As a receptor for the specific angiogenic peptide vascular endothelial growth factor (VEGF), KDR/flk-1 has a central role in the formation of blood vessels in normal development of the cardio-vascular system and in pathological neovascularization. Spatial/temporal expression patterns and the results of gene deletion studies in mice demonstrate a critical developmental role for this gene. In addition, because KDR/flk-1 is broadly expressed among endothelial cells yet remains highly specific for this cell type, the study of its regulation may provide a paradigm for understanding endothelial cell-specific gene expression. The research design of this proposal exploits well-characterized aspects of gene regulation, such as DNA-protein interactions and alterations in chromatin structure, as tools to explore the mechanisms of KDR/flk-1 expression. Methods to be used include transient transfection assays, studies of DNA-protein interaction in vitro and in vivo, analyses of gene expression in transgenic mice, and nuclear protein identification and isolation. Using these methods, specific DNA sequences (cis-acting elements) that regulate KDR/flk-1 will be identified (AIM I) and DNA-protein interactions involving these elements will be characterized (AIM II). Identified regulatory elements will be tested carefully in vivo using transgenic mice expressing KDR/flk-1 promoter reporter gene constructs (AIM III). After defining these cis-acting elements in vitro and in vivo, the DNA sequences will be used to identify and/or clone the DNA-binding proteins that confer cell type-specific expression to KDR/flk-1 (AIM IV). It is hypothesized that these DNA-binding proteins will be novel and will have a central role in endothelial cell differentiation from hemangioblastic precursors. The goal of this proposal is to allow the applicant to investigate the molecular bases of KDR/flk-1 regulation. Given the essential role of KDR/flk-1 in vascular endothelial cell growth, understanding its regulation should yield crucial information about endothelial cell development from multipotent precursors, blood vessel formation in health and disease, and endothelial cell-type specific gene expression. In addition, mechanisms for targeted gene delivery to endothelial cells and for disruption of angiogenesis in its pathologic forms may be revealed. Given the broad implications of this proposal, the rich interdisciplinary research program within the Sealy Center and the applicant's clinical association with the Division of Cardiology will provide the applicant with important resources for resolving fundamental issues in vascular biology.
Showing the most recent 10 out of 34 publications
