MMP-9调控巨噬细胞极化影响急性心梗诱导的心肌纤维化中内皮间质转化的作用及机制研究

Myocardial fibrosis is the common pathological basis of various heart diseases. The study found that cardiac endothelium transition (EndoMT) promotes the development and progression of myocardial fibrosis. Macrophage polarization is also involved in myocardial fibrosis, but whether macrophage polarization affects EndoMT involvement in myocardial fibrosis is still unclear. Our previous study found that in early stage of acute myocardial infarction in mice, fibrosis occurred in the infarcted area, accompanied by a large number of inflammatory macrophage infiltration and myocardial fibrosis. In vitro co-culture system, macrophages can promote mouse coronary artery EndoMT occurring in endothelial cells, whereas deletion of matrix metalloproteinase-9 (MMP-9) in macrophages inhibits macrophage transformation to pro-inflammatory M1 and ameliorate macrophage-induced endothelial cell interstitial transformation. Therefore, in this project, we intend to further confirm the role of macrophages in cardiac EndoMT and detect myocardial fibrosis by macrophage clearance and in vitro transfusion; macrophage polarization and TGF-β secretion after detection of MMP-9 deletion to elucidate whether MMP-9 regulates EndoMT by TGF-β, which affects macrophage secretion activity. This study is expected to reveal the mechanism of EndoMT in myocardial fibrosis, providing a new experimental basis for the treatment of myocardial fibrosis, and provide new ideas for the pathogenesis of other types of diseases such as pulmonary fibrosis, renal fibrosis and liver fibrosis.

心肌纤维化是多种心脏疾病共同病理基础。近期发现心脏内皮间质转化（EndoMT）和巨噬细胞极化后分泌因子均促进心肌纤维化发生发展，但二者之间如何相互作用参与心肌纤维化目前尚无明确报道。我们前期研究发现小鼠急性心梗早期心梗区发生纤维化，伴有大量炎性巨噬细胞浸润，且体外共培养显示巨噬细胞能够促进小鼠冠脉内皮细胞发生EndoMT，而基质金属蛋白酶9（MMP-9）缺失则能抑制巨噬细胞向促炎M1型转化，并缓解巨噬细胞诱导的EndoMT。本项目将从临床患者、原代细胞及基因敲除动物不同角度，通过巨噬细胞清除和体外回输、过表达及敲除MMP-9手段，检测MMP-9是否影响巨噬细胞极化及其分泌的TGF-β，探讨MMP-9调控巨噬细胞诱导的EndoMT在心肌纤维化中具体作用机制。本研究从全新角度揭示心肌纤维化中EndoMT发生机制，为心肌纤维化治疗提供新的实验依据，同时为其他纤维化类型疾病发病机制提供新思路。

近年研究对心肌纤维化发病机制提供新见解，但目前心肌纤维化疾病仍缺乏有效治疗药物。本研究发现，心脏内皮间质转化和巨噬细胞极化后分泌因子均促进心肌纤维化发生发展，小鼠急性心梗早期心梗区发生纤维化，伴有大量炎性巨噬细胞浸润，且体外共培养显示巨噬细胞能够促进小鼠冠脉内皮细胞发生内皮间质转化，而MMP-9缺失则能抑制巨噬细胞向促炎M1型转化，并缓解巨噬细胞诱导的内皮间质转化。MMP-9敲除小鼠梗死区M1型巨噬细胞比例显著降低，M2型巨噬细胞比例有一定程度升高。分离培养梗死区巨噬细胞，收集条件培养基与小鼠原代冠脉内皮细胞共培养，发现野生小鼠而非MMP-9敲除小鼠心脏巨噬细胞上清诱导内皮细胞表达成纤维细胞特异标志α-SMA。梗死后第四周野生型小鼠心脏血管内皮细胞有α-SMA的表达，梗死后第八周α-SMA表达进一步增强，而MMP-9基因敲除小鼠心脏血管内皮细胞α-SMA呈阴性。在此基础上，我们筛选到天然小分子化合物并证明该化合物对不同心肌纤维化模型均产生很好治疗效果。这项研究从全新角度阐释心梗后心肌纤维化发病机制，不但鉴定及发现心肌纤维化治疗潜在新靶点，而且设计合成新药硫化氢释放杂化体13-E，其可以在体内减少TAC诱导的心脏肥大，减轻心肌纤维化并恢复心功能。其调节AMPK信号通路并影响脂肪酸代谢过程，这可能归因于其心脏保护活性。此外，作为上述工作延伸及深化，我们揭示H2S在心肌纤维化发病中的作用，特别是调节RIP1/RIP3/MLKL信号通路的机制。