The goal of this renewal proposal is to gain an understanding of mechanism(s) whereby thrombospondin-1 (TSP1) and -2 (TSP2) control angiogenesis. The hypothesis is that one or more of the self-folding modules of the TSPs, in native conformation(s), interact(s) with cell surface receptor(s) via specific amino acid sequence motif(s). The keys to achieving the goal are identification of module(s) that possess the pro-and anti-angiogenic activities of intact TSP1 and TSP2, analysis of structure-function relationships, and identification of cell surface receptors that interact with the active module(s). The work plan includes six aims: (1) preparation of overlapping recombinant segments of human TSP1 and TSP2; (2) structural characterization of the recombinant segments by low resolution techniques (ultracentrifugation, circular dichroism, intrinsic fluorescence, microcalorimetry) and comparison of the structures with those of the same segments in intact TSP1 or TSP2; (3) preparation of polyclonal and monoclonal antibodies to specific segments and epitopes of human TSP1 and TSP2; (4) identification of the module(s) of TSP1 and TSP2 that possess pro- and anti-angiogenic activities using the recombinant segments and segment-specific antibodies; (5) characterization of the structural features of the pro- and anti-angiogenic module(s) that are important of activity; and (6) characterization of the interactions of the pro- and anti-angiogenic module(s) with vascular cells. The results will allow the activities of TSPs to be compared and contrasted with other modulators of angiogenesis and lead to more rational therapies directed towards manipulation of neovascularization.
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