Mechanisms Of S1PR2 Mediating Smooth Muscle Cell Proliferation And Endothelial Barrier Damage In Atherosclerosis

Objective: VSMCs proliferation and ECs dysfunction are early key factors leading to AS.S1 P is a metabolite of sphingomyelin,which involved in the development of AS by binding to S1 PRs.However,it is unclear whether S1 PRs regulate the function of VSMCs and ECs in AS.In this study,Apoe-/-spontaneously atherosclerotic mice,Ox-LDL-treated HAVSMCs and HUVECs were used to investigate the role of S1 PRs on the function of two types of target cells(VSMCs and ECs)in AS for the first time,and elucidated their mechanisms.Methods:(1)When the function damage and the expression changes of S1 PRs were studied in cell experiments,HAVSMCs and HUVECs were divided into normal group and Ox-LDL group;when the effect of S1PR2 was studied,HAVSMCs were divided into Ad-Null group,Ad-S1PR2 group,Ox-LDL+ Ad-Null group,Ox-LDL+Ad-S1PR2 group,HUVECs were divided into negative si RNA group,S1PR2 si RNA group,OxLDL+negative si RNA group,and Ox-LDL+S1PR2 si RNA group;when exploring the downstream mechanism,HAVSMCs were divided into control group,rapamycin group,Ox-LDL group,Ox-LDL+rapamycin group,and HUVECs were divided into negative si RNA group,ROCK1 si RNA group,Ox-LDL+negative si RNA group,Ox-LDL+ROCK1 si RNA group.The proliferation of HAVSMCs was detected by Ed U incorporation assay,the barrier function of HUVECs was detected by FITC-labeled dextran permeability,and S1PR1,S1PR2,S1PR3,p-Akt,Akt,p-m TOR,m TOR,PCNA,Rho A,ROCK1,VE-cadherin were detected by western blot,the expression of VE-cadherin was also detected by immunofluorescence.(2)When the expression of S1PR2 was detected in animal experiments,Apoe-/-mice were atherosclerosis group,and littermate wildtype mice were control group;when the effect of S1PR2 was studied,Apoe-/-mice were randomly divided into atherosclerosis group,S1PR2 agonist group and S1PR2 antagonist group,the wild-type littermates were considered control group,the mice were intervened for 8 weeks.Immunofluorescence was used to detect the expression of S1PR2 in aortic SMCs and ECs in vivo,aortic PCNA expression was detected by immunohistochemistry to evaluate SMCs proliferation,Evans staining was used to evaluate ECs barrier function,and oil red staining was used to evaluate lipid plaques.Result:(1)In AS mice,the expression of S1PR2 in aortic SMCs decreased and the proliferation increased,activation of S1PR2 can significantly improve the abnormal proliferation of SMCs.The expression of S1PR2 in ECs increased and the barrier function was impaired,and inhibition of S1PR2 can significantly improve ECs barrier damage.(2)The expression of S1PR2 was decreased in HAVSMCs treated with Ox-LDL,overexpression of S1PR2 inhibited the activation of Akt/m TOR and the increase of PCNA expression,and inhibited the abnormal proliferation of HAVSMCs,further inhibition of m TOR could also inhibit the increase of PCNA expression and inhibit abnormal proliferation.(3)The expression of S1PR2 was increased in HUVECs treated with Ox-LDL,silencing S1PR2 inhibited the activation of Rho A/ROCK1,upregulated the expression of VE-cadherin,and improved the cell barrier function,further silencing of ROCK1 could also up-regulate the expression of VE-cadherin and improve the cell barrier function.Conlusion:(1)The decreased expression of S1PR2 in aortic SMCs in atherosclerotic mice resulted in increased proliferation,and the increased expression of S1PR2 in aortic ECs resulted in impaired barrier function.(2)Overexpression of S1PR2 ameliorated the abnormal proliferation of SMCs induced by Ox-LDL by down-regulating Akt/m TOR/PCNA.(3)Silencing S1PR2 ameliorated Ox-LDL-induced ECs barrier damage by down-regulating Rho A/ROCK1 and up-regulating VEcadherin expression.