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Luciana Ferrara, Andrea De Erkenez, Elizabeth Fassbender, Siyuan Shen, Amber Woolfenden, Yubin Qiu, Stephen Poor, Karen Anderson, Bruce Jaffee; EphA2 Stimulation of Angiogenesis is Dependent on VEGFR2. Invest. Ophthalmol. Vis. Sci. 2013;54(15):5613. doi: https://doi.org/.
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The goal of this study was to determine the specific contribution of EphA2 to angiogenesis, independent of VEGF signaling. EphA2 is a member of the Eph receptors tyrosine kinases family. Inhibition of EphA2 signaling has been shown to block endothelial cell migration and assembly in vitro, and reduce pathological retinal and corneal neovascularization in vivo.
HUVEC and HRMVEC (Human Retinal Microvascular Endothelial Cells) were used at passage 5. Cells were treated with recombinant human VEGF or EphrinA1 and proliferation (BrdU incorporation, Roche) or migration (Transwell system, BD) was measured at 48h and 22h, respectively. Tube formation was determined in HUVECs after 11 days of treatment (IncuCyte, Essen BioScience). Ca2+ flux was measured using FLIPR Tetra (Molecular Devices). Rat aortic rings were sectioned from 8-week old rats and embedded in collagen I matrix. Rings were treated for 7 days and then vessel growth was visualized under phase contrast. Choroidal angiogenesis in murine laser-induced CNV model was performed on 8-week old C57/bl6 female mice by laser injury. VEGF, EphrinA1 or EphA2-Fc was administered subretinally immediately post-laser injury. Seven days post laser, mice were injected intravenously with FITC concanavalin A and CNV lesion size was quantified.
EphrinA1 stimulated proliferation, migration, tube formation and Ca2+ flux in HUVEC and HRMVEC, and angiogenesis in rat aortic rings, with a maximum effect comparable to VEGF. EphrinA1 also induced EphA2 and VEGFR2 phosphorylation, however, VEGF did not induce EphA2 phosphorylation. In order to determine the role of EphA2 in angiogenesis independent of VEGF, VEGFR2 was inhibited with axitinib, a tyrosine kinase inhibitor, and the functional response to EphrinA1 was evaluated. Our data suggest that blocking VEGFR2 signaling completely abrogates the functional response to EphrinA1 in endothelial cells (proliferation, Ca2+ flux). Therefore it is not possible to distinguish EphA2 contribution from VEGFR2. The role of EphA2 in angiogenesis was also evaluated in vivo in the mouse laser CNV model. EphrinA1 did not affect laser-induced neovascularization, while a soluble EphA2-Fc receptor, which had been reported to inhibit angiogenesis in vivo, exacerbated the CNV area (60% increase).
EphrinA1-induced activation of EphA2 stimulates angiogenesis in vitro, but not in vivo, and the effect is dependent on VEGFR2 signaling.
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