The Mechanism Of NOX4-mediated Arterial Smooth Muscle Cells Migration In Promoting Intimal Hyperplasia Of Vein Graft Anastomosis

Aims: Intimal hyperplasia is the major pathophysiological process in vein graft anastomosis.Vascular smooth muscle cells(VSMCs)are the main components of neointima.However,the origin of VSMCs and the specific mechanism of neointima formation remains unclear.NADPH oxidase 4(NOX4)formed vesicles and located to actin filaments,and promoted VSMCs migration.So far,the role of NOX4 in intimal hyperplasia at the arteriovenous anastomosis has not been reported.The purpose of this study is to explore the mechanism of NOX4 in the migration of VSMCs,and initially clarify the mechanism of intimal hyperplasia at the vein graft anastomosis.Material and Methods:1.Green fluorescent protein(GFP)transgenic and wild-type rat jugular vein-carotid artery allograft models were established.Morphological analysis was examined by HE staining.Cell types and the origin of neointima,NOX4 localization were detected by immunofluorescence staining.NOX4 protein level was measured by Western blot.2.Primary VSMCs were isolated from rat thoracic aorta.Platelet derived growth factor-BB(PDGF-BB)induced VSMCs migration and proliferation were detected by wound healing assay and Ki67 immunofluorescent staining.The protein and messenger RNA(m RNA)level of NOX4 were detected by Western blot and quantitative real-time PCR(q PCR).The role of NOX4 during VSMCs proliferation and migration were investigated using cells pretreated with small interfering RNA(si RNA)during PDGF stimulation.3.NOX4 si RNA was attached to the adventitia of autografts to examine the role of NOX4 in intimal hyperplasia.Reactive oxygen species were detected by dihydroethidium probe.Results:1.Vein graft neointima was mainly composed of artery derived VSMCs,with a high level of NOX4 expression and strong proliferative activity.2.PDGF-BB stimulation increased the protein but decreased the m RNA expression of NOX4.PDGF-BB promoted VSMCs proliferation and migration,and these effects could be inhibited by NOX4 gene silencing.3.Local NOX4 gene silencing in vivo could not alleviate intimal hyperplasia of vein graft anastomosis.Conclusion: The neointima of vein graft anastomosis is mainly composed of artery-derived VSMCs.NOX4 mediates the proliferation and migration of VSMCs and the formation of neointima.But local NOX4 gene silencing in vivo poorly inhibits intimal hyperplasia.