Cholesterol Efflux In Human Macrophages Research Articles

Cholesterol efflux is LXRα isoform-dependent in human macrophages.

BackgroundThe nuclear receptor liver X receptor (LXR) has two isoforms: LXRα and LXRβ. LXR activation promotes cholesterol efflux in macrophages, but the relative importance of each LXR isoform in mediating cholesterol efflux remains elusive.MethodsWe evaluated the ability of different doses of LXRs agonist T0901317 to affect cholesterol efflux in human macrophages and its relationship with mRNA and protein levels of several well-characterized proteins involved in cholesterol efflux, including ABCA1, ABCG1, SR-BI, LXRβ and LXRα, using quantitative real-time PCR, Western blotting, and siRNA techniques.ResultsHere we show that LXRα rather than LXRβ sustains baseline cholesterol efflux in human blood-derived macrophages. Treatment of human macrophages with a non-isoform-specific LXR agonist T0901317 substantially increased HDL- and apoA-I-mediated cholesterol efflux, which was associated with increased mRNA and protein expression levels of ABCA1, ABCG1, SR-BI, LXRα and LXRβ. The siRNA- mediated silencing of LXRα, but not LXRβ significantly reduced the protein levels of ABCA1,ABCG1, and SR-BI as wellas HDL- and ApoA1-mediated cholesterol in human macrophages.ConclusionsThese findings imply that LXRα- rather than LXRβ- specific agonists may promote reverse cholesterol transport in humans.

MiR-206 controls LXRα expression and promotes LXR-mediated cholesterol efflux in macrophages

Liver X receptors (LXRα and LXRβ) are key transcription factors in cholesterol metabolism that regulate cholesterol biosynthesis/efflux and bile acid metabolism/excretion in the liver and numerous organs. In macrophages, LXR signaling modulates cholesterol handling and the inflammatory response, pathways involved in atherosclerosis. Since regulatory pathways of LXR transcription control are well understood, in the present study we aimed at identifying post-transcriptional regulators of LXR activity. MicroRNAs (miRs) are such post-transcriptional regulators of genes that in the canonical pathway mediate mRNA inactivation. In silico analysis identified miR-206 as a putative regulator of LXRα but not LXRβ. Indeed, as recently shown, we found that miR-206 represses LXRα activity and expression of LXRα and its target genes in hepatic cells. Interestingly, miR-206 regulates LXRα differently in macrophages. Stably overexpressing miR-206 in THP-1 human macrophages revealed an up-regulation and miR-206 knockdown led to a down-regulation of LXRα and its target genes. In support of these results, bone marrow-derived macrophages (BMDMs) from miR-206 KO mice also exhibited lower expression of LXRα target genes. The physiological relevance of these findings was proven by gain- and loss-of-function of miR-206; overexpression of miR-206 enhanced cholesterol efflux in human macrophages and knocking out miR-206 decreased cholesterol efflux from MPMs. Moreover, we show that miR-206 expression in macrophages is repressed by LXRα activation, while oxidized LDL and inflammatory stimuli profoundly induced miR-206 expression. We therefore propose a feed-back loop between miR-206 and LXRα that might be part of an LXR auto-regulatory mechanism to fine tune LXR activity.

HMG-CoA Reductase Inhibitors, Simvastatin and Atorvastatin, Downregulate ABCG1-mediated Cholesterol Efflux in Human Macrophages

Recent studies showed that statins reduce ATP-binding cassette transporter A1 expression and ATP-binding cassette transporter A1-mediated cholesterol efflux in macrophages, whereas the effect of statins on ATP-binding cassette transporter G1 (ABCG1), another important lipid transporter, is still unclear. Here, we investigated the expression and functionality of ABCG1 on statins in THP-1-derived macrophages and human peripheral blood mononuclear cells. Simvastatin and atorvastatin were used in this study. Treatment with statins significantly decreased ABCG1-mediated cholesterol efflux in human macrophages (from 33.8% ± 2.8% of control to 22.9% ± 1.7% of 10 µM simvastatin or to 23.3% ± 3.3% of 10 µM atorvastatin; P < 0.01, n = 4), whereas the protein expression of ABCG1 remained unaltered on statins. Further analysis revealed that 2 major ABCG1 isoforms responded to statins differently. The expression of ABCG1-S, which exhibited higher activity in cholesterol efflux than ABCG1-L, was significantly decreased on statins compared with increased expression of ABCG1-L. The results suggested that the proportion change of ABCG1 isoform expressions could contribute to reduced ABCG1 functionality under treatment with statins. The effects of statins on ABCG1 isoform expression and functionality were reversed by low-dose liver X receptor agonist, TO-901317, indicating that statins' downregulation of ABCG1 functionality was likely through liver X receptor-dependent pathway. In conclusion, simvastatin and atorvastatin decreased ABCG1-mediated cholesterol efflux in human macrophages without alteration of total ABCG1 protein level. The proportion change of ABCG1 isoforms expressions may be involved in the down-regulation of ABCG1 functionality by statins, which provided a novel mechanism for the regulation of ABCG1 activity.

Atherogenic Lipids Induce High-Density Lipoprotein Uptake and Cholesterol Efflux in Human Macrophages by Up-Regulating Transmembrane Chemokine CXCL16 without Engaging CXCL16-Dependent Cell Adhesion

Atherosclerosis is a complex pathologic process in which chemokine-mediated leukocyte accumulation in arterial walls is thought to be an important mechanism of pathogenesis. An interesting exception to this paradigm is the chemokine CXCL16, also known as the scavenger receptor for phosphatidylserine and oxidized low density lipoprotein, which is highly expressed in mouse and human atherosclerotic lesions, yet appears to be atheroprotective. In this study, we address potential mechanisms responsible for this activity. Consistent with its presence in atherosclerotic plaque, we found that atherogenic lipids up-regulated CXCL16 in primary human monocyte-derived macrophages. However, the same lipids down-regulated the CXCL16-targeted protease ADAM10, resulting in preferential expression of CXCL16 as the transmembrane form, not the shed form. Although transmembrane CXCL16 is known to mediate cell-cell adhesion by binding its receptor CXCR6, and atherogenic lipids are known to stimulate macrophage adhesion to coronary artery smooth muscle cells, we found that heterotypic adhesion of these cell types occurred in a CXCL16-independent manner. Instead we found that in macrophages, CXCL16 promoted internalization of both oxidized low density lipoprotein and high density lipoprotein, as well as release of cholesterol. Moreover, CXCL16 deficiency in macrophages interfered with oxidized low density lipoprotein-induced up-regulation of atheroprotective genes: adenosine triphosphate-binding cassette transporter A1 and G1 as well as apolipoprotein E. Thus, our findings support the hypothesis that CXCL16 mediates atheroprotection through its scavenger role in macrophages and not by cell-cell adhesion.

Trans-9,trans-11-CLA is a potent liver X receptor agonist and enhances cholesterol efflux in human macrophages

trans-9,trans-11-CLA is a potent liver X receptor agonist and enhances cholesterol efflux in human macrophages

17beta-estradiol enhances the flux of cholesterol through the cholesteryl ester cycle in human macrophages.

Estrogens have been shown to have many positive effects on the function of arterial wall, and recent evidence suggest that 17beta-estradiol has a direct action in reducing the accumulation of cholesteryl ester in macrophages. The mechanisms underlying the effects of 17beta-estradiol on foam cell formation, however are poorly understood. The aim of this study is to investigate the role of 17beta-estradiol in the regulation of the cholesteryl ester cycle and cholesterol efflux in human macrophages. In addition, the influence of 17beta-estradiol on apolipoprotein E (apoE) and lipoprotein lipase (LDL) secretion by the cells was also tested. Human Monocyte Derived Macrophages (HMDM), matured in the presence or the absence of 17beta-estradiol, were loaded with [3H]-cholesteryl ester-labeled-acetyl LDL (low density lipoprotein) and the efflux of radioactivity into the medium was measured. The effect of 17beta-estradiol on cellular activities of acyl coenzyme A: cholesterol acyl transferase (ACAT), and both neutral and acid cholesteryl ester hydrolase (CEH) and the secretion of apoE and LDL into the medium, were also studied. The results indicate that 17beta-estradiol induces an increase in the amount of labeled cholesterol released from the cells and, the data obtained from the measurements of ACAT and CEH activities showed that, in estrogen-treated HMDM, the cholesteryl ester cycle favors the hydrolysis of lipoprotein cholesterol by CEH in comparison with its acylation by ACAT. In particular, for the first time a strong enhancement of neutral and acid CEH in human macrophages by 17beta-estradiol, was demonstrated. ApoE and LDL secretion increased during the maturation of monocytes to macrophages, and was not modified by 17beta-estradiol. In contrast, loading the cells with cholesterol by incubation in the presence of acetylated or oxidized LDL produced an increase in the levels of apoE secreted by both estrogen-treated and control macrophages. The activity of LPL found in the cell medium, on the other hand, in lipid loaded cells tended to be increased only in estrogen treated macrophages, suggesting that the effects of estrogen on unloaded macrophages are different from those produced on lipid-loaded macrophages. On the whole, the present findings suggest that one of the mechanisms by which 17beta-estradiol acts to reduce cholesterol accumulation in macrophages is by increasing reverse cholesterol transport through the enhancement of the cholesteryl ester cycle, so that the generation of intracellular unesterified cholesterol for excretion from the cells is favored.

PPAR-alpha and PPAR-gamma activators induce cholesterol removal from human macrophage foam cells through stimulation of the ABCA1 pathway.

Peroxisome proliferator-activated receptors (PPARs) are nuclear receptors that regulate lipid and glucose metabolism and cellular differentiation. PPAR-alpha and PPAR-gamma are both expressed in human macrophages where they exert anti-inflammatory effects. The activation of PPAR-alpha may promote foam-cell formation by inducing expression of the macrophage scavenger receptor CD36. This prompted us to investigate the influence of different PPAR-activators on cholesterol metabolism and foam-cell formation of human primary and THP-1 macrophages. Here we show that PPAR-alpha and PPAR-gamma activators do not influence acetylated low density lipoprotein-induced foam-cell formation of human macrophages. In contrast, PPAR-alpha and PPAR-gamma activators induce the expression of the gene encoding ABCA1, a transporter that controls apoAI-mediated cholesterol efflux from macrophages. These effects are likely due to enhanced expression of liver-x-receptor alpha, an oxysterol-activated nuclear receptor which induces ABCA1-promoter transcription. Moreover, PPAR-alpha and PPAR-gamma activators increase apoAI-induced cholesterol efflux from normal macrophages. In contrast, PPAR-alpha or PPAR-gamma activation does not influence cholesterol efflux from macrophages isolated from patients with Tangier disease, which is due to a genetic defect in ABCA1. Here we identify a regulatory role for PPAR-alpha and PPAR-gamma in the first steps of the reverse-cholesterol-transport pathway through the activation of ABCA1-mediated cholesterol efflux in human macrophages.