Soluble ephrin-B2 mediates apoptosis in retinal neovascularization and in endothelial cells

Purpose: EphB4 receptors and their ephrinB2 ligands are essential for vascular development, but also play a role in pathological neovascularization (NV). We previously reported that soluble (s) forms of EphB4 and ephrinB2 significantly reduced retinal NV in a model of oxygen-induced retinopathy. This study investigates if these molecules suppress retinal NV by stimulation of endothelial cell (EC) apoptosis. Methods: C57BL/6 mice at postnatal day 7 (P7) were exposed to 75% oxygen for 5 days (P12) and allowed to recover in room air to induce retinal NV. One eye was injected intravitreally with 150 ng in 1.5 μL of sEphB4 or sEphrinB2 on P12 and P14, while contralateral eyes were injected with IgG antibody as control. Eyes were enucleated for histological analysis. At P16 TUNEL analysis and caspase-3 immunohistochemistry was performed on retinal sections to compare the apoptotic response between sEphB4 or sEphrinB2 injected eyes and controls. In vitro studies were performed with human retinal microvascular EC (HREC). Results: Quantification of TUNEL positive vascular cells, located in areas of retinal NV, revealed approximately 2.5-fold increase in apoptosis in sEphrinB2 injected eyes compared to control eyes. Immunohistochemistry studies revealed co-localization of both TUNEL positive cells and caspase-3 positive cells with the endothelial marker, von Willebrand factor. Cultured HREC demonstrated significantly higher caspase-3 activity after a 3 h stimulation with sEphrinB2 ± VEGF compared to IgG control ± VEGF (P < 0.005). sEphB4 stimulation had no significant effect on caspase-3 activity in HREC cultures. Conclusions: These data suggest that modulation of the endogenous ephrin signaling mechanism by sEphrinB2 may induce suppression of retinal NV via induction of apoptosis. Results of the in vitro studies suggest that sEphrinB2 may directly induce apoptosis of EC during pathological neovascularization.