Thrombospondin 1 and 2 (TSPs) are naturally occurring regulators of angiogenesis that bind to CD36, the receptor that mediates the anti-angiogenic effects of TSPs on EC in vitro and in vivo. CD36 and its short cytoplasmic tail associate with src kinases, paxillin and unphosphorylated FAK. When expressed in TSP1- negative bEND cells (a transformed, tumorigenic EC line), TSP1 reverts the cells to a normal phenotype and alters expression of many genes involved in angiogenesis including PECAM1. TSP1 and PECAM1 regulate one another s expression in a reciprocal manner suggesting that they are components of a switch that regulates the balance between the angiogenic and differentiated states of EC. Thus TSP/CD36 do not simply inhibit angiogenesis, they promote the differentiated or vessel state of EC. Here we propose to (1) determine the dynamics and localization of CD36 expression during angiogenesis and development and assess the role of the peroxisome proliferator-activated receptor gamma (PPARgamma) in CD36 expression. (2) Use microvascular EC and non-CD36 expressing cells transfected with CD36 expression constructs to define the recognition or binding motif for CD36, identify CD36 associated proteins, both intracellular signaling proteins and other receptors, localize wild type and mutant CD36 in cells, elucidate the signaling mechanism of CD36 which inhibits motility and proliferation and leads to either apoptosis or differentiation depending on other coincident signals received by cells. (3) Determine the regulatory linkage between TSP/CD36 and PECAM1 expression and define its mechanism, and (4) Define the extent of the genetic program that characterizes the differentiated vs. the angiogenic state of EC using DNA microarray methtods. The sum of these experiments will provide a great deal of information about the regulation of angiogenesis as well as the compounds and information with which to initiate therapeutic trials. The principle of angiostatic therapy has already been established in the treatment of cancer using animal models. The TSP/CD36 system provides a normally functioning entre into the control of angiogenesis in which the ligand and receptor are well defined and whose mechanism will be elucidated as indicated above.
