Abstract
BackgroundIntegrins mediate cell adhesion, migration, and survival by connecting the intracellular machinery with the surrounding extracellular matrix. Previous studies demonstrated the interaction between αvβ3 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 αvβ3 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 αvβ3/VEGFR2 cross-talk and its downstream pathways.MethodsHuman 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 αvβ3/VEGFR2 receptors and the activation of intracellular signaling pathways were analyzed by western blotting. Morphological alterations were observed and quantified by fluorescence confocal microscopy.ResultsDisBa-01 treatment of endothelial cells inhibited critical steps of VEGF-mediated angiogenesis such as migration, invasion and tubulogenesis. The blockage of αvβ3/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 αvβ3 integrin and VEGFR2 in treated cells.ConclusionsDisintegrin inhibition of αvβ3 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.
Highlights
Integrins mediate cell adhesion, migration, and survival by connecting the intracellular machinery with the surrounding extracellular matrix
The number of viable endothelial cells was increased by vascular endothelial growth factor (VEGF) (34.5%) after a 24-h incubation period as expected, and DisBa-01 alone had no effect on cell viability (Fig.1a)
We showed that DisBa-01 does not affect focal adhesion kinase (FAK) or non-receptor protein tyrosine kinase (Src) phosphorylation; FAK and Src activation by VEGF appears to be insufficient to fully activate downstream signaling pathways, such as extracellular-signal-regulated kinase-1/2 (ERK1/2) or phosphatidylinositol 3-kinase (PI3K)
Summary
Migration, and survival by connecting the intracellular machinery with the surrounding extracellular matrix. VEGF-A/VEGFR2 signaling stimulates a myriad of intracellular signaling pathways such as activation of phosphatidylinositol 3-kinase (PI3K), extracellular-signal-regulated kinase (Erk) pathway, focal adhesion kinase (FAK), c-Src family and paxillin [9, 10] The activation of such pathways results in a wide range of cell responses including increased vessel permeability and remodeling, endothelial cell proliferation, migration, tubulogenesis, secretion of matrix metalloproteinases (MMPs), nitric oxide (NO) and prostanoid synthesis [9, 11]. This synchronized signaling network, associated with cross talking to integrin receptors, modulates the angiogenic response and it is fundamental for tumor blood supply and growth [12, 13]. A direct association between the cytoplasmic tails of β3 integrin and of VEGFR2 was demonstrated [14], including crosslinks mediated by the transglutaminase Factor XIII (FXIII) of the coagulation cascade [7]
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