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 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.


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-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 angiogenic signals on endothelial cells.
Iruela-Arispe
M L
ML
Department of Molecular, Cell, and Developmental Biology and the Molecular Biology Institute, UCLA, Los Angeles, Calif 90095, USA. arispe@mbi.ucla.edu
Lombardo
M
M
Krutzsch
H C
HC
Lawler
J
J
Roberts
D D
DD
D000595:Amino Acid Sequence / Q000235:genetics; D000818:Animals; D002455:Cell Division / Q000187:drug effects; D002642:Chick Embryo; D016228:Endothelial Growth Factors / Q000037:antagonists & inhibitors / Q000502:physiology; D004730:Endothelium, Vascular / Q000166:cytology / Q000187:drug effects; D005321:Extraembryonic Membranes / Q000098:blood supply / Q000187:drug effects; D005260:Female; D016222:Fibroblast Growth Factor 2 / Q000037:antagonists & inhibitors / Q000502:physiology; D008222:Lymphokines / Q000037:antagonists & inhibitors / Q000502:physiology; D015674:Mammary Neoplasms, Animal / Q000473:pathology; D051379:Mice; D008819:Mice, Nude; D008969:Molecular Sequence Data; D009389:Neovascularization, Pathologic / Q000517:prevention & control*; D010446:Peptide Fragments / Q000235:genetics / Q000494:pharmacology; D011994:Recombinant Proteins; D020449:Repetitive Sequences, Amino Acid; D015398:Signal Transduction / Q000187:drug effects; D019700:Thrombospondin 1 / Q000235:genetics* / Q000494:pharmacology*; D014183:Transplantation, Heterologous; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors
D016228:Endothelial Growth Factors; D008222:Lymphokines; D010446:Peptide Fragments; D011994:Recombinant Proteins; D019700:Thrombospondin 1; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors; D016222:Fibroblast Growth Factor 2