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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 multicomponent cytosolic Nox2 has an obligatory component, Ras-related C3 botulinum toxin substrate 1 (Rac1); in diabetes, Rac1 is functionally and transcriptionally active. Diabetes also facilitates many epigenetic modifications, and activates both DNA methylating (Dnmts) and hydroxymethylating (Tets) enzymes. Our aim was to investigate the role of epigenetics in Rac1 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 Dnmt and Tets were quantified at the Rac1 promoter. The effect of inhibition of Dnmts/Tets (pharmacological inhibitors or short interfering RNA [siRNA]) on glucose-induced activation of Rac1-ROS production was evaluated. Results were confirmed in retinal microvessels from streptozotocin-induced diabetic mice receiving intravitreally Dnmt1-siRNA., Results: Despite high glucose-induced increased binding of Dnmt1, 5mC levels remained subnormal at Rac1 promoter. But, at the same site, 5hmC levels and transcription factor nuclear factor (NF)-kB binding were increased. Inhibition of Dnmts/Tets prevented increase in 5hmC and NF-kB binding, and attenuated Rac1 activation. Similarly, in mouse retinal microvessels, Dnmt1-siRNA ameliorated diabetes-induced increase in Rac1 transcripts and activity, and decreased ROS levels., Conclusions: Thus, despite Dnmts activation, concomitant increase in Tets rapidly hydroxymethylates 5mC, allowing NF-κB to bind and activate Rac1. These results imply a critical role of an active DNA methylation in cytosolic ROS regulation in the development of diabetic retinopathy.

Purpose: Diabetes activates matrix metalloproteinase-9 (MMP-9), and MMP-9 via damaging retinal mitochondria, activates capillary cell apoptosis. MMP-9 promoter has binding sites for many transcription factors, and in diabetes its promoter undergoes epigenetic modifications, including histone modifications and DNA methylation. Enhancer of Zeste homolog 2 (Ezh2), which catalyzes dimethylation/trimethylation of histone 3 lysine 27 (H3K27me2 and me3), is also associated with DNA methylation. Our aim was to investigate link(s) between histone and DNA modifications in the regulation of MMP-9., Methods: Using human retinal endothelial cells, and also retinal microvessels from diabetic rats, effect of hyperglycemia on H3K27me3, and recruitment of Ezh2 at the MMP-9 promoter were quantified by chromatin-immunoprecipitation technique. Role of H3K27 trimethylation in regulating DNA methylation-transcription of MMP-9 was determined by regulating Ezh2 by its specific siRNA and also a pharmacologic inhibitor., Results: Hyperglycemia elevated H3K27me3 levels and the recruitment of Ezh2 at the MMP-9 promoter, and increased the enzyme activity of Ezh2. Inhibition of Ezh2 attenuated recruitment of both DNA methylating (Dnmt1) and hydroxymethylating (Tet2) enzymes and 5 hydroxymethyl cytosine at the same region of the MMP-9 promoter, and prevented increase in MMP-9 transcription and mitochondrial damage., Conclusions: Activation of Ezh2 in diabetes, via trimethylation of H3K27, facilitates recruitment of the enzymes responsible for regulation of DNA methylation of the MMP-9 promoter, resulting in its transcriptional activation. Thus, a close crosstalk between H3K27 trimethylation and DNA methylation in diabetes plays a critical role in the maintenance of cellular epigenetic integrity of MMP-9.