Unravelling the Effects of a Recombinant Snake Venom VEGF on Endothelial Barrier Integrity

Snake venoms contain bioactive proteins with potent pharmacological activities. However, low yields of native toxins often limit their functional characterization. Heterologous expression in yeast enables recombinant protein production for biological studies. In Crotalus durissus terrificus snake venom, a family of proteins named VEGFs (Vascular Endothelial Growth Factors) promotes angiogenesis and increases vascular permeability through receptor interactions. This study investigated the effects of recombinant VEGF (rCdtVEGF) on endothelial barrier integrity using human umbilical vein endothelial cells (HUVECs). For confocal microscopy, HUVECs (2 × 10⁵ cells/well) were seeded onto gelatine-coated coverslips and stimulated with rCdtVEGF (1 nM) for 5–60 min. Cells were stained for VE-cadherin and actin and analyzed by confocal microscopy. We observed rapid alterations in actin organization, discontinuous VE-cadherin staining, zigzag patterns, and intercellular gaps, indicating destabilization of adherens junctions. We then assessed endothelial permeability using FITC-dextran (4 kDa) and FITC-albumin (66 kDa). HUVECs (6 × 10⁴ cells/well) were cultured on gelatine-coated inserts with 0.4-µm pores until approximately 90% confluence. After incubation with fluorescent tracers (1 mg/mL), cells were stimulated with rCdtVEGF (1 nM), and samples were collected from the lower compartment at 0, 30, 60, 90, and 120 min and analyzed by fluorescence. Both tracers showed progressive passage across the endothelial monolayer, indicating increased permeability over time. These findings suggest that snake venom VEGFs may modulate endothelial permeability, potentially promoting a microenvironment conducive to tissue regeneration without prolonged disruption of vascular barrier integrity.