The inhibition of laser induced choroidal neovascularization in rats by soluble EphB4-HSA

Abstract

Purpose: EphB4 receptor and its ligand (EphrinB2) are critical regulators of new vessel formation. Inhibition of EphB4-Ephrin-B2 signaling suppress angiogenesis.The purpose of this study was to evaluate the effect of intravitreal injection of soluble EphB4 fused to human serum albumin (sEphB4-HSA) on the pathogenesis of laser induced choroidal neovascularization (CNV).

Methods: Focal laser photocoagulation (Diode green laser, 140 mW, 0.05 s, and 75 microns) was applied to Brown Norway pigmented rats. Four laser burns were created in the posterior pole of each retina. sEphB4-HSA (0.2, 0.6, 2 or 6 micrograms), or vehicle was injected into the vitreous, 3 and 7 days after laser photocoagulation. Fluorescein angiograms (FA) and Choroidal flat-mount with fluorescein-conjugated isolectin B4 staining was used to assess the volume of the CNV lesions using confocal microcopy. Yeast two hybrid screen was performed to study the interaction of EphB4 with other proteins. The interactions of EphB4 with candidate interacting proteins were verified in yeast two hybrid system and also using 293T cell co-immunoprecipitation experiment.

Results: sEphB4-HSA injection brought about a dose-dependent inhibition of leakage of FA. CNV volume was significantly reduced by the injection of sEphB4-HSA from 0.3 to 6 ug; the strongest inhibition of CNV was seen in the application of the highest dose of sEphB4-HSA. EphB4 showed interaction with three fibronectin coding clones in yeast two hybrid screen. The interaction was further confirmed by co-expression of tagged EphB4 and fibronectin. Work to define the minimum domain of fibronectin and the interaction domain of EphB4 is ongoing.

Conclusions: Intra-vitreous injection of sEphB4-HSA is able to suppress laser induced CNV. sEphB4- HSA efficacy is considered to be particularly high due to the observed binding to fibronectin that localizes to the vessel wall. Furthermore previous work shows long retention of sEphB4 in the vitreous and choroid, which is likely benefited from its binding to fibronectin. Early promising safety data of systemic sEphB4-HSA administration in humans and the efficacy in the laser injury model suggest that sEphB4-HSA should be investigated in the treatment of CNV.

Keywords: 453 choroid: neovascularization • 637 pathology: experimental