PEDF抑制血管内皮细胞EndMT抗心肌纤维化作用及机制

Myocardial fibrosis after myocardial infarction (MI) often leads to heart failure. It has reported that endothelial to mesenchymal transition (EndMT) is an important reason for promoting myocardial fibrosis. However, there is no effective method to inhibit EndMT in the ischemic heart so far. .Pigment epithelium- derived factor （PEDF） is an important environmental factor in the heart. Our preliminary experiments found that PEDF has a significantly protective effect on cardiac function after AMI; PEDF inhibits myocardial fibrosis and reduces the occurrence of EndMT in rat ischemic heart. Additionally, PEDF inhibits TGF-β1-induced EndMT and reduces β-catenin nuclear translocation in vitro. But the detailed mechanism is still uncertain. It is well documented that EndMT is closely related to intracellular β-catenin nuclear translocation, and VE-cadherin/ β-catenin complexes may be involved in this process. Therefore, we presume that PEDF reduces Src kinase activity, which down-regulates level of VE-cadherin phosphorylation, facilitates VE-cadherin/ β-catenin complexes stable and prevents the critical process of EndMT from β-catenin nuclear translocation, resulting in a reduction of myocardial fibrosis. .In this study, we try to validate the detailed mechanisms that PEDF inhibits EndMT by interrupting β-catenin nuclear translocation in cardiac endothelial cells in vitro and in vivo. The completion of this study will provide valuable experimental basis for clarifying the mechanisms of PEDF against myocardial fibrosis and reveal a novel target for myocardial fibrosis therapy.

心肌梗死后心肌纤维化可引起心功能衰竭，内皮细胞-间质细胞转化（EndMT）是促进心肌纤维化的重要因素。目前缺乏有效方法抑制心脏血管内皮细胞EndMT。.PEDF是心脏中重要的内源性细胞因子。申请者预实验发现：PEDF可抑制大鼠心梗模型中EndMT并减少心肌纤维化；PEDF能抑制TGF-β1诱导的内皮细胞EndMT并减少β-catenin核转位。有研究表明，内皮细胞中β-catenin核转位与EndMT发生有关；胞膜VE-cadherin/β-catenin复合体与β-catenin核转位有关。我们推测PEDF可通过抑制Src激酶活性下调VE-cadherin磷酸化水平，稳定胞膜VE-cadherin/β-catenin复合体，阻止β-catenin核转位，抑制EndMT，发挥抗心肌纤维化作用。.本项目拟从细胞和动物水平，过表达或沉默表达PEDF验证上述假说，为心肌纤维化寻找新的治疗靶点。

内皮细胞-间质细胞转化（EndMT）是促进心肌纤维化的重要因素。目前缺乏有效方法抑制心脏血管内皮细胞EndMT。色素上皮衍生因子（PEDF）由于在心血管疾病中具有重要保护作用而成为新的治疗靶点。本项目通过建立大鼠冠状动脉结扎心肌梗死模型，Masson染色观察心肌纤维化程度；天狼星红染色观察心肌纤维化程度；免疫荧光双标检测不同时间点内皮细胞的EndMT；首次发现PEDF可通过抑制心肌梗死后血管周围EndMT发生减少心肌梗死后血管周围纤维化。通过建立TGF-β1诱导血管内皮细胞EndMT模型，应用慢病毒载体过表达或沉默表达PEDF，构建β-catenin突变体及β-catenin敲减表达，利用病毒转染、RNA干扰、细胞培养、免疫印迹、免疫组化、免疫共沉淀、免疫细胞化学等技术手段发现PEDF可通过抑制Src激酶磷酸化从而减少VE-cadherin-β-catenin复合物解聚，通过阻止β-catenin核转位抑制TGF-β1诱导的血管内皮细胞EndMT发生。最终证明PEDF抗心肌纤维化的作用机制，即PEDF通过抑制Src激酶下调VE-cadherin磷酸化水平，维持细胞膜VE-cadherin/β-catenin复合体的稳定，阻止β-catenin由细胞膜-胞浆-胞核的转位，抑制梗死心脏的EndMT过程，从而减少梗死心肌纤维化。本研究为心肌纤维化提供新的治疗靶点和有益的实验依据。