细胞外基质蛋白CILP-1通过endoglin调控心肌间质纤维化的机制研究

Altered expression and function of extracellular matrix protein contributes to the development of pathological cardiac remodeling. Based on the data about the expression, distribution, and cellular resource of cartilage intermediate layer protein-1 (CILP-1) from our preliminary experiments, we will explore the effect of CILP-1 on myocardial interstitial fibrosis and reveal the possible molecular mechanism. This study will investigate the myocardial expression of CILP-1 in mice underwent transverse aortic constriction (TAC) and examine the myocardial interstitial remodeling in CILP-1 knock-out mice after TAC. In cultured adult mice cardiac fibroblasts, we will determine the effect of CILP-1 on the transforming growth factor (TGF)-β signaling pathway and the subsequent profibrotic response.To clarify whether endoglin is the direct target molecule of CILP-1 in this process, we will observe the effect of CILP-1 on endoglin activation and examine whether overexpression of endoglin in cardiac fibroblasts will abolish the inhibitory effect of CILP-1 on the profibrotic response induced by TGF-β1. The interaction between CILP-1 and endoglin will be clarified by use of GST pull-down, immunoprecipitation, and fluorescence resonance energy transfer (FRET) technology. Furthermore, the domain-domain interaction will be revealed. This study will identify CILP-1 as an extracellular matrix protein protecting against myocardial interstitial fibrosis and offer a new therapeutic strategy for preventing and treating myocardial interstitial remodeling.

细胞外基质蛋白的表达改变及功能异常是病理性心室重塑进行性发展的重要影响因素。申请者的前期工作揭示软骨间层蛋白-1（CILP-1）在心脏的表达、分布及细胞来源,但CILP-1对心肌间质纤维化的调控作用及机制有待研究。本课题拟在压力负荷性心室重塑的小鼠模型检测CILP-1的表达变化;利用基因敲除小鼠在整体水平探讨CILP-1对心肌间质重塑的调控作用,在心脏成纤维细胞检测CILP-1对TGF-β信号通路活化及其介导的促纤维化效应的影响。将endoglin作为CILP-1的直接作用靶点,揭示CILP-1对endoglin活性的调节以及过表达endoglin对CILP-1抑纤维化效应的影响,通过GST pull-down、免疫共沉淀及FRET等技术明确CILP-1与endoglin的相互作用和关键结构域。本研究将揭示CILP-1是抑制心肌间质纤维化的细胞外基质蛋白,并为干预心肌纤维化进程提供新策略。

细胞外基质蛋白的表达改变及功能异常是病理性心室重塑进行性发展的重要影响因素。细胞外基质软骨间层蛋白-1（CILP-1）在多种因素导致的病理性心室重塑过程中表达上调，但其对病理性心室重塑的影响尚不明确。本研究结果显示，CILP-1主要由心肌细胞产生并分泌，且在压力负荷过度所致的重塑心肌中表达显著增加。注射携带CILP-1 shRNA的腺相关病毒9（AAV9）降低心肌组织中CILP-1的表达可使压力超负荷所致的心肌肥大及间质纤维化进一步加重；注射携带CILP-1编码基因质粒的AAV9增加心肌组织中CILP-1的表达可改善压力超负荷所致的病理性心室重塑。进一步的研究证实，CILP-1 N端片段通过直接结合并抑制TGF-β1、C端片段通过激活Akt并促进Akt与Smad3的结合抑制TGF-β1的促纤维化效应。本研究揭示CILP-1是抑制心肌间质纤维化作用的细胞外基质蛋白，可为干预心肌纤维化进程提供新策略。