Soluble Forms of EphB4 and EphrinB2 Reduce Neovascular Tuft Formation in the Mouse Model of ROP: Novel Targets to Inhibit Angiogenesis

Abstract

Abstract: : Purpose: EphB4 (B4) receptors and their EphrinB2 (B2) ligands are key regulators of cell migration during development and help regulate venule–arteriole boundaries. We hypothesize that these molecules play a role in neovascularization (NV) in the mouse model of oxygen–induced retinopathy and that soluble versions of these molecules can potentially alter the pathologic neovascular response in this disease. Methods: C57BL/6 mice at postnatal day 7 (P7) were exposed to 75% oxygen (O₂) for 5 days (P12) and allowed to recover in room air to induce retinal NV. Retinas from O₂ exposed mice (n=4) and unexposed control mice (n=4) were obtained at Ps: 7, 8, 10, 12, 14, 17, 21, 24 and analyzed for B4 and B2 mRNA expression by RT–PCR. Some O₂ exposed mice had one eye–injected intravitreally with 150ng in 1.5µls of soluble B4/IgG–Fc chimera (n=12) or B2/IgG–Fc chimera (n=13) during transition to room air (P12) and reinjected at P14. Contralateral eyes were injected with IgG antibodies as control (n=25). All injected eyes were collected during peak disease (P17) and processed for quantification of preretinal nuclei and labeling of blood vessels with anti–type IV collagen antibodies. Results: B4 mRNA was detected in the retinas of control mice at all time points, peaking at P12–P14, and decreasing back to initial levels by P17–P24. Interestingly, exposing mice to O₂ shifted B4’s peak expression to P14–P21. By P24, B4 mRNA levels were the same in O₂ and control mouse retinas. In contrast, B2 mRNA remained constant through P7–P24 in both O₂ and control mouse retinas. The number of preretinal nuclei in O₂ exposed mice was reduced by 66% (p<0.05) in B4 injected eyes and by 69% (p<0.05) in B2 injected eyes, as compared to control injections. There was no apparent alteration of intraretinal vascular development by the injections. Conclusions: B4 and B2 are expressed in the developing mouse retina, with B4’s expression being altered in O₂ injured retinas. Treatment with either soluble B4 or B2 significantly reduced the extent of O₂ induced NV. These results support the hypothesis that B4 and B2 are regulators of retinal NV during ROP and may serve as novel targets for therapeutic intervention.

Keywords: retinal neovascularization