LIMK介导微血管内皮间质转化、促进心肌纤维化的机制研究

Myocardial fibrosis is highly associated with the poor prognosis in patients with heart failure. However, the mechanisms remain poorly understood. Emerging evidence indicates endothelial-to-mesenchymal transition (EndMT) plays an important role in myocardial fibrosis, especially perivascular fibrosis. We recently found that inhibition of LIM kinase (LIMK), a conservative regulator protein of cytoskeletal homeostasis, resulted in suppressed EndMT induced by transforming growth factor (TGF)- β1 in human microvascular endothelial cells (HMEC). In pressure overload model, LIMK-knockout mice showed an attenuated EndMT process and resistance to myocardial fibrosis comparing to wild-type animals. These were accompanied by echocardiographically preserved left ventricular function in LIMK-knockout mice. We thus hypothesize that LIMK mediates the myocardial fibrosis through regulating EndMT in microvascular endothelial cells. We will further use LIMK knockout mice to focus on the association between LIMK expression and EndMT during the different.stages of fibrosis, verifying the role of LIMK/cofilin/actin pathway in regulating EndMT process by loss and gain of function study. We will also dissect the molecular mechanisms of the transactivation of LIMK by TGF- β1 signaling pathway, and transcriptomically screen the upstream microRNAs that potentially regulate LIMK. Finally, we will investigate the association between LIMK expression level, extent of EndMT and the extent of myocardial fibrosis as well as the clinical severity of heart failure in human ventricular biopsies. This study will improve the understanding of the underlying mechanisms of myocardial fibrosis, shed light upon a novel mechanism of EndMT and ultimately provide novel molecular targets in gene therapy of myocardial fibrosis.

心肌纤维化是导致心力衰竭不良预后的重要原因，但机制尚未明确。近年来发现微血管内皮间质转化（EndMT）在促进心肌纤维化中起重要作用。申请人前期研究发现，在微血管内皮细胞中干扰LIM激酶（LIMK）表达可抑制TGF-β诱导的间质细胞表型。在主动脉缩窄（TAC）模型中，我们也发现LIMK基因敲除小鼠心脏微血管EndMT现象显著减少，伴心肌纤维化、心功能较野生型明显改善。故申请人提出LIMK通过介导微血管EndMT、促进心肌纤维化的假说。本项目拟用LIMK基因敲除小鼠，动态观察LIMK与EndMT的关系、明确其在早期纤维化中的作用。通过功能缺失/回复实验证实LIMK/cofilin通路在EndMT中的作用，探讨TGF-β1旁路调控LIMK的信号途径，微阵列筛选调控LIMK上游调控分子，并在心衰患者心肌组织内证实LIMK表达水平、EndMT程度与心力衰竭严重性的相关性。

心肌纤维化是导致心力衰竭不良预后的重要原因，但机制尚未明确。近年来发现微血管内皮间质转化（EndMT）在促进心肌纤维化中起重要作用。LIMK（LIM kinases）属于丝氨酸/苏氨酸激酶，位于小分子GTP酶家族信号通路的下游，是调控肌动蛋白细胞骨架的关键蛋白。然而LIMK能否调控EndMT、促进心肌纤维化尚不清楚。本项目利用LIMK1/2-/-基因敲除小鼠构建主动脉缩窄模型，首次发现LIMK基因敲除能显著抑制压力负荷小鼠心肌纤维化、减轻心肌EndMT，同时改善左室收缩功能。体外实验表明，LIMK1 siRNA及LIMK2 siRNA均能有效抑制TGF-β1刺激下人微血管内皮细胞（HCMECs）迁移及侵袭能力，同时抑制间充质相关基因表达、减少胶原合成。LIMK通过调控内皮细胞G-actin/F-actin转换，降低TGF-β1诱导的EndMT。通过微阵列筛选调控LIMK上游调控分子，在主动脉狭窄患者心肌组织内证实LIMK表达水平与EndMT的相关性。该成果为寻找心力衰竭的药物治疗靶点提供新的思路。