Abstract
VEGF and VEGFR antibodies have been used as a therapeutic strategy to inhibit angiogenesis in many diseases; however, frequent and repeated administration of these antibodies to patients induces immunogenicity. In previous studies, we demonstrated that aggretin, a heterodimeric snake venom C-type lectin, exhibits pro-angiogenic activities via integrin α2β1 ligation. We hypothesised that small-mass aggretin fragments may bind integrin α2β1 and act as antagonists of angiogenesis. In this study, the anti-angiogenic efficacy of a synthesised aggretin α-chain C-terminus (AACT, residue 106–136) was evaluated in both in vitro and in vivo angiogenesis models. The AACT demonstrated inhibitory effects on collagen-induced platelet aggregation and HUVEC adhesion to immobilised collagen. These results indicated that AACT may block integrin α2β1−collagen interaction. AACT also inhibited HUVEC migration and tube formation. Aortic ring sprouting and Matrigel implant models demonstrated that AACT markedly inhibited VEGF-induced neovascularisation. In addition, induction of FAK/PI3K/ERK1/2 tyrosine phosphorylation and talin 1/2 associated with integrin β1 which are induced by VEGF were blocked by AACT. Similarly, tyrosine phosphorylation of VEFGR2 and ERK1/2 induced by VEGF was diminished in integrin α2-silenced endothelial cells. Our results demonstrate that AACT is a potential therapeutic candidate for angiogenesis related-diseases via integrin α2β1 blockade.
Highlights
Angiogenesis is the growth of blood vessels from pre-existing vasculature and plays an important role in wound healing, tumour growth/metastasis and inflammation-related diseases[1]
To investigate the inhibitory effect of Aggretin α–chain C-terminus (AACT) on integrin α2β1 activation, we examined the effect of AACT on collagen-induced platelet aggregation
To confirm integrin α2β1 as the major target for AACT-mediated HUVEC-collagen attachment, we examined the involvement of integrin α2 in cell adhesion
Summary
Angiogenesis is the growth of blood vessels from pre-existing vasculature and plays an important role in wound healing, tumour growth/metastasis and inflammation-related diseases[1]. Upon integrin α2β1-expressing cell adhesion to collagen, many physiological functions are activated, including extracellular matrix remodelling and the ERK pathway. E7820, a potent angiogenesis inhibitor, has been shown to reduce integrin α2 mRNA expression and inhibit basic fibroblast growth factor/VEGF-induced HUVEC proliferation and tube formation[16,17]. We demonstrate that aggretin α-chain C-terminal (AACT, 31 amino acid residues) inhibits collagen-induced platelet aggregation and HUVEC adhesion predominantly via integrin α2β1 ligation. We unveiled the anti-angiogenic activities of AACT by demonstrating its inhibitory effects on HUVEC migration, Matrigel-induced capillary tube formation and aortic ring sprouting in ex vivo assays and reducing neovascularisation in Matrigel implant angiogenesis assays in vivo. VEGF-induced VEFGR2 and ERK1/2 activation were abolished by integrin α2 siRNA transfection These results demonstrate that AACT inhibits angiogenesis in response to VEGF via α2β1 integrin blockade
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