The Role And Mechanism Of Sox9 Regulating Vascular Smooth Muscle Cell Phenotypic Modulation And Transplant Arteriosclerosis

Objective: Chronic rejection is a leading cause of late allograft failure after organ transplantation.The common histopathological feature of chronic rejection in all transplanted organs is generalized transplant arteriosclerosis(TA).The phenotypic modulation of vascular smooth muscle cells(SMCs)is a key event for the initiation and progression of neointimal formation and TA.However,the molecular mechanisms underlying this process remain largely unknown.This study aims to explore the role and underlying mechanism of transcript factor Sox9 in vascular SMC phenotypic modulation and TA.Methods: We employed lentivirus-mediated gene transfer of short hairpinRNA(shRNA) targeting/against Sox9(Sox9-KD)in rat aortic allografts,whose selective knockdown in medial SMCs is modulated by aminimal SM22? promoter,and the rat model of aortic transplantation was established to detect Sox9 expression and its role in phenotypic modulation and neointimal formation in vivo.In vitro,cultured aortic SMCs or 293T cells were treated with HMGB1 and/or infected with shRNA or plasmids to explore the role and molecular mechanism of Sox9 in vascular SMC phenotypic modulation.Results: Increased expression of Sox9 was found in aortic allografts as well in cultured,HMGB1-activated vascular SMCs,accompanied by a reduction in the expression of Calponin and SM22?,2 differentiation markers of vascular SMCs.Lentivirus-mediated shRNA transfection effectively inhibited Sox9 expression in medial SMCs while restoring the expression of vascular SMC differention genes,associated with impaired neointimal formation in aortic allografts.In cultured vascular SMCs,shRNA knockdown f Sox9(Sox9-KD)markedly improved HMGB1 induced downregulation of ifferentiation genes accompanied by reduced cell proliferation and migration.Moreover,p27 repressed Sox9 transcriptional expression through binding to its promoter.The development of autophagosome and autophagolysosome induced by HMGB1 stimulation contributed to p27 protein ubiquitination and subsequently degradation,leading to the upregulation of Sox9.Inhibition of autophagy by bafilomycin A1 or shRNA knockdown of Atg5 strikingly upregulated p27 protein expression and differentiation genes,while repressed the transcription and expression of Sox9 even in the stimulation of HMGB1.Importantly,shRNA transfection of Atg5 in medial SMCs also significantly reduced neointimal formation in aortic allografts.Conclusions: Sox9 acts as an important downstream factor of autophagy/p27 signaling triggered by allogeneic transplantation that controls phenotypic modulation of vascular SMC and neointimal formation.Modulating Sox9 in vascular SMC may represent a new therapeutic approach to treat TA after transplantation.