Characterization of Azurocidin as a Permeability Factor in the Retina:Involvement in the Leukocyte–Mediated and VEGF–Induced Blood Retina Barrier Breakdown

Abstract

Purpose: : Azurocidin (AZ) is an inactive serine protease, which is released by neutrophils during inflammatory leukocyte–endothelial interaction. AZ is the main cause of neutrophil–evoked vascular hyperpermeability, however, its role in the eye is unknown. Therefore we investigated the role of AZ in retinal vascular permeability and in the VEGF–induced blood retina barrier (BRB) breakdown.

Methods: : Brown Norway rats received intravitreal injections of AZ (10µg) in one eye and PBS in the contralateral eye. BRB breakdown was quantified using the Evans Blue (EB) technique at 1, 3, and 24h after intravitreal injections. Firm leukocyte adhesion was quantified at 3 and 24h post AZ injection, using the conconavalin assay. To block AZ, aprotinin (30kKIU) was injected intravenously prior to the intravitreal injections. To investigate whether AZ plays a role in the VEGF–induced BRB breakdown, rats were treated intravenously with aprotinin (50kKIU q8h) or intravitreally with polyclonal anti–AZ antibody (1µg), followed by intravitreal injection of 50ng recombinant murine VEGF₁₆₄ in one eye and PBS in the other eye. BRB breakdown and retinal leukostasis were quantified 24h after VEGF injection.

Results: : AZ increases retinal vascular permeability in vivo. Intravitreal injection of AZ induced a 7.7 fold increase of vascular permeability compared to control eyes at 1–3h (n=8, p=0.03), a 2.7 fold increase at 3–5h (n=11, p=0.006) and a 1.7 fold increase at 24h (n=6, p=0.04). Our preliminary results suggest that AZ injection does not induce retinal leukostasis at 3 or 24h (n=4 and 5, p=0.67 and 0.95, respectively) post injection suggesting that AZ–induced BRB breakdown is predominantly through a direct effect on the retinal endothelium. Aprotinin inhibited AZ induced BRB breakdown by >95% (n=6, p=0.03). Treatment with aprotinin (n=6) or an anti–AZ antibody (n=8) suppressed VEGF–induced BRB breakdown by 93% (p=0.03) and 65% (p=0.03), respectively. Our preliminary data suggest that aprotinin does not affect VEGF–induced leukostasis (n=7) 24h post VEGF injection (p=0.13).

Conclusions: : Our data suggest that AZ is an important mediator of vascular permeability in the retina and that its action is downstream of VEGF–induced leukocyte–endothelial interaction.

Keywords: retina • inflammation • retinal adhesion