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Objective: The hypothesis that cholesterol that enters the cell within low-density lipoprotein (LDL) particles rapidly equilibrates with the regulatory pool of intracellular cholesterol and maintains cholesterol homeostasis by reducing cholesterol and LDL receptor synthesis was validated in the fibroblast but not in the hepatocyte. Accordingly, the present studies were designed to compare the effects of cholesterol that enters the hepatocyte within an LDL particle with those of cholesterol that enters via other lipoprotein particles., Approach and Results: We measured cholesterol synthesis and esterification in hamster hepatocytes treated with LDL and other lipoprotein particles, including chylomicron remnants and VLDL. Endogenous cholesterol synthesis was not significantly reduced by uptake of LDL, but cholesterol esterification (280%) and acyl CoA:cholesterol acyltransferase 2 expression (870%) were increased. In contrast, cholesterol synthesis was significantly reduced (70% decrease) with other lipoprotein particles. Furthermore, more cholesterol that entered the hepatocyte within LDL particles was secreted within VLDL particles (480%) compared with cholesterol from other sources., Conclusions: Much of the cholesterol that enters the hepatocyte within LDL particles is shunted through the cell and resecreted within VLDL particles without reaching equilibrium with the regulatory pool.

Purpose of Review: Even with the aggressive reduction of LDL-cholesterol, the risk of cardiovascular events in patients with coronary artery disease remains substantial. The Armed Forces Regression Study was a randomized, double-blind, placebo-controlled trial of combination drug therapy aimed at raising HDL-cholesterol in patients with angiographically evident coronary artery disease. Drug therapy ultimately resulted in regression of the angiographic lesions and a reduction in cardiovascular events. This review places the Armed Forces Regression Study within the context of other recent studies., Recent Findings: In the past few years a number of other important papers have further defined the important role HDL-cholesterol plays in the pathobiology of atherosclerosis. These studies have focused on three general areas: HDL-cholesterol metabolism and the reverse cholesterol transport pathway; novel therapeutic interventions and their effects on coronary artery disease as assessed through non-invasive imaging modalities; and finally a re-analysis of previous outcomes trials with established HDL-cholesterol modifying agents., Summary: The results of the Armed Forces Regression Study fit nicely within the evolving paradigm of targeting HDL-cholesterol in patients at risk of cardiovascular events. The use of niacin and well-tolerated fibrates as an adjunct to statins or as primary therapy in patients intolerant of statins appears reasonable in patients with low levels of HDL-cholesterol and at high risk of cardiovascular events. The further development of novel therapeutic approaches, in addition to broadening our pharmacological armamentarium, should further advance our understanding of HDL-cholesterol.

Objective: Inhibition of 2,3-oxidosqualene:lanosterol cyclase (OSC), an enzyme in the cholesterol synthesis pathway, has the unique ability to inhibit cholesterol synthesis while simultaneously enhancing oxysterol synthesis. Our objectives were to determine, in vivo, if a novel OSC inhibitor reduced low-density lipoprotein (LDL) cholesterol and to define the mechanism(s) involved., Methods and Results: Miniature pigs received the OSC inhibitor RO0717625 or placebo and a diet containing fat (34% of energy) and 400 mg per day of cholesterol. Treatment decreased plasma total cholesterol (-20%) and LDL cholesterol (-29%). Apolipoprotein B (apoB) kinetic parameters were determined. Very low-density lipoprotein (VLDL) apoB pool size decreased 22% because of inhibition of VLDL production (-43%). LDL apoB pool size decreased 22% because of a 1.5-fold increase in fractional catabolic rate (FCR). The increased FCR was associated with a 2-fold increase in hepatic LDL receptor mRNA. Hepatic total and microsomal cholesterol were reduced by 16% and 27%, respectively. Plasma lathosterol concentrations decreased 57%, reflecting inhibition of hepatic cholesterol synthesis. Treatment reduced plasma plant sterols and decreased postprandial cholesterol transport in chylomicrons., Conclusions: A novel OSC inhibitor, RO0717625, decreased VLDL and LDL apoB100 through decreased VLDL production and enhanced LDL clearance. Thus, OSC represents a potential therapeutic target for dyslipidemia.

Purpose of Review: Mammalian somatic cells do not catabolize cholesterol and therefore need to export it for sterol homeostasis at the levels of cells and whole bodies. This mechanism may reduce intracellularly accumulated cholesterol in excess, and thereby would contribute to the prevention or cure of the initial stage of atherosclerotic vascular lesions., Recent Findings: HDL is thought to play a main role in this reaction on the basis of epidemiological evidence and in-vitro experimental data. Two independent mechanisms have been identified for this reaction. One is non-specific diffusion-mediated cholesterol 'efflux' from the cell surface, and cholesterol is trapped by various extracellular acceptors including lipoproteins. Extracellular cholesterol esterification on HDL provides a driving force for the net removal of cell cholesterol, and some cellular factors may enhance this reaction. The other mechanism is an apolipoprotein-mediated process to generate HDL by removing cellular phospholipid and cholesterol. This reaction is mediated by a membrane protein ABCA1, and lipid-free or lipid-poor helical apolipoproteins recruit cellular phospholipid and cholesterol to assemble HDL particles. The reaction is composed of two elements: the assembly of HDL particles with phospholipid by apolipoprotein, and cholesterol enrichment in HDL. ABCA1 is essential for the former step, and the latter step requires further intracellular events., Summary: ABCA1 is a rate-limiting factor of HDL assembly and is regulated by transcriptional factors and posttranscriptional factors. Posttranscriptional regulation of ABCA1 involves the modulation of its calpain-mediated degradation.

Purpose of Review: ATP-binding cassette transporter AI (ABCA1)-mediated assembly of phospholipid and free cholesterol with apoA-I plays an important role in HDL biogenesis. This review focuses on recent progress in ABCA1-mediated HDL formation and regulation of ABCA1 expression., Recent Findings: Studies of hepatic ABCA1 overexpression suggest that the liver is a major site for HDL formation. Lipidation of apoA-I by ABCA1 increases its potential for reverse cholesterol transport based on the following findings: (1) apoA-I/lipid complexes formed by ABCA1 are better acceptors of cellular lipid via non-ABCA1-mediated efflux pathways than lipid-free apoA-I in vitro and (2) lipidation of apoA-I prevents it from rapidly associating with plasma HDL in vivo, resulting in more available nascent pre-beta HDL for cellular lipid efflux. Several novel regulatory mechanisms for ABCA1 at the post-transcriptional level have been identified recently. Interaction of apoA-I with ABCA1 prevents phosphorylation of a sequence rich in proline, glutamic acid, serine and threonine in a cytoplasmic domain of ABCA1, resulting in less degradation by calpain proteolysis and increased surface expression of ABCA1. In addition, destabilization and decreased cellular surface expression of ABCA1 protein by unsaturated fatty acids have been identified., Summary: Initial lipidation of apoA-I by hepatic ABCA1 is critical for plasma HDL formation because it enables pre-beta HDL to function more efficiently as a cholesterol acceptor for other pathways of cholesterol efflux in the reverse cholesterol transport pathway and prevents apoA-I from rapidly associating with preexisting plasma HDL particles, resulting in greater availability of pre-beta HDL particles for cholesterol efflux.

Objective: Human serum paraoxonase 1 (PON1) activity is inversely related to the risk of developing an atherosclerotic lesion, which contains cholesterol-loaded macrophage foam cells. To assess a possible mechanism for this relationship, we analyzed the effect of PON1 on cellular cholesterol biosynthesis., Methods and Results: Mouse peritoneal macrophages (MPMs) were harvested from PON1-deficient mice (PON1o and PON1o/Eo mice on the genetic background of C57BL/6J and Eo mice, respectively). PON1o/Eo mice exhibited a significantly 51% increased atherosclerotic lesion area and 35% increased macrophage cholesterol content compared with control E degrees mice. In parallel, macrophage cholesterol biosynthesis rates were increased in PON1-deficient mice MPMs by 50% compared with their controls. Incubation of macrophages with human PON1 revealed a dose-dependent inhibitory effect (up to 84%) on macrophage cholesterol biosynthesis. We demonstrated a PON1 phospholipase-A2-like activity on MPMs, evidenced by release of polyunsaturated fatty acids and formation of lysophosphatidylcholine. On incubation of macrophages with lysophosphatidylcholine, a dose-dependent inhibition (up to 40%) of cellular cholesterol biosynthesis was noted. The inhibitory effect of PON1 on macrophage cholesterol biosynthesis was shown to be downstream to mevalonate, probably at the lanosterol metabolic point., Conclusions: PON1 inhibits macrophage cholesterol biosynthesis and atherogenesis probably through its phospholipase-A2-like activity.

Background: HDL cholesterol levels are inversely correlated with coronary heart disease risk in humans, and in animal studies, HDL elevation decreases formation and progression of foam-cell lesions. The potential for HDL to affect preexisting advanced atherosclerotic lesions is not known. To approach this issue, we used a novel mouse aortic transplantation model., Methods and Results: ApoE-deficient (EKO) mice were fed a Western-type diet for 6 months, and thoracic aortic segments containing advanced lesions replaced segments of the abdominal aorta of 4-month-old EKO syngeneic mice not expressing (plasma HDL cholesterol approximately 26 mg/dL) or expressing (HDL approximately 64 mg/dL) a human apoAI (hAI) transgene. Both types of recipients had comparable non-HDL cholesterol levels. Five months after transplantation, mice were killed and grafts analyzed. Compared with lesion area in pretransplant mice (0.14+/-0.04 mm(2), mean+/-SEM), there was progression in the EKO recipients (0.39+/-0.06 mm(2), P<0.01). Compared with EKO recipients, hAI/EKO recipients had retarded progression (0.24+/-0.04 mm(2), P<0.05). Immunostaining for CD68 and other macrophage-associated proteins, monocyte chemoattractant protein-1, acyl coenzyme A:cholesterol acyltransferase, and tissue factor, in lesions of pretransplant and EKO recipient mice showed abundant macrophages. In contrast, compared with any other group, lesional macrophage area in hAI/EKO mice decreased >80% (P<0.003), and smooth muscle cell content (alpha-actin staining) increased >300% (P<0.006). The decrease in macrophages and increase in smooth muscle cells was primarily in the superficial subendothelial layer., Conclusions: Increasing HDL cholesterol levels in EKO mice retards progression of advanced atherosclerotic lesions and remodels them to a more stable-appearing phenotype.

gamma-Tocotrienol (gamma-T3), a naturally occurring analog of tocopherol (vitamin E), has been shown to have a hypocholesterolemic effect in animals and humans. Unlike tocopherol, it has also been shown to reduce plasma apoB levels in hypercholesterolemic subjects. The aim of this study was to define the mechanism of action of gamma-T3 on hepatic modulation of apoB production using cultured HepG2 cells as the model system. HepG2 cells preincubated with gamma-T3 were initially shown to inhibit the rate of incorporation of [14C]acetate into cholesterol in a concentration- and time-dependent manner, with a maximum 86+/-3% inhibition at 50 micromol/L observed within 6 hours. gamma-T3, on the other hand, had no significant effect on the uptake of [14C]glycerol into pools of cellular triacylglycerol and phospholipid relative to untreated control. The rate of apoB synthesis and secretion was then studied by an [35S]methionine pulse-labeling experiment and quantified by immunoprecipitating apoB on chasing up to 3 hours. An average reduction of 24+/-3% in labeled apoB in the media was apparent with gamma-T3 despite a 60+/-2% increase in apoB synthesis. Fractionation of secreted apoB revealed a relatively denser lipoprotein particle, suggesting a less stable particle. Using a digitonin-permeabilized HepG2 cell system, the effects of gamma-T3 on apoB translocation and degradation in the endoplasmic reticulum were further investigated. The generation of a specific N-terminal 70-kDa proteolytic fragment proved to be a sensitive measure of the rate of apoB translocation and degradation. The abundance of this fragment increased significantly in gamma-T3-treated cells relative to untreated control cells (50+/-21%) after 2 hours of chase. In addition, the presence of gamma-T3 resulted in an average decrease of 64+/-8% in intact apoB. Taken together, the data suggest that gamma-T3 stimulates apoB degradation possibly as the result of decreased apoB translocation into the endoplasmic reticulum lumen. It is speculated that the lack of cholesterol availability reduces the number of secreted apoB-containing lipoprotein particles by limiting translocation of apoB into the endoplasmic reticulum lumen.