Angiogenesis, or blood vessel formation, is a finely regulated multi-step process. There is little angiogenesis after a dynamic period of developmental activity, yet in adult animals it can be stimulated within minutes by the appropriate stimuli. Among the initial steps in blood vessel formation are the chemotactic and mitogenic responses of endothelial cells to angiogenic factors, but the regulation of these responses is not well-understood. The goal of the proposed research is to analyze the role of angiogenic factor expression on normal and pathological blood vessel formation in vivo. Both in vitro assays and bioassays have been used to study angiogenesis and to identify angiogenic factors. The effects of expressing such factors at specific places and times during development, however, have not been assessed. The experiments described here seek to develop transgenic mouse models to examine and manipulate expression of angiogenic factors in vivo. The results of these experiments should lead to advances in our understanding of the role of these angiogenic molecules in blood vessel formation, and with these mouse model both classical mouse genetics and targeted manipulations of endogenous genes will be available tools for further studies of angiogenesis. Moreover, knowledge of the regulation of angiogenesis will help to elucidate its involvement in physiological and disease processes such as ovulation, wound healing, and tumorigenesis. Several approaches will be used to accomplish this goal. (i) To determine whether endothelial tumor cells from transgenic mice retain properties of normal endothelial cells (and thus can model normal angiogenic responses), cultured murine endothelial cells will be derived from tumors that arise in transgenic mice carrying polyoma oncogenes. Tumors and cultured tumor cells will be analyzed for the expression of angiogenic factors and proteases implicated in angiogenesis, and the responses of tumor cells to exogenous factors will be assayed. (ii) To determine whether sustained inappropriate production of angiogenic factors will perturb normal angiogenesis, simple mouse models are sought in which angiogenic factors are expressed in a specific tissue that is chosen to optimize for a non-lethal and easily identifiable phenotype. To this end transgenic mice carrying gene fusions of tissue specific promoters and angiogenic factor genes that direct expression to the postnatal brain or to the avascular lens will be generated and analyzed. (iii) To determine whether endothelial cells can be marked in vivo by targeted gene expression, the expression pattern of several potential endothelial cell promoters linked to a reported gene will be analyzed in transgenic mice. The developmental expression pattern of endothelial cell promoters identified in this way will also be analyzed.
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