Sterol efflux mediated by endogenous macrophage ApoE expression is independent of ABCA1.

Sterol efflux importantly contributes to preservation of cellular cholesterol homeostasis, and multiple pathways may be involved for mediating such efflux. Recently, an important role has been ascribed to ABCA1 in facilitating lipid efflux from cells, including macrophages, to extracellular lipid-free apolipoproteins. Macrophages are relatively unique among cells because they express apoprotein E (apoE) as a major protein product, and this endogenous expression of apoE increases sterol and phospholipid efflux from macrophages. The studies in this article were designed to test whether the sterol efflux mediated by the endogenous expression of apoE in macrophages was dependent on ABCA1 expression. These studies were facilitated by comparing apoE-expressing J774 cells (J774E(+)) with nonexpressing parental cells (J774E(-)). Sterol efflux was higher from J774E(+) cells compared with J774E(-) cells, but the increment in efflux between these cell types was not increased by induction of ABCA1 expression with cAMP. Induction of ABCA1 with cAMP, however, did increase sterol efflux to exogenously added apoA1 from both cell types. Inhibitors of ABCA1 activity significantly reduced (by 40% to 50%) sterol efflux from both J774E(+) and J774E(-) cells treated with cAMP and apoA1. This inhibitor did not, however, reduce the increment in sterol efflux due to the expression of endogenous apoE. The results of these studies indicate that the increment in sterol efflux mediated by the endogenous expression of apoE in macrophages does not depend on ABCA1 expression or activity.

ATP-binding cassette transporter A1 mediates cellular secretion of alpha-tocopherol.

Alpha-tocopherol (alpha-TOH) is associated with plasma lipoproteins and accumulates in cell membranes throughout the body, suggesting that lipoproteins play a role in transporting alpha-TOH between tissues. Here we show that secretion of alpha-TOH from cultured cells is mediated in part by ABCA1, an ATP-binding cassette protein that transports cellular cholesterol and phospholipids to lipid-poor high density lipoprotein (HDL) apolipoproteins such as apoA-I. Treatment of human fibroblasts and murine RAW264 macrophages with cholesterol and/or 8-bromo-cyclic AMP, which induces ABCA1 expression, enhanced apoA-I-mediated alpha-TOH efflux. ApoA-I lacked the ability to remove alpha-TOH from Tangier disease fibroblasts that have a nonfunctional ABCA1. BHK cells that lack an active ABCA1 pathway markedly increased secretion of alpha-TOH to apoA-I when forced to express ABCA1. ABCA1 also mediated a fraction of the alpha-TOH efflux promoted by lipid-containing HDL particles, indicating that HDL promotes alpha-TOH efflux by both ABCA1-dependent and -independent processes. Exposing apoA-I to ABCA1-expressing cells did not enhance its ability to remove alpha-TOH from cells lacking ABCA1, consistent with this transporter participating directly in the translocation of alpha-TOH to apolipoproteins. These studies provide evidence that ABCA1 mediates secretion of cellular alpha-TOH into the HDL metabolic pathway, a process that may facilitate vitamin transport between tissues and influence lipid oxidation.

ABCA1. The gatekeeper for eliminating excess tissue cholesterol.

It is widely believed that HDL functions to transport cholesterol from peripheral cells to the liver by reverse cholesterol transport, a pathway that may protect against atherosclerosis by clearing excess cholesterol from arterial cells. A cellular ATP-binding cassette transporter (ABC) called ABCA1 mediates the first step of reverse cholesterol transport: the transfer of cellular cholesterol and phospholipids to lipid-poor apolipoproteins. Mutations in ABCA1 cause Tangier disease (TD), a severe HDL deficiency syndrome characterized by accumulation of cholesterol in tissue macrophages and prevalent atherosclerosis. Studies of TD heterozygotes revealed that ABCA1 activity is a major determinant of plasma HDL levels and susceptibility to CVD. Drugs that induce ABCA1 in mice increase clearance of cholesterol from tissues and inhibit intestinal absorption of dietary cholesterol. Multiple factors related to lipid metabolism and other processes modulate expression and tissue distribution of ABCA1.Therefore, as the primary gatekeeper for eliminating tissue cholesterol, ABCA1 has a major impact on cellular and whole body cholesterol metabolism and is likely to play an important role in protecting against cardiovascular disease.

Novel approaches to treating cardiovascular disease: lessons from Tangier disease.

Atherosclerotic cardiovascular disease (CVD) remains the leading cause of morbidity and mortality in Western societies. Although cholesterol is a major CVD risk factor, therapeutic interventions to lower plasma cholesterol levels have had limited success in reducing coronary events. Thus, novel approaches are needed to reduce or eliminate CVD. A potential therapeutic target is a newly discovered ATP binding cassette transporter called ABCA1, a cell membrane protein that is the gateway for secretion of excess cholesterol from macrophages into the high density lipoprotein (HDL) metabolic pathway. Mutations in ABCA1 cause Tangier disease, a severe HDL deficiency syndrome characterised by accumulation of cholesterol in tissue macrophages and prevalent atherosclerosis. Studies of Tangier disease heterozygotes revealed that the relative activity of ABCA1 determines plasma HDL levels and susceptibility to CVD. Drugs that induce ABCA1 in mice increase clearance of cholesterol from tissues and inhibit intestinal absorption of dietary cholesterol. Thus, ABCA1-stimulating drugs have the potential to both mobilise cholesterol from atherosclerotic lesions and eliminate cholesterol from the body. By reducing plaque formation and rupture independently of the atherogenic factors involved, these drugs would be powerful agents for treating CVD.

Membrane lipid domains distinct from cholesterol/sphingomyelin-rich rafts are involved in the ABCA1-mediated lipid secretory pathway.

Efflux of excess cellular cholesterol mediated by lipid-poor apolipoproteins occurs by an active mechanism distinct from passive diffusion and is controlled by the ATP-binding cassette transporter ABCA1. Here we examined whether ABCA1-mediated lipid efflux involves the selective removal of lipids associated with membrane rafts, plasma membrane domains enriched in cholesterol and sphingomyelin. ABCA1 was not associated with cholesterol and sphingolipid-rich membrane raft domains based on detergent solubility and lack of colocalization with marker proteins associated with raft domains. Lipid efflux to apoA-I was accounted for by decreases in cellular lipids not associated with cholesterol/sphingomyelin-rich membranes. Treating cells with filipin, to disrupt raft structure, or with sphingomyelinase, to digest plasma membrane sphingomyelin, did not impair apoA-I-mediated cholesterol or phosphatidylcholine efflux. In contrast, efflux of cholesterol to high density lipoproteins (HDL) or plasma was partially accounted for by depletion of cholesterol from membrane rafts. Additionally, HDL-mediated cholesterol efflux was partially inhibited by filipin and sphingomyelinase treatment. Apo-A-I-mediated cholesterol efflux was absent from fibroblasts with nonfunctional ABCA1 (Tangier disease cells), despite near normal amounts of cholesterol associated with raft domains and normal abilities of plasma and HDL to deplete cholesterol from these domains. Thus, the involvement of membrane rafts in cholesterol efflux applies to lipidated HDL particles but not to lipid-free apoA-I. We conclude that cholesterol and sphingomyelin-rich membrane rafts do not provide lipid for efflux promoted by apolipoproteins through the ABCA1-mediated lipid secretory pathway and that ABCA1 is not associated with these domains.

Tangier disease and ABCA1.

Tangier disease is an autosomal recessive genetic disorder characterized by a severe high-density lipoprotein (HDL) deficiency, sterol deposition in tissue macrophages, and prevalent atherosclerosis. Mutations in the ATP binding cassette transporter ABCA1 cause Tangier disease and other familial HDL deficiencies. ABCA1 controls a cellular pathway that secretes cholesterol and phospholipids to lipid-poor apolipoproteins. This implies that an inability of newly synthesized apolipoproteins to acquire cellular lipids by the ABCA1 pathway leads to their rapid degradation and an over-accumulation of cholesterol in macrophages. Thus, ABCA1 plays a critical role in modulating flux of tissue cholesterol and phospholipids into the reverse cholesterol transport pathway, making it an important therapeutic target for clearing excess cholesterol from macrophages and preventing atherosclerosis.

ABCA1 is the cAMP-inducible apolipoprotein receptor that mediates cholesterol secretion from macrophages.

Lipid-poor high density lipoprotein apolipoproteins remove cholesterol and phospholipids from cells by an active secretory pathway controlled by an ABC transporter called ABCA1. This pathway is induced by cholesterol and cAMP analogs in a cell-specific manner. Here we provide evidence that increased plasma membrane ABCA1 accounts for the enhanced apolipoprotein-mediated lipid secretion from macrophages induced by cAMP analogs. Treatment of RAW264 macrophages with 8-bromo-cAMP caused parallel increases in apoA-I-mediated cholesterol efflux, ABCA1 mRNA and protein levels, incorporation of ABCA1 into the plasma membrane, and binding of apoA-I to cell-surface ABCA1. All of these parameters declined to near base-line values within 6 h after removal of 8-bromo-cAMP, indicating that ABCA1 is highly unstable and is degraded rapidly in the absence of inducer. Thus, ABCA1 is likely to be the cAMP-inducible apolipoprotein receptor that promotes removal of cholesterol and phospholipids from macrophages.

ABCA1-mediated transport of cellular cholesterol and phospholipids to HDL apolipoproteins.

Lipid-poor apolipoproteins remove cellular cholesterol and phospholipids by an active transport pathway controlled by an ATP binding cassette transporter called ABCA1 (formerly ABC1). Mutations in ABCA1 cause Tangier disease, a severe HDL deficiency syndrome characterized by a rapid turnover of plasma apolipoprotein A-I, accumulation of sterol in tissue macrophages, and prevalent atherosclerosis. This implies that lipidation of apolipoprotein A-I by the ABCA1 pathway is required for generating HDL particles and clearing sterol from macrophages. Thus, the ABCA1 pathway has become an important therapeutic target for mobilizing excess cholesterol from tissue macrophages and protecting against atherosclerosis.

Apolipoprotein binding to protruding membrane domains during removal of excess cellular cholesterol.

High-density lipoproteins (HDL) are believed to protect against cardiovascular disease by removing excess cholesterol from cells. Lipid-free HDL apolipoproteins remove cellular cholesterol and phospholipids by an active, Golgi-dependent process that is still poorly understood. Here we characterized the morphology of apolipoprotein binding sites on cultured cells by immunogold electron microscopy. After 6 h incubations with lipid-free apoA-I or apoE, immunogold-labeled apolipoproteins were distributed sparsely along the planar surface of human fibroblasts and THP-1 macrophages. Overloading these cells with cholesterol led to a several-fold increase in the concentration of immunogold-labeled apoA-I and apoE on the cell surface, and over 80% of these gold particles were associated with novel electron-opaque structures protruding from the plasma membrane. Protrusions binding apoE were larger (100-200 nm) than those binding apoA-I (10-60 nm), and similar apoA-I-binding structures appeared when cells were incubated with either purified apoA-I or HDL particles. These structures were formed and enlarged by a time-dependent process inhibited by the Golgi disruptor brefledin A, the energy poison NaF, and low temperature. Moreover, formation of these structures was nearly absent in fibroblasts from a subject with Tangier disease, cells that lack a functioning apolipoprotein-mediated lipid removal pathway. Thus, formation of novel apolipoprotein binding structures protruding from the cell surface is an intermediate step in the cellular pathway by which apolipoproteins remove excess cholesterol.

The Tangier disease gene product ABC1 controls the cellular apolipoprotein-mediated lipid removal pathway.

The ABC1 transporter was identified as the defect in Tangier disease by a combined strategy of gene expression microarray analysis, genetic mapping, and biochemical studies. Patients with Tangier disease have a defect in cellular cholesterol removal, which results in near zero plasma levels of HDL and in massive tissue deposition of cholesteryl esters. Blocking the expression or activity of ABC1 reduces apolipoprotein-mediated lipid efflux from cultured cells, and increasing expression of ABC1 enhances it. ABC1 expression is induced by cholesterol loading and cAMP treatment and is reduced upon subsequent cholesterol removal by apolipoproteins. The protein is incorporated into the plasma membrane in proportion to its level of expression. Different mutations were detected in the ABC1 gene of 3 unrelated patients. Thus, ABC1 has the properties of a key protein in the cellular lipid removal pathway, as emphasized by the consequences of its defect in patients with Tangier disease.

Reduction in apolipoprotein-mediated removal of cellular lipids by immortalization of human fibroblasts and its reversion by cAMP: lack of effect with Tangier disease cells.

High density lipoprotein (HDL) phospholipids and apolipoproteins remove cellular lipids by two distinct mechanisms, but their relative contribution to reverse cholesterol transport is unknown. Whereas phospholipid-mediated cholesterol efflux from cultured cells reflects the activity of the HDL receptor SR-BI, apolipoprotein-mediated lipid removal is regulated in response to changes in cellular cholesterol content (positive) and cell proliferation rates (negative). Here we show that immortalization of human skin fibroblast lines with the papillomavirus E6/E7 oncogenes increased their proliferation rates and selectively reduced the activity of the apolipoprotein-mediated lipid removal pathway. This reduction was accompanied by a decrease in cellular cAMP levels and was reversed by treatment with a cAMP analog. The stimulatory effect of cAMP was independent of changes in cellular phenotype or activities of cholesteryl ester cycle enzymes. The severely impaired apolipoprotein-mediated lipid removal pathway in Tangier disease fibroblasts, which persisted after immortalization, was not improved by treatment with a cAMP analog, implying that the cellular defect in Tangier disease is upstream from this cAMP-dependent signaling pathway.These results indicate that papillomavirus-induced immortalization of fibroblasts selectively reduces the activity of the apolipoprotein-mediated lipid removal pathway by a cAMP-dependent process, perhaps to prevent loss of cellular lipids needed for continual membrane synthesis.

Phospholipid transfer protein enhances removal of cellular cholesterol and phospholipids by high-density lipoprotein apolipoproteins.

High-density lipoprotein (HDL) apolipoproteins remove excess cholesterol from cells by an active transport pathway that may protect against atherosclerosis. Here we show that treatment of cholesterol-loaded human skin fibroblasts with phospholipid transfer protein (PLTP) increased HDL binding to cells and enhanced cholesterol and phospholipid efflux by this pathway. PLTP did not stimulate lipid efflux in the presence of albumin, purified apolipoprotein A-I, and phospholipid vesicles, suggesting specificity for HDL particles. PLTP restored the lipid efflux activity of mildly trypsinized HDL, presumably by regenerating active apolipoproteins. PLTP-stimulated lipid efflux was absent in Tangier disease fibroblasts, induced by cholesterol loading, and inhibited by brefeldin A treatment, indicating selectivity for the apolipoprotein-mediated lipid removal pathway. The lipid efflux-stimulating effect of PLTP was not attributable to generation of prebeta HDL particles in solution but instead required cellular interactions. These interactions increased cholesterol efflux to minor HDL particles with electrophoretic mobility between alpha and prebeta. These findings suggest that PLTP promotes cell-surface binding and remodeling of HDL so as to improve its ability to remove cholesterol and phospholipids by the apolipoprotein-mediated pathway, a process that may play an important role in enhancing flux of excess cholesterol from tissues and retarding atherogenesis.

Cellular cholesterol transport and efflux in fibroblasts are abnormal in subjects with familial HDL deficiency.

Familial high density lipoprotein (HDL) deficiency (FHD) is a genetic lipoprotein disorder characterized by a severe decrease in the plasma HDL cholesterol (-C) level (less than the fifth percentile). Unlike Tangier disease, FHD is transmitted as an autosomal dominant trait. FHD subjects have none of the clinical manifestations of Tangier disease (lymphoid tissue infiltration with cholesteryl esters and/or neurological manifestations). Plasmas from FHD subjects contain pre-beta-migrating HDLs but are deficient in alpha-migrating HDLs. We hypothesized that a reduced HDL-C level in FHD is due to abnormal transport of cellular cholesterol to the plasma membrane, resulting in reduced cholesterol efflux onto nascent HDL particles, leading to lipid-depleted HDL particles that are rapidly catabolized. Cellular cholesterol metabolism was investigated in skin fibroblasts from FHD and control subjects. HDL3- and apolipoprotein (apo) A-I-mediated cellular cholesterol and phosphatidylcholine efflux was examined by labeling cells with [3H]cholesterol and [3H]choline, respectively, during growth and cholesterol loading during growth arrest. FHD cells displayed an approximately 25% reduction in HDL3-mediated cellular cholesterol efflux and an approximately 50% to 80% reduction in apoA-I-mediated cholesterol and phosphatidylcholine efflux compared with normal cells. Cellular cholesterol ester levels were decreased when cholesterol-labeled cells were incubated with HDL3 in normal cells, but cholesterol ester mobilization was significantly reduced in FHD cells. HDL3 binding to fibroblasts and the possible role of the HDL binding protein/vigilin in FHD were also investigated. No differences were observed in 125I-HDL3 binding to LDL-loaded cells between FHD and control cells. HDL binding protein/vigilin mRNA levels and its protein expression were constitutively expressed in FHD cells and could be modulated ( approximately 2-fold increase) by elevated cellular cholesterol in normal cells. In conclusion, FHD is characterized by reduced HDL3- and apoA-I-mediated cellular cholesterol efflux. It is not associated with abnormal cellular HDL3 binding or a defect in a putative HDL binding protein.

High-density lipoprotein-binding protein (HBP)/vigilin is expressed in human atherosclerotic lesions and colocalizes with apolipoprotein E.

Accumulation of cholesteryl esters within cells of the arterial intima is a hallmark of atherosclerosis. A small number of proteins have been shown in vitro to be upregulated by cellular cholesterol loading, including apolipoprotein E (apoE) and the recently cloned HDL-binding protein (HBP), but only apoE has been shown to be upregulated in cholesterol-loaded cells in atherosclerosis. To determine whether HBP (also called vigilin) might be expressed in human atherosclerosis, immunohistochemistry and in situ hybridization were performed on coronary arteries of 18 patients. HBP/vigilin was detected on all endothelial cells. HBP/vigilin mRNA and protein also were detected on a subset of macrophages and occasionally on smooth muscle cells (SMC) in atherosclerotic plaques but were not detected on these cell types in nondiseased coronary intima. The majority of HBP/vigilin-expressing macrophages were foam cells, but HBP/vigilin expression also was detected rarely in nonfoam cell macrophages. Foam cell macrophage HBP/vigilin expression was present in 100% of atherosclerotic quadrants, and nonfoam cell macrophage HBP/vigilin expression was present in 6% of atherosclerotic quadrants. HBP/vigilin-expressing human plaque cells also expressed apoE. However, HBP/vigilin was detected in cardiac myocyte foam cells of an apoE-deficient mouse, demonstrating that HBP/vigilin expression can occur independently of apoE. These results suggest that in vivo HBP/vigilin expression is upregulated by intracellular cholesterol loading but also that other factors present in atherosclerotic plaques may upregulate HBP/vigilin. Although the exact function of HBP/vigilin is unknown, its expression in plaque macrophages suggests a role for this molecule in atherogenesis.

Phosphoproteins regulated by the interaction of high-density lipoprotein with human skin fibroblasts.

Interaction of HDL with cells activates protein kinase C (PKC), a process that may be important in stimulating efflux of excess cellular cholesterol. Here we report that HDL treatment of cholesterol-loaded fibroblasts increases 32P labeling of three acidic phosphoproteins. These phosphoproteins, called pp80, pp27, and pp18 based on apparent M(r) in kD, were also phosphorylated by acute treatment of cells with phorbol myristate acetate, suggesting that they are regulated in response to PKC activation. The HDL-stimulated phosphorylation of pp80 and pp18 was significant after only 30 seconds and was sustained for at least 30 and 120 minutes, respectively, while increased phosphorylation of pp27 was transient, reaching a maximum at 10 minutes. Both pp27 and pp18 were phosphorylated on serine/threonine residues, whereas pp80 was phosphorylated on serine/threonine and tyrosine residues. Immunoprecipitation studies suggested that pp80 is the myristoylated alanine-rich C kinase substrate protein, but the identities of pp27 and pp18 are unknown. HDL and trypsin-digested HDL stimulated phosphorylation of pp80 and pp27, while purified apoA-I, apoA-II, or apoE had no stimulatory effects, indicating that the active component in HDL was trypsin resistant and unlikely to be an apolipoprotein. Conversely, HDL, apoA-I, apoA-II, and apoE all stimulated pp18 phosphorylation, while trypsin-digested HDL had less effect, consistent with pp18's being responsive to HDL apolipoproteins. Treatment of cholesterol-depleted cells with apoA-I also stimulated phosphorylation of pp18, but only transiently. These results suggest that HDL interaction with cells activates diverse PKC-mediated pathways that target different phosphoproteins. Of these three phosphoproteins, only pp18 has a phosphorylation response consistent with its being involved in apolipoprotein-mediated lipid transport.

High density lipoproteins stimulate mitogen-activated protein kinases in human skin fibroblasts.

Protein kinase C (PKC) seems to play an important role in many of HDL effects on cells, including removal of excess cholesterol. HDL removes cholesterol by at least two mechanisms. One mechanism involves desorption/diffusion of cholesterol from the plasma membrane onto the acceptor particle, whereas the second is mediated by apolipoproteins and may involve intracellular translocation of cholesterol to the plasma membrane for subsequent efflux. In this report, we examined the possibility that mitogen-activated protein (MAP) kinase is one of the downstream events from HDL activation of PKC. Using a gel kinase assay with myelin basic protein incorporated into the gel, HDL (50 micrograms protein/mL) stimulated multiple kinases of 42, 50, 52, 58, and 60 kDa. The 42-kDa protein kinase, corresponding to the unresolved MAP kinases ERK1 and ERK2 based on immunoblotting, was activated over 2-fold by HDL. HDL activated all identified kinases in a concentration- and time-dependent manner, which became maximal within 5 to 10 minutes and remained activated for at least 60 minutes. HDL activation of MAP kinase seems to be partially mediated by PKC, because down-regulation of PKC and known PKC inhibitors inhibited the HDL effect by 40 to 50%. Free apolipoproteins A-I (10 micrograms/mL) and A-II (10 micrograms/mL) had no significant effect on MAP kinase activation. Moreover, modifying HDL with trypsin or tetranitromethane, which abolishes apolipoprotein-mediated cholesterol efflux, had no effect on HDL activation of MAP kinase. These results suggest that HDL activates MAP kinase via multiple signal transduction pathways that are likely involved in an HDL effect unrelated to apolipoprotein-mediated cholesterol translocation and efflux.

Limited proteolysis of high density lipoprotein abolishes its interaction with cell-surface binding sites that promote cholesterol efflux.

High-density lipoprotein (HDL) components remove cholesterol from cells by two independent mechanisms. Whereas HDL phospholipids pick up cholesterol that desorbs from the plasma membranes, HDL apolipoproteins appear to interact with cell-surface binding sites that target for removal pools of cellular cholesterol that feed into the cholesteryl ester cycle. Here we show that mild trypsin treatment of HDL almost completely abolishes this apolipoprotein-mediated cholesterol removal process. When HDL was treated with trypsin for various periods of time and then incubated with cholesterol-loaded fibroblasts, treatment for only 5 min reduced the ability of HDL to remove excess cholesterol from cellular pools that were accessible to esterification by the enzyme acyl CoA:cholesterol acyltransferase. This mild treatment digested less than 20% of HDL apolipoproteins and did not alter the lipid composition, size distribution, or electrophoretic mobility of the particles. Trypsin treatment of HDL for up to 1 h caused no further reduction in its ability to remove cellular cholesterol despite a greater than 2-fold increase in apolipoprotein digestion. Trypsin treatment of HDL also reduced its ability to deplete the cholesteryl ester content of sterol-laden macrophages. Promotion of cholesterol efflux from the plasma membrane by HDL phospholipids was unaffected by even extensive proteolysis. In parallel to the loss of cholesterol transport-stimulating activity, trypsin treatment of HDL for only 5 min nearly abolished its interaction with high-affinity binding sites on cholesterol-loaded fibroblasts. Reconstitution of trypsin-modified HDL with isolated apo A-I or apo A-II restored the cholesterol transport-stimulating activity of the particles. Thus a minor trypsin-labile fraction of HDL apolipoproteins is almost exclusively responsible for the apolipoprotein-dependent component of cholesterol efflux mediated by HDL particles.

Oxidative tyrosylation of HDL enhances the depletion of cellular cholesteryl esters by a mechanism independent of passive sterol desorption.

It is believed that HDL protects against atherosclerosis by removing excess cholesteryl esters from cells of the artery wall. Previous studies have suggested that HDL depletes cells of cholesteryl esters both by stimulating cholesterol efflux from the plasma membrane and by activating transport processes that divert cholesterol from the cholesteryl ester cycle, but it is unknown if these are independent processes. We previously found that HDL oxidized by tyrosyl radical has a markedly enhanced ability to promote the removal of cholesterol from cultured cells [Francis, G. A., et al. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 6631-6635]. Here we show that incubation of cholesterol-loaded human fibroblasts with low concentrations of tyrosylated HDL depleted cells of cholesteryl esters and increased cellular free cholesterol without increasing efflux of cholesterol into the medium as compared to incubation with untreated HDL. Cells preincubated with tyrosylated HDL and then exposed to a variety of cholesterol acceptors exhibited significantly higher rates of free cholesterol efflux than did cells preincubated with HDL. This effect was observed in the presence or absence of an inhibitor of acyl CoA:cholesterol acyltransferase (ACAT) and was independent of cholesteryl ester hydrolysis, suggesting that alterations in cholesteryl ester cycle enzymes were not responsible for the loss of cholesteryl esters. In contrast to the reduction of cholesteryl esters, the rates of cholesterol and phospholipid efflux from the plasma membranes of cells exposed to tyrosylated HDL and HDL were identical. These results suggest for the first time that a mechanism exists to deplete cellular cholesteryl esters and the cholesterol substrate pool for esterification by ACAT prior to the removal of cholesterol from the plasma membrane. Identification of products in tyrosylated HDL responsible for this redistribution of cellular cholesterol may provide important insights into mechanisms of intracellular cholesterol trafficking and the ability of modified forms of HDL to protect the artery against wall pathological cholesterol accumulation.

Apolipoprotein-mediated removal of cellular cholesterol and phospholipids.

It is widely believed that high density lipoprotein (HDL) protects against cardiovascular disease by removing excess cholesterol from cells of the artery wall. Recent cell culture studies have provided evidence that a major pathway for removing cholesterol and phospholipids from cells is mediated by the direct interactions of HDL apolipoproteins (apo) with plasma membrane domains. These interactions efficiently clear cells of excess sterol by targeting for removal pools of cholesterol that feed into the cholesteryl ester cycle. The precursors for this pathway in vivo are likely to be lipid-free or lipid-poor apolipoproteins generated either by dissociation from the surface of HDL particles or by de novo synthesis. Fibroblasts from subjects with a severe HDL deficiency syndrome called Tangier disease have a cellular defect that prevents apolipoproteins from removing both cholesterol and phospholipids from cells. This defect is associated with a near absence of plasma HDL, markedly below normal low density lipoprotein (LDL) levels, and the appearance of macrophage foam cells in tissues. Thus, an inability of nascent apoA-I to acquire cellular lipids results in a rapid clearance of apoA-I from the plasma, decreased production and increased clearance of LDL, and sterol deposition in tissue macrophages. Although the molecular properties of this pathway are still poorly understood, these studies imply that the apolipoprotein-mediated pathway for removal of cellular lipids is a major source of plasma cholesterol and phospholipids and plays an important role in clearing excess cholesterol from macrophages in vivo.