) The metazoan circulatory system undergoes development and remodeling through the processes of vasculogenesis, angiogenesis, and arteriogenesis. Angiogenesis is now recognized as a process central to embryonic development, organogenesis and regenerative tissues proliferation, and tumor growth. Enhancement of angiogenesis and vasculogenesis is a current goal in the treatment of ischemic syndromes. Inhibition of angiogenesis, in contrast, is a current goal in the development of new adjunct therapies for cancer and for inflammatory or other benign disorders of hyperproliferation. Folkman and colleagues First postulated and confirmed the necessity of new capillary and microvessel growth for sustained tumor growth beyond a critical, usually nonlethal, mass determined by supply of nutrients and oxygen, and removal of metabolic waste products. Among the many substances Folkman and others subsequently identified as activators and inhibitors of angiogenesis have been proteolytic fragments of proteins with other functions. Objects of much recent attention due to their efficacy and lack of toxicity have been angiostatin, a carboxy-terminal fragment of the procoagulant, fibrinogen, endostatin, a carboxy-terminal fragment of collagen XVIII. Both were originally identified as inhibitors of human and murine tumor growth in mice, and subsequent biological investigation of these molecules has largely focused on their effects on angiogenesis in model system, and their effect on proliferation, cell progression, and apoptosis in tissue culture cells. Additional experiments, have described candidate proteolytic pathways for their biosynthesis and, more recently, structure determinations have been published. Still little studied has been the molecular mechanism by which angiostatin and endostatin interact with endothelial (and perhaps other target) cells and/or with matrix, and the hypothesized signaling cascades triggered by these putative binding interactions. Intracellular ions serve as important cellular second messengers and modulators in a wide variety of cell types and signaling pathways. We have therefore taken this approach to the study of angiogenesis inhibitors, and have discovered that both angiostatin and endostatin trigger acute Ca2+ transients in primary cultures of endothelial cells derived from both large and small-caliber vessels. Such transients are reduced or absent among a small panel of non-endothelial cells. More prolonged exposure to angiostatin and endostatin leads to attenuation of the Ca2+ transients produced by the angiogenic VEGF and FGF-2. In addition, endostatin triggers acute endothelial cell alkalinization. These observations form the basis of this two-year R2I proposal, in which we propose to study in greater detail the ionic signaling pathways elicited in endothelial cells by endostatin and angiostatin, and to use them to expression clone endothelial cell surface receptors for endostatin and, should time permit, for angiostatin as well. We will accomplish these objectives by pursuit of the following Specific Aims: 1. To clone cDNAs encoding endothelial cell receptors for endostatin and (time permitting) angiostatin, using parallel strategies enabled by the ability of these ligands to trigger elevations in intracellular [Ca2+]. 2. To extend our mechanistic characterization of Ca2+ signaling by endostatin and (time permitting) angiostatin, including interactions with endothelial responses to mechanical and aniosmotic perturbations. 3. To determine the molecular basis of pH-signaling by endostatin and (time permitting) angiostatin.
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