KLK8促进内皮细胞间质转化在糖尿病心肌病心肌间质纤维化中的作用及其机制研究

组织激肽释放酶相关肽酶8(KLK8)是丝氨酸蛋白酶家族成员。我们采用KLK8转基因大鼠首次发现KLK8过表达可导致心脏组织中发生内皮细胞间质转化（EndMT）和间质纤维化。高糖诱导EndMT已知是糖尿病心肌间质纤维化病理发生的重要机制，而我们发现高糖处理可以促使内皮细胞KLK8表达增加，敲低KLK8表达则可部分逆转高糖诱导的EndMT现象。结合免疫共沉淀、质谱分析和细胞水平验证工作，我们发现血管内皮钙粘蛋白（VE-cadherin）-连环蛋白（catenin）复合物可能是KLK8在内皮细胞上的作用靶点。在此基础上，本项目首先将利用KLK8转基因和KLK8基因敲除两种模式动物，证实KLK8表达增高在糖尿病心肌病心脏组织EndMT和间质纤维化过程中的关键作用，进而将主要围绕KLK8/VE-cadherin/catenin信号途径深入探讨KLK8促进心脏组织EndMT和间质纤维化的分子机制。

The tissue kallikrein-related peptidase family (KLK) is a group of trypsin- and chymotrypsin-like serine proteases. In our preliminary study, we found for the first time that transgenic-mediated KLK8 overexpression led to development of endothelial-to-msenchymal transition (EndMT) and fibrosis in the hearts. It has recently been found that high glucose-induced EndMT plays an important role in the pathogenesis of cardiac fibrosis in diabetic cardiomyopathy. We demonstrated that high glucose led to significant increase of KLK8 expression in human coronary artery endothelial cells (HCAECs). Moreover, KLK8 knockdown partly reversed high glucose-induced EndMT in HCAECs. Using co-immunoprecipitation and mass spectrometry, we obtained a number of potential target proteins which might be associated with KLK8. We then determined that vascular endothelial (VE)-cadherin/catenin complex might associate with KLK8 in endothelial cells, as indicated by IP of KLK8, which yielded VE-cadherin and γ-catenin. On the basis of these results, the present proposal will firstly verify that upregulation of KLK8 does contribute to the development of EndMT and cardiac fibrosis in diabetic cardiomyopathy, by using both KLK8 transgenic and KLK8 knockout rodent models. Since KLK8 associates with VE-cadherin/catenin complex in endothelial cells, we will then clarify whether upregulation of KLK8 leads to degradation of VE-cadherin and disassembly of VE-cadherin/catenin complex, thus resulting in nuclear translocation of β-catenin, γ-catenin and p120 catenin. Finally, the role of KLK8/VE-cadherin/catenin signaling pathway in EndMT and cardiac fibrosis in diabetic cardiomyopathy will be elucidated. The present study will provide reliable evidence to elucidate the mechanisms involved in the pathogenesis of cardiac fibrosis in diabetic cardiomyopathy. Blockade of KLK8/VE-cadherin/catenin signaling pathway may become one of the future treatment options of cardiac fibrosis in diabetic hearts.