Sox9在血管平滑肌细胞表型转化和移植物动脉硬化中的作用及机制研究

Solid-organ transplantation is currently the most effective therapy for patients with end-stage organ failure. Chronic allograft rejection is the leading cause of chronic allograft dysfunction following transplantation. Transplant arteriosclerosis is a common histopathological characteristic of chronic rejection in different transplants including heart, kidney and liver. It has been demonstrated that phenotypic modulation of vascular smooth muscle cells (SMCs) from contractile phenotype to synthetic phenotype is a central event in the initiation and progression of neointimal formation and transplant arteriosclerosis after transplantation. However, so far the molecular mechanism underlying SMC phenotypic modulation in allografts remains to be elucidated. Our preliminary studies indicated that Sox9 that acts as a downstream transcription factor of HMGB1 signaling triggered by allogeneic transplantation may play a critical role in inducing SMC phenotypic modulation and subsequent transplant arteriosclerosis.. In this project, to explore the role of Sox9 in the regulation of SMC phenotypic modulation and its possible mechanism, rat aortic SMCs will be cultured in vitro and exposed to HMGB1 preceded by treatment with specific siRNA targeting to Sox9 gene or inhibitors of various signaling molecules. In vitro experiments are focused to elucidating the signaling mechanism underlying the modulation of Sox9 expression and SMC phenotypic modulation stimulated by HMGB1. Moreover, to gain better insights into the functions of Sox9 in SMC phenotypic modulation following allogeneic transplantation, animal model of aortic transplantation will be established between BN and Lewis rats, in which aortic allografts will be infected with lentivirus carrying shRNA-Sox9 whose specific expression in vascular SMCs is controlled by a minimal SM22α promoter. This project aims to elucidate the precise role and its underlying mechanism for Sox9 of vascular SMCs in regulating phenotypic modulation and transplant arteriosclerosis after transplantation. These findings will provide novel evidence for the development of new therapeutic strategies to prevent chronic allograft rejection after transplantation.

移植物慢性排斥反应的主要病理特征是移植物动脉硬化，它是导致远期移植物失功的主要原因。研究表明，血管平滑肌细胞(SMC)表型转化是移植物动脉硬化形成的核心环节，但是移植损伤引起SMC表型转化的分子机制至今未明。我们的前期研究提示，Sox9作为HMGB1信号通路下游的关键转录因子，可能在移植物动脉SMC表型转化和动脉硬化中发挥重要作用。 . 本项目拟培养SMCs，制备shRNA-Sox9-SM22α启动子慢病毒，转染移植动脉建立动脉移植大鼠模型，采用药物干预和基因转染方法，从细胞和整体水平，探讨自噬介导HMGB1调控Sox9表达和SMC表型转化的信号通路，研究Sox9调控移植动脉SMC表型转化和内膜增生的作用机制。本研究将阐明Sox9在移植物动脉SMC表型转化和动脉硬化中的作用和分子机制，有助于进一步明确慢性移植物失功的发生机制，并有望为临床改善器官移植的远期疗效提供新的治疗靶点。

项目的研究背景：移植物慢性排斥反应的主要病理特征是移植物动脉硬化，是导致远期移植物失功的重要原因。研究表明，血管平滑肌细胞（SMC）表型转化是移植物动脉硬化的始动和进展的关键环节，但是移植损伤引起血管SMC表型转化的分子机制目前尚不清楚。我们前期研究提示，Sox9作为HMGB1信号通路下游的关键转录因子，可能在移植物动脉SMC表型转化和动脉硬化中发挥重要作用。. 研究内容和重要结果：体外培养大鼠动脉SMCs，采用药物干预和基因转染等方法，探讨自噬介导HMGB1调控Sox9表达和SMC表型转化的信号转导机制。结果显示，HMGB1可显著诱导Sox9表达上调，并引起血管SMC表型转化。采用siRNA转染诱导Sox9基因沉默可有效阻断HMGB1诱导的血管SMC表型转化，并伴随着血管SMC增殖和迁移能力减弱。此外，p27蛋白可通过结合在Sox9的启动子区域，抑制Sox9的表达，维持SMC分化标志基因的表达。HMGB1刺激可引起p27蛋白泛素化，并诱导血管SMC自噬活化，导致p27蛋白的泛素化降解。当采用 Atg5 siRNA 转染和药物抑制剂Bafilomycin抑制自噬体及自噬溶酶体的产生， p27蛋白表达上调，Sox9基因的转录及表达被限制, 并可显著阻断HMGB1诱导的SMC 分化标志基因表达下调。在异系移植动脉SMCs中Sox9的表达水平较对照组明显升高，而 Calponin及SM22α的表达水平显著降低。用慢病毒介导特异性敲低移植动脉中膜SMCs中Sox9的表达可显著抑制移植损伤引起的Calponin和SM22α表达下调，同时减少异系移植动脉新生内膜形成。. 结论及科学意义：HMGB1通过诱导SMCs中自噬活化，促进p27蛋白的泛素化降解，解除p27 蛋白对Sox9基因的转录抑制，上调Sox9蛋白表达，进而引起SMC表型转化。在大鼠动脉移植模型中，异系移植动脉SMCs中Sox9的选择性敲低可有效抑制SMC表型转化，进而抑制移植动脉新生内膜的形成和动脉硬化。因此，以Sox9为干预靶点并抑制其信号通路可有效改善移植物动脉硬化的形成和进展，并有望为临床防治慢性移植物失功提供新思路和新靶点。