The Study On The Inhibitory Effects And Mechanisms Of Cenerimod On PDGF-BB-induced Proliferation And Migration Of Vascular Smooth Muscle Cells

Background:Cardiovascular disease has become the leading cause of death worldwide and coronary heart disease is the primary cause of death in cardiovascular disease.Percutaneous coronary intervention is an important treatment for coronary heart disease,but restenosis after stenting seriously affects its long-term efficacy.Proliferation,migration and phenotypic transformation of vascular smooth muscle cells are the important pathological basis of atherosclerosis and restenosis after interventional operation.Studies have shown that non-selective s1p1-5 receptor modulator Fingolimod can inhibit the proliferation and migration of vascular smooth muscle cells induced by PDGF-BB,and the Hippo-YAP pathway plays an important role in the proliferation,migration and phenotypic transformation of vascular smooth muscle cells.But does selective S1P1 receptor modulator have an inhibitory effect on proliferation and migration of vascular smooth muscle cells?Is the mechanism related to the Hippo-YAP pathway?It's not clear.The purpose of this study was to investigate the effect of Cenerimod,a selective S1P1 receptor modulator,on PDGF-BB-induced proliferation and migration of vascular smooth muscle cells,and to explore the molecular mechanism of its effect by taking the Hippo-YAP pathway as a starting point.Methods:PDGF-BB?25ng/mL?was used to induce Human Aortic Smooth Muscle Cells?HASMC?and Rat Aortic Smooth Muscle Cells?RASMC?to establish a model of proliferation and migration of Smooth Muscle Cells,Cenerimod?7.5umol/L?treatment was given,and control was set simultaneously.CCK8 assay was used to detect the effect of Cenerimod on PDGF-BB-induced smooth muscle cell proliferation.Transwell assay was used to detect the effect of Cenerimod on smooth muscle cell migration induced by PDGF-BB.RT-qPCR assay was performed to detect the mRNA expression of LATs and YAP,key genes of the Hippo pathway.The protein expressions or phosphorylation levels of LATs1,pLATs1,YAP,pYAP,MCP-1,CyclinA1 and P21,key genes of the Hippo pathway,were detected by Western blot.Bioinformatics was used to predict the transcription factors bound to the YAP promoter region,and Western blot assay was used to detect the influence of Cenerimod in HASMC and RASMC on the expression of YAP and the predicted transcription factor protein.After the transcription factor siRNA was used to interfere with the expression of transcription factor,RT-qPCR and Western blot were used to detect the influence of transcription factor interference on the expression of YAP m RNA and protein.Results:Cell proliferation assay showed that Cenerimod could significantly inhibit PDGF-BB induced human aortic smooth muscle cell proliferation?P<0.01?.Transwell assay found that Cenerimod could reduce the migration of human aortic smooth muscle cells induced by PDGF-BB?P<0.01?.RT-qPCR showed that Cenerimod could down-regulate the expression levels of LATs1 mRNA and YAP mRNA induced by PDGF-BB?P<0.05?.Western blot analysis showed that Cenerimod significantly down-regulated the expression levels of LATS1,pLATS1,YAP and pYAP proteins in PDGF-BB-induced HASMCs?P<0.05?,down-regulated the expression levels of MCP-1and CyclinA1 proteins in PDGF-BB-induced HASMCs?P<0.01?,and up-regulated the expression levels of P21 and pP21 proteins in PDGF-BB-induced HASMCs?P<0.01?.Through bioinformatics prediction,it was found that TBP,c-jun and P53 may be transcription factors of YAP.Western Blot showed that the expression of YAP,TBP,c-jun and P53 proteins in HASMC nucleus decreased after Cenerimod treatment?P<0.01?.The expression levels of total YAP protein and related target transcription factor proteins?TBP,c-jun and P53?in RASMC cells decreased significantly?P<0.001?.RT-qPCR and Western blot showed that after transfection of TBP si RNA to RASMC,the expression levels of YAP mRNA and protein in the NC siRNA group were similar to those in the control group?P>0.05?.The expression levels of YAP mRNA and protein in TBP siRNA group were significantly lower than that in NC siRNA group?P<0.01?.The expression levels of YAP mRNA and protein in the TBP siRNA group were significantly higher than those in the Cenerimod group?P<0.0001 and P<0.05?.RT-qPCR and Western blot showed that after Jun si RNA transfection to RASMC,the expression levels of YAP mRNA and protein in NC siRNA group were basically the same as that in control group?P>0.05?.The expression levels of YAP mRNA and protein in Jun siRNA group were significantly lower than that in NCsi RNA group?P<0.01?.The expression levels of YAP mRNA and protein in Jun siRNA group were slightly higher than that in Cenerimod group?P<0.05?.Conclusion:Cenerimod,a novel highly selective S1P₁ receptor modulator,can reduce the expression of LATS and YAP,the key factors of the Hippo-YAP pathway,so as to block the Hippo-YAP pathway and inhibit the proliferation and migration of PDGF-BB-induced VSMCs.Cenerimod may inhibit the proliferation and migration of human vascular smooth muscle cells by activating P21,down-regulating the expression of McP-1 and Cyclin A1.Cenerimod down-regulates the expression of YAP and transcription factors TBP,c-jun and P53 in vascular smooth muscle cells,and transcription factors TBP and c-jun may be involved in regulating the inhibition of Cenerimod on the expression of YAP.