IL-6 Is a Mediator of Retinal Neovascularization During Ischemic Retinopathy

Abstract

Purpose: : Retinal neovascularization is a major cause of blindness in proliferative diabetic retinopathy and retinopathy of prematurity. Interleukin-6 (IL-6) has been found to be increased in vitreous samples from patients with proliferative diabetic retinopathy, but the specific role of IL-6 in retinal neovascularization is not known. The goal of this study was to directly test whether IL-6 expression is required for retinal neovascularization during ischemic retinopathy.

Methods: : Oxygen-induced ischemic retinopathy (OIR) was induced by maintaining neonatal mice in 70% oxygen from postnatal day (P)7 to P12 and room air from P12 to P17. Control mice were kept in room air. C57BL/6 wild type (WT) or IL-6 +/- or IL6-/- mice were used in this study. Retinas were prepared for analysis of mRNA for IL-6, IL-6 receptor, and VEGF by quantitative RT-PCR. Superoxide was assayed by dihydroethidium (DHE) labeling and quantified by the Metamorph Image System. The retinal vasculature was examined by fluorescence microscopy after labeling retinal wholemounts with isolectin-B4. Morphometric data were quantified using NIH imageJ.

Results: : Levels of IL-6, IL-6 receptor and VEGF mRNA were markedly and progressively increased in the retinas of OIR mice as compared with the controls (P<0.05). Associated with the increases in IL-6 signaling components, the retina from OIR mice displayed increased superoxide formation as determined by DHE labeling (P<0.05). This increase was blocked by IL-6 knockout (P<0.05). The specificity of the staining was verified by quenching superoxide with PEG-SOD or inhibition of NADPH oxidase with apocynin. To address the role of IL-6 in ischemic neovascularization, areas of neovascular tufts and vascular dropout were determined in IL-6 +/- and IL-6 -/- littermates. There was no difference in the vascular dropout area at P12. However, at P17 the neovascular tuft area was decreased by ~50% in IL-6 -/- mice as compared IL-6 +/- mice (P<0.01). In contrast, physiological revascularization was increased by IL-6ko. The capillary-free area was reduced by 20.2% in IL-6 -/- as compared with IL-6 +/- mice (P<0.001).

Conclusions: : Lack of IL6 prevents oxidative stress and retinal neovascularization while enhancing physiological revascularization during ischemic retinopathy. This result indicates that IL6 is critically involved in the progress of retinal neovascularization during ischemic retinopathy.

Keywords: retinal neovascularization • cytokines/chemokines • hypoxia