Lentivirus Vector-Mediated Knockdown of Erythropoietin-Producing Hepatocellular Carcinoma Receptors B4 Inhibits Laser-Induced Choroidal Neovascularization

Abstract

To evaluate the efficacy of erythropoietin-producing hepatocellular carcinoma receptors B4 (EphB4) knockdown on the development of laser-induced choroidal neovascularization (CNV) in vivo. We constructed recombinant lentiviral vectors (Lv) Lv-shRNA-EphB4 to specifically knock down the expression of EphB4. The mRNA and protein expression of EphB4 was investigated by real-time reverse transcription-polymerase chain reaction (RT-PCR) and western blot. CNV was induced by laser photocoagulation in C57BL/6 mice. The mice were then randomly assigned to be intravitreally injected with phosphate-buffered saline (PBS), Lv-shRNA-EphB4 recombinant lentivirus, or an unrelated shRNA recombinant lentivirus (pFU LV-shRNA-NC). An uninjected group was used as the control. Fundus fluorescein angiography (FFA), histologic analysis, and choroidal flat mounts analysis were applied to evaluate the inhibition of CNV after an intravitreal injection. Transfection of Lv-shRNA-EphB4 led to the knockdown of EphB4, and EphB4 mRNA was down-regulated by about 80%. FFA and histologic analysis revealed that the leakage areas and the mean thickness of CNV were much smaller in the Lv-shRNA-EphB4 group than in the PBS-treated, pFU Lv-shRNA-NC group and the non-injection group. Choroidal flat mounts showed significantly less leakage and smaller leakage areas in the Lv-shRNA-EphB4 group than those in other groups. Knocking down the expression of EphB4 exerts an inhibitory effect on CNV in vivo. It may provide a potential strategy for the treatment of CNV.