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). Apolipoprotein B-depleted (high-density lipoprotein-fraction) serum from individuals with unfavorable lipids (median [interquartile range]; high-density lipoprotein-cholesterol=39 [32-42], low-density lipoprotein-cholesterol=109 [97-137], TGs=258 [184-335] mg/dL; n=13) promoted greater ATP-binding cassette transporter A1-mediated [1,2-(3H)] cholesterol efflux (3.8±0.3%/4 hour versus 1.2±0.4%/4 hour; P<0.0001) from cyclic 3',5'-amp(CTP-amp)-treated J774 macrophages than from individuals with favorable lipids (high-density lipoprotein-cholesterol=72 [58-88], low-density lipoprotein-cholesterol=111 [97-131], TGs=65 [56-69] mg/dL; n=10). Thus, high TGs associated with more ATP-binding cassette transporter A1 acceptors. Efflux of cholesterol mass (µg free cholesterol/mg cell protein per 8 hour) to serum was also higher (7.06±0.33 versus 5.83±0.48; P=0.04). However, whole sera from individuals with unfavorable lipids promoted more influx (5.14±0.65 versus 2.48±0.85; P=0.02) and lower net release of cholesterol mass (1.93±0.46 versus 3.36±0.47; P=0.04). The pattern differed when mass flux was measured using apolipoprotein B-depleted serum rather than serum. Although individuals with favorable lipids tended to have greater influx than those with unfavorable lipids, efflux to apolipoprotein B-depleted serum was markedly higher (6.81±0.04 versus 2.62±0.14; P<0.0001), resulting in an efflux:influx ratio of ≈3-fold. Thus both serum and apolipoprotein B-depleted serum from individuals with favorable lipids promoted greater net cholesterol mass release despite increased ATP-binding cassette transporter A1-mediated efflux in samples of individuals with high TGs/unfavorable lipids. CONCLUSIONS: When considering the efficiency of serum specimens to modulate cell cholesterol content, both influx and efflux need to be measured.

Mechanisms responsible for the compositional heterogeneity of nascent high density lipoprotein.

Apolipoprotein (apo) A-I-containing nascent HDL particles produced by the ATP binding cassette transporter A1 have different sizes and compositions. To understand the molecular basis for this heterogeneity, the HDL particles produced by apoA-I-mediated solubilization of phospholipid (PL)/free (unesterified) cholesterol (FC) bilayer membranes in cell and cell-free systems are compared. Incubation of apoA-I with ATP binding cassette transporter A1-expressing baby hamster kidney cells leads to formation of two populations of FC-containing discoidal nascent HDL particles. The larger 11-nm diameter particles are highly FC-enriched (FC/PL = 1.2/1 mol/mol) relative to the smaller 8 nm particles and the cell plasma membrane (FC/PL = 0.4/1). ApoA-I-mediated spontaneous solubilization of either multilamellar or unilamellar vesicles made of a membrane-PL mixture and FC yields discoidal HDL particles with diameters in the range 9-17 nm and, as found with the cell system, the larger particles are relatively enriched in FC despite the fact that all particles are created by solubilization of a common FC/PL membrane domain. The size-dependent distribution of FC among HDL particles is due to varying amounts of PL being sequestered in a boundary layer by interaction with apoA-I at the disc edge. The presence of a relatively large boundary layer in smaller discoidal HDL promotes preferential distribution of phosphatidylserine to such particles. However, phosphatidylcholine and sphingomyelin which are the primary PL constituents of nascent HDL do not exhibit selective incorporation into HDL discs of different sizes. This understanding of the mechanisms responsible for the heterogeneity in lipid composition of nascent HDL particles may provide a basis for selecting subspecies with preferred cardio-protective properties.

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. This task was accomplished by incubating radiolabeled J774 cells with increasing concentrations of lipoprotein-depleted sera (LPDS) or components present in serum as shuttles and a constant amount of LDL, small unilamellar vesicles, or red blood cells (RBC) as sinks. Synergistic efflux was measured as the difference in fractional efflux in excess of that predicted by the addition of the individual efflux values of sink and shuttle alone. Synergistic efflux was obtained when LPDS was incubated with cells and LDL. When different components of LPDS were used as shuttles, albumin produced synergistic efflux, while apoA-I did not. A synergistic effect was also obtained when RBC was used as the sink and albumin as shuttle. The previously observed negative association of albumin with coronary artery disease might be linked to reduced cholesterol shuttling that would occur when serum albumin levels are low.

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. To understand this role we employed cytochalasin D (cyt.D) to promote actin depolymerization in J774 macrophages. Incubating J774 with acetylated LDL creates foam cells having a 4-fold increase in cellular cholesterol content (30-40% cholesterol present as cholesteryl ester (CE)) in cytoplasmic droplets. Lipid droplets formed in the presence of cyt.D are smaller in diameter. CE-deposition and -hydrolysis are decreased when cells are cholesterol-enriched in the presence of cyt.D or latrunculin A, another cytoskeleton disrupting agent. However, when lipid droplets formed in the presence of cyt.D are isolated and incubated with an exogenous CE hydrolase, the CE is more rapidly metabolized compared to droplets from control cells. This is apparently due to the smaller size and altered lipid composition of the droplets formed in the presence of cyt.D. Cytoskeletal proteins found on CE droplets influence droplet lipid composition and maturation in model foam cells. In J774 macrophages, cytoskeletal proteins are apparently involved in facilitating the interaction of lipid droplets and a cytosolic neutral CE hydrolase and may play a role in foam cell formation. This article is part of a Special Issue entitled Advances in High Density Lipoprotein Formation and Metabolism: A Tribute to John F. Oram (1945-2010).

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. Using under- and overexpression approaches, we provide multiple lines of evidence that establish ES-4 as a bona fide endogenous nCEH that can account for the majority of cholesteryl ester hydrolysis in transformed rat hepatic cells and primary rat hepatocytes.

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. We have developed an in vivo assay to measure cholesterol mass changes in atherosclerotic foam cells. METHODS AND RESULTS: Macrophages are entrapped in semipermeable (pore size 0.2 µm) hollow fibers and surgically implanted into the peritoneum of recipient mice. The fibers are removed from the peritoneum 24 hours after implantation. This method allows the complete recovery of the macrophages for quantification of changes in cholesterol mass and cellular protein. In wild-type mice we measured a significant reduction in total cell cholesterol (TC) when hollow fibers containing cholesterol-enriched macrophage cells were implanted (TC before implantation=105±18 µg/mg cell protein, TC 24 hours after implantation=60±16 µg/mg protein). Additionally, there was an increase in cholesterol content when hollow fibers containing cholesterol-normal macrophages were implanted in an atherogenic mouse model (LDLr/apobec dko) compared to a wild-type mouse (initial TC content=57±24 µg/mg protein, TC 24 hours after implantation: wild-type mice=52±10 µg/mg protein; LDLr/apobec dko mice=118±27 µg/mg protein). CONCLUSIONS: This assay can quantify in vivo both cholesterol mass accumulation, and reduction, in macrophages. This method permits quantitative analysis of the progression and regression of foam cells.

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. We compared efflux using a fluorescent sterol (boron dipyrromethene difluoride linked to sterol carbon-24, BODIPY-cholesterol) with that of [(3)H]cholesterol in J774 macrophages. Fractional efflux of BODIPY-cholesterol was significantly higher than that of [(3)H]cholesterol when apo A-I, HDL(3), or 2% apoB-depleted human serum were used as acceptors. BODIPY-cholesterol efflux correlated significantly with [(3)H]cholesterol efflux (p < 0.0001) when apoB-depleted sera were used. The BODIPY-cholesterol efflux correlated significantly with preß-1 (r(2) = 0.6) but not with total HDL-cholesterol. Reproducibility of the BODIPY-cholesterol efflux assay was excellent between weeks (r(2) = 0.98, inter-assay CV = 3.31%). These studies demonstrate that BODIPY-cholesterol provides an efficient measurement of efflux compared with [(3)H]cholesterol and is a sensitive probe for ABCA1-mediated efflux. The increased sensitivity of BODIPY-cholesterol assay coupled with the simplicity of measuring fluorescence results in a sensitive, high-throughput assay that can screen large numbers of sera, and thus establish the relationship between cholesterol efflux and atherosclerosis.

Update on HDL receptors and cellular cholesterol transport.

Efflux is central to maintenance of tissue and whole body cholesterol homeostasis. The discovery of cell surface receptors that bind high-density lipoprotein (HDL) with high specificity and affinity to promote cholesterol release has significantly advanced our understanding of cholesterol efflux. We now know that 1) cells have several mechanisms to promote cholesterol release, including a passive mechanism that depends on the physico-chemical properties of cholesterol molecules and their interactions with phospholipids; 2) a variety of HDL particles can interact with receptors to promote cholesterol transport from tissues to the liver for excretion; and 3) interactions between HDL and receptors show functional synergy. Therefore, efflux efficiency depends both on the arrays of receptors on tissue cells and HDL particles in serum.

Aldosterone production in human adrenocortical cells is stimulated by high-density lipoprotein 2 (HDL2) through increased expression of aldosterone synthase (CYP11B2).

Adrenal aldosterone production is regulated by physiological agonists at the level of early and late rate-limiting steps. Numerous studies have focused on the role of lipoproteins including high-density lipoprotein (HDL) as cholesterol providers in this process; however, recent research suggests that HDL can also act as a signaling molecule. Herein, we used the human H295R adrenocortical cell model to study the effects of HDL on adrenal aldosterone production and CYP11B2 expression. HDL, especially HDL2, stimulated aldosterone synthesis by increasing expression of CYP11B2. HDL treatment increased CYP11B2 mRNA in both a concentration- and time-dependent manner, with a maximal 19-fold increase (24 h, 250 µg/ml of HDL). Effects of HDL on CYP11B2 were not additive with natural agonists including angiotensin II or K(+). HDL effects were likely mediated by a calcium signaling cascade, because a calcium channel blocker and a calmodulin kinase inhibitor abolished the CYP11B2-stimulating effects. Of the two subfractions of HDL, HDL2 was more potent than HDL3 in stimulating aldosterone and CYP11B2. Further studies are needed to identify the active components of HDL, which regulate aldosterone production.

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 3.5% apolipoprotein (apo) B depleted human serum, HDL(3), and apo A-I. Net cell-cholesterol influxes were observed when cholesterol-normal J774 cells were incubated with the same acceptors except apo A-I. When incubated with 2.5% individual sera, cholesterol mass efflux in free cholesterol (FC)-enriched J774 cells correlated with the HDL-cholesterol (HDL-C) concentrations (r(2) = 0.4; P=0.003), whereas cholesterol mass influx in cholesterol-normal J774 cells correlated with the LDL cholesterol (LDL-C) concentrations (r(2) = 0.6; P<0.0001) of the individual sera. A positive correlation was observed between measurements of [(3)H]cholesterol efflux and reductions in cholesterol mass (r(2) = 0.4; P=0.001) in FC-enriched J774 cells. In conclusion, isotopic efflux measurements from cholesterol-normal or cholesterol-enriched cells provide an accurate measurement of relative ability of an acceptor to remove labeled cholesterol under a specific set of experimental conditions, i.e., efflux potential. Moreover, isotopic efflux measurements can reflect changes in cellular cholesterol mass if the donor cells are enriched with cholesterol.

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. Analysis of the individual components of the RCT pathway demonstrated that the apoA-I(M)-expressing mice promoted ATP-binding cassette transporter A1 (ABCA1)-mediated cholesterol efflux as efficiently as WT apoA-I but that apoA-I(M) had a reduced ability to promote cholesterol esterification via lecithin cholesterol-acyltransferase (LCAT). This resulted in reduced cholesteryl ester (CE) and increased free cholesterol (FC) levels in the plasma of mice expressing apoA-I(M) compared to WT apoA-I. These differences did not affect the rate of delivery of labeled cholesterol to the liver via SR-BI-mediated selective uptake or its subsequent excretion in the feces. CONCLUSIONS: Within the limits of the in vivo assay, WT apoA-I and apoA-I(M) are equally efficient at promoting macrophage RCT, suggesting that if apoA-I(M) is more atheroprotective than WT apoA-I it is not attributable to an enhancement of 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 264.7), which are stably transfected with human 15-LO-1. The RAW 15-LO-1 cells were incubated with acetylated low density lipoprotein to generate foam cells. 15(S)-hydroxyeicosatetraenoic acid [15(S)-HETE], the major 15-LO-1 metabolite of arachidonic acid, was produced in the 15-LO-1 RAW but not in the mock transfected cells when incubated with arachidonic acid. Lipid droplets were isolated from the cells and incubated with arachidonic acid, and production of 15(S)-HETE was measured over 2 h. 15(S)-HETE was produced in the incubations with the lipid droplets, and this production was attenuated when the lipid droplet fraction was subjected to enzyme inactivation through heating. Efflux of 15(S)-HETE from cholesteryl ester-enriched 15-LO RAW cells, when lipid droplets are present, was significantly reduced compared with that from cells enriched with free cholesterol (lipid droplets are absent). We propose that 15-LO-1 is present and functional on cytoplasmic neutral lipid droplets in macrophage foam cells, and these droplets may act to accumulate the anti-inflammatory lipid mediator 15(S)-HETE.

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. Additional experiments revealed an increase in mRNA and protein levels of ABCA1 and ABCG1 in the RAW expressing 15-LO-1 compared to controls. Efforts to examine whether the arachidonic acid metabolite of 15-LO-1, (15S)-hydroxyeicosatetraenoic acid (HETE), was responsible for the enhanced efflux revealed this eicosanoid metabolite did not play a role. Enhanced steryl ester hydrolysis was observed in 15-LO-1 overexpressing cells suggesting that the CE produced in the 15-LO-1 expressing cells was readily mobilized. To measure RCT, RAW macrophages overexpressing 15-LO-1 or mock-transfected cells were cholesterol enriched by exposure to acetylated low-density lipoprotein and [(3)H]-cholesterol. These macrophages were injected into wild-type animals and RCT was measured as a percent of injected dose of (3)H appearing in the feces at 48 hours. We found 7% of the injected (3)H in the feces of mice that received macrophages overexpressing 15-LO-1 and 4% in the feces of mice that received mock-transfected cells. CONCLUSIONS: These data are consistent with a model in which overexpression of human 15-LO-1 in RAW macrophages promotes RCT through increased CE hydrolysis and ABCA1-mediated cholesterol efflux.

The role of reverse cholesterol transport in animals and humans and relationship to atherosclerosis.

Reverse cholesterol transport (RCT) is a term used to describe the efflux of excess cellular cholesterol from peripheral tissues and its return to the liver for excretion in the bile and ultimately the feces. It is believed to be a critical mechanism by which HDL exert a protective effect on the development of atherosclerosis. In this paradigm, cholesterol is effluxed from arterial macrophages to extracellular HDL-based acceptors through the action of transporters such as ABCA1 and ABCG1. After efflux to HDL, cholesterol may be esterified in the plasma by the enzyme lecithin:cholesterol acyltransferase and is ultimately transported from HDL to the liver, either directly via the scavenger receptor BI or after transfer to apolipoprotein B-containing lipoproteins by the cholesteryl ester transfer protein. Methods for assessing the integrated rate of macrophage RCT in animals have provided insights into the molecular regulation of the process and suggest that the dynamic rate of macrophage RCT is more strongly associated with atherosclerosis than the steady-state plasma concentration of HDL cholesterol. Promotion of macrophage RCT is a potential therapeutic approach to preventing or regressing atherosclerotic vascular disease, but robust measures of RCT in humans will be needed in order to confidently advance RCT-promoting therapies in clinical development.

Characterization and properties of pre beta-HDL particles formed by ABCA1-mediated cellular lipid efflux to apoA-I.

The contribution of ABCA1-mediated efflux of cellular phospholipid (PL) and cholesterol to human apolipoprotein A-I (apoA-I) to the formation of pre beta 1-HDL (or lipid-poor apoA-I) is not well defined. To explore this issue, we characterized the nascent HDL particles formed when lipid-free apoA-I was incubated with fibroblasts in which expression of the ABCA1 was upregulated. After a 2 h incubation, the extracellular medium contained small apoA-I/PL particles (pre beta 1-HDL; diameter = 7.5 +/- 0.4 nm). The pre beta 1-HDL (or lipid-poor apoA-I) particles contained a single apoA-I molecule and three to four PL molecules and one to two cholesterol molecules. An apoA-I variant lacking the C-terminal alpha-helix did not form such particles when incubated with the cell, indicating that this helix is critical for the formation of lipid-poor apoA-I particles. These pre beta 1-HDL particles were as effective as lipid-free apoA-I molecules in mediating both the efflux of cellular lipids via ABCA1 and the formation of larger, discoidal HDL particles. In conclusion, pre beta 1-HDL is both a product and a substrate in the ABCA1-mediated reaction to efflux cellular PL and cholesterol to apoA-I. A monomeric apoA-I molecule associated with three to four PL molecules (i.e., lipid-poor apoA-I) has similar properties to the lipid-free apoA-I molecule.

Wild-type ApoA-I and the Milano variant have similar abilities to stimulate cellular lipid mobilization and efflux.

OBJECTIVE: The present study is a comparative investigation of cellular lipid mobilization and efflux to lipid-free human apoA-I and apoA-I(Milano), reconstituted high-density lipoprotein (rHDL) particles containing these proteins and serum isolated from mice expressing human apoA-I or apoA-I(Milano). METHODS AND RESULTS: Cholesterol and phospholipid efflux to these acceptors was measured in cell systems designed to assess the contributions of ATP-binding cassette A1 (ABCA1), scavenger receptor type BI (SRBI), and cellular lipid content to cholesterol and phospholipid efflux. Acceptors containing the Milano variant of apoA-I showed no functional increase in lipid efflux in all assays when compared with wild-type apoA-I. In fact, in some systems, acceptors containing the Milano variant of apoA-I promoted significantly less efflux than the acceptors containing wild-type apoA-I (apoA-I(wt)). Additionally, intracellular cholesteryl ester hydrolysis in macrophage foam cells was not different in the presence of either apoA-I(Milano) or apoA-I(wt). CONCLUSION: Collectively these studies suggest that if the Milano variant of apoA-I offers greater atheroprotection than wild-type apoA-I, it is not attributable to greater cellular lipid mobilization.

Measurement of cholesterol bidirectional flux between cells and lipoproteins.

We developed an assay that quantitates bidirectional cholesterol flux between cells and lipoproteins. Incubating Fu5AH cells with increasing concentrations of human serum resulted in increased influx and efflux; however, influx was 2- to 3-fold greater at all serum concentrations. With apolipoprotein B (apoB)-depleted serum, the ratio of influx to efflux (I/E) was close to 1, indicating cholesterol exchange. The apoB fraction of serum induced influx and little efflux, with I/E > 1. Using block lipid transport-1 to block scavenger receptor class B type I (SR-BI)-mediated flux with different acceptors, we determined that 50% to 70% of efflux was via SR-BI. With HDL, 90% of influx was via SR-BI, whereas with LDL or serum, 20% of influx was SR-BI-mediated. Cholesterol-enriched hepatoma cells produced increased efflux without a change in influx, resulting in reduced I/E. The assay was applied to cholesterol-normal and -enriched mouse peritoneal macrophages exposed to serum or LDL. The enrichment enhanced efflux without shifts in influx. With cholesterol-enriched macrophages, HDL efflux was enhanced and influx was greatly reduced. With all lipoproteins, cholesterol enrichment of murine peritoneal macrophages led to a reduced I/E. We conclude that this assay can simultaneously and accurately quantitate cholesterol bidirectional flux and can be applied to a variety of cells exposed to isolated lipoproteins or serum.

Cytotoxic cellular cholesterol is selectively removed by apoA-I via ABCA1.

Excess intracellular free cholesterol (FC) is cytotoxic. This study examines prevention of FC-induced cytotoxicity in J774 macrophage foam cells by incubation with apolipoprotein AI (apoA-I). J774 were cholesterol enriched using acetylated low-density lipoprotein and FC/phospholipid (PL) dispersions. Treatment with an acyl coenzyme-A:cholesterol acyltransferase (ACAT) inhibitor, in the absence of extracellular acceptors, produced hydrolysis of stored esterified cholesterol (EC) and FC-induced cytotoxicity. Incubation of cells with ACAT inhibitor plus apoA-I resulted in FC efflux (0.39 +/- 0.02%/h) along with a reduction in cytotoxicity (26.30 +/- 5.80%), measured by adenine release. Small unilamellar vesicles (SUV) caused greater FC efflux (0.53 +/- 0.02%/h, P = 0.001), but a modest reduction in cytotoxicity (8.40 +/- 2.70%, P = 0.008). Co-incubation of ACAT inhibitor plus the cholesterol transport inhibitor U18666A or the antioxidant Probucol reduced efflux to apoA-I, but not to SUV. Pre-treatment of J774 foam cells with CTP-cAMP upregulates hormone sensitive lipase (HSL) and further upregulates ATP binding cassette A1 (ABCA1). Using mouse serum as a cholesterol acceptor, CTP-cAMP caused greater protection against FC-induced cytotoxicity compared to cells without pre-treatment, suggesting a role of ABCA1 in removal of cytotoxic FC. We conclude that a cytotoxic pool of FC is located in the plasma membrane, is readily available for efflux to apoA-I, and removal of cytotoxic cholesterol may involve ABCA1.

Enhanced efflux of cholesterol from ABCA1-expressing macrophages to serum from type IV hypertriglyceridemic subjects.

Since elevated plasma triglycerides (TGs) are an independent cardiovascular risk factor, we have compared the cholesterol efflux potential of sera from asymptomatic hypertriglyceridemic (HTG) type IIb, type IV or normolipidemic (NLP) individuals using two different cell systems. In both type IIb and IV HTG, the efflux of cholesterol from SR-BI-rich Fu5AH cells was similar to that obtained with NLP. The maintenance of efflux efficiency in spite of reduced HDL-cholesterol levels can be mainly attributed to the relative enrichment of HDL with phospholipid. In the J774 macrophage cell system, pretreatment with cAMP, which upregulates ABCA1, induced a markedly higher increase in efflux to type IV sera compared with type IIb or NLP. In addition, type IV sera exhibited two-fold higher pre-beta HDL relative concentration (percentage of total apo AI) compared with NLP. Moreover, positive correlations were established between ABCA1-mediated efflux and the serum pre-beta HDL levels or TG concentrations. Thus, the hyperTGemia is associated with a higher fraction of apo AI recovered as pre-beta HDL which appear to be partly responsible for enhanced efflux obtained upon the cAMP stimulation of J774 cells. In conclusion, we demonstrated for the first time that the ABCA1-expressing J774 cell system is responsive to the percent of apo AI present in human serum as pre-beta HDL. Our results suggest that high-plasma TG, accompanied by low HDL may not result in an impaired cholesterol efflux capacity.

Testing the role of apoA-I, HDL, and cholesterol efflux in the atheroprotective action of low-level apoE expression.

Low levels of transgenic mouse apolipoprotein E (apoE) suppress atherosclerosis in apoE knockout (apoE-/-) mice without normalizing plasma cholesterol. To test whether this is due to facilitation of cholesterol efflux from the vessel wall, we produced apoA-I-/-/apoE-/- mice with or without the transgene. Even without apoA-I and HDL, apoA-I-/-/apoE-/- mice had the same amount of aorta cholesteryl ester as apoE-/- mice. Low apoE in the apoA-I-/-/apoE-/- transgenic mice reduced aortic lesions by 70% versus their apoA-I-/-/apoE-/- siblings. To define the free cholesterol (FC) efflux capacity of lipoproteins from the various genotypes, sera were assayed on macrophages expressing ATP-binding cassette transporter A1 (ABCA1). Surprisingly, ABCA1 FC efflux was twice as high to sera from the apoA-I-/-/apoE-/- or apoE-/- mice compared with wild-type mice, and this activity correlated with serum apoA-IV. Immunodepletion of apoA-IV from apoA-I-/-/apoE-/- serum abolished ABCA1 FC efflux, indicating that apoAI-V serves as a potent acceptor for FC efflux via ABCA1. With increasing apoE expression, apoA-IV and FC acceptor capacity decreased, indicating a reciprocal relationship between plasma apoE and apoA-IV. Low plasma apoE (1-3 x 10(-8) M) suppresses atherosclerosis by as yet undefined mechanisms, not dependent on the presence of apoA-I or HDL or an increased capacity of serum acceptors for FC efflux.