The Preliminary Study Of Effect Of PPAR-γ On The Phenotypic Transformation Of Vascular Smooth Muscle Cells Induced By Angiotensin Ⅱ And Its Mechanism

Background Cerebrovascular disease has become the second leading cause of death in the world.In China,ischemic stroke accounts for about 85% of all cerebrovascular diseases.According to the classic(TOAST)classification of ischemic stroke etiology,the intracranial large-artery atherosclerosis is the most common cause.The extensive resea rch on the pathogenesis of atherosclerosis indicated that vascular smooth muscle cells(VSMCs)participate in the middle and late stages of atherosclerosis.In addition,the migration,proliferation and secretion of extracellular matrix of VSMCs are involve d in the development and progression of atherosclerosis.It has been suggested that the phenotype transformation of VSMCs leads to excessive cell proliferation and migration.VSMCs show two distinct phenotypes,the ’synthetic’ and ’contractile’ phenotype.Under physiological conditions,it shows ’contractile’ phenotype with lower migration and proliferation ability.However,under the stimulation of various pathological factors,such as mechanical stimulus,the contractile type could transform into synthetic type,and the ability of migration/proliferation and secretion of extra-cellular matrixis enhanced.The phenotype transformation of VSMCs play an important role in the process of atherosclerosis,vascular restenosis and hypertension.Angiotensin Ⅱ(Ang Ⅱ),as an effector molecule of RAS system,could induce phenotype transformation of VSMCs.Peroxisome proliferator activated-receptor gamma(PPAR-γ)is a member of the nuclear hormone receptor superfamily and is a ligand dependent transcription factor.PPAR-γ has a variety of biological effects.In vivo and in vitro studies suggests that activation of PPAR-γ not only exerts a protective effect but also antagonizes the negetive effects of angiotensin Ⅱ on blood vessels.Objective To investigate the effect and mechanisms of activated PPAR-γ on the phenotypic transformation of VSMCs induced by angiotensin Ⅱ and its mechanism.Method Mouse aortic smooth muscle cells were cultured in vitro.In order to observe the effects of angiotensin Ⅱ and activated PPAR-γ on the VSMCs function and actin cytoskeleton,the VSMCs samples were divided into control group,Ang Ⅱ treatment group,rosiglitazone pretreatment + Ang Ⅱ stimulation group and /or rosiglitazone group.Cell migration was detected by Transwell assay,MTT assay was applied to detect the proliferation of cells,and the level of collagen type I was detected by ELISA assay.The morphology of microfilaments was observed under confocal laser scanning microscope.In order to investigate the effect and mechanism of activated PPAR-γ on the phenotype transformation of VSMCs induced by AngⅡ,the VSMCs samples were divided into control group,AngⅡ treatment group,rosiglitazone pretreatment + AngⅡtreatment group and/or rosiglitazone treatment group.The PPAR-γ,PKGΙα and SM22α m RNA and protein levels were detected by RT-qPCR and Western blot.To investigate the effect of rosiglitazone on the regulation of PKGΙα,the level of cGMP which is upstream activating molecule targeting PKG was detected by ELISA.The protein levels of PKGΙα and SM22α were detected by Western blot after PPAR-γ inhibitor GW9662 pretreatment to investigate whether rosiglitazone regulated the expression of PKGΙα and phenotypic transformation of VSMCs through PPAR-γ pathway.Results1)With the stimulation of angiotensin Ⅱ,the proliferation(0.68±0.03 vs.0.72±0.02,P<0.05)and migration(18.47±2.03 vs.25.13±3.38,P<0.05)of VSMCs were increased.Rosiglitazone pretreatment inhibited the migration(25.13±3.38 vs.16.87±2.85,P<0.05)and proliferation(0.72±0.02 vs.0.65±0.05,P<0.05)of VSMCs induced by angiotensin Ⅱ.2)With the stimulation of angiotensin Ⅱ,the morphology of VSMCs was irregular and cells typically produced localized protrus.Meanwhile,the number of microfilaments and the fluorescence intensity increased.After pretreatment with rosiglitazone(20μmol/L),the cells were long spindle shaped,with the disappearance of protrus.The microfilament dissolved into punctate and the fluorescence intensity decreased.3)Rosiglitazone(20μmol/L)decreased the level of collagen type Ⅰ of VSMCs(98.73±13.19 vs.75.86±27.06,P<0.05).4)The results of RT-q PCR showed that the transcription level of SM22α and PKGΙα decreased with the stimulation of angiotensin Ⅱ.Rosiglitazone(20μmol/L)pretreatment inhibited the down-regulation of PKGΙα(0.52± 0.13 vs.1.46±0.23,P<0.05)and SM22α(0.65±0.10 vs.1.18±0.30,P<0.05)m RNA level induced by angiotensin Ⅱ.5)The results of Western blot showed that the expression level of SM22α(1±0 vs.0.63±0.11,P<0.05)and PKGΙα(1±0 vs.0.76± 0.10,P<0.05)decreased with the stimulation of angiotensin Ⅱ.Rosiglitazone(20μmol/L)pretreatment inhibited the down-regulation of SM22α(0.63±0.11 vs.1.13±0.07,P<0.05)and PKGΙα(0.76± 0.10 vs.1.31±0.12,P<0.05)protein level induced by angiotensin Ⅱ.PPAR-γinhibitor GW9662 pretreatment antagonized the effects of rosiglitazone on SM22α and PKGΙα.6)Rosiglitazone had no significant effect on intracellular cGMP level of VSMCs(33.15±3.43 vs.35.55±6.18,P>0.05).Conclusions Our study suggested that PPAR–γ agonist could inhibit the phenotype transformation of VSMCs induced by angiotensin Ⅱ to suppress VSMCs proliferation,migration,secretion of extracellular matrix and actin cytoskeleton remodeling,which may be achieved by PKGΙα pathway.The activation of PKGΙα pathway induced by PPAR–γ agonist does not depend on the increment of cGMP.