Antiangiogenic Vitreous Proteins After Penetrating Ocular Injury

Abstract

Abstract: : Purpose: Intravitreal injection has been historically used in pre–clinical trials to screen potential pharmacologic agents and it has been recently used in clinical trials because it eliminates complications associated with bioavailability. A consistent and significant angiostatic effect of vehicle injection has been observed when testing potentially therapeutic agents in a rat model of retinopathy of prematurity (ROP). The purpose of the present study is to test the anti–mitotic activity of fractionated vitreous samples from sham injected and uninjected rat eyes on human retinal microvascular endothelial cells (HRMEC) and to identify any potential angiostatic factor(s). Methods: Adult Sprague–Dawley rats received a dry needle retinal injury by penetrating the temporal or nasal pole of one eye. The opposite non–punctured eye served as a control. Rats were sacrificed a day later and the vitreous harvested. Rat vitreous from either injured or uninjured eyes was homogenized and fractionated by strong cation exchange chromatography. The anti–angiogenic activity of fractions from injured and uninjured eyes was assessed by testing the inhibition of HRMEC proliferation with the BrdU assay. Results: Several of the fractionated vitreous protein samples from eyes injured by dry needle puncture significantly reduced HRMEC proliferation. Fractions with a more pronounced anti–mitotic activity demonstrated dose–dependency and were further characterized by 2D gel electrophoresis and LC–GC–Mass spectrometry. Potent anti–mitotic activity in fractionated vitreous samples from uninjured eyes was not observed. Conclusions: Preliminary results indicate that fractionated vitreous extracts from eyes injured by dry needle puncture yield dramatic differences in inhibition of HRMEC proliferation that are related to differences in protein constituency determined by proteomic analysis. Similar differences in anti–mitotic activity in fractions from non–injured eyes were not observed.

Keywords: neovascularization • protein purification and characterization • vitreous