Regulation of coronary vascular development by differential tissue hypoxia

描述（由申请人提供）：主要冠状动脉在定型位置形成。异常左冠状动脉与肺动脉（ALCAPA）的位置偏离具有严重的临床后果，包括猝死。本研究的总体目的是描述引导冠状动脉血管分化和组织的机制。我们检测到差异高水平的缺氧和缺氧诱导因子-1a（HIF-1a）的核定位在发育中的心脏在特定的网站，主要冠状动脉血管将发展。我们的假设是，这些差异水平的缺氧在胚胎心脏冠状动脉血管的发展是至关重要的步骤，通过HIF-1a转录调控下游基因。我们的方法将是改变HIF-1a介导的转录活性的影响，迫使显性负或组成型活性HIF-1a的表达，特别是在心肌或心外膜的禽胚胎在卵内使用病毒载体的特点。我们将破坏HIF-1a的活性，并分析其对发育后期冠状动脉结构的影响。在不同的发育阶段，将使用先进的定量方法评估正常血管模式的破坏，这可能使我们能够预测涉及哪些下游因素。这种复杂的分析将需要我们的合作者提供的三维重建和新的定量分析。接下来，我们将重点关注改变HIF-1a活性对冠状动脉前体细胞行为的影响，这些前体细胞来自于体外系统和卵内禽类胚胎的心外膜和心外膜。我们将检测分化标志物的表达、上皮间质转化、增殖、凋亡和心肌侵袭。最后一个具体的目标将是确定HIF-1a如何调节维尔姆斯肿瘤抑制基因产物Wt 1在心外膜细胞中使用体外系统的活性。将鉴定由HIF-1a和/或Wt 1直接调控的基因。这些研究将阐明微环境缺氧如何协调主要冠状动脉血管在何时何地发育。公共卫生相关性：这项研究的临床意义在于，它将增加对影响婴儿、儿童和成人发病率和死亡率的冠状动脉血管异常的了解。这些发现不仅可以更早地预测缺陷，而且可以导致预防缺陷的策略。这些研究的更广泛意义在于，它们还可能导致用于患病心脏组织、移植组织或来自干细胞或基因治疗的新形成组织的受控血运重建的策略。

DESCRIPTION (provided by applicant): The major coronary arteries form in stereotyped locations. Abnormalities in location such as in Anomalous Left Coronary Artery from the Pulmonary Artery (ALCAPA) have serious clinical consequences including sudden death. The overall aim of this study is to delineate the mechanisms that guide coronary vessel differentiation and organization. We detected differentially high levels of hypoxia and hypoxia inducible factor -1a (HIF-1a) nuclear localization in the developing heart at specific sites where major coronary vessels will develop. Our hypothesis is that these differential levels of hypoxia within the embryonic heart are critical for steps in coronary vessel development through HIF-1a transcriptional regulation of downstream genes. Our approach will be to characterize the consequences of altering HIF-1a-mediated transcriptional activity by forcing the expression of dominant negative or constitutively active HIF-1a specifically in the myocardium or the epicardium of avian embryos in ovo using viral vectors. We will disrupt HIF-1a activity and assay for consequences to the architecture of the coronary arteries at late stages in development. Disruption of normal vascular patterns will be assessed at different developmental stages using advanced quantitative methods that may allow us to predict which downstream factors are involved. This complex analysis will require 3- D reconstruction and novel quantitative analyses provided by our collaborators. We will next focus on the effects of altering HIF-1a activity on the behavior of coronary precursor cells originating from the proepicardium and epicardium using in vitro systems as well as avian embryos in ovo. We will assay for expression of differentiation markers, epithelial mesenchymal transition, proliferation, apoptosis and myocardial invasion. The last specific aim will be to determine how HIF-1a regulates the activity of the Wilms' tumor suppressor gene product Wt1 in epicardial cells using in vitro systems. Genes directly regulated by HIF-1a and/or Wt1 will be identified. These studies will elucidate how microenvironmental hypoxia, coordinates where and when the major coronary vessels develop. PUBLIC HEALTH RELEVANCE: The clinical significance of this study is that it will increase understanding of coronary vascular anomalies that impact the morbidity and mortality of infants, children, and adults. The findings may serve not only to predict defects earlier, but to lead to strategies to prevent defects. The broader significance of these studies is that they may also lead to strategies for the controlled revascularization of diseased cardiac tissues, transplanted tissues, or newly formed tissues from stem cell or gene therapy.