The Role of the Inflammatory Mediators TNF‐α and IFN‐γ in the Hyperglycemia‐induced PECAM‐1 Ubiquitination and Degradation in Diabetic Retinopathy

Abstract

ObjectiveThe aim of this study was to investigate the role of TNF‐α and IFN‐γ in PECAM‐1 ubiquitination and subsequent degradation under hyperglycemic conditions in the diabetic retina.IntroductionInflammation is a major contributor to the development of diabetic retinopathy (DR). Two of the key mediators of inflammation, tumor necrosis factor‐α (TNF‐α) and interferon‐γ (IFN‐γ), have been shown to contribute to the pathophysiological changes leading to the progression of DR. However, their role in the hyperglycemia‐induced loss of retinal PECAM‐1 is not known. Platelet endothelial cell adhesion molecule‐1 (PECAM‐1) is a cell surface protein that is heavily expressed on vascular endothelial cells, and has an important role in the maintenance of the endothelium and its functions. We have previously found a significant decrease in PECAM‐1 levels in the diabetic retina and cultured rat retinal microvascular endothelial cells (RRMECs) grown under high glucose conditions. Thus, an investigation into the possible mechanism/s of PECAM‐1 loss may help in understanding the molecular processes leading to DR, and provide a possible therapeutic target.Materials and MethodsFor our in vitro studies, primary RRMECs were grown in normal glucose (NG, 5 mM glucose) or high glucose (HG, 25 mM) media for six days, with TNF‐α and/or IFN‐γ (20 ng/ml and 30 ng/ml, respectively) added 24 hrs prior to harvesting cells for analysis. For our animal studies, age‐matched male Wistar rats were injected with either vehicle (control), or Streptozotocin (STZ) (diabetic, 30 mg/kg/day) for three consecutive days in a model of type I diabetes. Retinas and plasma were collected eight weeks post‐STZ injection for analysis. PECAM‐1, TNF‐α, IFN‐γ, and ubiquitin levels were assessed using western blotting, ELISA, Immunofluorescence, and immunoprecipitation assays.ResultsTotal ubiquitination levels were significantly increased in the diabetic retina and in RRMECs grown under HG conditions (35% and 40% increase, respectively, p<0.05). Moreover, PECAM‐1 ubiquitination levels were significantly increased in RRMECs grown under hyperglycemic conditions (3‐fold, p<0.05). TNF‐α levels were significantly increased in diabetic STZ plasma and RRMECs grown under HG conditions (31%, 74.4% increase, respectively, p<0.05). Additionally, IFN‐γ levels were significantly increased in the diabetic retina (35% increase, p<0.05). PECAM‐1 levels were significantly decreased in RRMECs when treated with TNF‐α and/or IFN‐γ (60–75% decrease, p<0.05), with a significant increase in total ubiquitination levels (20–28% increase, p<0.05).ConclusionThese results indicate a possible role for TNF‐α and IFN‐γ in the hyperglycemia‐induced loss of PECAM‐1 in retinal endothelial cells, possibly by upregulating PECAM‐1 ubiquitination and subsequent degradation.Support or Funding InformationThis work was supported by funding from the National Institutes of Health (NIH) EY025632This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.