(Circulation. 1999;100:1423-1431.)
© 1999 American Heart Association, Inc.
Basic Science Reports |
Inhibition of Angiogenesis by Thrombospondin-1 Is Mediated by 2 Independent Regions Within the Type 1 Repeats
From the Department of Molecular, Cell, and Developmental Biology and the Molecular Biology Institute, UCLA, Los Angeles, Calif (M.L.I.-A., M.L.); the Department of Pathology, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Mass (J.L.); and the Laboratory of Pathology, National Cancer Institute, NIH, Bethesda, Md (H.C.K., D.D.R.).
Correspondence to Dr Luisa Iruela-Arispe, Molecular Biology Institute, UCLA, 611 Charles Young Dr E, Los Angeles, CA 90095. E-mail arispe{at}mbi.ucla.edu
Background—Suppression of tumor growth by thrombospondin-1 (TSP-1) has been associated with its ability to inhibit neovascularization. The antiangiogenic activity of TSP-1, as defined by cornea pocket assays, was previously mapped to the amino-terminal portion of the protein within the procollagen region 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 chorioallantoic membrane (CAM) angiogenesis and endothelial cell proliferation assays. In both assays, fragments containing the second and third type 1 repeats but not the procollagen region inhibited angiogenesis and endothelial cell proliferation. To further define the sequences responsible for the angiostatic effect of TSP-1, we used synthetic peptides. The CAM assay defined 2 sequences that independently suppressed angiogenesis. The amino-terminal end of the type 1 repeats showed higher potency for inhibiting angiogenesis driven by basic fibroblast growth factor (FGF-2), whereas the second region equally blocked angiogenesis driven by either FGF-2 or vascular endothelial growth factor (VEGF). Modifications of the active peptides revealed the specific amino acids required for the inhibitory response. One sequence included the conserved tryptophan residues in the amino-terminal end of the second and third type 1 repeats, and the other involved the amino acids that follow the CSVTCG sequence in the carboxy-terminus of these repeats. Both inhibition in the CAM assay and inhibition of breast tumor xenograft growth in nude mice were independent of the TGF-ß–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 angiogenic signals on endothelial cells.
Key Words: angiogenesis • endothelium • vessels
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