SR-BI associates with ABCG1 and inhibits ABCG1-mediated cholesterol efflux from cells to high-density lipoprotein 3

Abstract Background The single and combined effects of scavenger receptor-BI (SR-BI), ATP-binding cassette transporter (ABC) A1 and G1 on cholesterol efflux from Chinese Hamster Ovary (CHO) cells were investigated. Results When apolipoproteinA-I (apoA-I) was used as an acceptor, ABCA1 overexpression led to an increase in total cholesterol (TC) in medium which is attributable to a 2-fold increase in free cholesterol (FC) content. When high-density lipoprotein 3 (HDL3) was used as an acceptor, SR-BI overexpression not only promoted FC efflux, but also promoted the uptake of cholesteryl ester (CE) into cells, resulting in no TC varieties in medium. Overexpression of ABCG1 increased both the FC and CE levels in medium. However, when apoA-I and HDL3 were both used as acceptors, coexpression of SR-BI has no effect on ABCA1-mediated increased FC and TC accumulation in medium. Interestingly, coexpression of SR-BI with ABCG1 blocked the ABCG1-mediated cholesterol efflux to HDL3, mostly by promoting the reuptake of CE from the medium. Furthermore, co-immunoprecipitation experiments revealed that SR-BI interacted with ABCG1 in BHK cells overexpressing ABCG1 and SR-BI. Conclusions We found SR-BI associates with ABCG1 and inhibits ABCG1-mediated cholesterol efflux from cells to HDL3.</p

Reverse-D-4F Increases the Number of Endothelial Progenitor Cells and Improves Endothelial Progenitor Cell Dysfunctions in High Fat Diet Mice

<div><p>Although high density lipoprotein (HDL) improves the functions of endothelial progenitor cells (EPCs), the effect of HDL ApoAI mimetic peptide reverse-D-4F (Rev-D4F) on EPC mobilization and repair of EPC dysfunctions remains to be studied. In this study, we investigated the effects of Rev-D4F on peripheral blood cell subpopulations in C57 mice treated with a high fat diet and the mechanism of Rev-D4F in improving the function of EPCs impaired by tumor necrosis factor-α (TNF-α). The high fat diet significantly decreased the number of EPCs, EPC migratory functions, and the percentage of lymphocytes in the white blood cells. However, it significantly increased the number of white blood cells, the percentage of monocytes in the white blood cells, and the level of vascular endothelial growth factor (VEGF) and TNF-α in the plasma. Rev-D4F clearly inhibited the effect of the high fat diet on the quantification of peripheral blood cell subpopulations and cytokine levels, and increased stromal cell derived factor 1α (SDF-1α) in the plasma. We provided in vitro evidence that TNF-α impaired EPC proliferation, migration, and tube formation through inactive AKT and eNOS, which was restored by Rev-D4F treatment. In contrast, both the PI3-kinase (PI3K) inhibitor (LY294002) and AKT inhibitor (perifosine) obviously inhibited the restoration of Rev-4F on EPCs impaired by TNF-α. Our results suggested that Rev-D4F increases the quantity of endothelial progenitor cells through increasing the SDF-1α levels and decreasing the TNF-α level of peripheral blood in high fat diet-induced C57BL/6J mice, and restores TNF-α induced dysfunctions of EPCs partly through stimulating the PI3K/AKT signal pathway.</p></div

The proportion of different leukocyte subsets in mice blood.

<p>A, Representative of the number of white blood cells per liter. B, C, and D Monocytes, neutrophils, and lymphocytes in percentage of white blood cells, respectively. Data are presented as mean±SD (n = 6). <i>**P</i> <0.01.</p

EPCs number and migratory functions in different groups of mice.

<p>A, EPCs numbers in different groups of mice. Numbers per high-power field in percentage of control. B, Different groups of mice mononuclear cells were isolated and cultured for 10 days to detect DiI-acLDL/lectin double-positive cells. Merge: green lectin, red Dil-acLDL. C, Mononuclear cells were isolated and cultured for 10 days to detect the migratory functions of EPCs using the transwell method. Numbers per high-power field in percentage of control. Scale bar represented 20μm. Data are presented as mean±SD (n = 6). <i>**P</i> <0.01.</p

Correlations between SDF-1α and TNF-α with EPCs subsets in different groups of mice, and Rev-D4F inhibited TNF-α-induced impairment of EPCs functions.

<p>A, The increased level of SDF-1α significantly correlated with the increased number of the CD34/FLK-1 subset. B, TNF-α levels inversely correlated with CD34/FLK-1 subset numbers. Samples were pretreated with a PI3K inhibitor LY294002 (30μM) or AKT inhibitor perifosine (5μM) for 2 h and incubated with Rev-D4F (50μg/ml) for 6h, EPCs were treated with TNF-α for 24h to detect the functions of viability (C and D), migration (E and F) and tube formation (G and H). EPCs tube formation ability was calculated by the average of complete tube lengths. Data are means ± SD from at least three independent experiments. <i>**P</i> <0.01 versus control, ^<i>##</i><i>P</i> <0.01 versus TNF-α, ^ΔΔ<i>P</i> <0.01 versus LY294002.</p

Rev-D4F decreased the expression of ICAM-1 and VCAM-1 in the arterial walls of C57BL/6J mice fed a high fat diet.

<p>Fluorescence intensity was calculated for the expression of ICAM-1 (A) and VCAM-1 (B). C, Representative of immunostained aortic sections with ICAM-1 and VCAM-1 antibodies. Relative fluorescence intensity in percentage of control. Scale bar represented 20μm. Data are presented as mean±SD (n = 6). <i>**P</i> <0.01.</p

The effect of Rev-D4F on plasma lipid level, NO, and cytokines (VEGF, TNF-α and SDF-1α) in different groups of mice.

<p>A, The level of total cholesterol was significantly increased in the high fat diet group of mice. B, C, and D show different groups of mice blood cytokine (SDF-1α, TNF-α and VEGF) levels. E, The blood NO level in different groups of mice was measured by an NO assay kit at 550nm. Data are presented as mean±SD (n = 6). <i>**P</i> <0.01.</p