Peptides derived from two separate domains of the matrix protein thrombospondin-1 have anti-angiogenic activity

Abstract

Abstract. Thrombospondin-1 (TSP1) is a large modular matrix protein containing three identical disulfidelinked 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 [~H]thymidine incorporation stimulated by bFGF in cultured capillary endothelial cells. Although TSP1 has been shown to bind active TGF/31, 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 EDs0'S of 0.6-7 #M. 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 sin 2 gle 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.

Downmodulation of bFGF-binding protein expression following restoration of p53 function

“…Both IGF -BP3 and TSP -1 have been implicated in the bystander effect in other systems. 67,68 The finding here, that the expression of neither TSP -1 nor IGF -BP3 is significantly altered in Av1p53 -treated cells, is in contrast to previous observations. 45,57,61,69 The discrepancy between these findings could be related to tissue specificity.…”

The Wilms' tumor gene product represses the transcription of thrombospondin 1 in response to overexpression of c-Jun

“…The e ect of TSP1 on tumor growth could result from its capacity to inhibit angiogenesis; thus TSP1 inhibits basic ®broblast growth factor-induced angiogenesis in a rat cornea model (Tolsma et al, 1993), exogenously added TSP1 blocks the ability of cultured capillary endothelial cells to organize into cords (Tolsma et al, 1997), and down regulation of endothelial TSP1 enhances angiogenesis in vitro (DiPietro et al, 1994). Good evidence for an antiangiogenic e ect of TSP1 in vivo is provided by a recent study showing that the tumorigenicity of human ®brosarcomas and B16 melanomas in nude mice varied inversely with the level of TSP1 produced by the ®brosarcomas or provided exogenously (Volpert et al, 1998).…”

Peptides derived from type I thrombospondin repeat-containing proteins of the CCN family inhibit proliferation and migration of endothelial cells

“…These peptides exhibited a biphasic (nonmonotonic) response: Their activity reached a maximum at an intermediate concentration and decreased as their concentration was increased. Some characteristic examples of such biphasic responses include the anti-proliferative effects of the full length endostatin in vitro and in vivo (Celik et al, 2005;O'Reilly et al, 1997) and its small fragment derivatives (Tjin Tham Sjin et al, 2005), the anti-angiogenic fragments derived from the thrombospondin-1 (Tolsma et al, 1993) and thrombospondin-2 (Lopes et al, 2003) proteins, anti-angiogenic fragments derived from SPARC (Chlenski et al, 2004;Sage et al, 2003) and urokinase (Y. Guo, Mazar, Lebrun, & Rabbani, 2002).…”

“…We also quantified the activity of the predicted peptide fragments in the proliferation experiments relative to known short peptides derived from the type I thrombospondin repeats of the thrombospondin 1 protein Mal II (SPWSSCSVTCGDGVITRIR) and Mal III (SPWDICSVTCGGGVQKRSK) (Dawson et al, 1999;Tolsma et al, 1993). Those short peptides exhibited maximum activity of 25% and 35%, respectively, at 10 μg/ml (data not shown).…”

“…Both can inhibit vascular endothelial growth factor (VEGF)-induced angiogenesis in the chick chorioallantoic membrane assay as well as fibroblast growth factor (FGF-2)-induced neo-vascularization in the corneal micro-pocket assay Vazquez et al, 1999). Also two peptides, Mal II and Mal III, derived from the type I thrombospondin repeats of the thrombospondin 1 protein have been shown to exert antiangiogenic activities both in vitro and in vivo (Dawson et al, 1999;Tolsma et al, 1993). There is also an increasing interest in using the thrombospondin-1 protein as a prototype for designing anti-angiogenic peptides with optimized activities (Haviv et al, 2005).…”