Molecular and Cellular Mechanisms in Coronary Artery Development and Anomalies

冠状动脉发育和异常的分子和细胞机制

• 批准号: 10595393
• 负责人: BIN ZHOU
• 金额: $ 76.48万
• 依托单位: ALBERT EINSTEIN COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-06-01 至 2023-09-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10595393
• 关键词: Address; Affect; Biological Assay; Birth; Breeding; Cardiac; Cells; Complement; Congenital Abnormality; Coronary; Coronary Circulation; Coronary Vessel Anomalies; Coronary Vessels; Coronary artery; Data; Data Analyses; Development; Disease; Embryo; Embryonic Development; Endocardium; Endothelium; Fluorescence-Activated Cell Sorting; Genes; Genetic; Goals; Growth; Heart; Histology; Hypoxia; Immunofluorescence Immunologic; In Situ Hybridization; Individual; Intervention; Knowledge; Label; Life; Mesenchymal; Metabolic; Molecular; Mus; Myocardial; Myocardial Ischemia; Myocardium; NOTCH1 gene; Names; Pathogenesis; Pathway Analysis; Perinatal; Phenotype; Process; Proliferating; Regulation; Reporter; Research Project Grants; Signal Transduction; Sinus; TGFB2 gene; TGFBR3 gene; Testing; Transforming Growth Factor Beta 2; VEGFA gene; Vascular Endothelial Growth Factor Receptor-3; Vascular Endothelial Growth Factors; Vascularization; Ventricular; Visualization; angiogenesis; biomarker validation; clinically significant; combat; data integration; differential expression; effective intervention; experimental study; gene network; in vivo; insight; loss of function; malformation; new therapeutic target; novel therapeutics; perinatal period; progenitor; single-cell RNA sequencing; stem cells; sudden cardiac death; translational medicine

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.

项目摘要 正常的冠状动脉形成对心脏的生长和功能至关重要。畸形冠状动脉是一种 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，发展机制， 正确的冠状动脉形成还不完全清楚，这阻碍了我们发展冠状动脉的能力。 针对这种毁灭性疾病的心脏特异性干预。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 调节因素，开发新的靶向治疗，以打击这种先天性疾病。我们已经解决 这一目标在上一次的调查中。我们的研究表明，胚胎冠状动脉在内部 致密心肌由心室内膜细胞通过血管生成形成，血管生成受VEGF- NOTCH信令。此外，我们的研究表明，这些胚胎冠状动脉经历了 围产期血管生成扩张，以将新血管添加到生长的致密心肌中。但在 相对于致密心肌的血管化，小梁心肌的血管化我们知之甚少 直到出生前仍基本无血管的心肌。我们最近发现了一个冠心病的亚群， 心室心内膜细胞中的祖细胞被定向到冠状动脉 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）， 它们来自致密心肌处的冠状动脉血管的第一波冠状动脉祖细胞（FCPs）。 SCP在胚胎发育早期通过以前未知的内皮细胞获得血管生成潜能 间充质转化（EMT）早在他们经历血管生成后，在围产期， 使小梁心肌血管化。在此更新申请中，我们建议将这种新的 血管生成EMT范式（angioEMT）。我们的首要假设是， SCP对心肌的作用是通过一种“二次打击”机制来调节的，该机制包括连续的血管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 使用基于谱系的单细胞RNA测序（scRNA-seq）分析和改良的 功能性血管EMT测定。目的2将使用以下方法定义SCP在早期命运决定中的血管EMT信号传导： 研究TGF β信号传导的遗传功能丧失方法。目标3将破译血管生成 SCPs的血管生成活化中的信号传导，集中于VEGFA-VEGFR 3和DLL 4-NOTCH 1信号传导。 将观察单个空白区的心肌小梁血管化和小梁致密化。 通过组织学、免疫染色和RNA原位杂交进行检查。SCP谱系的变化 将通过scRNA-seq分析确定，而两次打击血管EMT过程的关键因素 将通过基因网络分析进行鉴定。通过实现这些目标，我们希望提供新的 对冠状动脉发育的机制性认识， 动脉异常和心室致密化不全