Role Of EphrinB2 In Retinal Neovascularization

OBJECTIVE: To investigate the role of EphrinB2 in retinal neovascularization and observe the effect of ephrinB2/Fc on experimental retinal neovascularization.METHOD: (1)C57BL/6J mice at postnatal day (P)7 were exposed to 75% oxygen (O₂) for 5 days (until P12) and allowed to recover in room air to induce retinal NV. Retinas from unexposed and hyperxia-exposed mice between P7 to P24 were analyzed specifically for the retinal avascular area of the model animal, pre-retinal neovascular tufts counting, detecting the ephrinB2 by immunohitochemistry(IHC), transcript expression of ephrinB2 by RT-PCR and the active ephrinB2 protein by Western blot assay. (2) A group of hyperxia-exposed mice had one eye injected intravitreally with 150 ng/1.5 μ L of soluble EphrinB2/Fc chimeras during transition from high O₂ to room air (P12) and injected again on P14. Contralateral eyes were injected with PBS as the control. Preretinal nuclei and retinal blood vessels were quantified at peak disease (P17). (3)After treated by ephrinB2/Fc and VEGF165, the proliferation of HUVEC was measured by MTT method to observed the effect of ephrinB2/Fc and VEGF respectively. And the cell migration were also studied by scratch healing test and Transwell cell supports system. (4) The influence of VEGF on HUVEC and the expression of active ephrinB2 were analyzed by Western blot assay.RESULTS: (1) After 5 days exposed in the hyperxia of 75% O₂ and back into air condition, the animals were induced typical experimental retinal neovascularization(RNV) . Parts of the retinas from RNV were staining by ADPase and for stretched preparation. The avascular area and pre-retinal nuclei of the retinas was taken respectively for statistic in exposed and unexposed animal. There were much more nuclei tufts and avascular area in the RNV model animal(p<0.0001) than that of the control, and the ephrinB2 was detected in strongly expressed by IHC, the mRNA transcript gene was harvested by RT-PCR, the western blot assay showed the ladder of ephrinB2. (2)The data showed that ephrinB2/Fc is therapeutically effective for the experimental RNV according to the pre-retinal NV nuclei, avascular area image measurement and the positive reaction of ephrinB2 by IMC. (3) No suppress was found with ephrinB2/Fc alone add to HUVEC, whereas the inhibition becomes strong when adding VEGF to the culture, and it would be stronger when the concentration of ephrinB2 is augmentation. (4)VEGF can induce the intensive expression of ephrinB2 on the HUVEC and ephrinB2/Fc just inhibits the expressingof ephrinB2.CONCLUSIONS: (1) The animal model which copied by add O₂ for 5 days from P7 is very stable and available for experimental RNV research. (2) The retinal staining method by ADPase is very suitable and concise. The ephrinB2 could be specially detected in the experimental RNV, besides, ephrinb2/Fc could decrease the neovascular disease greatly. So it revealed that the ephrinB2 would be the important factor involved RNV, and maybe interacted with VEGF in the process of pathologic NV.