The mechanisms responsible for the inverse relationship between plasma high-density lipoprotein (HDL) levels and atherosclerotic cardiovascular disease are poorly understood. The ATP-binding cassette transporter A1 (ABCA1) mediates efflux of cellular cholesterol to lipid-poor apolipoproteins but not to HDL particles that constitute the bulk of plasma HDL. We show that two ABC transporters of unknown function, ABCG1 and ABCG4, mediate isotopic and net mass efflux of cellular cholesterol to HDL. In transfected 293 cells, ABCG1 and ABCG4 stimulate cholesterol efflux to both smaller (HDL-3) and larger (HDL-2) subclasses but not to lipid-poor apoA-I. Treatment of macrophages with an liver X receptor activator results in up-regulation of ABCG1 and increases cholesterol efflux to HDL. RNA interference reduced the expression of ABCG1 in liver X receptor-activated macrophages and caused a parallel decrease in cholesterol efflux to HDL. These studies indicate that ABCG1 and ABCG4 promote cholesterol efflux from cells to HDL. ABCG1 is highly expressed in macrophages and probably mediates cholesterol efflux from macrophage foam cells to the major HDL fractions, providing a mechanism to explain the relationship between HDL levels and atherosclerosis risk.A major theory to account for the inverse relationship between high-density lipoprotein (HDL) levels and cardiovascular risk is that HDL promotes the efflux of cholesterol from arterial wall macrophage foam cells and decrease atherosclerosis. This hypothesis appeared to be supported by the discovery that Tangier disease, a disorder characterized by very low HDL levels, macrophage foam cell accumulation, and increased atherosclerosis, is caused by mutations in the ATP-binding cassette transporter, ABCA1 (1-4). ABCA1 mediates efflux of cellular phospholipids and cholesterol to lipid-poor apolipoproteins, such as apoA-I and apoE (5, 6), initiating the formation of HDL. However, ABCA1 interacts poorly with HDL-2 and HDL-3 particles (5, 7) that constitute the bulk of the plasma HDL, and ABCA1 variants are not likely to account for a major part of the genetic variation in HDL levels in the general population (8). Thus, the activity of ABCA1 does not readily account for cholesterol efflux from foam cells to HDL, and the mechanism underlying the inverse relationship between HDL levels and atherosclerosis risk remains uncertain.The oxysterol-activated transcription factors liver X receptor͞ retinoid X receptor (LXR͞RXR) induce the expression of ABCA1, as well as a number of other molecules involved in cellular cholesterol efflux, transport, and excretion (9, 10). Treatment of macrophages with LXR activators increased net cholesterol efflux to HDL-2, suggesting the presence of unique LXR target genes mediating cholesterol efflux to HDL (11). Some ABCG family members are also LXR͞RXR targets, such as ABCG5 and ABCG8, the defective genes in sitosterolemia (12)(13)(14). ABCG family members are half-transporters, largely of unknown function. These considerations led us to investigate the ...

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Genetic deficiency or inhibition of cholesteryl ester transfer protein (CETP) leads to a marked increase in plasma levels of large HDL-2 particles. However, there is concern that such particles may be dysfunctional in terms of their ability to promote cholesterol efflux from macrophages. Recently, the ATP-binding cassette transporter ABCG1, a macrophage liver X receptor (LXR) target, has been shown to stimulate cholesterol efflux to HDL. We have assessed the ability of HDL from subjects with homozygous deficiency of CETP (CETP-D) to promote cholesterol efflux from macrophages and have evaluated the role of ABCG1 and other factors in this process. CETP-D HDL-2 caused a 2-to 3-fold stimulation of net cholesterol efflux compared with control HDL-2 in LXR-activated macrophages, due primarily to an increase in lecithin:cholesterol acyltransferase-mediated (LCAT-mediated) cholesteryl ester formation in media. Genetic knockdown or overexpression of ABCG1 showed that increased cholesterol efflux to CETP-D HDL was ABCG1 dependent. LCAT and apoE contents of CETP-D HDL-2 were markedly increased compared with control HDL-2, and increased cholesterol esterification activity resided within the apoE-HDL fraction. Thus, CETP-D HDL has enhanced ability to promote cholesterol efflux from foam cells in an ABCG1-dependent pathway due to an increased content of LCAT and apoE.

Objectives-This study examines the ABCG1-mediated cholesterol efflux and intracellular cholesterol transport by studying the ABCG1 localization and function in macrophages. Methods and Results-HEK 293 cell overexpressing ABCG1, RNA interference, or macrophages from ABCG1 or ABCG4 knockout mice were used. ABCG1 but not ABCG4 had a major role in the increased cholesterol mass efflux produced by treatment of macrophages with LXR activators. In 293 cells, ABCG1 was found in the plasma membrane, Golgi, and recycling endosomes. In contrast, in basal macrophages, ABCG1 was predominantly intracellular, and redistributed to the plasma membrane after LXR activation. LXR activation increased macrophage cholesterol efflux to high-density lipoprotein (HDL), low-density lipoprotein (LDL), and cyclodextrin in an ABCG1-dependent fashion. Suppression of ABCG1 expression increased cholesteryl ester formation and decreased SREBP2 target gene expression in macrophages, even in the absence of HDL acceptors. Conclusions-LXR

Objective-This study examines the effects of pharmacological inhibition of cholesteryl ester transfer protein (CETP) on the ability of high-density lipoprotein particles (HDL) to promote net cholesterol efflux from human THP-1 macrophage foam cells. Methods and Results-Two groups of 8 healthy, moderately hyperlipidemic subjects received the CETP inhibitor torcetrapib at 60 or 120 mg daily for 8 weeks. Torcetrapib increased HDL cholesterol levels in both groups by 50% and 60%, respectively. Compared with baseline, torcetrapib 60 mg daily increased HDL-mediated net cholesterol efflux from foam cells primarily by increasing HDL concentrations, whereas 120 mg daily torcetrapib increased cholesterol efflux both by increasing HDL concentration and by causing increased efflux at matched HDL concentrations. There was an increased content of lecithin:cholesterol acyltransferase (LCAT) and apolipoprotein E (apoE) in HDL-2 only at the 120 mg dose. ABCG1 activity was responsible for 40% to 50% of net cholesterol efflux to both control and T-HDL. Conclusions-These data indicate that inhibition of CETP by torcetrapib causes a modest increase in the ability of HDL to promote net cholesterol efflux at the 60 mg dose, and a more dramatic increase at the 120 mg dose in association with enhanced particle functionality. The HDL-mediated removal of excess free cholesterol (FC) from macrophage foam cells is thought to play a major role in the protection against the development of atherosclerosis. 2 However, current therapies for raising HDL are limited. Despite the favorable effects of statins on coronary heart disease, these agents have only modest effects on HDL-C levels. 3,4 Fibrates and niacin can raise HDL-C, but the increases are rarely Ͼ30% and only some of the fibrate trials have shown prevention of coronary events in patients with low HDL, and niacin is often not well tolerated. 5,6 Novel targets to raise HDL-C have emerged from the recent understanding of HDL synthesis, maturation, and catabolism. In humans, cholesteryl ester (CE) generated by the lecithin:cholesterol acyltransferase (LCAT) enzyme in HDL is transferred to apoB-lipoproteins by the cholesteryl ester transfer protein (CETP). CETP promotes the removal of cholesteryl ester (CE) from antiatherogenic HDL to atherogenic apoB-containing particles in exchange for triglycerides (TGs). 7 The marked increase in HDL cholesterol associated with human deficiency of CETP has suggested CETP inhibition as a potential strategy to treat atherosclerotic disease. 8,9 However, there has been concern that HDL particles accumulating in CETP deficiency might be dysfunctional. 10 We recently reported that large CE-rich HDL particles from 4 subjects with complete CETP deficiency (CETP-D) showed increased ability to promote cholesterol efflux from macrophage foam cells. 11 Central to this observation was the key role of LCAT and apolipoprotein E (apoE) present at high levels in CETP-D HDL driving net cholesterol efflux. Cholesterol efflux from cells to HDL can occur by passive diffusion...