NOTCH signaling on the underdeveloped cardiac vascularization of hypoplastic left heart syndrome in the hiPSC-derived vascularized cardiac organoids

在 hiPSC 衍生的血管化心脏类器官中，NOTCH 信号传导对左心发育不全综合征的不发达心脏血管化的影响

• 批准号: 10439137
• 负责人: Huaxiao Yang
• 金额: $ 43.82万
• 依托单位: UNIVERSITY OF NORTH TEXAS
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-09-08 至 2025-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10439137
• 关键词: BMP10 gene; Binding; Blood Vessels; Blood capillaries; CRISPR/Cas technology; Calcium; Cardiac; Cardiac Myocytes; Cardiovascular system; Cell Line; Cell model; Cell physiology; Cells; Complex; Data; Defect; Development; EGF gene; ERBB2 gene; Endothelial Cells; Endothelium; Fetal Heart; Functional disorder; Gene Expression; Goals; Growth Factor; Heart; Heart Diseases; Heart Valves; Human; Hypoplastic Left Heart Syndrome; Image; Knock-out; Left ventricular structure; Ligands; Link; Measures; Mediating; Mesoderm; Modeling; Molecular; Mutation; Myocardial; Myocardium; NOTCH1 gene; NRG1 gene; Newborn Infant; Nitric Oxide; Organoids; Palliative Surgery; Pathogenesis; Pathway interactions; Patients; Postpartum Period; Process; Proteome; Proteomics; Protocols documentation; Regulation; Signal Pathway; Signal Transduction; Smooth Muscle Myocytes; Structure; Study models; Tissues; Transgenic Mice; Up-Regulation; Validation; Vascularization; Ventricular; angiogenesis; ascending aorta; cardiac vasculature; cardiogenesis; congenital heart disorder; density; endothelial dysfunction; endothelial stem cell; extracellular; gamma secretase; genetic variant; genome editing; induced pluripotent stem cell; inhibitor; malformation; progenitor; receptor; receptor binding; single-cell RNA sequencing; small molecule; three dimensional cell culture

PROJECT SUMMARY Hypoplastic left heart syndrome (HLHS) is one of the most fatal congenital heart diseases (CHD) that occurs in 1 of every 4,344 newborns. The HLHS patients’ hearts show the severe underdevelopment of the left ventricle, heart valves, and ascending aorta, and specifically lower capillary density in the myocardium. Moreover, the genetic variant of NOTCH1 is implicated in HLHS patients showing the relevance to endocardial defects, endothelial dysfunction, and NOTCH1 signaling pathways. However, how the NOTCH1 signaling determines the underdevelopment of cardiac vasculature in the HLHS patients is still inconclusive. There is still a missing link between the pathogenesis in the cardiovascular underdevelopment and dysfunction found in the HLHS hearts and the NOTCH1 mutation. In this proposal, the genome-edited NOTCH1+/- hiPSCs will be co- differentiated in an already optimized protocol into the vascularized cardiac organoid (VCO) with cardiomyocytes, endothelial cells, smooth muscle cells, and other cardiac cells to recapitulate the underdevelopment and dysfunction of the cardiovascular system in the early development of HLHS heart with NOTCH1 mutation and to further delineate the pathogenesis of HLHS in the dynamic multicellular crosstalk of cardiovascular cells via NOTCH-DLL-JAG receptor-ligand interactions. In the preliminary results, we observed a reduced vascular formation with a smaller myocardial-like ring in VCO when NOTCH signaling was inhibited by g-secretase inhibitor, and smaller organoid size was noticed in the NOTCH1+/- VCO compared to the isogenic wildtype VCO. We will further explore the NOTCH1 signaling on vascular and cardiac formation in VCOs by measuring the cardiovascular structure, proliferation, function, and corresponding gene expressions (Aim 1). Moreover, our preliminary study also shows upregulation of NOTCH1 signaling via the NOTCH-DLL- JAG ligand-receptor interactions in the VCO compared to the non-vascularized cardiac organoid by the single- cell RNA-seq and ligand-receptor analysis. We will further investigate the ligand-receptor interactions and pseudotime trajectories of NOTCH1 signaling in the process of NOTCH1+/- VCO formation. The proteome profile over the HLHS VCO development will be uncovered by the proteomic analysis in correlation and validation with the single-cell RNA-seq data (Aim 2). The completion of the proposed project will be for better determining the NOTCH1 signaling-mediated multicellular crosstalk and downstream pathways of underdeveloped cardiac vascularization in HLHS.

项目摘要 左心发育不全综合征（HLHS）是一种常见的先天性心脏病， 每4，344名新生儿中就有1名。HLHS患者的心脏显示左心室严重发育不全， 心脏瓣膜和升主动脉，特别是降低心肌中的毛细血管密度。而且 NOTCH 1的遗传变异体与HLHS患者有关，显示出与内分泌缺陷相关， 内皮功能障碍和NOTCH 1信号通路。然而，NOTCH 1信令如何确定 HLHS患者的心脏脉管系统发育不全仍不确定。还有一个失踪的 在HLHS中发现的心血管发育不全和功能障碍的发病机制之间的联系 心脏和NOTCH 1突变。在该提案中，基因组编辑的NOTCH 1 +/-hiPSC将与基因组编辑的NOTCH 1 +/-hiPSC共表达。 在已经优化的方案中分化为血管化的心脏类器官（VCO）， 心肌细胞、内皮细胞、平滑肌细胞和其他心肌细胞，以概括 HLHS心脏早期发育中心血管系统发育不全和功能障碍， NOTCH 1突变，并进一步阐明HLHS的发病机制，在动态多细胞串扰， 心血管细胞通过NOTCH-DLL-JAG受体-配体相互作用。在初步结果中，我们观察到 当NOTCH信号被抑制时，VCO中的血管形成减少，心肌样环较小 通过g-分泌酶抑制剂，和较小的类器官的大小，注意到在NOTCH 1 +/- VCO相比， 等基因野生型VCO。我们将进一步探讨NOTCH 1信号在血管和心脏形成中的作用。 通过测量心血管结构、增殖、功能和相应的基因表达， (Aim 1）。此外，我们的初步研究还表明，通过NOTCH-DLL-1上调NOTCH 1信号传导。 VCO中的JAG配体-受体相互作用与非血管化的心脏类器官相比， 细胞RNA-seq和配体-受体分析。我们将进一步研究配体-受体相互作用， 在NOTCH 1 +/- VCO形成过程中NOTCH 1信令的伪时间轨迹。蛋白质组 HLHS VCO开发过程中的蛋白质组学分析将揭示相关性， 使用单细胞RNA-seq数据进行验证（目标2）。完成拟议的项目将是更好的 确定NOTCH 1信号传导介导的多细胞串扰和下游途径 HLHS中心脏血管发育不全。