Recombinant disintegrin targets α(v) β(3) integrin and leads to mediator production

Abstract

Integrin αvβ3 is most likely the foremost modulator of angiogenesis among all known integrins. Recombinant disintegrin DisBa-01, originally obtained from snake venom glands, binds to αvβ3, thereby significantly inhibiting adhesion and generating in vivo anti-metastatic ability. However, its function in mediator production is not clear. Here, we observed that the mediators VEGF-A, IL-8, and TGF-β are not produced by human umbilical vein endothelial cells (HUVEC cell line) or monocyte/macrophage cells (SC cell line) when cells adhered to vitronectin. However, when exposed to DisBa-01, HUVECs produced higher levels of TGF-β, and SC cells produced higher levels of VEGF-A. Nonetheless, HUVECs also showed an enhancement of apoptosis after losing adherence when exposed to disintegrin, which is a characteristic of anoikis. We propose that disintegrin DisBa-01 could be used to modulate integrin αvβ3 functions.

Keywords: DisBa; HUVEC; IL-8; TGF; VEGF; cancer; endothelial cell; integrin αvβ3; macrophage.

Figure 1. Cell viability measured by MTT salt after exposure to DisBa-01. Cell viability was observed after HUVECs or SC cells in culture were exposed to adhesion buffer (C), phorbol 12-myristate 13-acetate (PMA), or to DisBa-01 (DB). The results are expressed as the mean and standard error of at least four independent experiments, in which C is considered to represent maximum possible viability. Data were analyzed using the Tukey-Kramer test; P > 0.05 in all comparisons.

Figure 2. Apoptosis in cells that did or did not maintain adhesion after exposure to DisBa-01. HUVECs and SC cells were exposed to DisBa-01 at 10 µM (DB) or adhesion buffer alone (C) and left to adhere to vitronectin-coated plates. Apoptosis was observed by (A) detection of externalized phosphatidylserine or (B) targeting DNA breaks. The results are expressed as the mean and standard deviation of three independent experiments. Data were analyzed using the Dunn test; *P < 0.05. All other comparisons are not statistically significant.

Figure 3. Mediator production after adhesion to vitronectin. Production of VEGF-A, IL-8, and TGF-β1 was observed in the supernatants of HUVECs or SC cells after exposure to vitronectin (Vit) or bovine serum albumin (C) for 24 h. The results are expressed as the mean and standard error of at least three independent experiments. Data were analyzed using the Tukey-Kramer test; P > 0.05 in all comparisons.

Figure 4. Production of mediators when cells were stimulated with DisBa-01. Production of VEGF-A, IL-8, and TGF-β was observed in supernatants after (A) HUVECs or (B) SC cells were exposed to adhesion buffer (C), phorbol 12-myristate 13-acetate (PMA), or DisBa-01 (DB). The results are expressed as the mean and standard error of at least three independent experiments. Data were analyzed using the Tukey-Kramer test of samples compared with C; *P < 0.05, **P < 0.01, ***P < 0.001.