心脏损伤后p53在间质-内皮转化中的分子调控机制

Heart disease is a leading cause of mortality and morbidity worldwide. The mammalian heart is unable to regenerate lost myocardium and heals primarily by fibrosis. This eventually leads to the development of heart failure. Both the Modulation of scar tissue and formation of new blood vessels remain an important goal of cardiac repair strategies. We have recently identified a previous unrecognized phenomenon Mesenchymal-Endothelial-Transition (MEndoT), which occurs in a p53 dependent manner and has been demonstrated to be a beneficial process for cardiac repair. However, little is known about the molecular mechanism of p53 in MEndoT..We demonstrated that the administration of a p53 activator, RITA (Reactivation of p53 and Induction of Tumor Apoptosis) can enhance MEndoT, augment vascularity in the injury region and decrease scar formation and ameliorate post injury decline in cardiac function. It suggests that manipulation of MEndoT to enhance vascularity represents a potential therapeutic strategy for cardiac repair..Our ChIP assay data show that p53 directly initiates transcription factors, which are associated with the adoption of an endothelial cell like phenotype, such as eNOS、Claudin 5, HoxA9 and HoxD3. Using loss and gain of function approaches we demonstrated these transcription factors are specifically regulated by p53. It suggests a new mechanism of p53 in MEndoT. Thus, to determine the critical downstream target of p53 is important question for application of MEndoT. .Wnt4 is another important downstream gene of p53, which was found by RNA-seq. By inhibiting and activating p53, we found that wnt4 was specifically regulated by p53. Using loss and gain function, we also observed that Wnt4 is related to the expression of endothelial associated gene such as Claudin 5 and Occludin. After cardiac injury, over-expression of wnt4 in heart by AAV virus administration can improve the cardiac function. We hypothesized that p53 could regulate MEndoT through multiple key downstream genes. .Besides the identification of these discovered downstream genes of p53 in MEndoT, we also proposed to find more potential critical downstream targets of p53 by using next generation sequencing. Our work will provide an insight into the molecular mechanisms of p53 regulating MEndoT. It will also accelerate the discovery of new regulation strategies for clinical application of MEndoT.

间质-内皮转化（Mesenchymal-Endothelial Transition，MEndoT）是我们首次发现有利于心脏损伤修复的现象，它依赖于p53的调控。心脏损伤后，提高成纤维细胞中p53的表达，可促进MEndoT、减少纤维化和增加新生血管生成，它代表一种新的心脏损伤修复策略。然而p53调控MEndoT的分子机制尚不清楚。通过RNA-seq我们首先发现p53下游基因Wnt4与内皮细胞基因的表达特异相关，并可促进心脏损伤后的修复。通过p53的ChIP，我们发现MEndoT中p53特异地结合eNOS、Claudin5、HoxA9和HoxD3等内皮细胞相关基因的启动子。因此，本项目核心目标是针对这些已发现的p53下游调控基因进行深入研究，同时继续发现并鉴定p53在MEndoT中的其他关键靶基因。本项目从转录水平和p53靶基因两个层面阐明p53调控MEndoT的分子机制，为临床转化奠定基础

间质-内皮转化（Mesenchymal-Endothelial Transition，MEndoT）是一种依赖于p53调控的利于心脏损伤后修复的现象，本项目旨在阐明p53调控MEndoT的分子机制，为特异调控该转分化进而用于心脏修复奠定基础。我们的研究结果显示，心肌缺血损伤时，Wnt4在心肌成纤维细胞中的表达随时间显著增加，体外诱导心肌成纤维细胞发生MEndoT与Wnt4表达相关且受到p53的特异调控，ChIP测序 和分析进一步揭示p53通过靶向结合下游Wnt4启动子进而诱导心肌成纤维细胞发生MEndoT。在心肌成纤维细胞中敲除wnt4可抑制MEndoT发生，过表达wnt4则促进MEndoT发生。体内心肌成纤维细胞敲除p53引起的心功能恶化可以被心肌成纤维细胞中特异过表达的Wnt4补救，这证明p53通过靶向下游Wnt4促进MEndoT以降低心肌缺血损伤的新分子机制，为MEndoT精准调控提供了新的靶点。.此外，我们围绕心肌成纤维细胞在心脏损伤后的命运调控尤其是MEndoT的调控进一步展开了一系列深入研究，以期建立系统深入的理论体系：1) 阐明了MEndoT在心肌肥厚损伤中的病理生理意义，同时阐明了MEndoT转分化的内皮样细胞与天然内源内皮细胞在分子上差异，为未来MEndoT细胞调控指明了方向；2) 利用10×单细胞测序技术揭示心肌成纤维细胞在损伤前后的多样性，发现了两个与内皮细胞关系密切的亚群和一个与心肌损伤应答密切相关的亚群，为更精准的靶向心肌成纤维细胞亚群，进而精准调控转分化奠定了基础；3) 发现了心肌成纤维细胞特异标志物之一DDR2，可以特异标记骨外膜成骨细胞和成骨祖细胞，表达DDR2的骨外膜细胞更具显著成骨分化能力，这对研究心脏钙化中心肌成纤维细胞的命运和角色有借鉴意义；综合以上结果为未来心脏损伤后基于心肌成纤维细胞命运的精准调控从而促进心肌损伤后修复奠定了理论基础。