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Maneesh Mailankot, Dawn Smith, Catherine Doller, Scott Howell, Ram H. Nagaraj; Inflammatory Cytokines Induce Expression Of Indoleamine 2,3-dioxygenase In Human Retinal Capillary Endothelial Cells: Implications For Diabetic Retinopathy. Invest. Ophthalmol. Vis. Sci. 2011;52(14):5934.
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Indoleamine 2,3-dioxygenase (IDO) is the key regulatory enzyme in the kynurenine pathway. Several studies including our own have shown that IDO is induced by interferon-gamma and that kynurenines produced through IDO-mediated oxidation of tryptophan are cytotoxic. It is now well established that inflammatory cytokines play a role in the pathogenesis of diabetic retinopathy, although the mechanisms are still not clear. In this study we investigated the effect of inflammatory cytokines on IDO and determined the biochemical mechanisms by which kynurenines induce apoptosis in human retinal capillary endothelial cells (HREC).
Human eyes were obtained from the Cleveland Eye Bank. HREC in culture were treated with TNF-α (0-20 ng/ml) or IL-1β (0-20 ng/ml) for 2 days. IDO activity was determined by HPLC, and IDO protein expression by immunocytochemistry and western blotting. Intracellular kynurenines levels and protein modification by kynurenines were assessed by HPLC and immunocytochemistry, respectively. The involvement of p38MAPK signaling pathway was studied by western blotting. ROS generation in cells was detected by a fluorescence assay and cell cycle analysis by flow cytometry. The role of IDO in cytokine-induced cytotoxicity was evaluated in the presence specific chemical inhibitors of the kynurenine pathway.
The IDO activity and protein expression were higher in the diabetic than in the non-diabetic human retina. The immunocytochemical analyses showed higher IDO expression in capillary endothelial cells of the diabetic retina. TNF-α and IL-1β dose dependently induced IDO protein expression and enzyme activity in HREC in culture. Chemical inhibition of p38MAPK activation blocked cytokine-induced IDO expression. The IDO induction was accompanied by an increase in kynurenine formation, protein modification by kynurenine, ROS generation and cell cycle perturbation. All these cytotoxic effects were blocked by 1-methyl D,L-tryptophan, a competitive inhibitor of IDO.
Our results suggest that IDO expression and activity are higher in the diabetic human retina and IDO-mediated kynurenine formation could be a potential mechanism for retinal capillary endothelial cell death in the diabetic retina.
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