Deciphering the roles of Nfatc1 in developmental coronary angiogenesis

项目摘要 本研究项目的目标是了解Nfatc 1在发育性冠状动脉疾病中的分子方面， 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。心室内膜细胞（VEC）的DCA 产生冠状动脉;因此，成功完成拟议的研究将具有重大意义 翻译的影响，通过揭示潜在的因素DCA，可以相关的病因学 先天性冠状动脉缺陷，猝死的主要原因之一。更好地理解DCA将 也为基于细胞的冠心病治疗提供了急需的发展基础 心脏病是导致心力衰竭和死亡的主要原因。DCA是一种心脏特异性过程， 在身体的其他器官中的血管生成。这一过程背后的因素尚未得到充分研究， 由于缺乏适当的动物模型和实验方法，大部分未知。该项目旨在填补 差距通过研究新产生的小鼠模型的缺陷DCA和冠状动脉，重点是一个 内皮细胞特异性转录因子，活化T细胞胞质核因子1（Nfatc 1）， DCA的VECs特异性基因调控网络（GRN）的调节。初步研究显示，Nfatc 1 是维持造血干细胞和多能造血干细胞基因表达所必需的。 心血管祖细胞和限制内皮分化相关基因的表达 和/或规格，敲除Nfatc 1导致异常DCA。基于这些结果，我们假设 Nfatc 1作为一个“分子检查点”，通过转录调节一个 组织特异性GRN用于祖VEC分化为冠状动脉内皮细胞。三 目的是为了检验这一假设。目的1将描述发育中心脏血管生成VEC的特征 通过它们在血管生成基因表达和细胞形状方面的变化，以及功能性血管生成测定。 目的2是确定Nfatc 1在DCA中的功能，并确定其是否与Vegf-Notch通路相互作用 这对于使用小鼠遗传学和体外功能测定的血管生成至关重要。目标3将确定 使用RNA-seq/ChIP-seq和生物信息学分析的Nfatc 1依赖性GRN和枢纽基因VEC，以及 通过表达在体内和通过血管生成测定在体外证实它们在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.