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Purpose: Early activation of cytosolic NADPH oxidase-2 (Nox2) in diabetes increases retinal ROS production, damaging their mitochondria. The assembly of Nox2 holoenzyme requires activation of a small molecular weight G protein Rac1. Rac1 activation is regulated by guanine exchange factors and guanine nucleotide-dissociation inhibitors, and post-translational modifications assist in its association with exchange factors and dissociation inhibitors. The goal of this study is to investigate the mechanisms of Rac1 activation in the development of diabetic retinopathy., Methods: The levels of the dissociation inhibitor, prenylating enzyme (farnesyltransferase, FNTA), and exchange factor Vav2 were quantified in human retinal endothelial cells, incubated in normal or high glucose for 96 h. The roles of prenylation and Vav2 in Rac1-Nox2-ROS mitochondrial damage were confirmed in FNTA-siRNA-transfected cells and using the Vav2 inhibitor EHop, respectively. Retinal histopathology and functional changes associated with diabetic retinopathy were analyzed in diabetic mice receiving EHop for 6 months. Key parameters of Rac1 activation were confirmed in the retinal microvasculature from human donors with diabetic retinopathy., Results: In HRECs, glucose increased FNTA and Vav2 and decreased the dissociation inhibitor. FNTA-siRNA and EHop inhibited glucose-induced activation of Rac1-Nox2-ROS signaling. In diabetic mice, EHop ameliorated the development of retinopathy and functional/structural abnormalities and attenuated Rac1-Nox2-mitochondrial damage. Similar alterations in Rac1 regulators were observed in retinal microvasculature from human donors with diabetic retinopathy. In diabetes, Rac1 prenylation and its interactions with Vav2 contribute to Nox2-ROS-mitochondrial damage, and the pharmacological inhibitors to attenuate Rac1 interactions with its regulators could have the potential to halt/inhibit the development of diabetic retinopathy. Graphical Abstract Activation of prenylating enzyme farnesyltransferase (FNTA) in diabetes, prenylates Rac1. The binding of Rac1 with guanine nucleotide-dissociation inhibitor (GDI) is decreased, but its association with the guanine exchange factor, Vav2, is increased, resulting in Rac1 activation. Active Rac1 helps in the assembly of Nox2 holoenzyme, and Nox2 activation increases cytosolic ROS production, damaging the mitochondria. Damaged mitochondria accelerate capillary cell apoptosis, and ultimately, results in the development of diabetic retinopathy.

Mitochondria are dynamic in structure, and undergo continuous fusion-fission to maintain their homeostasis. In diabetes, retinal mitochondria are swollen, their membrane is damaged and mitochondrial fusion protein, mitofusin 2 (Mfn2), is decreased. DNA methylation machinery is also activated and methylation status of genes implicated in mitochondrial damage and biogenesis is altered. This study aims to investigate the role of mitochondrial fusion in the development of diabetic retinopathy, and to illustrate the molecular mechanism responsible for Mfn2 suppression. Using human retinal endothelial cells, manipulated for Mfn2, we investigated the role of fusion in mitochondrial structural and functional damage in diabetes. The molecular mechanism of its suppression in diabetic milieu was determined by investigating Mfn2 promoter DNA methylation, and confirmed using molecular and pharmacological inhibitors of DNA methylation. Similar studies were performed in the retinal microvasculature (prepared by hypotonic shock method) of diabetic rats, and human donors with documented diabetic retinopathy. Overexpression of Mfn2 prevented glucose-induced increase in mitochondrial fragmentation, decrease in complex III activity and increase in membrane permeability, mtDNA damage and apoptosis. High glucose hypermethylated Mfn2 promoter and decreased transcription factor (SP1) binding, and Dnmt inhibition protected Mfn2 promoter from these changes. In streptozotocin-induced diabetic rats, intravitreal administration of Dnmt1-siRNA attenuated Mfn2 promoter hypermethylation and restored its expression. Human donors with diabetic retinopathy confirmed Mfn2 promoter DNA hypermethylation. Thus, regulating Mfn2 and its epigenetic modifications by molecular/pharmacological means will protect mitochondrial homeostasis in diabetes, and could attenuate the development of retinopathy in diabetic patients., (Copyright © 2019. Published by Elsevier B.V.)

Aims: Diabetes increases oxidative stress in the retina and dysfunctions their mitochondria, accelerating capillary cell apoptosis. A 66 kDa adaptor protein, p66Shc, is considered as a sensor of oxidative stress-induced apoptosis. In the pathogenesis of diabetic retinopathy, a progressive disease, reactive oxygen species (ROS) production by activation of a small molecular weight G-protein (Ras-related C3 botulinum toxin substrate 1 [Rac1])-Nox2 signaling precedes mitochondrial damage. Rac1 activation is facilitated by guanine exchange factors (GEFs), and p66Shc increases Rac1-specific GEF activity of Son of Sevenless 1 (Sos1). p66Shc also possesses oxidoreductase activity and can directly stimulate mitochondrial ROS generation. Our aim was to investigate the role of p66Shc in the development of diabetic retinopathy and mechanism of its transcription., Results: High glucose increased p66Shc expression in human retinal endothelial cells, and elevated acetylated histone 3 lysine 9 (H3K9) levels and transcriptional factor p53 binding at its promoter. Glucose also augmented interactions between Rac1 and Sos1 and activated Rac1-Nox2. Phosphorylation of p66Shc was increased, allowing it to interact with peptidyl prolyl isomerase to facilitate its localization inside the mitochondria, culminating in mitochondrial damage. P66shc-small interfering RNA (siRNA) inhibited glucose-induced Rac1 activation and mitochondrial damage. Similar results are observed in retinal microvessels from diabetic rats., Innovation: This is the first report identifying the role of p66Shc in the development of diabetic retinopathy and implicating increased histone acetylation in its transcriptional regulation., Conclusion: Thus, p66Shc has dual role in the development of diabetic retinopathy; its regulation in the early stages of the disease should impede Rac1-ROS production and, in the later stages, prevent mitochondrial damage and initiation of a futile cycle of free radicals.

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.

Diabetic retinopathy is a multifactorial disease, and the exact mechanism of its pathogenesis remains obscure. Sirtuin 1 (Sirt1), a multifunctional deacetylase, is implicated in the regulation of many cellular functions and in gene transcription, and retinal Sirt1 is inhibited in diabetes. Our aim was to determine the role of Sirt1 in the development of diabetic retinopathy and to elucidate the molecular mechanism of its downregulation. Using Sirt1 -overexpressing mice that were diabetic for 8 months, structural, functional, and metabolic abnormalities were investigated in vascular and neuronal retina. The role of epigenetics in Sirt1 transcriptional suppression was investigated in retinal microvessels. Compared with diabetic wild-type mice, retinal vasculature from diabetic Sirt1 mice did not present any increase in the number of apoptotic cells or degenerative capillaries or decrease in vascular density. Diabetic Sirt1 mice were also protected from mitochondrial damage and had normal electroretinography responses and ganglion cell layer thickness. Diabetic wild-type mice had hypermethylated Sirt1 promoter DNA, which was alleviated in diabetic Sirt1 mice, suggesting a role for epigenetics in its transcriptional suppression. Thus strategies targeted to ameliorate Sirt1 inhibition have the potential to maintain retinal vascular and neuronal homeostasis, providing opportunities to retard the development of diabetic retinopathy in its early stages., (© 2018 by the American Diabetes Association.)

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.