title: Peptides derived from two separate domains of the matrix protein thrombospondin-1 have anti-angiogenic activity.
abstract: Thrombospondin-1 (TSP1) is a large modular matrix protein containing three identical disulfide-linked 180-kD chains that inhibits neovascularization in vivo (Good et al., 1990). To determine which of the structural motifs present in the 180-kD TSP1 polypeptide mediate the anti-angiogenic activity, a series of protease-generated fragments were tested using several in vitro and in vivo assays that reflect angiogenic activity. The majority of the anti-angiogenic activity of TSP1 resides in the central 70-kD stalk region which alone could block neovascularization induced by bFGF in the rat cornea in vivo and inhibit both migration in a modified Boyden chamber and [3H]thymidine incorporation stimulated by bFGF in cultured capillary endothelial cells. Although TSP1 has been shown to bind active TGF beta 1, this cytokine could not account for the inhibitory effects of the stalk region of TSP1 on cultured endothelial cells. Peptides and truncated molecules were used to further localize inhibitory activity to two domains of the central stalk, the procollagen homology region and the properdin-like type 1 repeats. Trimeric recombinant TSP1 containing NH2-terminal sequences truncated after the procollagen-like module inhibited endothelial cell migration in vitro and corneal neovascularization in vivo whereas trimeric molecules truncated before this domain were inactive as was the NH2-terminal heparin-binding domain that is present in both recombinant molecules. A series of peptides from the procollagen-like region, the smallest of which consisted of residues 303-309 of TSP1, inhibited angiogenesis in vivo in the rat cornea and the migration of endothelial cells in vitro. A 19-residue peptide containing these sequences blocked vessel formation in the granulation tissue invading a polyvinyl sponge implanted into the mouse. Nineteen residue peptides derived from two of the three type 1 repeats present in the intact TSP1 molecule blocked neovascularization in vivo in the rat cornea and inhibited the migration of cultured endothelial cells with ED50's of 0.6-7 microM. One of these peptides, containing residues 481-499 of TSP1, also inhibited vessel formation in granulation tissue invading sponges in vivo. These results suggest that the large TSP1 molecule employs at least two different structural domains and perhaps two different mechanisms to accomplish a single physiological function, the inhibition of neovascularization. The definition of short peptides from each of these domains that are able to block the angiogenic process may be of use in designing targeted inhibitors of the pathological neovascularization that underlies many diseases.
authors.affiliation: Department of Microbiology-Immunology, Northwestern University Medical School, Chicago, Illinois 60611.
authors.forename: S S
authors.initials: SS
authors.lastname: Tolsma
authors.forename: O V
authors.initials: OV
authors.lastname: Volpert
authors.forename: D J
authors.initials: DJ
authors.lastname: Good
authors.forename: W A
authors.initials: WA
authors.lastname: Frazier
authors.forename: P J
authors.initials: PJ
authors.lastname: Polverini
authors.forename: N
authors.initials: N
authors.lastname: Bouck
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D015815:Cell Adhesion Molecules / Q000737:chemistry / Q000502:physiology*; D002465:Cell Movement / Q000187:drug effects; D002478:Cells, Cultured; D003315:Cornea / Q000098:blood supply; D004730:Endothelium, Vascular / Q000166:cytology* / Q000187:drug effects; D016222:Fibroblast Growth Factor 2 / Q000494:pharmacology; D008969:Molecular Sequence Data; D009389:Neovascularization, Pathologic; D010446:Peptide Fragments / Q000737:chemistry / Q000494:pharmacology*; D010980:Platelet Membrane Glycoproteins / Q000737:chemistry / Q000502:physiology*; D011347:Procollagen / Q000737:chemistry; D011414:Properdin / Q000737:chemistry; D051381:Rats; D019699:Thrombospondins; D013936:Thymidine / Q000378:metabolism
chemical_list: D015815:Cell Adhesion Molecules; D010446:Peptide Fragments; D010980:Platelet Membrane Glycoproteins; D011347:Procollagen; D019699:Thrombospondins; D016222:Fibroblast Growth Factor 2; D011414:Properdin; D013936:Thymidine