Enhanced P65-mediated Vascular Inflammation Accelerates Atherosclerosis In VSMC-specific SCAP Over-expressed Mice

Objective:Rather a part of individuals with atherosclerosis?AS?resulting in stroke or heart attack have normal serum lipids level,but inflammation persists through all stages of AS.Sterol Regulatory Element Binding Protein?SREBPs?Cleavage Activating Protein?SCAP?is a cellular cholesterol sensor which plays an important role in maintaining intracellular cholesterol homeostasis,to date,its influence on inflammation has been poorly reported.Besides,vascular smooth muscle cells?VSMCs?are the second most part form of foam cells composing AS plaque and participate in all events during AS progress.Therefore,we set in vivo and in vitro experiments to investigate the influence of SCAP on VSMCs inflammation and AS progress.Methods:We used VSMC-specific SCAP over-expressed transgene mice(scap^Tg/+)and normal mice(scap^+/+)to do controlled experiments.The mRNA levels of SCAP in aorta,liver and kidney were quantified by Quantitative Real-Time PCR?RT-PCR?.The protein level of SCAP in aorta,liver and kidney were presented by Immunohistochemistry?IHC?Staining.The aorta lipids levels were visualized by Oil Red O staining.The serum lipids levels were measured by Enzymatic Method.The serum inflammatory cytokines were detected by Enzyme linked immunosorbent assay?Elisa?Method.The mRNA levels in aorta or primary cultured VSMCs of inflammatory cytokines IL-6?TNF-?and IL-1?,besides alpha smooth muscle actin??-SMA?,were quantified by Quantitative Real-Time PCR?RT-PCR?.The protein level of Monocyte chemotactic protein-1?MCP-1?in aorta was presented by Immunohistochemistry?IHC?Staining.The chemotactic effect of VSMCs on macrophages was tested by Transwell Method.To predict the binding site of SREBP2 on p65 gene transcription,we referred to Bioinformatics methods,and then the interaction of SREBP2 and p65 gene was determined by Chromatin Immunoprecipitation?ChIP?Method.Results:Compared to scap^+/+mice,the expression of SCAP was increased in scap^Tg/+mice aorta,there were no significantly differences of SCAP in mice liver or kidney.Compared to scap^+/+mice,the lipids accumulation was increased in scap^Tg/+mice aorta after feeding with high cholesterol diets for 12 weeks,the changes of lipids level in scap^Tg/+mice serum had no significant difference,the inflammatory cytokines in scap^Tg/+mice serum were significantly elevated.The mRNA and protein leves of inflammatory cytokines in scap^Tg/+mice aorta or primary cultured VSMCs were significantly elevated than control mice,while the?-SMA decreased.The primary cultured scap^Tg/+mice VSMCs had enhanced chemotactic effect on macrophages than scap^+/+mice VSMCs.SREBP2 protein had binding ability with p65 gene at its promoter region and the transcription of p65 gene was regulated by SCAP-SREBP2signaling pathway.Conclusions:The SCAP-SREBP2 signaling pathway directly regulates the transcription of inflammatory gene p65 and then the expression of all kinds of inflammatory cytokines,resulting in enhanced vascular inflammatory response,thus accelerating atherosclerosis progress.