成纤维细胞转录因子Krüppel-样因子4（Klf4）调控心脏纤维化重塑的作用与机制研究

Cardiac fibrosis is a common pathogenic process of various cardiovascular diseases, and always leads to heart failure and arrhythmia in the end. Myofibroblasts activation and inflammatory infiltration play a pivotal role in fibrosis, but the key regulatory molecules and mechanisms are unclear in this process. Our published works and preliminary results showed that the transcription factor Krüppel-like factor 4 (Klf4) was elevated in fibrotic hearts, and it was located in fibroblasts. Moreover, myofibroblast-specific Klf4 knockout inhibited cardiac fibrosis which was induced by angiotensin II perfusion. With Chromatin Immunoprecipitation (ChIP)-seqencing and RNA-seqencing analysis, we found angiopoietin-like 2 (Angptl2) was a potential target gene of Klf4. Consequently, we hypothesize that fibroblast-expressed Klf4 transcriptionally regulates the expression of Angptl2, which in turn promotes fibroblast-to-myofibroblast differentiation, and increases the secretion of chemokines which mediated macrophage infiltration, and eventually leading to cardiac fibrotic remodeling induced by hypertension. In this study, we plan to validate our proposal in vivo by using fibroblast-specific Klf4 knockout mice and combine with overexpression of Angptl2 in mouse by lentivirus injection. Furthermore, transcriptional activity of Klf4 on Angptl2 in fibroblasts, and co-culture of fibroblasts and macrophages will be applied to investigate the mechanism underlying our preliminary findings and to explore the role of Klf4 in cardiac fibrosis.

心脏纤维化是多种心血管疾病的共同病理过程，导致心衰和心律失常。肌成纤维细胞活化及炎症浸润是纤维化的重要因素，但其关键调控分子和机制并不清楚。申请人已发表工作和预实验提示Krüppel-样因子4 (Klf4)在纤维化心脏中表达升高，定位于成纤维细胞；肌成纤维细胞Klf4敲除可抑制血管紧张素II灌注造成的高血压心脏纤维化。通过ChIP-seq结合RNA-seq找到了Klf4可能的关键靶基因血管生成素样蛋白2（Angptl2）。据此我们推测成纤维细胞Klf4通过转录调控Angptl2，促进成纤维细胞转分化为肌成纤维细胞，并分泌趋化因子增加巨噬细胞浸润，介导高血压心脏纤维化重塑。本课题拟用成纤维细胞Klf4敲除小鼠结合慢病毒介导Angptl2过表达进行整体研究，在细胞中通过Klf4对Angptl2转录活性分析、成纤维与巨噬细胞共培养等手段进行机制探讨，揭示Klf4在心脏纤维化中的作用。

心脏受到压力负荷等诸多因素刺激会引发细胞外基质异位沉积，导致心脏纤维化，这一病理变化与远期左心室重构、心律失常、心力衰竭甚至猝死的发生密切相关，因而探究其机制，寻找干预靶点具有积极意义。成纤维细胞的活化和转分化在心脏纤维化过程中发挥核心作用，但其关键的调控分子和机制尚有待研究，并且纤维化进程中存在肌成纤维细胞的衰老，促进其早期衰老有助于抑制纤维化。.Krüppel-样因子4(Klf4)作为转录因子参与调节细胞的存活、分化、凋亡等生物过程，与诸多心血管重塑疾病发生相关。本课题中我们利用血管紧张素II(AngII)灌注和主动脉弓缩窄(TAC)建立心脏纤维化和心力衰竭模型，检测到Klf4在心脏成纤维细胞中表达上调。进一步构建在Col1a2+成纤维细胞中和αSMA+的肌成纤维细胞和平滑肌细胞中特异敲除Klf4的小鼠，发现在Col1a2+细胞中敲除Klf4可促进压力负荷诱导的心脏纤维化和心衰，而在αSMA+细胞中敲除Klf4结果相反。为研究机制，我们在成纤维细胞系过表达Klf4，通过RNA-seq和ChIP-seq结合TAC模型RNA-seq及心力衰竭患者单细胞RNA-seq数据分析，发现并验证了Klf4对肽酶抑制分子Pi16的直接转录调控，且Pi16高表达在成纤维细胞；进而在诱导衰老的成纤维细胞中我们检测到Klf4和Pi16的表达上调；鉴于Pi16调控HDAC1对心脏纤维化发挥抑制作用，而HDAC1调节细胞衰老，因而Pi16可能为介导Klf4调控成纤维细胞衰老从而抑制纤维化的关键分子。此外，我们的工作还探讨了细胞外基质沉积和降解的调节机制，阐明了细胞DNA损伤、补体活化C3a-C3a受体信号和Spp1在此过程中的作用。.总之，本课题揭示了成纤维细胞Klf4抑制压力负荷导致的心脏纤维化和心力衰竭的作用和机制，并阐述了细胞外基质降解和沉积的调控机制，为心脏纤维化疾病的治疗提供潜在靶点。