| The proliferation and migration of vascular smooth muscle cell(VSMC) play a major role in the development and progression of many cardiovascular diseases,including atherosclerosis(AS)and restenosis (RS)after percutaneous coronary intervention(PCI).During the early stages of AS or arterial wall injury,VSMC migrates into the intimal layer of arterial wall in response to platelet activation,thrombin generation,and the release of various growth factors and cytokines, causing Neointima(NI)thickening.Particularly,the cytokine tumor necrosis factor(TNF)-αis secreted by VSMC in the neointima as well as by macrophages accumulated in atherosclerotic lesions and it markedly induces proliferation and migration of VSMC.In addition,Prior studies reported that arterial injury induces expression of matrix metalloproteinase(MMP)-9 to degrade extracellular matrix(ECM),a prerequisite for VSMC proliferation and migration.The synthesis and secretion of MMP-9 can be stimulated by treatment of VSMC with TNF-α,or by arterial injury.The signaling pathways and transcriptional mechanisms that regulate MMP-9 expression in response to arterial injury or TNF-αin vitro are the subject of intense research but remain elusive.Our previous findings demonstrated that protein kinase C(PKC)β, an upstream regulator of early growth response(Egr)-1,contributed to VSMC proliferation in expanding neointima upon vascular injury,and regulated VSMC proliferation and migration upon stimulation with the prototypic stimulus of PKCβ,phorbol 12-myristate 13-acetate(PMA). In this study,we tested the hypothesis that genetic deletion of PKCβor Egr-1 would attenuate activation of MMP-9 in pathological processes consequent to vascular injury in vivo or stimulation with TNF-αin vitro;and further investigated the signaling pathway involved in PKCβ-mediated Egr-1 expression.Endothelial denudation injury was performed in murine femoral artery.Primary cultured VSMCs were isolated from WT or transgenetic mouse aorta by enzyme dispersal.Activity of MMP-9 was detected by zymography;MMP-9 antigen and MAPK signaling by Western blot;mRNA expression of MMP-9 and Egr-1 by real-time PCR(RT-PCR).In vivo,Activity/antigen of MMP-9 was induced in a time-dependent manner upon denuding injury in wild type(WT)mice.A peak activity/antigen of MMP-9 in vessels of WT mice was observed at 8h after denuding injury.In contrast,Activity/antigen of MMP-9 in vessels from PKCβor Egr-1 null mice was significantly lower than that in vessels from WT mice at 8h after injury(P＜0.001).In primary cultured aortic SMC from WT mice,secretion/expression of MMP-9 was induced in a time-dependent manner upon TNF-αstimulation.A peak activity/antigen of MMP-9 in the conditioned culture media of WT VSMC was observed at 24h,and the peak mRNA expression of MMP-9 at 12h after TNF-αstimulation.In contrast,induction of MMP-9 stimulated by TNF-αwas attenuated in PKCβor Egr-1 null VSMC(P＜0.001).Upon TNF-αstimulation,Egr-1 mRNA expression showed a rapid induction within minutes and rapid decay in one hour,which was attenuated by genetic deletion of PKCβ.Upon denuding injury in WT mice or stimulation by PMA in WT VSMC, phosphorylation of both ERK1/2 and JNK was significantly increased, which was attenuated by genetic deletion of PKCβ. These data critically link PKCβand Egr-1 to MMP-9 activation in vascular injury and cultured VSMC,and highlight new targets for therapeutic intervention to limit restenosis.
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