ABCA1 and ABCG1 or ABCG4 act sequentially to remove cellular cholesterol and generate cholesterol-rich HDL

Recent developments in lipid metabolism have shown the importance of ATP binding cassette transporters (ABCs) in controlling cellular and total body lipid homeostasis. ABCA1 mediates the transport of cholesterol and phospholipids from cells to lipid-poor apolipoprotein A-I (apoA-I), whereas ABCG1 and ABCG4 mediate the transport of cholesterol from cells to lipidated lipoproteins. ABCA1, ABCG1, and ABCG4 are all expressed in cholesterol-loaded macrophages, and macrophages from ABCA1 and ABCG1 knockout mice accumulate cholesteryl esters. Here, we show that the lipidated particles generated by incubating cells overexpressing ABCA1 with apoA-I are efficient acceptors for cholesterol released from cells overexpressing either ABCG1 or ABCG4. The cholesterol released to the particles was derived from a cholesterol oxidase-accessible plasma membrane pool in both ABCG1 and ABCG4 cells, which is the same pool of cholesterol shown previously to be removed by high density lipoproteins. ABCA1 cells incubated with apoA-I generated two major populations of cholesterol- and phospholipid-rich lipoprotein particles that were converted by ABCG1 or ABCG4 cells to one major particle population that was highly enriched in cholesterol. These results suggest that ABCG1 and ABCG4 act in concert with ABCA1 to maximize the removal of excess cholesterol from cells and to generate cholesterol-rich lipoprotein particles.     

Structural modification of plasma HDL by phospholipids promotes efficient ABCA1-mediated cholesterol release

It has been suggested that ABCA1 interacts preferentially with lipid-poor apolipoprotein A-I (apoA-I). Here, we show that treatment of plasma with dimyristoyl phosphatidylcholine (DMPC) multilamellar vesicles generates prebeta(1)-apoA-I-containing lipoproteins (LpA-I)-like particles similar to those of native plasma. Isolated prebeta(1)-LpA-I-like particles inhibited the binding of (125)I-apoA-I to ABCA1 more efficiently than HDL(3) (IC(50) = 2.20 +/- 0.35 vs. 37.60 +/- 4.78 microg/ml). We next investigated the ability of DMPC-treated plasma to promote phospholipid and unesterified (free) cholesterol efflux from J774 macrophages stimulated or not with cAMP. At 2 mg DMPC/ml plasma, both phospholipid and free cholesterol efflux were increased ( approximately 50% and 40%, respectively) in cAMP-stimulated cells compared with unstimulated cells. Similarly, both phospholipid and free cholesterol efflux to either isolated native prebeta(1)-LpA-I and prebeta(1)-LpA-I-like particles were increased significantly in stimulated cells. Furthermore, glyburide significantly inhibited phospholipid and free cholesterol efflux to DMPC-treated plasma. Removal of apoA-I-containing lipoproteins from normolipidemic plasma drastically reduced free cholesterol efflux mediated by DMPC-treated plasma. Finally, treatment of Tangier disease plasma with DMPC affected the amount of neither prebeta(1)-LpA-I nor free cholesterol efflux. These results indicate that DMPC enrichment of normal plasma resulted in the redistribution of apoA-I from alpha-HDL to prebeta-HDL, allowing for more efficient ABCA1-mediated cellular lipid release. Increasing the plasma prebeta(1)-LpA-I level by either pharmacological agents or direct infusions might prevent foam cell formation and reduce atherosclerotic vascular disease.     

Identification of an ABCA1-dependent phospholipid-rich plasma membrane apolipoprotein A-I binding site for nascent HDL formation: implications for current models of HDL biogenesis

It is well accepted that both apolipoprotein A-I (apoA-I) and ABCA1 play crucial roles in HDL biogenesis and in the human atheroprotective system. However, the nature and specifics of apoA-I/ABCA1 interactions remain poorly understood. Here, we present evidence for a new cellular apoA-I binding site having a 9-fold higher capacity to bind apoA-I compared with the ABCA1 site in fibroblasts stimulated with 22-(R)-hydroxycholesterol/9-cis-retinoic acid. This new cellular apoA-I binding site was designated "high-capacity binding site" (HCBS). Glyburide drastically reduced (125)I-apoA-I binding to the HCBS, whereas (125)I-apoA-I showed no significant binding to the HCBS in ABCA1 mutant (Q597R) fibroblasts. Furthermore, reconstituted HDL exhibited reduced affinity for the HCBS. Deletion of the C-terminal region of apoA-I (Delta187-243) drastically reduced the binding of apoA-I to the HCBS. Interestingly, overexpressing various levels of ABCA1 in BHK cells promoted the formation of the HCBS. The majority of the HCBS was localized to the plasma membrane (PM) and was not associated with membrane raft domains. Importantly, treatment of cells with phosphatidylcholine-specific phospholipase C, but not sphingomyelinase, concomitantly reduced the binding of (125)I-apoA-I to the HCBS, apoA-I-mediated cholesterol efflux, and the formation of nascent apoA-I-containing particles. Together, these data suggest that a functional ABCA1 leads to the formation of a major lipid-containing site for the binding and the lipidation of apoA-I at the PM. Our results provide a biochemical basis for the HDL biogenesis pathway that involves both ABCA1 and the HCBS, supporting a two binding site model for ABCA1-mediated nascent HDL genesis.     

Nascent HDL formation in hepatocytes and role of ABCA1, ABCG1, and SR-BI

To study the mechanisms of hepatic HDL formation, we investigated the roles of ABCA1, ABCG1, and SR-BI in nascent HDL formation in primary hepatocytes isolated from mice deficient in ABCA1, ABCG1, or SR-BI and from wild-type (WT) mice. Under basal conditions, in WT hepatocytes, cholesterol efflux to exogenous apoA-I was accompanied by conversion of apoA-I to HDL-sized particles. LXR activation by T0901317 markedly enhanced the formation of larger HDL-sized particles as well as cellular cholesterol efflux to apoA-I. Glyburide treatment completely abolished the formation of 7.4 nm diameter and greater particles but led to the formation of novel 7.2 nm-sized particles. However, cells lacking ABCA1 failed to form such particles. ABCG1-deficient cells showed similar capacity to efflux cholesterol to apoA-I and to form nascent HDL particles compared with WT cells. Cholesterol efflux to apoA-I and nascent HDL formation were slightly but significantly enhanced in SR-BI-deficient cells compared with WT cells under basal but not LXR activated conditions. As in WT but not in ABCA1-deficient hepatocytes, 7.2 nm-sized particles generated by glyburide treatment were also detected in ABCG1-deficient and SR-BI-deficient hepatocytes. Our data indicate that hepatic nascent HDL formation is highly dependent on ABCA1 but not on ABCG1 or SR-BI.     

Endothelial expression of human ABCA1 in mice increases plasma HDL cholesterol and reduces diet-induced atherosclerosis

The role of endothelial ABCA1 expression in reverse cholesterol transport (RCT) was examined in transgenic mice, using the endothelial-specific Tie2 promoter. Human ABCA1 (hABCA1) was significantly expressed in endothelial cells (EC) of most tissues except the liver. Increased expression of ABCA1 was not observed in resident peritoneal macrophages. ApoA-I-mediated cholesterol efflux from aortic EC was 2.6-fold higher (P < 0.0001) for cells from transgenic versus control mice. On normal chow diet, Tie2 hABCA1 transgenic mice had a 25% (P < 0.0001) increase in HDL-cholesterol (HDL-C) and more than a 2-fold increase of eNOS mRNA in the aorta (P < 0.04). After 6 months on a high-fat, high-cholesterol (HFHC) diet, transgenic mice compared with controls had a 40% increase in plasma HDL-C (P < 0.003) and close to 40% decrease in aortic lesions (P < 0.02). Aortas from HFHC-fed transgenic mice also showed gene expression changes consistent with decreased inflammation and apoptosis. Beneficial effects of the ABCA1 transgene on HDL-C levels or on atherosclerosis were absent when the transgene was transferred onto ApoE or Abca1 knockout mice. In summary, expression of hABCA1 in EC appears to play a role in decreasing diet-induced atherosclerosis in mice and is associated with increased plasma HDL-C levels and beneficial gene expression changes in EC.     

On the hepatic mechanism of HDL assembly by the ABCA1/apoA-I pathway

The mechanism for the assembly of HDL with cellular lipid by ABCA1 and helical apolipoprotein was investigated in hepatocytes. Both HepG2 cells and mouse primary culture hepatocytes produced HDL with apolipoprotein A-I (apoA-I) whether endogenously synthesized or exogenously provided. Probucol, an ABCA1 inactivator, inhibited these reactions, as well as the reversible binding of apoA-I to HepG2. Primary cultured hepatocytes of ABCA1-deficient mice also lacked HDL production regardless of the presence of exogenous apoA-I. HepG2 cells secreted apoA-I into the medium even when ABCA1 was inactivated by probucol, but it was all in a free form as HDL production was inhibited. When a lipid-free apoA-I-specific monoclonal antibody, 725-1E2, was present in the culture medium, production of HDL was suppressed, whether with endogenous or exogenously added apoA-I, and the antibody did not influence HDL already produced by HepG2 cells. We conclude that the main mechanism for HDL assembly by endogenous apoA-I in HepG2 cells is an autocrine-like reaction in which apoA-I is secreted and then interacts with cellular ABCA1 to generate HDL.     

Heterogeneity of high density lipoprotein generated by ABCA1 and ABCA7

The assembly of HDL by helical apolipoprotein and cellular lipid was studied using HEK293 cells to which ecdysone-inducible human ABCA1 or human ABCA7 was transfected. Expression of both ABCA1 and ABCA7 was induced linearly proportional to ponasterone A concentration in the medium. In the experimental conditions used, the ABC protein expression levels limited the rate of lipid release when the apolipoprotein concentration was high, and the apolipoprotein concentration was rate-limiting when the ABC protein expression levels were high. When ABCA1 expression increased in conditions in which it was rate-limiting, relative cholesterol content to phospholipid increased in the HDL produced. In contrast, it was constant when ABCA7 expression increased. To investigate the background mechanism, the HDL particles were analyzed by density gradient ultracentrifugation and high performance lipid chromatography. The ABCA1-mediated reaction produced two distinct HDLs, large cholesterol-rich and small cholesterol-poor particles, and the ABCA7-mediated reaction generated mostly small cholesterol-poor particles. The increase of HDL assembly with the increase of ABCA1 expression was predominant in large cholesterol-rich particles, whereas only small cholesterol-poor HDL increased as ABCA7 expression increased. We conclude that ABCA1 generates cholesterol-rich and cholesterol-poor HDL and that the former is more prominently dependent on the increase of ABCA1 expression. ABCA7 produces this HDL subfraction only as a very minor component.     

Direct detection of ABCA1-dependent HDL formation based on lipidation-induced hydrophobicity change in apoA-I

ABCA1 mediates the efflux of cholesterol and phospholipids into apoA-I to form HDL, which is important in the prevention of atherosclerosis. To develop a novel method for the evaluation of HDL formation, we prepared an apoA-I-POLARIC by labeling the specific residue of an apoA-I variant with a hydrophobicity-sensitive fluorescence probe that detects the environmental change around apoA-I during HDL formation. apoA-I-POLARIC possesses the intact ABCA1-dependent HDL formation activity and shows 4.0-fold higher fluorescence intensity in HDL particles than in the lipid-free state. Incubation of apoA-I-POLARIC with ABCA1-expressing cells, but not ABCA1-non-expressing cells, caused a 1.7-fold increase in fluorescence intensity. Gel filtration analysis demonstrated that the increase in fluorescence intensity of apoA-I-POLARIC represents the amount of apoA-I incorporated into the discoidal HDL particles rather than the amount of secreted cholesterol. THP-1 macrophage-mediated HDL formation and inhibition of HDL formation by cyclosporine A could also be measured using apoA-I-POLARIC. Furthermore, HDL formation-independent lipid release induced by microparticle formation or cell death was not detected by apoA-I-POLARIC. These results demonstrate that HDL formation by ABCA1-expressing cells can be specifically detected by sensing hydrophobicity change in apoA-I, thus providing a novel method for assessing HDL formation and screening of the HDL formation modulator.     

Denaturing high-performance liquid chromatography in the detection of ABCA1 gene mutations in familial HDL deficiency

Mutations in the ABCA1 gene are the cause of familial high density lipoprotein deficiency (FHD). Because these mutations are spread over the entire gene, their detection requires the sequencing of all 50 exons. The aim of this study was to validate denaturing high-performance liquid chromatography (DHPLC) in mutation detection as an alternative to systematic sequencing. Exons of the ABCA1 gene were amplified using primers employed for sequencing. Temperatures for DHPLC were deducted from a software and empirically defined for each amplicon. To assess DHPLC reliability, we tested 30 sequence variants found in FHD patients and controls. Combined DHPLC and sequencing was applied to the genotyping of new FHD patients. Most of the amplicons required from two to five temperature conditions to obtain partially denatured DNA over the entire amplicon length. Twenty-nine of the variants found by sequencing were detected by DHPLC (97% sensitivity). The detection of the last variant (in exon 40) required different primers and amplification conditions. DHPLC and sequencing analysis of new FHD patients revealed that all amplicons showing a heteroduplex DHPLC profile contained sequence variants. No variants were detected in amplicons with a homoduplex profile. DHPLC is a sensitive and reliable method for the detection of ABCA1 gene mutations.     

Apolipoprotein A-I activates Cdc42 signaling through the ABCA1 transporter

It has been suggested that the signal transduction initiated by apolipoprotein A-I (apoA-I) activates key proteins involved in cholesterol efflux. ABCA1 serves as a binding partner for apoA-I, but its participation in apoA-I-induced signaling remains uncertain. We show that the exposure of human fibroblasts to ABCA1 ligands (apolipoproteins and amphipathic helical peptides) results in the generation of intracellular signals, including activation of the small G-protein Cdc42, protein kinases (PAK-1 and p54JNK), and actin polymerization. ApoA-I-induced signaling was abrogated by glyburide, an inhibitor of the ABC transporter family, and in fibroblasts from patients with Tangier disease, which do not express ABCA1. Conversely, induction of ABCA1 expression with the liver X receptor agonist, T0901317, and the retinoid X receptor agonist, R0264456, potentiated apoA-I-induced signaling. Similar effects were observed in HEK293 cells overexpressing ABCA1-green fluorescent protein (GFP) fusion protein, but not ABCA1-GFP (K939M), which fails to hydrolyze ATP, or a nonfunctional ABCA1-GFP with a truncated C terminus. We further found that Cdc42 coimmunoprecipitates with ABCA1 in ABCA1-GFP-expressing HEK293 cells exposed to apoA-I but not in cells expressing ABCA1 mutants. We conclude that ABCA1 transduces signals from apoA-I by complexing and activating Cdc42 and downstream kinases and, therefore, acts as a full apoA-I receptor.     

Differential effects of HDL subpopulations on cellular ABCA1- and SR-BI-mediated cholesterol efflux

Our objective was to evaluate the associations of individual apolipoprotein A-I (apoA-I)-containing HDL subpopulation levels with ABCA1- and scavenger receptor class B type I (SR-BI)-mediated cellular cholesterol efflux. HDL subpopulations were measured by nondenaturing two-dimensional gel electrophoresis from 105 male subjects selected with various levels of apoA-I in pre-beta-1, alpha-1, and alpha-3 HDL particles. ApoB-containing lipoprotein-depleted serum was incubated with [(3)H]cholesterol-labeled cells to measure efflux. The difference in efflux between control and ABCA1-upregulated J774 macrophages was taken as a measure of ABCA1-mediated efflux. SR-BI-mediated efflux was determined using cholesterol-labeled Fu5AH hepatoma cells. Fractional efflux values obtained from these two cell systems were correlated with the levels of individual HDL subpopulations. A multivariate analysis showed that two HDL subspecies correlated significantly with ABCA1-mediated efflux: small, lipid-poor pre-beta-1 particles (P=0.0022) and intermediate-sized alpha-2 particles (P=0.0477). With regard to SR-BI-mediated efflux, multivariate analysis revealed significant correlations with alpha-2 (P=0.0004), alpha-1 (P=0.0030), pre-beta-1 (P=0.0056), and alpha-3 (P=0.0127) HDL particles. These data demonstrate that the small, lipid-poor pre-beta-1 HDL has the strongest association with ABCA1-mediated cholesterol even in the presence of all other HDL subpopulations. Cholesterol efflux via the SR-BI pathway is associated with several HDL subpopulations with different apolipoprotein composition, lipid content, and size.     

HDL from apoA1 transgenic mice expressing the 4WF isoform is resistant to oxidative loss of function

HDL functions are impaired by myeloperoxidase (MPO), which selectively targets and oxidizes human apoA1. We previously found that the 4WF isoform of human apoA1, in which the four tryptophan residues are substituted with phenylalanine, is resistant to MPO-mediated loss of function. The purpose of this study was to generate 4WF apoA1 transgenic mice and compare functional properties of the 4WF and wild-type human apoA1 isoforms in vivo. Male mice had significantly higher plasma apoA1 levels than females for both isoforms of human apoA1, attributed to different production rates. With matched plasma apoA1 levels, 4WF transgenics had a trend for slightly less HDL-cholesterol versus human apoA1 transgenics. While 4WF transgenics had 31% less reverse cholesterol transport (RCT) to the plasma compartment, equivalent RCT to the liver and feces was observed. Plasma from both strains had similar ability to accept cholesterol and facilitate ex vivo cholesterol efflux from macrophages. Furthermore, we observed that 4WF transgenic HDL was partially (∼50%) protected from MPO-mediated loss of function while human apoA1 transgenic HDL lost all ABCA1-dependent cholesterol acceptor activity. In conclusion, the structure and function of HDL from 4WF transgenic mice was not different than HDL derived from human apoA1 transgenic mice.     

Serum amyloid A generates high density lipoprotein with cellular lipid in an ABCA1- or ABCA7-dependent manner

Serum amyloid A (SAA) is an amphiphilic helical protein that is found associated with plasma HDL in various pathological conditions, such as acute or chronic inflammation. Cellular lipid release and generation of HDL by this protein were investigated, in comparison with the reactions by apolipoprotein A-I (apoA-I) and several types of cells that appear with various specific profiles of cholesterol and phospholipid release. SAA mediated cellular lipid release from these cells with the same profile as apoA-I. Upregulation of cellular ABCA1 protein by liver X receptor/retinoid X receptor agonists resulted in an increase of cellular lipid release by apoA-I and SAA. SAA reacted with the HEK293-derived clones that stably express human ABCA1 (293/2c) or ABCA7 (293/6c) to generate cholesterol-containing HDL in a similar manner to apoA-I. Dibutyryl cyclic AMP and phorbol 12-myristate 13-acetate, which differentiate apoA-I-mediated cellular lipid release between 293/2c and 293/6c, also exhibited the same differential effects on the SAA-mediated reactions. No evidence was found for the ABCA1/ABCA7-independent lipid release by SAA. Characterization of physicochemical properties of the HDL revealed that SAA-generated HDL particles had higher density, larger diameter, and slower electrophoretic mobility than those generated by apoA-I. These results demonstrate that SAA generates cholesterol-containing HDL directly with cellular lipid and that the reaction is mediated by ABCA1 and ABCA7.     

Proteomic analysis of HDL from inbred mouse strains implicates APOE associated with HDL in reduced cholesterol efflux capacity via the ABCA1 pathway

Cholesterol efflux capacity associates strongly and negatively with the incidence and prevalence of human CVD. We investigated the relationships of HDL’s size and protein cargo with its cholesterol efflux capacity using APOB-depleted serum and HDLs isolated from five inbred mouse strains with different susceptibilities to atherosclerosis. Like humans, mouse HDL carried >70 proteins linked to lipid metabolism, the acute-phase response, proteinase inhibition, and the immune system. HDL’s content of specific proteins strongly correlated with its size and cholesterol efflux capacity, suggesting that its protein cargo regulates its function. Cholesterol efflux capacity with macrophages strongly and positively correlated with retinol binding protein 4 (RBP4) and PLTP, but not APOA1. In contrast, ABCA1-specific cholesterol efflux correlated strongly with HDL’s content of APOA1, APOC3, and APOD, but not RBP4 and PLTP. Unexpectedly, APOE had a strong negative correlation with ABCA1-specific cholesterol efflux capacity. Moreover, the ABCA1-specific cholesterol efflux capacity of HDL isolated from APOE-deficient mice was significantly greater than that of HDL from wild-type mice. Our observations demonstrate that the HDL-associated APOE regulates HDL’s ABCA1-specific cholesterol efflux capacity. These findings may be clinically relevant because HDL’s APOE content associates with CVD risk and ABCA1 deficiency promotes unregulated cholesterol accumulation in human macrophages.     

ABCA1 contributes to macrophage deposition of extracellular cholesterol

We previously reported that cholesterol-enriched macrophages excrete cholesterol into the extracellular matrix. A monoclonal antibody that detects cholesterol microdomains labels the deposited extracellular particles. Macro­phage deposition of extracellular cholesterol depends, in part, on ABCG1, and this cholesterol can be mobilized by HDL components of the reverse cholesterol transport process. The objective of the current study was to determine whether ABCA1 also contributes to macrophage deposition of extracellular cholesterol. ABCA1 functioned in extracellular cholesterol deposition. The liver X receptor agonist, TO901317 (TO9), an ABCA1-inducing factor, restored cholesterol deposition that was absent in cholesterol-enriched ABCG1^−/− mouse macrophages. In addition, the ABCA1 inhibitor, probucol, blocked the increment in cholesterol deposited by TO9-treated wild-type macrophages, and completely inhibited deposition from TO9-treated ABCG1^−/− macrophages. Lastly, ABCA1^−/− macrophages deposited much less extracellular cholesterol than wild-type macrophages. These findings demonstrate a novel function of ABCA1 in contributing to macrophage export of cholesterol into the extracellular matrix.