High glucose sensitizes human retinal endothelial cells for IFN‐g‐mediated apoptosis

Abstract

Abstract Purpose The biochemical mechanisms by which inflammatory cytokines cause damage in the diabetic retina are poorly understood. Indoelamine 2, 3‐dioxygenase (IDO) is an inducible by IFN‐γ enzyme and is the first enzyme of the kynurenine pathway, which produces cytotoxic kynurenines. In this study we have investigated the role of IDO in apoptosis of human retinal capillary endothelial cells (HREC) under hyperglycemic conditions. Methods HREC were cultured in medium containing high glucose (25 mM) or low glucose (7.5 mM) and incubated with 1‐100 U/ml of IFN‐γ. IDO activity was measured by an HPLC assay. Expression of IFN‐γ receptor 1, and activation of the JAK‐STAT signaling pathway along with activation of PKC‐δ was assessed by Western blotting. HREC apoptosis was measured by Hoechst staining. The role of IDO in HREC apoptosis was evaluated in the presence specific chemical inhibitors of the kynurenine pathway. Results IFN‐γ dose‐dependently activated JAK‐STAT signaling and PKC‐δ, and upregulated IDO. The IDO‐mediated tryptophan oxidation led to formation of kynurenines, which was followed by chemical modification of proteins by kynurenines in HREC. These changes were accompanied by production of reactive oxygen species (ROS) and depletion of protein‐free thiols. IFN‐γ inhibited cell cycle at low concentrations and caused caspase‐3‐mediated apoptosis and at higher concentrations, and those effects were amplified in the presence of high glucose in HREC. We found that IFN‐γ mediated cytotoxicity in HREC was primarily due to ROS generated by 3‐hydroxykynurenine. Conclusion Our results suggest that high glucose sensitizes HREC to deleterious effects IFN‐γ and provide a novel mechanistic pathway for retinal capillary endothelial cell death in diabetes.