The proposed studies will focus on members of thrombospondin family and are based on the findings that thrombospondin 1 and 2 (TSP1 and TSP2) modulate growth and differentiation of endothelial cells. The applicants propose to express full-length, truncated, or in vitro mutagenized TSP1, TSP2, or TSP3 and also segments of TSP1. Production will be in Spodoptera frugiperda cells with recombinant baculoviruses. The recombinant proteins will be characterized, used to make antibodies, and tested in angiogenesis assays. The first specific aim is to learn the mechanism(s) by which TSP1 and TSP2 inhibit growth of bovine aortic endothelial (BAE) cells. They will define both the active site(s) in the TSPs and the receptor(s) on BAE cells. The second specific aim is to learn whether TSPs also inhibit growth of endothelial cells cultured from normal or malignant mouse mammary tissue. The third specific aim is to extend the analysis of the effects of TSP1 and TSP2 to the entire process of angiogenesis, including in vitro correlates such as cell migration and tube formation, and especially angiogenesis in vivo. Special emphasis will be placed on mammary gland- and mammary tumor- associated angiogenesis and on identification of TSP derivatives that are anti-angiogenic in all assays. The fourth specific aim is to learn how the anti-angiogenic activity of TSP1 and TSP2 may synergize or antagonize other determinants of angiogenesis in mammary tumors. In particular, the applicants will relate the antiangiogenic activity to other relevant activities of TSPs, especially activation of latent TGF- beta, inhibition of proteases, and modulation of cell adhesion. Longer- range goals will be to evaluate the hypothesis that expression of TSP1 and/or TSP2 will correlate inversely with density of microvessels in human breast carcinoma and then to plan experiments to evaluate the hypothesis that the TSP pathway is a suitable target for treatment of breast carcinoma based on inhibition of vascularization.
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