蛋白酶体激活促进腹主动脉瘤发生的分子机制研究

Abdominal aortic aneurysm (AAA) is a leading cause of sudden death in aging men. Among the numerous pathophysiologic mechanisms, inflammation, vascular smooth muscle cell (VSMC) phenotypic modulation play pivotal role. 26S proteasome, the central proteolytic unit of the the ubiquitin-proteasome system (UPS), plays a critic role in the regulation of the mechanisms mentioned above. β5 is one of three functional constitutive subunits of proteasome, mediating the chymotrypsin-like activity. By the action of the intended proinflammatory cytokines, the β5 subunits can be transferred into β5i, composing the immunoproteasome and mediating the enhanced chymotrypsin-like activity. And β5i plays important role in inflammatory response and autoimmune diseases. Recently, we have discovered the enhancement of β5 and β5i function in human and mice AAA tissues. Proteasome inhibitor Bortezomib could attenuate development of AAAs in Ang II-induced ApoE-/- mice, especially by suppressing β5 and β5i function. But the underlying mechanisms of proteasome activation on AAA formation have not been well defined. And how β5 and β5i respectively regulate VSMC phenotypic modulation and inflammation, and the correspondence patterns of the two mechanisms still remain unknown. On the basis of preliminary experiments, the project intends to solve the problems and shed light on the underlying interaction of inflammation response and VSMC phenotypic modulation in AAA formation. All experiments will be performed at the level of animal model and cell culture, and the specific role of β5i will be tested by specific inhibitor or gene knockout. We will use immunohistochemistry, qPCR, Western blot analysis, et al, to investigate the precise molecular mechanisms. In this way, we aim to find a potential target for future strategies designed to monitor and combat AAAs.

炎症反应和血管平滑肌细胞（VSMC）表型转换是调节腹主动脉瘤（AAA）发生主要机制。泛素-蛋白酶体系统（UPS）在调节T淋巴细胞介导的炎症和VSMC表型转换中发挥重要作用。我们前期研究表明T淋巴细胞参与AAA的发生，同时发现人及AopE-/-小鼠AAA组织中蛋白酶体亚基β5、β5i表达水平均明显增高，应用蛋白酶体抑制剂Bortezomib处理可显著抑制AopE-/-小鼠AAA的发生。然而β5、β5i参与AAA发生的分子机制仍不清楚。因此，申请人拟应用特异性抑制剂及基因敲除动物，进行体内、体外实验，采用免疫染色、Western blot、qPCR 等方法，观察β5、β5i对AAA发生率、病变程度、炎症反应等指标的影响，明确β5、β5i调节炎症、VSMC表型转换及AAA发生的分子机制。本项目将阐明蛋白酶体激活介导AAA的发生的分子机制，为寻找新的药物靶点提供理论依据。

腹主动脉瘤（AAA）是老龄化社会的常见病，主要发病机制包括炎症反应和血管平滑肌细胞（VSMC）表型转换等。本课题组研究发现AAA形成过程中泛素-蛋白酶体系统（UPS）的异常激活，包括蛋白酶体功能亚单位β1、β2、β5表达上调，相应蛋白水解酶活性增加，其中以β5亚单位的糜蛋白酶活性增加更为显著。体内实验表明，蛋白酶体抑制剂Bortezomib（BTZ）可通过抑制β5亚单位活性降低小鼠AAA的发生率及严重程度，主要机制包括：1、BTZ可抑制组织炎症细胞浸润、炎性因子分泌，抑制AAA形成；2、BTZ可抑制Myocardin降解，促进VSMC向合成亚型分化，抑制AAA形成；3、BTZ可抑制腹主动脉组织炎性重塑及弹力纤维断裂；4、BTZ可抑制腹主动脉组织中基质金属蛋白酶活化介导的细胞外基质降解；5、BTZ可抑制NF-κB信号通路激活，减少黏附因子表达，抑制炎症细胞在组织的黏附聚集。本课题组进一步研究发现，在AAA形成过程免疫蛋白酶体亚单位LMP7（β5i）表达上调，进一步体内实验表明，特异性敲除LMP7可抑制AAA发生发展，主要机制包括：1、LMP7敲除可抑制组织中T淋巴细胞向Th17亚型分化，抑制AAA形成；2、LMP7敲除可抑制腹主动脉组织炎性重塑及弹力纤维断裂。采用特异性LMP7抑制剂PR-957可抑制腹主动脉壁炎症反应，抑制AAA发生发展。本课题揭示了UPS激活在AAA形成中的作用及分子机制，发现BTZ、PR-957等药物对于AAA的治疗作用，为AAA临床转化提供了新的思路。此外，本课题组发现GLP-1和ghrelin可通过抑制JNK1/2和p38信号通路抑制高糖、高脂状态下微循环内皮细胞的凋亡及衰老；同时还发现了Ehlers-Danlos综合征新的突变位点COL3A1，提示E-D综合征相关致病因素在分子层面诊断的复杂性。课题在研期间，本课题组共发表相关SCI论著5篇，累计影响因子17.515，核心期刊文章5篇。指导8名博士研究生毕业。