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。

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.