The endothelial lining of cardiac micro- and macrovessels is composed of morphologically and functionally distinct cell populations. The molecular pathways that are responsible for establishing and maintaining these phenotypes are poorly understood. As one approach to this problem we have focused on the in vitro and in vivo approaches, we have focused on the transcriptional mechanisms underlying endothelial cell gene expression in the heart. Using a number of in vitro and in vivo approaches, we have demonstrated that a fragment of the von Willebrand factor promoter directs expression to the microvascular bed of the heart while the 1600 bp 5' flanking region of the endothelial nitric oxide synthase (eNOS) promoter contains information for expression in the endothelial cell lining of both cardiac microvessels and epicardial arteries. We have further shown that microvascular expression of eNOS and vWF in the heart mediated, in part, by the interaction of PDGF-AB with cell surface PDGF alpha receptor. Given the importance of the vWF and eNOS genes in mediating diverse processes such as hemostasis., vasodilation, and angiogenesis, the PDGF signal transduction pathway is likely to be a key regulator of cardiac homeostasis and pathophysiology. The overall goal of this proposal is to dissect the transcriptional basis of eNOS expression in the micro- and macrovascular beds of the heart. In the first aim, we will employ transient and stable transfection assays to map the myocyte-responsive elements of the eNOS promoter. The results of these studies will be confirmed in a novel in vivo system. Specifically, deletant and mutant eNOS promoter constructs will be targeted to the Hprt locus on embryonic stem cells and the recombinant cells used to generate chimeric mice. In the second aim, we will delineate the vascular bed-specific DNA-protein interactions and clone the transcription factor(s) involved in mediating expression in the cardiac microcirculation. In the third aim, we will determine how the microvascular circuit is activated and maintained by studying the molecular basis of the PDGF- Ralpha switch. In the fourth and final aim, we will delineate the epicardial-specific promoter regions of the eNOS gene and study the environmental influence of shear stress and smooth muscle cells on expression levels. Taken together, the studies outlined in this proposal are designed to provide insight about local endothelial cell circuits within the heart. The results should provide an important foundation for understanding cell subtype-specific control of cardiac homeostasis in both health and disease states.
Showing the most recent 10 out of 53 publications
