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Review Article |
Dyslipoproteinemia and Atherosclerosis Research Unit, National Institute for Health and Medical Research, Hôpital de la Pitié, Paris, France
Abstract
Abstract I. Introduction A. Inflammation and Oxidative Stress in the Progression of Atherosclerosis II. Functional High-Density Lipoprotein A. Structure, Composition, and Heterogeneity B. Metabolism C. Biological Activities 1. Cholesterol Efflux Capacity. 2. Antioxidative Activity. 3. Anti-Inflammatory Activity. 4. Antiapoptotic, Vasodilatory, Antithrombotic, and Anti-Infectious Activities. III. Functionally Defective High-Density Lipoprotein in Dyslipidemic and Inflammatory States A. Altered High-Density Lipoprotein Composition and Enzymatic Activities in Dyslipidemic and Inflammatory States 1. Apolipoproteins. 2. Enzymes with Antioxidative and Anti-Inflammatory Properties. 3. Lipid Components. B. Abnormal High-Density Lipoprotein Metabolism in Dyslipidemic and Inflammatory States C. Impaired High-Density Lipoprotein Biological Activities in Dyslipidemic and Inflammatory States 1. Cholesterol Efflux Capacity. 2. Antioxidative Activity. 3. Anti-Inflammatory Activity. IV. Physiological Relevance of Defective High-Density Lipoprotein Function in Dyslipidemia and Metabolic Disease V. Functionally Defective Small, Dense High-Density Lipoprotein as a Therapeutic Target A. Cholesteryl Ester Transfer Protein Inhibitors B. Niacin C. Fibrates D. Statins E. Reconstituted High-Density Lipoprotein F. Apolipoprotein-Mimetic Peptides G. Combination Therapy VI. Conclusions
High-density lipoproteins (HDL) possess key atheroprotective biological properties, including cellular cholesterol efflux capacity, and anti-oxidative and anti-inflammatory activities. Plasma HDL particles are highly heterogeneous in physicochemical properties, metabolism, and biological activity. Within the circulating HDL particle population, small, dense HDL particles display elevated cellular cholesterol efflux capacity, afford potent protection of atherogenic low-density lipoprotein against oxidative stress and attenuate inflammation. The antiatherogenic properties of HDL can, however be compromised in metabolic diseases associated with accelerated atherosclerosis. Indeed, metabolic syndrome and type 2 diabetes are characterized not only by elevated cardiovascular risk and by low HDL-cholesterol (HDL-C) levels but also by defective HDL function. Functional HDL deficiency is intimately associated with alterations in intravascular HDL metabolism and structure. Indeed, formation of HDL particles with attenuated antiatherogenic activity is mechanistically related to core lipid enrichment in triglycerides and cholesteryl ester depletion, altered apolipoprotein A-I (apoA-I) conformation, replacement of apoA-I by serum amyloid A, and covalent modification of HDL protein components by oxidation and glycation. Deficient HDL function and subnormal HDL-C levels may act synergistically to accelerate atherosclerosis in metabolic disease. Therapeutic normalization of attenuated antiatherogenic HDL function in terms of both particle number and quality of HDL particles is the target of innovative pharmacological approaches to HDL raising, including inhibition of cholesteryl ester transfer protein, enhanced lipidation of apoA-I with nicotinic acid and infusion of reconstituted HDL or apoA-I mimetics. A preferential increase in circulating concentrations of HDL particles possessing normalized antiatherogenic activity is therefore a promising therapeutic strategy for the treatment of common metabolic diseases featuring dyslipidemia, inflammation, and premature atherosclerosis.
According to the recent estimates of the World Health Organization, approximately one-third of all deaths (16.7 million people) around the globe resulted from cardiovascular (CV1) disease in 2002 (World Health Organization, 2004
). As shown in the recent INTERHEART study, which enrolled 29,972 subjects in 52 countries worldwide, the most strongly predictive CV risk factors for myocardial infarction were dyslipidemia, smoking, hypertension, diabetes, abdominal obesity, psychosocial factors, consumption of fruits, vegetables, and alcohol, and lack of regular physical activity (Yusuf et al., 2004). Collectively, these factors accounted for most (
90%) of the risk of myocardial infarction in both sexes and at all ages in all regions.
Atherosclerosis represents the pathological process that typically underlies CV morbidity and mortality, formation of plaques in the intima and media of the arterial wall. Such atherosclerotic plaques result from the progressive accumulation of cholesterol and diverse lipids in native and oxidized forms, extracellular matrix material, and inflammatory cells. Atherogenic dyslipidemia, a highly prominent CV risk factor, is intimately associated with premature atherosclerosis and corresponds to an imbalance between excess circulating levels of cholesterol in the form of pro-atherogenic apolipoprotein (apo) B-containing lipoproteins compared with subnormal levels of antiatherogenic apoA-I-containing lipoproteins (Fig. 1). Indeed, apoB is the predominant protein component of proatherogenic, cholesterol-rich low-density lipoprotein (LDL), triglyceride (TG)-rich very-low density lipoproteins (VLDL), VLDL remnants and intermediate-density lipoprotein (IDL), whereas apoA-I is the major protein component of antiatherogenic high-density lipoprotein (HDL). In the INTERHEART study, dyslipidemia was assessed as an elevated ratio of plasma levels of proatherogenic apoB to antiatherogenic apoA-I (5:1) (Yusuf et al., 2004), and as such, represented a direct estimate of atherogenic potential in any individual.
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Elevated circulating concentrations of LDL-cholesterol (LDL-C) occur frequently as hypercholesterolemia, a common form of atherogenic dyslipidemia (Wilson, 1990). LDL is the major vehicle for transport of cholesterol not only to peripheral tissues but also to the arterial wall (Lusis, 2000); indeed, ionic interaction of positively charged domains of apoB with negatively charged proteins of the extracellular matrix, including proteoglycans, collagen, and fibronectin, leads to intimal retention of apoB-containing lipoproteins, a major initiating factor in atherogenesis (Khalil et al., 2004).
Among factors other than LDL-C that are associated with dyslipidemia, a low level of HDL-cholesterol (HDL-C) is now most recognized (Gotto and Brinton, 2004). Several prospective epidemiological studies, including the Framingham Heart Study, US Physicians' Health Study, Prospective Cardiovascular Münster (PROCAM) Study, and Atherosclerosis Risk in Communities (ARIC) Study, have found that low serum HDL-C concentrations (defined as <40 mg/dl in both sexes or as <40 mg/dl in men and <50 mg/dl in women) (Chapman et al., 2004)) constitute an independent risk factor for coronary heart disease (CHD) in both nondiabetic and diabetic subjects (Maron, 2000; Sharrett et al., 2001; Gotto and Brinton, 2004). Moreover, low HDL-C is characteristic of atherogenic dyslipidemia and increased CV risk in patients with metabolic diseases such as type 2 diabetes and metabolic syndrome (MetS). In this context, it is of special relevance that the World Health Organization has estimated that the population of individuals with type 2 diabetes will have increased worldwide to 250 millions or more by 2025 (World Health Organization, 2004).
Prospective studies have revealed that CHD risk is elevated by 3% in women and 2% in men for each decrement of 1 mg/dl in HDL-C (Wilson, 1990). Conversely, a decreased risk of CV events is frequently observed in subjects with elevated HDL-C levels (Maron, 2000; Doggen et al., 2004; Gotto and Brinton, 2004); in addition, high concentrations of HDL-C (>60 mg/dl) are typically associated with longevity (Barzilai et al., 2003; Barter, 2004). The prevalence of low HDL-C levels can vary from 20% in a general population to up to 60% in patients with established CHD (Franceschini, 2001). Not only are low HDL-C levels associated with an increased incidence of CHD but also with a greater risk for carotid atherosclerosis and ischemic stroke mortality and with a more aggressive progression of angiographically defined coronary artery disease (CAD) (Maron, 2000; Gotto and Brinton, 2004). Finally, it is noteworthy that in the recent Myocardial Ischemia Reduction with Aggressive Cholesterol Lowering (MIRACL) trial in patients with acute coronary syndromes treated with atorvastatin, baseline HDL-C levels, rather than those of LDL-C, predicted the occurrence of CV events (Olsson et al., 2005).
A. Inflammation and Oxidative Stress in the Progression of Atherosclerosis
The imbalance between circulating levels of cholesterol transported in HDL relative to that in apoB-containing particles is intimately associated with induction of both endothelial dysfunction and oxidative stress in the arterial wall, which are in turn closely related to inflammation (Chisolm and Steinberg, 2000; Lusis, 2000); as a result, dyslipidemia, oxidative stress, and inflammation are closely interrelated in the development of atherosclerosis.
Oxidative stress is defined as an imbalance between prooxidant and antioxidant factors in favor of prooxidants and is central to the pathophysiology of atherosclerosis and CV disease (Fig. 2). Analysis of plaque composition has revealed products of protein and lipid oxidation, such as oxidized, chlorinated, and nitrated amino acids, lipid hydroperoxides (LOOH), short-chain aldehydes, oxidized phospholipids (PL), F2
-isoprostanes, and oxysterols, thereby suggesting the presence of local oxidative stress (Heinecke, 1998). The preferential retention of LDL in the arterial wall makes this lipoprotein a major substrate for oxidation by prooxidants produced by arterial wall cells. Various oxidative systems potentially contribute to LDL oxidation in vivo, and these include NAD(P)H oxidases, xanthine oxidase, myeloperoxidase, uncoupled nitric oxide synthase (NOS), lipoxygenases, and the mitochondrial electron transport chain (Madamanchi et al., 2005; Mueller et al., 2005). Accordingly, reactive oxygen, chlorine and nitrogen species, and lipid-derived free radicals are major prooxidants involved in the formation of oxidized LDL (oxLDL) in vivo. Significantly, production of both chlorine- and nitrogen-containing prooxidants is increased at sites of inflammation (Heinecke, 1998), suggesting that focal inflammation significantly contributes to the initiation of LDL oxidation at early stages of plaque formation. Consistent with the role of oxidative stress and oxidative modification of LDL in atherosclerosis, both urinary levels of F2-isoprostanes, currently the most robust and integrative marker of oxidative stress in vivo in humans (Morrow, 2005) and plasma levels of oxLDL constitute strong and independent risk factors for CHD (Schwedhelm et al., 2004; Meisinger et al., 2005).
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Inflammation is a systemic body response aimed to decrease the toxicity of harmful agents and repair damaged tissue. Chronic inflammation, which may be measured as circulating levels of an acute-phase protein, such as C-reactive protein (CRP), represents a major CV risk factor (Ridker et al., 2004b; Willerson and Ridker, 2004; Verma et al., 2005). A key feature of the inflammatory response involves activation of phagocytic cells involved in host defense, which produce an oxidative burst of reactive oxygen, chlorine, and nitrogen species, with subsequent creation of a highly prooxidative environment to combat invading pathogens. Local and systemic infections, arterial wall injury, and excessive retention of LDL may all potentiate activation of macrophages in the arterial wall, thereby triggering excessive production of prooxidant species (Hansson, 2005). As a result, oxidation of proteoglycan-bound LDL may occur in the extracellular space of the arterial intima (Memon et al., 2000).
OxLDL particles exhibit multiple atherogenic properties, which include uptake and accumulation in macrophages, as well as proinflammatory, immunogenic, apoptotic, and cytotoxic activities (Chisolm and Steinberg, 2000). In contrast to unmodified LDL, oxLDL is taken up through macrophage scavenger receptor pathways that are not down-regulated by excess ligand and lead to the formation of cholesterol-loaded foam cells, characteristic components of atherosclerotic plaques. The proinflammatory activities of oxLDL include chemoattractant action on circulating monocytes, induction of the expression of adhesion molecules on endothelial cells, promotion of monocyte differentiation into macrophages, induction of the production and release of proinflammatory cytokines and chemokines from macrophages, and inhibition of macrophage motility (Chisolm and Steinberg, 2000; Lusis, 2000). Most of the proinflammatory properties of oxLDL arise from products of LDL lipid peroxidation, such as 1-palmitoyl-2(5-oxovaleroyl)-sn-glycero-3-phosphorylcholine, 1-palmitoyl-2-glutaroyl-sn-glycero-3-phosphorylcholine, cholesteryl linoleate hydroperoxide, 7
-hydroperoxycholesterol, hydroxyoctadecadienoic acid, and 4-hydroxynonenal (Chisolm and Steinberg, 2000; Lusis, 2000; Van Lenten et al., 2001a). As a result, LDL oxidation further propagates the inflammatory process in the arterial wall, thereby accelerating atherogenesis (Lusis, 2000). Atherosclerosis can therefore be regarded as a chronic inflammatory disease of the arterial wall mediated by oxLDL in concert with a spectrum of additional proinflammatory agents.
HDL particles are distinguished from atherogenic apoB-containing lipoproteins by their capacity to exert a wide spectrum of antiatherogenic biological activities, including 1) their capacity to mediate cellular cholesterol efflux by acting as primary acceptors, thereby facilitating reverse cholesterol transport (RCT) from the arterial wall and peripheral tissues to the liver, 2) the protection of LDL against oxidative stress, 3) anti-inflammatory actions on arterial wall cells, and 4) antiapoptotic, 5) vasodilatory, 6) antithrombotic, and 7) anti-infectious activities. In this review, we will consider recent evidence for the heterogeneity of the atheroprotective properties of HDL particle subpopulations with emphasis on their ability both to protect against accumulation of lipids and to attenuate oxidative stress and inflammation in the arterial wall. Furthermore, new findings on functionally defective HDL will be discussed in the context of metabolic diseases associated with elevated CV risk; these data indicate that the potent antiatherogenic activities of small, dense HDL particles are impaired in the dyslipidemic and inflammatory state associated with type 2 diabetes and MetS. Finally, we will critically appraise innovative therapeutic strategies to normalize defective functionality of small, dense HDL particles; these exciting developments open new horizons for the treatment of atherogenic dyslipidemia in metabolic disease.
II. Functional High-Density Lipoprotein
A. Structure, Composition, and Heterogeneity
Functional plasma HDL are spherical or discoidal particles of high hydrated density (1.063-1.21 g/ml) due to elevated protein content (>30% by weight) compared with other lipoproteins (Fig. 3) (Asztalos and Schaefer, 2003; Barter et al., 2003b). Discoidal HDL are small particles consisting primarily of apoA-I embedded in a lipid monolayer constituted of PL and free cholesterol (Segrest et al., 1999, 2000). Spherical HDL are larger and additionally contain a hydrophobic core formed by cholesteryl esters (CE) and small amounts of TG. ApoA-I (molecular mass 28 kDa) is the major structural HDL apolipoprotein and accounts for 
70% of total HDL protein, whereas the second major HDL apolipoprotein, apoA-II, represents 20%. Minor HDL protein components (typically <10% of the HDL protein moiety) include apoE, apoA-IV, apoA-V, apoJ, apoC-I, apoC-II, and apoC-III (Asztalos and Schaefer, 2003; Barter et al., 2003b; Karlsson et al., 2005). In small discoidal HDL, two molecules of apoA-I adopt a "double belt" orientation with their helixes oriented parallel to the plane of the disc and perpendicular to the lipid acyl chains in such a way that they wrap around the lipid bilayer disc forming two stacked rings in an antiparallel orientation (Segrest et al., 1999, 2000; Silva et al., 2005); furthermore, apoA-I molecules appear to slide in relation to each other between two stable conformations (Silva et al., 2005). Plasma HDL particles also carry enzymes involved in lipid metabolism, including lecithin/cholesterol acyltransferase (LCAT), enzymes with plausible antioxidative activities, such as platelet-activating factor-acetyl hydrolase (PAF-AH, also called lipoprotein-associated phospholipase A2), paraoxonase 1 (PON1) and glutathione selenoperoxidase (GSPx) (Navab et al., 2004b), and other proteins and peptides, such as serum amyloid A (SAA), a major positive acute-phase reactant (Uhlar and Whitehead, 1999), -1-antitrypsin, a potent inhibitor of serine proteinases (Karlsson et al., 2005), or amyloid-, the principal constituent of senile plaques in Alzheimer's disease (Kontush, 2004).
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Plasma HDL particles are highly heterogeneous in their physicochemical properties, metabolism, and biological activity (Fig. 3) (Asztalos and Schaefer, 2003; Barter et al., 2003b). Such heterogeneity results from differences in relative contents of apolipoproteins and lipids in HDL and is intimately related to the amphipathic helical structure of human apoA-I (Reschly et al., 2002; Maiorano et al., 2004); these helixes possess a hinge domain that allows apoA-I to switch between two conformations corresponding to HDL particles of different size. When fractionated by ultracentrifugation, human HDL is typically separated into two major subfractions, HDL2 (d 1.063-1.125 g/ml) and HDL3 (d 1.125-1.21 g/ml) (Chapman et al., 1981). Nondenaturing polyacrylamide gradient gel electrophoresis has been used to separate HDL into five distinct subpopulations of decreasing size, HDL2b, 2a, 3a, 3b, and 3c (Anderson et al., 1977); equivalent subpopulations can be quantitatively isolated using isopycnic density gradient ultracentrifugation (Fig. 3) (Tall et al., 1982; Goulinet and Chapman, 1997; Guerin et al., 2000a). Other separation methods, such as two-dimensional electrophoresis, allow identification of more than 10 HDL subspecies in which spherical -HDL predominate (Asztalos et al., 1993; Asztalos and Schaefer, 2003); each subspecies may, however, be heterogeneous in physicochemical properties, as in the case of ultracentrifugally isolated subfractions. HDL can also be immunoseparated on the basis of apolipoprotein composition into particles containing only apoA-I (LpA-I) and both apoA-I and apoA-II (LpA-I/A-II) (Duriez and Fruchart, 1999). In most human subjects, apoA-I is distributed approximately equally between LpA-I and LpA-I/A-II, whereas virtually all apoA-II is in LpA-I/A-II. Finally, ultrafiltration (Atmeh, 1990; Atmeh and Abd Elrazeq, 2005) and size-exclusion chromatography (Nanjee and Brinton, 2000) allow isolation of small, protein-rich HDL particles of low molecular mass (40-70 kDa). Given the complexity of HDL particle heterogeneity, small, dense HDL will be defined for present purposes as lipid-poor and protein-rich discoidal and spherical HDL particles of small size (
9 nm), low molecular mass (200 kDa), and high density (1.125-1.24 g/ml). Depending on the fractionation method, small, dense HDL may include HDL3a, 3b, and 3c and very high-density lipoprotein separated by ultracentrifugation and pre--HDL separated by gradient gel electrophoresis.
On a particle basis, HDL are the most numerous per unit volume of plasma and are present at the highest (micromolar) levels compared with other lipoproteins. Concentrations of major HDL2 and HDL3 subfractions typically are in the range of 2 to 6 µM corresponding to 50 to 200 mg total mass/dl (Kontush et al., 2003, 2004, 2005; Hansel et al., 2004; Nobecourt et al., 2005).
The clinical relevance of circulating levels of individual HDL subfractions to atherosclerosis and CV disease is, however, unclear. Concentrations of HDL2-C and HDL3-C as estimates for plasma levels of the two major HDL subfractions were measured in several studies, which differed in separation methods (polyanion precipitation versus ultracentrifugation). Conflicting results were obtained, with evidence that either HDL2-C or HDL3-C constitutes a strong predictor of CHD or CV risk factors (Johansson et al., 1991; Drexel et al., 1992, 1994, 1996; Skinner, 1994; Robins et al., 2001; Alagona et al., 2002; Barter et al., 2003a; Yu et al., 2003; Desai et al., 2005). Furthermore, plasma levels of either large (Rosenson et al., 2002) or small (Mackey et al., 2002) HDL were reported to be associated with the progression of coronary atherosclerosis. Similarly controversial is the clinical significance of pre--HDL, pre--HDL, and LpA-I/A-II levels. By contrast, plasma levels of 1-HDL and LpA-I are typically associated with protection from atherosclerosis (Duriez and Fruchart, 1999; Asztalos et al., 2003; Asztalos and Schaefer, 2003).
Discordance in these data reflects complex relationships between HDL subfractions separated by different methods. For example, immunoisolated LpA-I/A-II is found predominantly in the HDL3 density range, whereas LpA-I is a prominent component of both HDL2 and HDL3 (Duriez and Fruchart, 1999). On the other hand, -migrating HDL predominate in both HDL2 and HDL3 subfractions, whereas pre--HDL coisolates with small, dense HDL particles (Asztalos and Schaefer, 2003). Another important example concerns ultracentrifugally isolated small, dense HDL3c, which does not precisely correspond to small HDL subpopulations as determined by other methods. Human HDL3c represents a minor subfraction accounting for approximately 6% of total HDL mass and 10% of apoA-I (Kontush et al., 2003, 2004, 2005; Hansel et al., 2004; Nobecourt et al., 2005). In two-dimensional electrophoresis, HDL3c reveals further heterogeneity and produces multiple signals corresponding to small 3-, pre-3- and pre-1-HDL (S. Chantepie, A. Kontush, and M. J. Chapman, unpublished data). By contrast, small HDL 3, pre-3, and pre-1 subfractions measured by two-dimensional electrophoresis in whole plasma account for approximately 37, 4, and 12% of apoA-I, respectively (Asztalos et al., 2004a); moreover, 3 together with 2 represent two major HDL subfractions in normolipidemic subjects. It is essential to emphasize that routine clinical measurement of plasma HDL-C primarily reflects levels of large, cholesterol-rich HDL particles and frequently lacks sensitivity to detect small cholesterol-poor HDL.
Spherical plasma HDL are mature particles generated by intravascular processes from lipid-free apoA-I or lipid-poor pre--HDL (Fig. 4) (Rye and Barter, 2004). These small HDL precursors are produced as nascent HDL by the liver or intestine, are also released as surface fragments from lipolysed TG-rich lipoproteins (VLDL and chylomicrons), and finally may be generated during the interconversion of HDL3 and HDL2 (von Eckardstein et al., 2001). Small nascent HDL are unstable and readily acquire lipids (Atmeh and Abd Elrazeq, 2005); their initial lipidation occurs at cellular membranes via the ATP-binding cassette transporter (ABC) A1-mediated efflux of cholesterol and PL from cells (Fig. 4) (Oram, 2002). ABCA1 is a major player in HDL metabolism; indeed, genetic defects in ABCA1 as occur in Tangier disease may result in low HDL-C levels, with cholesterol accumulation in peripheral tissues and premature atherosclerosis (Oram, 2002).
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Subsequent LCAT-mediated esterification of cell-derived cholesterol generates large spherical HDL2 particles with a neutral lipid core of CE and TG (Jonas, 2000); such particles undergo further remodeling via particle fusion and surface remnant transfer mediated by phospholipid transfer protein (PLTP) (van Tol, 2002). Large HDL2 can be converted in turn to small HDL3 upon cholesteryl ester transfer protein (CETP)-mediated transfer of CE from HDL to apoB-containing lipoproteins, upon scavenger receptor type BI (SR-BI)-mediated selective uptake of CE by the liver and steroidogenic organs and hepatic lipase (HL) and upon endothelial lipase-mediated hydrolysis of core TG (Fig. 4) (von Eckardstein et al., 2001). When CETP-mediated transfer of CE occurs between HDL and TG-rich lipoproteins, TG-rich HDL are generated (Le Goff et al., 2004), which can be further hydrolyzed by HL to small, TG-rich HDL particles (Santamarina-Fojo et al., 2004). The concerted action of CETP and HL promotes reduction in HDL size, formation of lipid-poor HDL particles and shedding from HDL of lipid-free apoA-I, which can interact with ABCA1 in the next lipidation cycle (Clay et al., 1992). HDL lipids are catabolized either separately from HDL proteins by selective uptake or via CETP transfer or as holoparticles primarily in the liver, via uptake through LDL receptors for apoE-containing HDL and through hitherto unidentified receptors for HDL holoparticles.
HDL particles possess multiple antiatherogenic activities (Stein and Stein, 1999; Nofer et al., 2002; Assmann and Nofer, 2003; Assmann and Gotto, 2004; Navab et al., 2004b). The central role of HDL in cellular cholesterol efflux and RCT is considered to form a basis for the capacity of HDL to attenuate atherogenesis (von Eckardstein et al., 2001; Nissen et al., 2003). However, compelling evidence has emerged that additional dimensions of the antiatherogenic action of HDL may be of major physiological and pathological relevance (Nofer et al., 2002; Assmann and Nofer, 2003; Assmann and Gotto, 2004; Navab et al., 2004b).
1. Cholesterol Efflux Capacity.
The cholesterol efflux capacity of HDL particles is related to their ability to remove cholesterol from membranes of peripheral cells and particularly macrophages and foam cells via interaction with the ABCA1 and ABCG1 transporters and/or SR-BI receptor. Lipid-free apoA-I, apoA-II, apoE, and other HDL apolipoproteins induce fast, saturable, unidirectional and LCAT-independent efflux of cellular cholesterol and PL (von Eckardstein et al., 2001; Lewis and Rader, 2005); as a result, HDL particles efficiently acquire cholesterol in the extravascular compartment (Nanjee et al., 2001). ApoA-I is thought to play a central role in cholesterol transport from macrophages to the liver, consistent with the demonstration of accelerated RCT in mice overexpressing human apoA-I (Zhang et al., 2003); apoA-II is also able to act as as a primary acceptor and to efficiently remove cholesterol from macrophages in vivo (Rotllan et al., 2005).
Apolipoprotein-mediated lipid efflux involves specific interactions with membrane proteins, desorption of membrane lipids from caveolae, lipidation of lipid-free apolipoproteins and production of small, lipid-poor HDL (Rothblat et al., 1999; von Eckardstein et al., 2001). Lipid-free apolipoproteins remove cholesterol and PL from macrophages, aortic smooth muscle cells, and normal human skin fibroblasts but not from fibroblasts of patients with Tangier disease (Brousseau et al., 2000; Oram, 2000). Defective ABCA1 transporter function in Tangier disease has provided clear evidence that ABCA1 has a central role in lipid efflux mediated by lipid-poor apolipoproteins. In support of this mechanism, apoA-I-mediated cholesterol efflux is severely decreased by inhibition of ABCA1 with either antisense oligonucleotides or pharmacological compounds but is increased by the overexpression of ABCA1 (Oram, 2002). Thus, ABCA1 is a pivotal regulator of cellular cholesterol efflux and of the lipidation of apoA-I, a key step in formation of mature, spherical HDL particles.
ABCA1 has two highly conserved cytoplasmic ATP binding cassettes and two transmembrane domains, each of which consists of six membrane-spanning segments (Langmann et al., 1999; Santamarina-Fojo et al., 2000). It has been suggested that ABCA1 forms a channel within the plasma membrane through which cholesterol and PL are transferred ("flopped") from the inner to the outer leaflet of the plasma bilayer membrane (Hamon et al., 1997, 2000). There the lipids may be picked up by lipid-free apolipoproteins or lipid-poor particles, which bind to ABCA1 (Oram et al., 2000; Wang et al., 2000).
In addition to ABCA1, there are several other sterol-regulated ABC transporters, including ABCG1 and ABCG4, which are involved in cholesterol efflux from macrophages to mature HDL2 and HDL3 particles (Nakamura et al., 2004; Wang et al., 2004; Kennedy et al., 2005). Within the plasma membrane, ABCG1 redistributes cell cholesterol to domains that interact preferentially with mature HDL particles but not with lipid-poor apolipoproteins (Vaughan and Oram, 2005). The relative quantitative importance of cholesterol efflux mediated by ABCA1 compared with ABCG1 in macrophages remains unclear.
In contrast to lipid-free apolipoproteins, lipid-containing HDL particles induce both specific and nonspecific forms of cholesterol efflux (von Eckardstein et al., 2001). Nonspecific cholesterol efflux can be also mediated by PL vesicles, synthetic cyclodextrins, albumin or partially proteolysed HDL; it is slow, unsaturable, and bidirectional and thus appears to occur by aqueous diffusion (Rothblat et al., 1999; von Eckardstein et al., 2001). It has been suggested that SR-BI mediates the bidirectional flux between mature HDL and plasma membranes through the binding of HDL particles and subsequent reorganization of lipids within cholesterol- and caveolae-rich domains in the plasma membrane (de la Llera-Moya et al., 1999; Yancey et al., 2004). The PL content of HDL is an important determinant of such SR-BI-mediated cholesterol efflux (Yancey et al., 2000).
Another mechanism implicated in HDL-mediated cholesterol efflux is retroendocytosis, i.e., the uptake of HDL into clathrin-coated endosomes followed by intracellular enrichment with lipids and resecretion (Heeren et al., 1999; Takahashi and Smith, 1999). Finally, HDL-mediated cholesterol efflux from macrophages may be facilitated by apoE secretion (Mazzone, 1996). Indeed, macrophage-derived apoE can associate with HDL and improve its cholesterol acceptor properties.
Distinct cholesterol efflux properties of lipid-free and lipid-containing HDL are indicative of functional heterogeneity of HDL particles. Indeed, a decrease in the lipid content of HDL is generally thought to increase its capacity to remove cellular cholesterol (Ohta et al., 1995; Sasahara et al., 1998); small, dense, lipid-poor, protein-rich HDL particles are therefore considered to represent more efficient cholesterol acceptors compared with their large, light, lipid-rich, protein-poor counterparts (Asztalos et al., 1997). For example, small, lipid-poor HDL predominate in rabbits expressing human apoA-I; in parallel, the cholesterol efflux capacity of rabbit serum increases (Duverger et al., 1996a,b).
Interestingly, pre-1-HDL, the initial product of apoA-I lipidation, is not essential for cellular cholesterol efflux (Sviridov et al., 2002), thereby suggesting that lipid-free, rather than lipid-poor, apolipoproteins function as primary cholesterol acceptors (Asztalos et al., 1997). Lipid-free and/or lipid-poor HDL apolipoproteins induce cholesterol uptake via interaction with ABCA1; consistent with this observation, plasma levels of small pre-1-HDL particles correlate with serum capacity to induce ABCA1-mediated cholesterol efflux from J774 macrophages (Asztalos et al., 2005). Conversely, large, lipid-rich HDL particles appear to represent a better ligand for cellular uptake of CE mediated by SR-BI compared with small, lipid-poor HDL (de Beer et al., 2001; Thuahnai et al., 2004), consistent with the role of these particles in RCT from peripheral cells to the liver (von Eckardstein et al., 2001; Asztalos et al., 2005).
2. Antioxidative Activity.
HDL antioxidative activity is typically observed as inhibition of LDL oxidation by HDL; LDL is thought to represent the major physiological target of HDL antioxidative action in vivo (Van Lenten et al., 2001a; Navab et al., 2004b). HDL is also able to inhibit generation of reactive oxygen species (ROS) in vitro under conditions of cell culture (Robbesyn et al., 2003; Lee et al., 2005) and in vivo in a rabbit model of acute arterial inflammation (Nicholls et al., 2005b). In addition, inhibitory actions of HDL on LDL oxidation have been reported in vitro upon their coincubation (Parthasarathy et al., 1990) and in vivo upon HDL injection (Klimov et al., 1993). HDL potently protects both lipid and protein moieties of LDL and inhibits accumulation of various oxidation products in LDL, including oxidized PL and short-chain aldehydes (Van Lenten et al., 2001a; Navab et al., 2004b).
The antioxidative activity of HDL is related to the presence of several apolipoproteins and enzymes with antioxidative properties in HDL particles. Apolipoproteins that possess antioxidative activity include apoA-I, apoE, apoJ, apoA-II, and apoA-IV. It appears that a major component of the antioxidative activity of HDL can be ascribed to apoA-I which can prevent and/or delay LDL oxidation by removing oxidized PL, including 1-palmitoyl-2(5-oxovaleroyl)-sn-glycero-3-phosphorylcholine and 1-palmitoyl-2-glutaroyl-sn-glycero-3-phosphorylcholine, from LDL and from arterial wall cells (Navab et al., 2000a,b). The capacity of apoA-I to remove oxidized lipids is not specific for arterial wall cells, because similar effects have been reported for erythrocytes and astrocytes (Klimov et al., 2001; Ferretti et al., 2003, 2004). Circulating HDL accumulate LOOH and have been proposed to function as a "sink" for oxidized lipids (Bowry et al., 1992), ensuring their efficient elimination from the circulation through the liver.
ApoE possesses established antiatherosclerotic activity, which is normally ascribed to its lipid transport properties (Davignon, 2005). However, the action of apoE goes beyond such activity. Indeed, apoE possesses distinct antioxidative properties (Miyata and Smith, 1996) and can promote regression of atherosclerosis independently of lowering plasma cholesterol levels (Thorngate et al., 2000; Tangirala et al., 2001; Raffai et al., 2005). HDL-associated apoJ can inhibit oxidation of LDL by artery wall cells (Navab et al., 1997); in addition, apoJ is cytoprotective at low physiological levels (Trougakos et al., 2005). The beneficial actions of apoJ may be related to its ability to maintain integrity of membrane and lipoprotein lipids via its hydrophobic-binding domains (Jordan-Starck et al., 1992). Antioxidative properties have also been reported for apoA-II (Boisfer et al., 2002) and apoA-IV (Ostos et al., 2001). The capacity of apoA-II to protect LDL from oxidation is, however, questionable, given the fact that overexpression of human apoA-II in dyslipidemic mice accelerates atherosclerosis, increases aortic accumulation of oxLDL, and reduces antioxidative activity of HDL (Ribas et al., 2004; Rotllan et al., 2005). Such proatherogenic actions of apoA-II may be related to the displacement of antiatherogenic apoA-I and PON1 by apoA-II from HDL particles (Ribas et al., 2004). Finally, HDL is able to function as a preventive antioxidant through its capacity to bind transition metal ions (Kunitake et al., 1992), which in free form are potent catalyzers of LDL oxidation. Intriguingly, plasma HDL carry amyloid- peptide, a major component of senile neuritic plaques and a strong chelator of transition metals (Kontush, 2004).
Major HDL enzymes possessing antioxidative activity are PON1, PAF-AH, LCAT, and GSPx (Van Lenten et al., 2001a; Navab et al., 2004b). PON1 is a component of HDL that is thought to hydrolyze LDL-derived short-chain oxidized PL once they are formed (Aviram et al., 1998). PON1 is anchored to lipids via its hydrophobic N terminus (Josse et al., 2002; Harel et al., 2004); the association of PON1 with HDL is a prerequisite for maintaining normal serum activity of the enzyme. HDL provides the optimal physiological acceptor complex for PON1, in terms of both stimulating enzyme secretion and stabilizing the secreted peptide (James and Deakin, 2004); PON1 interaction with apoA-I is critical for enzyme stability (Gaidukov and Tawfik, 2005). HDL and, less efficiently, VLDL but not LDL promote PON1 secretion from cells; the differences between these lipoproteins are related to differences in their lipid composition (Deakin et al., 2005).
PAF-AH and LCAT can also hydrolyze LDL-derived short-chain oxidized PL; the relationship between the hydrolyzing activities of PON1, PAF-AH, and LCAT toward oxidized PL remains unclear. Recent data question the ability of PON1 to hydrolyze oxidized PL and suggest that PAF-AH, rather than PON-1, is the oxidized PL hydrolase in HDL (Marathe et al., 2003; Connelly et al., 2005). Consistent with this conclusion, HDL-associated PAF-AH is thought to play an antiatherogenic role, in contrast to the LDL-associated enzyme (Quarck et al., 2001; Tsimihodimos et al., 2003; Zalewski and Macphee, 2005). Indeed, local arterial expression of PAF-AH reduces accumulation of oxLDL and inhibits inflammation, shear stress-induced thrombosis, and neointima formation in balloon-injured carotid arteries of nonhyperlipidemic rabbits (Arakawa et al., 2005).
The antioxidative activity of PON1 purified from human serum has recently been ascribed to the presence of detergents or some other unidentified proteins (Teiber et al., 2004). Interestingly, PON1 has been reported to catalyze the hydrolysis of a variety of lactones, including homocysteine thiolactone, suggesting that its native activity is as a lactonase (Jakubowski, 2000; Draganov et al., 2005; Khersonsky and Tawfik, 2005). Plasma levels of homocysteine are a strong CV risk factor (Duell and Malinow, 1997); by detoxifying homocysteine thiolactone, PON1 could protect against homocysteinylation, a post-translational modification of proteins associated with attenuated biological activity and a potential contributing factor to atherosclerosis.
In addition, HDL-associated PON1 enhances cholesterol efflux from macrophages via increased HDL binding mediated by ABCA1 (Rosenblat et al., 2005). PON1-induced cellular accumulation of lysophosphatidylcholine, which stimulates cholesterol efflux via the ABCA1 pathway, may account for this effect (Hara et al., 1997). One can hypothesize that both lactonase activity and an RCT-related mechanism may contribute to the antiatherosclerotic effects of PON1 observed in vivo (Shih et al., 1998; Tward et al., 2002).
Another HDL enzyme, GSPx, can reduce LOOH to corresponding hydroxides and thereby detoxify them (Maddipati and Marnett, 1987; Arthur, 2000; Chen et al., 2000). LOOH-reducing activity mediated by Met residues of apoA-I and apoA-II has also been reported (Sattler et al., 1994; Garner et al., 1998). Finally, upon HDL oxidation with peroxynitrite, apoA-I increases generation of PL core aldehydes that are subsequently hydrolyzed by HDL-associated enzymes, such as PAF-AH and/or PON1, with formation of lysophospholipids (Ahmed et al., 2001). Such a PAF-AH/PON1-coupled protective function of apoA-I can effectively divert proatherogenic LOOH to less harmful products (Van Lenten et al., 2001a; Tselepis and Chapman, 2002; Navab et al., 2004b).
Apolipoproteins and enzymes with antioxidative activities are nonuniformly distributed across HDL subfractions. In vivo PON1 is preferentially associated with large HDL but can be displaced to small, dense particles upon ultracentrifugation (Cabana et al., 2003; Kontush et al., 2003; Bergmeier et al., 2004). The size and shape of HDL seem to be critical for PON1 binding (Josse et al., 2002). By contrast, apoJ is associated with a subset of small HDL, which also contains PON1 (Kelso et al., 1994). Similarly, LCAT activity (Kontush et al., 2003), PAF-AH activity (Kontush et al., 2003), and apoA-IV (Bisgaier et al., 1985) are enriched in small, dense HDL isolated by ultracentrifugation. As a consequence, HDL particles are heterogeneous in their antioxidative activity. Under mild oxidative stress induced by an azo initiator 2,2'-azobis-(2-amidinopropane) hydrochloride or Cu2+, the antioxidative activity of HDL subfractions isolated by density gradient ultracentrifugation against LDL oxidation increases with increment in density in the order: HDL2b < HDL2a < HDL3a < HDL3b < HDL3c, thereby establishing that small, dense HDL act as potent protectors of LDL from oxidative stress (Kontush et al., 2003). Similarly, HDL3 is a more potent protector of LDL from in vitro oxidation compared with HDL2 (Yoshikawa et al., 1997; Huang et al., 1998). The antioxidative activity of small, dense HDL is related to the inactivation of proatherogenic products of LDL lipid peroxidation, primarily LOOH (Kontush et al., 2003). Mechanistically, this activity may arise from synergy in inactivation of oxidized lipids by enzymatic (hydrolysis) and nonenzymatic (physical removal) mechanisms, in part reflecting distinct intrinsic physicochemical properties of the small, dense HDL3c subfraction (Kontush et al., 2003).
The relative importance of HDL antioxidative activity in the overall cardioprotective effect of HDL compared with other biological actions remains indeterminate. A recent study proposed that the antioxidative activity of HDL is less important than cholesterol efflux capacity, as suggested by the absence of antioxidative effects of human apoA-I expression in apoE-/- mice accompanied by delayed atherosclerosis (Choudhury et al., 2004). The increase in non-HDL-C levels observed in this animal model, however, renders interpretation of these results complex. By contrast, both cholesterol efflux capacity and antioxidative activity of HDL were impaired in parallel to a similar extent in apoA-I-/- mice in another recent study (Moore et al., 2005).
3. Anti-Inflammatory Activity.
The anti-inflammatory activity of HDL is illustrated by the ability of HDL to decrease cytokine-induced expression of adhesion molecules on endothelial cells and to inhibit monocyte adhesion to these cells. HDL efficiently inhibit expression of the vascular cell adhesion molecule-1, intercellular adhesion molecule-1, and E-selectin in vitro induced by tumor necrosis factor- (TNF-), interleukin (IL)-1, or endotoxin (Cockerill et al., 1995; Calabresi et al., 1997; Baker et al., 1999). Moreover, this potent anti-inflammatory activity observed in vitro can be translated into inhibition of adhesion molecule expression and a decrease in neutrophil infiltration in the arterial wall by reconstituted HDL (rHDL) in a rabbit model of acute arterial inflammation (Nicholls et al., 2005b). The ability of HDL to inhibit adhesion molecule expression may be related to the presence of apoA-I, apoA-II, apoA-IV, and/or distinct molecular species of PL, including sphingosine-1-phosphate (S1P) and sphingosylphosphorylcholine (Baker et al., 1999; Recalde et al., 2004; Nofer and Assmann, 2005). The anti-inflammatory action of HDL involves inhibition of TNF--stimulated activation of sphingosine kinase and production of S1P, which induces adhesion molecule expression in endothelial cells (Xia et al., 1999); transforming growth factor may function as an important mediator of the anti-inflammatory activity (Norata et al., 2005). In addition, HDL attenuate IL-6 production in endothelial cells exposed to proinflammatory stimuli, such as TNF- or endotoxin (Gomaraschi et al., 2005).
The anti-inflammatory action of HDL also involves hydrolysis of oxidized lipids by HDL-associated enzymes (PAF-AH and PON1) and is mechanistically similar to the antioxidative activity of HDL (Van Lenten et al., 2001a; Navab et al., 2004b; Recalde et al., 2004). Oxidized PL possess potent proinflammatory activities and can trigger arterial inflammation (Furnkranz et al., 2005). Inactivation of oxidized lipids by HDL may be associated with decreased expression of adhesion molecules in and decreased macrophage adhesion to endothelial cells (Theilmeier et al., 2000; Navab et al., 2004b).
Direct interaction of apoA-I with T lymphocytes, which can block subsequent activation of monocytes by lymphocytes, represents another plausible mechanism of HDL anti-inflammatory action (Burger and Dayer, 2002). In addition, apoA-I has been reported to diminish neutrophil activation in vitro (Liao et al., 2005). The anti-inflammatory activity of HDL in vivo is consistent with elevated levels of CRP in subjects with hypoalphalipoproteinemia (Sampietro et al., 2002), with negative correlation between plasma levels of CRP and HDL-C (Pirro et al., 2003) but also between plasma levels of intercellular adhesion molecule-1 and HDL-C and particularly small, dense HDL3-C (Kent et al., 2004).
The potential heterogeneity of HDL anti-inflammatory activity remains poorly characterized. HDL3 has been reported to be superior to HDL2 in terms of its capacity to inhibit vascular cell adhesion molecule-1 expression in endothelial cells (Ashby et al., 1998), a finding that is consistent with the potent antioxidative activity of small, dense HDL3 particles (Yoshikawa et al., 1997; Huang et al., 1998; Kontush et al., 2003).
4. Antiapoptotic, Vasodilatory, Antithrombotic, and Anti-Infectious Activities.
Other antiatherogenic activities of HDL include antiapoptotic and vasodilatory actions, mitogenic activity in endothelial cells, attenuated platelet activation, and anticoagulant and anti-infectious activities (Calabresi et al., 2003).
HDL potently inhibit apoptosis in endothelial cells induced by oxLDL (Suc et al., 1997; Robbesyn et al., 2003) or TNF- (Sugano et al., 2000); this effect is paralleled by decreased intracellular generation of ROS and diminished levels of apoptotic markers, suggesting that it can be related to the intracellular antioxidative actions of HDL or HDL components (Suc et al., 1997; Sugano et al., 2000; Robbesyn et al., 2003). Indeed, HDL contain bioactive lysophospholipids, including S1P (Nofer and Assmann, 2005; Zhang et al., 2005), a potent antiapoptotic agent, which may mediate the antiapoptotic effect of HDL via increased NO production (Kwon et al., 2001).
Similarly, HDL vasodilatory activity may be related to the stimulation of NO release by endothelial cells mediated by intracellular Ca2+ mobilization and phosphorylation of NOS upon association with apoA-I (Drew et al., 2004; Nofer et al., 2004). Such activation of NO production involves HDL binding to SR-BI with a subsequent increase in intracellular ceramide levels (Yuhanna et al., 2001; Li et al., 2002). Furthermore, HDL can stimulate production of prostacyclin, which possesses potent vasorelaxing activity (Beitz and Forster, 1980; Norata et al., 2004). Again, the vasoactive effects of HDL can be mediated by S1P acting via the lysophospholipid receptor S1P3 (Nofer et al., 2004). S1P may be equally important for mitogenic effects of HDL in endothelial cells and for the inhibitory action of HDL on the migration of vascular smooth muscle cells (Kimura et al., 2003; Nofer and Assmann, 2005; Tamama et al., 2005).
Similarly, increased production of NO may form a basis for the inhibitory action of HDL on platelet aggregation (Chen and Mehta, 1994). The antithrombotic activity of HDL is observed as inhibitory actions on factors that promote blood coagulation, including tissue factor, factors X, Va, and VIIIa (Nofer et al., 2002; Calabresi et al., 2003). Mechanistically, this effect may be related to the presence of cardiolipin and phosphatidylethanolamine, two minor anionic PL with potent anticoagulant properties that are enriched in the HDL fraction (Deguchi et al., 2000). In addition, HDL acts via its protein moiety to enhance the anticoagulant activity of protein S and activated protein C (Griffin et al., 1999).
Finally, HDL play a major role in the binding and clearance of circulating endotoxin to the bile and thereby inhibit endotoxin-induced cellular activation, resulting in potent anti-infectious activity (Pajkrt et al., 1996; Levels et al., 2001; Stoll et al., 2004). The inactivation of endotoxin by HDL is mediated by direct interaction with apoA-I (Ma et al., 2004) and involves reduced CD14 expression on monocytes as a key step (Pajkrt et al., 1996). In addition, human HDL possess specific trypanosome-lytic activity, which selectively protects humans from Trypanosome brucei brucei (Hajduk et al., 1989).
The potential heterogeneity of these antiatherogenic activities among HDL particles is indeterminate. The anticoagulant activity of tissue factor pathway inhibitor in human plasma has been reported to be preferentially associated with dense subspecies of HDL and LDL (Lesnik et al., 1993). Similarly, the trypanosome-lytic activity is associated with a minor large and dense HDL subfraction with a molecular mass of 490 kDa (Hajduk et al., 1989). Finally, our recent data suggest that small, dense HDL potently inhibit apoptosis induced in endothelial cells by oxLDL (Suc et al., 1997; Robbesyn et al., 2003; J. de Souza, M. J. Chapman, and A. Kontush, unpublished data).
III. Functionally Defective High-Density Lipoprotein in Dyslipidemic and Inflammatory States
HDL is known to undergo dramatic modification in structure and composition as a result of the concerted actions of the acute-phase response and inflammation (Khovidhunkit et al., 2004b; Esteve et al., 2005). The close association between inflammation, oxidative stress, dyslipidemia, and atherosclerosis suggests that such HDL alterations play a significant role in disease progression. As a result, HDL particles progressively lose normal biological activities and acquire altered properties. Such altered HDL particles have been termed "dysfunctional HDL" (Navab et al., 2001b), and HDL has been proposed to possess "chameleon-like properties" (Navab et al., 1996; Van Lenten et al., 2001a). It is essential to emphasize that the degree of loss of normal HDL function compared with the absence of this function depends on the assay used to characterize HDL functionality. Indeed, HDL can be dysfunctional (with total loss of function) in cell-based or cell-free assays aimed at measuring anti-inflammatory activity (Navab et al., 2001b; Ansell et al., 2003), whereas measurements of antioxidative activity (Kontush et al., 2003, 2004, 2005; Hansel et al., 2004; Nobecourt et al., 2005) or cholesterol efflux capacity (Banka et al., 1995; Cavallero et al., 1995; Brites et al., 2000; Khovidhunkit et al., 2001) reveal a deficiency in normal HDL function rather than a complete dysfunction.
A. Altered High-Density Lipoprotein Composition and Enzymatic Activities in Dyslipidemic and Inflammatory States
1. Apolipoproteins.
Both the plasma levels and apolipoprotein content of HDL can be significantly altered during the acute phase as well as during acute and chronic inflammation. Levels of apoA-I and apoA-II decrease, whereas those of apoA-IV, apoA-V, apoJ, and apoE increase (Khovidhunkit et al., 2004a,b). The decrease in HDL apoA-I levels in inflammatory states is related to both decreased apoA-I synthesis in the liver and apoA-I replacement in HDL particles by SAA (Fig. 5) (Khovidhunkit et al., 2004b; Esteve et al., 2005). SAA is a 12-kDa acute-phase protein whose circulating levels can be induced up to 1000-fold (Malle et al., 1993). HDL is a major carrier of SAA in human, rabbit, and murine plasma (Hoffman and Benditt, 1982a; Marhaug et al., 1982; Cabana et al., 1996). In the circulation, SAA does not exist in a free form and associates with non-HDL lipoproteins in the absence of HDL (Cabana et al., 2004). In the presence of HDL, SAA is specifically associated with small, dense HDL3 subspecies (Benditt and Eriksen, 1977; Hoffman and Benditt, 1982a; Coetzee et al., 1986; Cabana et al., 1996) via its N-terminal domain (Liang et al., 1996), but it is also present in large and intermediate HDL (Coetzee et al., 1986; Cabana et al., 1996).
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; Coetzee et al., 1986); as a result, plasma levels of apoA-I decrease (Cabana et al., 1996). In dense HDL, SAA can account for up to 80% of total protein; such enrichment can further increase HDL protein content and density (Cabana et al., 1989). Elevated plasma levels of SAA are accompanied by elevated levels of lipid-free apoA-I, probably due to the dissociation of apoA-I from HDL (Cabana et al., 1996). In rabbits and mice, SAA can completely replace apoA-I in a subset of small, dense HDL particles, thereby functioning as a structural apolipoprotein (Cabana et al., 1996, 1999). In such SAA-only HDL, 20 molecules of SAA have been estimated to replace all 3 molecules of apoA-I in each HDL particle. SAA is mainly produced by the liver but also by arterial wall cells and adipocytes (Hoffman and Benditt, 1982b; Malle et al., 1993). Primary murine hepa
