Cellular Cholesterol Efflux Research Articles

CETP Inhibition in CVD Prevention: an Actual Appraisal.

By virtue of their effects on low-density lipoprotein cholesterol, high-density lipoprotein cholesterol, and cellular cholesterol efflux, there is considerable interest in the potential use of pharmacological inhibitors of cholesteryl ester transfer protein (CETP) as a novel approach for cardiovascular disease prevention. This is supported by observations from genetic and animal studies suggesting that less CETP activity has favorable cardiovascular effects. Despite the adverse effects of the first CETP inhibitor to move forward in clinical development, torcetrapib, there remains considerable interest in developing alternative CETP inhibitors without the off-target effects of torcetrapib. The clinical development programs leading to a number of promising CETP inhibitors will be reviewed.

Dietary cholesterol effects on adipose tissue inflammation.

Adipose tissue is a critical endocrine and immunological organ that regulates systemic energy homeostasis. During the pathogenesis of obesity, adipocyte hypertrophy is accompanied by adipose tissue inflammation, impeding insulin sensitivity and endocrine function of adipose tissue and other tissues. Adipocyte cholesterol accumulates in proportion to triglyceride as adipocytes undergo hypertrophy. Recent studies suggest that dietary cholesterol contributes to increased adipocyte cholesterol. However, how dietary cholesterol accumulates in adipocytes and its metabolic consequences are poorly understood. This review summarizes recent advances in knowledge of adipocyte cholesterol balance and highlights the emerging role of dietary cholesterol in adipose tissue cholesterol balance, inflammation, and systemic energy metabolism. Perturbation of cholesterol balance in adipocytes alters intracellular cholesterol distribution and modulates adipocyte insulin and proinflammatory signaling. Adipocyte cholesterol levels are maintained by a balance between dietary cholesterol uptake from triglyceride-enriched lipoproteins and cellular cholesterol efflux to HDL. Recent animal studies established a critical role for dietary cholesterol in promoting adipose tissue inflammation, thereby worsening obesity-mediated metabolic complications. Recent studies identified high dietary cholesterol as a potentiator of adipose tissue inflammation and dysfunction. Reducing excessive dietary cholesterol intake is suggested as a simple, but novel, way to attenuate obesity-associated metabolic diseases.

ABCA1 agonist peptides for the treatment of disease.

The review summarizes information pertaining to the preclinical development of new apolipoprotein (apo) E mimetic peptides that stimulate cellular cholesterol efflux. Small α-helical peptides based on the C-terminal domain of apoE have been developed for therapeutic applications. These peptides stimulate cellular cholesterol efflux via the ATP-binding cassette transporter A1 (ABCA1) with high potency, like native apolipoproteins on a molar basis. This potent activity has been related to the unique ability of these peptides to maintain α-helix structure upon dilution. Recent structure-activity studies improving the safety features of these mimetic peptides have greatly improved their potential for clinical use. These studies have identified structural features of the class A α-helix motif that induce muscle toxicity and hypertriglyceridemia, which may have implications for the design of other HDL mimetic peptides. ABCA1 is an integral membrane protein that plays a central role in biology. Its principal function is to mediate the efflux of cholesterol and phospholipid from cells to extracellular apo, preventing a build-up of excess cholesterol in membranes. This process generates HDL particles that perform a variety of functions to protect against disease. A number of these functions can be viewed as directly or indirectly supporting ABCA1 activity, thus constituting a positive feedback system to optimize cellular lipid efflux responses and disease prevention. Consequently, therapeutic approaches that mimic the activities of apos may prove highly effective to combat disease. One such approach involves the use of peptides. The broad biological relevance of ABCA1 suggests these apo mimetic peptides may be useful for the treatment of a number of diseases, such as atherosclerosis, diabetes, and Alzheimer's disease.

Correlation Between ABCA1 Gene Polymorphism and aopA-I and HDL-C in Abdominal Aortic Aneurysm.

BackgroundAs the most common type of aneurysm, abdominal aortic aneurysm (AAA) has an unfavorable prognosis due to the high frequency of rupture. Studies have indicated a close relationship between the pathogenesis and progression of AAA and abnormal serum lipid levels. ATP-binding cassette transport protein A1 (ABCA1) is a cell-surface protein facilitating cellular efflux of cholesterol. The single-nucleotide polymorphism (SNP) of ABCA1 gene has been suggested to be correlated with abnormal metabolism of lipids. Therefore, this study aimed to investigate the relationship between ABCA1 polymorphism and apoA-I and HDL-C in an attempt to elucidate its correlation with AAA occurrence.Material/MethodsWe included 126 AAA patients and 119 healthy controls in this study. PCR and restriction fragment length polymorphism (RFLP) were used to detect the SNP pattern of ABCA1 gene at locus rs2230806 from both AAA patients and healthy controls. The distribution pattern and correlation with apoA-I and HDL-C was analyzed.ResultsThe distribution of KK/RR genotype of ABCA1 gene had significant difference between disease and control group, with lower rates of RR genotype and R allele in the disease group (p<0.05). Levels of apoA-I and HDL-C, but not triglyceride and LDL-C levels, in AAA patients who carried R allele in ABCA1 gene (including RR and RK genotypes) were higher than in non-carriers (p<0.05). The R allele of ABCA1 gene was shown to be related with the occurrence of AAA (p<0.05).ConclusionsPolymorphism of ABCA1 gene is correlated with AAA occurrence, possibly via the regulation of serum lipid metabolism by R allele.

Effects of Porphyromonas gingivalis lipopolysaccharide on the expression of key genes involved in cholesterol metabolism in macrophages.

IntroductionCardiovascular diseases are positively correlated with periodontal disease. However, the molecular mechanisms linking atherosclerosis and periodontal infection are not clear. This study aimed to determine whether Porphyromonas gingivalis lipopolysaccharide (Pg-LPS) altered the expression of genes regulating cholesterol metabolism in macrophages in the presence of low-density lipoprotein (LDL).Material and methodsTHP-1-derived macrophages were exposed to different concentrations (0.1, 1, 10 µg/ml) of LPS in the presence of 50 µg/ml native LDL. Macrophages were also incubated with 1 µg/ml LPS for varying times (0, 24, 48, or 72 h) in the presence of native LDL. Foam cell formation was determined by oil red O staining and cholesterol content quantification. CD36, lectin-like oxidized LDL receptor-1 (LOX-1), ATP-binding cassette G1 (ABCG1), and acetyl CoA acyltransferase 1 (ACAT1) expression levels were measured by western blot and qRT-PCR.ResultsFoam cell formation was induced in a time- and concentration-dependent manner as assessed by both morphological and biochemical criteria. Pg-LPS caused downregulation of CD36 and ABCG1 but upregulation of ACAT1, while LOX-1 expression was not affected (p = 0.137).ConclusionsPg-LPS appears to be an important link in the development of atherosclerosis by mechanisms targeting cholesterol homeostasis, namely, excess cholesterol ester formation via ACAT1 and reduced cellular cholesterol efflux via ABCG1.

MiR-27b inhibits LDLR and ABCA1 expression but does not influence plasma and hepatic lipid levels in mice

RationaleRecently, there has been significant interest in the therapeutic administration of miRNA mimics and inhibitors to treat cardiovascular disease. In particular, miR-27b has emerged as a regulatory hub in cholesterol and lipid metabolism and potential therapeutic target for treating atherosclerosis. Despite this, the impact of miR-27b on lipid levels in vivo remains to be determined. As such, here we set out to further characterize the role of miR-27b in regulating cholesterol metabolism in vitro and to determine the effect of miR-27b overexpression and inhibition on circulating and hepatic lipids in mice. Methods and resultsOur results identify miR-27b as an important regulator of LDLR activity in human and mouse hepatic cells through direct targeting of LDLR and LDLRAP1. In addition, we report that modulation of miR-27b expression affects ABCA1 protein levels and cellular cholesterol efflux to ApoA1 in human hepatic Huh7 cells. Overexpression of pre-miR-27b in the livers of wild-type mice using AAV8 vectors increased pre-miR-27b levels 50–fold and reduced hepatic ABCA1 and LDLR expression by 50% and 20%, respectively, without changing circulating and hepatic cholesterol and triglycerides. To determine the effect of endogenous miR-27b on circulating lipids, wild-type mice were fed a Western diet for one month and injected with 5 mg/kg of LNA control or LNA anti-miR-27b oligonucleotides. Following two weeks of treatment, the expression of ABCA1 and LDLR were increased by 10–20% in the liver, demonstrating effective inhibition of miR-27b function. Intriguingly, no differences in circulating and hepatic lipids were observed between treatment groups. ConclusionsThe results presented here provide evidence that short-term modulation of miR-27b expression in wild-type mice regulates hepatic LDLR and ABCA1 expression but does not influence plasma and hepatic lipid levels.

Abstract 14630: Elevated CETP Activity is Associated With Low, Dysfunctional HDL in High Cardiovascular Risk South Asians

Background and Rationale: Low HDL in South Asians (SA) contribute greatly to their high coronary heart disease (CHD) risk relative to European Caucasians (EC), however, the cause of low HDL in SA is unknown. Here we investigated whether cholesteryl-ester transfer protein (CETP) plays a role in mediating low HDL in SA. Methods: Serum was obtained from metabolically well characterized individuals of SA and EC descent (N=244 and 238, respectively) and measured for CETP activity. HDL lipid and protein contents were quantified and HDL size and particle number were measured via NMR. The capacity of HDL to mediate cholesterol efflux was assessed in J774 macrophages with apoB-depleted patient serum. Results: CETP activity was elevated in SA compared with EC (26.5±0.45 vs. 18.5±0.45 pmol/uL/hr, P&lt; 0.0001). HDL from SA was pro-atherogenic - that is, triglyceride-enriched, cholesteryl ester-depleted, reduced in apoAI and smaller in size than in EC (P&lt; 0.05 for all). SA HDL, moreover, showed a reduced capacity to mediate macrophage cholesterol efflux (by 20%, P&lt; 0.0001), even in a subset of younger SA (&lt;40 years, P&lt;0.01). Composition and size changes in HDL were independent predictors of carotid artery intima-media thickness (IMT) in SA (P&lt; 0.05). Conclusions: We have identified for the first time a mechanism to account for low HDL in SA. Elevated CETP in SA was associated with triglyceride-enriched, cholesteryl ester-depleted HDL, smaller in size and reduced in apoA-I content, producing HDL that is less stable in the circulation and less atheroprotective. This was confirmed with the lower relative capacity of HDL from SA to mediate cellular cholesterol efflux and independent correlation of their HDL compositional changes with carotid IMT.

Abstract 17107: Distribution of Anti-inflammatory Activity During Remodeling of High Density Lipoprotein: Studies With CSL112

Elevated levels of circulating inflammatory markers predict an unfavorable cardiovascular outcome in acute coronary syndrome patients. CSL112 is human apolipoprotein A-I (apoA-I), reconstituted with phosphatidylcholine to form HDL particles suitable for infusion. Addition of CSL112 to stimulated human whole blood ex vivo strongly reduces pro-inflammatory cytokine production. Infusion of CSL112 into human subjects or addition to human plasma ex vivo causes remodeling of endogenous HDL. Similar remodeling occurs upon incubation of CSL112 with purified HDL3 and results in accumulation of three HDL species: enlarged HDL (HDL2), a smaller, dense species (HDL3c), and lipid-poor apoA-I (pre-β1 HDL). Study aim was to determine the anti-inflammatory activity of remodeled HDL species. CSL112 was incubated with HDL3 and the products of particle remodeling were purified by ultracentrifugation. The inhibitory effects on pro-inflammatory cytokine production were examined using human peripheral blood mononuclear cells (PBMC) stimulated with phytohemagglutinin-M (PHA-M) in vitro. Lipid-poor apoA-I, HDL3c as well as parent CSL112 exerted powerful inhibitory effects on secretion of pro-inflammatory mediators (&gt; 89.2% + 4.0% inhibition of TNF-α, IL-1β, IL-6 and Mip-1β); HDL3 and HDL2 were much less effective (&lt; 54.1% + 3.9% inhibition). The extent of inhibition correlated positively with induction of the transcription repressor ATF3, a negative regulator of pro-inflammatory cytokine production, with lipid-poor apoA-I and HDL3c inducing higher protein levels of ATF3 in PHA-stimulated PBMC compared to control medium, HDL3 or HDL2. Anti-inflammatory activity of the remodeled species also correlated with their ability to support cellular cholesterol efflux via the ABCA1 transporter: lipid-poor apoA-I and HDL3c were potent acceptors of cholesterol; HDL2 was inactive. The ability to generate HDL species with high cholesterol efflux and anti-inflammatory activity makes CSL112 a promising candidate for removing cholesterol and reducing inflammation in atherosclerotic plaque, thus reducing the high risk of early recurrent atherothrombotic events following acute MI (AMI). A Phase IIb trial (AEGIS-I; NCT02108262) of CSL112 in AMI patients is ongoing.

Therapeutic applications of reconstituted HDL: When structure meets function.

Reconstituted forms of HDL (rHDL) are under development for infusion as a therapeutic approach to attenuate atherosclerotic vascular disease and to reduce cardiovascular risk following acute coronary syndrome and ischemic stroke. Currently available rHDL formulations developed for clinical use contain apolipoprotein A-I (apoA-I) and one of the major lipid components of HDL, either phosphatidylcholine or sphingomyelin. Recent data have established that quantitatively minor molecular constituents of HDL particles can strongly influence their anti-atherogenic functionality. Novel rHDL formulations displaying enhanced biological activities, including cellular cholesterol efflux, may therefore offer promising prospects for the development of HDL-based, anti-atherosclerotic therapies. Indeed, recent structural and functional data identify phosphatidylserine as a bioactive component of HDL; the content of phosphatidylserine in HDL particles displays positive correlations with all metrics of their functionality. This review summarizes current knowledge of structure-function relationships in rHDL formulations, with a focus on phosphatidylserine and other negatively-charged phospholipids. Mechanisms potentially underlying the atheroprotective role of these lipids are discussed and their potential for the development of HDL-based therapies highlighted.

GW26-e2272 D-4F Reduced Cardiac Hypertrophy and Improved Heart Function in LDLR-Deficient Mice Treated With Western Diet by Increasing ABCA1 and LXRs Levels

Epidemiological studies and mouse models have suggested that hypercholesterolemia is an independent determinant of increased left ventricular (LV) mass and is related to cardiac hypertrophy and fibrosis. Since HDL particles and its major apolipoprotein apoA-I mediate cellular cholesterol efflux and

Assessment of the clinical effects of cholesteryl ester transfer protein inhibition with evacetrapib in patients at high-risk for vascular outcomes: Rationale and design of the ACCELERATE trial

Potent pharmacologic inhibition of cholesteryl ester transferase protein by the investigational agent evacetrapib increases high-density lipoprotein cholesterol by 54% to 129%, reduces low-density lipoprotein cholesterol by 14% to 36%, and enhances cellular cholesterol efflux capacity. The ACCELERATE trial examines whether the addition of evacetrapib to standard medical therapy reduces the risk of cardiovascular (CV) morbidity and mortality in patients with high-risk vascular disease. ACCELERATE is a phase 3, multicenter, randomized, double-blind, placebo-controlled trial. Patients qualified for enrollment if they have experienced an acute coronary syndrome within the prior 30 to 365 days, cerebrovascular accident, or transient ischemic attack; if they have peripheral vascular disease; or they have diabetes with coronary artery disease. A total of 12,092 patients were randomized to evacetrapib 130 mg or placebo daily in addition to standard medical therapy. The primary efficacy end point is time to first event of CV death, myocardial infarction, stroke, hospitalization for unstable angina, or coronary revascularization. Treatment will continue until 1,670 patients reached the primary end point; at least 700 patients reach the key secondary efficacy end point of CV death, myocardial infarction, and stroke, and the last patient randomized has been followed up for at least 1.5 years. ACCELERATE will establish whether the cholesteryl ester transfer protein inhibition by evacetrapib improves CV outcomes in patients with high-risk vascular disease.

The role of the lymphatic system in cholesterol transport.

Reverse cholesterol transport (RCT) is the pathway for removal of peripheral tissue cholesterol and involves transport of cholesterol back to liver for excretion, starting from cellular cholesterol efflux facilitated by lipid-free apolipoprotein A1 (ApoA1) or other lipidated high-density lipoprotein (HDL) particles within the interstitial space. Extracellular cholesterol then is picked up and transported through the lymphatic vasculature before entering into bloodstream. There is increasing evidence supporting a role for enhanced macrophage cholesterol efflux and RCT in ameliorating atherosclerosis, and recent data suggest that these processes may serve as better diagnostic biomarkers than plasma HDL levels. Hence, it is important to better understand the processes governing ApoA1 and HDL influx into peripheral tissues from the bloodstream, modification and facilitation of cellular cholesterol removal within the interstitial space, and transport through the lymphatic vasculature. New findings will complement therapeutic strategies for the treatment of atherosclerotic vascular disease.

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.

Asymptomatic individuals with high HDL-C levels overexpress ABCA1 and ABCG1 and present miR-33a dysregulation in peripheral blood mononuclear cells

Considering the growing knowledge and perspectives on microRNAs (miRNAs) that control high-density lipoprotein cholesterol (HDL-C) levels and metabolism, this study aimed at evaluating whether hsa-miR-33a and hsa-miR-128a are differentially expressed in peripheral blood mononuclear cells from asymptomatic individuals with low and high HDL-C, as well as at investigating the potential relationships with ATP binding cassete transporter A1 (ABCA1) expression, cholesterol efflux capacity and other parameters related with reverse cholesterol transport. In addition, the associations with cardiovascular risk were investigated by carotid-intima media thickness (cIMT). Asymptomatic volunteers of both genders (n=51) were classified according to HDL-C (mg/dL) in hypoalphalipoproteinemics (hypo, HDL-C≤39), hyperalphalipoproteinemics (hyper, HDL-C≥68) and controls (CTL, HDL-C≥40<68). cIMT, lipids, lipoproteins, HDL size and volume, C reactive protein and insulin were determined, as well as the activities of several proteins and enzymes related to HDL metabolism. In a subgroup of 19 volunteers the cellular cholesterol efflux and HDL composition were determined. Total RNA was extracted from peripheral blood mononuclear cells for relative quantification experiments. Hypo volunteers presented significantly higher levels of triglycerides, VLDL-C and insulin; in addition, HDL size and volume decreased when compared with CTL and hyper. Regarding gene expression analysis, the hyper group presented a decrease of 72% in hsa-miR-33a and higher mRNA expression of ABCA1 and ABCG1 when compared with CTL. No significant differences in hsa-miR-128a expression, cholesterol efflux, cIMT or plaques were found. Further studies are necessary to elucidate the mechanisms underlying the complex miRNA network, regulating cellular cholesterol homeostasis in humans and its clinical repercussions.

Glycated human serum albumin isolated from poorly controlled diabetic patients impairs cholesterol efflux from macrophages: an investigation by mass spectrometry.

Advanced glycation end-products impair ABCA-1-mediated cholesterol efflux by eliciting inflammation, the generation of reactive oxygen species and endoplasmatic reticulum (ER) stress. The glycation level of human serum albumin (HSA) from type 1 and type 2 diabetic patients was determined by matrix assisted laser desorption/ionization (MALDI) mass spectrometry and related to possible impairment of ER function and cellular cholesterol efflux. Comparison of the MALDI spectra from healthy and diabetic subjects allowed us to determine an increased HSA mean mass of 1297 Da for type 1 and 890 Da for type 2. These values reflect a mean condensation of at least 8 glucose units and 5 glucose units, respectively. Mouse peritoneal macrophages were treated with HSA from control, type 1 and type 2 diabetic subjects in order to measure the expression of Grp78, Grp94, protein disulfide isomerase (PDI), calreticulin (CRT) and ABCA-1. (14)C-cholesterol overloaded-J774 macrophages were treated with HSA from control and diabetic subjects and further incubated with apo A-1 to determine the cholesterol efflux. Combined analyses comprising HSA from type 1 and type 2 diabetic patients were performed in cellular functional assays. In macrophages, PDI expression increased 89% and CRT 3.4 times in comparison to HSA from the control subjects. ABCA-1 protein level and apo A-I mediated cholesterol efflux were, respectively, 50% and 60% reduced in macrophages exposed to HSA from type 1 and type 2 diabetic patients when compared to that exposed to HSA from control subjects. We provide evidence that the level of glycation that occurs in albumin in vivo damages the ER function related to the impairment in macrophage reverse cholesterol transport and so contributes to atherosclerosis in diabetes.

Abstract 14: Identification of Linc-OSBPL6 as a Competing Endogenous RNA that Regulates Cholesterol Homeostasis

Long non-coding RNAs (lncRNAs) have emerged as important regulators of gene expression in diverse biological contexts. Although several thousand lncRNAs have been identified in humans, only a small number have been fully characterized. Here we identify a novel human lncRNA, linc-OSBPL6, that functions as a competing endogenous RNA to regulate cellular cholesterol homeostasis. Linc-OSBPL6 has 7 variants that are broadly expressed in various cell types and tissues, and are controlled at the transcriptional level by the cholesterol-sensing nuclear receptor liver X receptor. We noted that each of the linc-OSBPL6 variants harbors binding sites for miR-27b and miR-33a/b, microRNAs recently identified as hubs controlling the expression of genes involved in cellular cholesterol efflux and HDL metabolism. Using linc-OSBPL6 knockdown and overexpression, combined with RNA profiling, we demonstrate that linc-OSBPL6 acts as a ‘microRNA sponge’ to limit availability of miR-27b and miR33a/b and inversely regulate expression of their target genes (ABCA1, OSBPL6, ANGPTL3, CROT, ABCB11). Consistent with the role of miR-27b and miR-33a/b in repressing cellular cholesterol export, we show that overexpression of linc-OSBPL6 increases cholesterol efflux, while shRNA-silencing of linc-OSBPL6 reduces cholesterol efflux from hepatocytes and macrophages. As cholesterol efflux in the liver plays a central role in HDL biogenesis, we investigated the relationship of hepatic linc-OSBPL6 to its microRNA-targets and plasma levels of HDL cholesterol in a cohort of 200 healthy individuals. We found that expression of linc-OSBPL6 is negatively correlated with levels of miR-27b and miR-33a/b in the liver, and positively correlated with levels of their target genes and plasma HDL cholesterol. These findings identify linc-OSBPL6 as a key regulatory component of the non-coding RNA circuitry that controls cellular cholesterol efflux and plasma HDL levels in humans.

Robust passive and active efflux of cellular cholesterol to a designer functional mimic of high density lipoprotein

The ability of HDL to support macrophage cholesterol efflux is an integral part of its atheroprotective action. Augmenting this ability, especially when HDL cholesterol efflux capacity from macrophages is poor, represents a promising therapeutic strategy. One approach to enhancing macrophage cholesterol efflux is infusing blood with HDL mimics. Previously, we reported the synthesis of a functional mimic of HDL (fmHDL) that consists of a gold nanoparticle template, a phospholipid bilayer, and apo A-I. In this work, we characterize the ability of fmHDL to support the well-established pathways of cellular cholesterol efflux from model cell lines and primary macrophages. fmHDL received cell cholesterol by unmediated (aqueous) and ABCG1- and scavenger receptor class B type I (SR-BI)-mediated diffusion. Furthermore, the fmHDL holoparticle accepted cholesterol and phospholipid by the ABCA1 pathway. These results demonstrate that fmHDL supports all the cholesterol efflux pathways available to native HDL and thus, represents a promising infusible therapeutic for enhancing macrophage cholesterol efflux. fmHDL accepts cholesterol from cells by all known pathways of cholesterol efflux: unmediated, ABCG1- and SR-BI-mediated diffusion, and through ABCA1.

Abstract 650: Characterization of Microparticles Formed by ABCA1

Microparticles (MPS) are lipoprotein-sized structures created by the ABCA1 transporter. Their biological roles in health and in disease remain unknown. Here, we study MPS released from baby hamster kidney (BHK) cells stably expressing ABCA1 and human THP-1 cells also expressing ABCA1. Media cell culture was first collected from BHK-ABCA1 expressing cells after (45min, 2, 4, 8, and 24h) incubation. After centrifugation (4000xg for 15min and 10000xg for 30min) to remove cell debris, the supernatant was passed through a 10kDa cutoff filter and subsequently subjected to analytical FPLC. FPLC analysis shows creation of a single peak in the presence of ABCA1 but not in mock-transfected BHK cells. In a time-course study, the estimated hydrodynamic diameter remained stable (≥20nm). After 8h incubation of BHK cell with apoA-I or an apo-E mimetic peptide, CS-6253 (1μM), ABCA1 mediated formation of MPS of similar size with a significant increase in 3[H]-FC content than those generated by ABCA1 alone, (373±23 % cpm, P&lt;0.05) and (277±60 % cpm, P&lt;0.05) respectively. This was associated with highly lipidated nHDL-CS-6253 compararely to nHDL-apoA-I (4535±72 % cpm, P&lt;0.001 versus 1059±14 % cpm) respectively. This data suggests that MPS formations are an integral component of cellular cholesterol efflux. Also, MPS do not contain CS-6253 when ABCA1 cells were incubated with the peptide as confirmed by western blotting similar to MPS generated from ABCA1 cells generated by apoA-I incubation. Cholesterol is effluxed more to nHDL-CS-6253 (14±7.68, % cpm, P&lt;0.01 than to MPS 1±0.10, % cpm, 24h; P&lt;0.01) similar to apoA-I. Depletion of membrane cholesterol by methyl-β-cyclodextrin treatment impaired HDL genesis and decrease MPS release. Detection of flotellin-2 protein enriched in MPS in total cell lysate indicated that these MPS may be related to exosomes. MPS generation was also characterized in THP-1 cells for further molecular characterization. We conclude that MPS are formed by ABCA1 in diverse cell types and the cholesterol content is dependent on activation by apo A-I or mimetic peptides. The physiological role of MPS remains to be understood. These particles may be transporters of lipids and nucleic acids.

Case report: A novel apolipoprotein A-I missense mutation apoA-I (Arg149Ser)Boston associated with decreased lecithin-cholesterol acyltransferase activation and cellular cholesterol efflux

We report a novel heterozygous apolipoprotein A-I (apoA-I) missense mutation (c.517C>A, p.Arg149Ser, designated as apoA-IBoston) in a 67-year-old woman and her 2 sons, who had mean serum high-density lipoprotein (HDL) cholesterol, apoA-I, and apoA-I in very large α-1 HDL that were 10%, 35%, and 16% of normal, respectively (all P < .05). The percentage of HDL cholesterol in the esterified form was also significantly (P < .05) reduced to 52% of control values. Cholesteryl ester tranfer protein (CETP) activity was normal. The mean global, adenosine triphosphate (ATP)-binding cassette transporter A1 and scavenger receptor B type I–mediated cellular cholesterol efflux capacity in apoB-depleted serum from affected family members were 41%, 37%, 47%, 54%, and 48% of control values, respectively (all P < .05). lecithin-cholesterol acyltransferase (LCAT) activity in plasma was 71% of controls, whereas in the cell-based assay, it was 73% of control values (P < .05). The data indicate that this novel apoA-I missense is associated with markedly decreased levels of HDL cholesterol and very large α-1 HDL, as well as decreased serum cellular cholesterol efflux and LCAT activity, but not with premature coronary heart disease, similar to other apoA-I mutations that have been associated with decreased LCAT activity.

Molecular complex composed of β-cyclodextrin-grafted Chitosan and pH-sensitive amphipathic peptide for enhancing cellular cholesterol efflux under acidic pH.

Excess of cholesterol in peripheral cells is known to lead to atherosclerosis. In this study, a molecular complex composed of β-cyclodextrin-grafted chitosan (BCC) and cellular cholesterol efflux enhancing peptide (CEEP), synthesized by modifying pH sensitive amphipathic GALA peptide, is introduced with the eventual aim of treating atherosclerosis. BCC has a markedly enhanced ability to induce cholesterol efflux from cell membranes compared to β-cyclodextrin, and the BCC-CEEP complex exhibited a 2-fold increase in cellular cholesterol efflux compared to BCC alone under weakly acidic conditions. Isothermal titration calorimetry and fluorescence spectroscopy measurements demonstrated that the random coil structure of CEEP at neutral pH converted to the α-helical structure at acidic pH, resulting in a three-order larger binding constant to BCC (K = 3.7 × 10(7) at pH 5.5) compared to that at pH 7.4 (K = 7.9 × 10(4)). Such high-affinity binding of CEEP to BCC at acidic pH leads to the formation of 100-nm-sized aggregate with positive surface charge, which would efficiently interact with cell membranes and induce cholesterol efflux. Since the cholesterol efflux ability of HDL is thought to be impaired under acidic environments in advanced atherosclerotic lesions, the BCC-CEEP complex might serve as a novel nanomaterial for treating atherosclerosis.