Epigenetics and Regulation of Oxidative Stress in Diabetic Retinopathy

PURPOSE. Oxidative stress plays a central role in the development of diabetic retinopathy, and in the pathogenesis of this blinding disease, activation of NADPH oxidase 2 (Nox2)-mediated. cytosolic reactive oxygen species (ROS) production precedes mitochondrial damage. The multicomponem cytosolic Nox2 has an obligatory component, Ras-related C3 botulinum toxin substrate 1 (Racl); in diabetes, Rae! is functionally and transcriptionally active. Diabetes also facilitates many epigenetic modifications, and activates both DNA methylating (Dmnts) and hydroxymethylating (Tets) enzymes. Our aim was to investigate the role of epigenetics in Rae] regulation in diabetes. METHODS: Using human retinal endothelial cells, exposed to high glucose, 5-methyl cytosine (5mC) and 5-hydroxy methyl cytosine (5hmC) levels, and binding of Dnm.t and 'lets were quantified at the Rae I promoter. The effect of inhibition of Dmnts/Tets (pharmacological inhibitors or short interfering RNA [siRNAD on glucose-induced activation of RacUROS production was evaluated. RESULTS were confirmed in retinal microvessels from streptozotociwinduced diabetic mice receiving intravitreally RESULTS. Despite high glucose-induced increased binding of Dnintl, 5mC levels remained subnormal at Rae] promoter. But, at the same site, 5hmC levels and transcription factor nuclear factor (NF)-kB binding were increased. Inhibition of Diunts/Tets prevented increase in 5hmC and NE-kB binding, and attenuated Racl activation. Similarly, in mouse retinal microvessels, Drina 1-siRNA ameliorated diabetes-induced increase in Rae] transcripts and activity, and decreased ROS levels. CONCLUSIONS. Thus, despite Dnmts activation, concomitant increase in s rapidly hydroxymethylates 5mC, allowing NF-KB to bind and activate Racl. These results imply a critical role of an active DNA methylation in cytosolic ROS regulation in the development of diabetic retinopathy.