Regulation of coronary vascular development by differential tissue hypoxia

描述（由申请人提供）：主要冠状动脉在固定位置形成。位置异常，如肺动脉左冠状动脉异常（ALCAPA）可导致包括猝死在内的严重临床后果。本研究的总体目的是描述指导冠状血管分化和组织的机制。我们在发育中的心脏中主要冠状血管发育的特定部位检测到不同水平的缺氧和缺氧诱导因子-1a （HIF-1a）核定位。我们的假设是，通过下游基因的HIF-1a转录调控，胚胎心脏内这些不同水平的缺氧对冠状血管发育的步骤至关重要。我们的方法将是通过使用病毒载体迫使HIF-1a显性阴性或组成性活性特异性表达在鸟蛋禽类胚胎的心肌或心外膜中，来表征改变HIF-1a介导的转录活性的后果。我们将破坏HIF-1a的活性，并在发展后期检测对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哪些下游因素参与其中。这种复杂的分析将需要三维重建和新颖的定量分析，我们的合作者提供。接下来，我们将关注改变HIF-1a活性对源自心外膜和心外膜的冠状动脉前体细胞行为的影响，并使用体外系统以及鸟蛋胚胎进行研究。我们将检测分化标志物、上皮间质转化、增殖、凋亡和心肌侵袭的表达。最后一个具体目标将是确定HIF-1a如何使用体外系统调节Wilms肿瘤抑制基因产物Wt1在心外膜细胞中的活性。HIF-1a和/或Wt1直接调控的基因将被鉴定。这些研究将阐明微环境缺氧如何协调主要冠状血管发育的地点和时间。公共卫生相关性：本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认识。这些发现不仅有助于更早地预测缺陷，而且有助于制定预防缺陷的策略。这些研究的更广泛意义在于，它们也可能导致通过干细胞或基因治疗对病变心脏组织、移植组织或新形成的组织进行有控制的血运重建的策略。

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.