ApoA-I Dissociated From Human HDL Retains its Acceptor Properties in ABCA1-mediated Cholesterol Efflux From RAW 264.7 Macrophages in Coronary Artery Disease.

It is generally thought that the large heterogeneity of human HDL confers antiatherogenic properties; however, the mechanisms governing HDL biogenesis and speciation are complex and poorly understood. Here, we show that incubation of exogenous apolipoprotein A-I (apoA-I) with fibroblasts, CaCo-2, or CHO-overexpressing ABCA1 cells generates only alpha-nascent apolipoprotein A-I-containing particles (alpha-LpA-I) with diameters of 8-20 nm, whereas human umbilical vein endothelial cells and ABCA1 mutant (Q597R) cells were unable to form such particles. Interestingly, incubation of exogenous apoA-I with either HepG2 or macrophages generates both alpha-LpA-I and prebeta1-LpA-I. Furthermore, glyburide inhibits almost completely the formation of alpha-LpA-I but not prebeta1-LpA-I. Similarly, endogenously secreted HepG2 apoA-I was found to be associated with both prebeta1-LpA-I and alpha-LpA-I; by contrast, CaCo-2 cells secreted only alpha-LpA-I. To determine whether alpha-LpA-I generated by fibroblasts is a good substrate for LCAT, isolated alpha-LpA-I as well as reconstituted HDL [r(HDL)] was reacted with LCAT. Although both particles had similar V(max) (8.4 vs. 8.2 nmol cholesteryl ester/h/microg LCAT, respectively), the K(m) value was increased 2-fold for alpha-LpA-I compared with r(HDL) (1.2 vs. 0.7 microM apoA-I). These results demonstrate that 1) ABCA1 is required for the formation of alpha-LpA-I but not prebeta1-LpA-I; and 2) alpha-LpA-I interacts efficiently with LCAT. Thus, our study provides direct evidence for a new link between specific cell lines and the speciation of nascent HDL that occurs by both ABCA1-dependent and -independent pathways.

HDL cholesterol (HDL-C) efflux function may be a more robust biomarker of coronary artery disease risk than HDL-C. To study HDL function, apoB-containing lipoproteins are precipitated from serum. Whether apoB precipitation affects HDL subspecies composition and function has not been thoroughly investigated. We studied the effects of four common apoB precipitation methods [polyethylene glycol (PEG), dextran sulfate/magnesium chloride (MgCl2), heparin sodium/manganese chloride (MnCl2), and LipoSep immunoprecipitation (IP)] on HDL subspecies composition, apolipoproteins, and function (cholesterol efflux and reduction of LDL oxidation). PEG dramatically shifted the size distribution of HDL and apolipoproteins (assessed by two independent methods), while leaving substantial amounts of reagent in the sample. PEG also changed the distribution of cholesterol efflux and LDL oxidation across size fractions, but not overall efflux across the HDL range. Dextran sulfate/MgCl2, heparin sodium/MnCl2, and LipoSep IP did not change the size distribution of HDL subspecies, but altered the quantity of a subset of apolipoproteins. Thus, each of the apoB precipitation methods affected HDL composition and/or size distribution. We conclude that careful evaluation is needed when selecting apoB depletion methods for existing and future bioassays of HDL function.

Background HDL is endowed with several metabolic, vascular, and immunoinflammatory protective functions. Among them, a key property is to promote reverse cholesterol transport from cells back to the liver. The aim of this study was to estimate the association of scavenger receptor class B type I (SR-BI)- and ATP binding cassette transporter A1 (ABCA1)-mediated cholesterol efflux (the two major routes for cholesterol efflux to HDL) with the presence, extent, and severity of coronary artery disease (CAD), vascular wall remodelling processes, coronary plaque characteristics, and the incidence of myocardial infarction in the different subgroups of patients from the CAPIRE study. Methods Patients (n = 525) from the CAPIRE study were divided into two groups: low-risk factors (RF), with 0–1 RF (n = 263), and multiple-RF, with ≥2 RFs; within each group, subjects were classified as no-CAD or CAD based on the segment involvement score (SIS) evaluated by coronary computed tomography angiography (SIS = 0 and SIS > 5, respectively). SR-BI- and ABCA1-mediated cholesterol efflux were measured using the plasma of all patients. Results SR-BI-mediated cholesterol efflux was significantly reduced in patients with CAD in both the low-RF and multiple-RF groups, whereas ABCA1-mediated cholesterol efflux was similar among all groups. In CAD patients, multivariable analysis showed that SR-BI-mediated cholesterol efflux <25th percentile predicted cardiovascular outcome (odds ratio 4.1; 95% CI: 1.3–13.7; p = .019), whereas ABCA-1-mediated cholesterol efflux and HDL-C levels significantly did not. Despite this finding, reduced SR-BI-mediated cholesterol efflux was not associated with changes in high-risk plaque features or changes in the prevalence of elevated total, non-calcified, and low-attenuation plaque volume. Conclusion SR-BI-mediated cholesterol efflux capacity is lower in patients with diffuse coronary atherosclerosis. In addition, a lower SR-BI-mediated cholesterol efflux capacity is associated with the worst clinical outcomes in patients with CAD, independently of atherosclerotic plaque features. Key Messages Increased cholesterol efflux capacity, an estimate of HDL function, is associated with a reduced CVD risk, regardless of HDL-C levels. HDL-C levels are significantly lower in patients with CAD. Lower SR-BI-mediated cholesterol efflux capacity is observed in patients with diffuse coronary atherosclerosis and is associated with the worst clinical outcomes in patients with CAD, independently of atherosclerotic plaque features.

Abnormal HDL metabolism among patients with diabetes and insulin resistance may contribute to their increased risk of atherosclerosis. ATP binding cassette transporter A1 (ABCA1) mediates the transport of cholesterol and phospholipids from cells to HDL apolipoproteins and thus modulates HDL levels and atherogenesis. Because fatty acids are increased in diabetes, we examined their effects on ABCA1 activity in cultured macrophages. cAMP analogs and ligands for the liver X receptor/retinoid X receptor (LXR/RXR) system can induce Abca1 transcription in murine macrophages. When induced by cAMP, unsaturated but not saturated long-chain fatty acids inhibit apolipoprotein-mediated lipid efflux by destabilizing ABCA1 protein. Here, we show that the saturated fatty acids palmitate and stearate also destabilize ABCA1 when Abca1 is induced by LXR/RXR ligands instead of cAMP. This was associated with increased palmitate and stearate desaturation by stearoyl-CoA desaturase (SCD), another gene product induced by LXR/RXR ligands. The SCD inhibitors conjugated linoleic acid and troglitazone nearly abolished ABCA1 destabilization by palmitate and stearate but not by linoleate. These results suggest that LXR/RXR ligands generate ABCA1-destabilizing monounsaturated fatty acids from their saturated precursors by activating SCD. Thus, with cholesterol-loaded macrophages exposed to saturated fatty acids, activated LXR/RXR may counteract the enhanced ABCA1 transcription by reducing the ABCA1 protein content.

Cholesterol is an essential component of eukaryotic cells; at the same time, however, hyperaccumulation of cholesterol is harmful. Therefore, the ABCA1 gene, the product of which mediates secretion of cholesterol, is highly regulated at both the transcriptional and post-transcriptional levels. The transcription of ABCA1 is regulated by intracellular oxysterol concentration via the nuclear liver X receptor (LXR)/retinoid X receptor (RXR); once synthesized, ABCA1 protein turns over rapidly with a half-life of 1-2 h. Here, we show that the LXRbeta/RXR complex binds directly to ABCA1 on the plasma membrane of macrophages and modulates cholesterol secretion. When cholesterol does not accumulate, ABCA1-LXRbeta/RXR localizes on the plasma membrane, but is inert. When cholesterol accumulates, oxysterols bind to LXRbeta, and the LXRbeta/RXR complex dissociates from ABCA1, restoring ABCA1 activity and allowing apoA-I-dependent cholesterol secretion. LXRbeta can exert an immediate post-translational response, as well as a rather slow transcriptional response, to changes in cellular cholesterol accumulation. Thus, we provide the first demonstration that protein-protein interaction suppresses ABCA1 function. Furthermore, we show that LXRbeta is involved in both the transcriptional and post-transcriptional regulation of the ABCA1 transporter.

A great majority of the morbidity and mortality worldwide can still be attributed to cardiovascular diseases, such as ischemic (coronary) heart disease, angina pectoris, and myocardial and cerebral infarction. Atherosclerosis, narrowing of the arteries because of arterial cholesterol deposition in macrophage foam cells, is the driving force behind the cardiovascular disease pathology. Water-soluble protein/lipid complexes called lipoproteins mediate the transport of cholesterol and other lipoid substances through the blood compartment. Relatively high levels of cholesterol associated with apolipoprotein B–containing low-density lipoprotein (LDL) particles predispose human subjects to the development of atherosclerosis and, thereby, increase the risk for cardiovascular disease.1,2 Apolipoprotein B–containing lipoproteins are, therefore, generally regarded as being proatherogenic factors. Cholesterol ester–rich high-density lipoprotein (HDL) particles use apolipoprotein A1 (apoA1) as their primary protein component. In sharp contrast to LDL, HDL is considered a potent anti-atherogenic agent. This notion is based on the fact that, in the general population, a strong inverse correlation exists between plasma levels of HDL cholesterol and the risk of cardiovascular disease.1 Of note, this inverse association seems to be independent of the level of cholesterol associated with proatherogenic LDL particles. As such, increasing plasma levels of HDL cholesterol has long been regarded a promising alternative therapy to supplement classical statin–based LDL cholesterol–lowering strategies that are able to reduce cardiovascular disease by only ≈30%.3 However, over the last decade, the enthusiasm for HDL as an interesting therapeutic target has been challenged by the HDL hypothesis critics because genetic association studies have excluded HDL cholesterol levels as determinants for cardiovascular disease risk.2,4 Furthermore, several therapeutic HDL-targeting approaches have proven insufficient to secure benefit for cardiovascular disease patients. Niacin is the most effective drug available in the clinic to raise plasma HDL cholesterol levels. Despite the fact that niacin is …

Relation of Increased Prebeta-1 High-Density Lipoprotein Levels to Risk of Coronary Heart Disease

Cardiovascular disease (CVD) is the most common cause of death worldwide.1 Major CVD risk factors are hypertension, smoking, physical inactivity, abnormal glucose levels/diabetes mellitus, and dyslipidemia. Among these, dyslipidemia characterized by a low level of HDL-C (high-density lipoprotein cholesterol) is strongly and inversely correlated with CVD risk,2–4 and low HDL-C levels is part of the atherogenic dyslipidemia complex associated with diabetes mellitus.5 These observations triggered intense interest in increasing HDL-C levels for therapeutic intervention of CVD.6,7 However, several recent lines of evidence now suggest that the association between HDL-C levels and CVD status may be indirect or more complicated than previously recognized.7 Thus, human genetic studies demonstrate that genetically altered HDL-C levels do not necessarily translate to an altered risk of CVD.8–12 Phase III clinical trials of drugs that elevate HDL-C, such as niacin and CETP (cholesteryl ester transfer protein) inhibitors, also have largely failed to reduce CVD events in statin-treated subjects with established CVD.13–15 For several CETP inhibitors, improvement in CVD prevention was unsuccessful because of off-target effects (torcetrapib) or lack of efficacy (dalcetrapib and evacetrapib).14,16–18 The exception is the recent REVEAL trial (Randomized Evaluation of the Effects of Anacetrapib Through Lipid-Modification), which included more subjects and was performed for longer than previous CETP inhibitor trials. The REVEAL demonstrated that the CETP inhibitor anacetrapib exhibited beneficial effects on cardiovascular outcomes on top of those of statin therapy, although the risk reduction was moderate19,20 and might have been due, at least in part, to a reduction in non-HDL-C rather than elevated HDL-C.21 Considering these cumulative observations, it remains uncertain whether increased HDL-C directly impacts atherosclerosis and the …

High-Density Lipoprotein Mediated Cellular Cholesterol Efflux in Acute Coronary Syndromes

The ATP binding cassette transporter A-1 (ABCA1) is critical for apolipoprotein-mediated cholesterol efflux, an important mechanism employed by macrophages to avoid becoming lipid-laden foam cells, the hallmark of early atherosclerotic lesions. It has been proposed that lipid-free apolipoprotein A-I (apoA-I) enters the cell and is resecreted as a lipidated particle via a retroendocytosis pathway during ABCA1-mediated cholesterol efflux from macrophages. To determine the functional importance of such a pathway, confocal microscopy was used to characterize the internalization of a fully functional apoA-I cysteine mutant containing a thiol-reactive fluorescent probe in cultured macrophages. ApoA-I was also endogenously labeled with (35)S-methionine to quantify cellular uptake and to determine the metabolic fate of the internalized protein. It was found that apoA-I was specifically taken inside macrophages and that a small amount of intact apoA-I was resecreted from the cells. However, a majority of the label that reappeared in the media was degraded. We estimate that the mass of apoA-I retroendocytosed is not sufficient to account for the HDL produced by the cholesterol efflux reaction. Furthermore, we have demonstrated that lipid-free apoA-I-mediated cholesterol efflux from macrophages can be pharmacologically uncoupled from apoA-I internalization into cells. On the basis these findings, we present a model in which the ABCA1-mediated lipid transfer process occurs primarily at the membrane surface in macrophages, but still accounts for the observed specific internalization of apoA-I.

In vitro experiments have demonstrated that exogenous phospholipid transfer protein (PLTP), i.e. purified PLTP added to macrophage cultures, influences ABCA1-mediated cholesterol efflux from macrophages to HDL. To investigate whether PLTP produced by the macrophages (i.e., endogenous PLTP) is also part of this process, we used peritoneal macrophages derived from PLTP-knockout (KO) and wild-type (WT) mice. The macrophages were transformed to foam cells by cholesterol loading, and this resulted in the upregulation of ABCA1. Such macrophage foam cells from PLTP-KO mice released less cholesterol to lipid-free apolipoprotein A-I (apoA-I) and to HDL than did the corresponding WT foam cells. Also, when plasma from either WT or PLTP-KO mice was used as an acceptor, cholesterol efflux from PLTP-KO foam cells was less efficient than that from WT foam cells. After cAMP treatment, which upregulated the expression of ABCA1, cholesterol efflux from PLTP-KO foam cells to apoA-I increased markedly and reached a level similar to that observed in cAMP-treated WT foam cells, restoring the decreased cholesterol efflux associated with PLTP deficiency. These results indicate that endogenous PLTP produced by macrophages contributes to the optimal function of the ABCA1-mediated cholesterol efflux-promoting machinery in these cells. Whether macrophage PLTP acts at the plasma membrane or intracellularly or shuttles between these compartments needs further study.

MicroRNAs (miRs) are small non-coding RNAs that regulate gene expression post-transcriptionally and are crucial in lipid metabolism. ATP-binding cassette transporter A1 (ABCA1) is essential for cholesterol efflux from cells to high-density lipoprotein (HDL). Dysregulation of miRs targeting ABCA1 can affect cholesterol homeostasis and contribute to coronary artery disease (CAD). This study aimed to investigate the expression of miRs targeting ABCA1 in human monocytes, their role in cholesterol efflux, and their relationship with CAD. We included 50 control and 50 CAD patients. RT-qPCR examined the expression of miR-33a-5p, miR-26a-5p, and miR-144-3p in monocytes. Logistic regression analysis explored the association between these miRs and CAD. HDL’s cholesterol acceptance was analyzed using the J774A.1 cell line. Results showed that miR-26a-5p (p = 0.027) and ABCA1 (p = 0.003) expression levels were higher in CAD patients, while miR-33a-5p (p < 0.001) levels were lower. Downregulation of miR-33a-5p and upregulation of ABCA1 were linked to a lower CAD risk. Atorvastatin upregulated ABCA1 mRNA, and metformin downregulated miR-26a-5p in CAD patients. Decreased cholesterol efflux correlated with higher CAD risk and inversely with miRs in controls. Reduced miR-33a-5p expression and increased ABCA1 expression are associated with decreased CAD risk. miR deregulation in monocytes may influence atherosclerotic plaque formation by regulating cholesterol efflux. Atorvastatin and metformin could offer protective effects by modulating miR-33a-5p, miR-26a-5p, and ABCA1, suggesting potential therapeutic strategies for CAD prognosis and treatment.

Cuando el yo y la carrera profesional de ese yo pasa a ser la maxima prioridad del directivo, por encima de la atencion a los demas e incluso situandose por delante de los objetivos de la empresa, el afectado padece los efectos del mal del ego. El ego, que podria parecer una virtud para ganar en seguridad personal e ir escalando laboralmente, se convierte poco a poco en un lastre que hara imposible que el directivo avance en el campo profesional. La obsesion por la imagen sera uno de los primeros sintomas que indicaran que se ha caido en brazos de este pecado directivo.

The cholesterol trafficking defect in Niemann-Pick type C (NPC) disease leads to impaired regulation of cholesterol esterification, cholesterol synthesis, and low density lipoprotein receptor activity. The ATP-binding cassette transporter A1 (ABCA1), which mediates the rate-limiting step in high density lipoprotein (HDL) particle formation, is also regulated by cell cholesterol content. To determine whether the Niemann-Pick C1 protein alters the expression and activity of ABCA1, we determined the ability of apolipoprotein A-I (apoA-I) to deplete pools of cellular cholesterol and phospholipids in human fibroblasts derived from NPC1+/+, NPC1+/-, and NPC1-/- subjects. Efflux of low density lipoprotein-derived, non-lipoprotein, plasma membrane, and newly synthesized pools of cell cholesterol by apoA-I was diminished in NPC1-/- cells, as was efflux of phosphatidylcholine and sphingomyelin. NPC1+/- cells showed intermediate levels of lipid efflux compared with NPC1+/+ and NPC1-/- cells. Binding of apoA-I to cholesterol-loaded and non-cholesterol-loaded cells was highest for NPC1+/- cells, with NPC1+/+ and NPC1-/- cells showing similar levels of binding. ABCA1 mRNA and protein levels increased in response to cholesterol loading in NPC1+/+ and NPC1+/- cells but showed low levels at base line and in response to cholesterol loading in NPC1-/- cells. Consistent with impaired ABCA1-dependent lipid mobilization to apoA-I for HDL particle formation, we demonstrate for the first time decreased plasma HDL-cholesterol levels in 17 of 21 (81%) NPC1-/- subjects studied. These results indicate that the cholesterol trafficking defect in NPC disease results in reduced activity of ABCA1, which we suggest is responsible for the low HDL-cholesterol in the majority of NPC subjects and partially responsible for the overaccumulation of cellular lipids in this disorder.

The ATP-binding cassette transporter A1 (ABCA1) is a key mediator of cellular cholesterol efflux and HDL maturation. ABCA1 mRNA has an unusually long 3’ untranslated region, which makes it highly susceptible to microRNA (miRNA) targeting and repression. As such, multiple miRNAs have been reported to directly target ABCA1, including miR-33a/b, miR-26, miR-106b, and miR-758. Many of these miRNAs participate in feed-forward or feedback networks in controlling cholesterol and lipid homeostasis. Antisense oligonucleotide-based inhibition of miR-33 was found to increase HDL-C levels and regress atherosclerosis in mice and non-human primates. In this edition of Circulation Research, two separate studies identified novel miRNA networks driven by nuclear receptor induced miR-144 targeting of ABCA1 and cholesterol efflux. The first study reports that miR-144 serves to buffer uncontrolled ABCA1 activation in response to high cholesterol states and liver X receptor (LXR) activation in macrophages and liver. The second study highlights the role of miR-144 and ABCA1 in promotion of bile acid secretion in response to farensoid X receptor (FXR) activation in the liver. These studies suggest that anti-miR-144, like anti-miR-33, could be a novel approach to targeting HDL and reverse cholesterol transport.