溶血磷脂酸激活炎症细胞促进心肌纤维化的研究

Lysophosphatidic acid (LPA) is an endogenous bioactive lipid molecule and plays an important role in atherosclerosis by regulating inflammatory cells. We have reported the expressions of LPA1, LPA2 and LPA3 were significantly enhanced in myocardial infarction (MI) rats when compared with sham controls and LPA dose-dependently induced proliferation and collagen synthesis in cardiac fibroblasts. Furthermore, in the preliminary experiment, we found the expression of autotoxin (ATX, enzyme for production of LPA) increased in active macrophages and the secretion of TGF-β were induced by LPA in these macrophages. These findings make us hypothesize that LPA signaling may promote cardiac fibrosis after MI through the activation of inflammatory cells. The present study includes ① confirming that LPA promotes cardiac fibrosis by activating Ly-6Chigh monocytes and M1 macrophages in vivo using LPA1, LPA2 and LPA3 knockout mice and siRNA technology; ② demonstrating that LPA triggers the release of the chemokine MCP-1 from endothelial cells through the activation of LPA1 and LPA3, and MCP-1 induces the adhesion of Ly-6Chigh monocytes to the vessel wall, then these monocytes migrate into the subendothelial space and transform into M1 macrophages, which play an important role in cardiac fibrosis; ③ exploring miR-16 and MMP9 secreted by M1 macrophages promote transdifferentiation of fibroblasts to myofibroblasts by regulating TGF-β;④ investigating M1 macrophages are the source of LPA promoting cardiac fibrosis after MI. The aim of this study is explore the role of LPA in the process of cardiac fibrosis after MI and its signaling pathway in activation of inflammatory cells and myofibroblasts, and the possibility of LPA receptors and ATX being the biological targets of drugs.

溶血磷脂酸（LPA）是内源性脂类信号分子，可调节炎症细胞促进动脉粥样硬化，我们发现3种LPA受体在心梗后晚期大鼠心脏表达上调。预实验发现活化的巨噬细胞（MФ）LPA生成酶ATX表达升高，且LPA促进MФ分泌TGFβ，推测LPA激活炎症细胞促进心梗后的心肌纤维化。本研究①利用3种LPA受体敲除鼠和siRNA技术，在整体动物确证LPA通过激活Ly-6Chigh单核细胞和M1型MФ（M1）促进心肌纤维化；②体外实验阐明LPA通过诱导内皮细胞分泌MCP-1，促进Ly-6Chigh迁移，增加促纤维化的M1数量；③细胞水平阐明LPA诱导M1中miR-16和MMP9表达，活化TGFβ，促使成纤维细胞向肌成纤维细胞转化；④细胞、动物、人群层面阐明促进心肌纤维化的LPA主要来自M1。本课题旨在探索LPA信号调节炎症细胞对心梗后心肌纤维化的影响及机制，提出LPA受体和ATX是改善心梗后心肌纤维化新的干预靶点。

心梗后持续的间质纤维化导致心肌僵硬、功能衰竭，使得心力衰竭发生率逐年增加。然而在心力衰竭的治疗方面仍没有特异性针对心肌纤维化的药物，因而我们需要进一步探索致心肌纤维化机制，寻找新的治疗靶点。本课题组以往研究表明溶血磷脂酸（LPA）可以促进心脏成纤维细胞增殖及胶原合成增加，且在大鼠心梗后其受体LPA1-3表达升高。本研究中我们在细胞水平发现，LPA促进单核巨噬细胞MMP-9表达和分泌，而MMP-9可使得组织纤维化加重。进一步机制研究发现，LPA通过LPA2调节NF-κB入核介导MMP-9的表达和分泌活化。在体动物实验，我们以3种LPA受体敲除小鼠为研究对象，利用小鼠心梗模型，观察心梗后8周心肌纤维化情况，发现LPA1杂合缺失不参与小鼠心梗后心功能以及重构的调节，LPA3缺失可以导致小鼠心功能恶化，但不影响心肌纤维化，而LPA2的缺失使得小鼠心梗后存活率下降，重构恶化，纤维化加重，表明LPA信号通过LPA2对抗心梗后心肌纤维化，这与原标书假设不符。进一步分析实验结果显示，LPA2缺失使得心梗后梗死周边区血管密度减少，推测LPA-LPA2可以促进血管新生，补充的小鼠下肢缺血实验初步证明了此新假设。综上所述，本研究表明，LPA-LPA2信号直接作用于成纤维细胞诱导其纤维化的发生，但在小鼠心肌梗死后LPA-LPA2信号可能通过促进梗死周边区血管新生，拮抗重构，保护心肌。详细机制，有待进一步研究。