ADAM9:A Regulator Between HCMV Infection And Changes In Smooth Muscle Cell Function

Background: A large number of epidemiological and clinical studies have shown that human cytomegalovirus(HCMV)infection is related to the pathogenesis of atherosclerosis.HCMV infection is associated with vascular restenosis after interventional surgery and vascular sclerosis after solid organ transplantation.However,the mechanisms that HCMV is involved in these vascular lesions are very limited.Vascular smooth muscle cell(VSMC)as the main component of the blood vessel wall,is the key target of HCMV infection.Changes in VSMC function are the basis of vascular lesions,such as increased cell proliferation and migration ability and phenotypic transformation.Metalloproteinases play an important role in vascular remodeling by participating in the degradation of extracellular matrix(ECM)and the processing,activation or inactivation of various soluble factors.In this study,we screened ADAM9 by detecting the effect of HCMV infection on the expression levels of metalloproteinase genes in VSMCs,and further investigated its regulatory role between HCMV infection and VSMC functional changes.These findings contribute to our understanding of the underlying pathological mechanisms of HCMV-induced vascular lesions.Methods:(1)Human VSMCs were infected with HCMV AD169 strain in vitro.The cytopathic effect was observed by inverted microscope,and the virus particles of HCMV AD169 were detected by electron microscope.The gene expression profiles of metalloproteinases in VSMCs after HCMV infection were detected by c DNA microarray,and the differential expression of metalloproteinase was verified by real-time quantitative PCR(RT-q PCR)and enzyme-linked immunosorbent assay(ELISA).(2)ADAM9 knockdown plasmid was transfected into VSMCs before HCMV infection.VSMCs were divided into blank group,HCMV infection group,HCMV infection + empty plasmid group and HCMV+Si-ADAM9 plasmid group.For different treatment groups,CCK8 assay was used to detect the proliferation ability of VSMCs,and wound healing assay and transwell assay were used to detect the migration ability of VSMCs.(3)RT-q PCR and ELISA were used to detect the expression levels of inflammatory cytokines TNF-α,IL-6 and CX3CL1 in each treatment group.Transwell experiment was applied to construct the co-culture system of VSMC cells and THP-1-derived macrophages,and then the chemotaxis of macrophages on VSMC was detected.(4)The expression levels of ACTA2 and OPN,the markers of VSMC phenotypic transformation,were determined by RT-q PCR and westernblot(WB)to explore whether the phenotypic transformation process of VSMC was regulated by ADAM9.Results:(1)c DNA microarray showed that the m RNA levels of various metalloproteinase genes changed differently in HCMV-infected VSMCs.Among them,the m RNA level of ADAM9 showed the most significant difference.RT-q PCR and ELSA confirmed that HCMV infection could significantly up-regulate ADAM9 expression level in VSMCs.(2)HCMV infection can improve the proliferation and migration ability of VSMCs.Knockdown of ADAM9 significantly reduced the proliferation and migration ability of HCMV-infected VSMCs.(3)HCMV promoted VSMC to secrete inflammatory cytokines TNF-α and CX3CL1 by up-regulating ADAM9 expression.Transwell showed that the chemotaxis of THP-1-derived macrophages to VSMCs was significantly increased after HCMV infection.(4)HCMV infection significantly increased the protein and RNA levels of OPN,a marker of VSMC’s synthetic phenotype,while decreased the protein and m RNA levels of ACTA2,a marker of VSMC’s contractile phenotype.High expression of ADAM9 after HCMV infection could inhibit the expression of ACTA2,but had no effect on OPN.Conclusion: ADAM9 mediates the proliferation and migration of VSMCs after HCMV infection,the release of inflammatory cytokines and the chemotaxis of macrophages to VSMCs,and the transformation of VSMC phenotype.