Chapter Literature Review I: Atherosclerosis and HDL Literature Review I: Atherosclersosis and HDL 2.1 Coronary Artery Disease (CAD) CAD is a leading cause of death in many industrialized nations and by 2020, based on current trends, it is projected to be the number one killer in developing countries (Lopez and Murray, 1998). In Singapore, between 2000 and 2003, CAD alone has accounted for a quarter of all case mortalities (Health Facts Singapore 2003, Ministry of Health, http://www.moh.gov.sg). CAD susceptibility is associated with a multitude of risk factors including cigarette smoking, diabetes mellitus, obesity, hypertension, age as well as plasma lipoproteins like elevated low density lipoprotein-cholesterol (LDL-C) and reduced high density lipoproteincholesterol (HDL-C). It can be modified by changes in behavior, such as diet, exercise, alcohol consumption and estrogen replacement therapy. Many patients with CAD not have elevated LDL-C but rather low HDL-C, either alone or accompanied by hypertriglyceraemia. Although CAD and HDL-C can be modifiable with lifestyle and pharmacological interventions, genetic components still contribute substantially to variation in HDL-C. 2.2 HDL Structure HDLs are the smallest (diameter 7-12 nm, molecular weight 200-400 kDa) and densest (1.063-1.25 g/ml) of the plasma lipoproteins (Barter et al., 2003). Each HDL particle consists of a hydrophobic core of mostly cholesterol esters (CE) and a small amount of triglycerides (TG) surrounded by a hydrophilic surface monolayer of phospholipids, unesterifed (free) cholesterol and apolipoproteins. Table 2.1 gives the breakdown of the composition of a HDL particle. Principal apolipoproteins include ApoAI (70% of total protein content) and ApoAII (20% of total protein content), with small amounts of the C Chapter Literature Review I: Atherosclerosis and HDL apolipoproteins (ApoCI-III), ApoAIV, ApoD, ApoE, ApoJ and ApoL. Also associated with the HDL particle are plasma factors involved in remodelling of lipoproteins: lecithincholesterol acyltransferase (LCAT), cholesterol ester transfer protein (CETP) and phospholipid transfer protein (PLTP). Remodelling activities account for much of the heterogeneity of HDL. Table 2.1 Composition of HDL Component Content Phospholipid 50% Cholesterol ester 30% Free cholesterol 10% Triglycerides 10% 2.2.1 HDL Subpopulations HDL is structurally heterogeneous and can be classified into various species according to size, lipid or protein content or a combination of these factors. Each HDL species behaves differently with respect to important physiological functions such as cholesterol efflux (von Eckardstein et al., 1994). Using modern ultracentrifugation techniques, HDLs are separated into two major subtractions, HDL2 (1.063-1.125 g/ml) and HDL3 (1.125-1.210 g/ml), as well as a minor subfraction, VHDL (1.12-1.25 g/ml) (Barter et al., 2003). HDL2 has a higher cholesterol ester but lower protein content than HDL3. Polyacrylamide gradient gel electrophoresis further resolves particles within HDL2 and HDL3 fractions: HDL2b (10.57 nm), HDL2a (9.16 nm), HDL3a (8.44 nm), HDL3b (7.97 nm), HDL3c (7.62 nm) (Blanche et al., 1981). After agarose gel electrophoresis followed by anti-ApoAI immunoblotting, α and pre-β fractions of HDL can be identified. The large, lipid-rich and spherical α-HDL, corresponding to HDL2 and HDL3, forms the bulk of the circulating form of HDL. The minor (5-15% of Chapter Literature Review I: Atherosclerosis and HDL plasma HDL), slower migrating pre-β HDL particles are small discs composed of mainly ApoAI and very little lipids. Relative to α-HDL, pre-β HDL particles are more abundant in the extravascular compartment where they are likely to be the major cholesterol accepting species of HDL (Assman and Nofer, 2003). Using two-dimensional electrophoresis with agarose gel in the first dimension and nondenaturing polyacrylamide gradient gel in the second dimension, pre-β HDL has been further resolved into preβ1, pre-β2, pre-β3 HDL species in increasing order of size. Pre-β HDL has a density of >1.21 g/mL (the VHDL fraction) and therefore it was not originally recovered in the HDL fraction isolated by traditional ultracentrifugation. On the basis of apolipoprotein content, specific species of HDLs are distinguishable. A1-HDL (floating in the HDL2 range; α mobility) contains only ApoAI and is without ApoAII, whereas A1/A2 HDL (HDL3 fraction; α mobility) contains both ApoAI and ApoAII. Other minor species contain both ApoAI and ApoAIV, or just ApoAIV. Heterogeneity in HDL particles also extends to the phospholipid composition (Rye et al., 1999). The major phospholipid found on the surface of HDL is phosphatidylcholine, with many species involved depending on the type of fatty acid residues. Most have a saturated fatty acid in the γ position, either C16 palmitic acid or C18 stearic acid, and an unsaturated fatty acid in the β position, such as oleic acid, linoleic acid or arachidonic acid (Subbaiah and Pritchard, 1989). Other HDL phospholipids include sphinogomyelin, phosphatidylserine, phosphatidylinositol and phosphatidylethanolamine (Rye et al., 1999). 2.2.2 Laboratory Determination of HDL In routine biochemical analysis, HDL in plasma is assayed as the total cholesterol content (free cholesterol and CE) following conversion of all CE to cholesterol, hence the term HDL-C. This is equivalent to the HDL band obtained by density gradient Chapter Literature Review I: Atherosclerosis and HDL ultracentrifugation and the α-HDL resolved by agarose gel electrophoresis. Nuclear magnetic spectroscopy provides a rapid and convenient method of measuring the levels of the different lipoprotein subclasses, thereby overcoming the limitations of existing laborious analytical methods that utilize centrifugation and gel electrophoresis (Otvos, 2000). 2.3 Beneficial Effects of HDL: Evidence from Epidemiological Studies and Clinical Trials A low level of HDL-C is a strong, independent risk factor for CAD. This observation was noted as early as in the 1950s but ignored until its rediscovery two decades later, possibly because HDL-C is normally a minor constituent (20-25%) of total plasma cholesterol (Breslow, 1995). Since then, many case-control, prospective and intervention studies have reaffirmed the inverse relationship between HDL and CAD. In the Framingham Heart Study, 2815 men and women between the ages of 49 and 82 were monitored for four years for development of CAD and lipid levels. The HDL-C level was found to be a potential risk factor, especially among older participants, and was significant even after other risk factors were considered (Gordon et al., 1977). The participants were followed up for another eight years, and again, low HDL was confirmed as an independent risk factor of CAD, even after accounting for multiple risk variables like total cholesterol, alcohol consumption, blood pressure, cigarette smoking and body mass index (BMI), and also after stratifying individuals according to total cholesterol levels (Castelli et al., 1986). The Prospective Cardiovascular Munster (PROCAM; Assman et al., 1996) and the Quebec Cardiovascular Studies (Despres et al., 2000) also supported the notion of an inverse relationship between HDL level and the risk of CAD. A metaanalysis of four prospective observational studies estimates that every mg/dL (0.025 Chapter Literature Review I: Atherosclerosis and HDL mM) reduction in HDL-C translates into a 2-3% increase in CAD risk whereas a mg/dL increment in LDL-C raises CAD risk by 1% (Gordon et al., 1989). While reducing LDL with statins has successfully reduced coronary events and cardiovascular risks, HDL remains an independent risk factor for CAD, as evidenced in several statin trials in which increases in HDL levels with statin therapy were associated with modest reductions in coronary events (Sacks, 2001). In the West of Scotland Coronary Prevention Study (WOSCOPS), pravastatin-treated patients were monitored for five years for the incidence of major coronary events. Despite the overall result that pravastatin treatment lowered the risk of coronary events, patients with a lower baseline of HDL still had higher risks than those with a higher baseline, suggesting the statin therapy did not alter the risk of low HDL. In the Helsinki Heart Study, the randomized, double-blind primary intervention trial of gemfibrozil administration to middle-aged men with primary dyslipidemia over five years showed a 11% increase in HDL and reduction in LDL-C was accompanied by a 34% reduction in CAD incidence, a rate much lower than that predicted based on the extent of LDL-C lowering (Frick et al., 1987). A similar effect of gemfibrozil has been demonstrated in the Veterans Affairs HDL Intervention Trial (VAHIT) in 2531 men with CAD selected for low HDL-C (average 40 mg/dL) and normal LDLC (140 mg/dL) (Rubins et al., 1999). After a five-year followup, the treatment group showed a 24% reduction in combined end points (defined as death from CAD, stroke, nonfatal myocardial infarction and stroke). Fibrates like gemfibrozil are potent agonists of peroxisome proliferator-activated receptor (PPAR)-α, a nuclear receptor with actions that include increased lipoprotein lipase (LPL)-mediated lipolysis leading to lower TG, and transcriptional induction of ApoAI and ApoAII culminating in higher HDL (Staels et al., 1998). These clinical trials independently demonstrate that HDL level is an independent risk factor of CAD and that the elevation of HDL has beneficial effects. The clinical Chapter Literature Review I: Atherosclerosis and HDL significance of HDL-C levels has been recognized by the National Cholesterol Education Program Adult Treatment Panel III with the latest guidelines raising the CAD risk threshold for low HDL-C from 35 to 40 mg/dL (Expert Panel on Detection, Evaluation, And Treatment of High Blood Cholesterol in Adults, 2001). 2.4 Atheroprotective Mechanisms of HDL Against the background of numerous epidemiological studies providing overwhelming evidence that HDL is atheroprotective, direct mechanistic explanations have been sought. The atheroprotective attributes of HDL are diverse and are reviewed below. 2.4.1 Protection against LDL Oxidation HDL, particularly the HDL3 fraction, protects against LDL oxidation. The appearance of oxidized LDL (oxLDL) species in the subendothelial matrix is a key initiating event in early atherogenesis. OxLDL serves as a chemoattractant for monocytes, transforms macrophages to foam cells, exerts cytotoxic effects on endothelium, activates platelets, stimulates movement and proliferation of smooth muscle cells, and antagonizes the vasorelaxation effect of nitric oxide (NO) (Nofer et al., 2002). The anti-oxidative properties of HDL are in part attributed to its associated enzymes such as paraoxonase (PON), platelet activating factor acetylhydrolase (PAFH) and glutathione peroxidase, as well as its structural components, ApoAI and certain lipid constituents (Assman and Nofer, 2003). PON acts by preventing formation of minimally oxidized LDL as well as destroying the biologically active oxidized phospholipids on LDL once they are formed (Watson et al., 1995; Navab et al., 2000). A direct evidence for the anti-atherogenic effect of PON comes from the finding that PON-deficient mice have larger aorta lesions than wild type and the HDL from these mice fails to prevent LDL oxidation in vitro (Shih et al., 1998). In animal models prone to atherosclerosis such as ApoE- or LDLR-knockout mice, raised levels of oxidation markers were accompanied by reduced paraoxonase activity (Shih et Chapter Literature Review I: Atherosclerosis and HDL al., 1996). PON colocalizes with the fraction of HDL that contains both ApoAI and ApoJ (Kelso et al., 1994). Apart from hydrolyzing platelet activating factor which is a potent lipid mediator with proinflammatory properties, PAFH, like PON and glutathione peroxidase, can destroy oxidized phospholipids on modified LDL (Assman and Nofer, 2003). In addition, natural antioxidants found on HDL such as ApoAI which possesses several methionine residues, α-tocopherol and other lipophilic molecules can scavenge reactive oxygen species, guarding against the formation and accumulation of lipid hydroperoxides and oxidized CE, seeding molecules that induce the oxidation of phospholipids non-enzymatically (Assman and Nofer, 2003). 2.4.2 Prevention of Platelet Aggregation HDL inhibits the propensity of platelets to aggregate, possibly mediated through phospholipase A2 formation of lysophosphatidyl from phosphatidylcholine (Yuan et al., 1995), attenuation of 12-lipooxygenase activity in platelets (Fujimoto et al., 1994) and increased NO production (see below). 2.4.3 Stimulation of Prostacyclin Synthesis HDL induces biosynthesis of prostacyclin, specifically via a cyclooxygenase-2-dependent mechanism involving the mitogen-activated protein kinase (MAPK) pathway (Vinals et al., 1999). Prostacyclin is a vasodilator prostaglandin synthesized by vascular smooth muscle cells. Its other atheroprotective actions include inhibition of platelet aggregation and adhesion, smooth muscle cell proliferation, leukocyte activation and adhesion, as well as reduction of CE accumulation in the vessel wall. 10 Chapter Literature Review I: Atherosclerosis and HDL 2.4.4 Modulation of Endothelial Adhesion An early event in atherosclerosis is the attachment of monocytes and lymphocytes but not neutrophils to the artery wall, triggered by minimally oxLDL. Cockerill et al. (1995) found that HDL modulates the mRNA and protein expression levels of cell adhesion molecules intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1) and E-selectin in stimulated vascular endothelial cells. E-selectin enables tethering and rolling of monocytes and lymphocytes along the endothelial surface while subsequent adhesion is mediated by ICAM-1 and VCAM-1. The marked inhibition is observed within the physiological range of HDL levels and is apparent with both native HDL and reconstituted discoidal HDL containing only ApoAI, phosphatidylcholine and free cholesterol. 2.4.5 Stimulation of Endothelial Nitric Oxide Synthase (eNOS) In endothelial cells, HDL binding to scavenger receptor BI (SR-BI) activates the tyrosine kinase receptor Src and phosphatidylinositol-3-kinase, triggering both the MAPK and Akt pathways and leading to phosphorylation of eNOS (Mineo and Shaul, 2003). eNOS activation generates NO which mediates a plethora of anti-atherogenic activities including vasorelaxation, attenuation of platelet adhesion and aggregation, inhibition of smooth muscle cell proliferation, reduced adhesion and migration of leukocytes to the vessel wall (Nofer et al., 2002). 2.4.6 Participation in Reverse Cholesterol Transport (RCT) Much of the anti-atherogenic property of HDL has been ascribed to its participation in RCT (Glomset, 1968). RCT is the pathway in which excess cholesterol from the peripheral cells is delivered to the liver for excretion in bile or for steroid hormone synthesis. In the non-growing adult animal, an amount of cholesterol equal to those 11 Chapter Literature Review I: Atherosclerosis and HDL synthesized and absorbed must be excreted daily. Humans synthesize about 10 mg cholesterol/day/kg (Dietschy et al., 1993). The appearance and accumulation of foam cells in the arterial wall, which are primarily macrophages engorged with CE, is a hallmark of atherosclerosis (Lusis, 2000). Scavenger receptors of class A and CD36 on the macrophage permit excessive uptake of modified lipoproteins (Linton and Fazio, 2001). Several steps can be identified in RCT: (i) cholesterol efflux; (ii) LCAT-mediated esterification of cholesterol; (iii) remodelling and HDL maturation in the plasma; (iv) delivery to liver and steroidogenic tissues. RCT is depicted schematically in Figure 2.1. The first step of RCT begins with cholesterol efflux, the movement of excess cellular cholesterol to external acceptors. Cholesterol efflux can proceed in both passive and active manners (Yancey et al., 2003). Aqueous diffusion of cholesterol, which occurs in all cells, is generally slow and inefficient, on the time scale of hours. SR-BI-facilitated efflux is more rapid but like passive diffusion, it proceeds via a concentration gradient and therefore cholesterol flux can proceed both ways. The principal cholesterol acceptors in aqueous diffusion and SRBI mediated efflux are phospholipid-containing molecules; in the former, the preferred acceptors are small, discoidal whereas SR-BI interacts with a wide range of acceptors such as HDL and LDL (Yancey et al., 2003). On the other hand, ABCA1 mediates cholesterol and phospholipid efflux to lipid-poor apolipoproteins (such as pre-β HDL regenerated from the remodelling activities in the plasma, free ApoAI or nascent HDL which are newly secreted by the liver and small intestine) in an energy-dependent and unidirectional manner (Yancey et al., 2003). Because ABCA1 has been identified as the molecular defect in Tangier disease, a disorder characterized by severe HDL-deficiency and impaired cholesterol efflux, it has been postulated that that ABCA1 accounts for the bulk of the cholesterol efflux in vivo. 12 Chapter Literature Review I: Atherosclerosis and HDL Following the efflux of cholesterol onto the surface of the nascent HDL particle, it is esterified by LCAT, an enzyme bound to HDL surface. The hydrophobic CE then partitions into the core, leaving the surface free to acquire more cholesterol, thus maintaining a cholesterol gradient from cells to HDL. As CE accumulates, the initially discoidal HDL is converted into the large, spherical HDL which is the predominant form of HDL in the vascular system. In the plasma, HDL undergoes further remodelling by several enzymes. CETP catalyzes the reciprocal exchange of CE and TG between HDL and TG-rich apoBcontaining lipoproteins (LDL, VLDL, chylomicron remnants). Another HDL-associated enzyme, PLTP, swaps CE of HDL for the phospholipids in TG-rich lipoproteins, regenerating pre-β HDL (van Tol, 2002). Hepatic lipase hydrolyses phospholipids and TG on HDL as well as VLDL remnants and LDL, generating smaller HDL particles (Santamarina-Fojo et al., 1998). Endothelial lipase, primarily a phospholipase AI, also remodels HDL to smaller particles (Choi et al., 2002). Much of the cholesterol initially received by HDL becomes re-partitioned to apoBcontaining lipoproteins which are subsequently taken up as whole particles by LDLRmediated endocytosis in the liver and steroidogenic tissues. The remaining CE in remnant HDL particles is catabolized by being selectively taken up by SR-BI in liver and steriodogenic tissues (Acton et al., 1996). Based on evidence from animal studies, it has been recently proposed that ABCA1 activity in the liver, and not peripheral macrophages, is a major source of plasma HDL-C (Haghpassand et al., 2001; Tall et al., 2001; Aiello et al., 2002; van Eck et al., 2002; Basso et al., 2003). This represents a major paradigm shift in the classical concept of RCT as a one-way pathway from the periphery to the liver. 13 Chapter Literature Review I: Atherosclerosis and HDL Figure 2.1 The reverse cholesterol transport (RCT) pathway. Nascent HDLs, secreted by the liver and small intestine, are discoidal in shape and devoid of core lipids with pre-β electrophoretic mobility. Excess cholesterol as well as PLs from peripheral cells are moved to the nascent HDLs in a process mediated by the ABCA1 transporter and ApoAI, and become converted into large spherical particles with a CE-rich core by LCAT. These spherical particles HDL2 and HDL3, are of α-electrophoretic mobility and form the bulk of plasma HDL-C. In one minor mechanism, CE from α-HDLs is directly delivered to the hepatocyte via selective uptake through SR-BI. In the second, major mechanism, CETP swaps the CEs from HDL with TG from ApoB-containing lipoproteins (VLDL, LDL), and the latter are subsequently taken up by LDLR-mediated endocytosis in the hepatocyte. In another remodelling activity in the plasma, PLTP, a product of the same gene family as CETP, transfers of PLs from ApoB-containing lipoproteins to HDL in exchange for CE. HL hydrolyses TGs and PLs in HDL, converting larger HDL2 to smaller HDL3. EL also hydrolyses the HDL-PL, generating lipid depleted HDL. CE=cholesterol ester, FC=free cholesterol, PL=phospholipids, TG=triglyerides, LCAT=lecithin-cholesterol acyltransferase, CETP=cholesterol ester transfer protein, PLTP=phospholipid transfer protein, EL=endothelial lipase, HL=hepatic lipase, HDL=high density lipoprotein, LDL=low density lipoprotein, VLDL=very low density lipoprotein, ApoAI=apolipoprotein AI, ApoB=apoliprotein B, ABCA1=ATP binding cassette transporter A1, LDLR=LDL receptor, SR-BI=scavenger receptor BI. 14 Chapter Literature Review I: Atherosclerosis and HDL 2.5 Genetics of HDL Although environmental and hormonal influences contribute a significant fraction of the phenotypic variation in HDL-C level, family studies show that genetic factors are important as well. For example, twin studies have indicated a high degree of heritability of HDL-C levels. These studies show a high correlation of HDL-C levels in monozygotic than dizygotic twins. In three different studies, the correlation of HDL-C levels in monozygotic twins ranged from 0.68 to 0.74 compared with only 0.34 to 0.46 in dizygotic twins (Feinleib et al., 1977; McGue et al., 1985; Austin et al., 1987). Other studies of familial aggregation of HDL-C levels have estimated the heritability of of HDL-C levels at 0.55, divided between genetic (0.36) and cultural (0.19) components. Together, these studies suggest a large genetic component in the determination of HDL-C variability. Numerous genes are known to regulate HDL function and metabolism. Candidate genes can be categorized in five groups (Wang and Paigen, 2002): (i) HDL-structural apolipoproteins (e.g. ApoAI, ApoAII, ApoCIII, ApoE); (ii) HDL-associated enzymes and transfer proteins (e.g. CETP, LCAT, PAFH, PLTP, PON); (iii) plasma- and cell-associated lipases (e.g. endothelial lipase, HL, LPL); (iv) cellular receptors and transporters (e.g. ABCA1, SR-BI); and (v) nuclear transcription factors (e.g. liver-X-receptor (LXR), retinoicX-receptor (RXR), PPAR). 2.6 Low HDL Genetic Disorders Despite the numerous candidate genes involved in the regulation of HDL metabolism, human pathology has only been consistently reported among a few in which structural alteration may cause low HDL (Genest et al., 1999). 15 Chapter Literature Review I: Atherosclerosis and HDL 2.6.1 ApoAI ApoAI is a potent, obligatory activator for LCAT activity in vivo as well an important mediator of cholesterol efflux. The mature human ApoAI is a 243 amino acid polypeptide synthesized as a prepro precursor in the liver and intestine and exists in a gene cluster with ApoCIII and ApoAIV on the long arm of chromosome 11q (Groenendijk et al., 2001). Generally, severe ApoAI deficiency owing to chromosomal aberrations or deletions leads to premature CAD. However, the impact of ApoAI genetic variants is more variable. Affected individuals with certain ApoAI missense gene mutations such as ApoAImilano and ApoAIparis show markedly reductions in HDL and ApoAI but may be free of signs of CAD despite their advanced age whereas others may be at increased CAD risk in the presence of other factors (Genest et al., 1999). Polymorphisms in ApoAI are also seen, with inconsistent associations (Groenendijk et al., 2001). Despite a large reduction in HDL-C, ApoAI-deficient mice remained relatively free of atherosclerosis (Li et al., 1993). Taken together, these findings show that deficiencies in ApoAI and HDL-C are by themselves inadequate predictors of atherosclerosis susceptibility. 2.6.2 LCAT LCAT transfers the sn-2 fatty acid of phosphatidylcholine to cholesterol, forming lysophosphatidylcholine and CE (Jonas, 2000). It is an essential player in RCT, helping to maintain a cholesterol gradient from the cell to HDL. Human genetic deficiency in LCAT is manifested as two clinically and biochemically distinct phenotypes (Kuivenhoven et al., 1997). In familial LCAT deficiency, esterification in all classes of lipoproteins is affected. Less common than familial LCAT deficiency is fish eye disease in which there is only a partial LCAT deficiency with absence of esterification activity in HDL but otherwise intact formation of CE from LDL-derived cholesterol. LCAT-knockout mice exhibit similar features (Ng et al., 1997). Despite the central role of LCAT in RCT, LCAT deficiency does 16 Chapter Literature Review I: Atherosclerosis and HDL not appear to be associated with protection against premature CAD in humans (Kuivenhoven et al., 1997). Over-expression of human LCAT gene in transgenic animals has been mixed, in part related to the type of animal model and extent of over-expression (Furbee et al., 2002). 2.6.3 ABCA1 and Tangier Disease In 1961, Donald Fredrickson and colleagues described Tangier disease in two siblings residing on the island of Tangier in Chespeake Bay off the coast of Virginia, USA. Tangier disease was the first HDL deficiency disorder described. Hallmark features of the disease include accumulation of CE in cells of the reticuloendothelial system, leading to the appearance of foam cells, and near absence of plasma HDL-C and ApoAI. Previous biochemical studies had shown aberrant cholesterol efflux to HDL in skin fibroblasts obtained from Tangier disease patients (Walter et al., 1994; Francis et al., 1995; Rogler et al., 1995). Since early investigations had ruled out defects in LCAT (Carlson et al., 1987) and ApoAI (Makrides et al., 1988) in these patients, a novel player involved in the first step of RCT was believed to underlie the disorder. In 1999, homozygous and compound heterozygous mutations in ABCA1 were identified in Tangier disease patients (Bodzioch et al., 1999; Brooks-Wilson et al., 1999; Rust et al., 1999), including the original kindred in whom the disease was first described (Remaley et al., 1999). Earlier work by Rust et al. (1998) had mapped the Tangier disease locus to chromosome 9q31, and subsequent finemapping by the same authors as well as Brooks-Wilson et al. (1999) led to the identification of the gene defect. ABCA1 was also confirmed to be the functional candidate using expression analysis (Langmann et al., 1999; Lawn et al., 1999). Heterozygous mutations in ABCA1 were also found in patients with familial hypoalphalipoproteinemia (FHA), a more common and less severe form of HDL deficiency (Brooks-Wilson et al., 1999; Marcil et al., 1999). The discovery of 17 Chapter Literature Review I: Atherosclerosis and HDL ABCA1 as the defect in these cholesterol efflux disorders represents a major advancement in the understanding of the RCT pathway. ABCA1 is a member of the ABC superfamily of transporters. Members of the ABC superfamily of transporters are involved in the energy-dependent transport of diverse substrates such as sugars, xenobiotics, ions, amino acids, peptides, vitamins, phospholipids and bile acids across membranes (Borst and Elferink, 2002). The eukaryotic ABC genes are organized either as full transporters containing two transmembrane (TM) domains and two nucleotide binding domains (NBDs), or as half transporters (Hyde et al. 1990). The latter must form either homodimers or heterodimers to constitute a functional transporter. ABC transporters can also form complexes with other proteins to act as channels. For example, ABCC8/SUR1 cooperates with KIR6.2 to act as ATP-dependent potassium channel. ABC genes are dispersed widely in eukaryotic genomes and are highly conserved between species, indicating that most of these genes have existed since the beginning of eukaryotic evolution. The genes can be divided into subfamilies based on structural similarity (half versus full transporters), order of the domains, and sequence homology in the NBD and TM domains. To date, there are seven mammalian ABC gene subfamilies encoding 49 ABC genes (http://nutrigene.4t.com/humanabc.htm). Eighteen ABC genes have been implicated in diverse human conditions including Tangier disease (ABCA1), cystic fibrosis (CFTR/ABCC7), sitosterolemia (ABCC5, ABCC8), adrenoleukodystrophy (ABCD1, ABCD2), several eye defects (ABCR/ABCA4), bile salt transport disorders (ABCB4, ABCB11), non-insulin-dependent diabetes mellitus (SUR1/ABCC8), immune deficiency (TAP1/ABCB2, TAP2/ABCB3) as well as multidrug resistance (MDR1/ABCB1, ABCC1-6, ABCG2). The ABCA1 gene spans about 149 kb on chromosome 9q31.1 and consists of 50 exons. It encodes an integral protein with a predicted size of 2261 amino acids 18 Chapter Literature Review I: Atherosclerosis and HDL (Santamarina-Fojo et al., 2000). Although the ABCA1 is ubiquitously expressed throughout the body, it shows the greatest activity in cells that accumulate cholesterol, consistent with the finding that derivatives of cholesterol are potent activators of ABCA1 gene transcription (Oram, 2002). Structurally the 250 kDa ABCA1 protein is organized as two similar halves arranged in tandem. Each half consists of a TM domain with six membrane spanning helices and a cytoplasmic NBD. The NBD carries the conserved Walker A and B motifs which are typically encountered in proteins that utilize ATP. Based on high sequence homology to ABCR (also known as ABCA4) in which mutations account for a number of retinal disorders, ABCA1 is predicted to have a cytoplasmic N-terminus and two large extracellular domains (ECDs) that are highly glycosylated and linked by disulphide bonds (Bungert et al., 2001). Two models have been proposed for the removal of cholesterol and phospholipids by the ABCA1 protein (Oram, 2003). In the exocytosis model, excess intracellular cholesterol is packaged in vesicles or rafts, perhaps in the Golgi apparatus, which then translocate to domains in the plasma membrane where ABCA1 is found. In the alternative retroendocytosis model, ABCA1- and apolipoprotein-containing vesicles endocytose to intracellular lipid deposits where ABCA1 promotes lipid transport into the vesicle lumen. The lipid is subsequently released by exocytosis. Despite severe reduction in HDL and ApoAI levels and massive CE accumulation in the peripheral tissues, two reports published prior to the time the ABCA1 gene defect in Tangier disease and FHA was identified found that CAD represents a significant clinical problem only in middle-aged and elderly Tangier disease patients (Schaefer et al., 1980; Serfaty-Lacrosniere et al., 1994). This apparent paradox is reinforced by animal studies showing no signs of atherosclerotic lesions in ABCA1-deficient mice fed on a regular or atherogenic diet. Moreover, there was also no further increase in atherosclerosis in 19 Chapter Literature Review I: Atherosclerosis and HDL ABCA1-deficient mouse that had been crossed to the atherosclerosis-prone ApoE- or LDLR-knockout mice (Aiello et al., 2002). The Wisconsin Hypo Alpha Mutant (WHAM) chicken, which harbours a naturally occurring missense mutation in ABCA1 (Attie et al., 2002), also shows no increase in atherosclerosis despite low plasma HDL and ApoAI levels (Poernama et al., 1992). Data from two transgenic mouse studies demonstrate that ABCA1 over-expression raises HDL-C (Vaisman et al., 2001; Singaraja et al., 2001). Joyce et al. (2002) provided first proof of a direct anti-atherogenic role for ABCA1: expression of human ABCA1 modulates aortic atherosclerosis outcome in a transgenic mouse. More recent observations in humans indicate a clear relationship with ABC1mediated cholesterol efflux and arterial-wall thickness, directly linking human ABCA1 function with protection from atherosclerosis (van Dam, et al., 2002). The combined results of these animal studies show that over-expression or a complete absence of ABCA1 need not always manifest as parallel changes in susceptibility to atherosclerosis. A plausible explanation is that any pro-atherogenic effect that might occur as a result of a defective ABCA1 is mitigated by a less atherogenic lipid profile, for instance, by a lowering in ApoB-containing lipoproteins. A relative lack of atherosclerosis risk has also been described in other rare genetically determined HDL deficiencies, such as familial LCAT deficiency syndromes (Kuivenhoven et al., 1997). Other explanations include differences in mouse strains and hepatic, not macrophage, expression of ABCA1 activity is an important source of HDL-C (Basso et al., 2003). 2.7 Non-Genetic Risk Factors for Low HDL Factors associated with low HDL-C include cigarette smoking, hypertension, obesity, physical inactivity and use of beta-adrenergic blockers (Barter et al., 2003). In addition, gender and hormones influence HDL-C levels. Adult males possess 20% higher levels of HDL-C than women; before puberty, boys and girls have equal HDL-C (Breslow, 1995). 20 Chapter Literature Review I: Atherosclerosis and HDL Exogenous estrogens are known to raise HDL-C (Krauss, 1982). Accordingly, simple lifestyle and pharmacological interventions can increase the levels of HDL-C. Some of these “environmental” components themselves have a significant genetic component, e.g. obesity. Therefore, gene-gene-environment as well as gene-environment interactions come into play in the regulation of HDL levels (Ordovas, 2002). 21 [...]... et al., 1997) Over-expression of human LCAT gene in transgenic animals has been mixed, in part related to the type of animal model and extent of over-expression (Furbee et al., 20 02) 2. 6.3 ABCA1 and Tangier Disease In 1961, Donald Fredrickson and colleagues described Tangier disease in two siblings residing on the island of Tangier in Chespeake Bay off the coast of Virginia, USA Tangier disease was... the ABCA1 protein (Oram, 20 03) In the exocytosis model, excess intracellular cholesterol is packaged in vesicles or rafts, perhaps in the Golgi apparatus, which then translocate to domains in the plasma membrane where ABCA1 is found In the alternative retroendocytosis model, ABCA1- and apolipoprotein-containing vesicles endocytose to intracellular lipid deposits where ABCA1 promotes lipid transport into... occurring missense mutation in ABCA1 (Attie et al., 20 02) , also shows no increase in atherosclerosis despite low plasma HDL and ApoAI levels (Poernama et al., 19 92) Data from two transgenic mouse studies demonstrate that ABCA1 over-expression raises HDL-C (Vaisman et al., 20 01; Singaraja et al., 20 01) Joyce et al (20 02) provided first proof of a direct anti-atherogenic role for ABCA1: expression of human... ABCB11), non-insulin-dependent diabetes mellitus (SUR1/ABCC8), immune deficiency (TAP1/ABCB2, TAP2/ABCB3) as well as multidrug resistance (MDR1/ABCB1, ABCC1-6, ABCG2) The ABCA1 gene spans about 149 kb on chromosome 9q31.1 and consists of 50 exons It encodes an integral protein with a predicted size of 22 61 amino acids 18 Chapter 2 Literature Review I: Atherosclerosis and HDL (Santamarina-Fojo et al., 20 00)... the ABCA1 is ubiquitously expressed throughout the body, it shows the greatest activity in cells that accumulate cholesterol, consistent with the finding that derivatives of cholesterol are potent activators of ABCA1 gene transcription (Oram, 20 02) Structurally the 25 0 kDa ABCA1 protein is organized as two similar halves arranged in tandem Each half consists of a TM domain with six membrane spanning... et al., 20 01) Despite a large reduction in HDL-C, ApoAI-deficient mice remained relatively free of atherosclerosis (Li et al., 1993) Taken together, these findings show that deficiencies in ApoAI and HDL-C are by themselves inadequate predictors of atherosclerosis susceptibility 2. 6 .2 LCAT LCAT transfers the sn -2 fatty acid of phosphatidylcholine to cholesterol, forming lysophosphatidylcholine and... of 17 Chapter 2 Literature Review I: Atherosclerosis and HDL ABCA1 as the defect in these cholesterol efflux disorders represents a major advancement in the understanding of the RCT pathway ABCA1 is a member of the ABC superfamily of transporters Members of the ABC superfamily of transporters are involved in the energy-dependent transport of diverse substrates such as sugars, xenobiotics, ions, amino... explanations include differences in mouse strains and hepatic, not macrophage, expression of ABCA1 activity is an important source of HDL-C (Basso et al., 20 03) 2. 7 Non-Genetic Risk Factors for Low HDL Factors associated with low HDL-C include cigarette smoking, hypertension, obesity, physical inactivity and use of beta-adrenergic blockers (Barter et al., 20 03) In addition, gender and hormones influence... subsequently taken up by LDLR-mediated endocytosis in the hepatocyte In another remodelling activity in the plasma, PLTP, a product of the same gene family as CETP, transfers of PLs from ApoB-containing lipoproteins to HDL in exchange for CE HL hydrolyses TGs and PLs in HDL, converting larger HDL2 to smaller HDL3 EL also hydrolyses the HDL-PL, generating lipid depleted HDL CE=cholesterol ester, FC=free... out defects in LCAT (Carlson et al., 1987) and ApoAI (Makrides et al., 1988) in these patients, a novel player involved in the first step of RCT was believed to underlie the disorder In 1999, homozygous and compound heterozygous mutations in ABCA1 were identified in Tangier disease patients (Bodzioch et al., 1999; Brooks-Wilson et al., 1999; Rust et al., 1999), including the original kindred in whom the . higher baseline, suggesting the statin therapy did not alter the risk of low HDL. In the Helsinki Heart Study, the randomized, double-blind primary intervention trial of gemfibrozil administration. phosphatidylethanolamine (Rye et al., 1999). 2. 2 .2 Laboratory Determination of HDL In routine biochemical analysis, HDL in plasma is assayed as the total cholesterol content (free cholesterol and CE) following. of human LCAT gene in transgenic animals has been mixed, in part related to the type of animal model and extent of over-expression (Furbee et al., 20 02) . 2. 6.3 ABCA1 and Tangier Disease In