Molecular and Cellular Mechanisms in Coronary Artery Development and Anomalies

项目概要 正常的冠状动脉形成对于心脏的生长和功能至关重要。畸形的冠状动脉是 具有临床意义的出生缺陷，可能导致危及生命的心脏并发症，包括心室 致密化不全、心肌缺血和心源性猝死。然而，驱动的发展机制 正确的冠状动脉形成尚未完全了解，这阻碍了我们开发冠状动脉的能力 针对这种毁灭性疾病的针对心脏的干预措施。因此，该项目的长期目标是揭示 冠状动脉发育的分子和细胞机制，以便我们可以确定关键的 开发新的靶向疗法来对抗这种先天性疾病的监管因素。我们已经解决了 在之前的调查期间实现了这一目标。我们的研究表明，胚胎冠状动脉的内层 致密心肌是由心室心内膜细胞通过 VEGF 调节的血管生成形成的。 NOTCH 信号。此外，我们的研究表明，这些胚胎冠状动脉经历 围产期血管生成扩张，将新生血管添加到不断生长的致密心肌中。然而，在 与致密心肌的血管化相反，我们对小梁的血管化知之甚少。 心肌在出生前基本上没有血管。我们最近发现了冠状动脉的一个亚群 心室心内膜细胞中的祖细胞，在心室中定向于冠状动脉 小梁心肌。我们将这些细胞命名为第二波冠状动脉祖细胞（SCP）以分离 它们来自致密心肌冠状血管的第一波冠状动脉祖细胞（FCP）。 SCP 在胚胎发育早期通过以前未知的心内膜获得血管生成潜力 早在围产期进行血管生成之前就发生了间充质转化（EMT） 使小梁心肌血管化。在这个更新申请中，我们建议描述这个新的 SCP 的血管生成-EMT 范例（angioEMT）。我们的首要假设是小梁的血管化 SCPs对心肌的调节是通过“二次打击”机制进行的，涉及连续的血管EMT和缺氧 发信号。我们计划在三个具体目标中检验这一假设。目标 1 将通过区分 SCP 来表征 使用基于谱系的单细胞 RNA 测序 (scRNA-seq) 分析和改良的方法从 FCP 中提取它们 功能性血管EMT测定。目标 2 将定义 SCP 在早期命运决定中的 angioEMT 信号传导 研究 TGFb 信号传导的遗传功能丧失方法。目标 3 将破译血管生成 SCPs 后期血管生成激活中的信号传导主要集中于 VEGFA-VEGFR3 和 DLL4-NOTCH1 信号传导。 小梁心肌的血管化以及个体空位中的小梁压实将是 通过组织学、免疫染色和RNAscope原位杂交进行检查。 SCP谱系的变化 将通过 scRNA-seq 分析确定，而二次打击 angioEMT 过程的关键因素 将通过基因网络分析来识别。通过完成这些目标，我们期望提供新的 对冠状动脉发育的机制见解，为冠状动脉的发育发病机制提供信息 动脉异常和心室致密化不全。

PROJECT SUMMARY Normal coronary artery formation is essential for heart growth and function. Malformed coronary arteries are a clinically significant birth defect that can cause life-threatening cardiac complications, including ventricular noncompaction, myocardial ischemia, and sudden cardiac death. Yet, developmental mechanisms that drive proper coronary artery formation are incompletely understood, which has hindered our ability to develop the heart-specific interventions for this devastating disease. The long-term goal of this project is therefore to reveal the molecular and cellular mechanisms underlying coronary artery development so that we may identify key regulatory factors for developing new targeted therapies to combat this congenital condition. We have addressed this goal during previous finding period. Our studies have shown that embryonic coronary arteries in the inner compact myocardium are formed by ventricular endocardial cells through angiogenesis regulated by the VEGF- NOTCH signaling. Furthermore, our studies have revealed that these embryonic coronary arteries undergo angiogenic expansion perinatally to add the neovessels to the growing compact myocardium. However, in contrast to the vascularization of the compact myocardium, we know little about vascularization of trabecular myocardium which remains largely avascular until birth. We have recently identified a subpopulation of coronary progenitor cells among ventricular endocardial cells which are committed to the coronary arteries in the trabecular myocardium. We named these cells as the second wave coronary progenitors (SCPs) to separate them from the first wave coronary progenitors (FCPs) for the coronary vessels at the compact myocardium. SCPs acquire angiogenic potential earlier in embryonic development through a previously unknown endocardial to mesenchymal transformation (EMT) long before they undergo angiogenesis later during perinatal periods to vascularize the trabecular myocardium. In this renewal application, we propose to characterize this new angiogenic-EMT paradigm (angioEMT) by SCPs. Our overarching hypothesis is that vascularization of trabecular myocardium by SCPs is regulated by a “two-hit” mechanism involving sequential angioEMT and hypoxia signaling. We plan to test this hypothesis in three Specific Aims. Aim 1 will characterize SCPs by distinguishing them from FCPs using a lineage-based single cell RNA-sequencing (scRNA-seq) analysis and a modified functional angioEMT assay. Aim 2 will define the angioEMT signaling in the early fate decision by SCPs using genetic loss-of-function approaches investigating the TGFb signaling. Aim 3 will decipher the angiogenic signaling in the later angiogenic activation of SCPs focusing on VEGFA-VEGFR3 and DLL4-NOTCH1 signaling. Vascularization of trabecular myocardium as well as trabecular compaction in the individual nulls will be examined by histology, immunostaining, and RNAscope in situ hybridization. The changes in the SCP lineages will be determined by scRNA-seq analysis, whereas the key factors underlying the two-hit angioEMT process will be identified through gene network analysis. By completing these aims, we expect to provide new mechanistic insights into coronary artery development that inform developmental pathogenesis of coronary artery anomalies and ventricular noncompaction.