Thrombospondin-1 (TSP-1) is a potent inhibitor of angiogenesis in vivo and of microvascular endothelial cell (MVEC) responses to angiogenic factors in vitro. The anti-angiogeneic activity of TSP-1 is contained in a structural domain known as the type 1 repeat or TSR-1. CD36 is the cellular receptor for TSP-1 on MV3EC and is necessary for its anti- angiogenic activity. Structure-function analyses have determined that binding of TSP-1 to CD36 is mediated by interaction of the TSR-1 domain of TSP with a conserved domain called CLESH-1 in CD36. The long-term goal of this project is to characterize the anti-angiogenic switch mediated by the CD36/TSR-1 system. One specific aim is to characterize the regulation of this switch. Studies will test the hypothesis that histidine-rich glycoprotein (HRGP), a soluble CLESH-1 domain containing plasma protein, modulates the TSR-1/CD36 anti-angiogenic response in vivo. The mechanisms that control expression of CD36 on MVEC will also be studied. Other studies will determine the role of CD36 in mediating the anti-angiogenic activities of TSR-1 containing proteins other than TSP-1, such as ADAMS-TS1 and TSP-2. These studies will include determination of the ability of TSR-1 containing proteins to bind CD36 and to effect endothelial cell function in vitro and in vivo. Another aim will determine the intracellular signaling mechanisms responsible for the CD36-mediated endothelial angiostatic response. These studies will take advantage of several unique reagents, including the CD36 null mouse strain generated in our lab and peptides derived from the cytoplasmic domain of CD36. Specific experiments will identify candidate signaling molecules and pathways by characterizing proteins that interact physically with CD36 in endothelial cells. Once candidate signaling partners are identified, their role in angiogenesis will be studied using in vitro assays of endothelial function and in vivo assays of angiogenesis. The last aim will use a murine model of wound angiogenesis and a murine endothelial progenitor cell transplant model (developed by Dr. Rafii; project 4) to study the role of the CD36/TSR-1 switch in vivo. Characterization of this novel anti-angiogenic system will increase our understanding of angiogenesis and will help define potential new therapeutic targets for blood and vascular diseases and cancer. This project involves extensive use of the program cores and collaborations with other investigators in the program project, including Dr. Rafii (Project 4) to study regulation of CD36 expression in EC progenitors, and Dr. Gershengorn (Project 3) to study the impact of CD36 signaling on endothelial ell G-protein coupled receptors.