The overall goal of this project is to elucidate the events, patterns and factors regulating coronary angiogenesis. We will utilize electron microscopy, morphometry, histochemistry, immunocytochemistry, in situ hybridization, and hemodynamic assessment to study prenatal rat and in ovo chick hearts.
The first aim i s to define the transmural and regional vascularization patterns in the ventricles of these species.
The second aim i s to establish the formation of the major coronary arteries. Since the extracellular matrix (ECM) modulates endothelial cell migration and tube formation, Aim 3 is to determine the role of selective ECM molecules in the regulation of coronary angiogenesis. Our underlying hypothesis is that key components of the ECM, i.e., laminin, fibronectin, and collagens I, III, and IV, are critical to the formation of coronary vessels.
Our fourth aim i s to test the hypothesis that fibroblast growth factors are essential in vivo regulators of coronary angiogenesis. Studies in Aims 3 and 4 will be based on avascular fetal rat hearts implanted in oculo where they become vascularized, innervated and beat spontaneously. To test these hypotheses we will introduce blocking agents for the extracellular molecules and growth factors in oculo along with the implant, and study implant vascularization at various time points. Localization of selected key ECM molecule mRNAs will be performed by in situ hybridization in order to establish whether their synthesis corresponds to sites of angiogenesis. In additional experiments we will document the effects of the blocking agents on endothelial cell migration and tube formation by utilizing avascular fetal rat hearts cultured in vitro.
Aims 5 and 6 will test the hypotheses that the early growth of the coronary vasculature is dependent upon hemodynamic load and cardiac mass. In chick hearts in ovo we will increase hemodynamic load and myocyte hyperplasia by constricting the outflow tract. In contrast, a decreased hemodynamic load and a decreased cardiac mass associated with hypoplasia of cardiocytes will be accomplished by chronic verapamil suffusion. These conditions will be imposed prior to vascularization, and the data compared to hearts not subjected to perturbations (Aim 1). Our studies are of particular significance because 1) there is a paucity of data on coronary angiogenesis, and 2) our novel models and approaches provide the opportunity to examine the role of key intrinsic and extrinsic factors in the regulation of coronary angiogenesis in vivo.
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