Extracellular ATP as regulator of hypoxia-induced vasa vasorum neovascularization

描述（由申请人提供）：病理性血管重构是体循环和肺循环血管疾病的关键组成部分，经常危及生命。在新生儿缺氧肺动脉高压模型中，我们之前报道过，与IPAH患者相似，缺氧诱导的肺动脉（PA）重塑与血管血管（VV）网络密度显著增加、循环祖细胞和炎症细胞向血管血管（VV）聚集和浸润相关，暗示VV与疾病发病机制有关。我们研究的长期目标是评估在缺氧诱导的肺血管重构中介导血管新生的细胞机制和内源性分子因子。细胞外腺嘌呤核苷酸越来越被认为是血管功能的重要调节因子。我们证明了外血管内皮细胞（VVEC）是细胞外ATP的有效来源，ATP作为自分泌/旁分泌因子介导缺氧诱导的VVEC血管生成。我们的数据表明，VVEC中夸大的核苷酸介导的血管生成反应涉及P2Y1和P2Y13嘌呤能受体、PI3K/Akt/mTOR、ERK1/2的激活，以及细胞质、核质和线粒体Ca2+的升高。此外，我们的初步数据表明，在VVEC培养物中存在表达高增殖潜力集落形成细胞（VVECFC）的P2Y13R亚群，这可能是血管生成VV扩增的原因。由于细胞能量代谢的重要性已被假设与细胞代谢状态和功能反应有关，在本提案中，我们将验证VV血管生成的嘌呤能调节涉及VVEC和VVECFC中有丝分裂和生物能量途径的激活，导致这些细胞的代谢重组/重编程和表型变化，以分化内皮表型。在三个具体目标中提出的研究将：(i)描述居住在VVEC中的集落形成细胞的层次结构；确定其表型、增殖和血管生成能力；（ii）检测嘌呤能受体介导的信号通路在VVEC和VVECFC的克隆生成、血管生成和信号反应中的作用；（iii）确定VVEC和VVECFC的生物能组织，并确定细胞外核苷酸和缺氧是否通过能量代谢和线粒体调节诱导这些细胞的血管生成和表型反应。最终，本研究计划希望将嘌呤能信号传导的基本问题转化为缺氧诱导血管生成的临床相关问题。了解VV向功能性血管网络扩张的代谢和信号调节的基本生物学机制，将有助于开发肺动脉高压和各种血管生成受损和病理性血管重塑的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.