- Email: [email protected]
Clinical significance of High-Density lipoproteins and the development of atherosclerosis: focus on the role of the adenosine triphosphate–Binding cassette protein A1 transporter
Clinical Significance of High-Density Lipoproteins and the Development of Atherosclerosis: Focus on the Role of the Adenosine Triphosphate–Binding Cassette Protein A1 Transporter H. Bryan Brewer, Jr.,
MD,
and Silvia Santamarina-Fojo,
MD, PhD
Low levels of high-density lipoprotein (HDL) cholesterol constitute a risk factor for coronary artery disease, and there is evidence that increasing HDL cholesterol levels reduces cardiovascular risk. The phenotype of low HDL cholesterol with or without elevated triglycerides is at least as common in patients hospitalized for cardiovascular disease as is hypercholesterolemia, and it is characteristic of diabetes and the metabolic syndrome, conditions associated with increased cardiovascular risk. Recent studies have elucidated mechanisms by which HDL acts to reduce cardiovascular risk, bolstering the rationale for targeting of HDL in lipid-modifying therapy. In particular, HDL (1) carries excess cholesterol from peripheral cells to the liver for removal in the process termed reverse cholesterol transport, (2) reduces oxidative modification of low-density lipoproteins (LDL), and (3) inhibits cytokine-induced expression of cellular adhesion molecules on endothelial cells. Studies of the newly described adenosine triphosphate– binding cassette protein A1 (ABCA1) transporter have established a crucial role for this transporter in modulating the levels of plasma HDL and intracellular cholesterol in the liver as
well as in peripheral cells. Elevated levels of intracellular cholesterol stimulate the liver X receptor pathway, enhancing the expression of ABCA1, which increases intracellular trafficking of excess cholesterol to the cell surface for interaction with lipid-poor apolipoprotein A-I to form nascent HDL. Nascent HDL facilitates the removal of additional excess cellular cholesterol, which is esterified by lecithin-cholesterol acyltransferase with conversion of the nascent HDL to mature spherical HDL. Overexpression of ABCA1 in mice on a regular chow or Western diet results in a marked increase in plasma HDL, increased LDL, and increased transport of cholesterol to the liver. On a high cholesterol/cholate diet, transgenic mice overexpressing ABCA1 have increased HDL, reduced LDL, increased HDL–mediated cholesterol flux to the liver, and reduced atherosclerosis. Ongoing investigation of mechanisms by which HDL acts to reduce the risk of atherosclerosis will provide several new targets for the development of drugs to decrease the risk of atherosclerosis. 䊚2003 by Excerpta Medica, Inc. Am J Cardiol 2003;92(suppl):10K–16K
uring the past 2 decades, research in atherosclerosis has focused on elucidating the roles of D lipoproteins as risk factors in disease pathogenesis.
indicating that increases in HDL cholesterol and decreases in triglycerides in the absence of change in LDL cholesterol are associated with reduced CAD risk.9,10 Whether HDL cholesterol constitutes a marker of risk or causally contributes to CAD risk and risk reduction continues to be debated. In part, this debate has been sustained by absence of knowledge on how HDL functions to reduce cardiovascular risk. Nevertheless, it has become increasingly clear that many patients with CAD have, as their primary lipid risk factor, the phenotype of low HDL cholesterol or low HDL cholesterol plus elevated triglycerides; indeed, this phenotype is at least as common as hypercholesterolemia among patients hospitalized with established cardiovascular disease.11 The growing awareness of the importance of HDL cholesterol in cardiovascular risk is reflected in the current National Cholesterol Education Program (NCEP) Adult Treatment Panel (ATP) III guidelines.12 These guidelines include low levels of HDL cholesterol as a positive risk factor and high levels of HDL cholesterol as a negative risk factor in risk scoring; the categorical low HDL cholesterol level is increased to ⬍40 mg/dL. Further, these guidelines establish diabetes as a CAD
Low-density lipoprotein (LDL) cholesterol has been identified as an important risk factor for premature coronary disease. In addition, over the past several years, primary and secondary prevention trials with statins have definitively established that reduction of LDL cholesterol is associated with reduction in coronary artery disease (CAD) risk.1– 6 However, the failure of treatment that primarily reduces LDL cholesterol to eliminate all excess risk of cardiovascular disease suggests that additional therapeutic approaches are needed. Epidemiologic data indicate that low levels of high-density lipoprotein (HDL) cholesterol constitute a risk factor for CAD,7,8 and there are data from the Veterans Affairs HDL Intervention Trial (VA-HIT) From the Molecular Disease Branch, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland, USA. Address for reprints: H. Bryan Brewer, Jr., MD, National Institutes of Health, Building 10, Magnuson CC, Room 7N115, 10 Center Drive, Bethesda, Maryland 20894. E-mail: [email protected].
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©2003 by Excerpta Medica, Inc. All rights reserved.
0002-9149/03/$ – see front matter doi:10.1016/S0002-9149(03)00769-0
TABLE 1 Fasting Lipid, Lipoprotein, and Apolipoprotein (Apo) Levels in hABCA1-tg Mice and Control Mice Fed a Regular Chow Diet Mean Concentration ⫾ SEM (mg/dL) hABCA1-tg (n ⫽ 9) Total cholesterol Triglycerides Non-HDL cholesterol Apo B HDL cholesterol Apo A-I
187 89 24 39 163 100
⫾ ⫾ ⫾ ⫾ ⫾ ⫾
10* 3* 8 2† 7* 6‡
Control (n ⫽ 18) 97 68 16 28 81 83
⫾ ⫾ ⫾ ⫾ ⫾ ⫾
4 5 9 1 4 4
hABCA1-tg ⫽ human adenosine triphosphate– binding cassette transporter A1 transgenic; HDL ⫽ high density lipoprotein. *p ⬍0.001. † p ⬍0.01. ‡ p ⬍0.05. Adapted from J Clin Invest.30
risk equivalent and emphasize the importance of the metabolic syndrome in cardiovascular risk. The low HDL cholesterol or low HDL cholesterol/elevated triglyceride phenotype is characteristic of both diabetes and the metabolic syndrome. Research over the past several years has provided considerable insight into mechanisms by which HDL provides protection from cardiovascular disease, particularly through elucidation of the functions of the adenosine triphosphate– binding cassette (ABC) transporters. These insights not only serve to improve the rationale for consideration of HDL cholesterol as a therapeutic target but also may suggest specific mechanisms by which HDL can be targeted in drug therapy.
ROLE OF THE ADENOSINE TRIPHOSPHATE–BINDING CASSETTE TRANSPORTERS IN CHOLESTEROL METABOLISM A primary mechanism whereby HDL might protect against atherosclerosis is via the reverse cholesterol transport process. In the classic model of reverse cholesterol transport, HDL acts to take up excess cholesterol from foam cells in the vessel wall and transports the cholesterol back to the liver for removal from the body.13,14 However, it has remained unclear precisely how cholesterol efflux from peripheral cells is achieved or how cholesterol is otherwise trafficked intracellularly, whether in the macrophage in the vessel wall, hepatocytes, or enterocytes. The discovery of the ABC transporters has contributed substantially to our understanding of the role of HDL in these processes. The ABCA1 transporter was discovered through analysis of patients with Tangier disease,15–20 a condition characterized by decreased plasma cholesterol, LDL, and HDL levels; increased triglyceride levels; increased tissue and macrophage cholesterol accumulation; and premature cardiovascular disease.21 The complete gene structure of ABCA1 has been elucidated,22 and the ABCA1 transporter is present in high concentration in the liver, macrophages, and other peripheral cells. Analysis of the promoter region of
the ABCA1 gene has identified several regulatory elements, including a liver X receptor.23–26 Currently, data indicate that a major mechanism for the regulation of the level of expression of the ABCA1 gene is the levels of intracellular cholesterol and oxysterols. Cholesterol after oxidation to oxysterols modulates ABCA1 expression by the liver X receptor pathway. Elevated levels of cellular cholesterol in these tissues stimulates the liver X receptor pathway, enhancing the expression of ABCA1, which increases intracellular trafficking of excess cholesterol to the cell surface for interaction with lipid-poor apolipoprotein A-I to form nascent HDL. Studies were recently conducted using adenovirus vectors carrying fluorescence-tagged ABCA1 transporters to analyze the cellular movement and function of the ABCA1 transporters within transfected cells.27 These studies established that the transporter rapidly recycles from the cell surface to the late endocytic compartment, facilitating the movement of intracellular cholesterol to the cell membrane. In addition, apolipoprotein A-I has been shown to have a recycling retroendocytic pathway with uptake and resecretion of the apolipoprotein A-I– containing lipoproteins by the cell.28 The changes in the cholesterol content in the cellular membrane induced by ABCA1 result in the ability of several poorly lipidated apolipoproteins to facilitate cholesterol efflux.29 The major apolipoprotein acceptor for ABCA1-mediated cholesterol efflux is poorly lipidated apolipoprotein A-I, which forms pre- or nascent HDL. Nascent HDL facilitates the removal of additional excess cellular cholesterol, and the cholesterol in nascent HDL is esterified by lecithin-cholesterol acyltransferase with conversion of the nascent HDL to mature spherical HDL. In an additional series of studies, we30 sought to determine whether overexpression of the ABCA1 transporters would result in increased levels of plasma HDL; others have also studied this issue.31,32 Transgenic (tg) mice were generated that overexpressed human ABCA1 in the liver (4- to 9-fold increase) and peripheral macrophages (3- to 6-fold increase). OverA SYMPOSIUM: DYSLIPIDEMIA MANAGEMENT
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FIGURE 1. Updated schematic overview of high-density lipoprotein (HDL) metabolism and reverse cholesterol transport. Model includes the liver as a major site of HDL cholesterol synthesis. ABCA1 ⴝ adenosine triphosphate– binding cassette transporter A1; ApoA-I ⴝ apolipoprotein A-I; B ⴝ apolipoprotein B; C ⴝ cholesterol; C-II ⴝ apolipoprotein C-II; CD36 ⴝ scavenger receptor–CD36; CE ⴝ cholesteryl ester; CETP ⴝ cholesteryl ester transfer protein; E ⴝ apolipoprotein E; FC ⴝ free cholesterol; GI ⴝ gastrointestinal; HL ⴝ hepatic lipase; IDL ⴝ intermediate-density lipoproteins; LCAT ⴝ lecithin-cholesterol acyltransferase; LDL ⴝ low-density lipoproteins; LDLr ⴝ LDL receptor; LPL ⴝ lipoprotein lipase; LRP ⴝ LDL receptor–related protein; PL ⴝ phospholipids; SR-A ⴝ scavenger receptor–A; SR-BI ⴝ scavenger receptor-BI; VLDL ⴝ very-low-density lipoproteins. TABLE 2 Fasting Lipid, Lipoprotein, and Apolipoprotein (Apo) Levels in hABCA1-tg and Control Mice After 15 Weeks on a High Cholesterol/Cholate Diet Mean Concentration ⫾ SEM (mg/dL) hABCA1-tg (n ⫽ 14) Total cholesterol Triglycerides Non-HDL cholesterol Apo B HDL cholesterol Apo A-I
247 43 196 109 51 145
⫾ ⫾ ⫾ ⫾ ⫾ ⫾
17* 3 20* 5† 4* 5*
Control (n ⫽ 11) 389 49 371 169 18 67
⫾ ⫾ ⫾ ⫾ ⫾ ⫾
33 4 37 16 1 6
hABCA1-tg ⫽ human adenosine triphosphate– binding cassette transporter A1 transgenic; HDL ⫽ high-density lipoprotein. *p ⬍0.0005. † p ⬍0.005. Adapted from Proc Natl Sci U S A.36 (Copyright 2003 National Academy of Sciences USA.)
expression of ABCA1 was associated with a marked increase in total cholesterol, HDL cholesterol, and apolipoprotein A-I, as well as a modest increase in non-HDL or LDL cholesterol and apolipoprotein B when compared with control mice (Table 1). In studies to determine whether these changes were associated with an increase in reverse cholesterol transport, we found that macrophages from the ABCA1-tg mice exhibited a 2-fold greater apolipoprotein A-I–mediated efflux of cholesterol compared with macrophages from control animals. The 2-fold increase in plasma HDL cholesterol in ABCA1-tg mice 12K THE AMERICAN JOURNAL OF CARDIOLOGY姞
was from both an increased HDL synthesis and a decreased HDL catabolism. In addition, it was found that the net hepatic delivery of exogenous radiolabeled cholesteryl ether HDL, which is dependent on the HDL cholesteryl ester pool size, was significantly increased by 1.5-fold in ABCA1-tg mice.30 These combined results indicated that activation of the ABCA1 transporter results in increased levels of plasma HDL, increased reverse cholesterol transport, and increased delivery of cholesterol back to the liver. The site of synthesis of the increased plasma HDL cholesterol in tg mice overexpressing ABCA1 in the
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FIGURE 2. Mean area of aortic atherosclerotic lesions in adenosine triphosphate– binding cassette transporter A1 transgenic (ABCA1tg) mice (n ⴝ 18) compared with control C57BL/6 mice (n ⴝ 11) on a high-cholesterol/cholate diet. (Reproduced with permission from Proc Natl Acad Sci U S A.36 Copyright 2003 National Academy of Sciences, USA.)
liver and macrophages was then evaluated. The ABCA1 transporter was localized to the basal-lateral side of the hepatocyte, consistent with the ABCA1mediated cholesterol efflux directed toward plasma rather than the bile.33 Selective hepatic expression of the ABCA1 transporter using an ABCA1 adenoviral vector resulted in a marked increase in plasma HDL cholesterol.34 The macrophage was eliminated as the synthesis site because recent studies have shown that the HDL cholesterol generated by macrophage overexpressing ABCA1 did not make a significant contribution to plasma HDL levels.35 These combined studies established the liver as the site of HDL cholesterol synthesis in the ABCA1-tg mice. The analysis of tg mice overexpressing ABCA1 in the liver provided the unique opportunity to discover that the hepatic ABCA1 transporter plays a major role in the determination of plasma HDL levels, as well as a pivotal mechanism for the regulation of the intracellular levels of hepatic cholesterol.34,36 An overview of our current working model of lipoprotein metabolism is shown in Figure 1. This new updated model illustrates the important role of the hepatic ABCA1 transporter in determining the plasma level of HDL cholesterol. To determine whether overexpression of the ABCA1 transporter reduced the development of atherosclerosis, we quantitated the atherosclerosis in ABCA1-tg and control mice on the classic high-cholesterol/cholate diet.36 On this diet, HDL cholesterol and apolipoprotein A-I were significantly increased; however, LDL cholesterol and apolipoprotein B were decreased when compared with control mice (Table 2).36 Most of the reduction in plasma cholesterol in tg mice was in fractions corresponding to very-low-density lipoproteins (VLDL), intermediate-density lipoproteins (IDL), and LDL, with decreased apoli-
poprotein B in the VLDL and IDL/LDL fractions and increased apolipoprotein A-I in HDL. Consistent with the reduced atherogenicity of the lipoprotein phenotype in the ABCA1-tg mice, assessment of proximal aortas in the mice after 15 weeks established that the mean atherosclerotic lesion area was significantly reduced by 65% in the ABCA1-tg mice compared with controls (Figure 2). We concluded from these results that hepatic and macrophage overexpression of the ABCA1 transporter in tg mice on the specific atherogenic high cholesterol/cholate diet was associated with a marked increase in HDL cholesterol, a reduction in LDL cholesterol, and decreased atherosclerosis. Plasma non-HDL cholesterol and apolipoprotein B were modestly increased in ABCA1-tg mice on the normal chow diet and decreased on the high-cholesterol/cholate diet. When ABCA1-tg mice were put on a Western diet, there was a marked increase in LDL levels and the apolipoprotein B– containing lipoproteins when compared with control mice or tg mice on a regular chow diet (Figure 3). Based on these results, we propose that the increased cholesterol absorption on the high-cholesterol diet resulted in an increase in flux of cholesterol to the liver and upregulation of the hepatic ABCA1 transporter with increased synthesis of HDL. The increase in cholesterol levels in the apolipoprotein B– containing lipoproteins is because of the rapid transfer of free cholesterol from HDL37–39 to the apolipoprotein B– containing lipoproteins. A schematic overview of the pathways is shown in Figure 4. Interchange of cholesterol between the HDL and apolipoprotein B pathways occurs by either free cholesterol or cholesteryl ester and cholesteryl ester transfer protein. The free cholesterol transferred to the apolipoprotein B– containing lipoproteins can be esterified by the -lecithin-cholesterol acyltransA SYMPOSIUM: DYSLIPIDEMIA MANAGEMENT
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FIGURE 3. Fast-performance liquid chromatography cholesterol elution profile of pooled plasma from adenosine triphosphate– binding cassette transporter A1 transgenic (ABCA1-tg) mice (squares) and control C57BL/6 mice (diamonds) on a Western diet. HDL ⴝ highdensity lipoprotein; LDL ⴝ low-density lipoprotein; VLDL ⴝ very-low-density lipoprotein.
FIGURE 4. Schematic overview of the very-low-density lipoprotein (VLDL)–intermediate-density lipoprotein (IDL)–low-density lipoprotein (LDL) 3 LDL receptor (LDLr) and nascent high-density lipoprotein (HDL)–HDL 3 scavenger receptor-BI (SR-BI) pathways. Both free cholesterol (C) and cholesteryl esters (CE) by cholesteryl ester transfer protein (CETP) can be transferred between the 2 pathways. ABCA1 ⴝ adenosine triphosphate– binding cassette transporter A1; A-I ⴝ apolipoprotein A-I; B ⴝ apolipoprotein B; C-II ⴝ apolipoprotein C-II; E ⴝ apolipoprotein E; HL ⴝ hepatic lipase; LCAT ⴝ lecithin-cholesterol acyltransferase; LPL ⴝ lipoprotein lipase; LRP ⴝ lipoprotein receptor–related protein.
ferase activity on the apolipoprotein B– containing lipoproteins. The combined results reviewed in this article raise the interesting possibility that overexpression of the hepatic ABCA1 may be associated with increased rather than decreased atherosclerosis, depending on the genetic makeup of the animal model or patient. 14K THE AMERICAN JOURNAL OF CARDIOLOGY姞
Hepatic ABCA1 overexpression with increased cholesterol in the apolipoprotein B– containing lipoproteins in LDL receptor deficiency may be associated with potentially increased atherosclerosis. In contrast, selective overexpression of the ABCA1 transporter in macrophages would be anticipated to result in decreased atherosclerosis.
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TARGETING HIGH-DENSITY LIPOPROTEINS IN THERAPY The studies of ABC transporters provide a more precise understanding of the important role of HDL in modulating atherosclerosis risk. Among the major mechanisms by which HDL is proposed to reduce atherosclerosis risk is the reduction of oxidized LDL by several mechanisms, including the transfer of oxidized lipids to HDL where they may be cleaved by enzymes present on HDL. Thus, HDL acts to block the proatherogenic oxidation of LDL in the vessel wall and decreases the conversion to modified LDL.40 HDL is believed also to reduce atherosclerotic risk through anti-inflammatory effects. Adhesion molecules, such as vascular cell adhesion molecule–1 and E-selectin, play a major role in regulating flux of monocytes into vessel walls and are upregulated in response to inflammatory cytokines.41 Exposure of endothelial cells to increasing concentrations of HDL results in increasing reductions in cytokine-induced expression of the adhesion molecules, suggesting reduced ability of macrophages to enter the vessel wall. The common presentation of low HDL cholesterol levels with or without elevated triglycerides in patients with diabetes and metabolic syndrome, and the frequency with which this phenotype is the predominant lipid abnormality in patients hospitalized with cardiovascular disease, argue for targeting of HDL cholesterol in lipid-modifying therapy along with LDL cholesterol reduction. The VA-HIT trial showed that fibrate therapy was associated with reduced risk of cardiovascular events in association with a relatively modest increase in HDL cholesterol.9,10 Statin therapy, which is recommended as first-line lipidmodifying therapy in individuals at risk for CAD, including those with diabetes and the metabolic syndrome, also produces modest but significant increases in HDL cholesterol. For example, simvastatin has been found to increase HDL cholesterol significantly more than atorvastatin at doses of 80 mg versus 40 mg and 80 mg of atorvastatin.42 Pooled data from the rosuvastatin phase 3 clinical trial program found rosuvastatin 10 mg to increase HDL cholesterol significantly more than atorvastatin 10 mg, simvastatin 20 mg, and pravastatin 10 mg.43
CONCLUSION The recent improvements in our understanding of the mechanisms by which HDL acts to prevent atherosclerosis suggest the potential for specific therapeutic targets to augment the HDL antiatherosclerotic function. Ongoing research in this area is likely to provide additional potential targets for drug development. 1. Heart Protection Study Collaborative Group. MRC/BHF Heart Protection
Study of cholesterol lowering with simvastatin in 20,536 high-risk individuals: a randomised placebo-controlled trial. Lancet 2002;360:7–22. 2. The Long-Term Intervention with Pravastatin in Ischaemic Disease (LIPID) Study Group. Prevention of cardiovascular events and death with pravastatin in patients with coronary heart disease and a broad range of initial cholesterol levels. N Engl J Med 1998;339:1349 –1357.
3. Sacks FM, Pfeffer MA, Moye LA, Rouleau JL, Rutherford JD, Cole TG, Brown L, Warnica JW, Arnold JM, Wun CC, Davis BR, Braunwald E, for the Cholesterol and Recurrent Events Trial Investigators. The effect of pravastatin on coronary events after myocardial infarction in patients with average cholesterol levels. N Engl J Med 1996;335:1001–1009. 4. Randomised trial of cholesterol lowering in 4444 patients with coronary heart disease: the Scandinavian Simvastatin Survival Study (4S). Lancet 1994;344: 1383–1389. 5. Shepherd J, Cobbe SM, Ford I, Isles CG, Lorimer AR, MacFarlane PW, McKillop JH, Packard, CJ, for the West of Scotland Coronary Prevention Study Group. Prevention of coronary heart disease with pravastatin in men with hypercholesterolemia. N Engl J Med 1995;333:1301–1307. 6. Stamler J, Vaccaro O, Neaton JD, Wentworth D. Diabetes, other risk factors, and 12-yr cardiovascular mortality for men screened in the Multiple Risk Factor Intervention Trial. Diabetes Care 1993;16:434 –444. 7. Gordon DJ, Rifkind BM. High-density lipoprotein: the clinical implications of recent studies. N Engl J Med 1989;321:1311–1316. 8. Miller NE. Associations of high-density lipoprotein subclasses and apolipoproteins with ischemic heart disease and coronary atherosclerosis. Am Heart J 1987;113:589 –597. 9. Rubins HB, Robins SJ, Collins D, Fye CL, Anderson JW, Elam MB, Faas FH, Linares E, Schaefer EJ, Schectman G, Wilt TJ, Wittes J, for the Veterans Affairs High-Density Lipoprotein Cholesterol Intervention Trial Study Group. Gemfibrozil for the secondary prevention of coronary heart disease in men with low levels of high-density lipoprotein cholesterol. N Engl J Med 1999;341:410 –418. 10. Robins SJ, Collins D, Wittes JT, Papademetriou V, Deedwania PC, Schaefer EJ, McNamara JR, Kashyap ML, Hershman JM, Wexler LF, Rubins HB. Relation of gemfibrozil treatment and lipid levels with major coronary events. VAHIT: a randomized controlled trial. JAMA 2001;285:1585–1591. 11. Genest JJ Jr, Martin-Munley SS, McNamara JR, Ordovas JM, Jenner J, Myers RH, Silberman SR, Wilson PW, Salem DN, Schaefer EJ. Familial lipoprotein disorders in patients with premature coronary artery disease. Circulation 1992; 85:2025–2033. 12. Expert Panel on Detection, Evaluation, and Treatment of High Blood Cholesterol in Adults. Executive summary of the third report of the National Cholesterol Education Program (NCEP) Expert Panel on Detection, Evaluation, and Treatment of High Blood Cholesterol in Adults (Adult Treatment Panel III). JAMA 2001;285:2486 –2497. 13. Glomset JA. The plasma lecithin: cholesterol acyltransferase reaction. J Lipid Res 1968;9:55–67. 14. Fielding CJ, Fielding PE. Molecular physiology of reverse cholesterol transport. J Lipid Res 1995;36:211–228. 15. Brooks-Wilson A, Marcil M, Clee SM, Zhang LH, Roomp K, van Dam M, Yu L, Brewer C, Collins JA, Molhuizen HO, et al. Mutations in ABC1 in Tangier disease and familial high-density lipoprotein deficiency. Nat Genet 1999;22:336 – 345. 16. Rust S, Rosier M, Funke H, Real J, Amoura Z, Piette JC, Deleuze JF, Brewer HB, Duverger N, Denefle P, Assmann G. Tangier disease is caused by mutations in the gene encoding ATP-binding cassette transporter 1. Nat Genet 1999;22: 352–355. 17. Bodzioch M, Orso E, Klucken J, Langmann T, Bottcher A, Diederich W, Drobnik W, Barlage S, Buchler C, Porsch-Ozcurumez M, et al. The gene encoding ATP-binding cassette transporter 1 is mutated in Tangier disease. Nat Genet 1999;22:347–351. 18. Remaley AT, Rust S, Rosier M, Knapper C, Naudin L, Broccardo C, Peterson KM, Koch C, Arnould I, Prades C, et al. Human ATP-binding cassette transporter 1 (ABC1): genomic organization and identification of the genetic defect in the original Tangier disease kindred. Proc Natl Acad Sci U S A 1999;96:12685– 12690. 19. Brousseau ME, Schaefer EJ, Dupuis J, Eustace B, Van Eerdewegh P, Goldkamp AL, Thurston LM, FitzGerald MG, Yasek-McKenna D, O’Neill G, et al. Novel mutations in the gene encoding ATP-binding cassette 1 in four Tangier disease kindreds. J Lipid Res 2000;41:433–441. 20. Lawn RM, Wade DP, Garvin MR, Wang X, Schwartz K, Porter JG, Seilhamer JJ, Vaughan AM, Oram JF. The Tangier disease gene product ABC1 controls the cellular apolipoprotein-mediated lipid removal pathway. J Clin Invest 1999;104:R25–R31. 21. Assman G, von Eckardstein A, Brewer HB Jr. Familial high-density lipoprotein deficiency: Tangier disease. In: Beaudet A, Childs B, Kinzler K, Scriver C, Sly W, Valle D, Vogelstein B, eds. The Metabolic and Molecular Basis of Inherited Disease. 8th ed. New York: McGraw Hill, Health Professions Division, 2001:2937–2980. 22. Santamarina-Fojo S, Peterson K, Knapper C, Qiu Y, Freeman L, Cheng JF, Osorio J, Remaley A, Yang XP, Haudenschild C, et al. Complete genomic sequence of the human ABCA1 gene: analysis of the human and mouse ATPbinding cassette A promoter. Proc Natl Acad Sci U S A 2000;97:7987–7992. 23. Venkateswaran A, Laffitte BA, Joseph SB, Mak PA, Wilpitz DC, Edwards PA, Tontonoz P. Control of cellular cholesterol efflux by the nuclear oxysterol receptor LXR alpha. Proc Natl Acad Sci U S A 2000;97:12097–12102. 24. Schwartz K, Lawn RM, Wade DP. ABC1 gene expression and ApoA-Imediated cholesterol efflux are regulated by LXR. Biochem Biophys Res Commun 2000;274:794 –802. 25. Costet P, Luo Y, Wang N, Tall AR. Sterol-dependent transactivation of the ABC1 promoter by the liver X receptor/retinoid X receptor. J Biol Chem 2000;275:28240 –28245.
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26. Chawla A, Boisvert WA, Lee CH, Laffitte BA, Barak Y, Joseph SB, Liao D, Nagy L, Edwards PA, Curtiss LK, Evans RM, Tontonoz P. A PPAR ␥-LXRABCA1 pathway in macrophages is involved in cholesterol efflux and atherogenesis. Mol Cell 2001;7:161–171. 27. Neufeld EB, Remaley AT, Demosky SJ, Stonik JA, Cooney AM, Comly M, Dwyer NK, Zhang M, Blanchette-Mackie J, Santamarina-Fojo S, Brewer HB Jr. Cellular localization and trafficking of the human ABCA1 transporter. J Biol Chem 2001;276:27584 –27590. 28. Takahashi Y, Smith JD. Cholesterol efflux to apolipoprotein AI involves endocytosis and resecretion in a calcium-dependent pathway. Proc Natl Acad Sci U S A 1999;96:11358 –11363. 29. Remaley AT, Stonik JA, Demosky SJ, Neufeld EB, Bocharov AV, Vishnyakova TG, Eggerman TL, Patterson AP, Duverger NJ, Santamarina-Fojo S, Brewer HB Jr. Apolipoprotein specificity for lipid efflux by the human ABCAI transporter. Biochem Biophys Res Commun 2001;280:818 –823. 30. Vaisman BL, Lambert G, Amar M, Joyce C, Ito T, Shamburek RD, Cain WJ, Fruchart-Najib J, Neufeld ED, Remaley AT, Brewer HB Jr, Santamarina-Fojo S. ABCA1 overexpression leads to hyperalphalipoproteinemia and increased biliary cholesterol excretion in transgenic mice. J Clin Invest 2001;108:303–309. 31. Cavelier LB, Qiu Y, Bielicki JK, Afzal V, Cheng JF, Rubin EM. Regulation and activity of the human ABCA1 gene in transgenic mice. J Biol Chem 2001;276:18046 –18051. 32. Singaraja RR, Bocher V, James ER, Clee SM, Zhang LH, Leavitt BR, Tan B, Brooks-Wilson A, Kwok A, Bissada N, et al. Human ABCA1 BAC transgenic mice show increased high density lipoprotein cholesterol and apoAI-dependent efflux stimulated by an internal promoter containing liver X receptor response elements in intron 1. J Biol Chem 2001;276:33969 –33979. 33. Neufeld EB, Demosky SJ Jr, Stonik JA, Combs C, Remaley AT, Duverger N, Santamarina-Fojo S, Brewer HB Jr. The ABCA1 transporter functions on the basolateral surface of hepatocytes. Biochem Biophys Res Commun 2002;297: 974 –979. 34. Basso F, Freeman L, Knapper CL, Remaley A, Stonik J, Neufeld EB, Tansey
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T, Amar MJ, Fruchart-Najib J, Duverger N, Santamarina-Fojo S, Brewer HB Jr. Role of the hepatic ABCA1 transporter in modulating intrahepatic cholesterol and plasma HDL cholesterol concentrations. J Lipid Res 2003;44:296 –302. 35. Haghpassand M, Bourassa PA, Francone OL, Aiello RJ. Monocyte/macrophage expression of ABCA1 has minimal contribution to plasma HDL levels. J Clin Invest 2001;108:1315–1320. 36. Joyce CW, Amar MJ, Lambert G, Vaisman BL, Paigen B, Najib-Fruchart J, Hoyt RF Jr, Neufeld ED, Remaley AT, Fredrickson DS, Brewer HB Jr, Santamarina-Fojo S. The ATP binding cassette transporter A1 (ABCA1) modulates the development of aortic atherosclerosis in C57BL/6 and apoE-knockout mice. Proc Natl Acad Sci U S A 2002;99:407–412. 37. Schwartz CC, Berman M, Vlahcevic ZR, Halloran LG, Gregory DH, Swell L. Multicompartmental analysis of cholesterol metabolism in man: the hepatic bile acid and biliary cholesterol precursor sites. J Clin Invest 1978;61:408 –423. 38. Schwartz CC, Vlahcevic ZR, Berman M, Meadows JG, Nisman RM, Swell L. Central role of high-density lipoprotein in plasma free cholesterol metabolism. J Clin Invest 1982;70:105–116. 39. Schwartz CC, Vlahcevic ZR, Halloran LG, Swell L. An in vivo evaluation in man of the transfer of esterified cholesterol between lipoproteins and into the liver and bile. Biochim Biophys Acta 1981;663:143–162. 40. Van Lenten BJ, Navab M, Shih D, Fogelman AM, Lusis AJ. The role of high-density lipoproteins in oxidation and inflammation. Trends Cardiovasc Med 2001;1:155–161. 41. Barter P. Effects of inflammation on high-density lipoproteins. Arterioscler Thromb Vasc Biol 2002;22:1062–1063. 42. Illingworth DR, Crouse JR III, Hunninghake DB, Davidson MH, Escobar ID, Stalenhoef AF, Paragh G, Ma PT, Liu M, Melino MR, et al. A comparison of simvastatin and atorvastatin up to maximal recommended doses in a large multicenter randomized clinical trial. Curr Med Res Opin 2001;17:43–50. 43. Blasetto J, Stein E, Brown WV, Chitra R, Raza A. Efficacy of rosuvastatin compared with other statins at selected starting doses in hypercholesterolemic patients and in special population groups. Am J Cardiol 2003;91(suppl 5A):3C– 10C.
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MD,
and Silvia Santamarina-Fojo,
MD, PhD
Low levels of high-density lipoprotein (HDL) cholesterol constitute a risk factor for coronary artery disease, and there is evidence that increasing HDL cholesterol levels reduces cardiovascular risk. The phenotype of low HDL cholesterol with or without elevated triglycerides is at least as common in patients hospitalized for cardiovascular disease as is hypercholesterolemia, and it is characteristic of diabetes and the metabolic syndrome, conditions associated with increased cardiovascular risk. Recent studies have elucidated mechanisms by which HDL acts to reduce cardiovascular risk, bolstering the rationale for targeting of HDL in lipid-modifying therapy. In particular, HDL (1) carries excess cholesterol from peripheral cells to the liver for removal in the process termed reverse cholesterol transport, (2) reduces oxidative modification of low-density lipoproteins (LDL), and (3) inhibits cytokine-induced expression of cellular adhesion molecules on endothelial cells. Studies of the newly described adenosine triphosphate– binding cassette protein A1 (ABCA1) transporter have established a crucial role for this transporter in modulating the levels of plasma HDL and intracellular cholesterol in the liver as
well as in peripheral cells. Elevated levels of intracellular cholesterol stimulate the liver X receptor pathway, enhancing the expression of ABCA1, which increases intracellular trafficking of excess cholesterol to the cell surface for interaction with lipid-poor apolipoprotein A-I to form nascent HDL. Nascent HDL facilitates the removal of additional excess cellular cholesterol, which is esterified by lecithin-cholesterol acyltransferase with conversion of the nascent HDL to mature spherical HDL. Overexpression of ABCA1 in mice on a regular chow or Western diet results in a marked increase in plasma HDL, increased LDL, and increased transport of cholesterol to the liver. On a high cholesterol/cholate diet, transgenic mice overexpressing ABCA1 have increased HDL, reduced LDL, increased HDL–mediated cholesterol flux to the liver, and reduced atherosclerosis. Ongoing investigation of mechanisms by which HDL acts to reduce the risk of atherosclerosis will provide several new targets for the development of drugs to decrease the risk of atherosclerosis. 䊚2003 by Excerpta Medica, Inc. Am J Cardiol 2003;92(suppl):10K–16K
uring the past 2 decades, research in atherosclerosis has focused on elucidating the roles of D lipoproteins as risk factors in disease pathogenesis.
indicating that increases in HDL cholesterol and decreases in triglycerides in the absence of change in LDL cholesterol are associated with reduced CAD risk.9,10 Whether HDL cholesterol constitutes a marker of risk or causally contributes to CAD risk and risk reduction continues to be debated. In part, this debate has been sustained by absence of knowledge on how HDL functions to reduce cardiovascular risk. Nevertheless, it has become increasingly clear that many patients with CAD have, as their primary lipid risk factor, the phenotype of low HDL cholesterol or low HDL cholesterol plus elevated triglycerides; indeed, this phenotype is at least as common as hypercholesterolemia among patients hospitalized with established cardiovascular disease.11 The growing awareness of the importance of HDL cholesterol in cardiovascular risk is reflected in the current National Cholesterol Education Program (NCEP) Adult Treatment Panel (ATP) III guidelines.12 These guidelines include low levels of HDL cholesterol as a positive risk factor and high levels of HDL cholesterol as a negative risk factor in risk scoring; the categorical low HDL cholesterol level is increased to ⬍40 mg/dL. Further, these guidelines establish diabetes as a CAD
Low-density lipoprotein (LDL) cholesterol has been identified as an important risk factor for premature coronary disease. In addition, over the past several years, primary and secondary prevention trials with statins have definitively established that reduction of LDL cholesterol is associated with reduction in coronary artery disease (CAD) risk.1– 6 However, the failure of treatment that primarily reduces LDL cholesterol to eliminate all excess risk of cardiovascular disease suggests that additional therapeutic approaches are needed. Epidemiologic data indicate that low levels of high-density lipoprotein (HDL) cholesterol constitute a risk factor for CAD,7,8 and there are data from the Veterans Affairs HDL Intervention Trial (VA-HIT) From the Molecular Disease Branch, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland, USA. Address for reprints: H. Bryan Brewer, Jr., MD, National Institutes of Health, Building 10, Magnuson CC, Room 7N115, 10 Center Drive, Bethesda, Maryland 20894. E-mail: [email protected].
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©2003 by Excerpta Medica, Inc. All rights reserved.
0002-9149/03/$ – see front matter doi:10.1016/S0002-9149(03)00769-0
TABLE 1 Fasting Lipid, Lipoprotein, and Apolipoprotein (Apo) Levels in hABCA1-tg Mice and Control Mice Fed a Regular Chow Diet Mean Concentration ⫾ SEM (mg/dL) hABCA1-tg (n ⫽ 9) Total cholesterol Triglycerides Non-HDL cholesterol Apo B HDL cholesterol Apo A-I
187 89 24 39 163 100
⫾ ⫾ ⫾ ⫾ ⫾ ⫾
10* 3* 8 2† 7* 6‡
Control (n ⫽ 18) 97 68 16 28 81 83
⫾ ⫾ ⫾ ⫾ ⫾ ⫾
4 5 9 1 4 4
hABCA1-tg ⫽ human adenosine triphosphate– binding cassette transporter A1 transgenic; HDL ⫽ high density lipoprotein. *p ⬍0.001. † p ⬍0.01. ‡ p ⬍0.05. Adapted from J Clin Invest.30
risk equivalent and emphasize the importance of the metabolic syndrome in cardiovascular risk. The low HDL cholesterol or low HDL cholesterol/elevated triglyceride phenotype is characteristic of both diabetes and the metabolic syndrome. Research over the past several years has provided considerable insight into mechanisms by which HDL provides protection from cardiovascular disease, particularly through elucidation of the functions of the adenosine triphosphate– binding cassette (ABC) transporters. These insights not only serve to improve the rationale for consideration of HDL cholesterol as a therapeutic target but also may suggest specific mechanisms by which HDL can be targeted in drug therapy.
ROLE OF THE ADENOSINE TRIPHOSPHATE–BINDING CASSETTE TRANSPORTERS IN CHOLESTEROL METABOLISM A primary mechanism whereby HDL might protect against atherosclerosis is via the reverse cholesterol transport process. In the classic model of reverse cholesterol transport, HDL acts to take up excess cholesterol from foam cells in the vessel wall and transports the cholesterol back to the liver for removal from the body.13,14 However, it has remained unclear precisely how cholesterol efflux from peripheral cells is achieved or how cholesterol is otherwise trafficked intracellularly, whether in the macrophage in the vessel wall, hepatocytes, or enterocytes. The discovery of the ABC transporters has contributed substantially to our understanding of the role of HDL in these processes. The ABCA1 transporter was discovered through analysis of patients with Tangier disease,15–20 a condition characterized by decreased plasma cholesterol, LDL, and HDL levels; increased triglyceride levels; increased tissue and macrophage cholesterol accumulation; and premature cardiovascular disease.21 The complete gene structure of ABCA1 has been elucidated,22 and the ABCA1 transporter is present in high concentration in the liver, macrophages, and other peripheral cells. Analysis of the promoter region of
the ABCA1 gene has identified several regulatory elements, including a liver X receptor.23–26 Currently, data indicate that a major mechanism for the regulation of the level of expression of the ABCA1 gene is the levels of intracellular cholesterol and oxysterols. Cholesterol after oxidation to oxysterols modulates ABCA1 expression by the liver X receptor pathway. Elevated levels of cellular cholesterol in these tissues stimulates the liver X receptor pathway, enhancing the expression of ABCA1, which increases intracellular trafficking of excess cholesterol to the cell surface for interaction with lipid-poor apolipoprotein A-I to form nascent HDL. Studies were recently conducted using adenovirus vectors carrying fluorescence-tagged ABCA1 transporters to analyze the cellular movement and function of the ABCA1 transporters within transfected cells.27 These studies established that the transporter rapidly recycles from the cell surface to the late endocytic compartment, facilitating the movement of intracellular cholesterol to the cell membrane. In addition, apolipoprotein A-I has been shown to have a recycling retroendocytic pathway with uptake and resecretion of the apolipoprotein A-I– containing lipoproteins by the cell.28 The changes in the cholesterol content in the cellular membrane induced by ABCA1 result in the ability of several poorly lipidated apolipoproteins to facilitate cholesterol efflux.29 The major apolipoprotein acceptor for ABCA1-mediated cholesterol efflux is poorly lipidated apolipoprotein A-I, which forms pre- or nascent HDL. Nascent HDL facilitates the removal of additional excess cellular cholesterol, and the cholesterol in nascent HDL is esterified by lecithin-cholesterol acyltransferase with conversion of the nascent HDL to mature spherical HDL. In an additional series of studies, we30 sought to determine whether overexpression of the ABCA1 transporters would result in increased levels of plasma HDL; others have also studied this issue.31,32 Transgenic (tg) mice were generated that overexpressed human ABCA1 in the liver (4- to 9-fold increase) and peripheral macrophages (3- to 6-fold increase). OverA SYMPOSIUM: DYSLIPIDEMIA MANAGEMENT
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FIGURE 1. Updated schematic overview of high-density lipoprotein (HDL) metabolism and reverse cholesterol transport. Model includes the liver as a major site of HDL cholesterol synthesis. ABCA1 ⴝ adenosine triphosphate– binding cassette transporter A1; ApoA-I ⴝ apolipoprotein A-I; B ⴝ apolipoprotein B; C ⴝ cholesterol; C-II ⴝ apolipoprotein C-II; CD36 ⴝ scavenger receptor–CD36; CE ⴝ cholesteryl ester; CETP ⴝ cholesteryl ester transfer protein; E ⴝ apolipoprotein E; FC ⴝ free cholesterol; GI ⴝ gastrointestinal; HL ⴝ hepatic lipase; IDL ⴝ intermediate-density lipoproteins; LCAT ⴝ lecithin-cholesterol acyltransferase; LDL ⴝ low-density lipoproteins; LDLr ⴝ LDL receptor; LPL ⴝ lipoprotein lipase; LRP ⴝ LDL receptor–related protein; PL ⴝ phospholipids; SR-A ⴝ scavenger receptor–A; SR-BI ⴝ scavenger receptor-BI; VLDL ⴝ very-low-density lipoproteins. TABLE 2 Fasting Lipid, Lipoprotein, and Apolipoprotein (Apo) Levels in hABCA1-tg and Control Mice After 15 Weeks on a High Cholesterol/Cholate Diet Mean Concentration ⫾ SEM (mg/dL) hABCA1-tg (n ⫽ 14) Total cholesterol Triglycerides Non-HDL cholesterol Apo B HDL cholesterol Apo A-I
247 43 196 109 51 145
⫾ ⫾ ⫾ ⫾ ⫾ ⫾
17* 3 20* 5† 4* 5*
Control (n ⫽ 11) 389 49 371 169 18 67
⫾ ⫾ ⫾ ⫾ ⫾ ⫾
33 4 37 16 1 6
hABCA1-tg ⫽ human adenosine triphosphate– binding cassette transporter A1 transgenic; HDL ⫽ high-density lipoprotein. *p ⬍0.0005. † p ⬍0.005. Adapted from Proc Natl Sci U S A.36 (Copyright 2003 National Academy of Sciences USA.)
expression of ABCA1 was associated with a marked increase in total cholesterol, HDL cholesterol, and apolipoprotein A-I, as well as a modest increase in non-HDL or LDL cholesterol and apolipoprotein B when compared with control mice (Table 1). In studies to determine whether these changes were associated with an increase in reverse cholesterol transport, we found that macrophages from the ABCA1-tg mice exhibited a 2-fold greater apolipoprotein A-I–mediated efflux of cholesterol compared with macrophages from control animals. The 2-fold increase in plasma HDL cholesterol in ABCA1-tg mice 12K THE AMERICAN JOURNAL OF CARDIOLOGY姞
was from both an increased HDL synthesis and a decreased HDL catabolism. In addition, it was found that the net hepatic delivery of exogenous radiolabeled cholesteryl ether HDL, which is dependent on the HDL cholesteryl ester pool size, was significantly increased by 1.5-fold in ABCA1-tg mice.30 These combined results indicated that activation of the ABCA1 transporter results in increased levels of plasma HDL, increased reverse cholesterol transport, and increased delivery of cholesterol back to the liver. The site of synthesis of the increased plasma HDL cholesterol in tg mice overexpressing ABCA1 in the
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FIGURE 2. Mean area of aortic atherosclerotic lesions in adenosine triphosphate– binding cassette transporter A1 transgenic (ABCA1tg) mice (n ⴝ 18) compared with control C57BL/6 mice (n ⴝ 11) on a high-cholesterol/cholate diet. (Reproduced with permission from Proc Natl Acad Sci U S A.36 Copyright 2003 National Academy of Sciences, USA.)
liver and macrophages was then evaluated. The ABCA1 transporter was localized to the basal-lateral side of the hepatocyte, consistent with the ABCA1mediated cholesterol efflux directed toward plasma rather than the bile.33 Selective hepatic expression of the ABCA1 transporter using an ABCA1 adenoviral vector resulted in a marked increase in plasma HDL cholesterol.34 The macrophage was eliminated as the synthesis site because recent studies have shown that the HDL cholesterol generated by macrophage overexpressing ABCA1 did not make a significant contribution to plasma HDL levels.35 These combined studies established the liver as the site of HDL cholesterol synthesis in the ABCA1-tg mice. The analysis of tg mice overexpressing ABCA1 in the liver provided the unique opportunity to discover that the hepatic ABCA1 transporter plays a major role in the determination of plasma HDL levels, as well as a pivotal mechanism for the regulation of the intracellular levels of hepatic cholesterol.34,36 An overview of our current working model of lipoprotein metabolism is shown in Figure 1. This new updated model illustrates the important role of the hepatic ABCA1 transporter in determining the plasma level of HDL cholesterol. To determine whether overexpression of the ABCA1 transporter reduced the development of atherosclerosis, we quantitated the atherosclerosis in ABCA1-tg and control mice on the classic high-cholesterol/cholate diet.36 On this diet, HDL cholesterol and apolipoprotein A-I were significantly increased; however, LDL cholesterol and apolipoprotein B were decreased when compared with control mice (Table 2).36 Most of the reduction in plasma cholesterol in tg mice was in fractions corresponding to very-low-density lipoproteins (VLDL), intermediate-density lipoproteins (IDL), and LDL, with decreased apoli-
poprotein B in the VLDL and IDL/LDL fractions and increased apolipoprotein A-I in HDL. Consistent with the reduced atherogenicity of the lipoprotein phenotype in the ABCA1-tg mice, assessment of proximal aortas in the mice after 15 weeks established that the mean atherosclerotic lesion area was significantly reduced by 65% in the ABCA1-tg mice compared with controls (Figure 2). We concluded from these results that hepatic and macrophage overexpression of the ABCA1 transporter in tg mice on the specific atherogenic high cholesterol/cholate diet was associated with a marked increase in HDL cholesterol, a reduction in LDL cholesterol, and decreased atherosclerosis. Plasma non-HDL cholesterol and apolipoprotein B were modestly increased in ABCA1-tg mice on the normal chow diet and decreased on the high-cholesterol/cholate diet. When ABCA1-tg mice were put on a Western diet, there was a marked increase in LDL levels and the apolipoprotein B– containing lipoproteins when compared with control mice or tg mice on a regular chow diet (Figure 3). Based on these results, we propose that the increased cholesterol absorption on the high-cholesterol diet resulted in an increase in flux of cholesterol to the liver and upregulation of the hepatic ABCA1 transporter with increased synthesis of HDL. The increase in cholesterol levels in the apolipoprotein B– containing lipoproteins is because of the rapid transfer of free cholesterol from HDL37–39 to the apolipoprotein B– containing lipoproteins. A schematic overview of the pathways is shown in Figure 4. Interchange of cholesterol between the HDL and apolipoprotein B pathways occurs by either free cholesterol or cholesteryl ester and cholesteryl ester transfer protein. The free cholesterol transferred to the apolipoprotein B– containing lipoproteins can be esterified by the -lecithin-cholesterol acyltransA SYMPOSIUM: DYSLIPIDEMIA MANAGEMENT
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FIGURE 3. Fast-performance liquid chromatography cholesterol elution profile of pooled plasma from adenosine triphosphate– binding cassette transporter A1 transgenic (ABCA1-tg) mice (squares) and control C57BL/6 mice (diamonds) on a Western diet. HDL ⴝ highdensity lipoprotein; LDL ⴝ low-density lipoprotein; VLDL ⴝ very-low-density lipoprotein.
FIGURE 4. Schematic overview of the very-low-density lipoprotein (VLDL)–intermediate-density lipoprotein (IDL)–low-density lipoprotein (LDL) 3 LDL receptor (LDLr) and nascent high-density lipoprotein (HDL)–HDL 3 scavenger receptor-BI (SR-BI) pathways. Both free cholesterol (C) and cholesteryl esters (CE) by cholesteryl ester transfer protein (CETP) can be transferred between the 2 pathways. ABCA1 ⴝ adenosine triphosphate– binding cassette transporter A1; A-I ⴝ apolipoprotein A-I; B ⴝ apolipoprotein B; C-II ⴝ apolipoprotein C-II; E ⴝ apolipoprotein E; HL ⴝ hepatic lipase; LCAT ⴝ lecithin-cholesterol acyltransferase; LPL ⴝ lipoprotein lipase; LRP ⴝ lipoprotein receptor–related protein.
ferase activity on the apolipoprotein B– containing lipoproteins. The combined results reviewed in this article raise the interesting possibility that overexpression of the hepatic ABCA1 may be associated with increased rather than decreased atherosclerosis, depending on the genetic makeup of the animal model or patient. 14K THE AMERICAN JOURNAL OF CARDIOLOGY姞
Hepatic ABCA1 overexpression with increased cholesterol in the apolipoprotein B– containing lipoproteins in LDL receptor deficiency may be associated with potentially increased atherosclerosis. In contrast, selective overexpression of the ABCA1 transporter in macrophages would be anticipated to result in decreased atherosclerosis.
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TARGETING HIGH-DENSITY LIPOPROTEINS IN THERAPY The studies of ABC transporters provide a more precise understanding of the important role of HDL in modulating atherosclerosis risk. Among the major mechanisms by which HDL is proposed to reduce atherosclerosis risk is the reduction of oxidized LDL by several mechanisms, including the transfer of oxidized lipids to HDL where they may be cleaved by enzymes present on HDL. Thus, HDL acts to block the proatherogenic oxidation of LDL in the vessel wall and decreases the conversion to modified LDL.40 HDL is believed also to reduce atherosclerotic risk through anti-inflammatory effects. Adhesion molecules, such as vascular cell adhesion molecule–1 and E-selectin, play a major role in regulating flux of monocytes into vessel walls and are upregulated in response to inflammatory cytokines.41 Exposure of endothelial cells to increasing concentrations of HDL results in increasing reductions in cytokine-induced expression of the adhesion molecules, suggesting reduced ability of macrophages to enter the vessel wall. The common presentation of low HDL cholesterol levels with or without elevated triglycerides in patients with diabetes and metabolic syndrome, and the frequency with which this phenotype is the predominant lipid abnormality in patients hospitalized with cardiovascular disease, argue for targeting of HDL cholesterol in lipid-modifying therapy along with LDL cholesterol reduction. The VA-HIT trial showed that fibrate therapy was associated with reduced risk of cardiovascular events in association with a relatively modest increase in HDL cholesterol.9,10 Statin therapy, which is recommended as first-line lipidmodifying therapy in individuals at risk for CAD, including those with diabetes and the metabolic syndrome, also produces modest but significant increases in HDL cholesterol. For example, simvastatin has been found to increase HDL cholesterol significantly more than atorvastatin at doses of 80 mg versus 40 mg and 80 mg of atorvastatin.42 Pooled data from the rosuvastatin phase 3 clinical trial program found rosuvastatin 10 mg to increase HDL cholesterol significantly more than atorvastatin 10 mg, simvastatin 20 mg, and pravastatin 10 mg.43
CONCLUSION The recent improvements in our understanding of the mechanisms by which HDL acts to prevent atherosclerosis suggest the potential for specific therapeutic targets to augment the HDL antiatherosclerotic function. Ongoing research in this area is likely to provide additional potential targets for drug development. 1. Heart Protection Study Collaborative Group. MRC/BHF Heart Protection
Study of cholesterol lowering with simvastatin in 20,536 high-risk individuals: a randomised placebo-controlled trial. Lancet 2002;360:7–22. 2. The Long-Term Intervention with Pravastatin in Ischaemic Disease (LIPID) Study Group. Prevention of cardiovascular events and death with pravastatin in patients with coronary heart disease and a broad range of initial cholesterol levels. N Engl J Med 1998;339:1349 –1357.
3. Sacks FM, Pfeffer MA, Moye LA, Rouleau JL, Rutherford JD, Cole TG, Brown L, Warnica JW, Arnold JM, Wun CC, Davis BR, Braunwald E, for the Cholesterol and Recurrent Events Trial Investigators. The effect of pravastatin on coronary events after myocardial infarction in patients with average cholesterol levels. N Engl J Med 1996;335:1001–1009. 4. Randomised trial of cholesterol lowering in 4444 patients with coronary heart disease: the Scandinavian Simvastatin Survival Study (4S). Lancet 1994;344: 1383–1389. 5. Shepherd J, Cobbe SM, Ford I, Isles CG, Lorimer AR, MacFarlane PW, McKillop JH, Packard, CJ, for the West of Scotland Coronary Prevention Study Group. Prevention of coronary heart disease with pravastatin in men with hypercholesterolemia. N Engl J Med 1995;333:1301–1307. 6. Stamler J, Vaccaro O, Neaton JD, Wentworth D. Diabetes, other risk factors, and 12-yr cardiovascular mortality for men screened in the Multiple Risk Factor Intervention Trial. Diabetes Care 1993;16:434 –444. 7. Gordon DJ, Rifkind BM. High-density lipoprotein: the clinical implications of recent studies. N Engl J Med 1989;321:1311–1316. 8. Miller NE. Associations of high-density lipoprotein subclasses and apolipoproteins with ischemic heart disease and coronary atherosclerosis. Am Heart J 1987;113:589 –597. 9. Rubins HB, Robins SJ, Collins D, Fye CL, Anderson JW, Elam MB, Faas FH, Linares E, Schaefer EJ, Schectman G, Wilt TJ, Wittes J, for the Veterans Affairs High-Density Lipoprotein Cholesterol Intervention Trial Study Group. Gemfibrozil for the secondary prevention of coronary heart disease in men with low levels of high-density lipoprotein cholesterol. N Engl J Med 1999;341:410 –418. 10. Robins SJ, Collins D, Wittes JT, Papademetriou V, Deedwania PC, Schaefer EJ, McNamara JR, Kashyap ML, Hershman JM, Wexler LF, Rubins HB. Relation of gemfibrozil treatment and lipid levels with major coronary events. VAHIT: a randomized controlled trial. JAMA 2001;285:1585–1591. 11. Genest JJ Jr, Martin-Munley SS, McNamara JR, Ordovas JM, Jenner J, Myers RH, Silberman SR, Wilson PW, Salem DN, Schaefer EJ. Familial lipoprotein disorders in patients with premature coronary artery disease. Circulation 1992; 85:2025–2033. 12. Expert Panel on Detection, Evaluation, and Treatment of High Blood Cholesterol in Adults. Executive summary of the third report of the National Cholesterol Education Program (NCEP) Expert Panel on Detection, Evaluation, and Treatment of High Blood Cholesterol in Adults (Adult Treatment Panel III). JAMA 2001;285:2486 –2497. 13. Glomset JA. The plasma lecithin: cholesterol acyltransferase reaction. J Lipid Res 1968;9:55–67. 14. Fielding CJ, Fielding PE. Molecular physiology of reverse cholesterol transport. J Lipid Res 1995;36:211–228. 15. Brooks-Wilson A, Marcil M, Clee SM, Zhang LH, Roomp K, van Dam M, Yu L, Brewer C, Collins JA, Molhuizen HO, et al. Mutations in ABC1 in Tangier disease and familial high-density lipoprotein deficiency. Nat Genet 1999;22:336 – 345. 16. Rust S, Rosier M, Funke H, Real J, Amoura Z, Piette JC, Deleuze JF, Brewer HB, Duverger N, Denefle P, Assmann G. Tangier disease is caused by mutations in the gene encoding ATP-binding cassette transporter 1. Nat Genet 1999;22: 352–355. 17. Bodzioch M, Orso E, Klucken J, Langmann T, Bottcher A, Diederich W, Drobnik W, Barlage S, Buchler C, Porsch-Ozcurumez M, et al. The gene encoding ATP-binding cassette transporter 1 is mutated in Tangier disease. Nat Genet 1999;22:347–351. 18. Remaley AT, Rust S, Rosier M, Knapper C, Naudin L, Broccardo C, Peterson KM, Koch C, Arnould I, Prades C, et al. Human ATP-binding cassette transporter 1 (ABC1): genomic organization and identification of the genetic defect in the original Tangier disease kindred. Proc Natl Acad Sci U S A 1999;96:12685– 12690. 19. Brousseau ME, Schaefer EJ, Dupuis J, Eustace B, Van Eerdewegh P, Goldkamp AL, Thurston LM, FitzGerald MG, Yasek-McKenna D, O’Neill G, et al. Novel mutations in the gene encoding ATP-binding cassette 1 in four Tangier disease kindreds. J Lipid Res 2000;41:433–441. 20. Lawn RM, Wade DP, Garvin MR, Wang X, Schwartz K, Porter JG, Seilhamer JJ, Vaughan AM, Oram JF. The Tangier disease gene product ABC1 controls the cellular apolipoprotein-mediated lipid removal pathway. J Clin Invest 1999;104:R25–R31. 21. Assman G, von Eckardstein A, Brewer HB Jr. Familial high-density lipoprotein deficiency: Tangier disease. In: Beaudet A, Childs B, Kinzler K, Scriver C, Sly W, Valle D, Vogelstein B, eds. The Metabolic and Molecular Basis of Inherited Disease. 8th ed. New York: McGraw Hill, Health Professions Division, 2001:2937–2980. 22. Santamarina-Fojo S, Peterson K, Knapper C, Qiu Y, Freeman L, Cheng JF, Osorio J, Remaley A, Yang XP, Haudenschild C, et al. Complete genomic sequence of the human ABCA1 gene: analysis of the human and mouse ATPbinding cassette A promoter. Proc Natl Acad Sci U S A 2000;97:7987–7992. 23. Venkateswaran A, Laffitte BA, Joseph SB, Mak PA, Wilpitz DC, Edwards PA, Tontonoz P. Control of cellular cholesterol efflux by the nuclear oxysterol receptor LXR alpha. Proc Natl Acad Sci U S A 2000;97:12097–12102. 24. Schwartz K, Lawn RM, Wade DP. ABC1 gene expression and ApoA-Imediated cholesterol efflux are regulated by LXR. Biochem Biophys Res Commun 2000;274:794 –802. 25. Costet P, Luo Y, Wang N, Tall AR. Sterol-dependent transactivation of the ABC1 promoter by the liver X receptor/retinoid X receptor. J Biol Chem 2000;275:28240 –28245.
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26. Chawla A, Boisvert WA, Lee CH, Laffitte BA, Barak Y, Joseph SB, Liao D, Nagy L, Edwards PA, Curtiss LK, Evans RM, Tontonoz P. A PPAR ␥-LXRABCA1 pathway in macrophages is involved in cholesterol efflux and atherogenesis. Mol Cell 2001;7:161–171. 27. Neufeld EB, Remaley AT, Demosky SJ, Stonik JA, Cooney AM, Comly M, Dwyer NK, Zhang M, Blanchette-Mackie J, Santamarina-Fojo S, Brewer HB Jr. Cellular localization and trafficking of the human ABCA1 transporter. J Biol Chem 2001;276:27584 –27590. 28. Takahashi Y, Smith JD. Cholesterol efflux to apolipoprotein AI involves endocytosis and resecretion in a calcium-dependent pathway. Proc Natl Acad Sci U S A 1999;96:11358 –11363. 29. Remaley AT, Stonik JA, Demosky SJ, Neufeld EB, Bocharov AV, Vishnyakova TG, Eggerman TL, Patterson AP, Duverger NJ, Santamarina-Fojo S, Brewer HB Jr. Apolipoprotein specificity for lipid efflux by the human ABCAI transporter. Biochem Biophys Res Commun 2001;280:818 –823. 30. Vaisman BL, Lambert G, Amar M, Joyce C, Ito T, Shamburek RD, Cain WJ, Fruchart-Najib J, Neufeld ED, Remaley AT, Brewer HB Jr, Santamarina-Fojo S. ABCA1 overexpression leads to hyperalphalipoproteinemia and increased biliary cholesterol excretion in transgenic mice. J Clin Invest 2001;108:303–309. 31. Cavelier LB, Qiu Y, Bielicki JK, Afzal V, Cheng JF, Rubin EM. Regulation and activity of the human ABCA1 gene in transgenic mice. J Biol Chem 2001;276:18046 –18051. 32. Singaraja RR, Bocher V, James ER, Clee SM, Zhang LH, Leavitt BR, Tan B, Brooks-Wilson A, Kwok A, Bissada N, et al. Human ABCA1 BAC transgenic mice show increased high density lipoprotein cholesterol and apoAI-dependent efflux stimulated by an internal promoter containing liver X receptor response elements in intron 1. J Biol Chem 2001;276:33969 –33979. 33. Neufeld EB, Demosky SJ Jr, Stonik JA, Combs C, Remaley AT, Duverger N, Santamarina-Fojo S, Brewer HB Jr. The ABCA1 transporter functions on the basolateral surface of hepatocytes. Biochem Biophys Res Commun 2002;297: 974 –979. 34. Basso F, Freeman L, Knapper CL, Remaley A, Stonik J, Neufeld EB, Tansey
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T, Amar MJ, Fruchart-Najib J, Duverger N, Santamarina-Fojo S, Brewer HB Jr. Role of the hepatic ABCA1 transporter in modulating intrahepatic cholesterol and plasma HDL cholesterol concentrations. J Lipid Res 2003;44:296 –302. 35. Haghpassand M, Bourassa PA, Francone OL, Aiello RJ. Monocyte/macrophage expression of ABCA1 has minimal contribution to plasma HDL levels. J Clin Invest 2001;108:1315–1320. 36. Joyce CW, Amar MJ, Lambert G, Vaisman BL, Paigen B, Najib-Fruchart J, Hoyt RF Jr, Neufeld ED, Remaley AT, Fredrickson DS, Brewer HB Jr, Santamarina-Fojo S. The ATP binding cassette transporter A1 (ABCA1) modulates the development of aortic atherosclerosis in C57BL/6 and apoE-knockout mice. Proc Natl Acad Sci U S A 2002;99:407–412. 37. Schwartz CC, Berman M, Vlahcevic ZR, Halloran LG, Gregory DH, Swell L. Multicompartmental analysis of cholesterol metabolism in man: the hepatic bile acid and biliary cholesterol precursor sites. J Clin Invest 1978;61:408 –423. 38. Schwartz CC, Vlahcevic ZR, Berman M, Meadows JG, Nisman RM, Swell L. Central role of high-density lipoprotein in plasma free cholesterol metabolism. J Clin Invest 1982;70:105–116. 39. Schwartz CC, Vlahcevic ZR, Halloran LG, Swell L. An in vivo evaluation in man of the transfer of esterified cholesterol between lipoproteins and into the liver and bile. Biochim Biophys Acta 1981;663:143–162. 40. Van Lenten BJ, Navab M, Shih D, Fogelman AM, Lusis AJ. The role of high-density lipoproteins in oxidation and inflammation. Trends Cardiovasc Med 2001;1:155–161. 41. Barter P. Effects of inflammation on high-density lipoproteins. Arterioscler Thromb Vasc Biol 2002;22:1062–1063. 42. Illingworth DR, Crouse JR III, Hunninghake DB, Davidson MH, Escobar ID, Stalenhoef AF, Paragh G, Ma PT, Liu M, Melino MR, et al. A comparison of simvastatin and atorvastatin up to maximal recommended doses in a large multicenter randomized clinical trial. Curr Med Res Opin 2001;17:43–50. 43. Blasetto J, Stein E, Brown WV, Chitra R, Raza A. Efficacy of rosuvastatin compared with other statins at selected starting doses in hypercholesterolemic patients and in special population groups. Am J Cardiol 2003;91(suppl 5A):3C– 10C.
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