TRAF5在血管内膜增生早期进程中的功能和机制研究

After endothelial cell injury and denudation, the infiltrations of monocyte-macrophages within subendothelium induce the vascular smooth muscle cells (VSMCs) behavior change through secreting multiple cytokines, which is well recognized as the important links in the early phrase of intimal hyperplasia. Thus, the investigation about the effect and underlying mechanism of macrophage polarization on VSMCs phenotypic switching plays an important role in attenuating the development of intimal hyperplasia. Tumor necrosis factor receptor-associated factor 5 (TRAF5) is an adaptor protein of the tumor necrosis factor (TNF) receptor superfamily and the interleukin-1 receptor/Toll-like receptor superfamily and plays important roles in regulating multiple signaling pathways. Our previous results demonstrated that TRAF5 showed a high level in the restenotic arteries and in vitro experiment exhibited that TRAF5 ablation decreased the classical macrophage (M1) but increased the alternative activation macrophage (M2). However, during the development of intimal hyperplasia, whether the regulation of macrophage polarization of TRAF5 can affect VSMCs phenotypic switching and neointima formation, as well as the underlying mechanism still unclear. The current project further explores the key genes change of TRAF5 downstream in intimal hyperplasia through gene microarray. More importantly, we urgently expect to clarify the interaction and functional regulation of TRAF5 on the key downstream proteins through mutual interaction proteomics analysis and ubiquification, as well as to verify the reverse effect of key protein on intimal hyperplasia mediated by TRAF5 through in vitro experiment. Collected, the present study would further expand and reveal the key pathological mechanism of intimal hyperplasia. More importantly, the research would provide a novel therapeutic target and theory for early diagnosis and early targeted therapy of intimal hyperplasia.

血管内皮损伤后，内膜下巨噬细胞的浸润能够分泌炎症因子诱导血管平滑肌细胞行为的改变，是血管内膜增生早期进程的重要环节。阐明巨噬细胞极化如何调控平滑肌细胞表型转化及潜在机制，对于阻遏内膜增生进展起着关键作用。我们前期研究结果证实TRAF5在增生血管组织中高表达，体外实验表明TRAF5敲除能够抑制M1、促进M2型巨噬细胞的转化。但是，TRAF5对巨噬细胞极化的调控作用是否影响平滑肌细胞表型转化、血管内膜新生及潜在机制尚未明确。本项目拟采用巨噬细胞特异性TRAF5敲除小鼠，通过基因芯片筛选TRAF5下游关键基因改变。进一步通过质谱分析、免疫共沉淀等互作蛋白质组学以及泛素化蛋白修饰手段，探寻其相互作用的关键靶蛋白以及对靶蛋白功能的调控。利用体外实验验证关键靶分子对TRAF5调控的血管内膜增生的逆转作用。从而，进一步揭示血管内膜增生的发病机制，为早期诊断，早期靶向治疗血管内膜增生提供重要的理论依据。

血管内皮损伤后，内膜下巨噬细胞的浸润，特别是巨噬细胞极化，能够影响平滑肌细胞表型转化，在调节血管内膜增生进展起着关键作用。本项目明确了TRAF5在病理状态，包括导丝损伤后的血管内膜以及LPS或者IL-4诱导的M1/M2型巨噬细胞中的表达情况。通过构建TRAF5敲低质粒转化的巨噬细胞，通过PCR、Western blot检测TRAF5敲低能够促进M1、抑制M2型巨噬细胞的转化。并通过共培养的方式，利用transwell、CCK8、PCR、Western blot明确了TRAF5敲低调控的巨噬细胞极化，能够调节平滑肌细胞的表型转化（增殖、迁移、去分化）。通过体内建立颈动脉血管导丝损伤动物模型，阐明了TRAF5敲除能够促进血管内膜增生及体内新生内膜中巨噬细胞M1型的极化。机制研究方面，揭示了TRAF5主要是通过影响PPARr来调控巨噬细胞极化。本研究阐明了调节血管内膜增生的新机制，为早期诊断，早期靶向治疗血管内膜增生提供重要的理论依据。