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The current model for reverse cholesterol transport proposes that HDL transports excess cholesterol derived primarily from peripheral cells to the liver for removal. However, recent studies in ABCA1 transgenic mice suggest that the liver itself may be a major source of HDL cholesterol (HDL-C). To directly investigate the hepatic contribution to plasma HDL-C levels, we generated an adenovirus (rABCA1-GFP-AdV) that targets expression of mouse ABCA1-GFP in vivo to the liver. Compared with mice injected with control AdV, infusion of rABCA1-GFP-AdV into C57Bl/6 mice resulted in increased expression of mouse ABCA1 mRNA and protein in the liver. ApoA-I-dependent cholesterol efflux was increased 2.6-fold in primary hepatocytes isolated 1 day after rABCA1-GFP-AdV infusion. Hepatic ABCA1 expression in C57Bl/6 mice (n ‫؍‬ 15) raised baseline levels of TC, PL, FC, HDL-C, apoE, and apoA-I by 150-300% ( P Ͻ 0.05 all). ABCA1 expression led to significant compensatory changes in expression of genes that increase hepatic cholesterol, including HMG-CoA reductase (3.5-fold), LDLr (2.1-fold), and LRP (5-fold) in the liver.These combined results demonstrate that ABCA1 plays a key role in hepatic cholesterol efflux, inducing pathways that modulate cholesterol homeostasis in the liver, and establish the liver as a major source of plasma HDL-C. The role of HDL in the removal of excess cholesterol from peripheral cells was postulated nearly 30 years ago and designated reverse cholesterol transport (RCT) (1, 2). In the current working model of RCT, excess cholesterol is removed from peripheral cells and esterified by lecithin cholesterol acyltransferase. The cholesteryl ester is then transported back to the liver for removal from the body, either directly by HDL or following transfer by cholesterol ester transfer protein to the apoB-containing lipoproteins. This model proposes that cholesterol and lipids used for the formation and maturation of HDL are derived from nonhepatic, peripheral cells as well as from the metabolism and remodeling of the triglyceride-rich, apoBcontaining lipoproteins. Previous reports indicate that the liver is an important source of apoA-I and thus contributes significantly to the plasma pool of nascent HDL (3, 4). However, although the liver is the most important modulator of cholesterol homeostasis in the body, it has not been implicated as a major in vivo source of cholesterol to lipidate HDL in the circulation .A major advance in our understanding of the first step in reverse cholesterol transport was the identification of the ABCA1 transporter as the genetic defect in patients with Tangier disease (5-10). ABCA1 is the major transporter that facilitates the efflux of cholesterol and phospholipids to poorly lipidated apoA-I to form nascent or pre ␤ HDL. In the absence of a functional ABCA1 transporter, patients with Tangier disease are unable to efflux cholesterol to apoA-I and accumulate cholesteryl esters in many tissues, including arterial macrophages. Attie et al. reported similar findings in the ...

Tangier disease is characterized by HDL hypercatabolism and increased deposition of cholesterol in tissues. Tangier disease skin fibroblasts have decreased apoA-I-mediated cholesterol and phospholipid efflux, which may lead to the excess accumulation of cellular cholesterol. The mechanism of apolipoprotein-mediated cholesterol efflux and the apolipoprotein acceptor specificity for cholesterol efflux from normal and Tangier disease fibroblasts was investigated. Normal cells readily effluxed cholesterol and phospholipid to apoA-I and to all of the other apolipoproteins tested (apoA-II, AIV, C-I, C-II, C-III). In contrast, Tangier cells were almost completely defective in cholesterol efflux to apoA-I and to all of the other apolipoproteins tested. HDL was also less effective, by approximately 50%, in stimulating cholesterol efflux from Tangier cells compared with normal cells. In addition, Tangier cells also showed significantly reduced phospholipid efflux to both apolipoproteins and HDL. A similar rate of cholesterol efflux, however, was observed from normal and Tangier cells when phospholipid vesicles or cyclodextrin were used as acceptors. In contrast to normal cells, only phospholipid vesicles and cyclodextrin and not apoA-I or HDL depleted intracellular cholesteryl esters from Tangier cells. Brefeldin, an inhibitor of intracellular vesicular trafficking, decreased HDL-mediated cholesterol efflux by approximately 40% but almost completely blocked both cholesterol and phospholipid efflux to apoA-I from normal cells. Brefeldin also inhibited cholesteryl ester depletion by apoA-I and HDL from normal cells. Brefeldin, however, had no significant effect on cholesterol efflux from Tangier cells to HDL. In summary, Tangier cells were found to be defective in both cholesterol and phospholipid efflux to HDL and apoA-I. The defect in apolipoprotein-mediated lipid efflux was not specific for apoA-I but also occurred for other apolipoproteins, and brefeldin blocked HDL-mediated lipid efflux from normal but not Tangier disease cells. On the basis of these results, a model is proposed whereby decreased cholesterol efflux by apolipoproteins in Tangier cells is the result of a defect in a brefeldin-sensitive pathway of lipid efflux.

Lipopolysaccharide (LPS) has recently been shown to facilitate macrophage foam cell formation and has been suggested to be a proatherogenic factor. The mechanism of LPS induced cholesterol accumulation, however, is unclear. In this report, using the macrophage-like RAW 264.7 cell line, we provide experimental evidence that LPS's proatherogenic effects may at least in part reflect altered cholesterol metabolism. Data presented demonstrate that in a dose-dependent manner, LPS is able to down regulate the mRNA expression of the two primary high-density lipoprotein (HDL) receptors, scavenger receptor B1 (SR-B1) and ATP binding cassette A1 (ABCA1), with a 50% inhibitory concentration of less than 0.2 ng/ml, as well as to decrease SR-B1 protein expression by 80%. We also found that LPS treatment resulted in a significant decrease (to 20% of the control level) of the specific 125 I-HDL binding as well as in 50% inhibition of the HDL-mediated cholesterol efflux compared to untreated cells. In addition, we compared the potencies of various modified LPS preparations and demonstrated that the phosphorylated lipid A portion of LPS, which is highly conserved among gram-negative microorganisms, including Chlamydia, is primarily responsible for the effects of LPS on SR-B1 and ABCA1 expression. Inhibitors of NF-B activation were observed to efficiently block the suppressive effect of LPS on SR-B1 and ABCA1, suggesting a mechanism involving NF-B. These data indicate that the LPS effects on cholesterol metabolism may contribute to the proatherogenic properties of LPS.

Intravenous administration of apolipoprotein (apo) A

Infusions of cholesterol-free reconstituted HDL (rHDL) particles have been shown to rapidly reverse atherosclerosis in a wide variety of animal models and in clinical trials of acute coronary syndrome patients ( 1-5 ). While a significant emphasis has been placed on investigating the HDL protein component, i.e., ApoA-I, ApoA-I mutants, and mimetic peptides, the importance of HDL phospholipid (PL) composition has not been systematically investigated. Lipid represents 50-80% of the total HDL mass and is known to affect particle stability in vivo, cholesterol effl ux from macrophages, the ability to interact with LCAT, and cholesterol elimination ( 6-11 ). Lipid composition also largely defi nes the size, net charge, and rigidity of the rHDL particles; all are important factors in the pharmacokinetic and pharmacodynamic properties of rHDL. The investigation of the effects of lipid composition on the resulting rHDL properties in vitro and in vivo is the focus of this article. By better defi ning the lipid effect of rHDL, we hope to be able to favorably alter the potency and safety of rHDL, and ultimately advance clinical translation of these potentially life-changing nanomedicines.The PL composition of endogenous HDL contains phosphatidylcholines (PCs), SM, and small amounts of lysophosphatidylcholine (LPC), phosphatidylethanolamine, Abstract The goal of this study was to understand how the reconstituted HDL (rHDL) phospholipid (PL) composition affects its cholesterol effl ux and anti-infl ammatory properties. An ApoA-I mimetic peptide, 5A, was combined with either SM or POPC. Both lipid formulations exhibited similar in vitro cholesterol effl ux by ABCA1, but 5A-SM exhibited higher ABCG1-and SR-BI-mediated effl ux relative to 5A-POPC ( P < 0.05). Injection of both rHDLs in rats resulted in mobilization of plasma cholesterol, although the relative potency was 3-fold higher for the same doses of 5A-SM than for 5A-POPC. Formation of pre ␤ HDL was observed following incubation of rHDLs with both human and rat plasma in vitro, with 5A-SM inducing a higher extent of pre ␤ formation relative to 5A-POPC. Both rHDLs exhibited antiinfl ammatory properties, but 5A-SM showed higher inhibition of TNF-␣ , IL-6, and IL-1 ␤ release than did 5A-POPC ( P < 0.05). Both 5A-SM and 5A-POPC showed reduction in total plaque area in ApoE ؊ / ؊ mice, but only 5A-SM showed a statistically signifi cant reduction over placebo control and baseline ( P < 0.01). The type of PL used to reconstitute peptide has signifi cant infl uence on rHDL's anti-infl ammatory and anti-atherosclerosis properties.