rapamycin诱导血管内皮、平滑肌细胞自噬的蛋白质组学研究

Rapamycin is widely used in drug eluting stents to prevent in-stent restenosis after percutaneous coronary intervention. Rapamycin suppresses the proliferation and migration of vascular smooth muscle cells. However, rapamycin also impairs the proliferation and migration of vascular endothelial cells, which leads to endothelial dysfunction and delayed re-endothelialization of stent struts, as well as higher rates of late stent thrombosis. This disadvantage limits the use of rapamycin in drug eluting stents. Recently, a number of studies have focused on the role of rapamycin in autophagy in endothelial cells. To systematically assess the molecular effect of rapamycin on vascular endothelial cells and smooth muscle cells, differential proteomic analysis was performed. Differential expression of 41 proteins are observed in rapamycin-treated human coronary artery endothelial cells. Some of the regulated proteins are involved in the processes of autophagy. Cofilin 1, a key regulator in cell migration and cancer cell invasion, is 18.91-fold down regulated by rapamycin treatment. Simultaneously, Cofilin 1 is inactivated by phosphorylation on Ser3 site. Knock-down of Cofilin 1 significantly induces autophagy in Hela cells. We therefore hypothesize that rapamycin impairs the vascular endothelial cells proliferation, migration, and cell tube formation by targeting Cofilin 1. This project aims to reveal the mechanisms underlying rapamycin induced Cofilin 1 inactive, enriching the mechanisms of autophagy processes, providing a new idea to attenuate the adverse effect of rapamycin in the use of drug eluting stents.

Rapamycin是洗脱支架常用药之一。但是其在抑制血管平滑肌细胞增殖迁移、有效防止介入术后再狭窄的同时，rapamycin损伤内皮功能、延缓内皮细胞覆盖包裹支架、增加了血栓风险。目前研究焦点集中在rapamycin的自噬诱导作用上。我们用蛋白质组学的方法，首次系统且有针对性的分别研究rapamycin对血管内皮和平滑肌细胞的分子作用。发现一批与细胞自噬相关的差异表达蛋白。其中促肿瘤细胞转移蛋白Cofilin 1在rapamycin诱导下表达下调，同时Ser3被磷酸化而失活。Knock-down Cofilin 1表达可以诱导细胞自噬形成。由此我们提出工作假设：rapamycin通过失活Cofilin 1，诱导内皮细胞过渡自噬损伤、抑制其增殖迁移和小管形成。本项目旨在阐明该机制及具体调控方式，以期为更全面的理解细胞自噬的分子机制，及为降低rapamycin临床使用的不良反应提供科学依据。

临床上发现rapamycin洗脱支架最常见的不良反应是支架内血栓的形成。其主要原因是rapamycin延缓了支架被正常内皮细胞覆盖包裹，暴露的支架增加了血栓风险。因此明确rapamycin诱导内皮细胞受损的具体分子机制是克服其不良反应的关键。为了系统探索rapamycin作用于血管内皮细胞的分子机制，我们采用蛋白质组学作为筛选手段，结合后期的具体分子功能分析进行研究。.我们利用2D-DIGE结合MALDI-ToF-ToF的手段，共发现rapamycin诱导的人冠状动脉内皮细胞HCAEC差异表达蛋白29种。同时，我们应用与传统的基于双向电泳的蛋白质组学方法形成相互补充、或者说更优的策略——iTRAQ蛋白质组学对课题进行进一步研究。发现≥1.5倍差异的蛋白质200种。在这些差异蛋白质中，既有与增殖密切相关的蛋白，如PCNA、EF2及CDK1等；也有与迁移密切相关的蛋白，如PCDH1、ICAM1等；还有与自噬直接相关的蛋白，如ATG9A、LAMP1及LAMP2等。.在两种蛋白质组学研究手段中，都发现有一个rapamycin诱导的下调的蛋白：Cofilin-1。Cofilin-1与细胞的增殖迁移密切相关。我们证实自噬时，Cofilin-1蛋白表达下调，而且Ser3被磷酸化，协同作用使Cofilin-1失活。在Hela细胞及HCAEC中，有效knock-down Cofilin-1后可以诱导自噬的形成。并且，knock-down Cofilin-1导致HCAEC的增殖迁移能力下降。这些结果表明rapamycin可以通过下调Cofilin-1诱导内皮细胞自噬、抑制内皮细胞增殖迁移能力。.该机制的阐明，将有助于更全面的理解细胞自噬的分子机制，也有助于更客观的评价rapamycin的临床使用、尤其是支架局部给药，为降低rapamycin的不良反应提供新的视角和科学依据。