EphrinA1 inhibits VEGF–induced intracellular signaling and suppresses retinal neovascularization and blood–retinal barrier breakdown.

Abstract

Abstract: : Purpose: We previously presented that ephrinA1 suppressed VEGF–induced proliferation and intracellular signaling cascades including protein kinase C (PKC)–extracellular signal–regulated kinase (ERK) pathway and Akt in cultured retinal vascular endothelial cells. In the present study, we further investigated this mechanism and examined the effect of ephrinA1 on retinal neovascularization and vascular permeability in animal models. Methods: The effect of ephrinA1 on VEGF–induced phosphorylation of intracellular signaling molecules was investigated by immunoprecipitation and Western blot analysis. The involvement of phosphatase in this mechanism was also investigated using dominant negative SHP1 and SHP2 adenoviruses. The effect of intraocular injection of ephrinA1 on retinal neovascularization and vascular permeability was investigated in ROP (retinopathy of prematurity) mouse model and in a rat model using Evans–blue method, respectively. Results: EphrinA1 inhibited VEGF–induced migration and tube formation activity of BRECs (bovine retinal microvascular endothelial cells) by 50.0±7.2% and 67.3±1.0%, respectively. Western blot analysis showed that ephrinA1 inhibited VEGF–induced phosphorylation of KDR/Flk–1 receptors by 63.8±16.5% followed by suppression of PLCγ–PKC–ERK pathway and Akt. SHP1DN and SHP2DN adenoviruses had no effect on this suppression by ephrinA1. Intraocular injection of ephrinA1 suppressed retinal neovascularization in ROP mouse by 36.0±14.9%. Intraocular injection of VEGF increased blood–retinal barrier breakdown by 3.3±0.6 fold in a rat model, which was suppressed by co–injection with ephrinA1 by 46.0±10.0%. Conclusions:It was suggested that ephrinA1 inhibited VEGF–induced intracellular signaling through KDR/Flk–1 receptor inhibition. Suppression of in vivo retinal neovascularization and vascular permeability by ephrinA1 indicates the therapeutic possibility of ephrinA1 for retinal neovascularization and vasopermeability abnormalities in ischemic retinal diseases including diabetic retinopathy.

Keywords: hypoxia • growth factors/growth factor receptors • retinal neovascularization