Objective-To study the acceptor specificity for human ABCG1 (hABCG1)-mediated cholesterol efflux. Methods and Results-Cells overexpressing hABCG1 were created in Chinese Hamster Ovary (CHO-K1) cells and characterized in terms of lipid composition. hABCG1 expressed in these cells formed homodimers and was mostly present intracellularly. Cholesterol efflux from hABCG1 cells to HDL 2 and HDL 3 was increased but not to lipid-free apolipoproteins. A range of phospholipid containing acceptors apart from high-density lipoprotein (HDL) subclasses were also efficient in mediating ABCG1-dependent export of cholesterol. Importantly, a buoyant phospholipidcontaining fraction generated from incubation of lipid-free apoA-I with macrophages was nearly as efficient as HDL 2 . The capacity of acceptors to induce ABCG1-mediated efflux was strongly correlated with their total phospholipid content, suggesting that acceptor phospholipids drive ABCG1-mediated efflux. Most importantly, acceptors for ABCG1-mediated cholesterol export could be generated from incubation of cells with lipid-free apoA-I through the action of ABCA1 alone. Conclusions-These

However, not all studies show a clear association between HDL-cholesterol and cardiovascular disease. 4,5 The observation that the capacity of serum to promote macrophage Molecular Medicine© 2015 American Heart Association, Inc. Objective: Our aims were to determine which HDL particle subfractions are most efficient in mediating cellular cholesterol efflux from foam cell macrophages and to identify the cellular cholesterol transporters involved in this process. Methods and Results:We used reconstituted HDL particles of defined size and composition, isolated subfractions of human plasma HDL, cell lines stably expressing ABCA1 or ABCG1, and both mouse and human macrophages in which ABCA1 or ABCG1 expression was deleted. We show that ABCA1 is the major mediator of macrophage cholesterol efflux to HDL, demonstrating most marked efficiency with small, dense HDL subfractions (HDL3b and HDL3c). ABCG1 has a lesser role in cholesterol efflux and a negligible role in efflux to HDL3b and HDL3c subfractions. Conclusions:

Objective-Maintenance of cholesterol homeostasis in human macrophages is essential to prevent foam cell formation. We evaluated the relative contribution of the ABCA1 and ABCG1 transporters to cholesterol efflux from human macrophages, and of the capacity of LXR agonists to reduce foam cell formation by stimulating export of cellular cholesterol. Methods and Results-ABCG1 mRNA levels were strongly increased in acLDL-loaded THP-1 macrophages and in HMDM on stimulation with LXR agonists. However, silencing of ABCG1 expression using ABCG1-specific siRNA indicated that ABCG1 was not essential for cholesterol efflux to HDL in cholesterol-loaded human macrophages stimulated with LXR agonists. Indeed, ABCA1 was solely responsible for the stimulation of cholesterol efflux to HDL on LXR activation, as this effect was abolished in HMDM from Tangier patients. Furthermore, depletion of cellular ATP indicated that the LXR-induced export of cholesterol was an ATP-dependent transport mechanism in human macrophages. Finally, use of an anti-Cla-1 blocking antibody identified the Cla-1 receptor as a key component in cholesterol efflux to HDL from cholesterol-loaded human macrophages. Conclusion-Our data indicate that stimulation of cholesterol efflux to HDL by LXR agonists in human foam cellsinvolves an ATP-dependent transport mechanism mediated by ABCA1 that it appears to be independent of ABCG1 expression. Key Words: ABC transporters Ⅲ LXR agonists Ⅲ cholesterol efflux Ⅲ macrophage Ⅲ foam cells I n atherosclerotic lesions, the accumulation of cellular cholesterol within macrophage "foam cells" drives lipid deposition and is a major contributor to lesion growth. It is understood that macrophages become foam cells as the result of a loss of the normal balance between cholesterol uptake (from lipoproteins) and cholesterol export. For this reason, the mechanisms by which macrophages export cellular cholesterol have been intensively investigated in recent years.It is now generally accepted that HDL and its apolipoproteins are the major initial acceptors of excess cellular cholesterol. Members of the ABC transporter family have been identified as key, and potentially complementary, cellular participants in export of cholesterol to these acceptors. ABCA1 mediates cholesterol efflux most efficiently to lipidpoor apolipoprotein AI (apoAI), whereas ABCG1 promotes cholesterol export to lipidated particles such as HDL. In addition, the SR-BI receptor pathway can also promote cholesterol export to HDL particles.The contributions of ABCA1 and ABCG1 to the maintenance of macrophage cholesterol homeostasis have been most convincingly demonstrated through the effects of targeted deletion of these transporters in mice. 1 In particular, the profound effects of combined deletion of both ABCA1 and ABCG1 in mouse macrophages on their accumulation of cholesterol in vivo and on their ability to export cholesterol to either apoAI or to HDL in vitro, suggests that the combined activity of both of these transporters is essential for macrophage cholester...

Objective-Cholesterol efflux from macrophages in the artery wall, a key cardioprotective mechanism, is largely coordinated by the nuclear oxysterol-activated liver X receptor, LXR␣. We investigated the effect of statins on LXR target gene expression and cholesterol efflux from human macrophages. Methods and Results-In human macrophages (THP-1 cell line and primary cells), the archetypal statin, compactin, greatly reduced mRNA levels of 2 LXR target genes, ABCA1 and ABCG1 mRNA, as well as decreased cholesterol efflux. Commonly prescribed statins also downregulated LXR target gene expression in THP-1 cells. We provide several lines of evidence indicating that statins decrease expression of LXR target genes by inhibiting the synthesis of an oxysterol ligand for LXR, 24(S),25-epoxycholesterol. When THP-1 cells were cholesterol-loaded via incubation with acetylated low-density lipoprotein, synthesis of 24(S),25-epoxycholesterol was greatly reduced and the downregulatory effect of compactin on ABCA1 mRNA levels and cholesterol efflux was lost. Key Words: statins Ⅲ human macrophage Ⅲ liver X receptor Ⅲ 24(S),25-epoxycholesterol Ⅲ THP-1 cells Ⅲ human monocyte-derived macrophages Ⅲ pleiotropic S tatins are highly effective in lowering serum cholesterol concentrations and in reducing the risk of coronary heart disease and stroke. 1 There is a growing body of clinical and experimental evidence that statins exert additional benefits beyond cholesterol reduction. 2,3 Intensive efforts are underway to study the pleiotropic, cholesterol-independent effects of statins on the vasculature. 2,3 Conclusions-Our See page 2209The macrophage is a key vascular cell involved in the development of atherosclerosis. Cholesterol-laden macrophages (foam cells) have essential functions in all phases of atherosclerosis, from development of the fatty streak to processes that ultimately contribute to plaque rupture and myocardial infarction. Because mammalian cells cannot degrade the sterol fused-ring structure, excess sterols undergo elimination from the body principally by biliary excretion. Therefore, macrophages must export cholesterol to extracellular acceptors for transport to the liver. 4 The liver X receptor (LXR), NR1H3 or LXR␣, plays an important role in coordinating cholesterol efflux from the macrophage. As a member of the nuclear receptor family of ligand-dependent transcription factors, LXR␣ activates transcription of target genes in response to the binding of certain oxidized forms of cholesterol (oxysterols). 5,6 LXR␣ forms a heterodimer with the retinoid X receptors to bind to specific response elements in the promoters or enhancers of target genes. The genes encoding several important proteins that facilitate cholesterol removal from macrophages are targets of LXR, including ABCA1, ABCG1, apolipoprotein E (apoE), and LXR␣ itself. ABCA1 and ABCG1 are members of the ATP-binding cassette superfamily of transporter proteins. Mutations in ABCA1 were recently identified as the basis for Tangier disease, a rare autosomal diso...

Abstract-Chemokines are important mediators of macrophage and T-cell recruitment in a number of inflammatory pathologies, and chemokines expressed in atherosclerotic lesions may play an important role in mononuclear cell recruitment and macrophage differentiation. We have analyzed the expression of the linked chromosome 16q13 genes that encode macrophage-derived chemokine (MDC/CCL22), thymus-and activation-regulated chemokine (TARC/ CCL17), and the CX 3 C chemokine fractalkine (CX 3 CL1) in primary macrophages and human atherosclerotic lesions by reverse transcription-polymerase chain reaction and immunohistochemistry. We show that macrophage expression of the chemokines MDC, fractalkine, and TARC is upregulated by treatment with the Th2-type cytokines interleukin-4 and interleukin-13. High levels of MDC, TARC, and fractalkine mRNA expression are seen in some, but not all, human arteries with advanced atherosclerotic lesions. Immunohistochemistry shows that MDC, fractalkine, and TARC are expressed by a subset of macrophages within regions of plaques that contain plaque microvessels. We conclude that MDC, fractalkine, and TARC, which are chromosome 16q13 chemokines, could play a role in mononuclear cell recruitment into atherosclerotic lesions and influence the subsequent inflammatory response. Macrophage-expressed chemokines upregulated by interleukin-4 may be useful surrogate markers for the presence of Th2-type immune responses in human atherosclerotic lesions. Key Words: chemokines Ⅲ atherosclerosis Ⅲ macrophages Ⅲ Th2-type T cells A therosclerosis is a major cause of morbidity and mortality in western countries. Atherosclerotic lesions within major blood vessels are responsible for myocardial infarction, strokes, and peripheral vascular disease. The initial event in the development of the atherosclerotic lesion is the adhesion of monocytes to the endothelial cells of the blood vessel wall and the extravasation of monocytes into the subendothelial space. 1,2 Recruited monocytes differentiate into macrophages and accumulate modified forms of LDL via scavenger receptors to yield lipid-laden foam cells. 3 Chronic recruitment of mononuclear cells into the developing lesion contributes to the growth of lesions and occlusion of the blood vessel. 4 Macrophages and T cells within atherosclerotic lesions produce an array of cytokines, growth factors, and inflammatory mediators, which are likely to play a role in continued mononuclear cell recruitment. 5,6 Chemoattractant cytokines (chemokines) are small disulfide-linked polypeptides of typically 60 to 70 amino acids in length that are potent chemoattractants for leukocytes. 7 The chemokine supergene family, which was unrecognized 10 years ago, now has Ͼ40 different members classified into different subfamilies on the basis of conserved structural features. 8,9 The CXC (or ␣) chemokines have a single amino acid separating the first 2 cysteine residues of the protein, whereas CC (or ␤) chemokines have no amino acid separating the signature C1 and C2 cysteines. The CC ch...

Cholesterol accumulation and removal are regulated by two different transcription factors. SREBP-2 (sterol-regulatory-elementbinding protein-2) is best known to up-regulate genes involved in cholesterol biosynthesis and uptake, whereas LXR (liver X receptor) is best known for up-regulating cholesterol efflux genes. An important cholesterol efflux gene that is regulated by LXR is the ATP-binding cassette transporter, ABCA1 (ATPbinding cassette transporter-A1). We have previously shown that statin treatment down-regulated ABCA1 expression in human macrophages, probably by inhibiting synthesis of the LXR ligand 24(S),25-epoxycholesterol. However, it was subsequently reported that ABCA1 expression is down-regulated by SREBP-2 through binding of SREBP-2 to an E-box element in ABCA1's proximal promoter. As statin treatment induces SREBP-2 activation, this may provide an alternative explanation for the statinmediated down-regulation of ABCA1. In the present study, we employed a set of CHO (Chinese-hamster ovary) mutant cell lines to investigate the role of SREBP-2 in the regulation of ABCA1. We observed increased ABCA1 mRNA levels in SREBP-2-overexpressing cells and decreased levels in cells lacking a functional SREBP-2 pathway, which were restored when the SREBP-2 pathway was reinstated. Moreover, ABCA1 gene expression was positively associated with synthesis of 24(S),25-epoxycholesterol in these cell lines. In studies using a human ABCA1 promoter reporter assay, mutation of the E-box motif had a similar response as the wild-type construct to either statin treatment or addition of 24(S),25-epoxycholesterol. By contrast, these responses were completely ablated when the DR4 element to which LXR binds was mutated. These results support the idea that 24(S),25-epoxycholesterol and statin treatment influence ABCA1 transcription via supply of an LXR ligand and not through an SREBP-2/E-boxrelated mechanism. In addition, our results indicate a critical role of SREBP-2 as a positive regulator of ABCA1 gene expression by enabling the generation of oxysterol ligands for LXR.

Apolipoprotein A-I (apoA-I)-mediated cholesterol efflux involves the binding of apoA-I to the plasma membrane via its C terminus and requires cellular ATP-binding cassette transporter (ABCA1) activity. ApoA-I also stimulates secretion of apolipoprotein E (apoE) from macrophage foam cells, although the mechanism of this process is not understood. In this study, we demonstrate that apoA-I stimulates secretion of apoE independently of both ABCA1-mediated cholesterol efflux and of lipid binding by its C terminus. Pulse-chase experiments using 35 S-labeled cellular apoE demonstrate that macrophage apoE exists in both relatively mobile (E m ) and stable (E s ) pools, that apoA-I diverts apoE from degradation to secretion, and that only a small proportion of apoA-I-mobilized apoE is derived from the cell surface. The structural requirements for induction of apoE secretion and cholesterol efflux are clearly dissociated, as C-terminal deletions in recombinant apoA-I reduce cholesterol efflux but increase apoE secretion, and deletion of central helices 5 and 6 decreases apoE secretion without perturbing cholesterol efflux. Moreover, a range of 11-and 22-mer ␣-helical peptides representing amphipathic ␣-helical segments of apoA-I stimulate apoE secretion whereas only the C-terminal ␣-helix (domains 220 -241) stimulates cholesterol efflux. Other ␣-helixcontaining apolipoproteins (apoA-II, apoA-IV, apoE2, apoE3, apoE4) also stimulate apoE secretion, implying a positive feedback autocrine loop for apoE secretion, although apoE4 is less effective. Finally, apoA-I stimulates apoE secretion normally from macrophages of two unrelated subjects with genetically confirmed Tangier Disease (mutations C733R and c.5220 -5222delTCT; and mutations A1046D and c.4629 -4630insA), despite severely inhibited cholesterol efflux. We conclude that apoA-I stimulates secretion of apoE independently of cholesterol efflux, and that this represents a novel, ABCA-1-independent, positive feedback pathway for stimulation of potentially anti-atherogenic apoE secretion by ␣-helix-containing molecules including apoA-I and apoE.

The T lymphocyte plasma membrane condenses at the site of activation but the functional significance of this receptor-mediated membrane reorganization is not yet known. Here we demonstrate that membrane condensation at the T cell activation sites can be inhibited by incorporation of the oxysterol 7-ketocholesterol (7KC), which is known to prevent the formation of raft-like liquid-ordered domains in model membranes. We enriched T cells with 7KC, or cholesterol as control, to assess the importance of membrane condensation for T cell activation. Upon 7KC treatment, T cell antigen receptor (TCR) triggered calcium fluxes and early tyrosine phosphorylation events appear unaltered. However, signaling complexes form less efficiently on the cell surface, fewer phosphorylated signaling proteins are retained in the plasma membrane and actin restructuring at activation sites is impaired in 7KC-enriched cells resulting in compromised downstream activation responses. Our data emphasizes lipids as an important medium for the organization at T cell activation sites and strongly indicates that membrane condensation is an important element of the T cell activation process.