Mechamisms Of Foam Cell Formation Caused By Dysfunction Of SCAP Under Inflammatory Stress

Objective:Cholesterol is derived from two pathways,one is the uptake of plasma via LDL receptor(LDLr)-mediated endocytosis,and the other is from cellular synthesis.In the pathway of cholesterol synthesis,HMGCoA reductase is the important rate-limking enzyme.Either HMGCoA reductase or LDLr activity is under control via a feedback system that is dependent on intracellular cholesterol concentration.We have demonstrated that inflammatory cytokines promote foam cell formation by increasing cholesterol uptake through disrupting cholesterol-mediated LDLr feedback regulation,inhibiting cholesterol efflux via reducing ABCA1 gene expression in peripheral cells,such as human mesangial cells(HMCs) and human vascular smooth muscle cells(VSMCs).The aim of this experiment sets out to investigate(1) if inflammatory stress disrupts LDLr-mediated feedback regulation in THP-1 macrophages,leading to excess uptake of native LDL and causing foam cell formation;(2) the mechanisms of abnormal translocation of SCAP-SREBP2 complex in VSMCs under inflammatory stress;(3) if there are differences between THP-1 macrophages and VSMCs regarding regulation of exogenous cholesterol uptake through LDLr under physiological and inflammation conditions.Materials and Methods:THP-1 macrophages and VSMCs were used in the present study.THP-1 macrophages were treated by no LDL loading, LDL loading alone,LDL plus lipopolysaccharide(LPS) co-loading and LPS loading alone,and VSMCs were treated by no LDL loading,LDL loading alone,LDL plus LPS co-loading,LDL plus LPS and swainsonine co-loading and LPS loading alone,then intracellular cholesterol content,tumor necrosis factor alpha(TNF-α) level of supernatants,mRNA and protein expression of LDLr,SREBP2,SCAP,SR-A and CD36 were assessed by Oil Red O staining,cholesterol enzymatic assay,enzyme-linked immunosorbent assay (ELISA),real time quantitative polymerase chain reaction(PCR),western blotting analysis and immunocytochemistry,respectively.SR-A protein expression in macrophages was examined by immunofluorescence method. Cell survival rate was measured by MTT method.Results:We demonstrated that LPS enhanced transformation of THP-1 macrophages into foam cells by increasing uptake of unmodified LDL via LDLr as evidenced by Oil Red O staining and direct assay of intracellular cholesterol.In the absence of LPS,LDL decreased LDLr mRNA and protein expression.However,LPS enhanced LDLr expression,overriding the suppression of LDLr induced by LDL and inappropriately increasing LDL uptake.Exposure to LPS also caused over-expression of mRNA and protein of SCAP and SREBP2.These observations indicate that LPS disrupts cholesterol-mediated LDLr feedback regulation,permitting intracellular accumulation of unmodified LDL and causing foam cell formation.In addition,we also showed that LPS enhanced transformation of VSMCs into foam cells by increasing uptake of unmodified LDL via LDLr as evidenced by Oil Red O staining and direct assay of intracellular cholesterol.The mechanism is that LPS caused over-expression of mRNA and protein of SCAP and SREBP2 and translocation of SCAP-SREBP2 complex from ER to Golgi.Furthermore,we demonstrated that the inflammatory stress by LPS induced the glycosylation of SCAP,and the modified SCAP returned to ER to escort more SREBP2 from ER to Golgi for activation.Alpha-mannosidaseⅡis the key enzyme of glycosylation of SCAP in Golgi enzymes,and its specific inhibitor swainsonine could reduce the abnormal translocation of SCAP-SREBP2 complex induced by LPS and downregulate the mRNA and protein expression of LDLr,SREBP2 and SCAP,restoring the LDLr-mediated feedback regulation.We proceeded to show that in the physiological condition LDL loading downregulated LDLr mRNA level in both THP-1 macrophages and VSMCs,but to a greater degree in VSMCs.In the inflammatory stress,LDLr mRNA levels were upregulated even in the presence of high concentration of LDL in both cells,but to a greater degree in THP-1 macrophages, suggesting that macrophages were more sensitive to LPS than VSMCs and easily transformated into foam cells.Conclusion.Taken together,these results imply that the normally tight sterol-dependent feedback regulation of LDLr in THP-1 macrophages is disrupted by LPS.Thus,in the presence of LPS,native LDL is taken up in excess via LDLr and results in massive cholesterol ester accumulation. These processes convert THP-1 macrophages into foam cells.The implications of these findings are that native LDL can be atherogenic without prior modification by oxidation in THP-1 macrophages and LDLr may be another important pathway of foam cell formation in macrophages under inflammatory stress.Under inflammation,SCAP was glycosylated byα-mannosidaseⅡof Golgi and abnormally circulated from ER to Golgi in VSMCs,which may be a target for treatment for atherosclerosis in the future. Furthermore,THP-1 macrophages are more sensitive to LPS than VSMCs and easily transformated into foam cells.