Empowering Cardiac Progenitor Cell-Mediated Repair of Injured Myocardium by Overexpressing P2Y2 Nucleotide Receptor

 描述（由申请人提供）：心力衰竭（HF）是美国的主要死因。目前的药物治疗和植入式心脏辅助装置可能有助于改善短期HF症状，但不足以恢复和维持长期心脏完整性和功能。自体干细胞疗法已被引入作为当前HF疗法的有前景的替代方法。然而，内源性或过继转移的干细胞的修复反应是有限的，由于极差的植入率和增加的易感性细胞死亡在不利的坏死环境。因此，已经对来自不同来源的干细胞（包括心脏来源的祖细胞（CPC））进行遗传修饰和离体扩增，以改善它们的生长和存活能力，并最终促进体内上级再生。心肌缺血期间释放的细胞外核苷酸通过激活多个跨膜核苷酸受体（包括P2Y2受体（P2Y2R），一种由ATP和UTP等能激活的G蛋白偶联受体）启动损伤心肌愈合所需的炎症和再生反应。除了其在啮齿动物模型和人心肌细胞中建立的心脏保护反应之外，P2Y2R激动剂ATP和UTP是人造血干细胞（hHSC）增殖和迁移的有效刺激剂。ATP还诱导人骨髓间充质干细胞（hMSCs）的分化。然而，细胞外核苷酸在CPC中的生理作用尚未得到充分解决。本研究的目的是探讨P2核苷酸受体介导的生理反应，重点是P2Y2R在CPC中，并确定其潜在的机制。研究策略包括使用药理学以及功能丧失和获得研究对小鼠和人CPC中P2Y2R介导的应答进行初步体外表征。通过在梗死小鼠心肌过继转移之前在人CPC中过表达P2 Y2 R，体外研究结果将在体内得到扩展，我们假设这可以提高CPC介导的心肌梗死后受损心肌的修复潜力。我们还假设P2Y2R诱导的CPC再生反应涉及已知由各种G蛋白偶联受体调控的Hippo信号通路的激活。这提供了心肌缺血期间释放的细胞外核苷酸、细胞外基质传感和Hippo信号传导之间的新型直接联系，Hippo信号传导最近被引入作为损伤后心脏再生的重要调节剂。

 DESCRIPTION (provided by applicant): Heart failure (HF) is a major cause of death in the United States. Current pharmacological treatments and implantable cardiac assist devices may contribute to improving symptoms of HF on the short term but they are insufficient to restore and maintain cardiac integrity and function on the long term. Autologous stem cell therapy has been introduced as a promising alternative approach to current therapies for HF. However, the reparative responses of either endogenous or adoptively transferred stem cells are limited due to extremely poor engraftment rates and increased susceptibility to cell death in the unfavorable necrotic environment. Hence, genetic modification and ex vivo expansion of stem cells from diverse origins, including cardiac-derived progenitor cells (CPCs), have been pursued to improve their growth and survival capabilities and eventually promote superior regeneration in vivo. Extracellular nucleotides released during cardiac ischemia initiate inflammatory and regenerative responses required for the healing of injured myocardium through the activation of multiple transmembrane nucleotide receptors, including P2Y2 receptor (P2Y2R), a G protein-coupled receptor that is equipotently activated by ATP and UTP. In addition to its established cardioprotective responses in both rodent animal models and human cardiomyocytes, P2Y2R agonists ATP and UTP are potent stimulants of human hematopoietic stem cell (hHSC) proliferation and migration. ATP also induces the differentiation of human bone-marrow derived mesenchymal stem cells (hMSCs). Nonetheless, the physiological roles of extracellular nucleotides in CPCs have not been adequately addressed. This proposal aims to explore the physiological responses mediated by P2 nucleotide receptors with a focus on the P2Y2R in CPCs and identify the underlying mechanisms. Research strategy involves initial in vitro characterization of P2Y2R-mediated responses in mouse and human CPCs using pharmacological as well as loss- and gain-of- function studies. The in vitro findings will be extended in vivo by overexpressing P2Y2R in human CPCs prior to adoptive transfer in infarcted mouse myocardium, which we hypothesize improves CPC-mediated reparative potential of injured myocardium following myocardial infarction. We also hypothesize that P2Y2R-induced regenerative responses in CPCs involve the activation of Hippo signaling pathway that is known to be regulated by various G protein-coupled receptors. This provides a novel direct link between extracellular nucleotides released during cardiac ischemia, extracellular matrix sensing and Hippo signaling that has been recently introduced as an important regulator for cardiac regeneration following injury.