Importance of evaluating cell cholesterol influx with efflux in determining the impact of human serum on cholesterol metabolism and atherosclerosis.

Objective: Cholesterol efflux relates to cardiovascular disease but cannot predict cellular cholesterol mass changes. We asked whether influx and net flux assays provide additional insights.

Approach And Results: Adapt a bidirectional flux assay to cells where efflux has clinical correlates and examine the association of influx, efflux, and net flux to serum triglycerides (TGs).

Acute consumption of walnuts and walnut components differentially affect postprandial lipemia, endothelial function, oxidative stress, and cholesterol efflux in humans with mild hypercholesterolemia.

Walnut consumption improves cardiovascular disease risk; however, to our knowledge, the contribution of individual walnut components has not been assessed. This study evaluated the acute consumption of whole walnuts (85 g), separated nut skins (5.6 g), de-fatted nutmeat (34 g), and nut oil (51 g) on postprandial lipemia, endothelial function, and oxidative stress.

Serum albumin acts as a shuttle to enhance cholesterol efflux from cells.

An important mechanism contributing to cell cholesterol efflux is aqueous transfer in which cholesterol diffuses from cells into the aqueous phase and becomes incorporated into an acceptor particle. Some compounds can enhance diffusion by acting as shuttles transferring cholesterol to cholesterol acceptors, which act as cholesterol sinks. We have examined whether particles in serum can enhance cholesterol efflux by acting as shuttles.

The effects of apolipoprotein F deficiency on high density lipoprotein cholesterol metabolism in mice.

Apolipoprotein F (apoF) is 29 kilodalton secreted sialoglycoprotein that resides on the HDL and LDL fractions of human plasma. Human ApoF is also known as Lipid Transfer Inhibitor protein (LTIP) based on its ability to inhibit cholesteryl ester transfer protein (CETP)-mediated transfer events between lipoproteins. In contrast to other apolipoproteins, ApoF is predicted to lack strong amphipathic alpha helices and its true physiological function remains unknown.

Cytoskeleton disruption in J774 macrophages: consequences for lipid droplet formation and cholesterol flux.

Macrophages store excess unesterified cholesterol (free, FC) in the form of cholesteryl ester (CE) in cytoplasmic lipid droplets. The hydrolysis of droplet-CE in peripheral foam cells is critical to HDL-promoted reverse cholesterol transport because it represents the first step in cellular cholesterol clearance, as only FC is effluxed from cells to HDL. Cytoplasmic lipid droplets move within the cell utilizing the cytoskeletal network, but, little is known about the influence of the cytoskeleton on lipid droplet formation.

A sensitive assay for ABCA1-mediated cholesterol efflux using BODIPY-cholesterol.

Studies have shown a negative association between cellular cholesterol efflux and coronary artery disease (CAD). Standard protocol for quantitating cholesterol efflux involves labeling cells with [(3)H]cholesterol and measuring release of the labeled sterol. Using [(3)H]cholesterol is not ideal for the development of a high-throughput assay to screen large numbers of serum as would be required in studying the link between efflux and CAD.

Novel in vivo method for measuring cholesterol mass flux in peripheral macrophages.

Objective: Reverse cholesterol transport is the process by which excess cholesterol is removed from peripheral tissue by HDL and delivered to the liver for excretion. Presently, methods of measuring in vivo reverse cholesterol transport do so by monitoring the appearance in the feces of labeled cholesterol that originated from peripheral macrophage foam cells. These methods do not account for changes in macrophage cholesterol mass.

Rat carboxylesterase ES-4 enzyme functions as a major hepatic neutral cholesteryl ester hydrolase.

Although esterification of free cholesterol to cholesteryl ester in the liver is known to be catalyzed by the enzyme acyl-coenzyme A:cholesterol acyltransferase, ACAT, the neutral cholesteryl ester hydrolase (nCEH) that catalyzes the reverse reaction has remained elusive. Because cholesterol undergoes continuous cycling between free and esterified forms, the steady-state concentrations in the liver of the two species and their metabolic availability for pathways, such as lipoprotein assembly and bile acid synthesis, depend upon nCEH activity. On the basis of the general characteristics of the family of rat carboxylesterases, we hypothesized that one member, ES-4, was a promising candidate as a hepatic nCEH.

Disruption of the murine procollagen C-proteinase enhancer 2 gene causes accumulation of pro-apoA-I and increased HDL levels.

Given the increased prevalence of cardiovascular disease in the world, the search for genetic variations that impact risk factors associated with the development of this disease continues. Multiple genetic association studies demonstrate that procollagen C-proteinase enhancer 2 (PCPE2) modulates HDL levels. Recent studies revealed an unexpected role for this protein in the proteolytic processing of pro-apolipoprotein (apo) A-I by enhancing the cleavage of the hexapeptide extension present at the N-terminus of apoA-I.

Thyroid hormone enhances the ability of serum to accept cellular cholesterol via the ABCA1 transporter.

Objective: The goal of this study was to examine the effects of thyroid hormone status on the ability of serum to accept cellular cholesterol.

Methods And Results: Sera from hypophysectomized rats treated ± T(3) was used to evaluate the role of thyroid hormone on serum efflux capacity. 2D-DIGE analysis of serum proteins showed that T(3) treated rats had increased ApoA-I, ApoA-IV and fetuin A levels with decreased Apo E levels.

Cholesterol efflux capacity, high-density lipoprotein function, and atherosclerosis.

Background: High-density lipoprotein (HDL) may provide cardiovascular protection by promoting reverse cholesterol transport from macrophages. We hypothesized that the capacity of HDL to accept cholesterol from macrophages would serve as a predictor of atherosclerotic burden.

Methods: We measured cholesterol efflux capacity in 203 healthy volunteers who underwent assessment of carotid artery intima-media thickness, 442 patients with angiographically confirmed coronary artery disease, and 351 patients without such angiographically confirmed disease.

Influence of apolipoprotein A-I domain structure on macrophage reverse cholesterol transport in mice.

Objective: The goal of this study was to determine the influence of apolipoprotein A-I (apoA-I) tertiary structure domain properties on the antiatherogenic properties of the protein. Two chimeric hybrids with the N-terminal domains swapped (human-mouse apoA-I and mouse-human apoA-I) were expressed in apoA-I-null mice with adeno-associated virus (AAV) and used to study macrophage reverse cholesterol transport (RCT) in vivo.

Methods And Results: The different apoA-I variants were expressed in apoA-I-null mice that were injected with [H(3)]cholesterol-labeled J774 mouse macrophages to measure RCT.

Importance of macrophage cholesterol content on the flux of cholesterol mass.

Net flux of cholesterol represents the difference between efflux and influx and can result in net cell-cholesterol accumulation, net cell-cholesterol depletion, or no change in cellular cholesterol content. We measured radiolabeled cell-cholesterol efflux and cell-cholesterol mass using cholesterol-normal and -enriched J774 and elicited mouse peritoneal macrophage cells. Net cell-cholesterol effluxes were observed when cholesterol-enriched J774 cells were incubated with 3.

Influence of apolipoprotein (Apo) A-I structure on nascent high density lipoprotein (HDL) particle size distribution.

The principal protein of high density lipoprotein (HDL), apolipoprotein (apo) A-I, in the lipid-free state contains two tertiary structure domains comprising an N-terminal helix bundle and a less organized C-terminal domain. It is not known how the properties of these domains modulate the formation and size distribution of apoA-I-containing nascent HDL particles created by ATP-binding cassette transporter A1 (ABCA1)-mediated efflux of cellular phospholipid and cholesterol. To address this issue, proteins corresponding to the two domains of human apoA-I (residues 1-189 and 190-243) and mouse apoA-I (residues 1-186 and 187-240) together with some human/mouse domain hybrids were examined for their abilities to form HDL particles when incubated with either ABCA1-expressing cells or phospholipid multilamellar vesicles.

Impact of combined deficiency of hepatic lipase and endothelial lipase on the metabolism of both high-density lipoproteins and apolipoprotein B-containing lipoproteins.

Rationale: Hepatic lipase (HL) and endothelial lipase (EL) are extracellular lipases that both hydrolyze triglycerides and phospholipids and display potentially overlapping or complementary roles in lipoprotein metabolism.

Objective: We sought to dissect the overlapping roles of HL and EL by generating mice deficient in both HL and EL (HL/EL-dko) for comparison with single HL-knockout (ko) and EL-ko mice, as well as wild-type mice.

Methods And Results: Reproduction and viability of the HL/EL-dko mice were impaired compared with the single-knockout mice.

High-density lipoprotein heterogeneity and function in reverse cholesterol transport.

Purpose Of Review: HDL is a cardioprotective lipoprotein, at least in part, because of its ability to mediate reverse cholesterol transport (RCT). It is becoming increasingly clear that the antiatherogenic effects of HDL are not only dependent on its concentration in circulating blood but also on its biological 'quality'. This review summarizes our current understanding of how the biological activities of individual subclasses of HDL particles contribute to overall HDL performance in RCT.

Adipocyte modulation of high-density lipoprotein cholesterol.

Background: Adipose harbors a large depot of free cholesterol. However, a role for adipose in cholesterol lipidation of high-density lipoprotein (HDL) in vivo is not established. We present the first evidence that adipocytes support transfer of cholesterol to HDL in vivo as well as in vitro and implicate ATP-binding cassette subfamily A member 1 (ABCA1) and scavenger receptor class B type I (SR-BI), but not ATP-binding cassette subfamily G member 1 (ABCG1), cholesterol transporters in this process.

The ability to promote efflux via ABCA1 determines the capacity of serum specimens with similar high-density lipoprotein cholesterol to remove cholesterol from macrophages.

Objective: We measured efflux from macrophages to apolipoprotein B-depleted serum from 263 specimens and found instances in which serum having similar high-density lipoprotein cholesterol (HDL-C) differed in their efflux capacity. Thus, we wanted to elucidate why efflux capacity could be independent of total HDL-C or apolipoprotein A-I (apoA-I).

Methods And Results: To understand why sera with similar HDL-C or apoA-I could differ in total efflux capacity, we assessed their ability to promote efflux via the pathways expressed in cAMP-treated J774 macrophages.

Pathways by which reconstituted high-density lipoprotein mobilizes free cholesterol from whole body and from macrophages.

Objective: Reconstituted high-density lipoprotein (rHDL) is of interest as a potential novel therapy for atherosclerosis because of its ability to promote free cholesterol (FC) mobilization after intravenous administration. We performed studies to identify the underlying molecular mechanisms by which rHDL promote FC mobilization from whole body in vivo and macrophages in vitro.

Methods And Results: Wild-type (WT), SR-BI knockout (KO), ABCA1 KO, and ABCG1 KO mice received either rHDL or phosphate-buffered saline intravenously.

Macrophage reverse cholesterol transport in mice expressing ApoA-I Milano.

Objective: To compare the abilities of human wild-type apoA-I (WT apoA-I) and human apoA-I(Milano) (apoA-I(M)) to promote macrophage reverse cholesterol transport (RCT) in apoA-I-null mice infected with adeno-associated virus (AAV) expressing either WT apoA-I or apoA-I(M).

Methods And Results: WT apoA-I- or apoA-I(M)-expressing mice were intraperitoneally injected with [H(3)]cholesterol-labeled J774 mouse macrophages. After 48 hours, no significant difference was detected in the amount of cholesterol removed from the macrophages and deposited in the feces via the RCT pathway between the WT apoA-I and apoA-I(M) groups.

Lecithin: cholesterol acyltransferase expression has minimal effects on macrophage reverse cholesterol transport in vivo.

Background: Lecithin:cholesterol acyltransferase (LCAT) catalyzes the formation of plasma cholesteryl ester, plays a key role in high-density lipoprotein metabolism, and has been believed to be critical in the process of reverse cholesterol transport (RCT).

Methods And Results: The role of LCAT in RCT from macrophages was quantified with a validated assay involving intraperitoneal injection in mice of (3)H-cholesterol-labeled J774 macrophages and monitoring the appearance of tracer in plasma, liver, bile, and feces. Human LCAT overexpression in human apolipoprotein A-I transgenic mice substantially increased plasma high-density lipoprotein cholesterol levels but surprisingly did not increase macrophage RCT.

15(S)-Lipoxygenase-1 associates with neutral lipid droplets in macrophage foam cells: evidence of lipid droplet metabolism.

15(S)-lipoxygenase-1 (15-LO-1) was present in the whole-cell homogenate of an acute human monocytic leukemia cell line (THP-1). Additionally, 15-LO-1 was detected on neutral lipid droplets isolated from THP-1 foam cells. To investigate if 15-LO-1 is active on lipid droplets, we used the mouse leukemic monocytic macrophage cell line (RAW 264.

Overexpression of human 15(S)-lipoxygenase-1 in RAW macrophages leads to increased cholesterol mobilization and reverse cholesterol transport.

Objective: The purpose of this study was to determine the effect of 15-lipoxygenase-1 (15-LO-1) on cholesterol mobilization from macrophages.

Methods And Results: Overexpression of human 15-LO-1 in RAW mouse macrophages led to enhanced cholesterol efflux, increased cholesteryl ester (CE) hydrolysis, and increased reverse cholesterol transport (RCT). Efflux studies comparing 15-LO-1 overexpressing cells to mock-transfected RAW macrophages resulted in a 3- to 7-fold increase in cholesterol efflux to apolipoprotein A-I and a modest increase in efflux to HDL.

Inflammation impairs reverse cholesterol transport in vivo.

Background: Inflammation is proposed to impair reverse cholesterol transport (RCT), a major atheroprotective function of high-density lipoprotein (HDL). The present study presents the first integrated functional evidence that inflammation retards numerous components of RCT.

Methods And Results: We used subacute endotoxemia in the rodent macrophage-to-feces RCT model to assess the effects of inflammation on RCT in vivo and performed proof of concept experimental endotoxemia studies in humans.