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.
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