Inhibition of angiogenesis by thrombospondin-1 is mediated by 2 independent regions within the type 1 repeats

Background-Suppression of tumor growth by thrombospondin-1 (TSP-1) has been associated with its ability to inhibit neovascularization. The antiangioge nic activity of TSP-1, as defined by cornea pocket assays, was previously m apped to the amino-terminal portion of the protein within the procollagen r egion and the type 1 repeats. Methods and Results-We evaluated the specificity and efficacy of different regions of TSP-1 using recombinant fragments of the protein on chorioallant oic membrane (CAM) angiogenesis and endothelial cell proliferation assays. In both assays, fragments containing the second and third type 1 repeats bu t not the procollagen region inhibited angiogenesis and endothelial cell pr oliferation. To further define the sequences responsible for the angiostati c effect of TSP-1, we used synthetic peptides. The CAM assay defined 2 sequ ences that independently suppressed angiogenesis. The amino-terminal end of the type 1 repeats showed higher potency for inhibiting angiogenesis drive n by basic fibroblast growth factor (FGF-2), whereas the second region;equa lly blocked angiogenesis driven by either FGF-2 or vascular endothelial gro wth factor (VEGF). Modifications of the active peptides revealed the specif ic amino acids required for the inhibitory response. One sequence included the conserved tryptophan residues in the amino-terminal end of the second a nd third type 1 repeats, and the other involved the amino acids that follow the CSVTCG sequence in the carboxy-terminus of these repeats. Both inhibit ion in the CAM assay and inhibition of breast tumor xenograft growth in nud e mice were independent of the TGF-beta-activating sequence located in the second type 1 repeat. Conclusions-These results indicate that the type 1 repeats of TSP-1 contain 2 subdomains that may independently inhibit neovascularization. They also identify 2 independent pathways by which TSP-1 can block FGF-2 and VEGF ang iogenic signals on endothelial cells.