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.
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.
