Deciphering the roles of Nfatc1 in developmental coronary angiogenesis

项目摘要 本研究项目的目的是了解NFATc1在发育冠状动脉中的分子方面 血管生成(DCA)。我们试图通过使用小鼠遗传学的协同方法来实现这一目标， 发育和分子生物学，以及系统生物学。心内膜细胞(VEC)的DCA 因此，成功完成拟议的研究将具有重大意义 通过揭示DCA背后可能与DCA的病因相关的因素来揭示翻译含义 先天性冠状动脉畸形是猝死的主要原因之一。更好地了解DCA将 也为基于细胞的冠心病治疗提供了迫切需要的发育基础 疾病，心力衰竭和死亡的主要原因。DCA是心脏特有的过程，不同于 身体其他器官的血管生成。这一过程背后的因素没有得到充分的研究，而且仍然存在 由于缺乏适当的动物模型和实验方法，很大程度上是未知的。该项目旨在填补 通过研究新产生的DCA和冠状动脉缺陷小鼠模型，重点研究了 心内膜特异转录因子、活化T细胞胞浆核因子1(NFATc1)的表达 DCA血管内皮细胞特异性基因调控网络(GRN)的调控。初步研究表明，NFATc1 是维持造血干细胞基因表达所必需的，并具有多潜能 心血管祖细胞与内皮分化相关基因的限制表达 和/或规范，且NFATc1基因敲除会导致DCA异常。基于这些结果，我们假设 NFATc1作为一个分子检查点，通过其转录调控来确定DCA的时间 组织特异性GRN诱导血管内皮细胞分化为冠状动脉内皮细胞三 AIMS旨在检验这一假设。目标1将描述发育中心脏的血管新生血管内皮细胞 通过改变血管生成基因的表达和细胞形态，并进行功能性血管生成实验。 目的2是确定NFATc1在DCA中的功能，并确定它是否与血管内皮生长因子-Notch途径相互作用 这对于利用小鼠遗传学和体外功能分析进行血管生成至关重要。目标3将确定 依赖NFATc1的GRN和HUB基因血管内皮细胞，使用RNA-SEQ/CHIP-SEQ和生物信息学分析，以及 通过体内表达和体外血管生成实验证实它们在DCA中的作用。

Project Summary The goal of this research project is to understand the molecular aspects of Nfatc1 in developmental coronary angiogenesis (DCA). We attempt to achieve the goal by using a synergistic approach of mouse genetics, developmental and molecular biology, and systems biology. DCA by the ventricular endocardial cells (VECs) gives rise to coronary arteries; therefore, successful completion of the proposed research will have significant translational implications by revealing the factors underlying DCA that can be relevant to the etiology of congenital coronary artery defects, one of major causes of sudden death. Better understanding of DCA will also provide the developmental basis that is critically needed for cell-based therapies for coronary heart disease, the leading cause of heart failure and death. DCA is a cardiac-specific process different from the vascular angiogenesis in other organs of body. Factors underlying this process are understudied and remain largely unknown due to the lack of proper animal models and experimental approaches. This project aims to fill the gap by studying newly generated mouse models of defective DCA and coronary arteries, focusing on an endocardial specific transcription factor, nuclear factor of activated T-cells cytoplasmic 1 (Nfatc1) in the regulation of a VECs specific gene regulatory network (GRN) of DCA. Preliminary studies revealed that Nfatc1 is required for maintaining the expression of genes for the hematopoietic stem cells and multi-potent cardiovascular progenitor cells and limiting the expression of genes involved in endothelial differentiation and/or specification, and knockout of Nfatc1 leads to abnormal DCA. Based on these results, we hypothesize that Nfatc1 functions as a `molecular checkpoint' for timing DCA through its transcriptional regulation of a tissue specific GRN for the differentiation of progenitor VECs into coronary arterial endothelial cells. Three Aims are designed to test this hypothesis. Aim 1 will characterize the angiogenic VEC in the developing heart by their changes in the expression of angiogenic genes and cell shape, and functional angiogenesis assays. Aim 2 is to ascertain Nfatc1 functions in DCA and determine whether it interacts with the Vegf-Notch pathway that is critical for angiogenesis using mouse genetics, and in vitro functional assays. Aim 3 will identify the Nfatc1-dependent GRN and hub genes VECs using RNA-seq/ChIP-seq and bioinformatics analysis, and confirm their roles in DCA in vivo by expression and in vitro by angiogenesis assays.