Regulatory Mechanism Of SRFBP1 On Phenotypic Transition Of Vascular Smooth Muscle Cells

Vascaular hyperplastic diseases including atherosclerosis(AS),pulmonary hypertension,have the largest impact on human health currently.It can cause a variety of clinical complications such as myocardial infarction,stroke and peripheral artery disease,and has a high incidence and mortality.Phenotypic switching and abnormal proliferation of vascular smooth muscle cells(VSMCs)are the major pathological basis.Therefore,exploring the mechanism of phenotypic switching of VSMCs has important theoretical significance for elucidating the pathogenesis of vascular proliferative diseases.VSMCs have both synthetic phenotype and contractile phenotype.Synthetic VSMCs are rich of mitochondria,Golgi apparatus and roμgh endoplasmic reticellular,and high efficient of synthesis.Neointimal formation and VSMCs proliferation can be induced by cellular stress such as mechanical injuries,PDGF-BB through pathways such as PI3 K,AKT,ERK1/2,and MAPK to activate VSMCs phenotype switching.Under normal physiological conditions,VSMCs are under contractile phenotype,cells are spindle-shaped and express abundant of α actin,Mhc11,SM22α,and Calponin,to keep contractility of VSMCs and elasticity of blood vessels.The phenotype switching of VSMCs is regulated by many factors,including serum response factor(SRF),Myocardin and its cofactors.In heart,serum response factor binding protein 1(SRFBP1)is a cofactor of SRF.SRFBP1 can regulate the development of heart and keep of the structure by regulatting the expression of SRF target genes.But the function and mechanism of SRFBP1 in VSMCs phenotype switching is not clear.In order to definde the influence of SRFBP1 on VSMCs phenotype switching,human aortic VSMCs were used as the cell lines.Rat carotid artery balloon injury models and different concentrations of PDGF-BB-induced VSMCs were used to analysis the expression of SRFBP1 and contractile VSMCs marker proteins in vivo and in vitro.We build the stable VSMCs cell lines with lentivirus infection carrying negative control(NC),SRFBP1 and SRFBP1-RNAi,respectively.The proliferation of three cell lines was detected with MTT with or without PDGF-BB induction.In addition,the expression of SRFBP1 and contractile proteins such as α actin,SM22α was analysized by Western Blotting.Dual-luciferase reporter assay system was used to detect the effect of SRFBP1 on transcription activity of contractile gene promoters.1.Rat carotid artery balloon injury model was identified by SEM and HE staining.Morphological observation showed the obviously neointimal formation and VSMCs proliferation.Western Blotting showed that the expression of SRFBP1 in the model group was significantly lower than that in the sham group,indicating that SRFBP1 is negatively correlated with the proliferation of VSMCs.The results of Western Blotting assays showed that the expression of SRFBP1,α actin and SM22α was decreased with the increase of PDGF-BB concentration.The results showed that SRFBP1 was positively correlated with the expression of contractile proteins,which suggest that SRFBP1 may keep the constrictile phenotype of VSMCs.2.In this experiment,we built NC,LV-SRFBP1(UP),LV-SRFBP1-RNAi(DOWN)stably VSMCs cell lines by lentivirus infection.MTT assays results showed that SRFBP1 inhibits the proliferation of VSMCs.Western Blotting showed that overexpression of SRFBP1 increased the expression of SM22α and α actin,and knocking down SRFBP1 significantly decreased the expression of SM22α and α actin,conversely.These results indicated that SRFBP1 can effectively keep the contractile phenotype of VSMCs and inhibit the proliferation of VSMCs.3.The SM22α promoter luciferase reporter gene vector was constructed.SRFBP1 and/or Myocardin expression vectors were co-transfected with the SM22α promoter reporter vector into the 293 T cells for luciferase activity analysis.The results showed that Myocardin can promote the transcriptional activation of SM22α,while SRFBP1 alone does not affect the transcriptional activity.SRFBP1 can promote Myocardin-induced transcriptional activation.These results showed that SRFBP1 can cooperate with Myocardin to activate the transcription of the contractile marker gene SM22α.Altogether,as a cofactor,SRFBP1 increases SM22α transcriptional activities in coordination with Myocardin.It is great significance for elucidating the pathogenesis of vascular hyperplastic diseases.