CatD在AGEs/RAGE介导的血管平滑肌细胞表型转化中的作用及其机制研究

The phenotypic transformation of vascular smooth muscle cells (VSMCs) from differentiation to dedifferentiation under injury factors plays a critical role in the progression of atherosclerosis. Advanced glycation end products (AGEs) are formed by the nonenzymatic glycation reaction of aldose sugars with amino groups of proteins, which can produce biological effects mainly through receptor for AGE (RAGE). AGEs/RAGE can induce phenotypic transformation of VSMCs and then lead to vascular complications of diabetes mellitus. Cathepsin D (CatD), one kind of hydrolase involving in autophagy, can degrade the accumulated AGEs-modified protein. In our previous studies, we first found that AGEs decreased CatD expression, and overexpression of CatD could suppress the proliferation of VSMCs induced by AGEs. Therefore, based on the previous results and literatures, we speculate that CatD is the major pathway of AGEs-mediated phenotypic transformation of VSMCs, AGEs suppress CatD via RAGE and its downstream signal pathway, then CatD regulate phenotypic transformation of VSMCs through autophagy. The current project aims to clarify the role of CatD in the phenotypic transformation of VSMCs and its potential mechanisms. This project will provide potential target for prevention/treatment of vascular complications of diabetes mellitus.

血管平滑肌细胞（VSMCs）在生理状态下呈收缩表型，但在损伤因素下会向合成表型转化，进而促进动脉粥样硬化进程。晚期糖基化终末产物（AGEs）是过量的糖和蛋白质结合形成的稳定共价化合物，主要通过AGE受体（RAGE）产生生物学效应，能够诱导VSMCs表型转化进而导致粥样硬化性糖尿病血管并发症。组织蛋白酶D（CatD）能够降解胞内蓄积的AGEs修饰蛋白，是介导自噬的重要酶。我们前期研究首次发现，AGEs能够显著抑制CatD表达，而过表达CatD能够降低AGEs诱导的VSMCs增殖。根据前期结果和文献报道我们推测，CatD是AGEs诱导VSMCs表型转化的重要途径，AGEs通过AGEs-RAGE轴及下游信号通路抑制CatD的表达，而CatD通过调控自噬水平影响VSMCs的表型转化。本项目旨在阐明这一科学假设，探讨CatD在VSMCs表型转化中的作用及机制，为糖尿病血管并发症的防治提供新靶点。

血管平滑肌细胞（VSMC）在损伤因素下会由收缩表型向合成表型转化，进而促进动脉粥样硬化进程。晚期糖基化终末产物（AGEs）是过量的糖和蛋白质结合形成的稳定共价化合物，能够诱导VSMC表型转化进而导致粥样硬化性糖尿病血管并发症。组织蛋白酶D（CatD）能够降解胞内蓄积的AGEs修饰蛋白。本项目旨在阐明CatD在AGEs介导的VSMC表型转化中的作用及其机制。首先，CCK-8和EdU染色证实AGEs能够显著促进VSMC的增殖；Western blotting结果显示AGEs使得合成表型基因OPN上调、收缩表型基因SM-22α和α-SMA下调。并且以上效果随着AGEs浓度增加而增加。由此可见，AGEs能够以浓度依赖性形式促进VSMC表型转化，同时伴随CatD的下调。为了验证AGEs的作用是否通过其受体（RAGE）而实现，我们选用特异性RAGE抑制剂FPS-ZM1。结果提示，FPS-ZM1可以逆转AGEs对VSMC细胞活力和增殖的促进作用，可以减弱AGEs对VSMC合成表型基因OPN的上调作用和对收缩表型基因SM-22α和α-SMA的下调作用，以及缓解AGEs对CatD的抑制。我们应用Flag标记的pCDNA3.1质粒构建CatD过表达模型，结果提示过表达CatD能够显著缓解AGEs对VSMC的以上效应，说明CatD是AGEs对VSMC发挥作用的关键环节。不仅如此，我们对CatD发挥作用的机制进行了研究。通过验证实验，我们发现CatD过表达能够降低AGEs引起的p-ERK激活，CatD敲减使得AGEs引起的p-ERK激活程度进一步增加。为了阐明CatD对p-ERK的调控机制，我们采用免疫共沉淀实验验证了二者之间存在内源性结合，考虑是蛋白酶与底物的作用关系。因此，CatD通过与p-ERK相互结合对其进行降解，进而调控AGE/RAGE轴介导的VSMC表型转化。综上，本项目完整地阐明了CatD在AGEs/RAGE介导的血管平滑肌细胞表型转化中的作用及机制。这些发现为糖尿病血管并发症的认识和防治提供了新的方向。