Inhibition of angiogenesis in vitro and in vivo: comparison of the relative activities of triflavin, an Arg-Gly-Asp-containing peptide and anti-alpha(v)beta3 integrin monoclonal antibody

Abstract

Disintegrin which contains the amino acid sequence Arg-Gly-Asp (RGD), has been implicated as a recognition site in interactions between extracellular matrix (ECM) and cell membrane receptors. Triflavin, a 7.5 kDa cysteine-rich polypeptide purified from Trimeresurus flavoviridis snake venom, belongs to a family of disintegrins. Integrin alpha(v)beta3 has recently been identified as a marker of angiogenic blood vessels and therefore anti-alpha(v)beta3 mAb may significantly inhibit angiogenesis. Therefore, this study was designed to compare the relative activity of triflavin and anti-alpha(v)beta3 mAb in human umbilical vein endothelial cell (HUVEC) adhesion and migration in vitro, and on angiogenesis induced by TNF(alpha) in chicken chorioallantoic membrane (CAM). In this study, it was shown that triflavin (0.1 to 0.4 microM) dose-dependently inhibited the adhesion of HUVECs to ECMs (i.e., vitronectin, fibronectin, laminin and collagen type IV). At a concentration of 10 microM, anti-alpha(v)beta3 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 collagen type IV. On the other hand, vitronectin and fibronectin promote a significantly greater extent of cell adhesion and migration than laminin or collagen type IV over a wide range of concentrations (5 to 15 microg/ml). In cell migration studies, triflavin (0.4 microM) inhibited more markedly vitronectin- and fibronectin-mediated migration than that mediated by laminin- and collagen type IV. Comparison of the relative effectiveness of triflavin with anti-alpha(v)beta3 mAb, showed that triflavin was at least twenty to thirty times more potent than anti-alpha(v)beta3 mAb at inhibiting cell adhesion and migration. Furthermore, we used TNF(alpha) as an inducer of angiogenesis in the CAM assay. Close examination of the effects of triflavin and anti-alpha(v)beta3 mAb on TNF(alpha)-induced angiogenesis revealed the presence of discontinuous and disrupted blood vessels. However, anti-alpha(v)beta3 mAb showed a significant effect only at a higher concentration (10 microM). These results suggest that the inhibition of angiogenesis may have been due to interference with the adhesion and migration of endothelial cells to ECMs. The results also indicate that triflavin has a more powerful inhibitory effect than anti-alpha(v)beta3 mAb on angiogenesis, suggesting that triflavin could theoretically be used as a reasonable therapeutic adjuvant for therapy or prevention of angiogenesis-induced diseases.