The Role Of Tumor Necrosis Factor-Alpha And Interferon-Gamma In The Hyperglycemia-Induced Ubiquitination And Loss Of Platelet Endothelial Cell Adhesion Molecule-1 In Rat Retinal Endothelial Cells

Objective: This study aimed to investigate the role of the hyperglycemia-induced increase in tumor necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma) in the ubiquitination and degradation of platelet endothelial cell adhesion molecule-1 (PECAM-1) in the diabetic retina.Methods: Type I diabetes was induced in rats by the injection of streptozotocin, with age-matched non-diabetic rats as controls. Primary rat retinal microvascular endothelial cells were grown in normal or high glucose media for 6 days or in normal glucose media for 24 h with addition of TNF-alpha and/or IFN-gamma. PECAM-1, TNF-alpha, IFN-gamma, and ubiquitin levels were assessed using Western blotting, immunofluorescence, and immunoprecipitation assays. Additionally, proteasome activity was assessed both in vivo and in vitro.Results: Under hyperglycemic conditions, total ubiquitination levels in the retina and RRMECs, and PECAM-1 ubiquitination levels in RRMECs, were significantly increased. Additionally, TNF-alpha and IFN-gamma levels were significantly increased under hyperglycemic conditions. PECAM-1 levels in RRMECs treated with TNF-alpha and/or IFN-gamma were significantly decreased. Moreover, there was a significant decrease in proteasome activity in the diabetic retina, hyperglycemic RRMECs, and RRMECs treated with TNF-alpha or IFN-gamma.Conclusion: Tumor necrosis factor-alpha and IFN-gamma may contribute to the hyperglycemia-induced loss of PECAM-1 in retinal endothelial cells, possibly by upregulating PECAM-1 ubiquitination.