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ATHEROGENESIS Accumulation of lipoprotein-derived lipids including cholesteryl ester (CE) and triglyceride (TG) within the arterial intima

is considered a fundamental factor in the initiation of atherogenesis. However, the process of atherosclerotic plaque formation is not a passive deposition of lipid within the arterial wall. It is a complex process which locally involves the coordinated response by immune cells and vascular smooth muscle cells to endothelial damage and lipid accumulation, in addition to the deposition of cholesterol,apoB, phospholipid and triglyceride. Physical stress in the local environment also contributes to lipid deposition. In regions of arteries where mechanical forces increase the exposure time of the lumen to lipoproteins, plaques are more likely to develop. Evidence of this is observed clinically as hypertension increases CVD risk by 2fold, independent of other risk factors. Studies in children with familial hypercholesterolemia and no other contributing risk factors, demonstrate that atherosclerosis occurs in proportion to blood lipid levels, suggesting high LDL-C is sufficient to cause disease. Arteries consist of three distinct layers: the intima (closest to the lumen of the vessel), the media and the adventitia. The inner surface of the intima is defined by endothelium while the internal elastic lamina delineates the outer surface. It contains a single layer of endothelial cells, smooth muscle cells and subendothelial connective tissue. Endotheial cells regulate vascular tone through the secretion of vasoactive substances including nitric oxide and endothelin. The media layer, defined by the external elastic lamina consists of smooth muscle cells which produce elastin, collagen and sulphated glucosaminoglycans. The adventitia or outermost layer contains collagen fibres, fibroblasts and progenitor cells. Normally, vascular endothelium prevents access to large molecules, including CE-rich LDL, however in response to endotheial dysfunction, this barrier increases adhesiveness and permeablility, allowing small, dense LDL particles to enter and accrue within the arterial intima. The upregulation of adhesion molecules including P-selectin, intracellular adhesion molecule (ICAM) and vascular cell adhesion molecule (VCAM) by the endothelium encourages the extravasation of monocytes and infiltration of macrophages into the intima. Composition of the atherosclerotic arterial wall Arteries contain three distinct layers, the intima which contains a layer of endothelial cells which normally provide a barrier against the entry of cholesterol-rich LDL particles, the media, which is contains smooth muscle cells and regulates vascular tone and the adventitia, which contains collagen and proteoglycans. In this image the intima is diseased and the plaque contains lipoproteins, macrophage foam cells and lymphocytes. The plaque is covered by a dense layer of smooth muscle cells and extracellular matrix proteins to prevent contact with the blood as the contents are highly thrombogenic. Lipid homeostasis in both macrophages and smooth muscle cells is dynamically regulated by uptake, storage and efflux of cholesterol in response to sterol content within the cell. When taken up in their native form by the LDL receptor (LDLR), CE from lipoprotein particles is hydrolyzed by lysosomal acid hydrolase to free cholesterol and transported by Niemman Pick C1 (NPC1) to the endoplasmic reticulum, where, it can be reesterified and stored in lipid droplets. Alternatively, cholesterol, can be effluxed as free cholesterol through the actions of ABC transporter A1 (ABCA1) to lipid poor apolipoprotein A1 (apoA1) or by ABCG1 to HDL particles . As cholesterol accumulates in the cell, expression of the LDLR and endogenous synthesis of cholesterol will decrease, and efflux from the cell will increase. However, upon entry into the intima, LDL can be oxidized and scavenged by macrophages. Macrophage scavenger receptors, which unlike the LDLR are not regulated by negative feedback by cholesterol, are responsible for the uptake of modified LDL. They include cluster of

differentiation (CD) 36, CD68 and scavenger receptor A l/lI (SRAI/II). As a result, macrophages accumulate large amounts of lipoprotein-derived CE within lipid droplets and develop into foam cells. It is through this process that earlystage lesions called fatty streaks are formed. They occur very early in life and may continue to develop from childhood onward or regress. Initial lesions or fatty streaks are characterized by the thickening of the intima and the accumulation of macrophages and lymphocytes, which secrete cytokines and chemokines, leading to increased leukocyte infiltration and proliferation. Monocyte infiltration has been shown to be crucial in early atherogenesis as mice with reduced levels of macrophages are protected from early lesion formation. Smooth muscle cells are normally quiescent within the arterial wall, and function to maintain vascular integrity and tone. As the lesion progresses, smooth muscle cells proliferate, accumulate lipid and secrete extracellular matrix proteins including collagen, Cholesterol trafficking within the macrophage. Lipid homeostasis in macrophages is maintained by regulation of uptake, storage and efflux. Triglyceride-rich lipoprotein particles are hydrolyzed by lipoprotein lipase (LPL) and fatty acids are taken up and re-esterified into lipid droplets. The regulated uptake of cholesteryl ester rich lipoproteins occurs through the low density lipoprotein receptor (LDLR). Upon hydrolysis in the endosome, cholesterol can be re-esterified in the endoplasmic reticulum and stored in cytosolic lipid droplets. Alternatively, cholesterol can be effluxed from the cell through the ATP binding cassette proteins, ABCA1 and ABCG1 which efflux cholesterol and phospholipids to apolipoprotein A1 and high density lipoprotein (HDL) respectively. Modified LDL is taken up by the scavenger receptors, CD36 and SRA l/lI. This type of lipid uptake is not regulated by the sterol content of the cell and results in the formation of macrophage foam cells. Atherosclerotic Lesion Characterization and Progression. Atherosclerosis is characterized as a chronic inflammatory disease. Initiating events in the development of atherosclerotic lesions include endothelial dysfunction and arterial deposition of lipid and lipoproteins. Lipid accumulation stimulates the influx of macrophages, which engulf the lipid leading to foam cell formation and the initiation of a fatty streak. In order to stabilize the growing lipid core, smooth muscle cells are stimulated to proliferate and they secrete and deposit extracellular matrix proteins leading to an increase in fibrous plaque. Lesions can regress or complications including vessel occlusion, rupture and thrombosis may occur. LIPOPROTEINS All lipoproteins are soluble, spherical lipid traffickers which consist of a hydrophobic triglyceride (TG) and CE core, surrounded by a surface layer of free cholesterol (FC), phospholipids and apolipoprotiens. Lipoprotein types are classified based on their density, lipid composition and association with apolipoproteins. The largest lipoproteins, chylomicrons, are formed in the intestine and transport dietary TG on an apolipoprotein B48 (apoB48) scaffold. They are also associated with apolipoproteins Al, All, AV, C and E. Very low density lipoproteins (VLDL) transport endogenous TG with a modest amount of CE on an apoBIOC scaffold and interact with apoEs and apoCs. VLDL undergoes a series of modifications and lipid exchanges to form CE-enriched LDL particles. Lipoprotein Classification

LIPOPROTEIN METABOLISM Whole body lipid homeostasis is maintained through a balance between exogenous cholesterol and fatty acid intake and endogenous synthesis. EXOGENOUS LIPOPROTEIN METABOLISM Cholesterol absorption is mediated by the transporter Niemann-Pick C1-like1 (NPC1L1). Approximately 50% of dietary cholesterol is absorbed by the intestine. Dietary fat is hydrolyzed by pancreatic lipase, absorbed very efficiently in the intestine and dictates the rate of formation of TG-rich chylomicrons . The protein scaffold for assembly of cholesterol and TG into chylomicron particles is apolipoprotein B. ApoB exists in two isoforms, which arise from editing of the APOB mRNA transcript. ApoB48 is the N-terminal 48% of the 550 kDa polypeptide chain which is edited by apoB-encoding mRNA (apobec). In the human intestine, apoB48 is the main isoform, while full length apoB100 is expressed exclusively in human liver. In mice, both the liver and intestine produce apoB48. TG and cholesterol, in addition to PL are packaged onto the apoB48 protein backbone by microsomal triglyceride transport protein (MTP) within intestinal enterocytes prior to secretion into the lymphatic system . Once chylomicrons enter the bloodstream, they become associated with apolipoproteins E and C. They then bind heparin sulfate proteoglycans, and apoCII is required for TG hydrolysis by lipoprotein lipase, an enzyme which is secreted by parenchymal cells of muscle and adipose and resides on the capillary endothelium. Released fatty acids and glycerol diffuse through the capillary walls to be taken up and utilized or reesterified and stored by peripheral tissues.CE rich remnant particles are taken up very efficiently by the liver through LDLR- and LDL related receptor (LRP)- mediated recognition of apoE ENDOGENOUS LIPOPROTEIN METABOLISM Since only a small component of circulating cholesterol is derived from the diet, the liver is the primary regulator of whole body cholesterol and lipid metabolism (Hegele, 2009). The liver is composed of hepatocytes, which perform the bulk of liver functions and non-parenchyma! cells including endothelial cells, Kupffer cells and other immune cells. Endothelial cells of capillaries in the liver allow free diffusion of small molecules between the blood and hepatocytes while filtering out larger molecules like chylomicrons, and thus aid in the regulation of lipid uptake by the liver (Bouwens et al., 1992). Endogenous and Exogenous Lipoprotein Metabolism Dietary triglycerides and cholesterol are packaged onto the apolipoprotein B48 scaffold and secreted as chylomicrons, initially into the lymphatics, and subsequently into the blood stream. The lipolysis of triglycerides by lipoprotein lipase (LPL) allows fatty acid uptake into peripheral tissues. The cholesteryl esterrich remnant particle is taken up by the liver through LDLR-mediated endocytosis. The liver secretes TG-rich VLDL particles, which also undergo hydrolysis by lipoprotein lipase. The subsequent CE-enriched particles, IDL and LDL may also be cleared by the liver through the LDLR. Cholesterol from peripheral tissues is effluxed to HDL and which is also taken up by the liver through SRB1 and secreted into bile. In addition to the uptake of dietary cholesterol and fatty acids, the endogenous synthesis of both lipids also occurs in the liver. Both cholesterol and triglyceride synthesis begin with the formation of acetyl-CoA, derived from the metabolism of glucose or fatty acids. Cholesterol synthesis is very complex, involving greater than 30 individual reactions. It requires the condensation of two acetyl-CoA molecules to create acetoacetyl-CoA which is subsequently converted to mevalonate by HMG-CoA reductase, the enzyme target of statin drugs. Through a series of condensation reactions, isoprene units are formed which condense to form squalene. Squalene is cyclized to form lanosterol, which is subsequently converted to cholesterol through a further 19 step process.

The synthesis of fatty acids begins when acetyl-CoA is carboxylated to form malonyl-CoA, which is subsequently elongated to palmitic acid by fatty acid synthase (FAS). Both cholesterol and triglyceride availability within the liver have been shown to regulate lipoprotein secretion. Assembly of the TG-rich core onto the apoB100 backbone happens exclusively in the liver and VLDL is the primary lipoprotein synthesized and secreted. Within the bloodstream, lipoprotein lipase (LPL) initiates lipolysis of TG within VLDL, resulting in the conversion of VLDL into intermediate-density lipoprotein (IDL), and fatty acid uptake via CD36 by peripheral tissues such as adipose and muscle. Once the VLDL are reduced in size, they can become enriched in apoE, which leads to uptake via recognition of apoE by the LDLR and LDLR related protein (LRP) (Hu et al., 2008). IDL can also be converted into LDL by further lipolysis and the addition of CE through the activity of CETP. These TG-enriched LDL particles are substrates for hepatic lipase, which further reduces particle size, resulting in small, dense LDL particles. Lipoprotein and fatty acid uptake can play a significant role in regulating lipid levels within tissues. The LDLR, which binds the apoB backbone of LDL, is expressed 19 ubiquitously and can mediate peripheral lipoprotein uptake. However both the LDLR and the LDLR related protein (LRP) are expressed predominantly in liver, which mediates the clearance of most lipoprotein particles from plasma (Espirito Santo et al., 2005). HEPATIC LIPOPROTEIN SYNTHESIS In order to be successfully secreted into plasma, apoB must be transcribed, fully translated and the polypeptide translocated, lipidated, properly folded and glycosylated. Thus the complexity of this process allows for multiple points of regulation during apoB particle synthesis and assembly

FLAVONOIDS Metabolic syndrome is a clustering of risk factors including dyslipidemia, hypertension, insulin resistance and visceral obesity for the development of type 2 diabetes and premature atherosclerosis . In light of the increasing prevalence of this disease, new therapies are required. In the search for new targets and interventions to improve risk factors and prevent the complications of atherosclerosis, attention has turned to non-traditional therapies including a group of naturally-occurring compounds known as flavonoid PROPERTIES OF FLAVONOIDS AND EPIDEMIOLOGIAL EVIDENCE Flavonoids are the most abundant polyphenols in the human diet and can be found in a variety of foods and beverages including soy, fruits, vegetables, nuts, seeds, cocoa, wine, coffee and tea . Several thousand have been identified and they can be divided into groups depending on their structural variation and degree of oxidation. These include flavonols, flavones, isoflavones, flavanones, flavan-3-ols and anthocyanidins. Epidemiological studies have established an inverse relationship between flavonoid consumption and many chronic diseases including coronary heart disease (CHD) and stroke. Participants in the top tertile of flavonoid intake demonstrated a 20% risk reduction in CHD mortality compared to individuals in the lower. Increases in the intake of fruits and vegetables have also been associated with reductions in a number of risk factors for heart disease including lower blood pressure , improved weight management and improved glucose tolerance .Most studies have focused on the ability of flavonoid rich foods to modulate endothelial function, flow-mediated dilatation, blood pressure and

lipids. However, in human studies, wide variability is observed in the effects of flavonoids on these biomarkers of cardiovascular disease risk. This heterogeneity in response reflects, in part, variation in the bioavailability of flavonoid subclasses. Furthermore, the most abundant flavonoids may not necessarily lead to the highest concentrations of biologically active compounds. More recently, attention has turned to purified flavonoids and the examination of their pharmacological properties. Flavonoids have the potential to prevent CVD through a number of mechanisms including improved endothelial function, blood pressure lowering, inhibition of lipoprotein oxidation and decreased plasma concentrations of apoB100-containing lipoproteins. Flavonoids by their structure are reducing agents and can serve as efficient chelators of transition metals involved in cellular oxidation reactions Therefore, much of the association between flavonoids and reduced CHD has been attributed to their antioxidant properties and linked to a reduction in oxidative stress. However, an increasingly number of studies have demonstrated that flavonoids are powerful biological agents capable of regulating many metabolic pathways through other mechanisms. FLAVONOIDS, LIPIDS AND LIPOPROTEINS In concert with their antioxidant effects, the lipid lowering properties of flavonoids have also been evaluated. Dyslipidemia is characterized by hepatic over secretion of apoBIOO-containing lipoproteins, hypertriglyceridemia, delayed clearance of LDL and low levels of HDL. Dietary studies in humans have demonstrated the beneficial effects of many flavonoids including those contained in soy protein isolate and green tea, both of which lower LDL cholesterol. However, studies with anthocyanidins, black tea, chocolate, red wine and grape demonstrated no beneficial effects on LDL-C or HDL-C in human studies. However, both animal and human studies have demonstrated that mixtures of specific bioactive flavonoids can decrease LDL-C. Treatment of fructose-induced, insulinresistant hamsters with a mixture of citrus polymethoxylated flavones improved dyslipidemia and glucose tolerance. However, the mechanism was not elucidated. Berberine, an alkaloid isolated from Chinese herbs, up-regulated LDL receptor expression in cultured hepatocytes through a MAPKerk dependent mechanism and reduced plasma lipids in a hyperlipidemic hamster model. Administration of berberine to hypercholesterolemic patients reduced LDL cholesterol by 25%, demonstrating the cholesterol lowering potential of berberine in humans.

FLAVONOIDS AND ATHEROSCLEROSIS The early stages of atherosclerosis are characterized by endothelial dysfunction and macrophage foam cell formation. Administration of pomegranate juice, rich in anthocyanidins and proanthocyanidins to apolipoprotein E-deficient (apoE1') mice resulted in dramatic reductions in lipid peroxides and macrophage CE accumulation, without significantly affecting plasma cholesterol. Following 3 months of supplementation, atherosclerosis was significantly reduced. Administration of pomegranate by-product (PBP) to apoE^mice also attenuated atherosclerosis development, as a result of decreased cellular uptake of oxidized LDL and macrophage oxidative stress. Using the same apoE1' mouse model, 4 month supplementation of a low fat diet with the polyphenolic compound resveratrol led to a reduction in total plasma cholesterol and LDL-C and an increase in HDL-C. The mechanism for the reduction in plasma cholesterol was through a reduction in hepatic cholesterol synthesis, which may have stimulated LDL receptor mediated uptake of LDL from plasma . Resveratrol also prevents lipid peroxidation and increases cholesterol

efflux from macrophages Through these mechanisms, resveratrol significantly reduced atherosclerotic plaque development in the aortic arch of apoE1' mice . CITRUS FLAVONOIDS The most common flavonoids in citrus fruits investigated for potential lipid lowering include naringenin, hesperitin, nobiletin and tangeritin. Administration of grapefruit or orange juice to hypercholesterolemic casein-fed rabbits reduced LDL-C and hepatic lipid accumulation suggesting components of citrus may have lipid lowering properties. Naringin, the glycoside form of naringenin found in citrus fruits, is hydrolyzed by the intestinal microflora to the flavanone naringenin. In streptozotocin-induced diabetic rats, i.p. injection of naringenin decreased blood glucose and improved dyslipidemia. In cholesterol-fed rats, naringenin lowered plasma cholesterol which was associated with inhibition of hepatic cholesterol synthesis and esterification . However, in none of these studies was the mechanism of action elucidated.

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