INHIBITION OF ANGIOGENESIS IN-VITRO AND IN-VIVO - COMPARISON OF THE RELATIVE ACTIVITIES OF TRIFLAVIN, AN ARG-GLY-ASP-CONTAINING PEPTIDE ANDANTI-ALPHA(V)BETA(3) INTEGRIN MONOCLONAL-ANTIBODY

Disintegrin which contains the amino acid sequence Arg-Gly-Asp (RGD), has been implicated as a recognition site in interactions between extr acellular matrix (ECM) and cell membrane receptors. Triflavin, a 7.5 k Da cysteine-rich polypep tide purified from Trimeresurus flavoviridis snake venom, belongs to a family of disintegrins. Integrin alpha(v) be ta(3) has recently been identified as a marker of angiogenic blood ves sels and therefore anti-alpha(v) beta(3) mAb may significantly inhibit angiogenesis. Therefore, this study was designed to compare the relat ive activity of triflavin and anti-alpha(v) beta(3) mAb in human umbil ical vein endothelial cell (HUVEC) adhesion and migration in vitro, an d on angiogenesis induced by TNF alpha in chicken chorioallantoic memb rane (CAM). In this study, it was shown that triflavin (0.1 to 0.4 mu M) dose-dependently inhibited the adhesion of HUVECs to ECMs (i.e., vi tronectin, fibronectin, laminin and collagen type IV). At a concentrat ion of 10 mu M, anti-alpha(v) beta(3) mAb almost completely inhibited the adhesion of cells to vitronectin, had a moderate inhibitory effect on fibronectin and laminin, but only a slight inhibitory effect on co llagen type IV. On the other hand, vitronectin and fibronectin promote a significantly greater extent of cell adhesion and migration than la minin or collagen type IV over a wide range of concentrations (5 to 15 mu g/ml). In cell migration studies, triflavin (0.4 mu M) inhibited m ore markedly vitronectin-and fibronectin-mediated migration than that mediated by laminin-and collagen type IV. Comparison of the relative e ffectiveness of triflavin with anti-alpha(v) beta(3) mAb, showed that triflavin was at least twenty to thirty times more potent than anti-al pha(v) beta(3) mAb at inhibiting cell adhesion and migration. Furtherm ore, we used TNFalpha as an inducer of angiogenesis in the CAM assay. Close examination of the effects of triflavin and anti-alpha(v) beta(3 ) mAb on TNFalpha-induced angiogenesis revealed the presence of discon tinuous and disrupted blood vessels. However, anti-alpha(v) beta(3) mA b showed a significant effect only at a higher concentration (10 mu M) . These results suggest that the inhibition of angiogenesis may have b een due to interference with the adhesion and migration of endothelial cells to ECMs. The results also indicate that triflavin has a more po werful inhibitory effect than anti-alpha(v) beta(3) mAb on angiogenesi s, suggesting that triflavin could theoretically be used as a reasonab le therapeutic adjuvant for therapy or prevention of angiogenesis-indu ced diseases. (C) 1997 Elsevier Science B.V.