CXCL12 Promotes Atherosclerosis By Downregulating Macrophage-Derived ABCA1 Expression

【Background and Aim】Overladen lipid induces macrophage to transform foam cell that contributing to the formation of atherosclerotic plaque,which is the hallmark of atherosclerosis.ATP-binding cassette-transporter A1（ABCA1）is a membrane protein and well known to be a cholesterol transporter.ABCA1-mediated the efflux of intracellular cholesterol to plasma apolipoprotein A-I（apoA-I）,followed by forming high density lipoprotein（HDL）,which is identified as the limited step of reverse cholesterol transport（RCT）.CXC chemokine ligand 12（CXCL12）belongs to CXC chemokine family and has various biological functions.It has been reported that CXCL12 involved the regulation of inflammatory cytokines secretion,platelet aggregation and the insulin resistance in diabetic rat.Recently,CXCL12 has been discovered to be associated with the plasma lipid profile of coronary heart disease（CHD）patients,suggesting an underlying effect of CXCL12 on cardiovascular disease（CAD）.In addition,silencing of CXCL12 could effectively reduced atherosclerotic plaques.However,it remains to be expounded the molecular mechanisms by which CXCL12-regulated the development and progression of atherosclerosis.Also,it was unclear about the distinct of role of CXCL12 in macrophage cholesterol efflux.Therefore,we aim to explore the effect of CXCL12 on lipid metabolism and its potential molecular mechanism by which further reveal the pro-atherogenic role of CXCL12.Our study is expected to reveal the molecular mechanism of CXCL12-aggravated atherosclerosis and provide a novel therapeutic target in future.【Methods】Incubation of 80 ng/ml CXCL12 with THP-1-derived macrophages,followed by related experiments.Detection of ABCA1expression using western blot,RT-PCR and immunofluorescence assay.High-performance liquid chromatography（HPLC）and liquid scintillation counter（LSC）were performed to examine the intracellular lipid contents and cholesterol efflux from cellular.Detection of TCF21 expression using western blot,and RT-PCR.Luciferase reporter gene and Chromatin immunoprecipitation assay were used to assess the effect of CXCL12 and TCF21 on ABCA1 promoter activity,as well as the role of CXCL12 in the binding of TCF21 to ABCA1 DNA region.Detection ofβ-catenin and p-β-catenin^T120 levels using western blot.Detection of ABCA1 and TCF21 expression using western blot and RT-PCR,after induction of cells withβ-catenin agonist SKL2001.Detection of GSK3βand p-GSK3βexpression using western blot.Detection of ABCA1 and TCF21expression using western blot and RT-PCR,and examination ofβ-catenin and p-β-catenin ^T120 levels by western blot,after induction of cells withβ-catenin agonist SKL2001.Transfection with CXCR4 siRNA or incubation of its inhibitor LY2510924 with cells,western blot and immunofluorescence assay were used to assess the interference efficient,and western blot was performed to examine the expression of TCF21,ABCA1,β-catenin,p-β-catenin ^T120,GSK3βand p-GSK3β.Apoe^-/-mice were injected with viral particles of LV-CXCL12 to overexpress CXCL12,followed by related experiments.ELISA kit was performed to detect plasma CXCL12 levels.HE,Oil Red O and Masson staining were used to assess the area,lipid and collagen contents within atherosclerotic plaques.Immunofluorescence assay was used to detect the expression of ABCA1and CD68 in atherosclerotic plaques.Also,immunohistochemistry was used to assess the macrophage infiltration.Detection of ABCA1expression in aorta and peritoneal macrophages isolated from Apoe^-/-mice using western blot and RT-PCR.Specific kits were used to measure the plasma TC,HDL-C,LDL-C and TG levels in Apoe^-/-mice.Liquid scintillation counter（LSC）was performed to examine the cholesterol efflux from peritoneal macrophages and the efficiency of RCT in Apoe^-/-mice.【Results】CXCL12 reduced ABCA1 expression and cholesterol efflux,as well as induced lipid deposition in THP-1-derived macrophages.Incubation of CXCL12 with cells can inhibit ABCA1 promoter activity,downregulate TCF21 expression and block the binding of TCF21 to ABCA1 promoter region.CXCL12 decreased nuclearβ-catenin contents by increasing the level of p-β-catenin^T120.In addition,β-catenin agonist SKL2001 partly inhibited the effect of CXCL12 on TCF21 and ABCA1expression.Also,GSK3βinhibitor TWS119 prevented CXCL12-induced phosphorylation ofβ-catenin^T120and inhibited the downregulation of TCF21 and ABCA1 expression by CXCL12.Meanwhile,CXCR4 siRNA or its inhibitor LY2510924 obviously blocked the effect of CXCL12 on GSK3β,β-catenin,p-β-catenin ^T120,TCF21 and ABCA1 expression。In vivo,overexpression CXCL12 significantly reduced the plasma HDL-C levels and expanded atherosclerotic plaques in Apoe^-/-mice,as well as aggravated macrophage infiltration within atherosclerotic plaques.Besides,we detected that CXCL12 markedly reduced the expression of ABCA1 in aortas and peritoneal macrophages in Apoe^-/-mice.Also,CXCL12 exerted an inhibitory effect on cholesterol efflux from peritoneal macrophages and RCT rate.【Conclusions】1.CXCL12 accelerates atherosclerosis by inhibiting cholesterol efflux from macrophage via reducing ABCA1 expression;2.CXCL12 activates GSK3β/β-catenin signaling that contributing to downregulating TCF21 and ABCA1 expression.