-associated nuclease 9) system is a new and more efficient genome engineering technology [105]. It allows targeting multiple genes at the same time by direct co-injection of RNA encoding the Cas9 nuclease and several gene-specific guide RNA into a one-cell embryo to generate mice with multiple modified genes [106]. Application of the CRISPR/Cas system will accelerate the production of mouse models carrying multiple modified genes to study complex disease such as hypercholesterolemia.

High responding opossums have naturally occurring genetic variants that predispose them to develop hypercholesterolemia when challenged with an HCHF diet. At least two major genes control the plasma cholesterol response to dietary challenge in high responding opossums. One has been identified as the *ABCB4* gene. Mutations in *ABCB*4 impair the ability of high responding opossums to secrete phospholipids into the bile. Because secretion of cholesterol into bile requires phospholipids, biliary cholesterol secretion is also impaired in high respond‐ ing opossums. As a consequence, plasma V+LDL cholesterol becomes elevated in high responders, and free and esterified cholesterol accumulates in their livers. However, some opossums that are homozygous for the *ABCB4* mutations are resistant to diet-induced hypercholesterolemia. The compensatory mechanism that allows these opossums to overcome the defect in biliary cholesterol secretion is not known. In light of the finding of a nonbiliary route of cholesterol excretion that functions in normal mice as well as in mice that have very low biliary cholesterol secretion, cholesterol excretion by the nonbiliary route may compensate for the defect in biliary excretion in opossums that are homozygous for the *ABCB4* mutations.

Phylogenetic similarities between humans and nonhuman primate models are core aspects for consideration in investigations of environmental and genetic factors that contribute to complex diseases/disorders, including hypercholesterolemia and atherosclerosis. Moreover, like humans, nonhuman primates exhibit diet-induced hypercholesterolemia and naturally develop atherosclerosis, making it possible to identify phenotypic variations without altering genetic background as is required for this line of research with mice. In addition, environ‐ mental factors, including diet, can be controlled for a prolonged period of time, invasive and terminal experiments can be conducted, and tissues and organs can be easily collected. These research activities are not attainable when working with human subjects.

Recent scientific advances have led to discovery of therapeutic regimes, including statins for lowering LDL cholesterol and retarding the development of atherosclerosis [107]. However, these therapies are limited by side effects and ineffectiveness in some individuals [108,109]. Thus, there is a need for continued searching for novel therapeutic agents. Diet-induced hypercholesterolemia in nonhuman primates provides an opportunity 1) to identify lipid profiles important for the development of atherosclerosis in primates in a controlled environ‐ ment, 2) to identify variation in responses to diet, and 3) to assess progression of and variation in development of atherosclerosis in response to dietary cholesterol and saturated fat. Identi‐ fication of these variations is essential for genetic analysis to develop novel therapeutic agents for lowering plasma cholesterol and biomarkers for detection of early atherosclerosis, a precursor for cardiovascular disease.

Nonhuman primate genetic resources for studying complex diseases are becoming increas‐ ingly sophisticated and available at SNPRC and Texas Biomedical Research Institute. These resources enable scientific collaborations to study human diseases using multidisciplinary approaches. Significant steps have been achieved in the identification of genetic causes of hypercholesterolemia and atherosclerosis, including discovery of QTL and genes and gene variants that influence plasma LDL and HDL cholesterol levels, and triglyceride levels.

Further improvement and enhancement of unique genetic resources for research with mice, laboratory opossums, and nonhuman primates will be critical for future research aimed at understanding genetic and epigenetic factors influencing human health and disease.

#### **Acknowledgements**

developing pharmacological agents to treat atherosclerosis. Traditional gene targeting using embryonic stem cells is a complex and time-consuming procedure to produce mutant mice and is limited to targeting one gene at a time. Mice carrying mutations in multiple genes are produced either by sequential gene targeting or intercrossing mice with a single mutation. The CRISPR (clustered regularly interspaced short palindromic repeat)-Cas9 (CRISPR-associated nuclease 9) system is a new and more efficient genome engineering technology [105]. It allows targeting multiple genes at the same time by direct co-injection of RNA encoding the Cas9 nuclease and several gene-specific guide RNA into a one-cell embryo to generate mice with multiple modified genes [106]. Application of the CRISPR/Cas system will accelerate the production of mouse models carrying multiple modified genes to study complex disease such

High responding opossums have naturally occurring genetic variants that predispose them to develop hypercholesterolemia when challenged with an HCHF diet. At least two major genes control the plasma cholesterol response to dietary challenge in high responding opossums. One has been identified as the *ABCB4* gene. Mutations in *ABCB*4 impair the ability of high responding opossums to secrete phospholipids into the bile. Because secretion of cholesterol into bile requires phospholipids, biliary cholesterol secretion is also impaired in high respond‐ ing opossums. As a consequence, plasma V+LDL cholesterol becomes elevated in high responders, and free and esterified cholesterol accumulates in their livers. However, some opossums that are homozygous for the *ABCB4* mutations are resistant to diet-induced hypercholesterolemia. The compensatory mechanism that allows these opossums to overcome the defect in biliary cholesterol secretion is not known. In light of the finding of a nonbiliary route of cholesterol excretion that functions in normal mice as well as in mice that have very low biliary cholesterol secretion, cholesterol excretion by the nonbiliary route may compensate for the defect in biliary excretion in opossums that are homozygous for the *ABCB4* mutations.

Phylogenetic similarities between humans and nonhuman primate models are core aspects for consideration in investigations of environmental and genetic factors that contribute to complex diseases/disorders, including hypercholesterolemia and atherosclerosis. Moreover, like humans, nonhuman primates exhibit diet-induced hypercholesterolemia and naturally develop atherosclerosis, making it possible to identify phenotypic variations without altering genetic background as is required for this line of research with mice. In addition, environ‐ mental factors, including diet, can be controlled for a prolonged period of time, invasive and terminal experiments can be conducted, and tissues and organs can be easily collected. These

Recent scientific advances have led to discovery of therapeutic regimes, including statins for lowering LDL cholesterol and retarding the development of atherosclerosis [107]. However, these therapies are limited by side effects and ineffectiveness in some individuals [108,109]. Thus, there is a need for continued searching for novel therapeutic agents. Diet-induced hypercholesterolemia in nonhuman primates provides an opportunity 1) to identify lipid profiles important for the development of atherosclerosis in primates in a controlled environ‐ ment, 2) to identify variation in responses to diet, and 3) to assess progression of and variation in development of atherosclerosis in response to dietary cholesterol and saturated fat. Identi‐ fication of these variations is essential for genetic analysis to develop novel therapeutic agents

research activities are not attainable when working with human subjects.

as hypercholesterolemia.

20 Hypercholesterolemia

This work was supported by National Institutes of Health Grants P01 HL028972, P01 HL028972-Supplement, P51 OD011133, R01 DK065058, and grants from the Robert J. Kleberg, Jr. and Helen C. Kleberg Foundation and Texas Biomedical Forum. This work was conducted in part in facilities constructed with support from Research Facilities Improvement Program Grant Numbers C06 RR013556 and C06 RR015456 from the National Center for Research Resources (now Office of Research Infrastructure Programs), National Institutes of Health.

#### **Author details**

Jeannie Chan1\*, Genesio M. Karere1 , Laura A. Cox2 and John L. VandeBerg2

\*Address all correspondence to: jchan@txbiomedgenetics.org

1 Department of Genetics, Texas Biomedical Research Institute, San Antonio, Texas, USA

2 Department of Genetics and Southwest National Primate Research Center, Texas Biomedical Research Institute, San Antonio, Texas, USA

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## **Hypercholesterolemia in Childhood**

Lorenzo Iughetti, Barbara Predieri and Patrizia Bruzzi

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/59760

**1. Introduction**

Hypercholesterolemia is a well-known risk factor for atherosclerosis in adulthood.

In the first years of life atherosclerosis is generally subclinical, but pathological studies demonstrate that, already in childhood, atherosclerotic vascular changes and their extend are associated with both the number of cardiovascular risk factors and their intensity [1, 2]. Fatty streaks and fibrous plaques at autopsy, presence of coronary artery calcium by electron-beam computed tomography, increased carotid-intima-media thickness, reduced arterial distensi‐ bility and compliance and endothelial dysfunction by ultrasound have been already associated with lipid abnormalities in youth [3]. Moreover, levels of cholesterol track strongly from childhood and adolescence over long follow-up period resulting in the progression of atherosclerosis process and increased cardiovascular disease (CVD) risk [4].

The mechanism by which hyperlipidemia contributes to atherogenesis includes several stages:


© 2015 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

In some conditions, characterized by altered lipid assessment or other evident disorders of risk factors, premature CVD could be manifest in the first decades of life. "Vascular age" is advanced in 73% of familial dyslipidemic children, independently from the presence of others atherosclerosis-promoting risk factors [8]. The American Heart Association (AHA), endorsed by the American Academy of Pediatrics (AAP), identified 8 high-risk pediatric diagnosis and developed practical recommendations for the management of cardiovascular risk [9]. The selected diseases comprise familial hypercholesterolemia (FH), together with diabetes mellitus (type 1 and 2), chronic kidney disease, heart transplantation, Kawasaki disease, congenital heart disease, chronic inflammatory disease and childhood cancer. Subclinical endothelial dysfunction, measured through non-invasive surrogate methods such as flow-mediated dilation (FMD), occurs early in FH children indicating an increased risk for premature CVD and reflecting the need for early initiation of anticholesterolemic treatment [10]. Moreover, increasing evidences indicate that, in high-risk conditions as well as in most children with a minor degree of vascular involvement, appropriate therapy could prevent and/or reverse the progression of these cardiovascular changes [11, 12]. Therefore, the identification and the management of hypercholesterolemia in children are of great consequence.

#### **2. Lipid concentrations in childhood and adolescence**

The thresholds for defining hypercholesterolemia and elevated low density lipoprotein cholesterol (LDL-C) in childhood and adolescence are not homogeneous. One set of values were derived from the National Cholesterol Education Program (NCEP) report (Table 1) [13]. An update, published in 2011, included values of apolipoprotein B (ApoB) and apolipoprotein A-1 (ApoA-1) coming from the National Health and Nutrition Examination Survey III. NCEP cutoffs seem to accurately estimate adult values of total cholesterol (TC), LDL-C and trigly‐ cerides (TG), while high lipoprotein cholesterol (HDL-C) levels are better predicted by National Health and Nutrition Examination Survey (NHANES) cutoff points [14].

Another set of values were derived from the Lipid Research Clinics Prevalence Study [15] and revised in 2008 by the AAP [16] (Table 2).

The lack of a consensus depends on: lipid variability during infancy, childhood and adoles‐ cence, the subsequent need of using percentiles instead of cut-off values (as used in adulthood) and the lack of studies that correlate lipid values in childhood with adult cardiovascular risk.

After birth, lipids and lipoproteins gradually increase up to 2 years of life, reaching values similar to adults: therefore, before the third year of life, the determination of the lipid profile is neither recommended nor useful. An increased stability with no significant differences between genders can be observed from 5 to 10 years: until pubertal activation, the use of reference values proposed by the AAP is recommended [16]. Taking into account the changes between genders occurring during puberty, the percentiles of reference proposed by Jolliffe and Janssen for males and females from 12 to 19 years could also be used: these percentiles are based on studies that correlate the values of lipid profile with the probability of subsequent clinical cardiovascular risk [17].


\*desiderable: > 65 mg/dl, 75° p

In some conditions, characterized by altered lipid assessment or other evident disorders of risk factors, premature CVD could be manifest in the first decades of life. "Vascular age" is advanced in 73% of familial dyslipidemic children, independently from the presence of others atherosclerosis-promoting risk factors [8]. The American Heart Association (AHA), endorsed by the American Academy of Pediatrics (AAP), identified 8 high-risk pediatric diagnosis and developed practical recommendations for the management of cardiovascular risk [9]. The selected diseases comprise familial hypercholesterolemia (FH), together with diabetes mellitus (type 1 and 2), chronic kidney disease, heart transplantation, Kawasaki disease, congenital heart disease, chronic inflammatory disease and childhood cancer. Subclinical endothelial dysfunction, measured through non-invasive surrogate methods such as flow-mediated dilation (FMD), occurs early in FH children indicating an increased risk for premature CVD and reflecting the need for early initiation of anticholesterolemic treatment [10]. Moreover, increasing evidences indicate that, in high-risk conditions as well as in most children with a minor degree of vascular involvement, appropriate therapy could prevent and/or reverse the progression of these cardiovascular changes [11, 12]. Therefore, the identification and the

management of hypercholesterolemia in children are of great consequence.

The thresholds for defining hypercholesterolemia and elevated low density lipoprotein cholesterol (LDL-C) in childhood and adolescence are not homogeneous. One set of values were derived from the National Cholesterol Education Program (NCEP) report (Table 1) [13]. An update, published in 2011, included values of apolipoprotein B (ApoB) and apolipoprotein A-1 (ApoA-1) coming from the National Health and Nutrition Examination Survey III. NCEP cutoffs seem to accurately estimate adult values of total cholesterol (TC), LDL-C and trigly‐ cerides (TG), while high lipoprotein cholesterol (HDL-C) levels are better predicted by

Another set of values were derived from the Lipid Research Clinics Prevalence Study [15] and

The lack of a consensus depends on: lipid variability during infancy, childhood and adoles‐ cence, the subsequent need of using percentiles instead of cut-off values (as used in adulthood) and the lack of studies that correlate lipid values in childhood with adult cardiovascular risk.

After birth, lipids and lipoproteins gradually increase up to 2 years of life, reaching values similar to adults: therefore, before the third year of life, the determination of the lipid profile is neither recommended nor useful. An increased stability with no significant differences between genders can be observed from 5 to 10 years: until pubertal activation, the use of reference values proposed by the AAP is recommended [16]. Taking into account the changes between genders occurring during puberty, the percentiles of reference proposed by Jolliffe and Janssen for males and females from 12 to 19 years could also be used: these percentiles are based on studies that correlate the values of lipid profile with the probability of subsequent

National Health and Nutrition Examination Survey (NHANES) cutoff points [14].

**2. Lipid concentrations in childhood and adolescence**

revised in 2008 by the AAP [16] (Table 2).

34 Hypercholesterolemia

clinical cardiovascular risk [17].

**Table 1.** Plasma lipid concentration in children and adolescence modified from NCEP, 1992 [13, 14]. Legend: p, percentile


**Table 2.** Lipid and Lipoprotein Distributions in Subjects Aged 5 to 19 Years [16]. Legend: p, percentile

Lipid concentrations vary also according to demographic variables (population specific): TC seems to be higher among Black children and adolescents than Caucasian ones [18]. Moreover, dietary habits together with current diseases and seasonality could transitionally influence cholesterol assessment.

Data from the National Health and Nutrition Examination Survey (NHANES) 1999 to 2006 for participants 6 to 17 years of age documented that 5.2%-6.6% (depending on the cut points used) and 9.6%-10.7% of them presented an elevated concentration of LDL-C and TC, respectively [19].

#### **3. Genetic Hypercholesterolemia**

Lipid abnormalities can be classified as primary disorders that encompass all genetic (mono‐ genic) forms of dyslipidemia, as summarized in Table 3 [20], and secondary disorders.


**Table 3.** Genetic Hypercholesterolemia [20]. Legend: LDLR, low density lipoprotein receptor; PCSK9, proprotein convertase subtilisin/kexin type 9

#### **4. Monogenic primary Hypercholesterolemia**

Lipid concentrations vary also according to demographic variables (population specific): TC seems to be higher among Black children and adolescents than Caucasian ones [18]. Moreover, dietary habits together with current diseases and seasonality could transitionally influence

Data from the National Health and Nutrition Examination Survey (NHANES) 1999 to 2006 for participants 6 to 17 years of age documented that 5.2%-6.6% (depending on the cut points used) and 9.6%-10.7% of them presented an elevated concentration of LDL-C and

Lipid abnormalities can be classified as primary disorders that encompass all genetic (mono‐ genic) forms of dyslipidemia, as summarized in Table 3 [20], and secondary disorders.

> Autosomal dominant 1:300-1:1.000.000

Autosomal recessive 1:100.000 (Sardinia)

Autosomal dominant 1:700 (North-Centre Europe)

**hyperlipidemia** Polygenic Premature CVD, Apo B elevated, TC 250-500

Autosomal recessive 1:1.000.000

**Table 3.** Genetic Hypercholesterolemia [20]. Legend: LDLR, low density lipoprotein receptor; PCSK9, proprotein

(40-60 y)

30 y)

Heterozygotes: TC 250-500 mg/dl (LDL-C > 135 mg/dl), xanthomas on the extensor tendons of the hands and feet, arcus cornea and premature CVD

Homozygotes: TC 500-1000 mg/dl, xanthomas

Variable, phenotype similar to homozygous FH, but generally less severe and more responsive to lipid-lowering therapy, large and bulky

xanthomas from early childhood, TC > 500 mg/dl,

Heterozygotes: TC 250-500 mg/dl, xanthomas, arcus senilis and premature CVD (50-60 y) Homozygotes: TC > 500 mg/dl, premature CVD (<

and very premature CVD (< 10 y)

in homozygotes: CVD < 30 y

mg/dl, TG 250-750 mg/dl

High vegetables sterols and LDL-C

**Name Genetic Defect Trasmission Clinical features**

LDLR, diminished LDL-C clearance

PCSK9, diminished LDL-C clearance

ARH adaptor protein absent or unable to interact with the LDLR, diminished LDL-C

Apo B, diminished LDL-C clearance

clearance

**Beta sitosterolemia** Carrier ABCG5/ABCG8

convertase subtilisin/kexin type 9

cholesterol assessment.

36 Hypercholesterolemia

TC, respectively [19].

**Classical Familial Hypercholesterolemia**

**Other autosomal dominant**

**hypercholesterolemia**

**Autosomal recessive hypercholesterolemia**

**Familial defective Apo**

**Familial combined**

**(FH)**

**(ARH)**

**B-100**

**3. Genetic Hypercholesterolemia**

Monogenic hypercholesterolemias are lifelong conditions that often present during childhood and adolescence with clinically and biochemically extreme phenotypes, due to a variety of gain-of-function or loss-of-function mutations in a range of candidate genes with important roles in lipid metabolism.

FH is an autosomal dominant monogenic condition. Homozygous familial hypercholestero‐ lemia (HoFH) is rare, with an occurrence of 1:1.000.000 individuals, but the heterozygous state (HeFH) is present in the general population with an incidence ranging from 1:300 to 1:500. On this basis we can affirm that HeFH is the most common monogenic disorder in North America and Europe. Causative FH mutation alters the function of LDL-receptor (LDLR) resulting in a reduced clearance of LDL-C particles from the circulation and consequently in an elevation of their plasma levels. In addition to LDLR defects, a similar phenotype can be caused by a number of mutations in the ApoB gene (that disrupt the binding of the LDL-C particle to the LDLR) and by gain of function mutations in the proprotein convertase subtilisin/kexin type 9 (PCSK9) gene (that increase LDLR degradation) [21]. So far, more than 1500 variants have been identified in the LDLR associated with FH, ranging from single-nucleotide substitutions to large deletions [22]. The clinical presentation of HeFH is characterized by two- to three-fold elevations in plasma LDL-C levels, by a family history positive for CVD, and, rarely in childhood, by the presence of physical symptoms of cholesterol deposits in tissues (tendon xanthomas, xanthelasma palpebrarum). Historically, left untreated, the cumulative risk of CVD in HeFH patient is greater than 50% in men by the age of 50 years and at least 30% in women by the age of 60 years. Homozygous and compound heterozygous FH subjects can experience serious cardiovascular events as early as in childhood: a six- to eight-fold increase in plasma LDL-C is found in these subjects and severe xanthomatosis and multiple types of xanthomas could occur.

Most people with FH are undiagnosed or only diagnosed after their first coronary event, but medical treatment seems to be effective and it could delay or prevent the onset of CVD. Therefore, early identification of affected individuals is crucial. Cholesterol levels alone are not sufficient to confirm a diagnosis of FH because of the extensive overlap in LDL–C levels existing between FH-causing mutation carries and non-carries (non-genetic polygenic hypercholesterolemia) and the high prevalence of modestly severe LDLR mutations that hampers the use of LDL-C cut-offs. Therefore, a diagnostic definition of FH, which supports cholesterol measurements with clinical signs and family history, has become widely used (Simon Broome Register Group definition of FH) [23]. According to this definition, a "definite" diagnosis of FH requires:

**a.** TC level above 260 mg/dl in children under 16 years of age

OR LDL-C levels above 160 mg/dl in children


A "possible" diagnosis of FH is suggested when (a) is present together with one of (d) or (e):


A similar diagnostic tool has been developed by the Dutch Lipid Clinic Network. This includes similar features to the Simon Broome criteria, but adds the calculation of a numeric score [24]. These criteria differ in DNA testing and in their diagnostic effectiveness. In summary, a child with significant and isolated elevation of LDL-C (≥160 mg/dl) should be considered to have FH, particularly if there is family history of early CVD.

After diagnosis of FH, cascade screening should occur in all first degree relatives after age 2 years. Cascade screening is a term used to describe searching for affected relatives of an inherited disorder once an affected person is known. In UK guidelines, DNA-based cascade testing is recommended in affected families; however, in about 60% of patients no mutations are found [25]. This finding leads to a great concern: assigning individuals to an "uncertain" category (when mutation is not identified) is unsatisfactory and provokes confusion and ambiguity both in children and adults.

After diagnosis, an appropriate treatment should be pursued, especially after CVD risk assessment, by a lipid specialist.

#### **5. Polygenic forms of Hypercholesterolemia**

Dyslipidemia may also result from interaction of synergic environmental and genetic factors developing the group of multifactorial or polygenic hypercholesterolemia. Because of this combination of causative factors, biochemical phenotype could be variable: LDL-C and TG may be high (rarely normal), HDL-C can be normal or reduced and there is significant production of small, dense LDL-C particles. Contrary to FH, it is less likely to be diagnosed in children because LDL-C elevation may occur frequently in adolescence, but strongly tracks into adulthood (70-75% of cases).

#### **6. Secondary Hypercholesterolemia**

The prevalence of lipid abnormalities in children is increasing because of the epidemic of obesity and subsequent metabolic syndrome (MS). Data from 1999-2004 NHANES demon‐ strated that approximately 10% of participants aged 8-19 years had high TC, 7% had low HDL-C, 9.7% had high TG and 7.6% had high LDL-C. In addition, prevalence of adverse lipid profile in youths with high adiposity was found significantly greater than participants without it [26]. Low HDL-C, high TG and small dense LDL-C, characterizing the so called "dyslipidemia of insulin resistance", are often associated with obesity and MS [27]. Promoting free-fatty acids release from visceral fat and altering the hepatic production of apolipoprotein, insulin resistance (IR) is the ethiological key of dyslipidemia in MS. Recently, the positive association demonstrated between PCSK9 activity and fasting glucose, insulin and homeostatic model assessment IR (in addition to lipid levels) suggests that PCSK9 could play a role in the development of dyslipidemia associated with the MS [28]. Moreover, the endocrine activity of adipose tissue produces inflammatory cytokines, such as adiponectin and tumor necrosis factor-alfa, influencing hepatic production of very low density lipoprotein (VLDL). All these findings highlight the importance to early counteract obesity to prevent the occurrence of dyslipidemia: the earlier the prevention begins, the better results are achieved.

Secondary dyslipidemia include also those caused by chronic disease, such as diabetes mellitus, chronic renal insufficiency, hypothyroidism, liver diseases and drugs (e.g. glucocor‐ ticoids, B-blockers, antiretroviral agents) (Table 4).


**Table 4.** Secondary dyslipidemia.

A "possible" diagnosis of FH is suggested when (a) is present together with one of (d) or (e): **d.** family history of myocardial infarction before the age of 50 years in grandparent, aunt,

**e.** family history of raised cholesterol in parents or siblings or levels above 290 mg/dl in

A similar diagnostic tool has been developed by the Dutch Lipid Clinic Network. This includes similar features to the Simon Broome criteria, but adds the calculation of a numeric score [24]. These criteria differ in DNA testing and in their diagnostic effectiveness. In summary, a child with significant and isolated elevation of LDL-C (≥160 mg/dl) should be considered to have

After diagnosis of FH, cascade screening should occur in all first degree relatives after age 2 years. Cascade screening is a term used to describe searching for affected relatives of an inherited disorder once an affected person is known. In UK guidelines, DNA-based cascade testing is recommended in affected families; however, in about 60% of patients no mutations are found [25]. This finding leads to a great concern: assigning individuals to an "uncertain" category (when mutation is not identified) is unsatisfactory and provokes confusion and

After diagnosis, an appropriate treatment should be pursued, especially after CVD risk

Dyslipidemia may also result from interaction of synergic environmental and genetic factors developing the group of multifactorial or polygenic hypercholesterolemia. Because of this combination of causative factors, biochemical phenotype could be variable: LDL-C and TG may be high (rarely normal), HDL-C can be normal or reduced and there is significant production of small, dense LDL-C particles. Contrary to FH, it is less likely to be diagnosed in children because LDL-C elevation may occur frequently in adolescence, but strongly tracks

The prevalence of lipid abnormalities in children is increasing because of the epidemic of obesity and subsequent metabolic syndrome (MS). Data from 1999-2004 NHANES demon‐ strated that approximately 10% of participants aged 8-19 years had high TC, 7% had low HDL-C, 9.7% had high TG and 7.6% had high LDL-C. In addition, prevalence of adverse lipid profile in youths with high adiposity was found significantly greater than participants without it [26]. Low HDL-C, high TG and small dense LDL-C, characterizing the so called "dyslipidemia of

uncle or before age 60 in parent or siblings

FH, particularly if there is family history of early CVD.

**5. Polygenic forms of Hypercholesterolemia**

grandparents, aunt or uncle.

38 Hypercholesterolemia

ambiguity both in children and adults.

assessment, by a lipid specialist.

into adulthood (70-75% of cases).

**6. Secondary Hypercholesterolemia**

#### **7. Diagnosing Hypercholesterolemia in childhood**

In 2011, the National Heart, Lung, and Blood Institute (NHLBI), backed by AAP, proposed an universal lipid screening to be performed with measurement of non fasting non-HDL-C (calculated by subtracting the HDL-C from the TC measurement) in all children between ages 9–11 and 17–21 years [14]. The normal variation in blood cholesterol levels within an individual over time is approximately 6%. Therefore, at least two elevated blood cholesterol measure‐ ments are required in a lapse of time between 15 days and 3 months, before a diagnosis of hypercholesterolemia can be made. In the remaining groups of age (2-8 and 12-16 years), NHLBI agrees with the use of selective screening, as proposed by the NCEP [13, 14]: lipid screening is recommended only in children with positive family history of premature CVD OR already known familial dyslipidemia OR unknown family history OR presence of multiple risk factors such as hypertension, diabetes and obesity OR overweight/obesity alone. Choles‐ terol screening modalities in youth have been debated for decades. The primary goal of universal screening is to identify those with FH. It has been shown that family history is incomplete in young individuals, since parents and even grandparents may be too young to have demonstrated early CVD [29]. The second goal of universal screening is to use cholesterol assessment to identify children with components of MS in an effort to highlight and prevent progression of additional components. Nevertheless, there are several critiques that comprise: the definition of risk-to-benefit ratio, especially regarding moderate dyslipidemia, the potential risk to determine anxiety in patients and families and, lastly, the financial costs [30].

### **8. Treatment of Hypercholesterolemia in childhood**

In 2010 the AHA, while developing the 2020 Impact Goals, defined the "cardiovascular health" concept and determined the metrics needed to monitor it over time [31]. In this way the first step proposed for management of children with identified lipid abnormalities is to assess their cardiovascular risk. It includes the collection of:


In children, the definition of ideal cardiovascular health includes several goals: avoid smoking, body mass index less than 85th percentile, more than 60 minutes of moderate- or vigorousintensity physical activity every day, health diet, TC values less than 170 mg/dl (6-19 years of age), blood pressure less than 90th percentile (8-19 years of age) and fasting plasma glucose less than 100 mg/dl (12-19 years of age) [31].

#### **8.1. Dietary treatment and lifestyle approach**

The cornerstone of lipid-lowering therapy is a healthy lifestyle [32].

Dietary recommendations emphasize the following pattern of nutrient intake:


risk factors such as hypertension, diabetes and obesity OR overweight/obesity alone. Choles‐ terol screening modalities in youth have been debated for decades. The primary goal of universal screening is to identify those with FH. It has been shown that family history is incomplete in young individuals, since parents and even grandparents may be too young to have demonstrated early CVD [29]. The second goal of universal screening is to use cholesterol assessment to identify children with components of MS in an effort to highlight and prevent progression of additional components. Nevertheless, there are several critiques that comprise: the definition of risk-to-benefit ratio, especially regarding moderate dyslipidemia, the potential risk to determine anxiety in patients and families and, lastly, the financial costs [30].

In 2010 the AHA, while developing the 2020 Impact Goals, defined the "cardiovascular health" concept and determined the metrics needed to monitor it over time [31]. In this way the first step proposed for management of children with identified lipid abnormalities is to assess their

**•** anamnestic data about familial premature cardiovascular disease (premature means before

**•** individual anamnestic risk factors as smoking habit, drugs and the presence of current high or moderate risk conditions such as hypercholesterolemia, diabetes, Kawasaki disease,

In children, the definition of ideal cardiovascular health includes several goals: avoid smoking, body mass index less than 85th percentile, more than 60 minutes of moderate- or vigorousintensity physical activity every day, health diet, TC values less than 170 mg/dl (6-19 years of age), blood pressure less than 90th percentile (8-19 years of age) and fasting plasma glucose less

**•** adequate nutrition should be achieved by eating a wide variety of foods low in saturated

**•** total caloric intake should be sufficient to support normal growth and development and

**•** total fat should provide an average of no more than 30% and no less than 20% of total calories

**•** physical examination that include blood pressure and body mass index.

The cornerstone of lipid-lowering therapy is a healthy lifestyle [32].

**•** saturated fatty acids should provide <10% of total calories

Dietary recommendations emphasize the following pattern of nutrient intake:

**8. Treatment of Hypercholesterolemia in childhood**

cardiovascular risk. It includes the collection of:

cancer treatment survivors...

40 Hypercholesterolemia

than 100 mg/dl (12-19 years of age) [31].

maintain desirable body weight

fat and cholesterol

**8.1. Dietary treatment and lifestyle approach**

55 years of age in males and before 65 in females)


All children with LDL-C level >130 mg/dl should receive targeted intervention and follow-up. The NCEP suggests for hypercholesterolemic patients a two level cholesterol-lowering diet [13]. In the Step 1 diet (Table 5) approximately 30% of calories derive from fat (10% from saturated fat) and the total intake of cholesterol should be limited to 300 mg/day. If the lipid values remain elevated after 6 weeks, the Step 1 diet should be reviewed to increase the compliance. If the diet for at least 3 months fails to achieve LDL-C concentrations <130 mg/dl (the ideal goal is to lower it to <110 mg/dl), a more aggressive dietary approach is needed (Step 2 diet). The two main differences between the Step 1 and Step 2 diets are that in the latter, the amount of saturated fat is reduced to 7% of total calories and the intake of cholesterol is decreased to 200 mg/day (Table 5). In order to implement this more stringent diet, advice from a nutritionist trained in dealing with children and disorders of lipids is needed. No restriction of fat or cholesterol is recommended for infants <2 years of age, when rapid growth and development require high energy intakes. Dietetic guidelines called Therapeutic Lifestyle Changes (TLC) replaced Step 1 and 2 diets. For higher risk people they recommended an adequate caloric intake, including an increased consume of whole grains, low-fat dairy products, fruits, vegetables and fish and a reduction of soft drinks and salt (Table 5) [33].


Legend: †The 25-35% fat recommendation allows for increased intake of unsaturated fat in place of carbohydrates in people with metabolic syndrome or diabetes.

**Table 5.** Characteristics and Differences between STEP 1, STEP 2 and TLC diets [33].

The improvement of dietary habits seems to be effective when hyperlipidemia is secondary to other conditions, such as obesity [34], but it is not sufficient in primary hypercholesterolemia. Nevertheless, also in the latter dietary restrictions have to be requested, in order to reduce the dose of medications and to avoid a further deterioration of the condition. No long-term (up to 10 years) adverse effects on growth and pubertal development have been documented [34-37].

In children with FH and polygenic hypercholesterolemia, additional benefit could derive from the introduction of soya protein [38] and/or plant stanols and sterol esters (2 g/day) in the diet [39]. Nevertheless, despite an improvement in TC and LDL-C levels, supplementation with stanols and sterols does not improve endothelial function, probably because they concomi‐ tantly reduce plasma carotenoids [39]. Recently, a significant reduction of small dense LDL-C has been demonstrated in 25 hypercholesterolemic children after the introduction in their diet of a yogurt-drink enriched with 2 gr/day plant sterols [40]. A large amount of sterol could also derive directly from fruits, vegetables and cereals (see table 6). Therefore, plant stanols and sterols have to be considered as beneficial, safe, tasteful, easy-accessible and low-cost lipidlowering strategy, especially until children at risk become eligible for a more aggressive therapy.


**Table 6.** Sterol content in food.

The efficacy of a cholesterol-lowering diet, started in childhood, upon reduction of CVD later in life, has not been firmly established yet [41].

Soluble fibers, including those from psyllium husk, have been shown to increase the choles‐ terol-lowering effects of a low-fat diet. In 2011 guidelines, the water soluble fiber psyllium can be added to a low fat low-saturated fat diet as cereal enriched with psyllium at a dose of 6 g/d for children 2-12 of age and 12 g/day for those ≥ 12 y of age [14]. Accordingly, it has been shown that glucomannan, a hydrosoluble fiber, may decrease TC and LDL-C levels, without changing HDL-C levels, in hypercholesterolemic children [42]. A novel pioneering approach in reducing the serum cholesterolemia could be represented by the exploitation of probiotics exerting cholesterol-lowering properties. A recent Italian randomized, double-blind, placebocontrol study evaluates the effects of a probiotic formulation containing three Bifidobacterium strains on lipid profiles in 39 children affected by primary dyslipidemia: compared to placebo, probiotics reduced TC by 3.9% and LCL-C by 3.8%. Moreover, this supporting therapy seems well tolerated [43].

Lifestyle change includes: regular physical activity, screen time less than 2 hours per day, attainment of ideal body weight (body mass index ≤85th centile for age and gender) and optimization of blood pressure. In adults the combination of intensive dietary restrictions and physical exercise improves lipid metabolism, IR and cardiorespiratory fitness, thereby diminishing cardiovascular risk factors [44]. Reported trials on benefits of physical activity on lipid-profile are scarce, rarely well—performed and their results are not optimistic. However, physical activity stimulates lipoprotein lipases and the function of some enzymes like the lecithin-cholesterol acyltransferase (LCAT), improving HDL-C formation and reducing their catabolism. Moreover, exercise reduced TG levels and is effective on size and density of LDL-C particles. Finally, exercise is safe and does not require any additional cost. Therefore, children should be encouraged to undertake 60 minutes or more of vigorous aerobic activity per day. The TLC diet itself recommends expending at least 200 kcal per day [33]. The abuse of tobacco and alcohol should be avoided. An early identification and correction of eating disorders should be recommended.

#### **8.2. Pharmacological treatment**

dose of medications and to avoid a further deterioration of the condition. No long-term (up to 10 years) adverse effects on growth and pubertal development have been documented [34-37].

In children with FH and polygenic hypercholesterolemia, additional benefit could derive from the introduction of soya protein [38] and/or plant stanols and sterol esters (2 g/day) in the diet [39]. Nevertheless, despite an improvement in TC and LDL-C levels, supplementation with stanols and sterols does not improve endothelial function, probably because they concomi‐ tantly reduce plasma carotenoids [39]. Recently, a significant reduction of small dense LDL-C has been demonstrated in 25 hypercholesterolemic children after the introduction in their diet of a yogurt-drink enriched with 2 gr/day plant sterols [40]. A large amount of sterol could also derive directly from fruits, vegetables and cereals (see table 6). Therefore, plant stanols and sterols have to be considered as beneficial, safe, tasteful, easy-accessible and low-cost lipidlowering strategy, especially until children at risk become eligible for a more aggressive

> **Sterol Content (mg/100 gr edible)**

therapy.

42 Hypercholesterolemia

**Cereals**

**Fats and Oils**

**Table 6.** Sterol content in food.

**Fruit and vegetables**

**Food**

Broccoli 44 Grean peas 25 Orange 24 Apple 13 Cucumber 6 Tomato 5

Wheat bran 200 Swedish knackebrot 89 Wholemeal bread 53 Rolled oats 39 Wheat Bread 29

Corn Oil 912 Rapeseed (canola) oil 668 Liquid margarine 522 Sunflower oil 213 Spreadable butter 153 Olive oil 154 If cholesterol does not reach acceptable levels through diet, the recourse to a pharmacological treatment is allowed and advised [45]. The NCEP recommends the administration of medica‐ tions only to children > 10 years of age (better if either at pubertal Tanner stage II or higher or after onset of menses in girls) and only when an aggressive diet lasting at least 6-12 months fails [13]. A lipid specialist should be consulted. Traditionally, bile acid sequestrants (BAR) were considered the first-line therapy in hypercholesterolemic children. Since AHA statement, statins replaced BAR: their use is now recommended in children younger than 10 years of age (from 8 years of age). Surprisingly, AAP encouraged the application of this revised therapeut‐ ical approach [16] causing many controversies among experts: up to now, the efficacy of statins on adult-onset of CVD is not clearly defined and data about their long-term safety, particularly because they interfere with the production of steroid hormones and liver function, are still lacking. Nevertheless, in 2002-2005, the use of lipid-lowering drugs increased in children by 15% [46] and the American Food and Drug Administration (FDA) and the European Medicines Agency (EMEA) approved pravastatin in children > 8 years, while simvastatin, lovastatin and atorvastatin were registered by the FDA for children > 10 years of age. Ezetimibe was also approved by the FDA and EMEA for pediatric use from the age of 10 years.

#### **8.3. 3-Hydroxy-3-MethylGlutaryl-CoA (HMG-Coa) reductase inhibitors**

Statins inhibit HMG-CoA reductase, an enzyme fundamental in de novo cholesterol synthesis. Their action consequently provokes an increase in hepatic production of LDLR determining an additional decrease in LDL-C levels. Statins are commonly safe and well tolerated. How‐ ever, because of the principal role of cholesterol in cellular structure and function, the use of statins is not allowed in prepubertal children. In HeFH children and adolescents, statins are effective in reducing levels of LDL-C by 20-40% and increasing HDL-C, but, because of the scarcity and the non-uniformity of trials shown by meta-analysis, no data about outcomes of different types, doses and length of therapy could be deduced [47,48]. In terms of safety, no differences in occurrence of adverse effects, alterations in sexual development and muscle or liver toxicity have been demonstrated in children treated with statins in comparison to placebotreated ones [49]. Nevertheless, while adult guidelines do not recommend routine screening of liver and muscle enzymes during treatment, pediatric guidelines indicate to start statin at the lowest dose with baseline measurements of alanine aminotransferase (ALT), aspartate aminotransferase (AST), and creatinine kinase (CK). These levels plus a fasting lipid profile should be repeated four and eight weeks after initiation of therapy and then every 3–6 months. If liver enzymes are above 3 times the upper limit of normal and/or CK is above 10 times the upper limit of normal and/or patient complains any adverse effects, medication should be stopped to determine if there is an improvement. Some researchers have suggested hydro‐ philic statins, such as fluvastatin, rosuvastatin and pravastatin are less potentially toxic than lipophilic statins, such as atorvastatin, lovastatin, and simvastatin; the risk of myopathy was suggested to be lowest with pravastatin and fluvastatin, probably because they are more hydrophilic [50].

Up to now, few studies have examined vascular efficacy of statins in children, confirming an increase of impaired FMD before and a significant improvement after treatment [11, 51], longterm effects have been less studied [52]. Recently, secretory phospholipase A2-IIA (sPLA2-IIA) receives increased interest because of its role in the inflammatory process of atherosclerosis: it induce LDL-C modification, foam cell formation and activation of various immune mecha‐ nisms. Published data demonstrated no effects of 2-years-long pravastatin therapy in reducing sPLA2-IIA mass or sPLA2 activity levels in 91 FH children compared to placebo [53].

Statins are contraindicated during pregnancy because of potential teratogenic risk. Because of this concern, statins should be used to treat adolescent FH females only if they are aware of the risk, under a close follow-up and on contraceptive therapy when indicated.

Target LDL-C is typically below 130 mg/dl, but ideally under 100 mg/dl in high risk popula‐ tions such as FH. If target levels are not achieved within 3 months, the dose of statin can be gradually increased to maximum dose. Occasionally, a second agent such as a BAR may be useful. Multiple drug therapy should be guided by a lipid specialist.

#### **8.4. BAR**

15% [46] and the American Food and Drug Administration (FDA) and the European Medicines Agency (EMEA) approved pravastatin in children > 8 years, while simvastatin, lovastatin and atorvastatin were registered by the FDA for children > 10 years of age. Ezetimibe was also

Statins inhibit HMG-CoA reductase, an enzyme fundamental in de novo cholesterol synthesis. Their action consequently provokes an increase in hepatic production of LDLR determining an additional decrease in LDL-C levels. Statins are commonly safe and well tolerated. How‐ ever, because of the principal role of cholesterol in cellular structure and function, the use of statins is not allowed in prepubertal children. In HeFH children and adolescents, statins are effective in reducing levels of LDL-C by 20-40% and increasing HDL-C, but, because of the scarcity and the non-uniformity of trials shown by meta-analysis, no data about outcomes of different types, doses and length of therapy could be deduced [47,48]. In terms of safety, no differences in occurrence of adverse effects, alterations in sexual development and muscle or liver toxicity have been demonstrated in children treated with statins in comparison to placebotreated ones [49]. Nevertheless, while adult guidelines do not recommend routine screening of liver and muscle enzymes during treatment, pediatric guidelines indicate to start statin at the lowest dose with baseline measurements of alanine aminotransferase (ALT), aspartate aminotransferase (AST), and creatinine kinase (CK). These levels plus a fasting lipid profile should be repeated four and eight weeks after initiation of therapy and then every 3–6 months. If liver enzymes are above 3 times the upper limit of normal and/or CK is above 10 times the upper limit of normal and/or patient complains any adverse effects, medication should be stopped to determine if there is an improvement. Some researchers have suggested hydro‐ philic statins, such as fluvastatin, rosuvastatin and pravastatin are less potentially toxic than lipophilic statins, such as atorvastatin, lovastatin, and simvastatin; the risk of myopathy was suggested to be lowest with pravastatin and fluvastatin, probably because they are more

Up to now, few studies have examined vascular efficacy of statins in children, confirming an increase of impaired FMD before and a significant improvement after treatment [11, 51], longterm effects have been less studied [52]. Recently, secretory phospholipase A2-IIA (sPLA2-IIA) receives increased interest because of its role in the inflammatory process of atherosclerosis: it induce LDL-C modification, foam cell formation and activation of various immune mecha‐ nisms. Published data demonstrated no effects of 2-years-long pravastatin therapy in reducing

Statins are contraindicated during pregnancy because of potential teratogenic risk. Because of this concern, statins should be used to treat adolescent FH females only if they are aware of

Target LDL-C is typically below 130 mg/dl, but ideally under 100 mg/dl in high risk popula‐ tions such as FH. If target levels are not achieved within 3 months, the dose of statin can be gradually increased to maximum dose. Occasionally, a second agent such as a BAR may be

sPLA2-IIA mass or sPLA2 activity levels in 91 FH children compared to placebo [53].

the risk, under a close follow-up and on contraceptive therapy when indicated.

useful. Multiple drug therapy should be guided by a lipid specialist.

approved by the FDA and EMEA for pediatric use from the age of 10 years.

**8.3. 3-Hydroxy-3-MethylGlutaryl-CoA (HMG-Coa) reductase inhibitors**

hydrophilic [50].

44 Hypercholesterolemia

Even if FDA never approved BARs in children, they were used in the past as first-line therapy in hypercholesterolemia. Colestipol, cholestyramine and colestilan bind bile salts in the intestine preventing their re-absorption and increasing their excretion and discharge from cholesterol pool. Increased emission of bile acids causes a large conversion of cholesterol into bile salts; in liver, cholesterol pool decreases and a compensatory rise in LDLR synthesis takes place. In children with HeFH, BARs determine a decrease in TC of 10-20%. These drugs are not absorbed systematically, but local side-effects as abdominal pain and nausea could nullify compliance. Recently, a novel bile acid sequestrant, the colesevelam hydrochloride, with enhanced binding capacity for bile acids has been evaluated in HeFH children, alone or in combination with statin therapy: the therapeutical compliance has increased (about 85%) together with an effective LDL-C reduction [54].

#### **8.5. Niacin and fibrates**

Niacin, a water-soluble B complex vitamin, increases HDL-C levels and significantly reduces hepatic production and release of VLDL but it is not commonly utilized in children because of lack of information about its safety. Adverse effects consist of flushing, hepatic insufficiency, myopathy, glucose intolerance and hyperuricemia. Therefore, niacin is limited both in HoFH children and in ones with stroke and increased lipoprotein A levels.

Fibrates (gemfibrazil, fenofibrate, bezafibrate and ciprofibrate) are mainly effective in lowering TG and in increasing HDL-C, while LDL-C levels seem only partially and variably influenced. They have a complex and poorly understood way of action and, due to the lack of data on safety, their use is restricted in HeFH children with high TG levels and an increased risk for pancreatitis.

#### **8.6. Ezetimibe**

Ezetimibe is a new selective cholesterol absorption inhibitor acting at the brush border of the small intestine with no effects on the absorption of TG and fat-soluble vitamins. Target pathways may consist in the Niemann-Pick C1-like protein and the annexin-caveolin 1 complex. In 2002, FDA approved ezetimibe in FH children older than 10 years of age, but long-term effects have not been extensively evaluated yet. In HoFH, ezetimibe has a synergic effect in reducing LDL-C, if associated with statins, without increasing adverse effects. Satisfying data in term of efficacy and tolerance have also been documented in children with polygenic hypercholesterolemia, HeFH and familial combined hyperlipidemia treated with ezetimibe [55].

These positive data are partially dampened by an absolute lack of knowledge about the longterm effects of ezetimibe. In fact, the combination of statin and ezetimibe may not restore endothelial dysfunction [56]. Moreover, ezetimibe dose not influence HDL-C, an independent risk factor for CVD. Future data from the ongoing IMPROVED-IT study, enlisting 18.000 adults affected by acute coronary syndrome on simvastatin either with or without ezetimibe, may clarify the role of ezetimibe in CVD prevention [57]. In addition, systemic effects of ezetimibe, in contrast to its minimal absorption have still to be clearly defined.

In the last years, new lipid-lowering drugs are coming out. Starting from the demonstration that PCSK9 loss-of-function mutations result in a significant drop in circulating LDL-C, subsequent studies demonstrated that PCSK9 binds the epidermal growth factor precursor homology domain-A on the surface LDLR and directs LDLR and PCSK9 for lysosomal degradation. A monoclonal antibody that binds circulating PCSK9 and blocks its interactions with surface LDLR and called Alirocumab has recently demonstrated a great potentiality in reducing LDL-C in adulthood. Nevertheless, there is no data in adolescence and no evidence on its capacity in improving CVD outcome yet [58].

#### **9. HoFH: Treatment in childhood**

HoFH has to be considered an almost exclusive pediatric disease. Because of the earlier risk of CVD, HoFH patients should started pharmacological therapy as soon as possible, as recom‐ mended by AHA [45]. LDL-C apheresis and/or liver transplant have been the historical treatment in this subset, although efficacy and success are variable [59]. LDL-C apheresis is an extracorporeal plasma-perfusion method that involves selective removal of LDL-C particles. The procedure takes three or more hours and is performed at 1- to 2-week intervals. Even if it results in the regression of coronary lesions and has been found to increase life expectancy, its use is limited by its availability, higher cost and difficulties in procedures [60]. As most of the LDLR are present in the liver, liver transplantation alone or in combination with pharmaco‐ therapy is effective in normalizing the plasma cholesterol levels. Nevertheless, the associated risks include the need of a life-long immunosuppressive therapy [61]. HoFH may also benefit from lipid-lowering drugs. However, statins require some residual LDLR function, thus they are not effective in receptor-negative HoFH. Higher risk patients will benefit from combination therapy: ezetimibe, but also niacin, fibrates, and BAR. Therapeutic efficacy, safety, medication adherence, and compliance should be monitored closely. Novel medical therapies for adults with HoFH have recently been approved in the US. These include inhibitors of PCSK9, microsomal triglyceride transfer protein and cholesteryl ester transfer protein (CETP), as well as mipomersen, an apolipoprotein B synthesis inhibitor [62, 63].

#### **10. Conclusions**

The role of pediatrician in the prevention of chronic and disabling diseases in adulthood is reinforced by the extensive scientific evidence that proves the beginning of causative processes, such as atherosclerosis, in childhood. Therefore, the identification of patients at risk of premature CVD has become, today, one of the primary aims of pediatricians. The evaluation of hypercholesterolemic children should not be based exclusively on lipid assessment: it is essential to quantify the overall cardiovascular risk through the collection of a full medical history (including familial history), the performance of an accurate physical examination, the assessment of eating habits and the identification of concomitant risk factors. Moreover, in hypercholesterolemic children, the monitoring over time of cardiovascular function through non-invasive methods can be useful.

Childhood could be considered as the best period of life to acquire a proper lifestyle and healthy eating habits, especially in patients at risk of premature CVD. The diet, low in saturated fat and cholesterol, should be the first therapeutic approach to be proposed in children with hypercholesterolemia. Early pharmacological treatment could be planned in cases of genetic hypercholesterolemia or when diet alone persistently fails. Several studies have demonstrated the short-term efficacy and safety of statins in childhood.

The definition of pediatric population-specific percentiles for lipid values, the achievement of a shared screening strategy and the demonstration of long-term safety and efficacy of statin therapy have to be considered the current priorities for improving the approach to childhood hypercholesterolemia.

#### **Nomenclature**

clarify the role of ezetimibe in CVD prevention [57]. In addition, systemic effects of ezetimibe,

In the last years, new lipid-lowering drugs are coming out. Starting from the demonstration that PCSK9 loss-of-function mutations result in a significant drop in circulating LDL-C, subsequent studies demonstrated that PCSK9 binds the epidermal growth factor precursor homology domain-A on the surface LDLR and directs LDLR and PCSK9 for lysosomal degradation. A monoclonal antibody that binds circulating PCSK9 and blocks its interactions with surface LDLR and called Alirocumab has recently demonstrated a great potentiality in reducing LDL-C in adulthood. Nevertheless, there is no data in adolescence and no evidence

HoFH has to be considered an almost exclusive pediatric disease. Because of the earlier risk of CVD, HoFH patients should started pharmacological therapy as soon as possible, as recom‐ mended by AHA [45]. LDL-C apheresis and/or liver transplant have been the historical treatment in this subset, although efficacy and success are variable [59]. LDL-C apheresis is an extracorporeal plasma-perfusion method that involves selective removal of LDL-C particles. The procedure takes three or more hours and is performed at 1- to 2-week intervals. Even if it results in the regression of coronary lesions and has been found to increase life expectancy, its use is limited by its availability, higher cost and difficulties in procedures [60]. As most of the LDLR are present in the liver, liver transplantation alone or in combination with pharmaco‐ therapy is effective in normalizing the plasma cholesterol levels. Nevertheless, the associated risks include the need of a life-long immunosuppressive therapy [61]. HoFH may also benefit from lipid-lowering drugs. However, statins require some residual LDLR function, thus they are not effective in receptor-negative HoFH. Higher risk patients will benefit from combination therapy: ezetimibe, but also niacin, fibrates, and BAR. Therapeutic efficacy, safety, medication adherence, and compliance should be monitored closely. Novel medical therapies for adults with HoFH have recently been approved in the US. These include inhibitors of PCSK9, microsomal triglyceride transfer protein and cholesteryl ester transfer protein (CETP), as well

The role of pediatrician in the prevention of chronic and disabling diseases in adulthood is reinforced by the extensive scientific evidence that proves the beginning of causative processes, such as atherosclerosis, in childhood. Therefore, the identification of patients at risk of premature CVD has become, today, one of the primary aims of pediatricians. The evaluation of hypercholesterolemic children should not be based exclusively on lipid assessment: it is essential to quantify the overall cardiovascular risk through the collection of a full medical history (including familial history), the performance of an accurate physical examination, the assessment of eating habits and the identification of concomitant risk factors. Moreover, in

in contrast to its minimal absorption have still to be clearly defined.

on its capacity in improving CVD outcome yet [58].

as mipomersen, an apolipoprotein B synthesis inhibitor [62, 63].

**10. Conclusions**

**9. HoFH: Treatment in childhood**

46 Hypercholesterolemia

AHA; American Heart Association AIDS; Acquired Immune Deficiency Syndrome ALT; Alanine aminotransferase Apo; Apolipoprotein AAP; American Academy of Pediatrics AST; Aspartate aminotransferase BAR; Bile acid sequestrants CK; Creatine phosphokinase CVD; Cardiovascular disease EMEA; European Medicines Agency FDA; Food and Drugs Administration FH; Familial Hypercholesterolemia FMD; Flow-mediated dilation HAART; Hightly active antiretroviral therapy HDL; High-density lipoprotein HDL-C; High-density lipoprotein cholesterol HeFH; Heterozygous familial hypercholesterolemia HIV; Human immunodeficiency virus

HMG-CoA; 3-Hydroxy-3-MethylGlutaryl-CoA HoFH; Homozygous familial hypercholesterolemia IR; insulin resistance LCAT; Lecithin-cholesterol acyltransferase LDL; Low-density lipoprotein LDL-C; Low-density lipoprotein cholesterol LDLR; Low-density lipoprotein receptor MS; Metabolic syndrome NCEP; National Cholesterol Education Program NHANES; National Health and Nutrition Examination Survey NHLBI; National Heart, Lung, and Blood Institute oxLDL; oxidized low-density lipoprotein PCSK9; Proprotein convertase subtilisin/kexin type 9 sPLA2-AII; Secretory phospholipase A2-IIA TC; Total cholesterol TG; Triglycerides TLC; Therapeutic lifestyle change VLDL; Very low density lipoprotein

#### **Author details**

Lorenzo Iughetti\* , Barbara Predieri and Patrizia Bruzzi

\*Address all correspondence to: iughetti.lorenzo@unimore.it

Department of Medical and Surgical Sciences of Mothers, Children and Adults, University of Modena & Reggio Emilia, Pediatric Unit, Modena, Italy

#### **References**

[1] Relationship of atherosclerosis in young men to serum lipoprotein cholesterol and smoking: a preliminary report from the Pathobiological Determinants of Atheroscle‐ rosis in Youth (PDAY) Research Group. JAMA 1990; 264: 3018-3024.

[2] Berenson GS, Srinivasan SR, Bao W, Newman WP III, Tracy RE, Wattigney WA. As‐ sociation between multiple cardiovascular risk factors and atherosclerosis in children and young adults: the Bogalusa Heart Study. N Engl J Med 1998; 338: 1650-1656.

HMG-CoA; 3-Hydroxy-3-MethylGlutaryl-CoA

LCAT; Lecithin-cholesterol acyltransferase

LDL-C; Low-density lipoprotein cholesterol

NCEP; National Cholesterol Education Program

NHLBI; National Heart, Lung, and Blood Institute

PCSK9; Proprotein convertase subtilisin/kexin type 9

NHANES; National Health and Nutrition Examination Survey

, Barbara Predieri and Patrizia Bruzzi

Department of Medical and Surgical Sciences of Mothers, Children and Adults, University

[1] Relationship of atherosclerosis in young men to serum lipoprotein cholesterol and smoking: a preliminary report from the Pathobiological Determinants of Atheroscle‐

rosis in Youth (PDAY) Research Group. JAMA 1990; 264: 3018-3024.

\*Address all correspondence to: iughetti.lorenzo@unimore.it

of Modena & Reggio Emilia, Pediatric Unit, Modena, Italy

LDLR; Low-density lipoprotein receptor

oxLDL; oxidized low-density lipoprotein

sPLA2-AII; Secretory phospholipase A2-IIA

TLC; Therapeutic lifestyle change VLDL; Very low density lipoprotein

IR; insulin resistance

48 Hypercholesterolemia

LDL; Low-density lipoprotein

MS; Metabolic syndrome

TC; Total cholesterol

TG; Triglycerides

**Author details**

Lorenzo Iughetti\*

**References**

HoFH; Homozygous familial hypercholesterolemia


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and meta-analysis. Atherosclerosis 2007;195: 339-347.

hypercholesterolaemia. Cochrane Database Syst Rev 2014;6: CD001918.

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52 Hypercholesterolemia

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diatr Cardiol 2007; 28: 8-13.

dren. Nutrition. 2014;30(7-8):831-6.


## **Role of Oxidized LDL in Atherosclerosis**

E. Leiva, S. Wehinger, L. Guzmán and R. Orrego

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/59375

#### **1. Introduction**

Nowadays, Atherosclerosis is the most important source of morbidity and mortality in the world, and is detected by the accumulation of lipids deposits (mainly cholesterol) in macro‐ phages located not only in large but also in medium sized arteries. Currently, the association between atherosclerosis and heightened oxidative stress is widely accepted. Nevertheless, despite numerous efforts the role of oxidative stress in the progression of Atherosclerosis is still not clear.

Oxidation is a biochemical process of loss of electrons, which is essential for life due to its involvement in the production of cellular energy. However, when oxidation is excessive causing cellular damage is when Oxidative Stress appears. This process is complex; therefore, it cannot be measured or defined by a single parameter. For this reason, currently the interest lies on developing antioxidant therapies and diets enriched with antioxidants that prevent or at least decrease cellular damage and atheromatous plaque formation originated by the excess of oxidative stress.

**Aim.** The aim of this review is to analyze the state of the art on oxidized LDL role within the pathogenesis of atherosclerosis.

This chapter will be developed according to the following titles,


© 2015 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and eproduction in any medium, provided the original work is properly cited.

The word "Atherosclerosis" comes from the ancient Greeks where "sclerosis" means harden‐ ing and "athere" is gruel or accumulation of lipid. The physiopathological process is charac‐ terized by the aggregation of cholesterol, infiltration of macrophages and the proliferation of smooth muscle cells (SMCs) as accumulation of connective tissues and thrombus creation. In early stages of the disease, the growth of the lesion starts in the sub endothelial space and its progress may even cause total cessation in blood flow with intermittent periods of quiescence. The accumulation of lipids and other organic molecules lead to a proliferation of certain cell types within the arterial wall that gradually impinge the vessel lumen and block up the blood flow in large and medium sized arteries. Furthermore, this disease tends to be more common in white than black men [1]. The magnitude of this problem is deep, because atherosclerosis claims more lives than all types of combined cancer and economic costs are considerably high[2]. Currently, the idea that atherosclerosis constitutes a state of high levels of oxidative stress is widely accepted and this phenomenon is associated with lipid and protein oxidation in the vascular wall[3, 4].

Despite the countless efforts made to explain the role of oxidative stress in progression of atherosclerosis, its predictive role is still not clear. Goldstein and Brown discovered the LDL incorporation process in peripheral cells as fibroblasts, macrophages and others -which meant for them the Nobel Prize- has been the basis for a series of subsequent discoveries, from 1979 up to now, which have intended to explain the development of the atherosclero‐ sis' process [5-8]. The hypothesis of oxidative modification in atherosclerosis, reviewed by Steinberg and others in several opportunities argues that the oxidation of low-density lipoprotein (LDL) is an early stage of the disease and that oxidized LDL (OxLDL) would contribute to atherogenesis [9-12].

Until 1991, the strength of the scientific evidence regarding the role of the oxidation of LDL in the phenomenon of atherosclerosis was such that the National Heart, Lung and Blood Institute recommended the initiation of clinical trials [8, 13-17]. In relationship to this hypothesis, based on *in vitro* assays, the evidence showed the following relevant aspects: 1) the LDL oxidation is the first event in the foam cell formations [18, 19] the LDL lipids in human arterial lesions are extensively oxidized and 3) the presence of Ox LDL is evident *in vivo*[20]. On the other hand, the existence of several structurally unrelated compounds such as probucol and vit E that inhibit atherosclerosis in animals, prevent the initiation of the disease due to a reduction of the oxidization of LDL [21]. In relationship to probucol, It seems to be a more effective protection against lesion formation on an early-stage of the disease than the statin-mediated lipidlowering effects [22].

The events involving the process of atherosclerosis begin with LDL oxidation in the vascular wall. This happens due to the production of reactive oxygen species (ROS) and nitrogen species (NOS) by endothelial cells, therefore, oxidative modifications would be crucial in the clinical aspect of coronary artery disease such as endothelial dysfunction and plaque disruption [23].

Although it is known by scientific evidence that LDL oxidation plays a central role in the pathophysiology of atherosclerosis, up to now there is no convincing proofs related to the protective effect of antioxidant therapy as a way to prevent the damage caused by that process on vital macromolecules such as lipids, proteins and DNA.This may be due to the discrepancies between human and animal studies that use antioxidant therapies either to try or to limit the atherosclerotic process and cardiovascular events. It is not clear if oxidative stress is cause or consequence of the atherogenic process. In this sense, it has been proposed that inflammation could be considered as a primary process and oxidative stress as a secondary event of atherosclerosis [24].

#### **2. LDL oxidation**

The word "Atherosclerosis" comes from the ancient Greeks where "sclerosis" means harden‐ ing and "athere" is gruel or accumulation of lipid. The physiopathological process is charac‐ terized by the aggregation of cholesterol, infiltration of macrophages and the proliferation of smooth muscle cells (SMCs) as accumulation of connective tissues and thrombus creation. In early stages of the disease, the growth of the lesion starts in the sub endothelial space and its progress may even cause total cessation in blood flow with intermittent periods of quiescence. The accumulation of lipids and other organic molecules lead to a proliferation of certain cell types within the arterial wall that gradually impinge the vessel lumen and block up the blood flow in large and medium sized arteries. Furthermore, this disease tends to be more common in white than black men [1]. The magnitude of this problem is deep, because atherosclerosis claims more lives than all types of combined cancer and economic costs are considerably high[2]. Currently, the idea that atherosclerosis constitutes a state of high levels of oxidative stress is widely accepted and this phenomenon is associated with lipid and protein oxidation

Despite the countless efforts made to explain the role of oxidative stress in progression of atherosclerosis, its predictive role is still not clear. Goldstein and Brown discovered the LDL incorporation process in peripheral cells as fibroblasts, macrophages and others -which meant for them the Nobel Prize- has been the basis for a series of subsequent discoveries, from 1979 up to now, which have intended to explain the development of the atherosclero‐ sis' process [5-8]. The hypothesis of oxidative modification in atherosclerosis, reviewed by Steinberg and others in several opportunities argues that the oxidation of low-density lipoprotein (LDL) is an early stage of the disease and that oxidized LDL (OxLDL) would

Until 1991, the strength of the scientific evidence regarding the role of the oxidation of LDL in the phenomenon of atherosclerosis was such that the National Heart, Lung and Blood Institute recommended the initiation of clinical trials [8, 13-17]. In relationship to this hypothesis, based on *in vitro* assays, the evidence showed the following relevant aspects: 1) the LDL oxidation is the first event in the foam cell formations [18, 19] the LDL lipids in human arterial lesions are extensively oxidized and 3) the presence of Ox LDL is evident *in vivo*[20]. On the other hand, the existence of several structurally unrelated compounds such as probucol and vit E that inhibit atherosclerosis in animals, prevent the initiation of the disease due to a reduction of the oxidization of LDL [21]. In relationship to probucol, It seems to be a more effective protection against lesion formation on an early-stage of the disease than the statin-mediated lipid-

The events involving the process of atherosclerosis begin with LDL oxidation in the vascular wall. This happens due to the production of reactive oxygen species (ROS) and nitrogen species (NOS) by endothelial cells, therefore, oxidative modifications would be crucial in the clinical aspect of coronary artery disease such as endothelial dysfunction and plaque disruption [23]. Although it is known by scientific evidence that LDL oxidation plays a central role in the pathophysiology of atherosclerosis, up to now there is no convincing proofs related to the protective effect of antioxidant therapy as a way to prevent the damage caused by that process on vital macromolecules such as lipids, proteins and DNA.This may be due to the discrepancies

in the vascular wall[3, 4].

56 Hypercholesterolemia

contribute to atherogenesis [9-12].

lowering effects [22].

Oxidation is a biochemical process of loss of electrons, which is essential for life due to its involvement in the production of cellular energy. Oxidative stress appears when oxidation is excessive. This apparently simple process is actually complex in all biological levels, and cannot be measured or defined by a single parameter.

The oxidation process of lipids and proteins is the result of an excess of free radical and other oxidant species derived from oxygen, nitrogen and other chemical elements in the body. Chemically, the oxidative stress is associated with an increased production of oxidizing species or a significant decrease in the effectiveness of antioxidants defenses such as reduced gluta‐ thione, catalase, peroxidases and others. The cell proliferation and death are key processes in the progression of atherosclerosis and severe oxidative stress can cause cell death and even mild oxidation can trigger cellular stress and apoptosis, while more intense stress may cause necrosis [25].

There is a constant production of ROS and other oxidative species derived from the normal and xenobiotic metabolism, ionizing radiation and smoke snuff exposure, among others. Oxidative molecules can exert positive or negative effects over cells and tissues, depending on their concentration. ROS plays an important role in several physiological cell processes, such as signaling and regulation cascades, however excesses can induce chemical and structural modifications which has been proven that alter the function of cellular components, inhibit protein function, induce DNA damage, viral activation and lipid peroxidation which can promote cell death (Figure 1).

In addition, redox systems such as gluthation peroxidase, thioredoxine reductase and pyridine nucleotide redox status can change their physiological function when modified by ROS and others reactive species, affecting the normal cell signaling including apoptotic cell death [26].

Today there are clear proofs that LDL oxidation plays a significant role in atherogenesis. In fact, this has been demonstrated throughout time. So, between 1985 and 1989, 62 papers about OxLDL were published; between 1992 and January 1997, the number of publications related to OxLDL went up to 727, and up to day only considering PubMed entry, it is possible to find over 7000 publications associated with the key words Oxidized LDL. This growing interest is supported by the large amount of evidence which confirms that oxidative modification of LDL plays a pivotal role in atherosclerosis and hence, makes it an obvious target for therapeutic approaches [10, 27].

**Figure 1.** The figure shows some sources and consequences of oxidative stress.

In 2002, Friedman et al. showed that the oxidized lipids from OxLDL are biologically active. Specifically, polyunsaturated fatty acids (PUFA) either free or bound to an ester from phos‐ pholipid are converted into hydroperoxides, which break down to form highly reactive molecules, such as malondialdehyde and 4-hydroxynonenal among other metabolic products. These reactive aldehydes can then form Schiff-bases, covalent Michael-type adducts with lysine residues of apolipoprotein B in LDL molecules. Besides, the sn-2 oxidized fatty acid fragments which can remain attached via ester bridges may also contain terminal reactive aldehydes. However, this reactive phospholipid also called "aldehyde phospholipid core" may also form adducts with Schiff-base lysine residues of apolipoprotein B and presumably also with other proteins and amines-containing phospholipids, such as phosphatidylethanolamine and phosphatidylserine (Figure 2).Finally, the authors proved that when LDL presents substantial oxidative modifications, a great number of neoepitopes are generated transforming it in a highly immunogenic LDL. Indeed, there are a variety of autoantibodies directed to epitopes of OxLDL derived from specific oxidation in animals and human, that appear to increase in individuals with clinical and morphological signs of atherosclerosis [28].

On the contrary, OxLDL is thought to promote atherosclerosis through complex inflammatory and immunologic mechanisms that lead to lipid dysregulation and foam cell formation. Matsuura et al (2006) proposed that in the intima of atherosclerotic lesions, the OxLDL forms complex with the Beta 2 glycoprotein I (beta2GPI) and / or C-reactive protein (CRP). In patients with systemic lupus erythematosus (SLE) and/or antiphospholipid syndrome (APS), anti-OxLDL/beta2GPI complex autoantibodies have been found which has been significantly related to arterial thrombosis. In a non-immunized animal model of APS (NZWxBXSB F1 mice), it was demonstrated that anti-OxLDL/beta2GPI complex IgG autoantibodies can emerge spontaneously. Moreover, a monoclonal autoantibody (WB-CAL-1; IgG2a) against a complex beta-2-GPI was derived from the same mice. WB-CAL-1 significantly increased the

**Figure 2.** Oxidative modifications in ApoB present in lipoproteins.

In 2002, Friedman et al. showed that the oxidized lipids from OxLDL are biologically active. Specifically, polyunsaturated fatty acids (PUFA) either free or bound to an ester from phos‐ pholipid are converted into hydroperoxides, which break down to form highly reactive molecules, such as malondialdehyde and 4-hydroxynonenal among other metabolic products. These reactive aldehydes can then form Schiff-bases, covalent Michael-type adducts with lysine residues of apolipoprotein B in LDL molecules. Besides, the sn-2 oxidized fatty acid fragments which can remain attached via ester bridges may also contain terminal reactive aldehydes. However, this reactive phospholipid also called "aldehyde phospholipid core" may also form adducts with Schiff-base lysine residues of apolipoprotein B and presumably also with other proteins and amines-containing phospholipids, such as phosphatidylethanolamine and phosphatidylserine (Figure 2).Finally, the authors proved that when LDL presents substantial oxidative modifications, a great number of neoepitopes are generated transforming it in a highly immunogenic LDL. Indeed, there are a variety of autoantibodies directed to epitopes of OxLDL derived from specific oxidation in animals and human, that appear to

**Figure 1.** The figure shows some sources and consequences of oxidative stress.

58 Hypercholesterolemia

increase in individuals with clinical and morphological signs of atherosclerosis [28].

On the contrary, OxLDL is thought to promote atherosclerosis through complex inflammatory and immunologic mechanisms that lead to lipid dysregulation and foam cell formation. Matsuura et al (2006) proposed that in the intima of atherosclerotic lesions, the OxLDL forms complex with the Beta 2 glycoprotein I (beta2GPI) and / or C-reactive protein (CRP). In patients with systemic lupus erythematosus (SLE) and/or antiphospholipid syndrome (APS), anti-OxLDL/beta2GPI complex autoantibodies have been found which has been significantly related to arterial thrombosis. In a non-immunized animal model of APS (NZWxBXSB F1 mice), it was demonstrated that anti-OxLDL/beta2GPI complex IgG autoantibodies can emerge spontaneously. Moreover, a monoclonal autoantibody (WB-CAL-1; IgG2a) against a complex beta-2-GPI was derived from the same mice. WB-CAL-1 significantly increased the

*in vitro* uptake of OxLDL/beta-2-GPI complex by macrophages, suggesting that these IgG auto antibodies are pro-atherogenic. As opposed, IgM antibodies to OxLDL found in pro- athero‐ genic mice ApoE (- / - ) and LDL -R (- / - ) seemed to be protective. In human beings it has been widely reported the presence of IgG anti-Ox - LDL antibodies, but their clinical significance is not clear yet [29].

In the beginning, OxLDL was characterized by its biological properties, specifically for being a ligand for acetyl LDL receptor instead of a native LDL receptor. The Acetyl LDL receptor, present in macrophages, uptakes the OxLDL much faster than the native receptors, favoring the excessive intracellular accumulation of cholesterol. LDL oxidation and its uptake can be accomplished *in vitro* by an overnight incubation with macrophages cultured on an appropri‐ ate medium with 5 - 10 μM Cu2 + (oxidant) for 8-16 h allowing the study of the mechanism and kinetics of OxLDL.[30]

LDL oxidative modification, produces numerous structural changes, resulting in an increment of electrophoretic mobility, higher density, a polipoprotein B degradation, hydrolysis of phosphatidylcholine, changes on the amino groups of lysine residues and generation of fluorescent adducts caused by the covalent binding of lipid oxidation products to Apo B[31].

*In vitro* assays have shown that oxidative modification of LDL can be mutated by cultured endothelial cells or by cupric ions, which results in an increase of the lipoprotein uptake into macrophages [32, 33]. Therefore, it seems to be obvious that LDL oxidation is a crucial step for macrophage-derived foam cells formation in early stages of an atherosclerotic lesion. More‐ over, LDL can be oxidized by specific enzymes such as lipooxygenase and phopholipase A2, even when these modifications are not necessarily identical to the endothelial cells-dependent modifications, they are still useful for studying oxidative alterations of LDL. In fact, in 1998, it was demonstrated that the oxidative modification of LDL by specific enzymes leads to an increased recognition by macrophages [32]. In conclusion, it is possible to say that oxidation of LDL in cells depends on at least three possibilities: (a) lipid oxidation by the action of lipoxygenase within the cells followed by the LDL exchange on its medium; (b) direct lipox‐ ygenase-dependent lipid oxidation during cell contact with LDL and (c) both possibilities mentioned above [34].

It is has been reported that the *in vitro* addition of acetyl groups to LDL (acetylation), generates a modified LDL which can induce cholesterol accumulation in macrophages. Indeed, acety‐ lated LDL is incorporated by "scavenger receptors" (SRA), which in contrast to the normal LDL receptor, are not "down regulated", so they induce a great intracellular lipid accumula‐ tion. Thus, acetylated LDL increases the formation of foam cells [35].

Another process that needs to be taken into account is the autoxidation of glucose or the early glycation products (carbonyl compounds) generated by oxygen free radicals (superoxide and hydroxyl) and hydrogen peroxide that can cause oxidative damage. Baynes et al in 1991 introduced the "glycoxidation hypothesis", which proposes that oxidative stress concomitant to glycation plays an important role in the stage of advanced glycation of proteins. Modifica‐ tions of lipoproteins by glycation and oxidation alter their structure to make them sufficiently immunogenic. In type 2 diabetes, high titles of antibodies have been found against glicosylat‐ ed-LDL and glicosylated-OxLDL. Immunogenic properties of glycosilated-OxLDL induce immune complex formation. It has been shown that glycosilated-OxLDL is trapped in the artery wall *in situ* [36-40].

Various pathologies can be originated by oxidative stress-induced apoptotic signaling which is a consequence of an increase of ROS and a decrease of other oxidative species and/or antioxidants, disruption of intracellular redox homeostasis and irreversible oxidative modifi‐ cations of lipids, proteins or DNA. A better understanding of redox control over the develop‐ ment of apoptotic process in the cell, could better guide the course of the therapeutic strategies associated with disorders related to oxidative stress [25].

A great number of diseases have been related to oxidative stress and generation of free radicals, for this reason, antioxidant therapies and diets (such as Mediterranean diet) rich or enriched with antioxidants are thought to be a promising way to prevent or at least to attenuate the organic deterioration originated by the excessive oxidative stress.

#### **3. OxLDL in atheromatous plaque formation**

Atherosclerosis is a chronic inflammatory disease of the arterial wall that culminates with the atheromatous plaque formation. At present, there is a consensus that oxidation of LDL in the endothelial wall is an early event in atherosclerosis, according to the oxidative hypothesis [24]. First, the circulating LDL particles are transported from the vascular space into the arterial wall, mainly across trancytosis[41]. LDL is retained in the extracellular matrix of subendothe‐ lial space, through the binding of basic aminoacids in a polipoprotein B100 to negatively charged sulphate groups of proteoglycans in the extracellular matrix (ECM) [42, 43], where it is prone to be oxidized by oxidative stress, generating OxLDL[21], as we previously mentioned in this article.

It is known that OxLDL participates actively in atheromatous plaque formation, where it is retained. Multiple studies provide evidence suggesting OxLDL contribute in atherosclerotic plaque formation in several ways. In fact, at least four mechanisms have been proposed, being they complementary to each other: a) endothelial dysfunction, b) foam cell formation, c) SMCs migration and proliferation and c) induction of platelet adhesion and aggregation.

#### **3.1. Endothelial dysfunction**

modifications, they are still useful for studying oxidative alterations of LDL. In fact, in 1998, it was demonstrated that the oxidative modification of LDL by specific enzymes leads to an increased recognition by macrophages [32]. In conclusion, it is possible to say that oxidation of LDL in cells depends on at least three possibilities: (a) lipid oxidation by the action of lipoxygenase within the cells followed by the LDL exchange on its medium; (b) direct lipox‐ ygenase-dependent lipid oxidation during cell contact with LDL and (c) both possibilities

It is has been reported that the *in vitro* addition of acetyl groups to LDL (acetylation), generates a modified LDL which can induce cholesterol accumulation in macrophages. Indeed, acety‐ lated LDL is incorporated by "scavenger receptors" (SRA), which in contrast to the normal LDL receptor, are not "down regulated", so they induce a great intracellular lipid accumula‐

Another process that needs to be taken into account is the autoxidation of glucose or the early glycation products (carbonyl compounds) generated by oxygen free radicals (superoxide and hydroxyl) and hydrogen peroxide that can cause oxidative damage. Baynes et al in 1991 introduced the "glycoxidation hypothesis", which proposes that oxidative stress concomitant to glycation plays an important role in the stage of advanced glycation of proteins. Modifica‐ tions of lipoproteins by glycation and oxidation alter their structure to make them sufficiently immunogenic. In type 2 diabetes, high titles of antibodies have been found against glicosylat‐ ed-LDL and glicosylated-OxLDL. Immunogenic properties of glycosilated-OxLDL induce immune complex formation. It has been shown that glycosilated-OxLDL is trapped in the

Various pathologies can be originated by oxidative stress-induced apoptotic signaling which is a consequence of an increase of ROS and a decrease of other oxidative species and/or antioxidants, disruption of intracellular redox homeostasis and irreversible oxidative modifi‐ cations of lipids, proteins or DNA. A better understanding of redox control over the develop‐ ment of apoptotic process in the cell, could better guide the course of the therapeutic strategies

A great number of diseases have been related to oxidative stress and generation of free radicals, for this reason, antioxidant therapies and diets (such as Mediterranean diet) rich or enriched with antioxidants are thought to be a promising way to prevent or at least to attenuate the

Atherosclerosis is a chronic inflammatory disease of the arterial wall that culminates with the atheromatous plaque formation. At present, there is a consensus that oxidation of LDL in the endothelial wall is an early event in atherosclerosis, according to the oxidative hypothesis [24]. First, the circulating LDL particles are transported from the vascular space into the arterial wall, mainly across trancytosis[41]. LDL is retained in the extracellular matrix of subendothe‐

tion. Thus, acetylated LDL increases the formation of foam cells [35].

associated with disorders related to oxidative stress [25].

**3. OxLDL in atheromatous plaque formation**

organic deterioration originated by the excessive oxidative stress.

mentioned above [34].

60 Hypercholesterolemia

artery wall *in situ* [36-40].

The Endothelial dysfunction is a pathological condition in which the endothelium presents an impairment of anti-inflammatory, anti-coagulant and vascular regulatory properties. Nowa‐ days, it is considered a key event in the atherosclerosis development. OxLDL formed and retained in the sub-entothelial space, activates endothelial cells (ECs) through the induction of the cell surface adhesion molecules which in turn, induce the rolling and adhesion of blood monocytes and T cells. It is reported that OxLDL induces the expression of intercellular adhesion molecule-1 (ICAM-1) and vascular-cell adhesion molecule-1 (VCAM-1), increasing the adhesive properties of endothelium in a similar manner to the effects of pro-inflammatory cytokines as interleukin 1 beta [44].

The blood leukocytes recruited migrate into the tunica intima, guided by chemokines. Indeed, OxLDL stimulates ECs and SMCs to secrete monocyte chemotactic protein-1 (MCP-1) and monocyte colony stimulating factor (mCSF) that induce the recruitment of monocytes into the endothelial wall [45-47]. On another hand, OxLDL can be chemotactic itself for monocytes and T lymphocytes (since it possesses lyso-phosphatidylcholine) and also for macrophages [48].

Nitric oxide (NO), is recognized as an important cardiovascular protective molecule, because exerts vasodilator properties and inhibits the adhesion of leucocytes and platelets to endothe‐ lium. This is generated in the vasculature by endothelial NO sythase (eNOS); the impairment of NO production and secretion by ECs is considered one of the most important characteristic of endothelial dysfunction [49].

The NO production from ECs is inhibited by OxLDL, given that the OxLDL is able to induce cholesterol depletion in the plasma membrane invaginations called caveolae, which causes the translocation of the protein caveolin and eNOS from the membrane domains, inhibiting eNOS activity in ECs [50]. Besides, another mechanisms to explain the inhibitory effect of OxLDL over NO production in ECs, has been proposed. It has been reported that OxLDL leads to an increased oxidative stress in ECs, producing significant amounts of superoxide, which chemically inactivates NO, forming peroxynitrite [51].

Lectin-like oxidized LDL receptor-1 (LOX-1), identified as the mayor OxLDL receptor in ECs, is expressed in several pro-inflammatory conditions and seems to play a crucial role in endothelial dysfunction induced by OxLDL[52]. Indeed, in human atherosclerotic lesions, LOX-1 overexpression in ECs has been reported, especially in the early stage of plaque formation [53]. It has been observed that the knockdown of LOX-1, inhibits the MCP-1 expression in human ECs stimulated with OxLDL and mitogen-activated protein kinase (MAPK) pathway would play a critical role [54]. Also, up-regulation of endothelial adhesion molecules as ICAM-1 and VCAM-1, can be induced by OxLDL in a LOX-1-dependent manner and this is mediated by the nuclear factor κB (NF-κB) [55]. Furthermore, the inhibitory effects of OxLDL over endothelial NO productions has been associated with LOX-1 function [51, 56]. Finally, it has been proposed that OxLDL can induce endothelial cell death through the activation of NF-κB and AP-1 pathways [57], worsening endothelial dysfunction and promot‐ ing the progression of the atherosclerotic plaque.

**Figure 3.** Role of OxLDL in endothelial dysfunction.

#### **3.2. Foam cell formation**

Once inside the sub-endothelial ECM, monocytes differentiate into macrophages that express several scavenger receptors (SRs) such as SR-AI/II, SR-BI, cluster of differentiation 36 (CD36) and LOX-1, and toll-like receptors (TLRs). It is important to remark that this phenotypic change, since the internalization of native LDL, occurs at a very low rate to account for foam cells formation and this process is prone to suffer down regulation of LDL receptor. In contrast, scavenger receptors have high affinity for OxLDL and they are not down regulated, leading to a massive intracellular lipid accumulation [20], which results in the foam cells formation [58, 59]. This differentiation into macrophages that promotes pro-inflammatory milieu, is part of a "macrophage trapping", a vicious circle that involves cell retention, oxidation of new LDL and the recruitment of more monocytes [18].

OxLDL also induce the expression of a number of genes associated to inflammation in macrophages: MCP-1, serum amyloid A, ceruloplasmin and hemeoxigenase-1 [60]. Moreover, macrophage activation induces the release of pro-inflammatory cytokines (interleukin 1-β, tumor necrosis factor), reactive oxygen species (ROS) and metalloproteases, which are associated with progression of inflammation [58].

Internalized OxLDL provides oxidized lipids as ligands for PPAR-γ pathway, upregulating CD36 expression, facilitating in turn, the internalization of more OxLDL[61, 62]. This inter‐ nalization activates the macrophage, inducing the secretion of cytokines that recruits immune cells to intima and the secretion of the enzymes myeloperoxidase and 12/15-lipoxigenase, which are thought to participate in the oxidization of new LDL, increasing the local pool of OxLDL[63, 64]. Also, the internalization of OxLDL by CD36 seems to induce the inhibition of macrophage migration, favoring cell spreading and the activation of focal adhesion kinase, in a process mediated by src-kinases and oxidative stress [65]. Besides, OxLDL-CD36 interaction induces the loss of cell polarization in macrophages, an essential process to cell migration [66]. Thus, the evidence suggests that OxLDL not only participates in monocyte differentiation and macrophage activation, but also macrophage retention.

As mentioned, LOX-1 is one of the SRs expressed in macrophages and when it occurs by the influence of pro inflammatory cytokines, OxLDL or other stimuli, the OxLDL uptake increases significantly favoring the foam cells formation [67, 68]. The accumulation of OxLDL can lead to foam cell apoptosis or necrosis, forming cellular debris deposited in the core of the athero‐ sclerotic plaque and contributing to inflammatory progression.

**Figure 4.** Role of OxLDL in foam cell formation.

formation [53]. It has been observed that the knockdown of LOX-1, inhibits the MCP-1 expression in human ECs stimulated with OxLDL and mitogen-activated protein kinase (MAPK) pathway would play a critical role [54]. Also, up-regulation of endothelial adhesion molecules as ICAM-1 and VCAM-1, can be induced by OxLDL in a LOX-1-dependent manner and this is mediated by the nuclear factor κB (NF-κB) [55]. Furthermore, the inhibitory effects of OxLDL over endothelial NO productions has been associated with LOX-1 function [51, 56]. Finally, it has been proposed that OxLDL can induce endothelial cell death through the activation of NF-κB and AP-1 pathways [57], worsening endothelial dysfunction and promot‐

Once inside the sub-endothelial ECM, monocytes differentiate into macrophages that express several scavenger receptors (SRs) such as SR-AI/II, SR-BI, cluster of differentiation 36 (CD36) and LOX-1, and toll-like receptors (TLRs). It is important to remark that this phenotypic change, since the internalization of native LDL, occurs at a very low rate to account for foam cells formation and this process is prone to suffer down regulation of LDL receptor. In contrast, scavenger receptors have high affinity for OxLDL and they are not down regulated, leading to a massive intracellular lipid accumulation [20], which results in the foam cells formation [58, 59]. This differentiation into macrophages that promotes pro-inflammatory milieu, is part of a "macrophage trapping", a vicious circle that involves cell retention, oxidation of new LDL

ing the progression of the atherosclerotic plaque.

62 Hypercholesterolemia

**Figure 3.** Role of OxLDL in endothelial dysfunction.

and the recruitment of more monocytes [18].

**3.2. Foam cell formation**

#### **3.3. Smooth muscle cell migration and proliferation**

The migration and subsequent focal proliferation of SMCs in tunica intima are some of the hallmarks of the atheromatous phenomenon and they play a critical role on it. The SMCs migrate from tunica media to the subendothelial space, where they proliferate in response to growth factors. The proliferation of SMCs can be stimulated by OxLDL, since these particles enhance platelet-derived growth factor (PDGF) and basic fibroblast growth factor (bFGF) expression and secretion [69, 70] by ECs and macrophages. On the other hand, OxLDL also induces the secretion of a variety of other growth factors and their receptors: insulin-like growth factor-1(IGF-1) and epidermal growth factor (EGF), all with mitogenic effects inducing SMCs proliferation [71].

OxLDL has also been shown to induce changes directly in SMCs. OxLDL increases migration and leads to changes in SMCs phenotype making them to produce large amounts of ECM [72]. The production of interstitial collagen and elastin leads to the building of a fibrous cap that covers the developing atherosclerotic plaque, forming a "necrotic core" containing foam cells, cellular debris, extracellular lipids and lysosomal enzymes [73]. Thus, OxLDL participate in the expansion of the atherosclerotic lesion size.

OxLDL also induce LOX-1 expression in SMCs and recently, it has been proposed that many of the named effects of OxLDL are mediated by LOX-1[71]. Another important effect mediated by LOX-1 is the increment of ROS generation induced by OxLDL in SMCs, which can induce the cell death, contributing to plaque instability and rupture in the final stage of atherosclerosis [74]. Taken together, the evidence suggests that OxLDL has a crucial role in the plaque instability and hence, in the development of its complications.

**Figure 5.** Role of OxLDL in smooth muscle cells proliferation and migration.

#### **3.4. Induction of platelet adhesion and aggregation**

**3.3. Smooth muscle cell migration and proliferation**

the expansion of the atherosclerotic lesion size.

instability and hence, in the development of its complications.

**Figure 5.** Role of OxLDL in smooth muscle cells proliferation and migration.

SMCs proliferation [71].

64 Hypercholesterolemia

The migration and subsequent focal proliferation of SMCs in tunica intima are some of the hallmarks of the atheromatous phenomenon and they play a critical role on it. The SMCs migrate from tunica media to the subendothelial space, where they proliferate in response to growth factors. The proliferation of SMCs can be stimulated by OxLDL, since these particles enhance platelet-derived growth factor (PDGF) and basic fibroblast growth factor (bFGF) expression and secretion [69, 70] by ECs and macrophages. On the other hand, OxLDL also induces the secretion of a variety of other growth factors and their receptors: insulin-like growth factor-1(IGF-1) and epidermal growth factor (EGF), all with mitogenic effects inducing

OxLDL has also been shown to induce changes directly in SMCs. OxLDL increases migration and leads to changes in SMCs phenotype making them to produce large amounts of ECM [72]. The production of interstitial collagen and elastin leads to the building of a fibrous cap that covers the developing atherosclerotic plaque, forming a "necrotic core" containing foam cells, cellular debris, extracellular lipids and lysosomal enzymes [73]. Thus, OxLDL participate in

OxLDL also induce LOX-1 expression in SMCs and recently, it has been proposed that many of the named effects of OxLDL are mediated by LOX-1[71]. Another important effect mediated by LOX-1 is the increment of ROS generation induced by OxLDL in SMCs, which can induce the cell death, contributing to plaque instability and rupture in the final stage of atherosclerosis [74]. Taken together, the evidence suggests that OxLDL has a crucial role in the plaque Platelets are important players in atherosclerosis plaque development, especially after the plaque rupture, where they promote thrombus formation. In this process, OxLDL also is implicated. The impairment of endothelial NO production by OxLDL has been associated with an increase in prostaglandin secretion and thus, platelet aggregation [73]. CD36 is expressed in resting platelets and its interaction with OxLDL has been implicated with platelet activation, evidenced by P-selectin expression and the activation of integrin αIIbβ3[75].

OxLDL seems to induce a hyperactive state in platelets, since when they are cultured with OxLDL, they show more sensitivity to the classic platelet-activator ADP, in a process mediated by JNK and Vav family members [76, 77]. OxLDL is able to promote shape change and fast platelet activation through the action of Src kinases and Rho kinase-signaling pathways [78]. The effects of OxLDL over platelets could account also for additional pro-atherogenic phe‐ nomena. Platelets exposed to OxLDL release chemokines that favors atherosclerotic develop‐ ment [79] and promote endothelial dysfunction and foam cells formation [80, 81].

LOX-1 is expressed in platelets once they are activated [82], where it contributes to OxLDL internalization together with CD36. Since LOX-1 is able to binds anionic phospholipids as the present in the surface of activated platelets, has been proposed that endothelial LOX-1 mediates platelet adhesion to ECs [68]. Indeed, platelet binding to LOX-1 enhances endothe‐ lin-1 (ET-1) release from ECs [83] and induces oxidative inactivation of NO in ECs [56], suggesting that LOX-1 participates in endothelial dysfunction also through activated platelets. Thus, OxLDL seems to play a pivotal role in the pro-atherosclerotic behavior of activated platelets.

**Figure 6.** Role of OxLDL in pro-atherosclerotic function of platelets.

#### **4. Mouse models for atherosclerosis**

Given the importance of knowing the role of oxidized LDL in the process of atherosclerotic plaque formation, the study of animal models has been an important tool, where the exami‐ nation of genetically modified mice has significantly contributed towards a better under‐ standing of the mechanisms involved in this pathology.

It is worth noting that the use of small animals in research benefits from easy availability and low cost compared to large animals like primates. In addition, working with small animals reduces ethical concerns and limits the quantity of new agents needed for *in vivo* studies.

Transgenic and knockout mouse models for atherosclerosis have also been instrumental in evaluating existing, finding and testing new atherosclerotic drugs [84]. Small-animal models have the advantage of a well-defined genetic characterization which opens the possibility to transform them into transgenic and gene knockout animals [85].

Atherosclerosis is it not developed by wild-type mice; in fact they have high levels of antiatherogenic high-density lipoprotein (HDL) and low levels of pro-atherogenic LDL and verylow-density lipoprotein (VLDL). Furthermore, mice do not express cholesteryl ester transfer protein (CETP), a plasma protein known to transfer cholesteryl ester from HDL particles and other lipoprotein fractions to pro-atherogenic apolipoprotein-B-containing lipoproteins LDL, VLDL and intermediate low-density lipoproteins (ILDL).

The current mouse models of atherosclerosis are based on disorders on the metabolism of lipoproteins through diets or genetic manipulations [84]. These perturbations have been made thanks to the current availability of genetic information, a variety of inbred strains and the development of molecular biology technics [86]. Atherosclerotic mice were first reported by Thompson et al., 1969, [87] using C57BL/6 inbred mice fed for five weeks with a diet containing a 50% of fat, whereas control mice were fed with a regular diet of 5% of fat. Nevertheless, this diet had a high percentage of mortality [86]. Paigen et al. modified the diet proposed by Thompson supplementing it with a regular diet containing 1.25% of cholesterol, 0.5% of cholic acid and 15% of fat. Nowadays, this diet is named the "Paiged diet" [88]. However, Ignatowski et al., [89] reported in 1980 the first evidence of atherosclerosis in the aorta of a rabbit model fed with a diet containing animal proteins like meat, eggs and milk.

Nowadays, the most used model of atherosclerosis in mice is based on the alteration of genes that codify the low-density lipoprotein receptor (LDLr) and the apolipoprotein E (ApoE), being both key elements for the lipid metabolism.

#### **5. Apolipoprotein E-deficient (***Apo E -/-)* **mice**

The ApoE, is a glycoprotein found in almost every lipoprotein with the exception of LDL. The purpose of this glycoprotein mainly synthesized in the brain and liver is to serve as a ligand for receptors that removes the VLDL and chylomicrons remnants. Since the ApoE can also be synthesized by macrophages and monocytes in the atherosclerotic vessels, is thought to have an important role on inflammatory processes and on the cholesterol homeostasis [90]. More‐ over, it has been reported that ApoE may function in the biliary excretion and in dietary absorption of cholesterol [91].

Plump et al., [92] in 1992, produced the first mice models deficient in apolipoprotein E (*ApoE-/-*). These animals were fed with a diet of 4.5% fat to develop a strong atherosclerosis model. This became an important tool in the research of atherosclerosis.

To inactivate the mice's *ApoE* gene, a homologous recombination of genes was made in embryonic stem cells. Two plasmids (pNMC109 and pJPB63) with a neomycin-resistance gene were used to disrupt the structure of the *ApoE* gene. Chimeric mice were generated by blastocyst injection with targeted lines [93]. The fact that homozygous animals were born at the expected frequency and that they appeared to be healthy, was of significant importance.

Currently, the *ApoE-/-*mice are available on Jackson Laboratories which are direct descendants of the original *ApoE-/* mouse created by the Maeda group (002052 B6.129P2-Apoetm1Unc).

Under a normal chow-fed diet, the mice developed a fatty streak observed in the aorta as early as a 3-month-old [93]. Foam cells at 10 weeks of age under the same diet were observed using a light microscopy. At 15 weeks of age, lesions containing SMCs and foam cells were observed, and at 20 weeks of age, fibrous plaques could be seen. It is worth mentioning that when a Western diet is used, the process is accelerated [94].

Although it is known that this model of *ApoE-/-* has considerable limitations, it has been used widely, because of the rapid development of atherosclerosis. A major drawback of the complete absence of ApoE protein is that most plasma cholesterol is confined to VLDL and not to LDL particles as in humans.

#### **6. LDL receptor-deficient (***LDLr***-/-) mice**

**4. Mouse models for atherosclerosis**

66 Hypercholesterolemia

standing of the mechanisms involved in this pathology.

transform them into transgenic and gene knockout animals [85].

VLDL and intermediate low-density lipoproteins (ILDL).

fed with a diet containing animal proteins like meat, eggs and milk.

both key elements for the lipid metabolism.

**5. Apolipoprotein E-deficient (***Apo E -/-)* **mice**

Given the importance of knowing the role of oxidized LDL in the process of atherosclerotic plaque formation, the study of animal models has been an important tool, where the exami‐ nation of genetically modified mice has significantly contributed towards a better under‐

It is worth noting that the use of small animals in research benefits from easy availability and low cost compared to large animals like primates. In addition, working with small animals reduces ethical concerns and limits the quantity of new agents needed for *in vivo* studies.

Transgenic and knockout mouse models for atherosclerosis have also been instrumental in evaluating existing, finding and testing new atherosclerotic drugs [84]. Small-animal models have the advantage of a well-defined genetic characterization which opens the possibility to

Atherosclerosis is it not developed by wild-type mice; in fact they have high levels of antiatherogenic high-density lipoprotein (HDL) and low levels of pro-atherogenic LDL and verylow-density lipoprotein (VLDL). Furthermore, mice do not express cholesteryl ester transfer protein (CETP), a plasma protein known to transfer cholesteryl ester from HDL particles and other lipoprotein fractions to pro-atherogenic apolipoprotein-B-containing lipoproteins LDL,

The current mouse models of atherosclerosis are based on disorders on the metabolism of lipoproteins through diets or genetic manipulations [84]. These perturbations have been made thanks to the current availability of genetic information, a variety of inbred strains and the development of molecular biology technics [86]. Atherosclerotic mice were first reported by Thompson et al., 1969, [87] using C57BL/6 inbred mice fed for five weeks with a diet containing a 50% of fat, whereas control mice were fed with a regular diet of 5% of fat. Nevertheless, this diet had a high percentage of mortality [86]. Paigen et al. modified the diet proposed by Thompson supplementing it with a regular diet containing 1.25% of cholesterol, 0.5% of cholic acid and 15% of fat. Nowadays, this diet is named the "Paiged diet" [88]. However, Ignatowski et al., [89] reported in 1980 the first evidence of atherosclerosis in the aorta of a rabbit model

Nowadays, the most used model of atherosclerosis in mice is based on the alteration of genes that codify the low-density lipoprotein receptor (LDLr) and the apolipoprotein E (ApoE), being

The ApoE, is a glycoprotein found in almost every lipoprotein with the exception of LDL. The purpose of this glycoprotein mainly synthesized in the brain and liver is to serve as a ligand for receptors that removes the VLDL and chylomicrons remnants. Since the ApoE can also be In humans, mutations in the *LDLr* gene cause familial hypercholesterolemia. The *LDLr*-/-mouse has a milder lipoprotein alteration than *ApoE*-/-mice when fed standard low-fat chow, with plasma cholesterol levels around 250 mg/dL due mainly to the accumulation of LDL [95].

In 1993, *LDLr*-/- mice were created by gene targeting of embryonic stem cells [96]. By feeding them with a 10% fat diet, an increase of total cholesterol level (2-fold) was observed on these mice, due mainly to the high levels of VLDL and LDL. When fed on a high-fat/high-cholesterol diet, *LDLr*-/- mice showed a rapid increase in the severity of hypercholesterolemia and atherosclerotic lesion development throughout the coronary arteries, aortic root, and aorta [85, 97]. The plasma lipoprotein profile of *LDLr*-/- mice resembled the one of humans, with the cholesterol being confined mainly to the LDL fraction. Nevertheless, this mice model of atherosclerosis is very responsive to the diet. In fact, their cholesterol levels rose up to 1500 mg/dL when they were under the Paigen diet [98]. The lesions produced in *LDLr-/-* mice were similar to the lesions produced in the *ApoE-/-* mice, in terms of their development of plaques in a time-dependent manner. On the contrary, the *LDLr-/-* mouse produced a more moderate murine model of atherosclerosis than the *ApoE-/-* mouse. This characteristic is produced mainly due to a milder degree of hyperlipidemia [99].

In 1998, a mouse model deficient in the Apo B mRNA editing activity (*Apobec1-/-)* and LDL receptor (*LDLr-/-*) were generated by Powell-Braxton et al [100]. The lipoprotein profile of this animal model resembles the human familial hypercholesterolemia and when fed with a chow diet, exhibited atherosclerosis at its 8-month of age. The characteristics of this animal model provided an advantage to study the interactions between the environment (high fat diet) and the gene response in the onset of atherosclerosis [101].

#### **7. ApoE3-leiden transgenic mice**

Mutations in the gene encoding ApoE can lead to a binding-defective ApoE, which mediates the binding of lipoproteins to LDL receptor and is an essential ligand for the receptor-mediated uptake of chylomicron and VLDL remnants by hepatic lipoprotein receptors. This results in a disturbed receptor-mediated clearance of lipoprotein remnants by the liver, as has been described for patients with familial dysbetalipoproteinemia[102]. Premature atherosclerosis is produced by a genetic disorder named familial dysbetalipoproteinemia, which presents high levels of plasmatic triglycerides and cholesterol, mainly due to the increase in the VLDL remnants and chylomicron. The ApoE3-Leiden, a genetic variant of ApoE, is related with an inherited familial dysbetalipoproteinemia [103].

The ApoE3-Leiden mutation it is characterized by a rare dominant-negative tandem duplica‐ tion of codons 120 to 126 in human *ApoE3* gene. Introducing a human ApoE3-Leiden gene construct into C57BL/6 mice has generated the ApoE3-Leiden transgenic mice. The ApoE3- Leiden gene consists of a construct with the *ApoC1*and *ApoE* genes with a promoter element to regulate the expression. While, this mice model of atherosclerosis still expresses ApoE protein, the clearance of lipoproteins containing ApoE is impaired, being less dramatic than the *ApoE-/-* mice model of atherosclerosis. The introduction of the *ApoC1* gene in transgenic mice has exhibited elevated levels of cholesterol and triglycerides owing to an accumulation of VLDL-size particles in the circulation, increasing plasma lipid levels by diminished lipolysis and VLDL uptake through both the LDLr and low density lipoprotein receptor-related protein (LRP) [84, 104].The ApoE3-Leiden mice have a hyperlipidemic phenotype, develop athero‐ sclerosis on being fed cholesterol, and are more sensitive to lipid-lowering drugs than *ApoE*-/ and *LDLr*-/- mice [105]. The ApoE3-Leiden mice model is very responsive to diets containing sugar, fat and cholesterol, developing high levels of plasma triglycerides and cholesterol, with a prominent increase in LDL and VLDL lipoproteins.

Compared with *ApoE*-/- and *LDLr*-/- mice, ApoE3-Leiden mice represent a moderate mouse model for hyperlipidemia. Therefore, diets and drugs that influence the production of VLDL and chylomicron also show parallel effects on plasma levels of triglycerides and cholesterol. In this sense, the ApoE3-Leiden mice are more responsive to hypolipidemic compounds than the *LDLr-/-* and *ApoE-/-* mice [84, 106].

#### **8. Double knockout mice models**

in a time-dependent manner. On the contrary, the *LDLr-/-* mouse produced a more moderate murine model of atherosclerosis than the *ApoE-/-* mouse. This characteristic is produced mainly

In 1998, a mouse model deficient in the Apo B mRNA editing activity (*Apobec1-/-)* and LDL receptor (*LDLr-/-*) were generated by Powell-Braxton et al [100]. The lipoprotein profile of this animal model resembles the human familial hypercholesterolemia and when fed with a chow diet, exhibited atherosclerosis at its 8-month of age. The characteristics of this animal model provided an advantage to study the interactions between the environment (high fat diet) and

Mutations in the gene encoding ApoE can lead to a binding-defective ApoE, which mediates the binding of lipoproteins to LDL receptor and is an essential ligand for the receptor-mediated uptake of chylomicron and VLDL remnants by hepatic lipoprotein receptors. This results in a disturbed receptor-mediated clearance of lipoprotein remnants by the liver, as has been described for patients with familial dysbetalipoproteinemia[102]. Premature atherosclerosis is produced by a genetic disorder named familial dysbetalipoproteinemia, which presents high levels of plasmatic triglycerides and cholesterol, mainly due to the increase in the VLDL remnants and chylomicron. The ApoE3-Leiden, a genetic variant of ApoE, is related with an

The ApoE3-Leiden mutation it is characterized by a rare dominant-negative tandem duplica‐ tion of codons 120 to 126 in human *ApoE3* gene. Introducing a human ApoE3-Leiden gene construct into C57BL/6 mice has generated the ApoE3-Leiden transgenic mice. The ApoE3- Leiden gene consists of a construct with the *ApoC1*and *ApoE* genes with a promoter element to regulate the expression. While, this mice model of atherosclerosis still expresses ApoE protein, the clearance of lipoproteins containing ApoE is impaired, being less dramatic than the *ApoE-/-* mice model of atherosclerosis. The introduction of the *ApoC1* gene in transgenic mice has exhibited elevated levels of cholesterol and triglycerides owing to an accumulation of VLDL-size particles in the circulation, increasing plasma lipid levels by diminished lipolysis and VLDL uptake through both the LDLr and low density lipoprotein receptor-related protein (LRP) [84, 104].The ApoE3-Leiden mice have a hyperlipidemic phenotype, develop athero‐ sclerosis on being fed cholesterol, and are more sensitive to lipid-lowering drugs than *ApoE*-/ and *LDLr*-/- mice [105]. The ApoE3-Leiden mice model is very responsive to diets containing sugar, fat and cholesterol, developing high levels of plasma triglycerides and cholesterol, with

Compared with *ApoE*-/- and *LDLr*-/- mice, ApoE3-Leiden mice represent a moderate mouse model for hyperlipidemia. Therefore, diets and drugs that influence the production of VLDL and chylomicron also show parallel effects on plasma levels of triglycerides and cholesterol. In this sense, the ApoE3-Leiden mice are more responsive to hypolipidemic compounds than

due to a milder degree of hyperlipidemia [99].

68 Hypercholesterolemia

the gene response in the onset of atherosclerosis [101].

**7. ApoE3-leiden transgenic mice**

inherited familial dysbetalipoproteinemia [103].

a prominent increase in LDL and VLDL lipoproteins.

the *LDLr-/-* and *ApoE-/-* mice [84, 106].

A model that develops severe hyperlipidaemia and atherosclerosis was obtained with an *ApoE* and *LDLr* double knockout (*ApoE*-/-/ *LDLr*-/- / DKO) [98]. It has been observed that, even on a regular chow diet, the atherosclerosis progression is generally more considerable than in mice only deficient in ApoE [107, 108]. Hence, the *ApoE-/-*/, *LDLr-/-*/, DKO mouse is appropriate to study the effect of compounds with anti-atherosclerotic activity without the need of athero‐ genic diets.

Besides, the role of the ApoE and the LDLr in the development of the atherosclerosis and dysregulation of the NOS system leading to impairment of NO bioavailability, has been documented for some time in atherosclerotic vessels of both experimental animals and humans [109]. To study the contribution of endothelial eNOS to lesion formation, Kuhlencordt et al. [110] created *ApoE*-/-/ *eNOS*-/-/double knockout mice (*ApoE*-/-/ *eNOS*-/- / DKO), which presents a more pronounced atherosclerosis than ApoE-/- mouse model. Besides, eNOS absence favors the development of peripheral coronary disease, chronic myocardial ischemia, heart failure and an array of other vascular complications not detected in *ApoE*-/- mice [111].

Additionally, key structural proteins like apoB100 and apoB48, are needed to assemble lipoproteins rich in triacylglycerol; moreover, these proteins are part of all classes of athero‐ genic lipoproteins [112]. Veniant et al., 1998, characterized *LDLr-/-* and *ApoE-/-* mice which were homozygous to the ApoB-100 allele, founding that the *LDLr-/-*/ApoB100/100 mice model develop an extensive atherosclerosis, even when were fed with a normal chow diet, [113]

In summary, the experimental evidences show that the oxidative stress plays a pivotal role in atherogenesis, having OxLDL as a crucial player. Nevertheless, the clinical trials that used antioxidants strategies have shown poor results in relationship to the development of athero‐ sclerosis, besides strong discrepancies between the different studies to establish the correlation between oxidative stress and atherogenic process. Therefore, the achievement of a successful therapy in humans based on the oxidative modification hypothesis is still a major challenge.

#### **Author details**

E. Leiva\* , S. Wehinger, L. Guzmán and R. Orrego

\*Address all correspondence to: eleivam@utalca.cl

Interdisciplinary Excellence Research Program on Healthy Aging (PIEI-ES), Universidad de Talca, Talca, Chile

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[84] Zadelaar, S.; Kleemann, R.; Verschuren, L.; de Vries-Van der Weij, J.; van der Hoorn, J.; Princen, H. M.; Kooistra, T. Mouse models for atherosclerosis and pharmaceutical

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[87] Thompson, J. S. Atheromata in an inbred strain of mice. *J Atheroscler Res* 10:113-122;

[88] Paigen, B.; Morrow, A.; Brandon, C.; Mitchell, D.; Holmes, P. Variation in susceptibil‐ ity to atherosclerosis among inbred strains of mice. *Atherosclerosis* 57:65-73; 1985. [89] Ignatowski A.C. Influence of animal food on the organism of rabbits. *S Peterb Izviest*

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**Chapter 4**

**A Practical Case-Based Approach to Dyslipidaemia in Light of the European Guidelines**

Olivier S. Descamps , Lale Tokgozoglu and Eric Bruckert

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/59481

#### **1. Introduction**

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[110] Kuhlencordt, P. J.; Gyurko, R.; Han, F.; Scherrer-Crosbie, M.; Aretz, T. H.; Hajjar, R.; Picard, M. H.; Huang, P. L. Accelerated atherosclerosis, aortic aneurysm formation, and ischemic heart disease in apolipoprotein E/endothelial nitric oxide synthase dou‐

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ble-knockout mice. *Circulation* 104:448-454; 2001.

2003.

78 Hypercholesterolemia

The European Atherosclerosis Society and the European Society of Cardiology have made new recommendations regarding the treatment of dyslipidaemia (1). Such recommendations build upon the "Joint European Societies' Task Force Guidelines on the Prevention of CVD in Clinical Practice" of 2007 [1, 2]. The most important changes include the redefinition of the risk categories and the addition of a 'very high-risk' category. For these new risk categories, the LDL-C targets have been redefined. In the highest risk individuals, the lowering of LDL to 70 mg/dl is recommended. Furthermore, HDL-c has been added to the new SCORE risk chart and non-HDL cholesterol is now considered to be a secondary target.

In this study, we illustrate how these guidelines can be used in clinical practice. We also give some tips on how to make them more user-friendly for clinicians (Figure 1). The discussed case (Clinical Case 1) was developed to combine a series of difficulties in therapeutic decisionmaking. We accepted the principle that any correctable secondary causes of dyslipidaemia had been excluded and that the patient had already received all the care to improve the other risk factors but without real (enough) success. These risk factors included smoking cessation, weight loss and/or blood pressure reduction. Unfortunately, this case is far from an exception. The reality is that it is often more difficult to quantitatively reduce risk factors, than it is to reduce cholesterol levels.

© 2015 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

**Figure 1.** Algorithm of dyslipidaemia management in cardiovascular prevention.

#### **Clinical Case 1. Presentation.**

*The patient,a woman of 59 years of age, has come to visit her doctor because she has reached the same age as when her sister suffered from a heart attack (two years ago).*

**Personal history:***Without particularity*.

**Family history:***Her mother is obese and has been treated for diabetes since the age of 60 years. Her sister has undertaken a coronary bypass surgery following a heart attack at 59 years of age.*

**Lifestyle-dietary habits.***Smokes (15 cigarettes per day), sedentary (works in an office, does not practice sport and only engages in interior recreation).*

**Clinical examination.***Weight: 92 kg; height: 1,74 m (Body mass index – BMI : 30,5 kg/m²); waist circumference: 99 cm ; blood pressure: 145 / 85 mm Hg, no xanthoma, no corneal arcus.*

**Biology:***Total cholesterol: 5.8 mmol/l; HDL cholesterol(HDL-C) : 1.0 mmol/l; triglycerides (TG): 3.5 mmol/l: cholesterol LDL (LDL-C) : 3.2 mmol/l (tableau 1); glycaemia : 112 mg/dl ; hs-CRP= 1,2 mg/dL; normal levels of creatinin, hepatic enzymes and thyroid hormones.*

#### **Clinical Case 2. What is the Cardiovascular Risk of this Patient?**

*Our patient has no cardiovascular history, diabetes or any severe risk factors. Thus, we should calculate the risk SCORE, i.e., the risk of cardiovascular mortality in 10 years (we should take into consideration that we are a country with a high-risk population).*

*- Based on the classical risk factors (gender, age, systolic blood pressure and total cholesterol levels) and according to the classical SCORE table, the risk SCORE is 4% (Figure 2).*

*- Based on the new way to calculate the risk, the risk of our patient is quite high. After adjusting the HDL-C (near 1 mmol/l), using the four tables or using multipliers plus the reference chart, the SCORE reached should be 6 -6,4%. Given her family history (her sister, a woman of 60 years), this must be multiplied by 1.7 (Figure 2). Thus, the calculation is 10-11%. In addition, the presence of other risk factors (high levels of triglycerides, obesity, sedentariness and high hs-CRP) justifies the multiplication by 1.5 (~1.46=1.1 x 1.1 x 1. 1 x 1.1). This gives us 15-16% (let us take 15% as a base to facilitate later calculations). Thus, in total, we estimate that the risk of the patient dying of cardiovascular disease in 10 years is likely to be 15 % (Table 1). The patient is, therefore, in the category of risk labelled very high. The global risk (fatal and non-fatal) can be calculated by multiplying this by three (as the patient is a woman of nearly 60 years of age), giving a value of ~ 45%, which is very high.*

#### **2. First question: What is the cardiovascular risk of this patient?**

To determine whether the patient is in the highest/high-risk category, we propose the acronym "C.A.R.D.I.A.SCORE". This will make it easy to remember (Figure 1).

#### **2.1. Acronym « C.A.R.D.I.A. SCORE »**

**Figure 1.** Algorithm of dyslipidaemia management in cardiovascular prevention.

*undertaken a coronary bypass surgery following a heart attack at 59 years of age.*

*99 cm ; blood pressure: 145 / 85 mm Hg, no xanthoma, no corneal arcus.*

*The patient,a woman of 59 years of age, has come to visit her doctor because she has reached the same age as when*

**Family history:***Her mother is obese and has been treated for diabetes since the age of 60 years. Her sister has*

**Lifestyle-dietary habits.***Smokes (15 cigarettes per day), sedentary (works in an office, does not practice sport and*

**Clinical examination.***Weight: 92 kg; height: 1,74 m (Body mass index – BMI : 30,5 kg/m²); waist circumference:*

**Biology:***Total cholesterol: 5.8 mmol/l; HDL cholesterol(HDL-C) : 1.0 mmol/l; triglycerides (TG): 3.5 mmol/l: cholesterol LDL (LDL-C) : 3.2 mmol/l (tableau 1); glycaemia : 112 mg/dl ; hs-CRP= 1,2 mg/dL; normal levels of*

**Clinical Case 1. Presentation.**

80 Hypercholesterolemia

*her sister suffered from a heart attack (two years ago).*

**Personal history:***Without particularity*.

*only engages in interior recreation).*

*creatinin, hepatic enzymes and thyroid hormones.*

If there is an history of **c**ardiovascular **(« C »)** disease, **c**oronary**, c**erebrovascular or any **a**rtery injuries **(« A ») in** general (peripheral arteries, aneurism, etc.); if there is **r**enal insufficiency **(« R »)** defined by a glomerular filtration below 60 ml/min/1,73 m²; if there is **d**iabetes **(« D »)**, either type I or II complicated of organ injuries (microalbuminuria) or type II without com‐ plications; if the patient is older than 40 years of age and displaying other risk factors (notice that, in diabetes, the three conditions of « **c**omplication, **a**ge and **r**isk factor give the acronym « CAR »), the patient should be considered as « very high-risk ».

For patients who show an isolated, yet severe, risk factor **(« I »),** like diabetes, without any other risk factor, severe hypercholesterolemia (cholesterol > 7.5 mmol/l and/or familial hypercholesterolemia) or severe hypertension (>180 mm Hg), they should be considered as **« high-risk »**. Furthermore, patients who have been diabetic since a young age **(« I »)** should also be considered as **« high-risk »**.

All other individuals who do not present one of the above characteristics should be examined using SCORE (6).

#### **2.2. SCORE with two novelties**

**First novelty.** It is now possible to qualify the risk SCORE quantitatively, according to the presence of a family history of early cardiovascular disease and the level of HDL cholesterol (1, 3and 4).We can do this by using either the four specific tables (one for each of the four HDL-C levels) (1) or the HDL-C specific multipliers to adjust the SCORE risk based on one reference table (the table for HDL-C = 0.8 mmol/l)(6) (Figure 2). In addition, we should consider the patient as higher risk if other risk factors are present (high levels of triglycerides, obesity, sedentariness, etc.). For these factors, the guidelines do not provide any multipliers but, as a rule of thumb, we should accept a minimum value of 1.1 as a multiplier for each additional factor. Indeed, in epidemiological studies, it is rare that a parameter can be identified as statistically independent and a clinically meaningful risk factor if it does not increase the risk to a minimum of 10%, after adjusting for other risk factors.

**Second novelty.** Patients should not be categorized according to a single "frontier of risk" (below or above 5%). Instead, they should be categorized based on the three "frontiers of risk": 1%, 5% and 10%. Thus, the risk SCORE should be defined as "**low**" if it is less than 1%,"**moderate**" if it is between 1% and 5%, "**high**" if it is between 5% and 10% and "**very high**" if it is over 10% (Figure 1).

Our case (Clinical Case 2) illustrates how a "moderate" 4% risk can be significantly enhanced in refining the other risk factors such as HDL-C, family history and so on. It is possible to calculate the global risk (fatal and non-fatal) simply by multiplying by three for men, by four for women and by a little less for older people. Taking this into consideration, for our female patient of 59 years, we should multiply by three. Thus, the overall risk is 45%. This is another way of expressing the CVD risk (with a higher number!). It should be used to increase our patient's awareness of CVD and encourage her to be more motivated to follow the regime and take her medication.

#### **3. Second question, how do we treat this patient?**

The LDL-C remains the primary target, as in the previous guidelines. The target LDL-C is determined as a function of the patient's risk.For each risk category, there is a different LDL GOAL. There is also a simplified chart to calculate the LDL reduction percentage to reach that goal. The evidence to decrease LDL-C to such low levels is supported by previous studies that indicate a possible regression of atherosclerotic plaques (i.e., a "rejuvenation" of the arteries) in this condition.

What if the level of LDL-C is unavailable because it is non-computable by the Friedewald formula (when TG levels are above 4.5 mmol/l)? In this case, we should use another target. The alternative target is not the level of total cholesterol, as proposed in the previous guide‐ lines. Instead, it is the level of non HDL-C that will become, in this case, the primary target (see §4).

**2.2. SCORE with two novelties**

82 Hypercholesterolemia

**high**" if it is over 10% (Figure 1).

take her medication.

in this condition.

(see §4).

to a minimum of 10%, after adjusting for other risk factors.

**3. Second question, how do we treat this patient?**

**First novelty.** It is now possible to qualify the risk SCORE quantitatively, according to the presence of a family history of early cardiovascular disease and the level of HDL cholesterol (1, 3and 4).We can do this by using either the four specific tables (one for each of the four HDL-C levels) (1) or the HDL-C specific multipliers to adjust the SCORE risk based on one reference table (the table for HDL-C = 0.8 mmol/l)(6) (Figure 2). In addition, we should consider the patient as higher risk if other risk factors are present (high levels of triglycerides, obesity, sedentariness, etc.). For these factors, the guidelines do not provide any multipliers but, as a rule of thumb, we should accept a minimum value of 1.1 as a multiplier for each additional factor. Indeed, in epidemiological studies, it is rare that a parameter can be identified as statistically independent and a clinically meaningful risk factor if it does not increase the risk

**Second novelty.** Patients should not be categorized according to a single "frontier of risk" (below or above 5%). Instead, they should be categorized based on the three "frontiers of risk": 1%, 5% and 10%. Thus, the risk SCORE should be defined as "**low**" if it is less than 1%,"**moderate**" if it is between 1% and 5%, "**high**" if it is between 5% and 10% and "**very**

Our case (Clinical Case 2) illustrates how a "moderate" 4% risk can be significantly enhanced in refining the other risk factors such as HDL-C, family history and so on. It is possible to calculate the global risk (fatal and non-fatal) simply by multiplying by three for men, by four for women and by a little less for older people. Taking this into consideration, for our female patient of 59 years, we should multiply by three. Thus, the overall risk is 45%. This is another way of expressing the CVD risk (with a higher number!). It should be used to increase our patient's awareness of CVD and encourage her to be more motivated to follow the regime and

The LDL-C remains the primary target, as in the previous guidelines. The target LDL-C is determined as a function of the patient's risk.For each risk category, there is a different LDL GOAL. There is also a simplified chart to calculate the LDL reduction percentage to reach that goal. The evidence to decrease LDL-C to such low levels is supported by previous studies that indicate a possible regression of atherosclerotic plaques (i.e., a "rejuvenation" of the arteries)

What if the level of LDL-C is unavailable because it is non-computable by the Friedewald formula (when TG levels are above 4.5 mmol/l)? In this case, we should use another target. The alternative target is not the level of total cholesterol, as proposed in the previous guide‐ lines. Instead, it is the level of non HDL-C that will become, in this case, the primary target

**Figure 2.** Table of risk SCORE, (the risk of cardiovascular death at 10 years) and the adjustments according to the level of HDL-C (8) as well as any family history of early cardiovascular disease (before 50 years of age in men and before 60 years of age in women). The table should be used as a reference chart; the table of SCORE at 0.8 mmol/l HDL-C, which contains the highest risk values. This allows the multipliers for the other HDL levels to be between 0 and 1 (which facilitates multiplication, e.g., to multiply by 0.4, multiply by 4 and divide by 10). Figure 2a. displays the SCORE in the high-risk population and Figure 2.b, in the low risk population.

#### **3.1. Which Statin, What Dose and How Fast**

Statin should always be the first line treatment (even for dyslipidaemia mixed with elevations of cholesterol and triglycerides, as we will demonstrate below (§4)). We should begin by prescribing a statin at a dosage that is the most likely to be effective in obtaining the correct reduction target. The rationale to choose the statin type and dosage is quite mathematical and is based on the baseline level, the LDL-C target and knowledge of the different statins' power (Figure 3). In terms of power (for a same dosage), these are fluvastatin < pravastatin < sim‐ vastatin <pitavastatin< atorvastatin < rosuvastatin [5]. Another rule is that, on average, doubling each of the statin dosage leads to a further decline in the rate of LDL-C of 4% to 6%. Another way to intensify the treatment is to associate the statin with one of the other antidyslipidaemia drugs (Figure 3). Among these, ezetimibe has the largest (20% to 25%) addi‐ tional reduction of LDL-C. When the risk is high and the target is not reached, it is important to adjust the treatment as quickly as possible. The compliance and satisfaction of the patient, as well as the physician, depends on it.

**Figure 3.** The choice and the statin dose should be based on a near mathematical criterion: the target reduction of LDL– C. The necessary reduction is easily estimated by the difference between the baseline LDL-C level and its target (ac‐ cording to the risk level) divided by the baseline LDL-C. The figure displays the power of different treatments in re‐ ducing LDL-C. For better precision of atorvastatin, we should use the following rule (dosage→ % LDL-C reduction): 10 mg → 38%, 20 mg → 45%, 40 mg → 49%, 80 mg → 52%). Other drugs also influence the HDL-C and TG levels. Statins have a small effect on the levels of HDL-C (slight ↑) and triglycerides (slight ↓). Fluva: fluvastatin; prava: pravastatin; pita: pitavasatin; simva: simvastatin; rosuva:rosuvastatin; atorva: atorvastatin.

#### **3.2. Before prescribing a statin, we will check**

Before the initiation of treatment, it is important to ensure that the CK (creatine phosphokinase) levels are not too high. If they are very high (> 5 times the upper limit of normal), it is better to delay and to check again six weeks later. Furthermore, it is important to determine the cause of these levels (intense physical exercise, trauma or recent intramuscular injection). To reduce the risk of muscle side effects, we have to be more vigilant in elderly patients or in cases where the simultaneous use of treatment is interfering (via cytochrome P 450) with the metabolism of the proposed statin. We also have to be particularly careful if the patient has previously suffered from hepatic or renal insufficiencies.

For our patient in the very high-risk category, the LDL-C target is below 1.8 mmol/l. To achieve this target, we should reduce the patient's LDL-C by 40% (Clinical Case 3).

#### **Clinical Case 3. How do we Treat Cholesterol in this Patient?**

*For our patient with a very high-risk,*

*- The target is < 1.8 mmol/l*

**3.1. Which Statin, What Dose and How Fast**

84 Hypercholesterolemia

as well as the physician, depends on it.

Statin should always be the first line treatment (even for dyslipidaemia mixed with elevations of cholesterol and triglycerides, as we will demonstrate below (§4)). We should begin by prescribing a statin at a dosage that is the most likely to be effective in obtaining the correct reduction target. The rationale to choose the statin type and dosage is quite mathematical and is based on the baseline level, the LDL-C target and knowledge of the different statins' power (Figure 3). In terms of power (for a same dosage), these are fluvastatin < pravastatin < sim‐ vastatin <pitavastatin< atorvastatin < rosuvastatin [5]. Another rule is that, on average, doubling each of the statin dosage leads to a further decline in the rate of LDL-C of 4% to 6%. Another way to intensify the treatment is to associate the statin with one of the other antidyslipidaemia drugs (Figure 3). Among these, ezetimibe has the largest (20% to 25%) addi‐ tional reduction of LDL-C. When the risk is high and the target is not reached, it is important to adjust the treatment as quickly as possible. The compliance and satisfaction of the patient,

**Figure 3.** The choice and the statin dose should be based on a near mathematical criterion: the target reduction of LDL– C. The necessary reduction is easily estimated by the difference between the baseline LDL-C level and its target (ac‐ cording to the risk level) divided by the baseline LDL-C. The figure displays the power of different treatments in re‐ ducing LDL-C. For better precision of atorvastatin, we should use the following rule (dosage→ % LDL-C reduction): 10 mg → 38%, 20 mg → 45%, 40 mg → 49%, 80 mg → 52%). Other drugs also influence the HDL-C and TG levels. Statins have a small effect on the levels of HDL-C (slight ↑) and triglycerides (slight ↓). Fluva: fluvastatin; prava: pravastatin;

Before the initiation of treatment, it is important to ensure that the CK (creatine phosphokinase) levels are not too high. If they are very high (> 5 times the upper limit of normal), it is better to delay and to check again six weeks later. Furthermore, it is important to determine the cause of these levels (intense physical exercise, trauma or recent intramuscular injection). To reduce

pita: pitavasatin; simva: simvastatin; rosuva:rosuvastatin; atorva: atorvastatin.

**3.2. Before prescribing a statin, we will check**

*- The necessary LDL-C reduction is: 3.0 mmol/l - 1.8 mmol/l = 1.2 mmol/l, which requires a reduction of 40% of the LDL-C (1.2 / 3.0 = 40%).*

*- The statins that are able to achieve a 40% reduction (Figure 3) are, for example, 40 mg simvastatin or 20 mg atorvastatin (there is very little chance of achieving such a reduction with pravastatin, even with 20 mg simvastatin).*

*- Prior to prescribing the drug, we should first verify the levels of CPK and liver enzymes, if this has not already been achieved.*

**Figure 4.** Shows how various LDL reductions decrease the risk of cardiovascular mortality up to 10 years (SCORE) and the risk of global cardiovascular risk (fatal and non-fatal) up to 10 years (this is calculated by multiplying the risk SCORE by three). (1) The current 0.9 mmol/l reduction of LDL-C relatively reduces CV events by 18%. Hence, the abso‐ lute risk of mortality CV decreases from 15% to 12%. Similarly, the global CV risk (multiplied by 3, see above) decreas‐ es from 45% to 36%. (2) A greater reduction below the target of 1.8 mmol/l allows a relative reduction in the amount of CV events by 32%. Hence, the absolute risk of mortality CV decreases from 15% to 10% and the CV global risk decreas‐ es from 45% to 30%.

#### **4. Third question, how to follow up the patient?**

In order to verify the effectiveness and safety of the patient's prescription, the improved guidelines recommend a patient follow up eight weeks later. Once the lipid levels have reached the target levels (according to the risk of the patient) and a safe level is maintained, an annual follow up will suffice.

#### **4.1. Tolerance monitoring**

Throughout the therapy, the monitoring of liver enzymes (Figure 5) is required. In subjects complaining of muscle pain, the muscle enzymes should be analysed. As long as the enzymes are not too high (< 3 x the upper limit of normal or ULN), we should continue the statin. However, if the enzymes exceed by 3 x ULN for liver enzymes or 5 X ULN for muscle enzymes (Figure 5), the statins should be stopped. Furthermore, the enzymes should be checked four to six weeks later (or two weeks later if the CK is very high). For high elevation of the CK, we should check the renal function. When the enzymes return to a normal value, treatment (or an alternative treatment) should be carefully reintroduced. In all cases, other common causes of the elevation of these enzymes should be excluded. In the instance of high CK, this means intensive muscle efforts and injuries (including intramuscular injections, etc.). For cases of high ALT, this means weight gain, excess of sugar, fat or alcohol, steatosis, hepatitis, lithiasis migration and other medications, etc.

**Figure 5.** Biological monitoring of liver and muscle enzymes. CK: Creatine kinase. ALT. Alanine aminotransferase (al‐ so called SGPT).

**Figure 6.** In the intensity of treatment, a rapid escalation is more beneficial than a slow progression.

#### **4.2. Efficacy monitoring**

**4. Third question, how to follow up the patient?**

follow up will suffice.

86 Hypercholesterolemia

**4.1. Tolerance monitoring**

migration and other medications, etc.

so called SGPT).

In order to verify the effectiveness and safety of the patient's prescription, the improved guidelines recommend a patient follow up eight weeks later. Once the lipid levels have reached the target levels (according to the risk of the patient) and a safe level is maintained, an annual

Throughout the therapy, the monitoring of liver enzymes (Figure 5) is required. In subjects complaining of muscle pain, the muscle enzymes should be analysed. As long as the enzymes are not too high (< 3 x the upper limit of normal or ULN), we should continue the statin. However, if the enzymes exceed by 3 x ULN for liver enzymes or 5 X ULN for muscle enzymes (Figure 5), the statins should be stopped. Furthermore, the enzymes should be checked four to six weeks later (or two weeks later if the CK is very high). For high elevation of the CK, we should check the renal function. When the enzymes return to a normal value, treatment (or an alternative treatment) should be carefully reintroduced. In all cases, other common causes of the elevation of these enzymes should be excluded. In the instance of high CK, this means intensive muscle efforts and injuries (including intramuscular injections, etc.). For cases of high ALT, this means weight gain, excess of sugar, fat or alcohol, steatosis, hepatitis, lithiasis

**Figure 5.** Biological monitoring of liver and muscle enzymes. CK: Creatine kinase. ALT. Alanine aminotransferase (al‐

If the target is not achieved and, especially, if the observed reduction is lower than expected, as is the case here (Clinical Case 4), we should first check whether the patient has been compliant. We know that, after one year, approximately half of patients do not correctly take their medication [6]. If the patient has been compliant with her medication, it is possible that the inadequate response is due to a resistance to the statin. This occurs in 10% of patients. If this is the case, we should adapt the treatment.

#### **Clinical Case 4. How do we Follow up the Patient?**

*Eight weeks after the prescription of 40 mg simvastatin, the patient is not complaining of myalgia. CPK levels have not been controlled and the GOT rates are normal. However, we are slightly disappointed with the lipid profile of the patient, which has become (Table 1):*

*Total cholesterol: 4.8 mmol/l; LDL-C: 2, 3 mmol/l; HDL-C: 1.1 mmol/l; Triglycerides: 3.1 mmol/l*

*The treatment appears to be well tolerated but the target has not been reached. Curiously, the reduction has not achieved the expected 40% (but only 28%!).*

#### *4.2.1. Why is it important that the target is reached?*

In a recent meta-analysis of the results of 118,000 subjects from 26 intervention trials with statins, there was evidence of a relationship between the reduction of LDL-C and the reduction of the incidence of CV disease in 4-5 years. Every decrease of 1 mmol/l for LDL - C by a statin (Figure 4) was associated with a relative reduction in about 20% coronary events, stroke and heart deaths [7]. This relationship was universal (regardless of age, sex and other risk factors). Furthermore, it was almost linear over the range of studied LDL-C levels.

The absolute benefit produced by statins not only depends on their capacity to lower LDL-C but also, on the initial CV risk. Thus, the greater the initial CV risk, the greater the reduction of absolute risk. In such cases, it is crucial to ensure the reduction of LDL-C. For our patient (Clinical Case 4 and Figure 4), we calculated that a reduction ofLDL-C to 1.6 mmol/l (below the target of 1.8 mmol/l), instead of the current reached level of 2.3 mmol/l (Clinical Case 4), should reduce the risk of mortality from 12% to 10% CV. This should also reduce the CV global risk (multiplied by three) from 36% to 30% (Figure 4). This means that if this target was not achieved in 100 patients, six (30-36%) patients would experience a CV event and two patients would die (10%-12%). This highlights the importance of achieving values below the target, even if it sometimes seems difficult.

#### *4.2.2. What to do if the target is not reached?*

In our patient's case (Clinical Case 5), we should try to obtain her compliance by reassuring her. If, in spite of this, the target is still not reached, we should adapt the treatment. This can be achieved by increasing the dosage of the statin (doubling each = 6% further LDL reduction) or replacing it with a more powerful statin. Another option is to combine the treatment with Ezetimibe (with an additional reduction of 20-25%) (Figure 3). The latter option is particularly useful when the observed reduction appears lower than expected, despite the patient's compliance with the treatment. Indeed, this suggests that the patient has a resistance to the statins. This usually affects all statins at all doses (the effect of each doubling statin dose does not allow the patient to achieve more than 4% or even 3%).

#### **Clinical Case 5. What do we do if the Target is not Reached?**

*Due to the disappointing result at the second visit, we ask the patient about her resistance to comply. The patient says that she is scared of the side effects of the medication and thus, she only takes half a pill and tends to forget to take her medication. We answer all of her questions. We put the patient's mind at ease and encourage her to correctly take her medicine. Thus, the patient finally complies. This is confirmed at her third visit. However, at this visit, the patient's LDL-C still remained above 1.8 mmol/l. This means that we should adapt the statin* (Table 1).

*Eight weeks later (visit 4, Table 1), the lipid profile of the patient becomes (Table 1):*

*Total cholesterol: 4.0 mmol/l; LDL-C : 1.6 mmol/l; HDL-C : 1.2 mmol/l; Triglycerides: 2.8 mmol/l.*

*The target of LDL - C (less than 1.8 mmol/l) has now been reached. However, elevated levels of triglycerides and low level of HDL – C still remain! Should we be concerned about them?*

#### *4.2.3. Reach the target« ASAP2 ».*

of the incidence of CV disease in 4-5 years. Every decrease of 1 mmol/l for LDL - C by a statin (Figure 4) was associated with a relative reduction in about 20% coronary events, stroke and heart deaths [7]. This relationship was universal (regardless of age, sex and other risk factors).

The absolute benefit produced by statins not only depends on their capacity to lower LDL-C but also, on the initial CV risk. Thus, the greater the initial CV risk, the greater the reduction of absolute risk. In such cases, it is crucial to ensure the reduction of LDL-C. For our patient (Clinical Case 4 and Figure 4), we calculated that a reduction ofLDL-C to 1.6 mmol/l (below the target of 1.8 mmol/l), instead of the current reached level of 2.3 mmol/l (Clinical Case 4), should reduce the risk of mortality from 12% to 10% CV. This should also reduce the CV global risk (multiplied by three) from 36% to 30% (Figure 4). This means that if this target was not achieved in 100 patients, six (30-36%) patients would experience a CV event and two patients would die (10%-12%). This highlights the importance of achieving values below the target,

In our patient's case (Clinical Case 5), we should try to obtain her compliance by reassuring her. If, in spite of this, the target is still not reached, we should adapt the treatment. This can be achieved by increasing the dosage of the statin (doubling each = 6% further LDL reduction) or replacing it with a more powerful statin. Another option is to combine the treatment with Ezetimibe (with an additional reduction of 20-25%) (Figure 3). The latter option is particularly useful when the observed reduction appears lower than expected, despite the patient's compliance with the treatment. Indeed, this suggests that the patient has a resistance to the statins. This usually affects all statins at all doses (the effect of each doubling statin dose does

*Due to the disappointing result at the second visit, we ask the patient about her resistance to comply. The patient says that she is scared of the side effects of the medication and thus, she only takes half a pill and tends to forget to take her medication. We answer all of her questions. We put the patient's mind at ease and encourage her to correctly take her medicine. Thus, the patient finally complies. This is confirmed at her third visit. However, at this visit, the*

*The target of LDL - C (less than 1.8 mmol/l) has now been reached. However, elevated levels of triglycerides and low*

*patient's LDL-C still remained above 1.8 mmol/l. This means that we should adapt the statin* (Table 1).

*Total cholesterol: 4.0 mmol/l; LDL-C : 1.6 mmol/l; HDL-C : 1.2 mmol/l; Triglycerides: 2.8 mmol/l.*

*Eight weeks later (visit 4, Table 1), the lipid profile of the patient becomes (Table 1):*

Furthermore, it was almost linear over the range of studied LDL-C levels.

even if it sometimes seems difficult.

88 Hypercholesterolemia

*4.2.2. What to do if the target is not reached?*

not allow the patient to achieve more than 4% or even 3%).

**Clinical Case 5. What do we do if the Target is not Reached?**

*level of HDL – C still remain! Should we be concerned about them?*

As mentioned above, the benefit of the LDL-C reduction can be seen within the first year treatment. The goal of "as quickly as possible" is all the more important in patients that are considered as high-risk. In a patient such as ours, there is a 15% risk of her dying from CVD in the next 10 years. Furthermore, there is a 45% (almost a "chance" on 2) risk of her having a global (fatal and non-fatal) CV event in the next 10 years. This means that there is a respective 1.5% risk and 4.5% (almost a "chance" on 20) risk of CV mortality and global CV risk per year. Although this may seem like a relatively small number, the delay in the necessary reduction of LDL-C for a year in 100 individuals (like our patient) would result in at least four to five CV events (including one death). However, in our practice, we should take note of the excuses given by the patient or by ourselves (anniversary cake, Christmas or Easter celebration, carnivals, holidays in all-inclusive hotels, etc., are a few days before blood sampling!). Such legions delay treatment adaption and obtaining satisfactory results. Taking this into consid‐ eration, it is clear that a more honest escalation in treatment would be more beneficial for the patient (Figure 6).

#### **5. Fourth question: Should we go beyond LDL?**

This leads us to three questions: (1) under statin therapy, does the patient still have a residual CV risk? (2) Is it necessary to target HDL-C and triglycerides? (3) Finally, is there scientific evidence that suggests further intervention would be beneficial? To answer these three questions, the guidelines respond affirmatively. Even after the reduction of LDL–C under the correct value target, a residual risk persists (between 60 and 80% of the initial risk). Part of this residual risk is attributed to the persistence of other alterations in a lipid profile.

Thus, if after the LDL-C correction, the patient still displays a high or very high-risk and has combined dyslipidaemia (triglycerides [TG] high > 1.7 mmol/l and HDL cholesterol [HDL - C] too low < 1.2 mmol/l for men or < 1.4 mmol/l for women), she may benefit from further improvement in her lipid profile.

The next question is: at this stage, how should a therapeutic target be set? Should we correct these levels of HDL-C and triglycerides or should we seek another target? In practice, it is often impossible to completely correct TG and HDL-C levels to achieve levels of 1.7 mmol/l for TG or 1.2 mmol/l in men and1.4 mmol/l in women for HDL-C. On the one hand, TG levels vary too much from one day to the next. On the other hand, HDL-C levels are difficult to increase. For example, a baseline HDL-C at 0.8 mmol/l gives a rise of 20%, equivalent to 0.16 mmol/l. This only allows the HDLL-C levels to reach 1 mmol/l - a difference barely perceptible, given the limited accuracy of laboratory measurements. Thus, the new guidelines recommend a more realistic target: the level of **non-HDL cholesterol** (non-HDL-C). This level is measured by a simple calculation:

In fact, as we can understand from the formula of Friedewald, this non-HDL-C is the sum [LDL-C + VLDL cholesterol]. The originality of this parameter is that it integrates all the potentially atherogenic lipoproteins, namely LDL and VLDL. These have a particularly high presence of low HDL-C and high TG (e.g., in the metabolic syndrome) [8].

The conditions and level of non-HDL-C targets are easy to deduct from the target levels of LDL–C. This is because they integrate the target value of LDL-C (< 1.8 or <2.5 mmol/l) plus the ideal value of VLDL-C (< 0.8 mmol/l; 0.8 is obtained by dividing the ideal 1.7 mmol/l level of TG by 2.2, see the Friedewald formula). Consequently, the level of non-HDL-C should be less than 2.5 mmol/l or 3.3 mmol/l as the risk is very high or high, respectively (Clinical Case 6).

#### **Clinical Case 6. Should we go Further in Correcting the Dyslipidaemia?**

*The presence of high levels of triglycerides and low levels of HDL –C signal the calculation of the level of***cholesterol non-HDL** *and the examination of the patient's residual risk*.

*At this stage, the calculated risk SCORE (with a total cholesterol of 4 mmol/l) is still very high. The SCORE is equal to 10.4%, taking into account the SCORE, HDL-C and family history, as well as the other risk factors such as TG, obesity, sedentariness and high hs-CRP (Table 1). Another way to calculate this risk is by considering the initial risk SCORE of 15% and removing the CV risk reduction produced by the LDL - C reduction (Figure 4). This leads to the same result.*

*Non-HDL cholesterol, which is equal to 2,8 mmol/l (tot chol – HDL-C = 4.0 – 1.2), should certainly be reduced below 2.5 mmol/l (= target for very high-risk). To do so, we can either strengthen the power of the statin or add ezetimibe to further reduce LDL–C. Another option is to add a fibrate or niacin to reduce TG (and LDL for niacin). Although these alternatives result in approximately the same reduction of the non - HDL cholesterol, they lead to different final lipid patterns (Table 1).*


A Practical Case-Based Approach to Dyslipidaemia in Light of the European Guidelines http://dx.doi.org/10.5772/59481 91


In fact, as we can understand from the formula of Friedewald, this non-HDL-C is the sum [LDL-C + VLDL cholesterol]. The originality of this parameter is that it integrates all the potentially atherogenic lipoproteins, namely LDL and VLDL. These have a particularly high

The conditions and level of non-HDL-C targets are easy to deduct from the target levels of LDL–C. This is because they integrate the target value of LDL-C (< 1.8 or <2.5 mmol/l) plus the ideal value of VLDL-C (< 0.8 mmol/l; 0.8 is obtained by dividing the ideal 1.7 mmol/l level of TG by 2.2, see the Friedewald formula). Consequently, the level of non-HDL-C should be less than 2.5 mmol/l or 3.3 mmol/l as the risk is very high or high, respectively (Clinical Case 6).

*The presence of high levels of triglycerides and low levels of HDL –C signal the calculation of the level of***cholesterol**

*At this stage, the calculated risk SCORE (with a total cholesterol of 4 mmol/l) is still very high. The SCORE is equal to 10.4%, taking into account the SCORE, HDL-C and family history, as well as the other risk factors such as TG, obesity, sedentariness and high hs-CRP (Table 1). Another way to calculate this risk is by considering the initial risk SCORE of 15% and removing the CV risk reduction produced by the LDL - C reduction (Figure 4). This leads to*

*Non-HDL cholesterol, which is equal to 2,8 mmol/l (tot chol – HDL-C = 4.0 – 1.2), should certainly be reduced below 2.5 mmol/l (= target for very high-risk). To do so, we can either strengthen the power of the statin or add ezetimibe to further reduce LDL–C. Another option is to add a fibrate or niacin to reduce TG (and LDL for niacin). Although these alternatives result in approximately the same reduction of the non - HDL cholesterol, they lead to different final*

Time line 0 2 months 6 months 10 months

LDL-C < 1,.8 mmol/l < 70 mg/dl

4,8 mmol/l (185 mg/dl)

**Visit 1 Visit 2 Visit 4 Visit 5 (2 alternatives)**

Second attempt to correct LDL-C


> 4,0 mmol/l (156 mg/dl)

Non HDL-C < 2.5 mmol/l (↓ LDL-C)

Same as visit 4 + ezetimibe 10 mg

> 3,6 mmol/l (140 mg/dl)

Alternative to ↓ non HDL-C < 2,5 mmol/l (↓ VLDL-C)

Same as visit 4 +fenofibrate 145 mg

> 3,7 mmol/l (141 mg/dl)

presence of low HDL-C and high TG (e.g., in the metabolic syndrome) [8].

**Clinical Case 6. Should we go Further in Correcting the Dyslipidaemia?**

**non-HDL** *and the examination of the patient's residual risk*.

Current treatment Baseline Simva 40 mg

(224 mg/dl)

Total cholesterol 5,8 mmol/l

*the same result.*

90 Hypercholesterolemia

*lipid patterns (Table 1).*

Therapeutic target(s)

The relative risk reduction (RRR) is calculated from the CTT regression (every LDL-C reduction of 1 mmol/l LDL-C leads to a 20% CVD risk reduction). \* The effect associated with the combination of statin and fibrate is expected to be greater than the effect of the LDL reduction. According to a recent study of diabetes (ACCORD), the addition of fibrate to statin in patients (including non-diabetic patients) with high TG and low HDL-C brings an additional 30% reduction in the risk of CVD. In our SCORE calculation, the smaller risk (8-9%) is due to the increase in HDL-C (the multiplier is 0.6 instead of 0.7) and the suppression of TG amongst the "other risk factors" (only obesity, sedentariness and hs-CRP remain, therefore, the multiplier for the other risk factor is 1.1 x 1.1 x 1.1 = 1.3 instead of 1.5). Simva: simvastatin; rosuva: rosuvastatin; atorva: atorvastatin.

**Table 1.** The evolution of the lipid profiles and CV risk in relation to our patient's increased treatment.

The reduction in the level of non-HDL-C should be achieved by an additional lowering of the LDL-C level. There are a number of ways to do this, including the prescription of a higher statin dosage, a stronger statin, a combination with ezetimibe (if intolerant) or by lowering the level of TG (and therefore, VLDL-C) via the association of the statin with fibrate or niacin (Clinical Case 6).

#### **6. Conclusions**

The new recommendations offer a practical approach. They are more precise in supporting the lipid profile of CV prevention. The four levels of risk and the possible adjustment of the new targets (non-HDL-C or apoB) next to the traditional targets of the LDL–C and HDL-C rate will allow better prescriptions of appropriate therapeutic drugs.

The present case illustrates step by step (visit by visit) the rationale for escalating treatment in order to achieve the best cardiovascular prevention. We hope that such an example can help give a better understanding of the EAS/ESC guidelines. The rigorous mathematical reasoning is, of course, only displayed here to better quantify the benefit of the various therapeutic choices. It is unlikely that it would be used in clinical practice. It is important to note that the practical implementation of guidelines requires the intuitive clinical skill of the practitioner, as well as open discussions with the patient. We would also like to highlight that, if the correction of the lipid profile is accepted as the cornerstone of CV prevention, the importance of lifestyle change (smoking, diet and physical activity) and the need to correct other risk factors should not be forgotten.

#### **Author details**

Olivier S. Descamps 1\*, Lale Tokgozoglu2 and Eric Bruckert3

\*Address all correspondence to: olivierdescamps@hotmail.com

1 Département de Médecine Interne, Centre de Recherche Médicale de Jolimont, Hôpital de Jolimont, Haine Saint-Paul, Belgium

2 Cardiology, Hacettepe University, Ankara, Turkey

3 Service d'endocrinologie-métabolisme, Hôpital de La Pitié Salpêtrière, Paris, France

#### **References**


[4] Descamps OS, et al. 'A Simple Multiplier to Calculate the Impact of HDL Cholesterol on Cardiovascular Risk Estimation Using SCORE'. Atherosclerosis (2012), http:// dx.doi.org/10.1016/j.atherosclerosis.2012.03.035.

**6. Conclusions**

92 Hypercholesterolemia

factors should not be forgotten.

Olivier S. Descamps 1\*, Lale Tokgozoglu2

Jolimont, Haine Saint-Paul, Belgium

[2] www.escardio.org/guidelines.

Cardiovasc Prev Rehabil 2009; 16: 304-314.

**Author details**

**References**

The new recommendations offer a practical approach. They are more precise in supporting the lipid profile of CV prevention. The four levels of risk and the possible adjustment of the new targets (non-HDL-C or apoB) next to the traditional targets of the LDL–C and HDL-C rate

The present case illustrates step by step (visit by visit) the rationale for escalating treatment in order to achieve the best cardiovascular prevention. We hope that such an example can help give a better understanding of the EAS/ESC guidelines. The rigorous mathematical reasoning is, of course, only displayed here to better quantify the benefit of the various therapeutic choices. It is unlikely that it would be used in clinical practice. It is important to note that the practical implementation of guidelines requires the intuitive clinical skill of the practitioner, as well as open discussions with the patient. We would also like to highlight that, if the correction of the lipid profile is accepted as the cornerstone of CV prevention, the importance of lifestyle change (smoking, diet and physical activity) and the need to correct other risk

and Eric Bruckert3

1 Département de Médecine Interne, Centre de Recherche Médicale de Jolimont, Hôpital de

[1] Graham I, Atar D, Borch-Johnsen K, Boysen G, Burell G, Cifkova R et al. 'European Guidelines on Cardiovascular Disease Prevention in Clinical Practice. Fourth Joint Task Force of the European Society on Cardiovascular Disease Prevention in Clinical

[3] Cooney MT, Dudina A, De Bacquer D, Fitzgerald A et al. 'How Much Does HDL Cholesterol Add to Risk Estimation? A report from the SCORE Investigators'. Eur J

3 Service d'endocrinologie-métabolisme, Hôpital de La Pitié Salpêtrière, Paris, France

Practice'. Eur J CardiovascPrevRehabil 2007; 14 (Suppl 2): S1-S112.

will allow better prescriptions of appropriate therapeutic drugs.

\*Address all correspondence to: olivierdescamps@hotmail.com

2 Cardiology, Hacettepe University, Ankara, Turkey


## **Hypercholesterolemia Effect on Potassium Channels**

Anna N. Bukiya and Avia Rosenhouse-Dantsker

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/59761

#### **1. Introduction**

Cholesterol is a major lipid component of the plasma membrane in mammalian cells consti‐ tuting up to 45 mol % with respect to other lipids [1, 2]. Yet, even a limited increase in blood and/or tissue cholesterol of up to 2-3 fold above the physiological level is cytotoxic [1-3] and is associated with the development of cardiovascular disease [4-6]. The underlying source for the effect of cholesterol on cellular functions is its ability to alter the function of multiple membrane proteins including ion channels (see, for example, reviews [7-9]).

In recent years, high cholesterol diet has been shown to affect the function of multiple ion channels. In this chapter we focus on the effect of dietary-induced increase in blood and tissue cholesterol levels on potassium channels. Potassium channels are among the largest and most complex types of ion channels. They are widely expressed in human tissues and are involved in many aspects of cell function including membrane excitability, regulation of heart rate, neuronal signaling, vascular tone, insulin release and salt flow across epithelia (see, for example, reviews [10-17]). In addition, they also play a critical role in the protection of neurons and muscle under metabolic stress. As a result, mutations in potassium channels lead to a wide range of disease in the brain (epilepsy, episodic ataxia), ear (deafness), heart (arrhythmia), muscle (myokymia, periodic paralysis), kidney (hypertension), pancreas (hyperinsulinemic hypoglycemia, neonatal diabetes). Therefore, the effect of hypercholesterolemia on potassium channel function has important pathophysiological implications.

The most common effect of a high-cholesterol diet on ion channels in general and potassium channels in particular is a decrease in channel activity. Yet, the activity of some channels is increased following a high cholesterol diet. For example, hypercholesterolemia suppressed the function of the Kir2 subfamily of inwardly rectifying potassium (Kir) channels in different cell types by ~2 fold [18-19]. However, atrial G-protein gated inwardly rectifying potassium channels (GIRK or Kir3) that underlie KACh currents in the heart are enhanced by a high-

© 2015 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and eproduction in any medium, provided the original work is properly cited.

cholesterol diet [19]. Several types of voltage gated (Kv) channels were sensitive to changes in the level of cellular membrane and dietary cholesterol [20-26]. The majority of the reports described suppression of channel function by high-cholesterol diet. Moreover, large conduc‐ tance calcium-activated potassium (BK) channels were often suppressed following a highcholesterol diet [27-31].

In this chapter, we will describe the implications of high-cholesterol dietary intake on members of three major families of potassium channels: voltage gated potassium (Kv) channels, calciumactivated potassium (KCa) channels of large conductance (BK) and inwardly rectifying potassium (Kir) channels. We will demonstrate that not only does high-cholesterol diet increase the levels of blood cholesterol but it also increases the level of cholesterol in tissues in which these types of channels are expressed. We will show that this cholesterol accumulation *in-vivo* is reflected in the function of potassium channels. Lastly, we will discuss the importance of the observed effects to organ function.

#### **2. High-cholesterol diet model**

Cholesterol-rich diet that is characteristic of Western societies critically controls blood lipid levels in several species, including humans [32-33]. Regression models have been reported for serum total cholesterol, triacylglycerol, and low-density-, high-density-, and very-lowdensity-lipoprotein cholesterol. In particular, correlations between increased levels of dietary cholesterol and these plasma lipids and lipoproteins were found to be 0.74, 0.65, 0.41, 0.14, and 0.34, respectively [32]. It has been predicted that compliance with the dietary recommendation to consume <300 mg cholesterol per day (with 30% of energy from fat, < 10% from saturated fat) will reduce plasma total and low-density-lipoprotein-cholesterol (LDL) concentrations by approximately 5% compared with amounts associated with the average American diet [32]. Restriction of dietary cholesterol intake represents a widely used preventative measure against numerous pathological conditions since increased total cholesterol and LDL levels are wellrecognized risk factors for several largely prevalent pathologies, including stroke [34-37], coronary heart disease [38-39], vascular dementia [40], and atherosclerosis [41]. Therefore, it is not surprising that a cholesterol-rich diet has been recreated in a research laboratory setting to study the deleterious effects of cholesterol-rich food intake.

High-cholesterol diet-induced hypercholesterolemia is widely used for studies on monkeys [42], hamsters [43], guinea pigs [44], rabbits [20, 27, 29, 45, 46], rats [19, 47-49] and mice [50]. The dietary-induced hypercholesterolemia model has several advantages. First, it mimics the high-cholesterol food intake that is characteristic to the US population, and which impacts cholesterol levels in the blood of human individuals [32-33]. Second, it does not require alteration of the genetic background of the animal. These advantages make high-cholesterol diet a useful tool to manipulate cholesterol levels in species in which genetic alterations to achieve hypercholesterolemia are challenging. For example, diet-induced changes in blood cholesterol level have been detected in primates besides humans, such as baboons *Papiospp* (reviewed by [51]) and Japanese monkeys *Macacafuscata* [42].

It should be noted that earlier studies have documented the existence of "hyper-" and "hyporesponders" to a cholesterol-rich diet in the human population. Trials with the same subjects demonstrated that the human population includes people with a consistently low or high response to increased dietary intake of cholesterol (reviewed by [52]). Similarly to humans, other primates also vary in their blood lipid responses to dietary lipid composition. Selective breeding of primates based on their individual responses to the composition of the diet resulted in lines that are characterized by low versus high responses to changes in dietary lipids. Thus, similarly to humans, changes in lipoprotein patterns in response to dietary cholesterol seem to be heritable in primates (reviewed by [51]). Further studies have shown that the differential response to cholesterol consumption in smaller laboratory animals also results in inbred strains of rabbits, rats, and mice that differ in their sensitivity to highcholesterol diet. Their responsiveness to high-cholesterol diet is largely influenced by the genetic background [52]. Compared to humans, changes in blood cholesterol and LDL levels induced by high-cholesterol diet in lab animals are robust and of high magnitude. Therefore, a high-cholesterol diet represents a useful and practical tool to induce an increase in blood cholesterol levels that ultimately leads to hypercholesterolemia in animal models.

cholesterol diet [19]. Several types of voltage gated (Kv) channels were sensitive to changes in the level of cellular membrane and dietary cholesterol [20-26]. The majority of the reports described suppression of channel function by high-cholesterol diet. Moreover, large conduc‐ tance calcium-activated potassium (BK) channels were often suppressed following a high-

In this chapter, we will describe the implications of high-cholesterol dietary intake on members of three major families of potassium channels: voltage gated potassium (Kv) channels, calciumactivated potassium (KCa) channels of large conductance (BK) and inwardly rectifying potassium (Kir) channels. We will demonstrate that not only does high-cholesterol diet increase the levels of blood cholesterol but it also increases the level of cholesterol in tissues in which these types of channels are expressed. We will show that this cholesterol accumulation *in-vivo* is reflected in the function of potassium channels. Lastly, we will discuss the importance

Cholesterol-rich diet that is characteristic of Western societies critically controls blood lipid levels in several species, including humans [32-33]. Regression models have been reported for serum total cholesterol, triacylglycerol, and low-density-, high-density-, and very-lowdensity-lipoprotein cholesterol. In particular, correlations between increased levels of dietary cholesterol and these plasma lipids and lipoproteins were found to be 0.74, 0.65, 0.41, 0.14, and 0.34, respectively [32]. It has been predicted that compliance with the dietary recommendation to consume <300 mg cholesterol per day (with 30% of energy from fat, < 10% from saturated fat) will reduce plasma total and low-density-lipoprotein-cholesterol (LDL) concentrations by approximately 5% compared with amounts associated with the average American diet [32]. Restriction of dietary cholesterol intake represents a widely used preventative measure against numerous pathological conditions since increased total cholesterol and LDL levels are wellrecognized risk factors for several largely prevalent pathologies, including stroke [34-37], coronary heart disease [38-39], vascular dementia [40], and atherosclerosis [41]. Therefore, it is not surprising that a cholesterol-rich diet has been recreated in a research laboratory setting

High-cholesterol diet-induced hypercholesterolemia is widely used for studies on monkeys [42], hamsters [43], guinea pigs [44], rabbits [20, 27, 29, 45, 46], rats [19, 47-49] and mice [50]. The dietary-induced hypercholesterolemia model has several advantages. First, it mimics the high-cholesterol food intake that is characteristic to the US population, and which impacts cholesterol levels in the blood of human individuals [32-33]. Second, it does not require alteration of the genetic background of the animal. These advantages make high-cholesterol diet a useful tool to manipulate cholesterol levels in species in which genetic alterations to achieve hypercholesterolemia are challenging. For example, diet-induced changes in blood cholesterol level have been detected in primates besides humans, such as baboons *Papiospp*

cholesterol diet [27-31].

96 Hypercholesterolemia

of the observed effects to organ function.

**2. High-cholesterol diet model**

to study the deleterious effects of cholesterol-rich food intake.

(reviewed by [51]) and Japanese monkeys *Macacafuscata* [42].

In a typical protocol for a high-cholesterol diet in animal models, the animal would be fed (*ad libitum* of via gavage) cholesterol-rich food. The ability of a high-cholesterol diet to increase blood cholesterol and LDL levels is well documented in various animal models. For instance, consumption of food containing 2% cholesterol for 20 months leads to up to a 2-fold increase in total cholesterol and LDL levels in the blood of a macaque [42]. An even more drastic increase was documented multiple times in rabbits: feeding animals with dietary cholesterol supple‐ mentation in the amount of 1 g per day for 4 weeks resulted in ~20-fold increase in total serum cholesterol concentration in rabbits [29], 1% cholesterol food for 8 weeks resulted in a nearly 30-fold increase in total serum cholesterol level [20], and 0.5% cholesterol for 12 weeks raised blood cholesterol level by nearly 33-fold [45]. Another study reported a 10-fold increase in plasma cholesterol concentration in rabbits fed by 0.5% cholesterol food for 16 weeks [27]. Mice and rats usually respond with a lower magnitude of increase in blood cholesterol and LDL levels in response to diet, yet these changes are still easily detected. In mice, consumption of 1, 000 mg/kg cholesterol per day for 8 weeks increased blood cholesterol and LDL levels by 2-3 times [50]. Significant increase in blood cholesterol and LDL levels have been documented following feeding with 4% cholesterol food for 4 weeks in Sprague-Dawley rats [48], 2% cholesterol diet for 20-24 weeks in the same strain [19], and 5% cholesterol diet for 3 weeks led to a significant increase in the plasma cholesterol level in Wistar rats [47].

In our rat model of a high-cholesterol diet, male Sprague-Dawley rats (50 g) were subjected to an *ab libitum* high-cholesterol diet (2% cholesterol). The total cholesterol plasma level remained unchanged for 11-12 weeks of diet, but significantly increased during weeks 18-23 and was increased even further during weeks 27-28 of the high-cholesterol diet compared to the control group (Figure 1A). After receiving a high-cholesterol diet for 11-12 weeks, the rats displayed a significant increase in the LDL plasma level, with the increase becoming more pronounced during weeks 18-23 on a high-cholesterol diet and reaching a nearly 5-fold increase during weeks 27-28 on a high-cholesterol diet when compared to the control group (Figure 1B). Highdensity-lipoproteins (HDL), however, seemed to be less sensitive to high-cholesterol diet intake, as HDL level increased significantly only during weeks 27-28 on a high-cholesterol diet (Figure 1C). Triglycerides followed the pattern of response of cholesterol and LDL: triglyceride level increased significantly during weeks 18-23 and became even higher during weeks 27-28 of the high-cholesterol diet (Figure 1D). Overall, changes in the blood lipid profile correlated with the duration of the high-cholesterol diet. Therefore, a high-cholesterol diet constitutes a valuable tool when the degree of change in the blood lipid profile needs to be controlled tightly.

**Figure 1. Blood lipid profile in Sprague-Dawley rats fed high-cholesterol diet**. (A) Serum total cholesterol. (B) Lowdensity-lipoprotein-cholesterol. (C) High-density-lipoprotein. (D) Triglycerides. Here and in all figures: significantly different from control is indicated by an asterisk (\*, p<0.05; \*\*, p<0.01).

#### **3. Effect of high-cholesterol diet on potassium channels.**

In view of the advantages of a high cholesterol diet described above and its ability to ade‐ quately represent the characteristics of high cholesterol food intake in the US, it is widely used for studies on ion channel function during dyslipidemia and hypercholesterolemia. For instance, dietary-induced hypercholesterolemia was shown to up-regulate the function of Ltype Ca2+-channels in detrusor smooth muscle [48], transient receptor potential channels 5 and 6 (TRPC5 and TRPC6) in aortic endothelial cells [53], cardiac G protein gated inwardly rectifying potassium channels [19], and epithelial Na+ channels (eNaC) [54].

density-lipoproteins (HDL), however, seemed to be less sensitive to high-cholesterol diet intake, as HDL level increased significantly only during weeks 27-28 on a high-cholesterol diet (Figure 1C). Triglycerides followed the pattern of response of cholesterol and LDL: triglyceride level increased significantly during weeks 18-23 and became even higher during weeks 27-28 of the high-cholesterol diet (Figure 1D). Overall, changes in the blood lipid profile correlated with the duration of the high-cholesterol diet. Therefore, a high-cholesterol diet constitutes a valuable tool when the degree of change in the blood lipid profile needs to be controlled tightly.

98 Hypercholesterolemia

**Figure 1. Blood lipid profile in Sprague-Dawley rats fed high-cholesterol diet**. (A) Serum total cholesterol. (B) Lowdensity-lipoprotein-cholesterol. (C) High-density-lipoprotein. (D) Triglycerides. Here and in all figures: significantly

In view of the advantages of a high cholesterol diet described above and its ability to ade‐ quately represent the characteristics of high cholesterol food intake in the US, it is widely used

different from control is indicated by an asterisk (\*, p<0.05; \*\*, p<0.01).

**3. Effect of high-cholesterol diet on potassium channels.**

In this chapter, we will focus on high-cholesterol diet-driven changes in the function of potassium channels. In particular, we will discuss the effect of an increase in the dietary intake of cholesterol on voltage gated (Kv) channels, calcium activated potassium channels (KCa) and inwardly rectifying potassium channels (Kir).

**Voltage-gated potassium (KV) channels**. Several reports describing the effect of a highcholesterol diet on potassium channel function came from studies on voltage-gated potassium channels (KV) [21]. These channels are transmembrane proteins that are located on the plasma membrane and sensitive to changes in the transmembrane potential [55]. Upon membrane depolarization, voltage-gated potassium channels conduct outward potassium currents as these channels exhibit the highest selectivity for K+ ions compared to other monovalent cations. Activation of KV channels usually results in decreased depolarization and the return of the plasma membrane potential to the resting level. Vertebrate KV channels are tetramers of four pore-forming subunits, each contributing to the wall of the K+ conducting pore. Each poreforming subunit is composed of six transmembrane α-helices with intracellular N- and Ctermini (Figure 2A). Helices S1-S4 contribute to voltage-sensing and S5-S6 form the pore region. Voltage-gated potassium channels play a key role in cellular excitability including vascular smooth muscle [55]. The effect of a high-cholesterol diet on KV channel function has been extensively studied in the cardiovascular system.

High-cholesterol diet failed to modulate KV channel function after a brief placement of an Ossabaw miniature swine model on a high-fat/high-cholesterol/high-fructose diet [26]. The animals were fed by the diet for 9 weeks. The authors considered the duration of diet admin‐ istration to be relatively short. As a result, only an early stage of a complex metabolic syndrome developed. The diet caused ~4-fold increase in blood cholesterol level in the group on diet compared to the control group on standard chow. Coronary arterioles from both groups were isolated and pressurized to 60 cmH2O for *in-vitro* pharmacological studies. Those include the assessment of the KV channel contribution to the arteriolar response to the vasodilator 2 chloroadenosine. Pharmacological blockade of KV channels by 4-aminopyridine reduced the arteriolar sensitivity to 2-chloroadenosine in both control and early stage metabolic syndrome groups. This result suggests that Kv channel regulation of the arteriolar diameter was still preserved at the early stage of the modeled metabolic syndrome. In contrast, blockade of Kir6 (KATP) channels (see below) with glibenclamide reduced the arteriolar sensitivity to 2-chlor‐ oadenosine in the control group only. Therefore, the involvement of KV channels in the arteriolar responses to 2-chloroadenosine is resistant to the described diet.

A more complex scenario was observed in work by Heaps *et al* on a Yucatan miniature swine model that was placed on high-fat/high-cholesterol diet for 20 weeks [22-23]. Already after 4 weeks, blood cholesterol and LDL levels were increased on the average by 6 times when compared to the pre-diet and to the group on control diet levels. At the end of a 20 week-long

**Figure 2. Schematic structures of Kv, KCa, and Kir channels.** (A) Schematic structure of voltage-gated and voltage-/ calcium gated potassium channel subunits. TM: transmembrane domain of b subunit; S: transmembrane domain of a subunit; RCK: regulator of conductance of potassium; EXT: extracellular media; INT: intracellular media. (B) Schematic structure of inwardly rectifying potassium channel subunit. TM1 is the outer transmembrane helix and TM2 is the in‐ ner transmembrane helix.

diet, *coronary vascular reactivity* was assessed. Coronary microvessels were removed, cannu‐ lated, pressurized at 40 mmHg and the luminal diameter was monitored. In this experimental setting, 4-aminopyridine attenuated adenosine-induced dilation of coronary arterioles in the control group, but did not affect adenosine-induced arteriole dilation in the group subjected to high-fat/high-cholesterol diet. Therefore, adenosine activation of KV channels is attenuated during hypercholesterolemia. In contrast, Bender *et al* did not detect diet-induced changes in KV channel contribution to the 2-chloroadenosine-induced dilation of swine coronary arterio‐ les [26]. This controversy may be attributed to several differences between the two studies. For instance, in the study by Bender et al., animals were subjected to a high-fructose diet in addition to high-fat/high-cholesterol food. Second, Bender *et al* were studying the consequences of brief (only 9 weeks) dieting as opposed to Heaps *et al* who placed the animals on a 20 weeks diet. It is therefore likely that a longer duration of diet is needed for KV channels to lose their sensitivity to adenosine application. Loss of KV channel contribution to adenosine-mediated vasodilation was studied further at the level of coronary arteriole smooth muscle KV currents. Outward K+ currents were recorded in whole-cell configuration from freshly isolated arteriole myocytes. Noteworthy, the intracellular calcium was chelated to eliminate the calciumdependent component from the whole-cell K+ currents. KV currents from myocytes in the highcholesterol diet group were significantly smaller compared to the control [22-23]. Therefore, reduction in K+ currents may underlie the attenuation of the KV component in adenosineinduced arteriole dilations. Treatment of membrane patches with different concentrations of the non-selective potassium channel blocker tetraethylammonium (TEA) revealed that highcholesterol diet primarily altered KV channel isoforms with high sensitivity to TEA. RT-PCR experiments determined that KV3.1 and KV3.3 channel isoforms were expressed in coronary arterioles. However, expression levels of KV3.1 and KV3.3 were not changed by the diet. In contrast, it was shown that arteriole dilation caused by the receptor-independent activator of adenylyl cyclaseforscolin was abolished in the high-fat/high-cholesterol diet group. Further‐ more, the KV channel blocker 4-aminopyridine and the non-selective blocker of potassium channels TEA significantly attenuated forscolin-mediated vasodilation in control, but not in the high-fat/high-cholesterol diet group. Taken together, these data suggest that hypercholes‐ terolemia-mediated ablation of adenosine-induced vasodilation of coronary arterioles could be attributed to the impairment of the adenylyl cyclase pathway coupled to highly TEAsensitive KV channel isoforms.

These data showing a reduced KV component in the whole-cell outward potassium current are consistent with another report that focused on potassium currents in swine coronary artery smooth muscle cells [25]. The animals were placed on a high-fat diet for 20 weeks. The diet significantly increased the total blood serum cholesterol level and triglycerides. Remarkably, the increase in both blood lipid components was higher in female swines. 4-aminopyridinsensitive component of the whole-cell outward potassium current recorded from the isolated coronary artery smooth muscle cells was significantly diminished in the high-cholesterol diet group of male swines. In females, however, no significant reduction in KV 4-aminopyridinesensitive (KV component) was detected [25]. This report suggests that the effect of highcholesterol diet on the function of KV channels may be gender-specific.

diet, *coronary vascular reactivity* was assessed. Coronary microvessels were removed, cannu‐ lated, pressurized at 40 mmHg and the luminal diameter was monitored. In this experimental setting, 4-aminopyridine attenuated adenosine-induced dilation of coronary arterioles in the control group, but did not affect adenosine-induced arteriole dilation in the group subjected to high-fat/high-cholesterol diet. Therefore, adenosine activation of KV channels is attenuated during hypercholesterolemia. In contrast, Bender *et al* did not detect diet-induced changes in KV channel contribution to the 2-chloroadenosine-induced dilation of swine coronary arterio‐ les [26]. This controversy may be attributed to several differences between the two studies. For instance, in the study by Bender et al., animals were subjected to a high-fructose diet in addition to high-fat/high-cholesterol food. Second, Bender *et al* were studying the consequences of brief (only 9 weeks) dieting as opposed to Heaps *et al* who placed the animals on a 20 weeks diet. It is therefore likely that a longer duration of diet is needed for KV channels to lose their sensitivity to adenosine application. Loss of KV channel contribution to adenosine-mediated

ner transmembrane helix.

100 Hypercholesterolemia

**Figure 2. Schematic structures of Kv, KCa, and Kir channels.** (A) Schematic structure of voltage-gated and voltage-/ calcium gated potassium channel subunits. TM: transmembrane domain of b subunit; S: transmembrane domain of a subunit; RCK: regulator of conductance of potassium; EXT: extracellular media; INT: intracellular media. (B) Schematic structure of inwardly rectifying potassium channel subunit. TM1 is the outer transmembrane helix and TM2 is the in‐

> Loss of KV channel current and its contribution to vasodilatory responses has not only been documented in coronary arteries, but also in the *middle cerebral artery.* In the latter work, New Zealand rabbits were placed on chow supplemented with 1% cholesterol for 8 weeks [20]. The total blood serum cholesterol increased close to 30 times at the end of the diet. In the vasodi‐ latory response study, the middle cerebral arteries were dissected, mounted and pre-contract‐ ed with a high-K+ (50 mM) physiologic saline solution. In the pre-contracted arteries isolated from control animals, acetylcholine produced artery relaxation. In the arteries from the highcholesterol diet group, similar concentrations of acetylcholine induced less relaxation. In the control group, neither TBA, an inhibitor of KCa channels, nor glibenclamide, an inhibitor of KATP channels, significantly affected the concentration-response to acetylcholine, whereas 4 aminopyridine, a blocker of KV channels, strongly inhibited this relaxation. In the highcholesterol diet group, TBA, glibenclamide or 4-aminopyridine did not significantly affect the response to acetylcholine [20]. Loss of 4-aminopyridine effect on acetylcholine-induced

cerebral artery dilation suggests that either the function of KV channels or their sensitivity to acetylcholine-activated pathway is diminished.

Apart from the vascular system, the effect of a high-cholesterol diet on KV channels was studied in the *heart* itself [24]. Rabbits were subjected to a high-cholesterol diet (1.5% cholesterol) for 8 weeks. The diet resulted in an approximately 14 times increase in blood serum cholesterol level. RT-PCR studies were used to determine the impact of the high-cholesterol diet on the left ventricular mRNA expression level of several potassium channels that play a key role in the contractility of the heart. High-cholesterol diet significantly decreased the expression of KV4.2, but did not alter the level of KV4.3. Notably, a significant decrease and a significant increase in mRNA levels were detected for KV11.1 (ERG1) and KV7.1 (KVLQT1) channels, respectively [24]. These data document that that a high-cholesterol diet differentially affects the expression of voltage-gated potassium channels, with decrease, increase and "no-effect" being detected for this group of channels. The authors suggested that the arrhythmogenic nature of the hypercholesterolemia may be mediated by changed expression levels of the examined channels.

**Calcium-activated potassium (KCa) channels**. The effect of a high-cholesterol diet on calciumsensitive potassium channels (KCa) is well described for calcium- and voltage-gated potassium channels of large conductance (BK). Fully functional BK channels result from the tetrameric association of 125-140 kDa polypeptides termed α, slo or slo1 subunits. Slo1 subunits share significant homology with KV channels of the six transmembrane domain (TM6) family, with the S1-S4 regions contributing to voltage-sensing and the S5-S6 helices forming the activation gate-pore region (Figure 2A) [12, 56-57]. Unlike purely voltage-gated KV channels, in addition to the S1-S6 core, BK slo1 subunits contain an additional transmembrane helix S0 leading to an extracellular N-terminus [58] and a large cytosolic tail domain (CTD) [59-61]. The CTD includes two domains that Regulate the Conductance of K+ (RCK). RCKs contain sites for sensing intracellular Ca2+ levels and allow BK channels to increase channel activity in response to an increase in intracellular Ca2+ physiological levels [59-60]. In most tissues, BK channels usually result from the association of slo1 proteins with auxiliary β subunits. Four types of BK β subunits, β1-4, have been identified. BK β subunits share an overall topology of two TM segments joined by a large extracellular loop, plus two short intracellular N and C termini [62]. Expression of β subunits is tissue-specific, with the β1 subunit being prevalent in smooth muscle, including vasculature [62-63]. The functional role of BK channels is similar to KV channels: upon membrane depolarization BK channels generate outward potassium currents to oppose depolarization and favor the return of the transmembrane voltage to its resting level.

Several animal models have been used to document that plasmalemma BK channels are sensitive to dietary-induced hypercholesterolemia [64]. Considering the ample evidence linking hypercholesterolemia to cardiovascular disease, and the key role of BK channels in regulating vascular tone [65], most of the studies describe the effect of a high-cholesterol diet on BK channel function in the vascular system. One of the early studies addressed changes in endothelium-dependent and independent components *in-vitro* relaxation of carotid artery rings obtained from rabbits following administration of a 12 week-long high-cholesterol diet (0.5% cholesterol). Evaluation of isometric tension in carotid artery rings was performed in the presence of nitric oxide (NO), sodium nitroprusside and 8-bromoguanosine 3′, 5′-cyclic monophosphate (8-Brc-GMP). The responses to all three chemicals remained unaltered by dietary-induced hypercholesterolemia [45]. After application of a BK channel blocker, NOmediated artery relaxation was significantly reduced in the hypercholesterolemic group. These findings could imply that BK channel contribution to the regulation of arterial tension during hypercholesterolemia is enhanced. It has been repeatedly suggested that dietary-induced hypercholesterolemia may lead to a compensatory increase in BK channel activity [45, 66]. Considering that smooth muscle BK channels are critical regulators of the arterial diameter, the compensatory increase in BK channel function during the course of high-cholesterol diet could take place in the arterial smooth muscle [45, 66]. Alternatively, BK channel activity could be reduced by hypercholesterolemia, in which case BK channels would be more available for activation [64], and would contribute more to an NO-induced decrease in vascular tension. However, the latter scenario is unlikely as an increase in vascular smooth muscle BK channel activity has been reported in cell-attached recordings from the area affected by the athero‐ sclerotic plaque formation [67]. This difference is lost when BK channel function is evaluated in cell-free patches excised from the cells [67]. Thus, increased smooth muscle BK channel activity associated with atherosclerotic plaque likely requires the involvement of the intracel‐ lular organelles and/or freely diffusible cytosolic signals.

cerebral artery dilation suggests that either the function of KV channels or their sensitivity to

Apart from the vascular system, the effect of a high-cholesterol diet on KV channels was studied in the *heart* itself [24]. Rabbits were subjected to a high-cholesterol diet (1.5% cholesterol) for 8 weeks. The diet resulted in an approximately 14 times increase in blood serum cholesterol level. RT-PCR studies were used to determine the impact of the high-cholesterol diet on the left ventricular mRNA expression level of several potassium channels that play a key role in the contractility of the heart. High-cholesterol diet significantly decreased the expression of KV4.2, but did not alter the level of KV4.3. Notably, a significant decrease and a significant increase in mRNA levels were detected for KV11.1 (ERG1) and KV7.1 (KVLQT1) channels, respectively [24]. These data document that that a high-cholesterol diet differentially affects the expression of voltage-gated potassium channels, with decrease, increase and "no-effect" being detected for this group of channels. The authors suggested that the arrhythmogenic nature of the hypercholesterolemia may be mediated by changed expression levels of the

**Calcium-activated potassium (KCa) channels**. The effect of a high-cholesterol diet on calciumsensitive potassium channels (KCa) is well described for calcium- and voltage-gated potassium channels of large conductance (BK). Fully functional BK channels result from the tetrameric association of 125-140 kDa polypeptides termed α, slo or slo1 subunits. Slo1 subunits share significant homology with KV channels of the six transmembrane domain (TM6) family, with the S1-S4 regions contributing to voltage-sensing and the S5-S6 helices forming the activation gate-pore region (Figure 2A) [12, 56-57]. Unlike purely voltage-gated KV channels, in addition to the S1-S6 core, BK slo1 subunits contain an additional transmembrane helix S0 leading to an extracellular N-terminus [58] and a large cytosolic tail domain (CTD) [59-61]. The CTD

sensing intracellular Ca2+ levels and allow BK channels to increase channel activity in response to an increase in intracellular Ca2+ physiological levels [59-60]. In most tissues, BK channels usually result from the association of slo1 proteins with auxiliary β subunits. Four types of BK β subunits, β1-4, have been identified. BK β subunits share an overall topology of two TM segments joined by a large extracellular loop, plus two short intracellular N and C termini [62]. Expression of β subunits is tissue-specific, with the β1 subunit being prevalent in smooth muscle, including vasculature [62-63]. The functional role of BK channels is similar to KV channels: upon membrane depolarization BK channels generate outward potassium currents to oppose depolarization and favor the return of the transmembrane voltage to its resting level.

Several animal models have been used to document that plasmalemma BK channels are sensitive to dietary-induced hypercholesterolemia [64]. Considering the ample evidence linking hypercholesterolemia to cardiovascular disease, and the key role of BK channels in regulating vascular tone [65], most of the studies describe the effect of a high-cholesterol diet on BK channel function in the vascular system. One of the early studies addressed changes in endothelium-dependent and independent components *in-vitro* relaxation of carotid artery rings obtained from rabbits following administration of a 12 week-long high-cholesterol diet (0.5% cholesterol). Evaluation of isometric tension in carotid artery rings was performed in the

(RCK). RCKs contain sites for

includes two domains that Regulate the Conductance of K+

acetylcholine-activated pathway is diminished.

examined channels.

102 Hypercholesterolemia

Hypercholesterolemia-driven increase in arterial KCa channel function compared to control chow was also reported in diabetic pigs receiving high-fat/high-cholesterol (2%) diet [21]. Patch-clamp recording of whole-cell outward potassium currents revealed increased density of the KCa-component in the high-cholesterol diet group. Western blot failed to detect a significant increase in the amount of the KCa pore-forming protein. In addition, intracellular calcium concentration did not differ in control versus high-cholesterol diet groups. The data indicated that diabetic hypercholesterolemia leads to an increased functional coupling between KCa and intracellular calcium release.

In contrast to the above *in-vitro* studies, *in-vivo* work conducted on hypercholesterolemic rabbits showed a reduction in NO-induced vasodilation as determined by monitoring the hindlimb vascular conductance in response to acetylcholine and bradykinin [27]. In this set of experiments, rabbits were fed high-cholesterol (0.5% cholesterol) diet for 16 weeks. The NOindependent vasodilation in response to acetylcholine and bradykinin, however, was larger in animals on high-cholesterol diet. Development of this NO-independent component of vasodilation was blocked by either TEA or by a mixture of the KCa channel blocker charybdo‐ toxin and the small conductance KCa channels blocker apamin. Thus, the authors concluded that hypercholesterolemia impaired KCa channel-mediated vasodilation [27].

Another study using hypercholesterolemic rabbits to test acetylcholine-induced vasorelaxa‐ tion focused on renal artery. Rabbits were subjected to a high-cholesterol diet (0.5% cholesterol) for 5 weeks. This diet resulted in an over 50 fold increase in the total blood cholesterol and an almost 40-fold increase in LDL-cholesterol [28]. Contrary to the findings in the hindlimb circulation, acetylcholine-induced dilation of phenylephrine pre-constricted renal arteries was not changed by the high-cholesterol diet. However, the NO-independent (N(G)-nitro-larginine-resistant) component of this relaxation was significantly enhanced in arteries from hypercholesterolemic animals. This component totally vanished after endothelial removal in both control and hypercholesterolemic groups, yet was only reduced significantly in the hypercholesterolemic group when an artery with endothelium was incubated in BK and the intermediate conductance KCa channel blocker charybdotoxin [28].

Studies on rat cerebral arteries yielded results that are in agreement with the conclusions obtained in the hindlimb of rabbits. Specifically, rat middle cerebral arteries obtained from a Sprague-Dawley strain on control versus high-cholesterol (2% cholesterol supplement for either 10 or 18-23 weeks) were dissected, de-endothelialized, cannulated and pressurized at 60 mm Hg. A blood lipid profile revealed a significant increase in the total serum cholesterol, LDL, and triglyceride levels only at 18-23 weeks of diet (Figure 1). The arterial responses to a depolarizing solution containing 60 mM KCl were similar in control and in all hypercholes‐ terolemic arteries. However, treatment of arteries from either one of the high-cholesterol diet groups with the selective BK channel blocker paxilline resulted in vasoconstriction that was significantly smaller compare to the control group (Figure 3A). BK channel function seemed to be altered rather selectively: arterial diameter responses to the KV channel blocker 4 aminopyridine were similar in control versus hypercholesterolemic animals (Figure 3A). First, these results demonstrated that the general contractile capability of the artery (as tested by a high KCl-containing solution) was largely preserved during the high-cholesterol diet. Second, endothelium-independent vasodilation that is mediated by the activity of smooth muscle BK channels was diminished during hypercholesterolemia [30, 31]. Moreover, the fact that reduced sensitivity to paxilline was observed after 10 weeks on a high-cholesterol diet, well before the changes in the blood lipid profile took effect (Figure 1) suggested that BK channels were highly sensitive to dietary cholesterol levels, independent of the increase in blood cholesterol.

Further experimentation took place in an effort to unveil molecular mechanisms that enable the sensitivity of BK channels to dietary cholesterol. First, it was shown that cholesterol accumulation in the wall of de-endothelialized cerebral arteries of hypercholesterolemic rats followed the pattern of increase in blood cholesterol level. In particular, the cholesterol level in cerebral artery tissue was only increased significantly during weeks 18-23 on diet, but not earlier (Figure 3B) [31]. Therefore, the direct accumulation of cholesterol in the vicinity of the BK channel might not be the sole reason for depressed BK channel sensitivity to paxilline during a high-cholesterol intake. The reduction in paxilline-induced cerebral artery constric‐ tion by hypercholesterolemia might result from a decreased number of BK channels in arterial smooth muscle. In particular, hypercholesterolemia may down-regulate accessory, smooth muscle-type β subunit (β1) (Figure 2A). Indeed, cerebral arteries of β1 (*KCNMB1)* knock-out mice were reported to be insensitive to selective BK channel block by the peptide blocker iberiotoxin [68]. Moreover, hypercholesterolemia-induced changes in BK β1 subunit level have been studied in circular smooth muscle strips from the sphincter of Oddi in rabbits fed by highcholesterol food (1 g cholesterol per day) for 4 weeks [29]. Immunohistochemical and Western blot protein analysis using a BK β1 subunit-specific polyclonal antibody showed a decreased level of the BK β1 protein in the cholesterol-fed group. Thus, in the sphincter of Oddi highcholesterol diet down-regulated BK β1 subunits [29]. However, in rat cerebral artery myocytes high-cholesterol diet did not down-regulate, but actually significantly increased the fluores‐ cent signal associated with selective labeling of BK β1 subunits (Figure 3C, left panel). Since β1 subunits themselves do not form functional channels, diminished responses of the artery to paxilline may be indicative of the reduction in the amount of the BK pore-forming (α) subunit protein. However, no significant differences between the BK α subunit-associated fluorescence signal in cerebral artery myocytes from rats on control versus high-cholesterol diet were detected (Figure 3C, right panel). Therefore, ablated paxilline-induced cerebral artery con‐ striction observed in pressurized cerebral arteries from hypercholesterolemic rats could not be attributed to down-regulation of BK protein surface presence on myocyte membranes. Alternatively, ablated paxilline-induced cerebral artery constriction may arise from diminish‐ ed functional properties of the BK channel itself. These may include but are not limited to the coupling (both, physical association and functional communication) between the BK poreforming α subunit and the accessory β1 subunits, changes in the gating pattern of the channel making the protein less responsive to paxilline binding. Alternatively, reduction in paxillineinduced cerebral artery constriction may not be a reflection of decreased BK channel function but rather be explained by changes in the affinity of the channel to paxilline. The latter possibility needs to be further studied as several BK channel blockers other than paxilline block BK channel with different efficacy depending on the presence of the accessory β1 and β4 subunits [69]. Theoretically, an increased amount of the BK β1 protein by high-cholesterol diet may preclude paxilline from reaching its site in the BK α subunit and therefore, may result in a decreased paxilline effect. The most unequivocal evidence of the effect of a high-cholesterol diet on BK channel function must come from direct electrophysiological evaluation of BK voltage- and calcium-dependent gating following a high-cholesterol diet. The intrinsic mechanism(s) that underlie(s) alteration in BK channel function during a high-cholesterol diet is currently under investigation.

hypercholesterolemic animals. This component totally vanished after endothelial removal in both control and hypercholesterolemic groups, yet was only reduced significantly in the hypercholesterolemic group when an artery with endothelium was incubated in BK and the

Studies on rat cerebral arteries yielded results that are in agreement with the conclusions obtained in the hindlimb of rabbits. Specifically, rat middle cerebral arteries obtained from a Sprague-Dawley strain on control versus high-cholesterol (2% cholesterol supplement for either 10 or 18-23 weeks) were dissected, de-endothelialized, cannulated and pressurized at 60 mm Hg. A blood lipid profile revealed a significant increase in the total serum cholesterol, LDL, and triglyceride levels only at 18-23 weeks of diet (Figure 1). The arterial responses to a depolarizing solution containing 60 mM KCl were similar in control and in all hypercholes‐ terolemic arteries. However, treatment of arteries from either one of the high-cholesterol diet groups with the selective BK channel blocker paxilline resulted in vasoconstriction that was significantly smaller compare to the control group (Figure 3A). BK channel function seemed to be altered rather selectively: arterial diameter responses to the KV channel blocker 4 aminopyridine were similar in control versus hypercholesterolemic animals (Figure 3A). First, these results demonstrated that the general contractile capability of the artery (as tested by a high KCl-containing solution) was largely preserved during the high-cholesterol diet. Second, endothelium-independent vasodilation that is mediated by the activity of smooth muscle BK channels was diminished during hypercholesterolemia [30, 31]. Moreover, the fact that reduced sensitivity to paxilline was observed after 10 weeks on a high-cholesterol diet, well before the changes in the blood lipid profile took effect (Figure 1) suggested that BK channels were highly sensitive to dietary cholesterol levels, independent of the increase in blood

Further experimentation took place in an effort to unveil molecular mechanisms that enable the sensitivity of BK channels to dietary cholesterol. First, it was shown that cholesterol accumulation in the wall of de-endothelialized cerebral arteries of hypercholesterolemic rats followed the pattern of increase in blood cholesterol level. In particular, the cholesterol level in cerebral artery tissue was only increased significantly during weeks 18-23 on diet, but not earlier (Figure 3B) [31]. Therefore, the direct accumulation of cholesterol in the vicinity of the BK channel might not be the sole reason for depressed BK channel sensitivity to paxilline during a high-cholesterol intake. The reduction in paxilline-induced cerebral artery constric‐ tion by hypercholesterolemia might result from a decreased number of BK channels in arterial smooth muscle. In particular, hypercholesterolemia may down-regulate accessory, smooth muscle-type β subunit (β1) (Figure 2A). Indeed, cerebral arteries of β1 (*KCNMB1)* knock-out mice were reported to be insensitive to selective BK channel block by the peptide blocker iberiotoxin [68]. Moreover, hypercholesterolemia-induced changes in BK β1 subunit level have been studied in circular smooth muscle strips from the sphincter of Oddi in rabbits fed by highcholesterol food (1 g cholesterol per day) for 4 weeks [29]. Immunohistochemical and Western blot protein analysis using a BK β1 subunit-specific polyclonal antibody showed a decreased level of the BK β1 protein in the cholesterol-fed group. Thus, in the sphincter of Oddi highcholesterol diet down-regulated BK β1 subunits [29]. However, in rat cerebral artery myocytes

intermediate conductance KCa channel blocker charybdotoxin [28].

cholesterol.

104 Hypercholesterolemia

Remarkably, dietary cholesterol does not only alter the paxilline-sensitivity of the channel but also protects against ethanol-induced BK channel-mediated constriction of cerebral arteries. It was shown that BK channels represent a major target for ethanol in the cerebral vessels. Upon ethanol application, BK channel function is diminished, and cerebral artery constriction is observed [70]. The protective role of a high-cholesterol diet against alcohol-induced constric‐ tion of cerebral arteries was demonstrated *in-vivo* using a closed cranial window on anesthe‐ tized rats receiving conventional chow versus high-cholesterol (2% cholesterol) diet for 18-23 weeks [31]. Control or ethanol-containing solutions were infused into the cerebral circulation via a catheter in the carotid artery of the rat, and the diameter of the pial arterioles was determined. Infusion of 50 mM ethanol (e.g. the amount of ethanol detected in the blood during moderate-to-heavy alcohol intake in humans) into the cerebral circulation of control rats rendered a significant, 20% decrease in the pial arteriole diameter (Figure 4A). In contrast to the control group, infusion of 50 mM ethanol into the cerebral circulation of rats on a highcholesterol diet resulted in no more that a 10% decrease in the pial arteriole diameter. To rule out the contribution of circulating factors in the observed protective effect of a high-cholesterol diet, the experiment was repeated using isolated cerebral arteries that were pressurized *invitro* at 60 mmHg. As observed with pial arterioles *in-vivo*, application of 50 mM ethanol to pressurized middle cerebral arteries from control rats resulted in up to 12% decrease in the

**Figure 3. Effect of high-cholesterol diet on rat cerebral artery BK channel function.** (A) Average data showing a de‐ crease in arterial diameter by the selective BK channel blocker paxilline. (B) Cholesterol level in de-endothelialized cer‐ ebral arteries from rats fed control versus high-cholesterol diet. (C) Representative confocal microscope images showing an isolated cerebral artery myocyte and its fluorescence labeling of BK β1 and α subunits. (D) Averaged fluo‐ rescence intensity associated with BK β1 subunit. (E) Averaged fluorescence intensity associated with BK α subunit. (With modifications from [94]).

arterial diameter. In contrast, application of 50 mM ethanol to pressurized middle cerebral arteries from rats on a high-cholesterol diet resulted in only a 6-7% decrease in the arterial diameter (Figure 4B). Remarkably, the protective effect of a high-cholesterol diet against ethanol-induced constriction of cerebral arteries was similar in arteries with intact endotheli‐ um and in de-endothelialized vessels. Accordingly, dietary cholesterol-driven protection did not require the presence of functional endothelium and/or endothelium-derived vasoactive factors. Moreover, accumulation of cholesterol within the arterial wall was shown to play a major role in the observed protection by high-cholesterol diet against ethanol-induced constriction of cerebral arteries. In particular, after removal of the cholesterol that was accumulated in the cerebral artery tissue in the course of a high-cholesterol diet, ethanolinduced constriction was restored and reached the control value (Figure 4C) [31]. The molec‐ ular mechanisms by which membrane cholesterol affects the function of a major ethanol target in the artery, namely the BK channel, are still under investigation and are discussed in great detail elsewhere [71].

**Inwardly rectifying potassium (Kir) channels**. The effect of high-cholesterol diet on Kir channel function has been demonstrated for several members of the Kir family. Kir channels regulate important functions including membrane excitability, heart rate, vascular tone, insulin release and salt flow across epithelia (see, for example, reviews [10, 11, 13]). Structur‐ ally, they are comprised of four homo- or heteromeric subunits, each with two membrane spanning helices and intracellular N- and C- termini (Figure 2B). Fifteen Kir channels have been identified and classified in seven subfamilies (Kir1–7) [72]. Among these, it has been shown that a high-cholesterol diet affects the function of Kir2 channels, Kir3.1/Kir3.4 (KACh) channels and Kir6 (KATP) channels.

The effect of a high-cholesterol diet on Kir2 channels has been determined in different animal models and cell types. In earlier studies of Kir2 channels expressed in endothelial cells, hypercholesterolemia was induced by administering an atherogenic diet (0.5% cholesterol, 10% lard, and 1.5% sodium cholate) to castrated male Yorkshire pigs [18]. The properties of endothelial Kir2 channels and the values of the membrane potentials were compared in porcine aortic endothelial cells freshly isolated from the pig aortas. Cells isolated from hypercholes‐ terolemic animals had significantly lower Kir currents than those isolated from control cells. Moreover, the membrane potential in hypercholesterolemic pigs was significantly more depolarized compared with that in control animals. More recently, the effect of a highcholesterol diet on Kir2 channels expressed in cardiac myocytes was determined using a rat model [19]. In these experiments a group of 25-day-old male Sprague-Dawley rats was placed on a high-cholesterol diet (2% cholesterol in standard rodent food). Another group of the same age was fed an isocaloric, cholesterol-free diet from the same supplier. The rats were sacrificed as atrial tissue donors after 20-24 weeks on control or high-cholesterol diet. Notably, as a result of the high-cholesterol diet, in addition to an approximately 2.5-fold increase in serum LDL levels (see Figure 1), there was also an approximately1.8-fold increase in cholesterol levels in the atrial tissue itself (see Figure 5A). This increase in cholesterol levels in the atrial myocytes following the high cholesterol diet resulted in an approximately 60% decrease in Kir2 currents in atrial cardiomyocytes [19].

**Figure 3. Effect of high-cholesterol diet on rat cerebral artery BK channel function.** (A) Average data showing a de‐ crease in arterial diameter by the selective BK channel blocker paxilline. (B) Cholesterol level in de-endothelialized cer‐ ebral arteries from rats fed control versus high-cholesterol diet. (C) Representative confocal microscope images showing an isolated cerebral artery myocyte and its fluorescence labeling of BK β1 and α subunits. (D) Averaged fluo‐ rescence intensity associated with BK β1 subunit. (E) Averaged fluorescence intensity associated with BK α subunit.

(With modifications from [94]).

106 Hypercholesterolemia

**Figure 4. Cholesterol control of ethanol-induced constriction of cerebral artery.** (A) Averaged data showing constric‐ tion of pial arterioles *in-vivo* in rats fed control versus high-cholesterol diet. (B) Ethanol-induced cerebral artery con‐ striction in pressurized cerebral arteries *in-vitro* obtained from rats on control versus high-cholesterol diet. (C) Averaged data showing ethanol-induced cerebral artery constriction *in-vitro* in arteries from rats on a high-cholesterol diet before and after removal of excessive cholesterol using the cholesterol carrier methyl-β–cyclodextrin.(From [94]).

In addition to Kir2 channels, atrial myocytes also express Kir3 channels. In particular, atrial KACh channels are heterotetrameric proteins that consist of Kir3.1 and Kir3.4 [73]. Recent studies [19] demonstrated that unexpectedly rats that were on a high-cholesterol diet for 18-22 weeks exhibited up to 2-3 fold increase in KACh currents that were sensitive to the selective IK, AChblocker tertiapin (Figure 5B-5E). The summary data in Figure 5D-E show that the highcholesterol diet affected both inward and outward currents in a similar manner. Thus, while the effect was more visible for the larger inward currents, the physiologically relevant smaller outward currents were also significantly affected by cholesterol. This result was surprising because an increase in channel function following an increase in cholesterol levels (as shown in Figure 5A) is rare. These data suggest that an increase in cholesterol levels in atrial myocytes may underlie the increase in KACh currents in hypercholesterolemic rats.

**Figure 5. ACh-induced** KACh**currents in atrial cardiomyocytes are enhanced in hypercholesterolemic rats.** (A) Choles‐ terol levels in atrial tissue of Sprague-Dawley rats fed a high-cholesterol diet compared to control. Tertiapin-sensitive currents (B) and I-V relationships (C) of ACh-induced current densities for atrial myocytes from control and hypercho‐ lesterolemic rats. Summary data: (D) inward ACh-induced current densities at -80 mV; (E) outward ACh-induced cur‐ rent densities at +40 mV. ((B)-(E) From [19]).

In addition to Kir2 channels, atrial myocytes also express Kir3 channels. In particular, atrial KACh channels are heterotetrameric proteins that consist of Kir3.1 and Kir3.4 [73]. Recent studies

**Figure 4. Cholesterol control of ethanol-induced constriction of cerebral artery.** (A) Averaged data showing constric‐ tion of pial arterioles *in-vivo* in rats fed control versus high-cholesterol diet. (B) Ethanol-induced cerebral artery con‐ striction in pressurized cerebral arteries *in-vitro* obtained from rats on control versus high-cholesterol diet. (C) Averaged data showing ethanol-induced cerebral artery constriction *in-vitro* in arteries from rats on a high-cholesterol diet before and after removal of excessive cholesterol using the cholesterol carrier methyl-β–cyclodextrin.(From [94]).

108 Hypercholesterolemia

Several studies were also carried out to determine the effect of high-cholesterol diet on Kir6 channels. ATP-sensitive K+ (KATP) channels are expressed in the sarcolemma of cardiomyocytes [74] and in the mitochondrial inner membrane [75]. Structurally, KATP channels are comprised of a pore forming Kir channel (Kir6.1 or Kir6.2) and an ATP-binding regulatory subunit, the sulfonylurea receptor (SUR1, SUR2A, or SUR2B).

Activation of KATP channels mediates coronary vasodilation during decreases in perfusion pressure within the autoregulatory range [76] and dilation of collateral and noncollateral vessels during ischemia [77]. However, dilation in response to the KATP channel activator aprikalim was not altered in monkeys following an atherogenic diet and reduction in dietary cholesterol [78].

In contrast, acidosis-induced coronary arteriolar dilation was impaired in hypercholesterole‐ mic rabbits. When myocardial ischemia takes place [79], the interstitial pH of the heart rapidly decreases followed by an immediate decrease in coronary resistance by microvascular dilation [80]. It was shown that acidosis-induced coronary arteriolar dilation is mediated via the activation of pertussis toxin-sensitive G protein and consequent opening of the KATP channel [81-82]. Since hypercholesterolemia produces structural and functional abnormalities in blood vessels [83], its impact on coronary microvascular response to acidosis was investigated [84]. Coronary arterioles isolated from rabbit hearts were cannulated to micropipettes in a vessel chamber and microvascular responses were observed. The effect of the KATP channel blocker glibenclamide on the acidosis-induced microvascular responses was examined. Coronary arterioles significantly dilated as the pH was reduced and the dilation was significantly inhibited by glibenclamide. In another set of experiments, rabbits were randomly assigned to normal chow or high-cholesterol diet. After 8 weeks, the responses of isolated coronary arterioles to acidosis were examined in the two groups. Acidosis-induced dilation in the highcholesterol group was significantly attenuated compared to the control group. These data suggest that KATP channels play an important role in the acidosis-induced dilation of rabbit coronary arterioles and that dilation of coronary arterioles is impaired in hypercholesterole‐ mia. Notably, the impairment occurs upstream of KATP channel opening.

KATP channels play a key role in endogenous cardioprotective mechanisms [85-88]. Specifi‐ cally, during cardiac ischemia, the levels of intracellular ATP may decrease. This would result in the opening of KATP channels that operate as molecular biosensors for coupling cellular energy metabolism and excitability [89]. The opening of KATP channels leads to increased influx of K+ , which then leads to shortening of the action potential duration and to reduction of the Ca2+ overload that occurs during ischemia-reperfusion induced injury [90-92]. Since hyperlipidemia has been shown to interfere with cardioprotective mecha‐ nisms, studies were carried out to investigate the interaction of hyperlipidemia with cardioprotection induced by pharmacological activators of KATP channels [93]. Hearts isolated from rats fed a 2% cholesterol-enriched or normal diet for 8 weeks were subject‐ ed to 30 min of global ischemia and 120 min of reperfusion in the presence or absence of KATP modulators. In normal diet-fed rats, activation of KATP channels either by the nonselec‐ tive KATP activator cromakalim or the selective mitochondrial KATP channel opener diazo‐ xide significantly decreased infarct size compared with vehicle-treated control rats. Moreover, the cardioprotective effect was abolished by blocking the channels using the nonselective KATP blocker glibenclamide or the selective mitochondrial KATP channel blocker 5-hydroxydecanoate. In contrast, in cholesterol-fed rats, the cardioprotective effect was not observed following administration of KATP channel activators, demonstrating that cardiopro‐ tection by KATP channel activators is impaired in cholesterol-enriched diet-induced hyperli‐ pidemia. Notably, whereas protein levels of Kir6.1 and Kir6.2 remained unchanged, cardiac expression of Kir6.1 was significantly downregulated in cholesterol-fed rats.

Together, these data demonstrate a wide range of effects of a high-cholesterol diet on the function of inwardly rectifying potassium channels and on their physiological implications. Whereas the function of Kir2 and Kir6 channels was suppressed in several cases following a high-cholesterol diet, atrial Kir3 channels were enhanced. Moreover, in the case of Kir6 channels, whereas KATP-mediated coronary vasodilation was not altered in atherosclerotic monkeys, a high-cholesterol diet resulted in impaired cardioprotection by KATP channel activators in rats and impaired KATP-mediated acidosis-induced coronary arteriolar dilation in rabbits.

#### **4. Conclusive remarks**

Several studies were also carried out to determine the effect of high-cholesterol diet on Kir6

[74] and in the mitochondrial inner membrane [75]. Structurally, KATP channels are comprised of a pore forming Kir channel (Kir6.1 or Kir6.2) and an ATP-binding regulatory subunit, the

Activation of KATP channels mediates coronary vasodilation during decreases in perfusion pressure within the autoregulatory range [76] and dilation of collateral and noncollateral vessels during ischemia [77]. However, dilation in response to the KATP channel activator aprikalim was not altered in monkeys following an atherogenic diet and reduction in dietary

In contrast, acidosis-induced coronary arteriolar dilation was impaired in hypercholesterole‐ mic rabbits. When myocardial ischemia takes place [79], the interstitial pH of the heart rapidly decreases followed by an immediate decrease in coronary resistance by microvascular dilation [80]. It was shown that acidosis-induced coronary arteriolar dilation is mediated via the activation of pertussis toxin-sensitive G protein and consequent opening of the KATP channel [81-82]. Since hypercholesterolemia produces structural and functional abnormalities in blood vessels [83], its impact on coronary microvascular response to acidosis was investigated [84]. Coronary arterioles isolated from rabbit hearts were cannulated to micropipettes in a vessel chamber and microvascular responses were observed. The effect of the KATP channel blocker glibenclamide on the acidosis-induced microvascular responses was examined. Coronary arterioles significantly dilated as the pH was reduced and the dilation was significantly inhibited by glibenclamide. In another set of experiments, rabbits were randomly assigned to normal chow or high-cholesterol diet. After 8 weeks, the responses of isolated coronary arterioles to acidosis were examined in the two groups. Acidosis-induced dilation in the highcholesterol group was significantly attenuated compared to the control group. These data suggest that KATP channels play an important role in the acidosis-induced dilation of rabbit coronary arterioles and that dilation of coronary arterioles is impaired in hypercholesterole‐

mia. Notably, the impairment occurs upstream of KATP channel opening.

KATP channels play a key role in endogenous cardioprotective mechanisms [85-88]. Specifi‐ cally, during cardiac ischemia, the levels of intracellular ATP may decrease. This would result in the opening of KATP channels that operate as molecular biosensors for coupling cellular energy metabolism and excitability [89]. The opening of KATP channels leads to

to reduction of the Ca2+ overload that occurs during ischemia-reperfusion induced injury [90-92]. Since hyperlipidemia has been shown to interfere with cardioprotective mecha‐ nisms, studies were carried out to investigate the interaction of hyperlipidemia with cardioprotection induced by pharmacological activators of KATP channels [93]. Hearts isolated from rats fed a 2% cholesterol-enriched or normal diet for 8 weeks were subject‐ ed to 30 min of global ischemia and 120 min of reperfusion in the presence or absence of KATP modulators. In normal diet-fed rats, activation of KATP channels either by the nonselec‐ tive KATP activator cromakalim or the selective mitochondrial KATP channel opener diazo‐ xide significantly decreased infarct size compared with vehicle-treated control rats.

, which then leads to shortening of the action potential duration and

(KATP) channels are expressed in the sarcolemma of cardiomyocytes

channels. ATP-sensitive K+

cholesterol [78].

110 Hypercholesterolemia

increased influx of K+

sulfonylurea receptor (SUR1, SUR2A, or SUR2B).

Different types of potassium channels have been shown to be affected by high-cholesterol diet in a variety of species. The modulation of potassium channel activity by high-cholesterol diet results in alterations of organ function *in-vitro* and *in-vivo*. Notably, the effect of high-choles‐ terol diet on potassium channels varies and may result in decreased or increased channel function. In a few cases, potassium channels were found to be insensitive to dietary cholesterol manipulation. Among the potassium channels that were affected by a high-cholesterol diet are included several voltage-gated potassium channels (KV), calcium-activated potassium (KCa) channels, and inwardly rectifying potassium (Kir) channels. Among the channels studied, only the currents of the heterotetrameric Kir3.1/Kir3.4 (KAch) channels were consistently enhanced by high-cholesterol dietary intake. The structural and molecular bases for the diverse effect of high-cholesterol diet on potassium channels remain largely unknown. Thus, considering the critical role of potassium channels in physiology and pathology, an important aspect of future studies will be to elucidate the intrinsic mechanisms leading to dietary cholesterol modulation of channel function.

#### **Acknowledgements**

This work was supported by a Scientist Development Grant 11SDG5190025 from the American Heart Association (to A.R.-D.), the Alcoholic Beverage Medical Research Foundation grant for New Investigator (A.B.), and NIH Support Opportunity for Addiction Research for New Investigators R03 AA020184 (A.B.)

#### **Author details**

Anna N. Bukiya3 and Avia Rosenhouse-Dantsker1,2\*

\*Address all correspondence to: dantsker@uic.edu

1 Department of Chemistry, University of Illinois at Chicago, Chicago, IL, USA

2 Departments of Medicine and Pharmacology, University of Illinois at Chicago, Chicago, IL, USA

3 Department of Pharmacology, The University of Tennessee Health Science Center, Mem‐ phis, TN, USA

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