ABCA1-mediated Cholesterol Efflux Research Articles

Abstract 389: Covalent Modifications of Apolipoproteins in High Density Lipoprotein by Oxidized Phospholipids Impair the Cholesterol Efflux Function of High Density Lipoprotein and are Detected in Hyperlipidemic Plasma

Oxidized phospholipids (oxPLs) accumulate at sites of oxidative stress and contribute significantly to atherosclerosis and thrombosis. Most oxPLs have electrophilic substituents and are highly likely to form covalent adducts with proteins, thus compromising protein function. Detection of covalent interactions between oxPLs and proteins could provide important information regarding the type of proteins preferentially modified by oxPLs. However, to date, such studies are extremely limited due to significant technical challenges. We now carry out systematic studies on the protein adduction by oxPLs formed in murine and human plasma. Plasma samples were exposed to a physiologically relevant myeloperoxidase/H 2 O 2 /NO 2 – oxidizing system of phagocytes. Protein adduction by the oxPLs generated in plasma was assessed using LC-MS/MS after tryptic digestion and peptide enrichment using a novel method that we developed. We found that HDL apolipoproteins are the major targets of modification by oxPLs in both murine and human plasma. For apoA-I, the most abundant apolipoprotein in HDL, the major modification sites of oxPLs were located in the region (AA144-186), which is critical for the ABCA1 mediated cholesterol efflux to HDL. We further demonstrated that human apoA-I was also heavily crosslinked by specific oxPLs via histidine and lysine residues located in the region (AA144-186) with apoA-I, or other apolipoproteins, including apoA-II, apoA-IV and apoC-I. In vitro experiments demonstrated that oxPLs modification on lipid free apoA-I or nascent HDL (HDL3) dramatically impairs their function as cholesterol efflux mediators. Using hyperlipidemic LDLr-/- mice, we detected a crosslink adduct of apoA-II with apoE by oxPL in murine plasma. To the best of our knowledge, this is the first report for the detection of endogenous protein adducts with oxPLs.

Poly(ADP-ribose) Polymerase 1 Represses Liver X Receptor-mediated ABCA1 Expression and Cholesterol Efflux in Macrophages

Liver X receptors (LXR) are oxysterol-activated nuclear receptors that play a central role in reverse cholesterol transport through up-regulation of ATP-binding cassette transporters (ABCA1 and ABCG1) that mediate cellular cholesterol efflux. Mouse models of atherosclerosis exhibit reduced atherosclerosis and enhanced regression of established plaques upon LXR activation. However, the coregulatory factors that affect LXR-dependent gene activation in macrophages remain to be elucidated. To identify novel regulators of LXR that modulate its activity, we used affinity purification and mass spectrometry to analyze nuclear LXRα complexes and identified poly(ADP-ribose) polymerase-1 (PARP-1) as an LXR-associated factor. In fact, PARP-1 interacted with both LXRα and LXRβ. Both depletion of PARP-1 and inhibition of PARP-1 activity augmented LXR ligand-induced ABCA1 expression in the RAW 264.7 macrophage line and primary bone marrow-derived macrophages but did not affect LXR-dependent expression of other target genes, ABCG1 and SREBP-1c. Chromatin immunoprecipitation experiments confirmed PARP-1 recruitment at the LXR response element in the promoter of the ABCA1 gene. Further, we demonstrated that LXR is poly(ADP-ribosyl)ated by PARP-1, a potential mechanism by which PARP-1 influences LXR function. Importantly, the PARP inhibitor 3-aminobenzamide enhanced macrophage ABCA1-mediated cholesterol efflux to the lipid-poor apolipoprotein AI. These findings shed light on the important role of PARP-1 on LXR-regulated lipid homeostasis. Understanding the interplay between PARP-1 and LXR may provide insights into developing novel therapeutics for treating atherosclerosis.

Abstract 391: Apolipoprotein E3, Apolipoprotein AI, and Apolipophorin III Chimeras Provide Insight Into the Domain Organization of Apolipoproteins

Apolipoproteins (apo) A-I and E are exchangeable apolipoproteins that play a dominant role in regulating lipoprotein metabolism. They are composed of a series of amphipathic α-helices, which are organized into an N-terminal (NT) helix bundle domain and a smaller C-terminal (CT) domain. The objective of the current study is to understand the functional and structural role of the domains of these proteins by “domain swapping” using apoE3 and apoAI, both of which bear a 4-helix bundle in their NT domain, and apolipophorin III (apoLp-III), a monomeric one-domain model apolipoprotein. A series of chimeric apolipoproteins were generate: apoE3-NT/apoAI-CT, apoAI-NT/apoE-CT and apoLp-III/apoA-I-CT. The α-helical content of the chimeras was comparable to that of the parent proteins. Unfolding studies indicated that addition of CT-apoE3 to NT-apoAI conferred more stability to apoAI. While addition of CT-apoAI to NT-apoE3 had no significant effect on the stability of apoE3, addition of NT-apoAI to apoLp-III increased the stability of apoLp-III. Moreover, apoLp-III switched from a monomeric to an oligomeric protein upon addition of CT-apoAI, showing that CT-apoAI has a strong tendency to self-associate. Addition of CT-apoAI to NT-apoE or apoLp-III increased the lipid binding activity by ~ 10-fold, while addition of apoE-CT to apoA-I-NT had no measurable effect on the lipid binding activity. All three chimeras promote ABCA1-mediated cholesterol efflux from J774 macrophages, with the efflux capability being highest for apoAI-NT/apoE-CT. Whereas apoE3-NT/apoAI-CT elicits LDLr binding ability, apoAI-NT/apoE-CT did not. These results suggest that CT of apoAI is a strong promoter of lipid binding, while CT of apoE3 can improve cholesterol efflux ability of apoAI.

Abstract 28: Adiponectin Stimulates Cholesterol Efflux Efficiently in Human THP-1 Macrophages and Modulates HDL-apoA-I Biogenesis

Introduction: Adiponectin (APN) is an anti-inflammatory and anti-atherogenic adipokine that is strongly correlated with circulating HDL levels. However, its role in macrophage lipid metabolism, a crucial process in atherogenesis, remains poorly investigated. We examined the effect of APN on cholesterol efflux from human THP-1 macrophages, elucidated its kinetics, and investigated its role in HDL biogenesis. Methods: APN dose-dependent (0.1 to 60 μM) and time-dependent (0.5 to 24 hours) cholesterol efflux studies were performed in 3 [H]-cholesterol labeled human THP-1 macrophages in the presence of apoA-I. Following efflux studies, the HDL fractions within media were concentrated (10kDa cut-off filter) and subjected to analytical FPLC and 2D-PAGGE technique to reveal HDL species. Results: APN stimulated ABCA1-mediated cholesterol efflux in a dose-dependent and time-dependent manner. Kinetics analysis revealed that increased molar doses of APN and apoA-I had similar Km efficiency of cholesterol efflux but greater velocity ( Km =3.24±0.71 μM, Vmax =4.90±0.07 efflux/6h) when compared to apoA-I alone ( Km =3.33±0.57 μM, Vmax =3.83±0.24 efflux/6h). Importantly, once APN was tested against a fixed dose of apoA-I (10 μg/mL), it promoted cholesterol efflux with Km = 0.17±0.06 μM. This was associated with a 75.7% decrease in intracellular free cholesterol in THP-1 cells in the presence of APN and apoA-I when compared to apoA-I alone (P<0.01). APN alone had no effect on the level of residual efflux (reached a level of 1%). The FPLC cholesterol profiles demonstrated that in the presence of APN and apoA-I there was increased lipidated nascent HDL (nHDL) during the process of cholesterol efflux, compared to apoA-I alone. This was associated with increased size of nHDL-apoA-1 pre-β and α species via 2D-PAGGE analyses. By immunoblotting for apoA-I and APN, APN oligomers exhibited a molecular weight range of 9 to 20 nm, appearing within the size range of nHDL-apoA-I. Conclusion: In addition to promoting macrophage cholesterol efflux in vitro , APN can modulate HDL-apoA-I biogenesis, by increasing the generation of nHDL particles. These findings suggest that APN may be of potential therapeutic value in the modulation of HDL’s protective role in atherosclerosis.

Lactobacillus acidophilus K301 Inhibits Atherogenesis via Induction of 24 (S), 25-Epoxycholesterol-Mediated ABCA1 and ABCG1 Production and Cholesterol Efflux in Macrophages.

Lactobacillus acidophilus species are well-known probiotics with the beneficial activity of regulating cholesterol levels. In this study, we showed that L. acidophilus K301 reduced the level of cholesterol through reverse transport in macrophages. L. acidophilus K301 upregulated the mRNA and protein levels of genes such as ATP-binding cassette A1 (ABCA1) and ATP-binding cassette G1 (ABCG1) under the control of liver X receptor (LXR), resulting in increased apoA-I-dependent cholesterol efflux in phorbol 12-myristate 13-acetate (PMA)-differentiated THP-1 cells. L. acidophilus K301 induced both ABCA1 and ABCG1 through the endogenous LXR agonist 24(S), 25-epoxcycholesterol, which is synthesized by intracellular cholesterol synthetic pathways. In vivo studies using L. acidophilus K301-treated ApoE-/- mice showed reduced accumulation of lipoproteins in the arterial lumen. The inhibitory effects of L. acidophilus K301 on accumulation of lipoprotein in atherosclerotic plaques were mediated by the induction of squalene reductase (SQLE) and oxidosqualene cyclase (OSC) and resulted in ABCA1-mediated cholesterol efflux. Taken together, our findings revealed that Lactobacillus acidophilus K301 regulates the expression of genes related to cholesterol reverse transport via the induction of endogenous LXR agonist, suggesting the therapeutic potential of Lactobacillus acidophilus K301 as an anti-atherosclerotic agent.

IL-19 Halts Progression of Atherosclerotic Plaque, Polarizes, and Increases Cholesterol Uptake and Efflux in Macrophages

Atherosclerosis regression is an important clinical goal, and treatments that can reverse atherosclerotic plaque formation are actively being sought. Our aim was to determine whether administration of exogenous IL-19, a Th2 cytokine, could attenuate progression of preformed atherosclerotic plaque and to identify molecular mechanisms. LDLR(-/-) mice were fed a Western diet for 12 weeks, then administered rIL-19 or phosphate-buffered saline concomitant with Western diet for an additional 8 weeks. Analysis of atherosclerosis burden showed that IL-19-treated mice were similar to baseline, in contrast to control mice which showed a 54% increase in plaque, suggesting that IL-19 halted the progression of atherosclerosis. Plaque characterization showed that IL-19-treated mice had key features of atherosclerosis regression, including a reduction in macrophage content and an enrichment in markers of M2 macrophages. Mechanistic studies revealed that IL-19 promotes the activation of key pathways leading to M2 macrophage polarization, including STAT3, STAT6, Kruppel-like factor 4, and peroxisome proliferator-activated receptor γ, and can reduce cytokine-induced inflammation invivo. We identified a novel role forIL-19 in regulating macrophage lipid metabolism through peroxisome proliferator-activated receptor γ-dependent regulation of scavenger receptor-mediated cholesterol uptake and ABCA1-mediated cholesterol efflux. These data show that IL-19 can halt progression of preformed atherosclerotic plaques by regulating both macrophage inflammation and cholesterol homeostasis and implicate IL-19 as a link between inflammation and macrophage cholesterol metabolism.

Eicosapentaenoic acid membrane incorporation impairs ABCA1-dependent cholesterol efflux via a protein kinase A signaling pathway in primary human macrophages

A diet rich in n-3/n-6 polyunsaturated fatty acids (PUFAs) is cardioprotective. Dietary PUFAs affect the cellular phospholipids composition, which may influence the function of membrane proteins. We investigated the impact of the membrane incorporation of several PUFAs on ABCA1-mediated cholesterol efflux, a key antiatherogenic pathway. Arachidonic acid (AA) (C20:4 n-6) and docosahexaenoic acid (DHA) (C22:6 n-3) decreased or increased cholesterol efflux from J774 mouse macrophages, respectively, whereas they had no effect on efflux from human monocyte-derived macrophages (HMDM). Importantly, eicosapentaenoic acid (EPA) (C20:5 n-3) induced a dose-dependent reduction of ABCA1 functionality in both cellular models (−28% for 70μM of EPA in HMDM), without any alterations in ABCA1 expression. These results show that PUFA membrane incorporation does not have the same consequences on cholesterol efflux from mouse and human macrophages. The EPA-treated HMDM exhibited strong phospholipid composition changes, with high levels of both EPA and its elongation product docosapentaenoic acid (DPA) (C22:5 n-3), which is associated with a decreased level of AA. In HMDM, EPA reduced the ATPase activity of the membrane transporter. Moreover, the activation of adenylate cyclase by forskolin and the inhibition of cAMP phosphodiesterase by isobutylmethylxanthine restored ABCA1 cholesterol efflux in EPA-treated human macrophages. In conclusion, EPA membrane incorporation reduces ABCA1 functionality in mouse macrophages as well as in primary human macrophages and this effect seems to be PKA-dependent in human macrophages.

Cystathionine γ-lyase(CSE)/hydrogen sulfide system is regulated by miR-216a and influences cholesterol efflux in macrophages via the PI3K/AKT/ABCA1 pathway

This study was designed to evaluate whether CSE/H2S system, which is regulated by miR-216a, regulated ABCA1-mediated cholesterol efflux and cholesterol contents in THP-1 macrophages-derived foam cells. Our qPCR and western blotting results showed that CSE/H2S significantly up-regulated the expression of ATP-binding cassette transporter A1 (ABCA1) mRNA and protein via PI3K/AKT pathway in foam cells derived from human THP-1 macrophages. The miR-216a directly targeted 3′ untranslated region of CSE. It significantly reduced CSE and ABCA1 expression, and also decreased the phosphorylation of PI3K and AKT. Additionally, cholesterol efflux decreased, and cholesterol levels increased in THP-1 macrophage-derived foam cells in response to treatment with miR-216a. Our study demonstrates that CSE/H2S system is regulated by miR-216a, and regulates ABCA1-mediated cholesterol efflux and cholesterol levels through the PI3K/AKT pathway.

Pleiotropic effects of antitumour alkylphospholipids on cholesterol transport and metabolism

BackgroundAlkylphospholipid (APL) analogs are a new class of membrane-directed synthetic compounds with a variety of biological actions and clinical applications. In particular, these agents are promising candidates in cancer treatment. We have demonstrated that after prolonged treatment APLs alter intracellular cholesterol traffic and metabolism in human tumor-cell lines, leading to an accumulation of cholesterol inside the cell. After further investigation concerning the mode of action of APLs, we have explored the influence of several APLs on novel aspects of cholesterol and lipoprotein homeostasis using hepatoma HepG2 cells and THP1-derived macrophages. MethodsQuantitative real-time PCR analysis with a pathway-focused PCR array system was performed to measure relative changes in the mRNA expression of a number of genes related to cholesterol transport and metabolism. We compared the gene-expression profiles of HepG2 cells treated with miltefosine, edelfosine or perifosine for 6h and 24h with the profile of control cells. We also analysed particular genes of interest in both HepG2 and macrophage-like THP1 cells using specific PCR assays. Immunoblots were used to confirm protein-expression changes. Measurement of ABCA1-mediated cholesterol efflux was determined using apoA1 as cholesterol acceptor. ResultsWe found global changes in gene-expression patterns to maintain cholesterol homeostasis after exposure of cells to APLs. The pathways for cholesterol biosynthesis and LDL-cholesterol uptake were both transcriptionally upregulated by the three APLs assayed. Conversely, major pathways involved in the catabolism of cholesterol to bile acids and lipoprotein-associated cholesterol export were impaired after APL incubation, which may well contribute to the higher cell-cholesterol levels induced by these compounds. ConclusionIncubation of cells with different APLs stimulated cholesterol biosynthesis and uptake at the same time as it depressed common pathways for excess cholesterol removal in tumor cells, ultimately leading to altered cholesterol homeostasis.

A single infusion of MDCO-216 (ApoA-1 Milano/POPC) increases ABCA1-mediated cholesterol efflux and pre-beta 1 HDL in healthy volunteers and patients with stable coronary artery disease.

AimsApolipoprotein A-1 (ApoA-1), based on epidemiology, is inversely associated with cardiovascular (CV) events. Human carriers of the ApoA-1 Milano variant have a reduced incidence of CV disease. Regression of atherosclerotic plaque burden was previously observed on intravascular ultrasound (IVUS) with ETC-216, a predecessor of MDCO-216. MDCO-216, a complex of dimeric ApoA-1 Milano and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, is being developed to reduce atherosclerotic plaque burden and CV events. We investigated the efficacy and safety of a single infusion of MDCO-216 in healthy volunteers and in patients with coronary artery disease (CAD).Methods and resultsTwenty-four healthy volunteers and 24 patients with documented CAD received a 2-h infusion of MDCO-216 in a randomized, placebo controlled, single ascending dose study. Five cohorts of healthy volunteers and four cohorts of CAD patients received ApoA-1 Milano doses ranging from 5 to 40 mg/kg. Subjects were followed for 30 days. Dose-dependent increases in ApoA-1, phospholipid, and pre-beta 1 HDL and decreases in ApoE were observed. Prominent and sustained increases in triglyceride, and decreases in HDL-C, endogenous ApoA-1 and ApoA-II occurred at doses >20 mg/kg and profound increases in ABCA1-mediated cholesterol efflux were observed. Other lipid and lipoprotein parameters were generally unchanged. MDCO-216 was well tolerated.ConclusionsMDCO-216-modulated lipid parameters profoundly increased ABCA1-mediated cholesterol efflux and was well tolerated. These single-dose data support further development of this agent for reducing atherosclerotic disease and subsequent CV events.

LXR-Mediated ABCA1 Expression and Function Are Modulated by High Glucose and PRMT2.

High cholesterol and diabetes are major risk factors for atherosclerosis. Regression of atherosclerosis is mediated in part by the Liver X Receptor (LXR) through the induction of genes involved in cholesterol transport and efflux. In the context of diabetes, regression of atherosclerosis is impaired. We proposed that changes in glucose levels modulate LXR-dependent gene expression. Using a mouse macrophage cell line (RAW 264.7) and primary bone marrow derived macrophages (BMDMs) cultured in normal or diabetes relevant high glucose conditions we found that high glucose inhibits the LXR-dependent expression of ATP-binding cassette transporter A1 (ABCA1), but not ABCG1. To probe for this mechanism, we surveyed the expression of a host of chromatin-modifying enzymes and found that Protein Arginine Methyltransferase 2 (PRMT2) was reduced in high compared to normal glucose conditions. Importantly, ABCA1 expression and ABCA1-mediated cholesterol efflux were reduced in Prmt2 -/- compared to wild type BMDMs. Monocytes from diabetic mice also showed decreased expression of Prmt2 compared to non-diabetic counterparts. Thus, PRMT2 represents a glucose-sensitive factor that plays a role in LXR-mediated ABCA1-dependent cholesterol efflux and lends insight to the presence of increased atherosclerosis in diabetic patients.

Increased cholesterol efflux capacity in metabolic syndrome: Relation with qualitative alterations in HDL and LCAT.

Metabolic syndrome (MetS) is associated with changes in HDL levels, composition and sub-fraction profile. Whether these alterations affect HDL anti-atherogenic function, specifically measured as its capacity to perform cholesterol efflux, is not yet clearly known. To evaluate the relation between serum cholesterol efflux capacity and the changes in HDL composition and sub-fraction profile in MetS. In 35 non-treated MetS patients and 15 healthy controls, HDL mediated cholesterol efflux was measured as the ability of apoB-depleted serum to accept cholesterol from cholesterol-loaded BHK cells expressing either ABCA1 or ABCG1. Additionally we determined: lipid profile, HDL sub-fractions (NMR) and LCAT mass (ELISA). Isolated HDL (δ:1.063-1.210 g/mL) was chemically characterized. Pre-β1-HDL was determined by 2D-electrophoresis in a sub-group of MetS and controls (n = 6 each). Surprisingly, MetS patients presented higher ABCA1 mediated cholesterol efflux (10.4 ± 1.8 vs. 8.7 ± 0.3%; p = 0.0001), without differences in ABCG1 efflux. In MetS, HDL showed reduction in particle size and number (p < 0.02) and lower large/small HDL ratio (p = 0.05), as well as triglyceride enrichment (p = 0.0001). Pre-β1-HDL was increased in MetS (p = 0.048) and correlated with ABCA1-cholesterol efflux (r = 0.64; p = 0.042). LCAT mass showed a tendency to reduction in MetS (p = 0.08), and inversely correlated with ABCA1-cholesterol efflux (r = -0.51; p = 0.001), independently of obesity and insulin-resistance (β = -0.40, p = 0.034). This is the first description of ABCA1 mediated cholesterol efflux in MetS. Regardless the reduced HDL-cholesterol, in vitro cholesterol efflux capacity by ABCA1 was enhanced, linked to increased pre-β1-HDL and slightly reduced in LCAT mass that would probably reflect a delay in reverse cholesterol transport occurring in MetS.

Association between the ABCA1-565C/T gene promoter polymorphism and coronary heart disease severity and cholesterol efflux in the Chinese Han population.

ABCA1 -565C/T gene promoter variants have been associated with the severity of coronary artery disease in Western populations. The purpose of our study was to investigate the association between the -565C/T gene polymorphism and coronary artery disease severity and cholesterol efflux in the Chinese Han population. A cohort of 298 acute coronary syndrome (ACS) patients and 541 healthy controls was genotyped using the highly sensitive ligase detection reaction. ABCA1 -565C/T genotype was correlated with the clinical features of 164 acute myocardial infarction (AMI) patients. Monocytes from patients with various -565C/T gene polymorphisms were isolated and differentiated into foam cells by coincubation with [(3)H]-labeled acetyl-low-density lipoprotein cholesterol. ABCA1 mRNA and protein expression levels were evaluated, as well as cellular cholesterol efflux. The frequency of the TT genotype in the -565C/T polymorphism of ACS patients was significantly increased when compared with controls (0.211 vs. 0.162, p<0.05). The TT genotype, but not the CT or CC genotypes, in the -565C/T gene polymorphism correlated with the severity of the coronary lesion observed in AMI patients. Patients with the TT homozygote genotype also exhibited significantly lower cellular cholesterol efflux (TT [6.37%±0.554%]) levels than controls and also had the lowest levels of ABCA1 mRNA and protein expression among the group of variants. In contrast, cholesterol efflux levels in AMI patients with CT [11.35%±3.975%] and CC ([15.32%±6.293%]) genotypes were not significantly different from controls. Impaired ABCA1-mediated cholesterol efflux in macrophages may be associated with the severity of the coronary lesions in AMI patients with the TT genotype at the -565C/T gene polymorphism.

Macrophage Mitochondrial Energy Status Regulates Cholesterol Efflux and Is Enhanced by Anti-miR33 in Atherosclerosis

Therapeutically targeting macrophage reverse cholesterol transport is a promising approach to treat atherosclerosis. Macrophage energy metabolism can significantly influence macrophage phenotype, but how this is controlled in foam cells is not known. Bioinformatic pathway analysis predicts that miR-33 represses a cluster of genes controlling cellular energy metabolism that may be important in macrophage cholesterol efflux. We hypothesized that cellular energy status can influence cholesterol efflux from macrophages, and that miR-33 reduces cholesterol efflux via repression of mitochondrial energy metabolism pathways. In this study, we demonstrated that macrophage cholesterol efflux is regulated by mitochondrial ATP production, and that miR-33 controls a network of genes that synchronize mitochondrial function. Inhibition of mitochondrial ATP synthase markedly reduces macrophage cholesterol efflux capacity, and anti-miR33 required fully functional mitochondria to enhance ABCA1-mediated cholesterol efflux. Specifically, anti-miR33 derepressed the novel target genes PGC-1α, PDK4, and SLC25A25 and boosted mitochondrial respiration and production of ATP. Treatment of atherosclerotic Apoe(-/-) mice with anti-miR33 oligonucleotides reduced aortic sinus lesion area compared with controls, despite no changes in high-density lipoprotein cholesterol or other circulating lipids. Expression of miR-33a/b was markedly increased in human carotid atherosclerotic plaques compared with normal arteries, and there was a concomitant decrease in mitochondrial regulatory genes PGC-1α, SLC25A25, NRF1, and TFAM, suggesting these genes are associated with advanced atherosclerosis in humans. This study demonstrates that anti-miR33 therapy derepresses genes that enhance mitochondrial respiration and ATP production, which in conjunction with increased ABCA1 expression, works to promote macrophage cholesterol efflux and reduce atherosclerosis.

Abstract 473: Development of High-throughput ABCA1, ABCG1 and SR-BI Mediated HDL Efflux Assays

Studies have shown an inverse association between HDL-efflux capacity and cardiovascular disease (CVD). The efflux of cholesterol from cells to HDL can occur by several mechanisms, including unmediated aqueous diffusion and ABCA1, ABCG1 and SR-BI specifically mediated process. We developed a high throughput assay to measure pathway specific HDL efflux capacity using BHK cells expressing inducible ABCA1, ABCG1, or SR-BI in 96-well plate format. We found that ABCA1-mediated cholesterol efflux in BHK cells correlated significantly with that of J774 macrophages when apoA-I, HDL or 2% apoB-depleted serum were used as acceptors (R 2 =0.98 to 0.99, p&lt;0.0001). Compared with J774 macrophages, ABCA1-mediated efflux using BHK cells is more sensitive, consistent and has better reproducibility (Intro-assay CV=3.0%, R 2 =0.99; Inter assay CV=3.2%, R 2 =0.98). The consistence and reproducibility for ABCG1 and SR-BI mediated cholesterol efflux were also excellent as judged by intro-assay and inter-assay CV and R 2 . When pathway specific assay was employed in a high-throughput format to measure HDL efflux capacity among subjects with or without CVD, we found that ABCA1-mediated cholesterol efflux was significantly lower in CVD subjects when compared with that of control (p&lt;0.01), while ABCG1 or SR-BI mediated cholesterol efflux was not significantly different among the two groups. ABCG1-mediated cholesterol efflux was significantly associated with HDL-C (R 2= 0.65, p&lt;0.01), but ABCA1 or SR-BI mediated cholesterol efflux was not correlated with HDL-C. Those studies demonstrate that BHK cells with inducible ABCA1, ABCG1, or SR-BI expression provide a sensitive, reproducible high throughput efflux assay that can be used to screen large number of serums in mechanistic studies or drug discovery program.

Abstract 323: ABCA1 Expression Promotes ApoAI Acidification on the Plasma Membrane via Recruitment of Vacuolar ATPase

Objective: We have shown that ABCA1 mediates unfolding of apoAI N-terminal helical hairpin on the cell surface. Others have shown that apoAI can solubilize liposomes made with egg PC:PS (8:2) only at acidic pH, where apoAI has unfolded to expose more hydrophobic surfaces. We postulated that ABCA1 may mediate the local acidification of the plasma membrane to promote apoAI unfolding and lipidation. Methods and Results: We found that apoAI unfolding in guanidine was facilitated at pH 5.0 vs. pH 7.5 (EC50 = 0.75 M and 0.94 M guanidine, respectively). We confirmed that apoAI solubilization of and binding to POPC:PS liposomes was only effective at pH &lt; 5.5. We then designed a fluorescent ratiometric apoAI pH indicator labeling apoAI with FITC and Alexa647, and validated it in solution assays showing that the FITC/Alexa647 ratio decreased as the pH decreased. Using flow cytometry in both RAW264.7 and BHK cell lines, we found that incubation of the apoAI pH indicator with ABCA1 expressing cells at 37 o C led to a large acidification of apoAI, which may be due to the uptake of apoAI into endosomes/lysosomes. However, incubation of the apoAI pH indicator with ABCA1 expressing cells at 21 o C or with an inhibitor of endocytosis, where the vast majority of apoAI is on the cell surface, still led to mild apoAI acidification. To elucidate the mechanism of ABCA1 effects on local acidification of the plasma membrane, we examined if ABCA1 regulates vacuolar ATPase (V-ATPase) proton pump, which is composed of a peripheral V 1 domain and an embedded V 0 domain. We observed that bafilomycin, the V-ATPase inhibitor, dose dependently inhibited ABCA1 mediated cholesterol efflux to apoAI, and also eliminated the pH shift of apoAI on plasma membrane. We showed V-ATPase was expressed on the cell surface by cell surface biotinylation. Although the total level of V 0 A1 subunit didn’t change in ABCA1 expressing cells, ABCA1 induced the cell surface level of the V 0 A1 subunit. Conclusion: Our results support that ABCA1 induces the levels of V-ATPase on the cell surface leading to local acidification of apoAI, which may promote apoAI unfolding and lipidation.

Abstract 319: Design of Novel Chimeras to Provide Insight Into Structure/Function Activity of Apolipoprotein E3 and Apolipoprotein AI

Apolipoprotein (apo) E3 and apoAI are exchangeable apolipoproteins that play a dominant role in regulating plasma cholesterol levels and are considered anti-atherogenic. ApoE3 (299 residues, ~34 kDa) serves as a ligand for the low density lipoprotein receptor (LDLr) family of proteins that mediate cellular uptake and clearance of plasma lipoproteins. The ability of apoE3 to function as a ligand for LDLr resides in the N-terminal domain (NT) of apoE3. ApoAI (243 residues, ~28 kDa) lowers cholesterol levels through its ability to promote cholesterol efflux from peripheral tissues such as macrophages, forming HDL, which transports cholesterol back to the liver. Residues located in the C-terminal domain (CT) of apoAI mediate this function of promoting cholesterol efflux. The objective of this study is to generate chimeric apolipoproteins composed of apoE3 and apoAI to better understand the role of the C-terminal tail of the parent proteins. Two chimeras were generated by “domain swapping”: apoE3-NT/apoAI-CT and apoAI-NT/apoE-CT. They were overexpressed in E. coli, isolated and purified. Western blot analysis revealed the presence of epitopes from both parent proteins. Circular dichroism spectroscopy revealed that the α-helical content of apoE3-NT/apoAI-CT and apoAI-NT/E-CT was 43 and 54 %, respectively, which is comparable to that of the parent proteins. Chemical denaturation was used to probe for changes in protein stability. Addition of CT of apoE3 to NT of apoAI conferred more stability to apoAI, while addition of CT of apoAI to NT of apoE3 had no significant effect on apoE3. The rate of apolipoprotein-induced conversion of DMPC vesicles to discoidal structures was followed to assess their lipid binding capability. ApoE3-NT/AI-CT displayed a rate constant that was ~10x higher compared to apoE3, while apoAI-NT/apoE-CT showed a rate constant similar to that of apoAI. Whereas apoE3-NT/apoAI-CT elicits the ability to bind to the LDLr, apoAI-NT/apoE-CT did not. Lastly, both chimeras promote ABCA1-mediated cholesterol efflux from J774 macrophages, with the efflux capability being 2-fold greater for apoAI-NT/apoE-CT. These results show that CT of apoAI can promote lipid binding of apoE3, while CT of apoE3 can improve cholesterol efflux ability of apoAI.

The implication of cigarette smoking and cessation on macrophage cholesterol efflux in coronary artery disease patients

We investigated ATP-binding cassette transporters A1/G1 expression and function in mediating cholesterol efflux by examining the macrophages of cigarette-smoking patients with coronary artery disease (CAD) before and after smoking abstinence. Peripheral blood monocyte cells were collected from nonsmokers (n = 17), non-CAD (NCAD) smokers (n = 35), and CAD smokers (n = 32) before and after 3 months of smoking cessation. We found that the ABCA1 expression level was lower in macrophages from NCAD and CAD smokers than from nonsmokers at baseline. The ABCA1 function of mediating cholesterol efflux was reduced in NCAD and CAD smokers as compared with nonsmokers. After 3 months of smoking cessation, ABCA1 expression and function were improved in CAD smokers. However, ABCG1 expression and function did not change after smoking cessation. Furthermore, ABCA1 expression was inhibited by tar in human acute monocytic leukemia cell line THP-1-derived macrophages through the inhibition of liver X receptors. Nicotine and carbon monoxide did not inhibit ABCA1 expression. Our results indicate that chronic cigarette smoking impaired ABCA1-mediated cholesterol efflux in macrophages and that tobacco abstinence reversed the function and expression of ABCA1, especially in CAD patients. It was tobacco tar, rather than nicotine or carbon monoxide, that played a major role in the tobacco-induced disturbance of cellular cholesterol homeostasis.

Inhibition of soluble epoxide hydrolase in mice promotes reverse cholesterol transport and regression of atherosclerosis

Adipose tissue is the body largest free cholesterol reservoir and abundantly expresses ATP binding cassette transporter A1 (ABCA1), which maintains plasma high-density lipoprotein (HDL) levels. HDLs have a protective role in atherosclerosis by mediating reverse cholesterol transport (RCT). Soluble epoxide hydrolase (sEH) is a cytosolic enzyme whose inhibition has various beneficial effects on cardiovascular disease. The sEH is highly expressed in adipocytes, and it converts epoxyeicosatrienoic acids (EETs) into less bioactive dihydroxyeicosatrienoic acids. We previously showed that increasing EETs levels with a sEH inhibitor (sEHI) (t-AUCB) resulted in elevated ABCA1 expression and promoted ABCA1-mediated cholesterol efflux from 3T3-L1 adipocytes. The present study investigates the impacts of t-AUCB in mice deficient for the low density lipoprotein (LDL) receptor (Ldlr−/− mice) with established atherosclerotic plaques. The sEH inhibitor delivered in vivo for 4 weeks decreased the activity of sEH in adipose tissue, enhanced ABCA1 expression and cholesterol efflux from adipose depots, and consequently increased HDL levels. Furthermore, t-AUCB enhanced RCT to the plasma, liver, bile and feces. It also showed the reduction of plasma LDL-C levels. Consistently, t-AUCB–treated mice showed reductions in the size of atherosclerotic plaques. These studies establish that raising adipose ABCA1 expression, cholesterol efflux, and plasma HDL levels with t-AUCB treatment promotes RCT, decreasing LDL-C and atherosclerosis regression, suggesting that sEH inhibition may be a promising strategy to treat atherosclerotic vascular disease.

Statin-induced decrease in ATP-binding cassette transporter A1 expression via microRNA33 induction may counteract cholesterol efflux to high-density lipoprotein.

Cholesterol efflux from macrophages to HDL, measured in vitro, is augmented by treatment with agents which raise HDL cholesterol. In vitro, cholesterol depletion by statins is known to trigger a positive feedback on the cholesterol synthetic pathway via sterol regulatory element-binding protein (SREBP) transcription and changes in expression of SREBP regulated genes including microRNA33 (miR33) which is co-transcribed with SREBPand down-regulates ABCA1 and ABCG1 expression. We investigated whether miR33 up-regulation, associated with SREBP increased transcription by statins, reduces macrophage ATP-binding cassette (ABC) transporter expression, thereby decreasing HDL-mediated cholesterol efflux at the tissue level. In human macrophage THP-1 cells cholesterol-loaded with acetylated LDL, incubation with 1μM atorvastatin increased miR33 by 33% (P < 0.05), and decreased ABCA1 messenger RNA (mRNA) and ABCG1 mRNA by 47% (P < 0.05) and 27% (NS), respectively. InJ774A.1mouse macrophage, labelled with 3H-cholesterol, ABCA1 mRNA and ABCA1-mediated cholesterol efflux were decreased by 1μM statin: simvastatin > pitavastatin > atorvastatin > rosuvastatin > pravastatin. HDL incubated with rhCETP and dalcetrapib increased ABCA1-mediated cholesterol efflux. However, incremental simvastatin concentrations decreased cholesterol efflux to HDL treated with rhCETP and dalcetrapib. When HDL was incubated with rhCETP, addition of dalcetrapib augmented ABCA1-mediated cholesterol efflux from J774A.1 macrophages. However, simvastatin ≥1μM virtually eliminated any HDL-ABCA1-mediated cholesterol efflux and any augmentation of that process by dalcetrapib. In vitro, statins increase miR33 expression, and decrease ABCA1 expression and cholesterol efflux from peripheral tissues; this may counteract the potential benefit of agents that raise HDL and apolipoprotein A-I in statin-treated patients.