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M. H. Davies, D. O. Zamora, J. R. Smith, M. R. Powers; Ephrin Molecules Mediate Apoptosis in Endothelial Cells and Retinal Neovascularization. Invest. Ophthalmol. Vis. Sci. 2007;48(13):1729. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
EphB4 (B4) receptors and their ephrinB2 (B2) ligands are essential for vascular development, but also play a role in pathological neovascularization (NV). We previously reported that soluble B4 or B2 significantly reduced retinal NV in a model of oxygen-induced retinopathy. This study investigates if soluble B4 and B2 suppresses retinal NV by induction of apoptosis in retinal endothelial cells (EC).
C57BL/6 mice at postnatal day 7 (P7) were exposed to 75% oxygen for 5 days (P12) and recovered in room air to induce retinal NV. One eye was injected intravitreally with 150ng in 1.5µl of soluble B4 or B2 on P12 and P14, while contralateral eyes were injected with IgG antibodies as control. Eyes were enucleated for histological analysis. At P16, TUNEL analysis was performed on retinal sections to compare the apoptotic response between B4 or B2 injected eyes and controls. In vitro studies were performed with human retinal microvascular EC (HREC), which are known to express B4 and B2. To examine the effect of B4 and B2 on HREC signaling pathways and apoptosis, cells were stimulated with 2µg/mL soluble B4, B2, or control IgG antibody in the presence or absence of VEGF (10ng/mL) for 0-3 hrs. Total protein was isolated and phospho-Erk1/2 and phospho-AKT levels were assessed by immunoblot analysis following 0, 2.5, 5, 15, 30 and 60 minutes of co-stimulation with B4, B2 or IgG ± VEGF. Additionally, a caspase 3 activation assay was performed as a measure of apoptosis following a 3hr stimulation.
Quantification of TUNEL positive vascular cells, located in areas of retinal NV, revealed a ~2.5-fold increase in apoptotic cells in the B4 and B2 injected mice compared to controls. Western blot analysis indicated that Erk1/2 phosphorylation was inhibited by ~50% after a 15 minute incubation with both B4 and B2 ± VEGF compared to IgG ± VEGF. There was no change in AKT phosphorylation following B4 or B2 stimulation compared to control. Furthermore, there was a significant increase in caspase 3 activity (~2-fold) after 3hr stimulation of B2 ± VEGF compared to IgG control ± VEGF (p<0.005). B4 stimulation had no effect on caspase 3 activity.
These data suggest that modulation of endogenous ephrinB2 and EphB4 signaling by soluble B4 and B2 can result in the suppression of retinal NV via induction of apoptosis. In vitro studies are consistent with a pro-apoptotic role for B2 and B4, either by modulation of VEGF-induced intracellular signals or, in the case of B2, by direct stimulation of EC apoptosis. These data illustrate the pleiotropic functions of B4 and B2, which may vary in response to cellular context.
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