Angiogenesis or neovascularization is an important process in development, wound healing, and cycling of the female reproductive system. Many diseases have a neovascular component which contributes to their pathogenesis. Notable among these is cancer in which the growth of tumors depends on a vascular input that also provides a route for metastasis to distant sites. Thrombospondin 1 (TS1) is a naturally occurring inhibitor of angiogenesis. Two peptides from the TS1 sequence have been identified which have angiostatic activity in vivo and in vitro. The long term goal of the proposed work is to understand the mechanism by which TS1 and these peptides derived from it inhibit angiogenesis and regulate EC phenotype. A line of polyoma middle T transformed microvascular endothelial cells (bEND) has been identified which expresses essentially no TS1. These cells grow rapidly with a nonendothelial habit and form large, cavernous hemangiomas in mice. Expression of TS1 in these cells results in a reversion of phenotype in vitro and prevents tumor formation in vivo. These cells can thus be used to assess the effects of """"""""adding back"""""""" TS1 expression and the results compared with normal, primary endothelial cells (EC). The effects of the peptide inhibitors in several in vitro systems which model critical steps of the angiogenic process will be assessed and the expression of important regulatory proteins will be determined in an in vitro three-dimensional model of capillary formation. The transformed bEND cells will be used as a test system in which a variety of mutants of TS1 will be expressed to localize in terms of domain and specific amino acid sequences the site(s) in the TS1 structure that regulate EC behavior. The receptors with which TS1 and its peptides interact will be identified and the candidate receptor CD36 will be tested to determine if it mediates some (or all) of the inhibitory effects of TS1 on EC. The intracellular signaling mechanisms used by known angiogenic factors will be examined and the effects of TS1 on these signal transduction pathways will be determined.
