Calpastatin/Calpain System Ameliorates Vascular Restenosis Through MMP2/TGF-?1 Pathway

Objective:The present study used over-expressing calpastatin mice and vascular smooth mucle cells cultured in vitro to investigate the role of Calpastatin/Calpain system in simvastatin attenuated PDGF-induced vascular restenosis and proliferation,migration and collagen synthesis in VSMCs.Methods:The whole experiment was divided into two parts:animal experiment and cell experiment.Part I:Animal experiment.1.Groups:WT,WT Ligation,TG,TG+Ligation.2.Animal Model:carotid bifurcation ligation.3.Analysis of the expression of Calpain-1,Calpain-2,Calpastatin mRNA in carotid by using qRT-PCR method.4.After 0,7,14,21,28 days,the carotid artery were stained with hematoxylin&eosin(H&E),the areas of intima and media of carotid artery were analyzed.5.Fourteen days after surgery,the expressions ofPCNA,Collagen I,Calpain-1,Calpain-2,Calpastatin,MMP2,MT1MMP,TIMP2 of carotid artery were determined by using immunohistochemical staining.Part I:Cell experiment1.Groups:WT,WT+PDGF-BB,WT+PDGF-BB+Simvastatin,TG,TG+PDG F-BB,TG+PDGF-BB+Simvastatin2.The proliferation of vascular smooth muscle cells in different groups was detected using CCK8 kit.3.The directional and no-directional migration of vascular smooth muscle cells in different groups was respectively evaluated using a Transwell chamber and wound healing.4.Analysis of the expression of Calpain-1,Calpain-2,Calpastatin,MMP2,MT1MMP,TIMP2,Collagen?,TGF-?1 mRNA of vascular smooth muscle cells in different groups were measured by qRT-PCR method.5.The expressions of PCNA,Collagen I,Calpain-1,Calpain-2,Calpastatin,MMP2,MT1MMP,TIMP2 of vascular smooth muscle cells in different groups were determined by using western blotting.Results:1.Expressions of calpain-1 and calpain-2 were increased in carotid restenosis,whereas expression of calpastatin was decreased.2.Over expression of calpastatin attenuated carotid restenosis,and expressions of calpain-1 and calpain-2 were decreased.3.Overexpression of calpastatin inhibited expressions of MMP2 and TGF-?1 in carotid restenosis.4.Overexpression of calpastatin suppressed PDGF-induced proliferation and migration of VSMCs and synthesis of collagen?and?via inhibiting calpain-1/2 expression and activity.5.Overexpression of calpastatin inhibited PDGF-induced expressions of MMP2 and TGF-?1.6.Simvastatin attenuated PDGF-induced proliferation and migration of VSMCs and synthesis of collagen?and?via calpastatin/calpain system mediated inhibition of MMP2/TGF-?1.Conclusion:1.Expressions of calpain-1 and calpain-2 were increased in pathological process of vascular restenosis,whereas expression of calpastatin was decreased.2.Over expression of calpastatin attenuated vascular restenosis.3.Expressions of MMP2 and TGF-?1 were regulated by Calpastatin/Calpain system via MT1MMP/TIMP2.4.Overexpression of calpastatin suppressed PDGF-induced proliferation and migration of VSMCs and synthesis of collagen?and?via inhibiting calpain-1/2 expression and activity.5.Calpastatin/calpain system was an important pathway to regulate simvastatin attenuated PDGF-induced vascular restenosis.