CTRP15通过抑制心脏Gli1+周细胞向肌成纤维细胞分化参与调控心肌纤维化的机制研究

Although the differentiation of cardiac Gli1+ pericytes into myofibroblasts is recently revealed as the predominant contributor to cardiac fibrosis, little is known about the factors and mechanisms that controll this process. Our preliminary data showed that complement C1q/tumor necrosis factor-related protein (CTRP) 15 originated mainly from cardiac microvascular endothelial cells. In this study, we will examine the development of cardiac fibrosis in the wild type mice and the transgenic mice with endothelial-specific overexpression of CTRP15 underwent transverse aortic constriction (TAC). This study will further reveal the effect of CTRP15 on the differentiation of cardiac Gli1+ pericytes into myofibroblasts in in vivo fate-tracing studies using the Gli1CreERt2; tdTomato mice. The direct action of CTRP15 on the differentiation of cardiac Gli1+ pericytes into myofibroblasts will be determined by the coculture of cardiac Gli1+ pericytes with human cardiac microvascular endothelial cells (HCMEC) in which CTRP15 is stably over-expressed or stably knocked-down. To clarify the key target of CTRP15 in regulating the myofibroblasts differentiation from cardiac Gli1+ pericytes, we will observe the effect of CTRP15 on hedgehog (Hh) signaling pathway in cardiac Gli1+ pericytes. The expression of Hh ligands, the activation of membrane receptors, and the transcription activity of Gli1 will be determined by using radioligand binding assay, fluorescence resonance energy transfer (FRET), AlphaLISA, and chromatin immunoprecipitation (ChIP). This study will identify CTRP15 as a novel endogenous cytokine protecting against the differentiation of cardiac Gli1+ pericytes into myofibroblasts and the pathogenesis of cardiac fibrosis. Our research also offers a new therapeutic strategy for preventing and treating cardiac fibrosis.

心脏Gli1+周细胞向肌成纤维细胞分化是促进心肌纤维化的重要新机制，但其影响因素和调控机制尚不明确。本课题在揭示微血管内皮细胞是心脏补体C1q/肿瘤坏死因子相关蛋白(CTRP)15 主要来源的基础上，利用内皮细胞特异性过表达CTRP15的转基因小鼠，观察CTRP15对心肌纤维化进程的影响；利用细胞谱系示踪技术以及将稳定过表达或敲除CTRP15的微血管内皮细胞与Gli1+周细胞共培养，明确CTRP15在调控Gli1+周细胞向肌成纤维细胞分化中的作用。将Hedgehog(Hh)信号通路作为CTRP15的关键作用靶点，利用放射性配基结合试验、FRET、AlphaLISA、ChIP等技术，从配体、膜受体及转录因子水平深入揭示CTRP15对Hh信号通路的影响。本研究将揭示CTRP15是抑制心脏Gli1+周细胞向肌成纤维细胞分化和心肌纤维化的新内源性保护因子，为临床防治心肌纤维化提供新的靶点与思路。

1. 背景.心肌纤维化是慢性心力衰竭时心室重构的一种重要形式，过度的心肌纤维化会增加心室壁的僵硬度，导致心室顺应性降低，引起心脏收缩和舒张功能障碍。心脏成纤维细胞是心肌中胶原蛋白等细胞外基质的主要来源，其向肌成纤维细胞的表型转化是心肌纤维化发生发展的重要环节。补体C1q/肿瘤坏死因子相关蛋白15（CTRP15）表达于成年小鼠心脏中，但其在心脏的生物学功能尚未见报道。本研究观察CTRP15在压力负荷所致心室重构中的作用，并重点探讨CTRP15对心肌间质纤维化的影响及相关分子机制。.2. 研究内容.利用主动脉缩窄术构建压力超负荷的心室重构模型，检测心室重构中CTRP15的表达变化规律。小鼠于术后尾静脉注射携带CTRP15编码基因的腺相关病毒9及携带空载体的对照病毒，检测心肌过表达CTRP15对心室重构、心肌纤维化及肌成纤维细胞转化的影响。过表达或敲低心肌细胞或心脏成纤维细胞中CTRP15的表达，揭示其可能的作用机制。.3. 重要结果和关键数据 .CTRP15主要表达于小鼠的心肌细胞，在成纤维细胞表达较少。TAC术后小鼠左心室舒张功能减退、出现心室肥厚及纤维化，心肌组织CTRP15表达明显减少；心脏过表达CTRP15可以减轻压力超负荷引起的心室重构、改善心室舒张功能，并抑制成纤维细胞向肌成纤维细胞表型转化。细胞实验证实：心肌细胞合成和分泌的CTRP15通过旁分泌作用于心脏成纤维细胞，抑制其向肌成纤维细胞转化。CTRP15通过激活IR/IRS-1/Akt通路抑制TGF-β1诱导的Smad3磷酸化，从而发挥抑制心肌纤维化作用。.4. 科学意义.本研究揭示了抑制肌成纤维细胞转化的内源性保护因子CTRP15，对于阐明心肌纤维化的调控机制及探索心肌纤维化的治疗策略具有重要意义。