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(Arteriosclerosis, Thrombosis, and Vascular Biology. 2001;21:13.)
© 2001 American Heart Association, Inc.

Brief Reviews

High Density Lipoproteins and Arteriosclerosis

Role of Cholesterol Efflux and Reverse Cholesterol Transport

Arnold von Eckardstein; Jerzy-Roch Nofer; Gerd Assmann

From the Institut für Klinische Chemie und Laboratoriumsmedizin (A.v.E., J.-R.N., G.A.), Zentrallaboratorium, Westfälische Wilhelms-Universität Münster, and the Institut für Arterioskleroseforschung an der Universität Münster (A.v.E., J.-R.N., G.A.), Münster, Germany.

Correspondence to Dr Arnold von Eckardstein, Institut für Klinische Chemie und Laboratoriumsmedizin, Zentrallaboratorium, Westfälische Wilhelms-Universität Münster, Albert-Schweitzer-Strasse 33, D-48129 Münster, Germany. E-mail vonecka{at}uni-muenster.de

	Abstract

Top Abstract Introduction Epidemiological Background HDL Subclasses HDL Metabolism Cholesterol Efflux HDL-Mediated Delivery of... Genetic Disturbances of HDL... Concluding Remarks References

 
Abstract—High density lipoprotein (HDL) cholesterol is an important risk factor for coronary heart disease, and HDL exerts various potentially antiatherogenic properties, including the mediation of reverse transport of cholesterol from cells of the arterial wall to the liver and steroidogenic organs. Enhancement of cholesterol efflux and of reverse cholesterol transport (RCT) is considered an important target for antiatherosclerotic drug therapy. Levels and composition of HDL subclasses in plasma are regulated by many factors, including apolipoproteins, lipolytic enzymes, lipid transfer proteins, receptors, and cellular transporters. In vitro experiments as well as genetic family and population studies and investigation of transgenic animal models have revealed that HDL cholesterol plasma levels do not necessarily reflect the efficacy and antiatherogenicity of RCT. Instead, the concentration of HDL subclasses, the mobilization of cellular lipids for efflux, and the kinetics of HDL metabolism are important determinants of RCT and the risk of atherosclerosis.

Key Words: apolipoproteins • lipid transfer proteins • lipases • ABC transporter • Tangier disease

	Introduction

Top Abstract Introduction Epidemiological Background HDL Subclasses HDL Metabolism Cholesterol Efflux HDL-Mediated Delivery of... Genetic Disturbances of HDL... Concluding Remarks References

 
Alow level of HDL cholesterol is an important cardiovascular risk factor.¹ Moreover, HDLs exert various potentially antiatherogenic properties.^{2 3 4} As a consequence, therapeutic modifications of HDL cholesterol (HDL-C) levels have attracted considerable interest. Drugs increasing HDL-C are sought for antiatherogenic therapies, and drugs decreasing HDL-C are suspected to increase cardiovascular risk.⁴

Reverse cholesterol transport (RCT) describes the metabolism and an important antiatherogenic function of HDL, namely, the HDL-mediated efflux of cholesterol from nonhepatic cells and its subsequent delivery to the liver and steroidogenic organs, in which it is used for the synthesis of lipoproteins, bile acids, vitamin D, and steroid hormones.^{2 3 4} Approximately 9 mg cholesterol per kilogram body weight is synthesized by peripheral tissues every day and must be moved to the liver for effective catabolism.⁵ Distortion of RCT can favor the deposition of cholesterol within the arterial wall and thereby contribute to the development of arteriosclerosis. This >30-year-old concept of John Glomset⁶ has been much revived only recently by the identification of several important players in this metabolic pathway, which is reviewed in the present article. Because of the limitation of space, additional potentially antiatherogenic functions of HDL will be reviewed by us in detail elsewhere.

	Epidemiological Background

Top Abstract Introduction Epidemiological Background HDL Subclasses HDL Metabolism Cholesterol Efflux HDL-Mediated Delivery of... Genetic Disturbances of HDL... Concluding Remarks References

 
Numerous clinical and epidemiological studies have demonstrated the inverse and independent association between HDL-C and the risk of coronary heart disease (CHD).¹ More than 40% of patients with myocardial infarction have low HDL-C as a cardiovascular risk factor.⁷ In the prospective and multicentric European Concerted Action on Thrombosis and Disabilities (ECAT) Angina Pectoris Study, we identified low HDL-C and low apoA-I as the most important biochemical risk factors for coronary events in patients with angiographically assessed CHD.⁸ By convention, the risk threshold value of HDL-C has been defined as 35 mg/dL (0.9 mmol/L) in men and 45 mg/dL (1.15 mmol/L) in women.^{9 10 11} Because of interaction, the strength of the association between HDL-C and cardiovascular risk depends on the presence of additional risk factors. Therefore, threshold values are higher in men with diabetes mellitus or hypercholesterolemia or in the presence of multiple risk factors.⁴ Low HDL-C has been identified as the most frequent familial dyslipoproteinemia in patients with premature myocardial infarction.¹² Finally, in the Helsinki Heart Study¹³ and the High-Density-Lipoprotein Cholesterol Intervention Trial of the Department of Veterans Affairs (VA-HIT) study,¹⁴ increases of HDL-C on treatment with gemfibrozil were correlated with the prevention of CHD events. Thus, HDL-C has become an important component of algorithms to assess the global cardiovascular risk of patients and also a target for therapeutic intervention and for the definition of treatment goals.^{9 10} However, in contrast to LDL cholesterol and smoking, it is as yet unknown whether the epidemiological association between HDL-C and CHD is causal.

Several arguments against a causal relationship must be taken into account: First, the association between HDL-C and cardiovascular mortality is U-shaped rather than linear; ie, cardiovascular death rates are higher in individuals with very high levels of HDL-C (eg, fifth quintile) than in individuals with intermediate levels of HDL-C (eg, third quintile)¹⁵ (Figure I, available online at http://atvb.ahajournals.org). Second, in ecological studies, considerable differences in HDL-C between ethnic populations did not explain the differences in cardiovascular morbidity and mortality (eg, differences between Germany and Israel).¹⁶ Third, in some but not all populations (eg, in Germans but not in Turks),¹⁷ low HDL-C is frequently found as a component of the metabolic syndrome, ie, together with hypertriglyceridemia, small dense LDL, glucose intolerance or overt diabetes mellitus, hypertension, overweight or obesity, and hyperinsulinemia. Insulin resistance is considered the common soil of these cardiovascular risk factors. As a consequence, insulin resistance and/or the proatherogenic components of the metabolic syndrome rather than an antiatherogenic function of HDL may underlie the inverse association between HDL-C and cardiovascular risk.¹⁸ The coincidence of low HDL-C with many additional risk factors puts patients with the metabolic syndrome at high global risk for coronary events. This is in contrast to individuals who have low HDL-C as an isolated risk factor.^{4 11} Fourth, as a negative acute-phase reactant, low HDL-C can serve as a surrogate marker for a systemic inflammation, which causes or aggravates atherosclerosis (eg, smoking or chronic infections), or as a disease marker for local inflammation, especially in unstable plaques.¹⁹ In support of the importance of this association, low HDL-C is a more powerful risk factor in short-term follow-ups than in long-term follow-ups of prospective epidemiological studies. For example, the relative risk of CHD events associated with HDL-C <35 mg/dL (<0.9 mmol/L) was 6.1 in a 2-year follow-up of the Prospective Cardiovascular Münster (PROCAM) study but only 2.1 to 2.7 in longer follow-ups (Figure II, available online at http://atvb.ahajournals.org). In the ECAT Angina Pectoris Study, HDL cholesterol had no association with coronary events when plasma levels of C-reactive protein or fibrinogen, which are positive acute-phase reactants, were low.⁸ Also, in the PROCAM study, HDL-C had a significant association with cardiovascular and overall mortality in smokers but not in nonsmokers (Figure I). Fifth, the elevation of HDL-C with statins, fibrates, or estrogens was correlated with the prevention of CHD events in some trials (ie, Helsinki Heart Study and VA-HIT)^{13 14} but not in other trials (Bezafibrate Infarction Prevention [BIP], Scandinavian Simvastatin Survival Study [4S], Cholesterol and Current Events [CARE], West of Scotland Pravastatin Trial [WOSCOP], Air Force/Texas Coronary Atherosclerosis Prevention Study [AFCAPS/TexCAPS], and the Heart and Estrogen/Progestin Replacement Study [HERS]).^{20 21 22 23 24} Moreover, most of these interventions have only moderate effects on HDL-C levels, and none of them is specific for HDL, but they exert a broad scope of mostly antiatherogenic but sometimes even proatherogenic effects on lipid metabolism, the hemostatic system, and inflammation.

In summary, the data from observational and interventional studies demonstrate that the increased coronary risk associated with low HDL-C does not necessarily reflect a defective antiatherogenic repair system and that therapeutically induced changes in HDL-C may not necessarily lead to expected changes in clinical outcomes. Despite this skepticism toward HDL-C as a suitable intermediary phenotype for the monitoring of the cardiovascular effects of therapeutic interventions, it is still believed that modulation of HDL metabolism and the RCT system might be an important target for antiatherosclerotic drug therapy.

	HDL Subclasses

Top Abstract Introduction Epidemiological Background HDL Subclasses HDL Metabolism Cholesterol Efflux HDL-Mediated Delivery of... Genetic Disturbances of HDL... Concluding Remarks References

 
HDL encompasses a heterogeneous class of lipoproteins, which have in common a high density (>1.063 g/mL) and a small size (Stoke’s diameter 5 to 17 nm).²⁵ The majority of the HDL particles contain apoA-I. Differences in the quantitative and qualitative content of lipids, apolipoproteins, enzymes, and lipid transfer proteins (Table 1) result in the presence of various HDL subclasses, which are characterized by differences in shape, density, size, charge, and antigenicity.²⁵ After agarose gel electrophoresis of plasma and anti–apoA-I immunoblotting, the majority of apoA-I is present in a fraction, which migrates with -electrophoretic mobility and is designated -LpA-I. This fraction contains eventually all of the cholesterol, which is quantified in the routine laboratory as HDL-C. About 5% to 15% of apoA-I in human plasma is associated with particles, which have electrophoretic pre-ß mobility and which can be further distinguished by subsequent polyacrylamide gradient gel electrophoresis into pre-ß₁-LpA-I, pre-ß₂-LpA-I, and pre-ß₃-LpA-I (Figure III, available online at http://atvb.ahajournals.org). Pre-ß₁-LpA-I is the smallest particle, discoidal in shape, and contains apoA-I either as a lipid-free apolipoprotein or in association with a few molecules of sphingomyelin and phosphatidylcholine.^{26 27} Similar lipid-poor particles contain only apoE (-LpE) or apoA-IV (LpA-IV) as their only apolipoproteins.^{28 29 30} It is also important to note that relative to the concentration of lipid-rich -HDL, the concentration of these lipid-poor particles is increased in extravasal compartments including the lymph, where RCT is initiated in vivo.^{27 31 32} -HDL can be further differentiated by ultracentrifugation according to density (HDL₂ and HDL₃), by nondenaturing polyacrylamide gradient gel electrophoresis according to size, or by immunoaffinity chromatography according to apolipoprotein composition (eg, LpA-I/A-II and LpA-I).²⁵

View this table: [in this window] [in a new window]   Table 1. Characteristics of Pivotal Genes/Proteins Involved in RCT

	HDL Metabolism

Top Abstract Introduction Epidemiological Background HDL Subclasses HDL Metabolism Cholesterol Efflux HDL-Mediated Delivery of... Genetic Disturbances of HDL... Concluding Remarks References

 
Lipid-rich -HDLs arise from lipid-poor particles or even lipid-free apolipoproteins.^{25 26 27 33} These lipid-poor HDL precursors are produced either as nascent HDL by hepatocytes³⁴ and the intestinal mucosa,³⁵ or they dissociate from chylomicrons and VLDL during lipoprotein lipase–mediated hydrolysis of triglycerides³⁶ or are generated by the interconversion of HDL₂ and HDL₃ by cholesteryl ester transfer protein (CETP),^{37 38} phospholipid transfer protein (PLTP),^{39 40} and hepatic lipase (HL)⁴¹ (Figure 1, Table 1). Lipid-free apolipoproteins or lipid-poor particles acquire phospholipids and unesterified cholesterol from hepatic and nonhepatic cells.⁴² It is not known whether this lipidation occurs intracellularly or extracellularly or both. On the one hand, lipid-free apolipoproteins were shown to induce phospholipid and cholesterol efflux from various cells, including hepatocytes and macrophages, which suggests extracellular assembly.^{43 44} On the other hand, macrophages, hepatocytes, and fibroblasts were shown to internalize lipid-free apolipoproteins, HDL, and chylomicron remnants and to resecrete lipidated apolipoproteins.^{45 46} This process has been termed retroendocytosis and was found to be defective in Tangier disease,⁴⁷ in which mutations in the ATP binding cassette transporter 1 (ABC1) interfere with cellular lipid efflux and lead to the absence of lipid-rich -HDL from plasma.^{48 49 50 51 52} Because ABC1 is expressed in many cells, including hepatocytes and enterocytes,^{53 54} this protein probably plays an important role not only in lipid efflux from peripheral cells but also in the hepatic and intestinal birth of HDL.

View larger version (40K): [in this window] [in a new window]   Figure 1. Pathways involved in the generation and conversion of HDL. Mature HDL3 and HDL2 are generated from lipid-free apoA-I or lipid-poor pre-ß1-HDL as the precursors. These precursors are produced as nascent HDL by the liver or intestine or are released from lipolysed VLDL and chylomicrons or by interconversion of HDL3 and HDL2. ABC1-mediated lipid efflux from cells is important for initial lipidation; LCAT-mediated esterification of cholesterol generates spherical particles that continue to grow on ongoing cholesterol esterification and PLTP-mediated particle fusion and surface remnant transfer. Larger HDL2 particles are converted into smaller HDL3 particles on CETP-mediated export of cholesteryl esters from HDL onto apoB-containing lipoproteins, on SR-BI–mediated selective uptake of cholesteryl esters into liver and steroidogenic organs, and on HL- and EL-mediated hydrolysis of phospholipids. HDL lipids are catabolized either separately from HDL proteins (ie, by selective uptake or via CETP transfer) or together with HDL proteins (ie, via uptake through as-yet-unknown HDL receptors or apoE receptors). The conversion of HDL2 into HDL3 and the PLTP-mediated conversion of HDL3 into HDL2 liberated lipid-free or poorly lipidated apoA-I. A part of lipid-free apoA-I undergoes glomerular filtration in the kidney and tubular readsorption through cubilin. For further details, see text and Table 1. Blue arrows represent lipid transfer processes, and red arrows represent protein transfer processes. TGRL indicates triglyceride-rich lipoproteins.

Lipid-poor HDL precursors become mature, lipid-rich, and spherical -LpA-I by the acquisition of phospholipids and unesterified cholesterol from either cells or apoB-containing lipoproteins, by the lecithin:cholesterol acyltransferase (LCAT)-mediated esterification of cholesterol, and by the association of additional apolipoproteins.^{33 55 56 57} The initial products are small HDL₃ particles, which on esterification of cholesterol through LCAT and fusion with other HDL₃ particles through PLTP and PLTP-mediated acceptance of surface remnants of triglyceride-rich lipoproteins grow into larger HDL₂ particles^{33 58 59 60} (Figure 1, Table 1).

Lipids or proteins of -HDL are removed from the circulation by at least 2 direct pathways, which involve the selective uptake of lipids by scavenger receptor B1 (SR-BI) and the holoparticle uptake by apoE or apoA-I receptors, respectively, and 2 indirect pathways, which involve the action of CETP, HL, and endothelial lipase (EL) (Figure 1, Table 1).^{61 62 63 64 65 66 67} (For details see below.)

The removal of lipids from HDL₂ by SR-BI, CETP, and HL, the subsequent conversion of HDL₂ into HDL₃, and the conversion of HDL₃ into HDL₂ by PLTP regenerate pre-ß₁-LpA-I or lipid-free apoA-I.^{26 33 37 38 39 40} These small apolipoproteins or particles can leave the plasma into the extravascular space,^{25 26 27} where they serve as acceptors of cellular lipids and thus again initiate the generation of HDL. In the kidney, these small particles are filtered and removed from the plasma. The reuptake of apoA-I from the proximal tubulus lumen is mediated by cubilin^{68 69 70} (Figure 1, Table 1).

	Cholesterol Efflux

Top Abstract Introduction Epidemiological Background HDL Subclasses HDL Metabolism Cholesterol Efflux HDL-Mediated Delivery of... Genetic Disturbances of HDL... Concluding Remarks References

 
Cellular Cholesterol Trafficking
The uptake of modified lipoproteins by macrophages of the vascular wall plays an important role in the pathogenesis of atherosclerosis because accumulation of lipids turns them into activated foam cells, which produce various growth factors, cytokines, and proteases and thereby influence the course of atherosclerosis. Macrophages internalize modified lipoproteins (especially by type A scavenger receptors) and cell debris (by phagocytosis). Neither process is regulated by cholesterol. In consequence and in contrast to other cells (which limit their cholesterol content by a finely tuned, sterol-regulated interplay of endogenous cholesterol synthesis and LDL receptor–mediated uptake of exogenous lipoproteins), macrophages accumulate lipids. Released from cholesteryl esters by lysosomal acid lipase, unesterified cholesterol is transferred to the endoplasmic reticulum (ER) either directly or indirectly via the plasma membrane. In the ER, cholesterol is esterified by acyl coenzyme A:cholesterol acyltransferase (ACAT) to protect the cell from the cytotoxicity of excess unesterified cholesterol.^{71 72 73} Cholesteryl esters formed by ACAT appear as cytosolic lipid droplets, which give lipid-laden macrophages their foamy appearance. Cytosolic cholesteryl esters can be hydrolyzed by neutral cholesteryl ester hydrolase (NCEH), which is activated by a cAMP-dependent protein kinase A.⁷⁴ Cholesterol released by NCEH is transferred to the cell membrane, where it can be transported to the ER for reesterification by ACAT.^{71 72 73} This cycle of cholesterol and cholesteryl esters between ACAT and NCEH is interrupted by the presence of extracellular cholesterol acceptors, including HDL, which cause cholesterol efflux.^{42 71 72 73 75 76}

The plasma membrane, from which cholesterol is ultimately released into the extracellular space, accounts for 60% to 90% of unesterified cholesterol in mammalian cells, 95% of which is localized in the cytofacial leaflet of the bilayer membrane.^{72 73} The lateral distribution of cholesterol within the plasma membrane is organized into microdomains.⁷⁷ Coated pits, ie, clathrin-stabilized plasma membrane invaginations that contain lipoprotein receptors, have less cholesterol and sphingolipids than the rest of the plasma membrane. In contrast, caveolae, detergent-resistant plasma membrane invaginations that are characterized by the presence of caveolins but the absence of clathrin, are enriched in cholesterol and sphingolipids. Relatively small amounts of unesterified cholesterol are found in intracellular organelles except in those that communicate with the plasma membrane (endosomes, lysosomes, and trans-Golgi network [TGN]).⁷² The TGN is an acceptor of newly synthesized cholesterol from the ER and exogenous cholesterol from endocytic vesicles, lysosomes, and caveolae^{72 73 78} and subsequently distributes cholesterol and phospholipids either in the form of detergent-resistant and caveolin-containing vesicles (rafts or cytolipoproteins) or as secretory detergent-soluble vesicles.^{72 73 79 80 81}

Diverse Cholesterol Efflux Pathways
Cholesterol efflux from cells is the result of unspecific and passive as well as specific and active processes (Figure 2).^{30 42 75 76}

View larger version (42K): [in this window] [in a new window]   Figure 2. Regulation of cholesterol efflux from cells. Aqueous diffusion of unesterified cholesterol (UC) from the plasma membrane onto lipid-rich lipoproteins, albumin, phospholipid vesicles, or cyclodextrins is slow. Binding of HDL to SR-BI leads to reorganization of cholesterol within the plasma membrane and facilitates cholesterol efflux. ABC1-mediated efflux of UC and phospholipids (PL) onto lipid-free apolipoproteins or lipid-poor particles is fast and involves the translocation of cholesterol from intracellular compartments to the plasma membrane. This transport process appears to involve signal transduction processes, ie, the cAMP-mediated activation of a protein kinase A (PKA) and the degradation of phosphatidylcholine by phospholipase C (PC-PLC) and phospholipase D (PC-PLD). The products DAG and phosphatidic acid (PA) act as second messengers, which activate intracellular transporters either directly or indirectly by activation of a PKC.

Protein-free phospholipid vesicles, synthetic cyclodextrins, albumin, and trypsinized HDL mediate a slow and unsaturable cholesterol efflux from all cell types.^{76 82} This form of cholesterol efflux is not prevented by partial proteolysis of plasma membranes or by inhibition of intracellular vesicular transport.^{76 81 83} It does not involve specific interactions with cell surface receptors or the specific activation of cellular transport processes but simply reflects aqueous diffusion of cholesterol out of the plasma membrane onto acceptor molecules.⁷⁶ Although the loss in cholesterol from the plasma membrane can be replenished, this form of cholesterol efflux has little effect in depleting cells of intracellularly stored cholesteryl esters.^{76 83}

By contrast, lipid-free apoA-I, apoA-II, apoA-IV, apoC, and apoE and also amphipathic synthetic peptides without sequence homology to these apolipoproteins cause an efflux of phospholipids and cholesterol that is fast, saturable, unidirectional, independent of LCAT, and efficient in reducing the content of cytosolic cholesteryl esters.^{28 42 75 76 84 85 86 87} Phospholipid efflux appears to precede cholesterol efflux.^{76 87} As a result, apolipoprotein-mediated lipid efflux lipidates apolipoproteins and hence produces HDL-like lipoproteins with electrophoretic pre-ß and pre- mobilities.^{43 85} Apolipoprotein-mediated lipid efflux involves unspecific desorption of plasma membrane lipids, ie, microsolubilization, and specific interactions with plasma membrane proteins.^{76 88} It is cell specific and takes place only in growth-arrested and cholesterol-enriched cells.^{51 89} Lipid-free apolipoproteins remove phospholipids and cholesterol from normal human skin fibroblasts, aortic smooth muscle cells, and macrophages^{28 75 76 83 84 85 86 87 90 91} but not from erythrocytes, rat or porcine smooth muscle cells, or fibroblasts of patients with Tangier disease.^{84 91 92 93} Moreover, lipid-free apolipoprotein-mediated cholesterol efflux is suppressed by low temperature, by partial proteolysis of cell membranes, or by interference with the regular function of the Golgi apparatus and with intracellular vesicular transport (eg, with monensin or brefeldin A).^{83 84} It has been suggested that lipid-free apolipoproteins and pre-ß₁-LpA-I specifically release cholesterol that is located in the caveolae.^{72 94} Activation of protein kinase C (PKC) enhances and inhibition of PKC suppresses apolipoprotein-mediated cholesterol efflux.^{90 95 96} cAMP was also found to increase apolipoprotein-induced cholesterol efflux from macrophage cell lines.^{45 97 98 99} In RAW264 cells, this was associated with internalization and resecretion of apoA-I⁴⁵ as well as with secretion of apoE.⁹⁸ It has been hypothesized that apoA-I binds to a signal-transducing cell-surface receptor and that this binding facilitates the translocation of cholesterol from intracellular compartments to the plasma membrane.^{42 75} The nature of this receptor is unknown. Because cholesterol efflux from cells and ABC1 are defective in Tangier disease, it is likely that ABC1 plays an important role in apolipoprotein-mediated efflux.^{48 49 50 51 52 84 92 93} In support of this possibility, apoA-I–mediated cholesterol efflux is severely decreased by the inhibition of ABC1 with either antisense oligonucleotides or pharmacological compounds and is increased by the overexpression of ABC1.^{51 100} In addition to ABC1, other ABC transporters appear to be involved in apolipoprotein-mediated lipid efflux. For example, inhibition of the sterol-regulated half-transporter ABC8 (ABCG1) impairs cholesterol efflux from cells.⁵⁵

Native and reconstituted lipid-rich HDLs induce specific and unspecific forms of cholesterol efflux. Partial proteolysis of either HDL or cells does not fully prevent HDL-mediated cholesterol efflux, which is slow, unsaturable, and bidirectional and thus appears to occur by aqueous diffusion.^{28 42 75 76} Esterification of the released cholesterol by LCAT prevents the rediffusion of cholesterol from HDL back to the plasma membrane and thus enhances net cholesterol efflux.¹⁰¹ Expression of SR-BI increases HDL-mediated cholesterol efflux.¹⁰² However, SR-BI–mediated cholesterol efflux does not simply reflect the binding of HDL to cells, inasmuch as binding of HDL to CD36, which is another HDL binding scavenger receptor, does not facilitate cholesterol efflux.¹⁰³ Therefore, it has been suggested that binding of HDL to SR-BI facilitates the bidirectional flux between HDL and plasma membrane by reorganization of lipids within cholesterol- and caveolae-rich domains within the plasma membrane.¹⁰³

HDL-mediated cholesterol efflux also shares some properties of lipid-free apolipoprotein-mediated cholesterol efflux. In the presence of HDL, intracellularly stored cholesteryl esters are removed.^{75 76} After incubation of lipid-enriched cells with brefeldin A or the PKC inhibitor sphingosine, HDL-mediated cholesterol efflux is reduced by 50%.^{84 96} Native and reconstituted HDL elicit various signal transduction pathways, which may activate intracellular lipid transfer processes.^{95 96 104 105 106 107 108} HDL induces the hydrolysis of phosphatidylcholine and phosphatidylinositol biphosphates by phospholipases C and D and thereby the generation of diacylglycerol (DAG), phosphatidic acid, and inositol phosphates. DAG activates PKC, which has been shown to stimulate the translocation of newly synthesized cholesterol to the plasma membrane as well as cholesterol efflux.^{95 96 104 108} By contrast, HDL-mediated breakdown of phosphatidylinositol biphosphate and the subsequent mobilization of intracellular calcium^{106 109} does not stimulate cholesterol efflux but rather mediates the mitogenic effects of HDL.¹¹⁰ In macrophages, HDL has been shown to increase the concentration of cAMP, resulting in the activation of protein kinase A, which may activate the hydrolysis of cytosolic cholesteryl esters by NCEH and the mobilization of cholesterol by ABC1.^{45 74 97 98 99 111} Another mechanism of HDL-mediated cholesterol efflux is retroendocytosis, ie, the uptake of HDL into clathrin-coated endosomes, which is followed by intracellular enrichment with lipids and resecretion.^{45 46 47}

ABC1 Is a Pivotal Regulator of Cholesterol Efflux
As a complete ABC transporter, ABC1 has 2 highly conserved cytoplasmic ATP binding cassettes and 2 transmembrane domains, each of which consists of 6 transmembrane-spanning segments.^{53 112} In analogy with the multidrug resistance (MDR) protein 1, whose low-resolution structure is known, it has been suggested that the transmembrane segments form the wall of an aqueous chamber within the plasma membrane. This chamber is opened to the extracellular space and to the lipid phase of the plasma membrane but not to the cytosol. Substrates of ABC1, ie, cholesterol, phospholipids, vitamins A, E, and K, anions, and interleukin-1ß, may be transported through this channel.^{113 114 115 116}

ABC1 is expressed in many organs. The highest expression was found in fetal tissues, placenta, liver, lung, and adrenal glands.^{53 54} In cell culture, ABC1 was expressed in confluent but not in growing fibroblasts, indicating that ABC1-mediated cholesterol efflux is important in cell quiescence and other conditions of low need for cholesterol.⁵¹

The promotor of the ABC1 gene has been analyzed.^{112 117} It contains binding motifs for several transcription factors, including the sterol regulatory binding protein, the liver X receptor/retinoid X receptor, and activator proteins. In agreement with a regulatory role of these transcription factors, ABC1 expression and lipid efflux are upregulated by cholesterol,^{51 53} oxysterols,¹¹⁷ rexinoids,¹¹⁸ and cAMP analogues.^{51 99} The linker segment of ABC1 has several serine and threonine residues that can be phosphorylated and thereby modulate ABC1 activity posttranslationally.⁵³

In analogy with MDRs, it has been suggested that ABC1 forms a channel within the plasma membrane through which phospholipids (sphingomyelin, phosphatidylserine, and phosphatidylinositol?) and cholesterol are transferred ("flopped") from the inner leaflet to the outer leaflet of the plasma bilayer membrane.^{100 115} There they may be picked up by lipid-free apolipoproteins or lipid-poor particles, which may even bind to ABC1.^{119 120}

HDL and apoA-I have been previously found to be internalized by macrophages into an endosomal compartment, from which they are resecreted together with lipids.^{45 46 47} Tangier macrophages appear to have a defect in resecretion and erroneously target internalized HDL to lysosomes for degradation.⁴⁷ For this reason and because of the presence of hyperplastic Golgi structures within lipid-laden Tangier macrophages,¹²¹ it has been suggested that ABC1 serves as a protein component of vesicles or rafts that target lipids and proteins between lipid-rich intracellular organelles (lysosomes and TGN) and the plasma membrane. In fact, several ABC transporters, such as cystic fibrosis transductance receptor, Rim, and MDRs, are important for the trafficking of proteins and lipids between intracellular organelles and the plasma membrane.^{122 123}

In addition to ABC1, there are several other sterol-regulated ABC transporters.^{54 124} One of them, ABC8, also appears to be involved in cholesterol efflux from macrophages.⁵⁴ It is a so-called half-transporter, inasmuch as it consists of only 1 ATP binding cassette and 1 transmembrane-spanning domain. To be operative, this half-transporter needs another half-transporter, which has not yet been identified.

Role of Endogenous ApoE for Cholesterol Efflux From Macrophages
Macrophages produce apoE, although to a much lesser extent than do hepatocytes.¹²⁵ Transplantation of wild-type bone marrow^{126 127} or the selective expression of a human apoE transgene in macrophages^{127 128} decreased atherosclerosis in apoE-deficient mice. Conversely, on a western diet, atherosclerosis was promoted in wild-type mice receiving bone marrow from apoE-deficient mice.¹²⁹ Because expression of a human apoE transgene in macrophages of apoE-deficient mice¹²⁸ and transplantation of apoE-deficient macrophage stem cells into wild-type mice¹²⁹ produced little changes in plasma lipid levels, macrophage-derived apoE exerts antiatherogenic properties largely independent of its effects on plasma lipoproteins.

One possible explanation for the antiatherogenicity of macrophage-derived apoE is its contribution to cholesterol efflux from these cells.¹²⁵ In vitro, lipid loading and activation of protein kinase A with 8-bromo-cAMP stimulates the synthesis and secretion of apoE in macrophages, which in turn facilitates cholesterol efflux from these cells.^{98 125 126 127 128 129 130 131 132 133 134} ApoE secretion is also stimulated in the presence of exogenous HDL and apoA-I.^{130 132 135 136} It was originally suggested that macrophage-derived apoE facilitates cholesterol efflux by improving the cholesterol acceptor properties of HDL.^{125 130 131 132} However, at least human monocyte–derived macrophages secrete apoE together with cellular lipids also independent of exogenous HDL.^{133 134} The efficacy of this pathway depends on the apoE genotype (ie, it is least efficient in apoE4 macrophages and most efficient in apoE2 macrophages).¹³⁴ Moreover, in the presence of excess exogenous apolipoproteins, including apoE cholesterol, efflux from apoE-producing mouse peritoneal macrophages or J774 macrophages transfected with human apoE was more effective than cholesterol efflux from apoE-deficient mouse macrophages and untransfected J774 macrophages, respectively.^{137 138} This suggests that endogenous apoE modulates cholesterol efflux and cholesteryl ester hydrolysis in macrophages by the regulation of intracellular transport processes.^{137 138}

	HDL-Mediated Delivery of Cholesterol to the Liver and Steroidogenic Organs

Top Abstract Introduction Epidemiological Background HDL Subclasses HDL Metabolism Cholesterol Efflux HDL-Mediated Delivery of... Genetic Disturbances of HDL... Concluding Remarks References

 
Lipids and proteins of mature -HDL are removed from the circulation by direct pathways, which involve the selective lipid uptake by SR-BI^{61 62} and the holoparticle uptake by apoE receptors, such as cubilin and probably also other as-yet-unknown HDL receptors,^{63 139} as well as by indirect pathways that involve the action of CETP,⁶⁴ HL,^{65 66} and EL.⁶⁷

Selective Cholesterol Uptake by SR-BI
SR-BI mediates the selective uptake of cholesteryl esters from HDL and also LDL into hepatocytes and steroid hormone–producing cells without internalizing HDL proteins.^{61 62 140} In contrast to holoparticle receptors (eg, members of the LDL receptor gene family or class A scavenger receptors), which reside in clathrin-coated pits of the plasma membrane and which direct internalized lipoproteins via endosomes to lysosomes for degradation, SR-BI is predominantly found in caveolae and targets internalized lipids into nonendosomal and nonlysosomal compartments.¹⁴¹ Epitopes recognized by SR-BI appear to include phospholipids and apolipoproteins. The mechanism of how SR-BI achieves the dissociation of lipids and proteins and the incorporation of cholesteryl esters into the plasma membrane is not yet understood. Binding of HDL appears to be a necessary but not sufficient prerequisite, because binding of HDL to CD36, another member of the SR-B gene family, does not result in selective lipid uptake.^{142 143} Thus, it has been suggested that SR-BI exerts a lipid transfer activity, eg, by forming a hydrophobic channel through which cholesterol diffuses from the HDL particle into the plasma membrane.¹⁴⁴ Alternatively or in addition, selective uptake by SR-BI may depend on the presence of cofactors. Thus, selective uptake of lipids by SR-BI is facilitated in the presence of HL. HL and other phospholipases may hydrolyze phospholipids of surface layers from HDL and plasma membrane and thereby enable the flux of cholesteryl esters from the lipoprotein core into the plasma membrane.^{145 146} Remodeling of HDLs by CETP also makes HDLs more viable for selective uptake.¹⁴⁶ Finally, selective uptake is reduced in apoE-deficient mice, suggesting that apoE is also involved in the interaction of HDL with SR-BI.¹⁴⁷

As discussed before, SR-BI also mediates cholesterol efflux from cells.^{102 103} The net movement of cholesterol into or out of the cell may depend on the relative activities of extracellular lipid transfer enzymes (LCAT, PLTP, and CETP) and intracellular enzymes, which metabolize cholesterol to cholesteryl esters, bile acids, lipoproteins, or steroid hormones.

Turnover studies have suggested that selective uptake accounts for 90% of cholesteryl ester elimination from HDLs of rodents like mice, which do not express CETP, and 20% of cholesteryl ester elimination from HDLs of species that express CETP.^{61 62} In agreement with the important role of SR-BI in the regulation of HDL metabolism are the results of studies in genetically modified mice. Knockout of the SR-BI gene resulted in a 2-fold elevation of HDL-C but an increase in atherosclerosis.¹⁴⁸ Overexpression of SR-BI enhanced selective cholesteryl ester uptake and HDL catabolism and severely decreased HDL-C levels.¹⁴⁹ In LDL receptor knockout mice or apoE knockout mice, overexpression of SR-BI also led to the disappearance of atherogenic lipoproteins^{150 151} and inhibited atherosclerosis. Hence, upregulation of SR-BI in the liver may be antiatherogenic by increasing the catabolism of lipids from HDL and atherogenic lipoproteins.

SR-BI is downregulated by cholesterol and estradiol and upregulated by polyunsaturated fatty acids in parenchymal liver cells, by adrenocorticotropic hormone in adrenocortical cells, by gonadotropins in theca interna and interstitial cells of the preovulatory ovary, in luteinized granulosa cells, and in the corpus luteum as well as in Leydig cells and less so in Sertoli cells of the testis.^{61 62} Interestingly, at least in rats, in contrast to their effects in hepatocytes, cholesterol and estradiol stimulate SR-BI in macrophage-like Kupffer cells.¹⁵²

Cholesteryl Ester Transfer Protein
CETP exchanges cholesteryl esters of HDL₂ with triglycerides of VLDL, IDL, and LDL. The HDL-derived cholesteryl esters are removed from the circulation via the LDL receptor pathway.^{64 153} Triglycerides in HDL are hydrolyzed by HL. The concerted action of CETP and HL converts larger HDL₂ into smaller HDL₃ and releases lipid-free apoA-I and/or pre-ß₁-LpA-I.³³ The metabolic importance of this pathway in humans is emphasized by the finding of retarded HDL catabolism and elevated HDL-C levels in individuals with CETP deficiency.¹⁵⁴ CETP also exchanges cholesteryl esters between LDL and VLDL. The direction of transfer is modulated by apoF, which inhibits the transfer between LDL and VLDL but activates the transfer from HDL to VLDL.^{153 155}

CETP gene expression is upregulated by cholesterol via activation of the sterol regulatory binding protein and through oxysterols via binding to the liver X receptor/retinoid X receptor.^{156 157 158} Responsiveness of CETP to sterols is probably the basis of reduced cholesteryl ester transfer activity in plasmas of patients treated with statins.¹⁵⁹ Cholesteryl ester transfer activity is increased in hypertriglyceridemic individuals independently of changes in CETP mass. This is considered to be an important mechanism that causes low HDL-C in patients with (postprandial) hypertriglyceridemia and thus in patients with the metabolic syndrome because of insulin resistance.^{18 160}

Because CETP enriches LDL with cholesterol and depletes HDL of cholesterol and because of the initial finding of CETP deficiency in Japanese families with longevity, CETP was originally considered to be a proatherogenic modulator of HDL metabolism. However, the generation of lipid-free apoA-I,^{33 37 38} the enhancement of RCT,¹⁵⁸ and the transfer of oxidized lipids¹⁶¹ also indicate antiatherogenic properties. Data of genetic studies in men and mice are in agreement with this more complex scenario.¹⁶² Despite high HDL-C, CETP deficiency in hypertriglyceridemic Japanese has been associated with an increased risk of coronary events.¹⁶³ Likewise, nonsynonymous alleles of the polymorphic CETP gene increased the risk of CHD events despite being associated with increased levels of HDL-C.^{164 165 166} In contrast, a Taq1 polymorphism of the CETP gene has repeatedly been associated with increased HDL-C and decreased risk of CHD.¹⁶⁷ Immunization of hypercholesterolemic rabbits with CETP decreased plasma CETP activity, increased HDL-C, and reduced atherosclerosis.¹⁶⁸ Overexpression of a human or a simian CETP transgene decreased HDL-C and resulted in increased atherosclerosis in some but not all mouse models (Table 2).^{169 170 171 172 173} Thus, whether inhibition of CETP prevents or enhances atherosclerosis appears to depend on the presence or absence of other dyslipidemias. CETP has been suggested to be proatherogenic in hypercholesterolemia (especially when the LDL receptor pathway is ineffective) and antiatherogenic in hypertriglyceridemia and hyperalphalipoproteinemia.¹⁶²

View this table: [in this window] [in a new window]   Table 2. Genetic Animal Models of HDL Metabolism and Atherosclerosis

HL and EL
HL hydrolyzes phospholipids and triglycerides in all lipoprotein classes.^{65 66} In agreement with the broad substrate and particle specificity of this enzyme, HL deficiency in men and knockout of the HL gene in mice result in the accumulation of apoB-containing remnant-like lipoproteins and phospholipid- and apoE-rich HDLs.^{174 175} ApoA-II inhibits HL so that HDLs devoid of apoA-II are the preferred HDL particle of HL.^{176 177} The concerted action of CETP-mediated cholesteryl ester transfer and the HL-mediated hydrolysis of triglycerides and phospholipids depletes the core of large HDL₂ and helps to form smaller HDL₃ as well as lipid-free apoA-I and/or pre-ß-HDL.³³ Independent of its lipolytic activity, HL appears to serve as a cofactor in the selective uptake of HDL lipids, which is mediated by SR-BI^{145 146} but also by an additional putative HDL binding site in hepatocytes.¹⁷⁸

In addition to regulation of SR-BI, regulation of HL is an important mechanism by which sex steroids affect HDL-C levels. HL activity is suppressed by estradiol and increased by testosterone.^{179 180} Experiences with HL deficiency in men and genetic animal models suggest that HL exerts both proatherogenic and antiatherogenic activities.^{66 175} Several HL-deficient men have suffered from premature CHD despite high HDL-C levels.¹⁷⁴ Despite the inverse correlation between HL activity and HDL-C, a low HL activity has been associated with the presence of atherosclerosis.¹⁸¹ Knockout of HL led to the occurrence of an atherogenic lipoprotein phenotype but reduced atherosclerosis in apoE-deficient mice.¹⁸² Transgenic overexpression of HL in either mice or rabbits decreased HDL-C levels but did not cause atherosclerosis.^{175 183} The controversial data from genetic animal models of HL (and SR-BI) demonstrate the difficulty of interpreting the estrogen-induced increase of HDL-C and the testosterone-induced decrease of HDL-C toward the effects on atherosclerosis.

By contrast to lipoprotein lipase and HL, EL uses phospholipids as its exclusive substrate.^{67 184 185} By hydrolysis of phospholipids in HDL, EL generates free fatty acids, which are taken up by endothelial cells. In addition, removal of phospholipids eventually generates smaller HDL particles, which thereby may become viable for the passage through the endothelial layer into the extravascular space.⁶⁷ Transient overexpression of EL lowers HDL-C.¹⁸⁴ Animal models are needed to evaluate proatherogenic or antiatherogenic effects of EL.

Catabolism of HDL Apolipoproteins
The removal of the protein constituents of HDL from the circulation is less well understood than the removal of HDL-associated lipids. Potential mechanisms for the catabolism of HDL holoparticles and lipid-free apolipoproteins are endocytosis into liver and kidney cells as well as into placenta and yolk sac during pregnancy.⁶⁸

The receptor for the intrinsic factor/vitamin B₁₂, cubilin, has recently been identified as an HDL/apoA-I binding site in epithelial cells of the proximal tubulus of the kidney and of the yolk sac.^{68 69 70} Small HDL particles with a size of <8 nm and lipid-free apoA-I are filtrated by renal glomeruli into the primary urine. Because cubilin-deficient men or dogs have increased excretion of apoA-I in their urine, cubilin has been suggested to mediate the uptake of apoA-I into proximal tubulus cells.^{68 69} However, cubilin has no transmembrane domain and can therefore not mediate the internalization of its ligands without coreceptors.^{68 69 70} Megalin, a member of the LDL receptor gene family, has been suggested to play this role in the kidney and the yolk sac.^{68 69 186} After internalization, the ligands of cubilin (HDL, apoA-I, and the vitamin B₁₂–intrinsic factor complex) are targeted to lysosomes for degradation. Thus, it is unlikely that apoA-I of the primary urine taken up by proximal tubulus cells reenters the plasma compartment and that cubilin is an important determinant of HDL-C plasma concentration.⁶⁸

ApoE-containing HDLs, which constitute the minority of HDLs, are internalized by hepatic apoE receptors (LDL receptor and LDL receptor–related protein).^{63 187} There is considerable evidence of the presence of additional HDL receptors on liver cells, which mediate the catabolism of apoE-free HDL. Ligand blotting studies have identified HDL binding sites of different size in liver cells, which are candidates for receptors mediating hepatic HDL holoparticle uptake.^{139 188} Morphological studies provided evidence for the binding and endocytosis of HDL as well. Some authors have demonstrated resecretion of HDL, which was internalized into liver cells.^{189 190} Interestingly, hepatic binding, uptake, degradation, and resecretion of HDL is disturbed in ob/ob mice, which are deficient in leptin.¹⁹¹ Because ob/ob mice have elevated HDL-C levels because of retarded HDL catabolism,¹⁹² Tall and colleagues have suggested the presence of a hepatic leptin-regulated HDL receptor, which regulates HDL-C levels by mediating holoparticle uptake into liver cells.¹⁹¹

	Genetic Disturbances of HDL Metabolism in Men and Animal Models

Top Abstract Introduction Epidemiological Background HDL Subclasses HDL Metabolism Cholesterol Efflux HDL-Mediated Delivery of... Genetic Disturbances of HDL... Concluding Remarks References

 
The pivotal role of apoA-I, ABC1, LCAT, PLTP, CETP, SR-BI, HL, and EL in the regulation of HDL metabolism is highlighted by the eventually tremendous changes of HDL-C levels in men with inborn errors of HDL metabolism or in genetically modified animal models (Tables 2 and 3). However, changes in HDL-C were not always associated with the expected inverse changes in atherosclerosis, especially when the catabolism of HDL was modified. This indicates that HDL-C levels lack both specificity and sensitivity in assessing the effect of an intervention on atherosclerosis. Rather, the concentration of various HDL subclasses and thereby the function of HDL as well as the kinetics of HDL metabolism appear to influence the course of atherosclerosis.

View this table: [in this window] [in a new window]   Table 3. Inborn Errors of HDL Metabolism and Atherosclerosis in Men

Many patients with apoA-I null alleles have suffered from CHD very early in their life.¹⁹³ Increased atherosclerosis was also found in one but not in another apoA-I–deficient mouse model.^{194 195} Plasmas of apoA-I–deficient men and mice have half-normal cholesterol efflux capacity.^{196 197} In mice and rabbits, transgenic overexpression of the apoA-I gene yielded the expected result, namely, increased HDL-C levels and reduced atherosclerosis.^{198 199 200 201 202 203} Somatic overexpression of apoA-I even induces the regression of preexisting lesions.^{202 203} In line with improved HDL function and accelerated RCT, expression of apoA-I transgenes in mice resulted in an increased cholesterol efflux capacity of plasma.²⁰⁴ In men, infusion of apoA-I increased fecal sterol excretion.²⁰⁵ Likewise, overexpression of a human apoA-IV transgene reduced atherosclerosis in mice.^{206 207} These data suggest that stimulated production of HDL precursors enhances RCT and protects from atherosclerosis.

Although they have reduced HDL-C levels, SR-BI transgenic mice show features of enhanced RCT, namely, increased biliary cholesterol excretion, and are protected from atherosclerosis.^{150 151} Vice versa, knockout of SR-BI causes atherosclerosis despite doubling the HDL-C levels in mice.¹⁴⁸

Other inborn errors and genetic manipulation of HDL metabolism in men and animal models, respectively, showed less conclusive relationships between HDL metabolism and atherosclerosis. Defective HDL maturation because of structural apoA-I variants, Tangier disease, LCAT deficiency, or fisheye disease caused premature atherosclerosis in some but not all patients.^{188 193 208 209} Heterozygotes for the underlying defects in the genes of apoA-I, LCAT, and ABC1 have half-normal levels of HDL-C, ie, usually below the cutoff of 35 mg/dL (0.9 mmol/L) but no increased risk of CHD. Likewise, knockout of LCAT caused HDL deficiency but not atherosclerosis in mice.²¹⁰ Overexpression of LCAT increased HDL-C in mice and rabbits but was antiatherogenic in rabbits and proatherogenic in mice.^{211 212} High HDL-C levels in CETP deficiency were originally found to be associated with reduced CHD risk but were later found to be associated with increased CHD risk in hypertriglyceridemic subpopulations.¹⁶³ Likewise, some genetic variants of CETP^{164 165 166} were associated with increases in HDL-C and CHD risk, whereas others showed the expected inverse association between HDL-C and CHD risk (Table 3). In agreement with the controversial data in men, overexpression of CETP decreased HDL-C but had proatherogenic or antiatherogenic effects in different mouse models.^{169 170 171 172 173} Likewise, human HL deficiency as well as transgenic overexpression or knockout of HL or apoA-II in mice unraveled the antiatherogenic and proatherogenic effects of HL and apoA-II, respectively.^{174 175 182 183 213 214 215 216} Thus, whether LCAT, CETP, or HL is proatherogenic or antiatherogenic appears to be strongly influenced by additional factors. In addition, modification of HDL metabolism can influence the metabolism of atherogenic apoB-containing lipoproteins. Thus, LCAT-deficient men, ABC1-deficient Tangier patients, and SR-BI–overexpressing mice, all of which do not develop atherosclerosis despite HDL deficiency, have low levels of apoB and LDL cholesterol.^{150 151 188 208} Vice versa, atherogenic apoB-containing lipoproteins accumulate in men and mice with HL deficiency and also in LCAT transgenic mice, which develop atherosclerosis despite high levels of HDL-C.^{174 175 211}

	Concluding Remarks

Top Abstract Introduction Epidemiological Background HDL Subclasses HDL Metabolism Cholesterol Efflux HDL-Mediated Delivery of... Genetic Disturbances of HDL... Concluding Remarks References

 
Enhancement of RCT has a great potential for antiatherosclerotic drug therapy. However, it is not the increase or decrease of plasma HDL-C concentration per se but rather the concentration of various HDL subclasses, the cellular mobilization and transport of lipids, and the kinetics of HDL metabolism that determine the efficacy of RCT and thus the risk of atherosclerosis. This challenging concept is also applicable to other antiatherogenic properties of HDL, such as antioxidative, anti-inflammatory, antiadhesive, antiaggregatory, and profibrinolytic effects. Importantly, the interpretation of data from recent trials on statins, fibrates, and estrogens should be cautious in view of the diverse functionality of HDL and the pleiotropic effects of these drugs. Moreover, functional and controlled interventional clinical outcome studies are needed at an early stage to prove the antiatherogenic effects of HDL-elevating drugs that are currently being developed by various pharmaceutical companies.

	Acknowledgments

 
Dr Arnold von Eckardstein is supported by a grant from the European Union on "The Antiatherogenicity of HDL" (grant No. BIOMED2 BMH4-CT98-3699).

Received May 22, 2000; accepted October 18, 2000.

	References

Top Abstract Introduction Epidemiological Background HDL Subclasses HDL Metabolism Cholesterol Efflux HDL-Mediated Delivery of... Genetic Disturbances of HDL... Concluding Remarks References

 

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