Extracellular ATP as regulator of hypoxia-induced vasa vasorum neovascularization

描述(申请人提供)：病理性血管重塑是体循环和肺循环血管疾病的关键组成部分，常常危及生命。在一个新生儿缺氧性肺动脉高压模型中，我们曾报道，与人类的缺氧性肺动脉高压相似，低氧诱导的肺动脉(PA)重塑与血管网络(VV)密度显著增加、循环前体细胞和炎性细胞向PA募集和渗透相关，提示VV参与了疾病的发病机制。我们研究的长期目标是评估在缺氧性肺血管重塑中介导血管新生的细胞机制和内源性分子因素。细胞外腺嘌呤核苷酸越来越被认为是重要的血管功能调节因子。我们证明外膜血管内皮细胞(VVEC)是细胞外ATP的有效来源，它作为自分泌/旁分泌因子介导低氧诱导的VVEC血管生成。我们的数据表明，在血管内皮细胞中，被夸大的核苷酸介导的血管生成反应包括激活PI3K/Akt/mTOR，以及胞浆、核质和线粒体钙升高。此外，我们的初步数据表明，在血管内皮细胞培养中存在表达高增殖潜能集落形成细胞(VVECFC)的P2Y13R亚群，这可能与血管生成VV的扩张有关。由于细胞能量代谢的重要性被认为是联系细胞代谢状态和功能反应的重要环节，在本提案中，我们将检验以下假设：嘌呤能调节VV血管生成涉及激活VVEC和VVECFC中的有丝分裂和生物能量通路，导致代谢重组/重编程和这些细胞的表型向分化的内皮表型转变。这些研究有三个具体目标：(I)确定存在于VVEC的集落形成细胞的等级；确定它们的表型、增殖和血管生成能力；(Ii)研究嘌呤能受体介导的VVEC和VVECFC中克隆、血管生成和信号反应的信号通路；(Iii)确定VVEC和VVECFC的生物能量组织，并确定细胞外核苷酸和低氧是否通过能量代谢和线粒体调节在这些细胞中诱导血管生成和表型反应。最终，这项研究计划致力于将嘌呤能信号的基本问题转化为低氧诱导的血管生成这一临床相关问题。了解VV扩张为功能性血管网络的代谢和信号调节的基本生物学机制，将有助于开发治疗肺动脉高压和涉及血管生成受损和病理性血管重塑的各种血管疾病的创新治疗方法。

DESCRIPTION (provided by applicant): Pathological vascular remodeling is a key component and frequently life-threatening consequence, of vascular diseases in both the systemic and pulmonary circulation. In a neonatal model of hypoxic pulmonary hypertension, we have previously reported tha We have previously reported that similar to humans with IPAH, hypoxia-induced pulmonary artery (PA) remodeling is associated with marked increases in the density of vasa vasorum (VV) network, recruitment and infiltration of circulating progenitor and inflammatory cells to the PA, implicating VV in disease pathogenesis. The long-term goal of our studies is to evaluate cellular mechanisms and endogenous molecular factors that mediate vasa vasorum neovascularization in hypoxia-induced pulmonary vascular remodeling. Extracellular adenine nucleotides are increasingly recognized as important regulators of vascular functions. We demonstrated that the adventitial vasa vasorum endothelial cells (VVEC) are a potent source of extracellular ATP, which acts as an autocrine/paracrine factor mediating hypoxia-induced VVEC angiogenesis. Our data demonstrated that exaggerated nucleotide-mediated angiogenic responses in VVEC involve activation of P2Y1, and P2Y13 purinergic receptors, PI3K/Akt/mTOR, ERK1/2, and the elevation of cytoplasmic, nucleoplasmic, and mitochondrial Ca2+. In addition, our preliminary data for this proposal we demonstrated the presence of subsets of P2Y13R expressing highly proliferative potential colony forming cells (VVECFC) in VVEC cultures, which might be responsible for angiogenic VV expansion. As the importance of cellular energy metabolism has been postulated to link cellular metabolic state and functional responses, in this proposal we will test the hypothesis that purinergic regulation of VV angiogenesis involves the activation of mitogenic and bioenergetic pathways in VVEC and VVECFC, leading to metabolic reorgnization/reprogramming and phenotypical changes in these cells towards differentiated endothelial phenotype. The studies proposed in three specific aims will: (i) Characterize a hierarchy of colony-forming cells residing in VVEC; determine their phenotype, proliferative and angiogenic capabilities; (ii) examine purinergic receptor-mediated signaling pathway in clonogenic, angiogenic, and signaling responses in VVEC and VVECFC; (iii) determine bioenergetic organization of VVEC and VVECFC and determine whether extracellular nucleotides and hypoxia induce angiogenic and phenotypic responses in these cells via energy metabolism and mitochondrial regulation. Ultimately, this research proposal aspires to translate fundamental questions of purinergic signaling to the clinically relevant problem of hypoxia-induced angiogenesis. Understanding of fundamental biological mechanisms of metabolic and signaling regulation of VV expansion to a functional vascular network will facilitate development of the innovative treatments for pulmonary hypertension and a variety of vascular diseases that involve impaired angiogenesis and pathologic vascular remodeling.