TNF-α mediated apoptosis plays an important role in the development of early diabetic retinopathy and long-term histopathological alterations

Purpose: The pathophysiology of diabetic retinopathy involves leukocyte adhesion to retinal vasculature, early bloodretinal barrier breakdown, capillary nonperfusion, and endothelial cell death. We investigated the involvement of tumor necrosis factor α (TNF-α) in diabetes-related histopathological changes in two relevant rodent models. Methods: In short-term studies, Long-Evans rats with streptozotocin–induced diabetes were treated with or without the TNF-α inhibitor, etanercept. For long-term studies, tumor necrosis factor receptor I (TNF-RI)-deficient mice and TNFRII-deficient mice, as well as C57/Bl6 wild-type mice, were fed 30% galactose for up to 20 months. The retinal histopathological alterations of hypergalactosemia were analyzed in trypsin digest preparations. Endothelial cell injury and apoptosis in rat retinas were evaluated by propidium iodide, TUNEL, CytoDeath staining, and DNA fragmentation ELISA. Caspase 3 and 8 activity was evaluated by immunoblotting and quantitative enzymatic activity assay. Results: Etanercept suppressed caspase activation, retinal cell injury, and apoptosis in short-term diabetic rats. Pericyte and endothelial cell loss were also reduced in long-term hypergalactosemic mice. Long-term studies demonstrated that pericyte loss and endothelial cell loss were reduced in comparison to wild-type diabetic controls. Conclusions: Our study identifies an important role for TNF-α in the pathogenesis of signature diabetic retinopathy pathologies and demonstrates that etanercept can inhibit retinal cell death and long-term complication of diabetes. Taken together, our results suggest that etanercept could prove beneficial in preventing both early and late vascular diabetic complications. Leukocyte adhesion to the diabetic retinal vasculature results in early blood-retinal barrier breakdown, capillary nonperfusion, endothelial cell injury, and cell death. We have previously shown that intercellular adhesion molecule-1 (ICAM-1) and the leukocyte integrin CD18 are required for these processes [1,2]. We also demonstrated that leukocytemediated retinal cell apoptosis is among the earliest pathological manifestations of diabetic retinopathy and results in the formation of acellular-occluded capillaries, microaneurysms, and vascular basement membrane thickening [3-6]. The diabetic eye responds to the progressive vascular occlusions with an increase of vascular permeability, leading to macula edema, or the formation of neovessels that finally proliferate into the vitreous [3,4]. Various mediators, such as vascular endothelial growth factor (VEGF) and tumor necrosis factor-α (TNF-α), contribute to the upregulation of the adhesive molecules of the endothelial cells and leukocytes. Although the role of VEGF in the development of diabetic