EphrinA1 Signaling Inhibits VEGF-induced ERK1/2 Phosphorylation and Retinal Endothelial Cell Proliferation

Abstract

Abstract: : Purpose: Retinal neovascularization is a very serious complication in ischemic ocular diseases such as diabetic retinopathy and retinopathy of prematurity. Eph receptors, a family of receptor tyrosine kinases, and ephrin ligands have been reported to play critical roles in embryonic angiogenesis and recently in adult neovascularization. VEGF is an endothelial cell-specific mitogenic factor, and is an important cytokine in various angiogenic processes such as retinal neovascularization. In the present study, we investigated the effects of Eph and ephrin ligands on action of vascular endothelial growth factor (VEGF). Methods: Northern blot analysis was performed to examine expression of Eph and ephrin on porcine retinal endothelial cells (PRECs) using radioactive probes of EphA2, B4, ephrinA1, B2, and B3. Western blot analysis was performed after stimulation with ephrinA1-Fc to evaluate the effect on VEGF signaling. DNA content of PRECs was measured to estimate the effect of ephrinA1 on PRECs growth. Results: Northern blot analysis indicated predominant expression of EphA2 receptor on PRECs. Western blot analysis showed tyrosine-phosphorylation of EphA2 receptor by ephrinA1-Fc stimulation. VEGF (25ng/ml) increased ERK1/2 phosphorylation by 8.5 fold 10 minutes after stimulation, which was inhibited by 5 minutes pretreatment with ephrinA1-Fc(1µg/ml) by 98.1. Basal phosphorylation level of ERK1/2 was decreased by 86 15minutes after ephrinA1-Fc stimulation. In the cell growth assay, VEGF increased PRECs growth (50±4.1, P0.001), which was completely inhibited by ephrinA1-Fc. Conclusions: These results suggest the therapeutic potential of Eph and ephrin for retinal neovascularization by inhibiting VEGF-induced ERK1/2 phosphorylation and retinal endothelial cell proliferation.

Keywords: retinal neovascularization • signal transduction • inhibitory receptors