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Abstract-The removal of excess free cholesterol from cells by HDL or its apolipoproteins is important for maintaining cellular cholesterol homeostasis. This process is most likely compromised in the atherosclerotic lesion because the development of atherosclerosis is associated with low HDL cholesterol. Multiple mechanisms for efflux of cell cholesterol exist. Efflux of free cholesterol via aqueous diffusion occurs with all cell types but is inefficient. Efflux of cholesterol is accelerated when scavenger receptor class-B type I (SR-BI) is present in the cell plasma membrane. Both diffusion-mediated and SR-BI-mediated efflux occur to phospholipid-containing acceptors (ie, HDL and lipidated apolipoproteins); in both cases, the flux of cholesterol is bidirectional, with the direction of net flux depending on the cholesterol gradient. The ATP-binding cassette transporter AI (ABCA1) mediates efflux of both cellular cholesterol and phospholipid. In contrast to SR-BI-mediated flux, efflux via ABCA1 is unidirectional, occurring to lipid-poor apolipoproteins. The relative importance of the SR-BI and ABCA1 efflux pathways in preventing the development of atherosclerotic plaque is not known but will depend on the expression levels of the two proteins and on the type of cholesterol acceptors available. Key Words: cholesterol efflux Ⅲ scavenger receptor class-BI Ⅲ ATP-binding cassette transporter AI Ⅲ reverse cholesterol transport H DL levels are inversely correlated with the incidence of coronary artery disease. 1-4 A long-standing hypothesis to explain this protective effect of HDL against atherosclerosis is the process of reverse cholesterol transport (RCT). 5 In RCT, HDL or its apolipoproteins mediate the removal of excess free cholesterol (FC) from peripheral cells and, after a series of reactions in plasma, the cholesterol is delivered via either LDL or HDL to the liver for excretion into the bile. The flux of FC between cells and extracellular acceptors is important at two points in the RCT pathway: (1) the removal of FC from peripheral cells and (2) the delivery of HDL FC to the liver. There are 3 known mechanisms of FC flux: (1) aqueous diffusion, (2) SR-BI-mediated FC flux, and (3) ABCA1-mediated efflux (Figure 1). The purpose of this review is to discuss each mechanism and the relative importance of each mechanism to RCT. Aqueous DiffusionCholesterol molecules are sufficiently water-soluble to be able to transfer from either model 6 or cell membranes 7 to an acceptor by the so-called aqueous diffusion mechanism. 8 This process involves desorption of cholesterol molecules from the donor lipid-water interface and diffusion of these molecules through the intervening aqueous phase until they collide with and are absorbed by an acceptor. At a constant donor particle concentration, there is a hyperbolic dependence of cholesterol transfer rate on the concentration of acceptor particles; the kinetics can be described in terms of the rate constants for At lower acceptor concentrations, the transfer rate is dependent on th...

, we showed that SR-BI-mediated cholesterol efflux was highly correlated (r 2 ‫؍‬ 0.985) with HDL phosphatidylcholine content. The effects of varying HDL phospholipid composition on SR-BI-mediated free cholesterol flux were not correlated with changes in either the K d or B max values for high affinity binding to SR-BI. We conclude that SR-BI-mediated free cholesterol flux is highly sensitive to HDL phospholipid composition. Thus, factors that regulate cellular SR-BI expression and the local modification of HDL phospholipid composition will have a large impact on reverse cholesterol transport.The deposition of cholesterol in peripheral cells is opposed by the process of reverse cholesterol transport (RCT) 1 where high density lipoproteins (HDL) remove free cholesterol (FC) from cells and deliver it back to the liver for excretion (1-3). The flux of FC between cells and HDL is bi-directional. Depending on the direction of the FC concentration gradient between cells and lipoproteins, either net efflux or net influx of cholesterol can occur (4, 5). The creation of a cholesterol gradient depends upon many properties of the acceptors and the cell plasma membrane. Such factors include the cholesterol and phospholipid content of the acceptors and plasma membrane (4, 5), the existence of cholesterol domains within the plasma membrane (6 -10), and the size, number, and composition of acceptor particles (11-13).Recent studies have shown that, when cells express scavenger receptor BI (SR-BI), the bi-directional flux of FC between cells and HDL is accelerated (8,14,15). The mechanism by which SR-BI mediates FC flux is uncertain. However, recent studies from our laboratory demonstrated that binding of the acceptor particles to SR-BI is not a requirement for SR-BImediated cholesterol efflux (7,8). Rather SR-BI induces a reorganization of the plasma membrane cholesterol, and this reorganization is linked to enhanced FC flux (7,8,16). Regardless of the mechanism, evidence is accumulating to support the importance of SR-BI-mediated FC flux in RCT. Recent studies of Ji and colleagues (17) showed that either attenuation or overexpression of hepatic SR-BI in mice led to significantly decreased or increased delivery of HDL FC into bile. In addition, the expression of SR-BI in peripheral cells and in foam cells of the arterial wall suggests a role for SR-BI in the removal of FC from the periphery (15, 18, 19).SR-BI-mediated FC flux requires phospholipid in the acceptor (15), and studies have shown that cholesterol efflux from cells is highly correlated with the concentration of HDL phospholipid in serum (20,21). Also, the stimulation of cholesterol efflux upon phospholipid supplementation of serum is closely linked to the levels of SR-BI among cell types (14). These observations are consistent with epidemiological data demonstrating that humans with low HDL phospholipid levels have a high incidence of coronary artery disease (22). These findings suggest that changes in HDL phospholipid content may alter SR-BI-mediated FC flux. The cu...

In this study, we compared the kinetics of cholesterol efflux from cells with 2-hydroxypropyl-␤-cyclodextrins and with discoidal high density lipoprotein (HDL) particles to probe the mechanisms governing the remarkably rapid rates of cyclodextrin-mediated efflux. The rate of cholesterol efflux was enhanced by shaking cells growing in a monolayer and further enhanced by placing cells in suspension to achieve maximal efflux rates. The extent of efflux was dependent on cyclodextrin concentration, and maximal efflux was observed at concentrations >50 mM. For several cell types, biexponential kinetics of cellular cholesterol efflux were observed, indicating the existence of two kinetic pools of cholesterol: a fast pool (half-time (t1 ⁄2 ) ϳ19 -23 s) and a slow pool with t1 ⁄2 of 15-30 min. Two distinct kinetic pools of cholesterol were also observed with model membranes (large unilamellar cholesterol-containing vesicles), implying that the cellular pools are in the plasma membrane. Cellular cholesterol content was altered by incubating cells with solutions of cyclodextrins complexed with increasing levels of cholesterol. The number of kinetic pools was unaffected by raising the cellular cholesterol content, but the size of the fast pool increased. After depleting cells of the fast pool of cholesterol, this pool was completely restored after a 40-min recovery period. The temperature dependence of cyclodextrinmediated cholesterol efflux from cells and model membranes was compared; the activation energies were 7 kcal/mol and 2 kcal/mol, respectively. The equivalent activation energy observed with apo-HDL-phospholipid acceptor particles was 20 kcal/mol. It seems that cyclodextrin molecules are substantially more efficient than phospholipid acceptors, because cholesterol molecules desorbing from a membrane surface can diffuse directly into the hydrophobic core of a cyclodextrin molecule without having to desorb completely into the aqueous phase before being sequestered by the acceptor.The first step in reverse cholesterol transport is the efflux of cellular cholesterol molecules to extracellular acceptors (1-3). This initial step is thought to be mediated by high density lipoproteins (HDL) 1 or by specific subpopulations of HDL (1-3). It is generally accepted that cholesterol efflux occurs by an aqueous diffusion mechanism whereby the cholesterol molecules desorb from the plasma membrane into the aqueous phase, diffuse, and are solubilized by an acceptor particle (2, 4).␤-Cyclodextrins are cyclic heptasaccharides consisting of ␤(1-4)-glucopyranose units (5). These water-soluble compounds contain a hydrophobic core capable of solubilizing nonpolar substances (5, 6). Thus, cyclodextrins have been used as vehicles to deliver hydrophobic drugs (5, 6). The ␤-cyclodextrins (7 glucose units), when compared with ␣ (6 glucose units) and ␥ (8 glucose units) cyclodextrins, have the highest affinity for encapsulating sterols, in particular cholesterol (7). Chemical modifications of the hydroxyl groups of cyclodextrins often enhance b...

Background Proprotein convertase subtilisin/kexin type 9 (PCSK9) modulates low-density lipoprotein (LDL) receptor (LDLR) degradation, thus influencing serum cholesterol levels. However, dysfunctional LDLR causes hypercholesterolemia without affecting PCSK9 clearance from the circulation. Methods and Results To study the reciprocal effects of PCSK9 and LDLR and the resultant effects on serum cholesterol, we produced transgenic mice expressing human (h) PCSK9. Although hPCSK9 was mainly expressed in the kidney, LDLR degradation was more evident in the liver. Adrenal LDLR levels were not affected, likely due to impaired PCSK9 retention in this tissue. In addition, hPCSK9 expression increased hepatic secretion of apoB-containing lipoproteins in an LDLR-independent fashion. Expression of hPCSK9 raised serum murine (m) PCSK9 levels by 4.3-fold in wild-type (WT) mice and not at all in LDLR−/− ice, where mPCSK9 levels were already 10-fold higher than in WT mice. In addition, LDLR+/− mice had 2.7-fold elevation in mPCSK9 levels and no elevation in cholesterol levels. Conversely, acute expression of hLDLR in transgenic mice caused a 70% decrease in serum mPCSK9 levels. Turnover studies using physiological levels of hPCSK9 showed rapid clearance in WT (half-life 5.2 min), faster in hLDLR transgenics (2.9 min), and much slower in LDLR−/− recipients (50.5 min). Supportive results were obtained using an in vitro system. Finally, up to 30% of serum hPCSK9 was associated with LDL regardless of LDLR expression. Conclusions Our results support a scenario where LDLR represents the main route of elimination of PCSK9, and a reciprocal regulation between these two proteins controls serum PCSK9 levels, hepatic LDLR expression, and serum LDL levels.

These findings suggest that abnormal HDL capacity to mediate cholesterol efflux is a key driver of excess CVD in patients on chronic hemodialysis and may explain why statins have limited effect to decrease CV events. The findings also suggest cellular cholesterol transporters as potential therapeutic targets to decrease CV risk in this population.

The mechanism(s) by which lipid-free apolipoprotein (apo) AI is able to stimulate efflux of cholesterol and phospholipid from cells in cultures has (have) been examined. This process was found to be enhanced when macrophages were enriched with cholesterol. There were 12- and 4-fold increases in cholesterol and phospholipid efflux, respectively, from cholesterol-enriched mouse macrophages when compared to cells not loaded with cholesterol. This enhancement in cholesterol efflux to lipid-free apo AI from macrophages enriched with cholesterol was found to be controlled by the level of free cholesterol in the cells. When cholesterol-enriched mouse macrophages were exposed to lipid-free apo AI at 20 micrograms/mL (706 nM), there was significant efflux of [14C]cholesterol and [3H]phospholipid (20% +/- 0.5%/24 h and 6% +/- 0.3%/24 h, respectively). In comparison, HDL at equivalent protein concentrations only stimulated 11% and 4% efflux of cholesterol and phospholipid, respectively. Synthetic peptides containing amphipathic helical segments that mimic those present in apo AI were used to examine the structural features of the apoprotein which stimulate lipid efflux. Peptides containing only one (18A) or two (37pA) amphipathic helical segments stimulated as much cholesterol efflux from both mouse macrophages and L-cells as apo AI. The order of efficiency, as assessed by the mass concentration at which half-maximal efflux was reached (EC50), was apo AI > 37pA > 18A, indicating that acceptor efficiency was dependent on the number of amphipathic helical segments per molecule. When the helical content of 18A was increased by neutralizing the charges at the ends of the peptide (Ac-18A-NH2), there was a substantial increase in the efficiency for cholesterol efflux (EC50 18A = 17 micrograms/mL vs Ac-18A-NH2 = 6 micrograms/mL). In contrast, when the amphipathicity of the helix in 18A was decreased by scrambling the amino acid sequence, thereby reducing its lipid affinity, cholesterol and phospholipid efflux were not stimulated. The efficiency with which the peptides stimulated cholesterol efflux was in order of their lipid affinity (37pA > Ac-18A-NH2 > 18A), and this order was similar for phospholipid efflux. The time course of lipid release from mouse macrophages and L-cells indicated that phospholipid appeared in the extracellular medium before cholesterol. These results suggest that the apo AI or peptides first interacted with the cell to form protein/phospholipid complexes, that could then accept cholesterol.

Abstract-Macrophage low-density lipoprotein receptor-related protein (LRP) mediates internalization of remnant lipoproteins, and it is generally thought that blocking lipoprotein internalization will reduce foam cell formation and atherogenesis. Therefore, our study examined the function of macrophage LRP in atherogenesis. We generated transgenic mice that specifically lack macrophage LRP through Cre/lox recombination. Transplantation of macrophage LRP Ϫ/Ϫ bone marrow into lethally irradiated female LDLR Ϫ/Ϫ recipient mice resulted in a 40% increase in atherosclerosis. The difference in atherosclerosis was not caused by altered serum lipoprotein levels. Furthermore, deletion of macrophage LRP decreased uptake of 125 I-very-low-density lipoprotein compared with wild-type cells in vitro. The increase in atherosclerosis was accompanied by increases in monocyte chemoattractant protein type-1, tumor necrosis factor-␣, and proximal aorta macrophage cellularity. We also found that deletion of macrophage LRP increases matrix metalloproteinase-9. This increase in matrix metalloproteinase-9 was associated with a higher frequency of breaks in the elastic lamina. Contrary to what was found with other lipoprotein receptors, deletion of LRP increases atherogenesis in hypercholesterolemic mice. Our data support the hypothesis that macrophage LRP modulates atherogenesis through regulation of inflammatory responses. (Circ Res. 2007;100:670-677.) Key Words: low-density lipoprotein receptor-related protein Ⅲ atherosclerosis Ⅲ lipoproteins Ⅲ metalloproteinase Ⅲ macrophage F irst defined as a complex for removal of ␣ 2 -macroglobulin, 1,2 and then later identified as the lowdensity lipoprotein receptor (LDLR)-related protein (LRP), 3 LRP is a 600-kDa membrane receptor linked to numerous cellular functions and intracellular signaling events. 4 It has multiple extracellular ligands, including apolipoprotein E (apoE), lipoprotein lipase, plasma proteases (urokinase-type and tissue plasminogen activators), fibrinolytic factors (IXa and VIIIa), thrombospondin 1 and 2, and chaperone proteins receptor associated protein and heat shock protein-96 (reviewed elsewhere 5 ). The cytoplasmic tail of LRP binds to multiple intracellular adapter and scaffold proteins including disabled-1 and FE65. 6,7 LRP is present in numerous cell types, including macrophages and hepatocytes, and its systemic expression is essential for embryonic development. 8 A fundamental role for hepatic LRP in the clearance of plasma remnants has been demonstrated, as conditional hepatic LRP deletion results in increased plasma triglyceride and chylomicron levels, particularly in the absence of the LDLR. 9 Furthermore, decreased expression of hepatic LRP causes delayed chylomicron remnant clearance, supporting a protective effect of hepatic LRP on atherogenesis via reduced plasma lipoprotein burden. 10 Besides the effect on lipoprotein remnants, hepatic LRP may provide additional vascular protection by mediating the clearance of other proinflammatory ligands including matrix...