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.

项目总结 正常的冠状动脉形成对心脏的生长和功能是必不可少的。畸形的冠状动脉是一种 临床上严重的出生缺陷，可导致危及生命的心脏并发症，包括脑室 致密化不全、心肌缺血和心脏性猝死。然而，推动发展的机制 正确的冠状动脉形成还不完全了解，这阻碍了我们发展冠状动脉的能力 针对这一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 冠脉发育的分子和细胞机制，以便我们可以识别关键 开发新的靶向治疗以对抗这种先天性疾病的监管因素。我们已经解决了 这一目标是在之前的发现阶段实现的。我们的研究表明，胚胎冠状动脉内部 致密心肌是由血管内膜细胞在血管内皮生长因子的调控下通过血管生成形成的。 凹槽信号。此外，我们的研究表明，这些胚胎冠状动脉经历了 围产期血管新生扩张，为正在生长的致密心肌增加新生血管。但是，在 与致密心肌的血管化相比，我们对小梁的血管化知之甚少 在出生前大部分保持无血管状态的心肌。我们最近发现了冠心病的一个亚群 心内膜细胞中定位于冠状动脉的祖细胞 小梁心肌。我们将这些细胞命名为第二波冠脉祖细胞(SCP)，以分离 它们来自致密心肌冠脉的第一波冠脉祖细胞(FCP)。 SCPS通过一种未知的心内膜在胚胎发育早期获得血管生成潜力 到间充质转化(EMT)，直到他们在围产期后期经历血管生成之前很久 使小梁心肌血管化。在此续订申请中，我们建议将这一新的 SCP的血管生成-EMT范式(AngioEMT)。我们的主要假设是小梁的血管化 SCPs对心肌的调节是一种“二次打击”机制，涉及顺序血管内皮细胞移植和低氧 发信号。我们计划在三个具体目标上检验这一假设。目标1将通过区分SCP来描述SCP 使用基于谱系的单细胞RNA测序(scRNA-seq)分析和改良的 功能性血管内皮细胞移植试验。目标2将在SCP的早期命运决策中定义AngioEMT信号，使用 研究转化生长因子b信号的遗传功能丧失方法。目标3将破译血管生成 在SCPs后期血管生成激活中的信号转导主要是VEGFA-VEGFR3和DLL4-NOTCH1信号转导。 小梁心肌的血管化以及单个骨小梁的致密化将是 组织学、免疫组织化学染色和RNAScope原位杂交检查。SCP谱系的变化 将由scRNA-seq分析确定，而两次击中血管内皮细胞移植过程背后的关键因素 将通过基因网络分析进行鉴定。通过实现这些目标，我们预计将提供新的 冠脉发育的机械学洞察为冠状动脉发育的发病机制提供信息 动脉畸形和心室致密化不全。