Inhibitory Efficacy Of Lentiviral Recombinants Expressing ShRNA Targeting EphB4Gene On Choroidal Neovascularization In A Laser-induced Mice Model

Background Choroidal neovascularization (CNV) leads to age-relatedmacular degeneration (AMD), which is the most common cause leading tovision loss. Also CNV will be found in a large number of patients who havepathologic myopia, the eye histoplasmosis syndrome， vascular-like stripedisease and idiopathic polyp-like choroidal vascular lesions. Pathophysiologyof performance generated by the CNV, including: activation of the choroidalvascular endothelial cells, changes in the extracellular matrix, while the complexprocess of endothelial cell proliferation, adhesion, migration, one of the manypositive or negative regulatory factor interactionseventually led to the CNV.Suppress the occurrence of CNV development and explore its pathogenesis hasbeen the hot spot of the ophthalmic scholars. The VEGF receptor, Angiopoietin (Tie) and Ephrin receptors (Ephs) belongsto the endothelial cell receptor tyrosine kinase (RTKs), RTKs is an importantregulator in the occurrence of vascularmolecules. On CNV study, the researchershad noted that VEGF is one of the most important factors in promoting CNVgenerated, while the Ephrin ligand and its receptor Eph specific cause vascularendothelial cell proliferation, migration, differentiation, angiogenesis.EphB receptors and EphrinB ligands in angiogenesis through signalingswhich are send out by each other. It can promote vascular network formation,and arteriovenous differentiation, and microvascular system in order to establisha non-symmetrical. Although the role of EphB4and EphrinB2in mature bloodvessels is still small, but they have played an important role in tissue damagecaused by physiological or pathological angiogenesis. Studies have shown thathuman retinal pigment epithelial cells (RPE) and bovine choroidalmicrovascular endothelial cells (CEC) were found to have EphrinB2ligands andEphB4receptor expression; By down-regulating the expression of VEGF inRPE cells the EphrinB2ligands and EphB4receptor expression is also reducedin the RPE and CEC co-culture system. He et al found that EphrinB2ligandsand EphB4receptor expressed in rat CNV, and the intravitreal injection ofrecombinant soluble EphB4extracellular domain of the unit composition(sEphB4) inhibited the formation of CNV, in order to further clarifyEphrinB2/EphB4impactting CNV has laid a solid foundation.We envision that by intravitreal injection of the lentiviral vector for EphB4gene in mice may inhibit the formation of CNV. In this study, we first constitutesa short hairpin targeting the mouse EphB4recombinant RNAs (vshRNA) byintravitreal injection into the laser-induced mouse CNV model, furtherobservation of CNV changes, and to prove the EphrinB2/EphB4mechanism participate in CNV formation.Objective: This study was to evaluate and investigate the inhibitory efficacy ofrecombinant lentiviral short hairpin RNA (shRNA) vector targeting the miceEphB4gene on experimental choroidal neovascularization (CNV).Methods: We developed intravitreal administered green fluorescent protein(GFP) co-expressed pGC FU-RNAi-NC-LV and EphB4-RNAi-LV. Thereal-time PCR and Weston blot were used to select the best one. CNV wasinduced by laser photocoagulation in96mice. The mice were then randomlyassigned to be injected intravitreally with phosphate-buffered saline (PBS), pGCFU-RNAi-NC-LV and LV-shRNA-EphB4respectively, and non-injection groupwas set as the control. Immunofluorescence staining, choroidal flatmount,fluorescein fundus angiography and histologic analysis were performed toevaluate the inhibitory efficacy on CNV.Results:(1) The Real-time PCR and Weston blot results showed that the thirdgeoup has the best Interference effection.(2)The fluorescein leakage areas ofCNV were significantly larger in the PBS, pGC FU-RNAi-NC-LV and non-injection group than in EphB4-RNAi-LV group (P<0.01). The mean thickness,breadth and area of the CNV lesions in the intravitreally EphB4-RNAi-LVtreated group was significantly smaller than other groups (P<0.01). No signs offunctional or ultrastructural destruction in retina were detected.Conclusion: EphB4may act as an important inhibit role on the formation ofexperimental CNV.