Aggretin, a snake venom-derived endothelial integrin a 2b 1 agonist, induces angiogenesis in vitro and in vivo via expression of vascular endothelial growth factor

Abstract number: P1218

Huang^* T. F., Chung^* C. H.

^*National Taiwan University, Taiwan

Aggretin, a heterodimeric platelet aggregation inducer purified from Calloselasma rhodostoma venom, was identified as a collagen-like a 2b 1 agonist. In this report, we investigated the angiogenic effects of aggretin both in vitro and in vivo experiments, and also its possible mechanisms of action. Exposure of human umbilical vein endothelial cells (HUVEC) to aggretin significantly increased cell proliferation as determined by tetrazolium assay. Moreover, HUVEC migration toward immobilized aggretin was increased in a modified Boyden chamber model (haptotaxis) and this effect was blocked by A2-IIE10, an antibody raised against integrin a 2. Aggretin elicited significant angiogenic effects both in vivo (CAM model) and in vitro (Matrigel tube formation) angiogenesis assays, and these effects were blocked by A2-IIE10 or VEGF mAb. Regarding the binding site of aggretin, the binding of fluorescein-5-isothiocyanate-conjugated aggretin to HUVEC was dose-dependent and saturable, which was specifically inhibited by A2-IIE10, as examined by flow cytometry. Furthermore, incubation of HUVECs with aggretin activated PI3K p85, Akt and ERK1/2 phosphorylation, and the VEGF level was also elevated. The Akt and ERK1/2 activation was abolished by A2-IIE10 pretreatment. The angiogenic effect induced by aggretin may be via the production of VEGF, since the VEGF mAb was able to inhibit the later phase of Akt/ERK1-2 activation and the in vivo angiogenesis induced by aggretin. PI3K inhibitor simultaneously inhibited aggretin-induced endothelial proliferation and ERK activation. Therefore the activation of PI3 kinase p85, Akt and ERK are likely related to induction of cell migration and proliferation. In conclusion, aggretin induces endothelial cell proliferation, migration and angiogenesis by interacting with integrin a 2b 1, leading to activation of PI3K, Akt and ERK kinase pathways, and the increased expression of VEGF may be responsible for its angiogenic activity.