Recombinant RGD-disintegrin DisBa-01 blocks integrin α_vβ₃ and impairs VEGF signaling in endothelial cells

Background: Integrins mediate cell adhesion, migration, and survival by connecting the intracellular machinery with the surrounding extracellular matrix. Previous studies demonstrated the interaction between α_vβ₃ integrin and VEGF type 2 receptor (VEGFR2) in VEGF-induced angiogenesis. DisBa-01, a recombinant His-tag fusion, RGD-disintegrin from Bothrops alternatus snake venom, binds to α_vβ₃ integrin with nanomolar affinity blocking cell adhesion to the extracellular matrix. Here we present in vitro evidence of a direct interference of DisBa-01 with α_vβ₃/VEGFR2 cross-talk and its downstream pathways.

Methods: Human umbilical vein (HUVECs) were cultured in plates coated with fibronectin (FN) or vitronectin (VN) and tested for migration, invasion and proliferation assays in the presence of VEGF, DisBa-01 (1000 nM) or VEGF and DisBa-01 simultaneously. Phosphorylation of α_vβ₃/VEGFR2 receptors and the activation of intracellular signaling pathways were analyzed by western blotting. Morphological alterations were observed and quantified by fluorescence confocal microscopy.

Results: DisBa-01 treatment of endothelial cells inhibited critical steps of VEGF-mediated angiogenesis such as migration, invasion and tubulogenesis. The blockage of α_vβ₃/VEGFR2 cross-talk by this disintegrin decreases protein expression and phosphorylation of VEGFR2 and β₃ integrin subunit, regulates FAK/SrC/Paxillin downstream signals, and inhibits ERK1/2 and PI3K pathways. These events result in actin re-organization and inhibition of HUVEC migration and adhesion. Labelled-DisBa-01 colocalizes with α_vβ₃ integrin and VEGFR2 in treated cells.

Conclusions: Disintegrin inhibition of α_vβ₃ integrin blocks VEGFR2 signalling, even in the presence of VEGF, which impairs the angiogenic mechanism. These results improve our understanding concerning the mechanisms of pharmacological inhibition of angiogenesis.