Regulation of coronary vascular development by differential tissue hypoxia

组织缺氧差异对冠状血管发育的调节

• 批准号: 7527668
• 负责人: MICHIKO WATANABE
• 金额: $ 51.89万
• 依托单位: CASE WESTERN RESERVE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-01 至 2011-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7527668
• 关键词: Adult; Apoptosis; Appearance; Architecture; Behavior; Biological Assay; Birds; Blood Vessels; Cardiac; Cells; Child; Clinical; Complex; Coronary; Coronary Vessels; Coronary artery; Defect; Development; Differentiation Antigens; Dominant-Negative Mutation; Embryo; Embryonic Heart; Ensure; Epicardium; Epithelial; Genes; Heart; Hypoxia; Hypoxia Inducible Factor; In Vitro; Infant Mortality; Lead; Location; Mediating; Mesenchymal; Methods; Microarray Analysis; Minor; Modeling; Molecular; Morbidity - disease rate; Myocardial; Myocardium; Nephroblastoma; Nuclear; Pathway interactions; Pattern; Proteins; Pulmonary artery structure; Regulation; Site; Staging; Stem cells; Stereotyping; Sudden Death; System; Thromboplastin; Time; Tissues; Transcriptional Activation; Transcriptional Regulation; Transplanted tissue; Tumor Suppressor Genes; Variant; Viral Vector; chromatin immunoprecipitation; clinically significant; gene therapy; in vivo; left coronary artery; novel; precursor cell; prevent; public health relevance; reconstruction; vasculogenesis

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.

描述（由申请人提供）：主要冠状动脉形成于固定位置。肺动脉左冠状动脉异常 (ALCAPA) 等位置异常会产生严重的临床后果，包括猝死。本研究的总体目标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将发育的特定部位检测到不同程度高水平的缺氧和缺氧诱导因子-1a (HIF-1a) 核定位。我们的假设是，胚胎心脏内这些不同程度的缺氧对于通过 HIF-1a 转录调节下游基因的冠状血管发育步骤至关重要。我们的方法是通过使用病毒载体强制显性失活或组成型活性 HIF-1a 在卵内禽胚胎的心肌或心外膜中表达，来表征改变 HIF-1a 介导的转录活性的后果。我们将破坏 HIF-1a 活性并分析对发育后期冠状动脉结构的影响。将使用先进的定量方法在不同的发育阶段评估正常血管模式的破坏，这可以让我们预测涉及哪些下游因素。这种复杂的分析需要我们的合作者提供 3D 重建和新颖的定量分析。接下来我们将重点研究改变 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，使用体外系统以及卵内禽胚胎。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。最后一个具体目标是利用体外系统确定 HIF-1a 如何调节心外膜细胞中 Wilms 肿瘤抑制基因产物 Wt1 的活性。将鉴定由 HIF-1a 和/或 Wt1 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血管发育的地点和时间。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血管异常的了解。这些发现不仅可以用于更早地预测缺陷，还可以用于制定预防缺陷的策略。这些研究更广泛的意义在于，它们还可能导致针对患病心脏组织、移植组织或通过干细胞或基因治疗新形成的组织进行受控血运重建的策略。