423 - Role of Oxidative Stress in Epigenetic Modification of Matrix Metalloproteinase-9 in the Development of Diabetic Retinopathy

Abstract

In the pathogenesis of diabetic retinopathy, damaged retinal mitochondria accelerate apoptosis of retinal capillary cells, and regulation of oxidative stress by manipulating mitochondrial superoxide dismutase ( Sod2 ) protects mitochondrial homeostasis and prevents the development of diabetic retinopathy. We have shown that diabetes activates retinal matrix metalloproteinase-9, and activated MMP-9 damages the mitochondria. Recent studies have shown that the machinery responsible for epigenetic modifications is also altered in diabetes, and a dynamic DNA methylation process plays an important role in regulation of retinal MMP-9 transcription. The aim of this study is to investigate the role of oxidative stress in retinal MMP-9 transcription in diabetes. Methods The effect of regulation of mitochondrial superoxide on DNA methylation of MMP-9 promoter was investigated in retinal endothelial cells incubated in the presence or absence of a MnSOD mimetic- MnTBAP, by quantifying the levels of 5 methyl cytosine (5mC) and hydroxyl-methyl cytosine (5hmC). The binding of DNA methylating and hydroxymenthylating enzymes (Dnmts and Tets, respectively) at MMP-9 promoter (by chromatin immunoprecipitation) and mitochondrial homeostasis (cytochrome c leakage in cytosol and mtDNA transcription) were also evaluated. The in vitro results were confirmed in the retina of diabetic mice overexpressing Sod2 . Results Addition of MnTBAP ameliorated glucose-induced decrease in 5mC levels and increase in Dnmt1 binding at the MMP-9 promoter. In the same cell preparations, MnTBAP also ameliorated alterations in 5hmC levels and Tet binding, regulated MMP-9 transcription and prevented mitochondrial damage. Similarly, mice overexpressing Sod2 were protected from diabetes-induced alteration in MMP-9 promoter methylation and its transcription, and mitochondrial dysfunction. Conclusions Thus, oxidative stress regulation by chemical/genetic approaches maintains retinal mitochondrial homeostasis by ameliorating epigenetic modifications of the MMP -9 promoter.