PEPTIDES DERIVED FROM 2 SEPARATE DOMAINS OF THE MATRIX PROTEIN THROMBOSPONDIN-1 HAVE ANTI-ANGIOGENIC ACTIVITY

Thrombospondin-1 (TSP1) is a large modular matrix protein containing t hree identical disulfide-linked 180-kD chains that inhibits neovascula rization in vivo (Good et al., 1990). To determine which of the struct ural motifs present in the 180-kD TSP1 polypeptide mediate the anti-an giogenic activity, a series of protease-generated fragments were teste d using several in vitro and in vivo assays that reflect angiogenic ac tivity. The majority of the anti-angiogenic activity of TSP1 resides i n the central 70-kD stalk region which alone could block neovasculariz ation induced by bFGF in the rat cornea in vivo and inhibit both migra tion in a modified Boyden chamber and [H-3]thymidine incorporation sti mulated by bFGF in cultured capillary endothelial cells. Although TSP1 has been shown to bind active TGFbeta1, this cytokine could not accou nt for the inhibitory effects of the stalk region of TSP1 on cultured endothelial cells. Peptides and truncated molecules were used to furth er localize inhibitory activity to two domains of the central stalk, t he 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 mole cules truncated before this domain were inactive as was the NH2-termin al heparin-binding domain that is present in both recombinant molecule s. A series of peptides from the procollagen-like region, the smallest of which consisted of residues 303-309 of TSP1, inhibited angiogenesi s 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 implan ted into the mouse. Nineteen residue peptides derived from two of the three type 1 repeats present in the intact TSP1 molecule blocked neova scularization in vivo in the rat cornea and inhibited the migration of cultured endothelial cells with ED50's of 0.6-7 muM. One of these pep tides, containing residues 481-499 of TSP1, also inhibited vessel form ation in granulation tissue invading sponges in vivo. These results su ggest that the large TSP1 molecule employs at least two different stru ctural domains and perhaps two different mechanisms to accomplish a si ngle 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 i nhibitors of the pathological neovascularization that underlies many d iseases.