TAK1介导细胞凋亡诱导血管平滑肌细胞表达SDF-1α促进移植物动脉硬化的作用机制

Chronic allograft rejection, which is characterized by transplant arteriosclerosis in the graft vasculature, is now recognized as the leading cause of long-term allograft dysfunction following organ transplantation. Emerging evidence from our laboratory suggests that medial smooth muscle cell apoptosis is a central event in the initiation and progression of transplant arteriosclerosis through inducing the production of SDF-1α from neighboring viable smooth muscle cells (SMCs). However, the potential mechanisms underlying the modulation of SDF-1α production by SMC apoptosis remain to be elucidated. Our previous studies indicated that TAK1 that acts as a key regulator in the signaling pathway triggered by SMC apoptosis may play an important role in inducing SDF-1α production and subsequent transplant arteriosclerosis. In this project, to explore the role of cell apoptosis in activating TAK1 signaling and its possible mechanism, rat aortic SMCs will be cultured and subjected to cell apoptosis induced by transfection with pro-apoptotic p53 gene or treated with etoposide in vitro. Additionally, to explore the role of TAK1 in SDF-1α production induced by cell apoptosis, selective TAK1 inhibitor or specific siRNA targeting to TAK1 gene will be utilized to treat cultured SMCs. In vitro experiments are focused on elucidating the signaling mechanisms by which SMC apoptosis modulate SDF-1α production. Furthermore, to detect the effect of blocking TAK1 on SDF-1α production and neointimal formation in aortic allograft following transplantation, rat model of aortic transplantation will be established between BN and Lewis rats, in which aortic allografts will be infected with lentivirus carrying TAK1 shRNA, whose specific expression in vascular SMCs is modulated by a minimal SM22α promoter. This project aims to elucidate the precise role and its underlying mechanism for TAK1 in modulating SDF-1α production and transplant arteriosclerosis. The findings will provide novel evidence for the development of new therapeutic strategies to prevent chronic allograft rejection.

以移植物动脉硬化为主要病理特征的慢性排斥反应是导致远期移植物功能丧失的重要原因。我们最近研究发现，血管平滑肌细胞凋亡通过诱导表达SDF-1α促进移植物动脉内膜增生和动脉硬化，但是细胞凋亡调控SDF-1α表达的分子机制至今未明。我们前期研究提示，作为细胞凋亡启动的关键信号分子，TAK1可能在调控SDF-1α表达及移植物动脉硬化中发挥重要作用。 本项目拟体外培养血管平滑肌细胞并诱导细胞凋亡，探讨细胞凋亡对TAK1的活化作用及分子机制；利用TAK1抑制剂和siRNA转染，研究其对SDF-1α表达的诱导作用，重点探讨调控SDF-1α表达的分子机制。建立大鼠动脉移植模型，用TAK1 shRNA-SM22α启动子慢病毒转染供体动脉，检测抑制中膜平滑肌细胞TAK1对SDF-1α表达和内膜增生的影响，探讨TAK1在SDF-1α诱导表达和动脉硬化中的作用机制，以探索移植动脉硬化和慢性排斥反应的有效防治策略。

项目的研究背景：以移植物动脉硬化为主要病理特征的慢性排斥反应是导致远期移植物功能丧失的重要原因。研究发现，血管平滑肌细胞凋亡通过诱导表达SDF-1α促进移植物动脉内膜增生和动脉硬化，但是细胞凋亡调控SDF-1α表达的分子机制至今未明。我们前期研究提示，作为细胞凋亡启动的关键信号分子，TAK1可能在调控SDF-1α表达及移植物动脉硬化中发挥重要作用。. 研究内容和结果：体外培养大鼠动脉SMCs并诱导细胞凋亡，采用药物干预和基因转染等方法，探讨细胞凋亡对TAK1的活化作用，并研究其对SDF-1α表达分泌的诱导作用及分子机制。结果显示，Ltv-p53转染或H2O2均可有效诱导SMC凋亡，产生IL-1、IL-6和TGF-β等细胞因子，其中IL-1和TGF-β介导细胞凋亡诱导TAK1活化，并上调SDF-1α的表达分泌。用TAK1抑制剂或siRNA转染阻断TAK1信号通路，均能抑制细胞凋亡对SDF-1α表达分泌的诱导作用。用IL-1α处理SMCs，结果发现，IL-1α以IKKβ信号依赖的方式通过上调C/EBPβ诱导SDF-1α的表达分泌，而TAK1通过减弱C/EBPβ表达负性调节IL-1α诱导的SDF-1α表达。用TGF-β处理SMCs，结果发现，TGF-β通过活化TAK1/HIF-1α信号通路促进SMCs表达分泌SDF-1α。再者，制备TAK1shRNA-SM22α启动子慢病毒，转染移植动脉并建立大鼠腹主动脉移植模型，WB及病理图像分析等显示，在异系移植动脉，慢病毒介导TAK1shRNA转染可选择性敲低移植动脉中膜细胞TAK1基因表达，有效阻断移植损伤引起的SDF-1α的表达分泌，减少炎症免疫细胞的浸润，显著抑制移植动脉新生内膜面积及内膜/中膜面积比。. 结论及科学意义：血管SMC凋亡通过产生IL-1和TGF-β激活TAK1信号通路，调控SDF-1α的表达分泌。在大鼠动脉移植模型中，TAK1介导移植损伤上调SDF-1α的表达分泌，诱导炎症免疫细胞浸润，从而促进移植动脉新生内膜形成和动脉硬化。因此，以TAK1为干预靶点抑制其信号通路可有效抑制移植物动脉硬化，这为防治移植物动脉硬化及慢性排斥反应的发生发展提供了新的靶点和治疗策略。