VEGF Dependent Leukocyte Infiltration and Blood-Retinal Barrier Breakdown in Endotoxin-Induced Uveitis

Abstract

Abstract: : Purpose: Recently, vascular endothelial growth factor (VEGF) has been recognized as a pro-inflammatory cytokine. At sites of inflammation, leukocyte recruitment is mediated through a multistep cascade of events involving sequential rolling along vessel wall, firm adhesion to the endothelium, and transmigration out of vasculature. The current study investigated the effects of VEGF on leukocyte infiltration and blood-retinal barrier (BRB) breakdown in the mouse endotoxin-induced uveitis (EIU) model. Methods: EIU was induced in C57BL/6J mice via the subcutaneous injection of lipopolysaccharide (LPS). At 0, 6, 12, and 24 hours after LPS injection, retinas were isolated and the retinal VEGF mRNA levels were evaluated with RT-PCR. Retinal VEGF protein was evaluated with ELISA and immunohistochemistry. To investigate the effects of VEGF inhibition on EIU, an anti-VEGF aptamer (EYE001) was administered via intraperitoneal injection at the time of LPS injection. Leukocyte rolling and adhesion were evaluated via acridine orange leukocyte fluorography and Concanavalin A lectin labeling, respectively. Leukocytes infiltrating the vitreous were counted in 4 µm histological sections. BRB breakdown was evaluated with the FITC-dextran leakage assay. Results: Retinal VEGF mRNA and protein levels increased during the development of EIU (P < 0.01). VEGF was detected in all layers of the retina in EIU mice, while a little VEGF was detected in the retinas of the control mice. Leukocyte rolling and adhesion, as well as leukocyte infiltration into vitreous were significantly increased in the EIU mice (P < 0.01, P < 0.01, and P < 0.01 respectively). Significant blood-retinal barrier breakdown was detected in the EIU mice. VEGF inhibition with EYE001 potently suppressed leukocyte infiltration into the vitreous (P < 0.01) and blood-retinal barrier breakdown (P < 0.01), but did not suppress leukocyte rolling and adhesion. Conclusions: VEGF is upregulated in the retinas of EIU mice and contributes to blood-retinal barrier breakdown and the transendothelial migration of leukocytes.

Keywords: uveitis-clinical/animal model • inflammation • cytokines/chemokines