TGF-β/Hippo通路调控前脂肪细胞转化为肌成纤维细胞促进大动脉炎血管纤维化的机制研究

Takayasu arteritis (TA) is a systematic vasculitis with fatal outcomes. It is featured by the vascular fibrosis in the whole three layers, especially, in the adventitia. However, the pathogenesis of TA has not been clarified so that it lacks effective treatment to improve the structural impairment. As for cellular level, myofibroblast is the most important kind of histocytes in the progress of vascular fibrosis in TA because it directly synthesizes abundant extracellular matrix (ECM) such as collagen type I, III and fibronectin, eta. Interestingly, the origins of myofibroblast have not been fully known. The perivascular adipose tissue (PVAT) closely wraps the aorta without any boundary and it keeps providing the adipocytes and preadipocytes, which makes them become the second most cells in the adventitia. Our previous study in vitro showed that TGF-β significantly promoted the relative expression of α-SMA and collagen type I on the mRNA and protein levels in preadipocytes. Furthermore, the high expression of TGF-β and a target protein of Hippo signaling pathway, CYR61, were detected in the PVAT lesion of TA patients by immumohistochemical staining. Thus, on the basis of above findings, the preadipocytes may be a crucial origin of myofibroblast in TA lesion wall and TGF-β/Hippo signaling pathway may play a key role in regulating this cellular phenotype transformation. But its underlying mechanism remains unclear. To verify this phenomenon, firstly, the confocal microscopy studies will be used to observe the colocalization of TGF-β, α-SMA and preadipocytes in the vascular tissue of TA patients. Secondly, the experimental techniques including RT-PCR, Western blot and ChIP will be used in a co-cultural system of TGF-β and preadipocytes to demonstrate the regulation of Hippo signaling pathway in the cellular transformation. Thirdly, the targeted interventions to the TGF-β/Hippo pathway will be explored in a TA mice model. In conclusion, the launch of this research aims to clarify the new sight of pathogenesis and search for the novel targets of treatment in TA.

大动脉炎(Takayasu arteritis, TA)是严重威胁人类生命的系统性血管炎，以血管全层纤维化为突出特征，肌成纤维细胞是最关键的致病细胞，但对其来源尚未充分认识。与主动脉紧密连接的血管周围脂肪组织富含前脂肪细胞，我们前期的体外实验显示TGF-β显著上调前脂肪细胞表达α-SMA，并在TA病变血管周围脂肪组织中发现TGF-β和Hippo通路的靶蛋白呈显著高表达。据此，本课题组提出前脂肪细胞是TA血管壁肌成纤维细胞的重要来源之一，TGF-β/Hippo通路可能调控这一细胞表型转化过程，但其机制不明。为此，我们拟测定TA病变组织中TGF-β、α-SMA的蛋白表达量以及与前脂肪细胞的共定位关系、构建TGF-β与原代人内脏前脂肪细胞共培养体系探索Hippo通路调控细胞表型转化的分子机制，并对TA模型小鼠开展靶向干预实验进行体内验证。本研究将为阐明TA的发病机制和寻找诊治新靶点提供新视角。

大动脉炎(Takayasu arteritis, TAK)是严重威胁人类生命的系统性血管炎，以血管全层纤维化为突出特征，肌成纤维细胞是最关键的致病细胞，但对其来源尚未充分认识。与主动脉紧密连接的血管周围脂肪组织富含前脂肪细胞，本课题采集大动脉炎患者主动脉周围组织、构建人前脂肪细胞体外培养体系，聚焦于TGF-β诱导人前脂肪细胞促纤维化的表型转化及其机制研究，并在华东地区大动脉炎注册队列(ECTA)中进行临床验证。主要研究结果包括(1) TGF-β在大动脉炎患者主动脉周围脂肪组织的纤维化区域高表达，并与血管外膜有一定的连续性，提示主动脉周围脂肪组织可能早期即参与大动脉炎血管病变进程，为后续机制研究提供关键基础；(2) 在体外人内脏前脂肪细胞培养体系中，发现TGF-β显著促进了α-SMA、I型胶原的表达，呈现出促纤维化的变化趋势，同时抑制了人前脂肪细胞向脂肪细胞分化的能力；（3）利用转录组测序（RNA-seq）分析TGF-β作用下人前脂肪细胞的基因表达水平，筛选出Hippo通路可能在TGF-β诱导人前脂肪细胞表型转化中起重要作用；（4）Hippo通路的关键分子YAP/TAZ在TGF-β刺激后迅速去磷酸化，并进入细胞核内，与TEAD结合上调促纤维因子的表达，siRNA干扰实验和维替泊芬抑制实验证实YAP/TAZ数量和功能均对促纤维因子的表达发挥作用；（5）ERK激酶调控了YAP/TAZ去磷酸化，为细胞内不同信号通路的交互作用提供新证据；（6）致炎因子IL-6显著增强了TGF-β诱导人前脂肪细胞表达促纤维化因子，而TNF-α反而抑制了I型胶原的表达，且只能被TGF-β部分逆转，因此，炎症微环境中的致炎细胞因子和TGF-β的交互作用表现出了复杂性，为探索精准诊疗提供可能的理论依据；（7）姜黄对TGF-β促纤维化的抑制作用，提示其在治疗大动脉炎上的临床应用潜力；（8）ECTA队列研究显示IL-6促进大动脉炎组织纤维化，TNF-α加重组织损伤的特点，与基础研究结果相符，CYR61与主动脉狭窄闭塞密切相关，姜黄能显著改善大动脉炎的疾病活动度。本项目为揭示大动脉炎血管纤维化的发生发展机制提供新的突破点，从主动脉周围脂肪组织入手，为大动脉炎“外膜起源”学说提供新证据，并为大动脉炎诊疗提供潜在的新靶点。