Qing Zhong

Epigenetic Changes in Mitochondrial Superoxide Dismutase in the Retina and the Development of Diabetic Retinopathy

OBJECTIVE To investigate the role of epigenetic regulation of the manganese superoxide dismutase gene (sod2) in the development of diabetic retinopathy and the metabolic memory phenomenon associated with its continued progression after hyperglycemia is terminated. RESEARCH DESIGN AND METHODS Streptozotocin-induced diabetic rats were maintained in poor glycemic control (PC, GHb ∼12%) or in good glycemic control (GC, GHb ∼7.0%) for 4 months, or were allowed to maintain PC for 2 months, followed by GC for 2 additional months (PC-Rev). For experimental galactosemia, a group of normal rats were fed a 30% galactose diet for 4 months or for 2 months, followed by a normal diet for 2 additional months. Trimethyl histone H4 lysine 20 (H4K20me3), acetyl histone H3 lysine 9 (H3K9), and nuclear transcriptional factor NF-κB p65 and p50 at the retinal sod2 promoter and enhancer were examined by chromatin immunoprecipitation. RESULTS Hyperglycemia (diabetes or galactosemia) increased H4K20me3, acetyl H3K9, and NF-κB p65 at the promoter and enhancer of retinal sod2, upregulated protein and gene expression of SUV420h2, and increased the interactions of acetyl H3K9 and NF-κB p65 to H4K20me3. Reversal of hyperglycemia failed to prevent increases in H4K20me3, acetyl H3K9, and NF-κB p65 at sod2, and sod2 and SUV420h2 continued to be abnormal. Silencing SUV420h2 by its small interfering RNA in retinal endothelial cells prevented a glucose-induced increase in H4K20me3 at the sod2 enhancer and a decrease in sod2 transcripts. CONCLUSIONS Increased H4K20me3 at sod2 contributes to its downregulation and is important in the development of diabetic retinopathy and in the metabolic memory phenomenon. Targeting epigenetic changes may serve as potential therapeutic targets to retard the development and progression of diabetic retinopathy.

Oxidative damage of mitochondrial DNA in diabetes and its protection by manganese superoxide dismutase

Abstract Retinal mitochondria become dysfunctional in diabetes and the production of superoxide radicals is increased; over-expression of MnSOD abrogates mitochondrial dysfunction and prevents the development of diabetic retinopathy. The mitochondrial DNA (mtDNA) is particularly prone to oxidative damage. The aim of this study is to examine the role of MnSOD in the maintenance of mtDNA. The effect of MnSOD mimic, MnTBAP or over-expression of MnSOD on glucose-induced alterations in mtDNA homeostasis and its functional consequence was determined in retinal endothelial cells. Exposure of retinal endothelial cells to high glucose increased mtDNA damage and compromised the DNA repair machinery. The gene expressions of mitochondrial-encoded proteins of the electron transport chain complexes were decreased. Inhibition of superoxide radicals by either MnTBAP or by over-expression of MnSOD prevented mtDNA damage and protected mitochondrial-encoded genes. Thus, the protection of mtDNA from glucose-induced oxidative damage is one of the plausible mechanisms by which MnSOD ameliorates the development of diabetic retinopathy.

Role of histone acetylation in the development of diabetic retinopathy and the metabolic memory phenomenon

Hyperglycemia is considered as one of the major determinants in the development of diabetic retinopathy, but the progression of retinopathy resists arrest after hyperglycemia is terminated, suggesting a metabolic memory phenomenon. Diabetes alters the expression of retinal genes, and this continues even after good glycemic control is re‐instituted. Since the expression of genes is affected by chromatin structure that is modulated by post‐translational modifications of histones, our objective is to investigate the role of histone acetylation in the development of diabetic retinopathy, and in the metabolic memory phenomenon. Streptozotocin‐induced rats were maintained either in poor glycemic control (PC, glycated hemoglobin, GHb >11%) or good glycemic control (GC, GHb <6%) for 12 months, or allowed to be in PC for 6 months followed by in GC for 6 months (PC‐GC). On a cellular level, retinal endothelial cells, the target of histopathology of diabetic retinopathy, were incubated in 5 or 20 mM glucose for 4 days. Activities of histone deacetylase (HDAC) and histone acetyltransferase (HAT), and histone acetylation were quantified. Hyperglycemia activated HDAC and increased HDAC1, 2, and 8 gene expressions in the retina and its capillary cells. The activity HAT was compromised and the acetylation of histone H3 was decreased. Termination of hyperglycemia failed to provide any benefits to diabetes‐induced changes in retinal HDAC and HAT, and histone H3 remained subnormal. This suggests “in principle” the role of global acetylation of retinal histone H3 in the development of diabetic retinopathy and in the metabolic memory phenomenon associated with its continued progression. J. Cell. Biochem. 110: 1306–1313, 2010.

Transcription Factor Nrf2-Mediated Antioxidant Defense System in the Development of Diabetic Retinopathy

PURPOSE Increase in reactive oxygen species (ROS) is one of the major retinal metabolic abnormalities associated with the development of diabetic retinopathy. NF-E2-related factor 2 (Nrf2), a redox sensitive factor, provides cellular defenses against the cytotoxic ROS. In stress conditions, Nrf2 dissociates from its cytosolic inhibitor, Kelch like-ECH-associated protein 1 (Keap1), and moves to the nucleus to regulate the transcription of antioxidant genes including the catalytic subunit of glutamylcysteine ligase (GCLC), a rate-limiting reduced glutathione (GSH) biosynthesis enzyme. Our aim is to understand the role of Nrf2-Keap1-GCLC in the development of diabetic retinopathy. METHODS Effect of diabetes on Nrf2-Keap1-GCLC pathway, and subcellular localization of Nrf2 and its binding with Keap1 was investigated in the retina of streptozotocin-induced diabetic rats. The binding of Nrf2 at GCLC was quantified by chromatin immunoprecipitation technique. The results were confirmed in isolated retinal endothelial cells, and also in the retina from human donors with diabetic retinopathy. RESULTS Diabetes increased retinal Nrf2 and its binding with Keap1, but decreased DNA-binding activity of Nrf2 and also its binding at the promoter region of GCLC. Similar impairments in Nrf2-Keap1-GCLC were observed in the endothelial cells exposed to high glucose and in the retina from donors with diabetic retinopathy. In retinal endothelial cells, glucose-induced impairments in Nrf2-GCLC were prevented by Nrf2 inducer tBHQ and also by Keap1-siRNA. CONCLUSIONS Due to increased binding of Nrf2 with Keap1, its translocation to the nucleus is compromised contributing to the decreased GSH levels. Thus, regulation of Nrf2-Keap1 by pharmacological or molecular means could serve as a potential adjunct therapy to combat oxidative stress and inhibit the development of diabetic retinopathy.

Abrogation of MMP-9 Gene Protects Against the Development of Retinopathy in Diabetic Mice by Preventing Mitochondrial Damage

OBJECTIVE In the development of diabetic retinopathy, mitochondrial dysfunction is considered to play an important role in the apoptosis of retinal capillary cells. Diabetes activates matrix metalloproteinase-9 (MMP-9) in the retina and its capillary cells, and activated MMP-9 becomes proapoptotic. The objective of this study is to elucidate the plausible mechanism by which active MMP-9 contributes to the mitochondrial dysfunction in the retina. RESEARCH DESIGN AND METHODS Using MMP-9 gene knockout (MMP-KO) mice, we investigated the effect of MMP-9 regulation on diabetes-induced increased retinal capillary cell apoptosis, development of retinopathy, mitochondrial dysfunction and ultrastructure, and mitochondrial DNA (mtDNA) damage. To understand how diabetes increases mitochondrial accumulation of MMP-9, interactions between MMP-9 and chaperone proteins (heat shock protein [Hsp] 70 and Hsp60) were evaluated. The results were confirmed in the retinal mitochondria from human donors with diabetic retinopathy, and in isolated retinal endothelial cells transfected with MMP-9 small interfering RNA (siRNA). RESULTS Retinal microvasculature of MMP-KO mice, diabetic for ∼7 months, did not show increased apoptosis and pathology characteristic of retinopathy. In the same MMP-KO diabetic mice, activation of MMP-9 and dysfunction of the mitochondria were prevented, and electron microscopy of the retinal microvasculature region revealed normal mitochondrial matrix and packed lamellar cristae. Damage to mtDNA was protected, and the binding of MMP-9 with Hsp70 or Hsp60 was also normal. As in the retina from wild-type diabetic mice, activation of mitochondrial MMP-9 and alterations in the binding of MMP-9 with chaperone proteins were also observed in the retina from donors with diabetic retinopathy. In endothelial cells transfected with MMP-9 siRNA, high glucose–induced damage to the mitochondria and the chaperone machinery was ameliorated. CONCLUSIONS Regulation of activated MMP-9 prevents retinal capillary cells from undergoing apoptosis by protecting mitochondrial ultrastructure and function and preventing mtDNA damage. Thus, MMP-9 inhibitors could have potential therapeutic value in preventing the development of diabetic retinopathy by preventing the continuation of the vicious cycle of mitochondrial damage.

Epigenetic Modification of Sod2 in the Development of Diabetic Retinopathy and in the Metabolic Memory: Role of Histone Methylation

PURPOSE Mitochondrial superoxide levels are elevated in the retina in diabetes, and their scavenging enzyme, MnSOD, becomes subnormal. The objective of this study is to investigate the role of histone methylation of Sod2, the gene that encodes MnSOD, in the development of diabetic retinopathy and in the metabolic memory phenomenon associated with its continued progression after termination of hyperglycemia. METHODS Effect of high glucose on monomethyl H3K4 (H3K4me1), dimethyl H3K4 (H3K4me2), and lysine-specific demethylase-1 (LSD1) was quantified at Sod2 by chromatin immunoprecipitation in isolated retinal endothelial cells. The role of histone methylation in the metabolic memory phenomenon was investigated in the retina of rats maintained in poor glycemic control (PC, approximately 12% glycated hemoglobin [GHb]) for 3 months followed by in good glycemic control (GC, approximately 6% GHb) for 3 months. RESULTS Hyperglycemia reduced H3K4me1 and -me2, and increased the binding of LSD1 and Sp1 at Sod2. Regulation of LSD1 by LSD1-siRNA ameliorated glucose-induced decrease in H3K4 methylation at Sod2, and prevented decrease in Sod2 gene expression. In rats, re-institution of GC failed to reverse decrease in H3K4me1 and -me2 at Sod2, and LSD1 remained active with increased binding of LSD1 and Sp1 at Sod2. Retina from human donors with diabetic retinopathy also had decreased H3K4me2 and increased LSD1 at Sod2. CONCLUSIONS Histone methylation of retinal Sod2 has an important role in the development of diabetic retinopathy and in the metabolic memory phenomenon associated with its continued progression. Targeting enzymes important for histone methylation may serve as a potential therapy to halt the development of diabetic retinopathy.

Regulation of matrix metalloproteinase-9 by epigenetic modifications and the development of diabetic retinopathy.

Diabetes activates retinal matrix metalloproteinase-9 (MMP-9), and MMP-9 damages the mitochondria and augments capillary cell apoptosis. Our aim is to elucidate the mechanism responsible for MMP-9 activation. Histone modifications and recruitment of the nuclear transcriptional factor-κB (p65 subunit) at the MMP-9 promoter and the activity of lysine-specific demethylase 1 (LSD1) were measured in the retina from streptozotocin-induced diabetic rats. The role of LSD1 in MMP-9 activation was investigated in isolated retinal endothelial cells transfected with LSD1 small interfering RNA (siRNA). The results were confirmed in the retina from human donors with diabetic retinopathy. Diabetes decreased histone H3 dimethyl lysine 9 (H3K9me2) and increased acetyl H3K9 (Ac-H3K9) and p65 at the retinal MMP-9 promoter. LSD1 enzyme activity and its transcripts were elevated. LSD1 siRNA ameliorated the glucose-induced decrease in H3K9me2 and increase in p65 at the MMP-9 promoter, and prevented MMP-9 activation, mitochondrial damage, and cell apoptosis. Human donors with diabetic retinopathy had similar epigenetic changes at the MMP-9 promoter. Thus, activated LSD1 hypomethylates H3K9 at the MMP-9 promoter and this frees up that lysine 9 for acetylation. Increased Ac-H3K9 facilitates the recruitment of p65, resulting in MMP-9 activation and mitochondrial damage. Thus, the regulation of LSD1 by molecular or pharmacological means has the potential to retard the development of diabetic retinopathy.

Matrix metalloproteinases in diabetic retinopathy: potential role of MMP-9.

Introduction: Diabetic retinopathy remains one of the most feared complications of diabetes. Despite extensive research in the field, the molecular mechanism responsible for the development of this slow progressing disease remains unclear. In the pathogenesis of diabetic retinopathy, mitochondria are damaged and inflammatory mediators are elevated before the histopathology associated with the disease can be observed. Matrix metalloproteinases (MMPs) regulate a variety of cellular functions including apoptosis and angiogenesis. Diabetic environment stimulates the secretion of several MMPs that are considered to participate in complications, including retinopathy, nephropathy and cardiomyopathy. Patients with diabetic retinopathy and also animal models have shown increased MMP-9 and MMP-2 in their retina and vitreous. Recent research has shown that MMPs have dual role in the development of diabetic retinopathy; in the early stages of the disease (pre-neovascularization), MMP-2 and MMP-9 facilitate the apoptosis of retinal capillary cells, possibly via damaging the mitochondria, and in the later phase, they help in neovascularization. Areas covered: This article reviews the literature to evaluate the role of MMPs, especially MMP-9, in the development of diabetic retinopathy, and presents existing evidence that the inhibitors targeted toward MMP-9, depending on the duration of diabetes at the times their administration could have potential to prevent the progression of this blinding disease, and protect the vision loss. Expert opinion: Inhibitors of MMPs could have dual role: in the early stages of the diseases, inhibit capillary cell apoptosis, and if the disease has progressed to the angiogenic stage, inhibit the growth of new vessels.

Diabetic Retinopathy and Damage to Mitochondrial Structure and Transport Machinery

PURPOSE Mitochondrial function is controlled by membrane structure. In diabetes, retinal mitochondria are dysfunctional, and reversal of hyperglycemia fails to inhibit such changes. The goal of this study was to use anatomic and molecular biologic techniques to investigate the effect of diabetes on mitochondrial membrane structure. METHODS Wistar rats were maintained in poor glycemic control (PC; GHb 11.2%) or good glycemic control (GC; GHb 5.5%) for 12 months or in PC for 6 months, followed by GC for an additional 6 months. The structure of the retinal mitochondria in the microvascular region was evaluated by electron microscopy (TEM) and gene expressions of mitochondrial structure-related proteins by rat mitochondrial PCR array. Representative genes were validated by real-time PCR, and their protein expression by Western blot. The results were confirmed in the retina obtained from human donors with diabetic retinopathy. RESULTS TEM showed enlarged mitochondria with partial cristolysis in the retinal microvasculature from PC rats, compared with those from normal rats. Among 84 genes, 6 retinal genes were upregulated and 12 were downregulated. PCR confirmed alternations in the gene expressions of fusion (Mfn2), carrier (Timm44 and Slc25a21), Akt1, and fission proteins (Dnm1l). Protein levels of Mfn2 and Dnm1l were consistent with their mRNA levels, but their mitochondrial abundance was decreased. Reversal of hyperglycemia failed to normalize these changes. Retinas from donors with diabetic retinopathy also presented similar patterns of changes in the gene and protein expressions. CONCLUSIONS Mitochondrial structural and transport proteins play an important role in the development of diabetic retinopathy and also in the metabolic memory phenomenon associated with its continued progression.

Diabetic retinopathy, superoxide damage and antioxidants.

Retinopathy, the leading cause of acquired blindness in young adults, is one of the most feared complications of diabetes, and hyperglycemia is considered as the major trigger for its development. The microvasculature of the retina is constantly bombarded by high glucose, and this insult results in many metabolic, structural and functional changes. Retinal mitochondria become dysfunctional, its DNA is damaged and proteins encoded by its DNA are decreased. The electron transport chain system becomes compromised, further producing superoxide and providing no relief to the retina from a continuous cycle of damage. Although the retina attempts to initiate repair mechanisms by inducing gene expressions of the repair enzymes, their mitochondrial accumulation remains deficient. Understanding the molecular mechanism of mitochondrial damage should help identify therapies to treat/retard this sight threatening complication of diabetes. Our hope is that if the retinal mitochondria are maintained healthy with adjunct therapies, the development and progression of diabetic retinopathy can be inhibited.