Diabetic Retinopathy and Regulation of Mitochondrial Glutathione-Glutathione Peroxidase Axis in Hyperhomocysteinemia.

Diabetic patients have elevated homocysteine levels, and hyperhomocysteinemia is shown to exacerbate mitochondrial damage, which plays a central role in diabetic retinopathy. Glutathione peroxidases (GPx) catalyze hydrogen peroxide (H2O2) reduction using glutathione (GSH) as a cofactor. GSH and GPx are mainly cytosolic but are also present in the mitochondria to neutralize H2O2 produced by superoxide dismutase, and in diabetes, they are downregulated. Hyperhomocysteinemia also disrupts the balance between S-adenosyl-L-homocysteine and S-adenosylmethionine (SAM); SAM is also a methyl donor for DNA methylation. The aim of this study was to investigate the role of homocysteine in mitochondrial GSH-GPx1 regulation in diabetic retinopathy. Human retinal endothelial cells in 20 mM D-glucose + high homocysteine were analyzed for ROS, GSH and GPx in the mitochondria, and SAM levels and GPx1 promoter DNA methylation were also studied (5-methylcytosine and MS-PCR). The results were confirmed in the retina from streptozotocin-induced hyperhomocysteinemic (cystathionine-ß-synthase-deficient) diabetic mice. High homocysteine exacerbated the glucose-induced decrease in GSH levels and GPx activity in the mitochondria and the downregulation of GPx1 transcripts and further increased SAM levels and GPx1 promoter DNA methylation. Similar results were obtained in a hyperglycemic-hyperhomocysteinemic mouse model. Thus, elevated homocysteine in diabetes hypermethylates GPx1 promoter, thus decreasing the mitochondrial GPx/GSH pool and exacerbating mitochondrial damage. Modulating hyperhomocysteinemia could be a potential therapeutic avenue to target mitochondrial dysfunction in diabetic retinopathy.

Homocysteine and mitochondrial quality control in diabetic retinopathy.

BACKGROUND: Diabetic retinopathy is a progressive disease, and one of the key metabolic abnormalities in the pathogenesis of diabetic retinopathy, mitochondrial damage, is also influenced by the duration of hyperglycemia. Mitochondrial quality control involves a coordination of mitochondrial dynamics, biogenesis and removal of the damaged mitochondria. In diabetes, these processes are impaired, and the damaged mitochondria continue to produce free radicals. Diabetic patients also have high homocysteine and reduced levels of hydrogen sulfide, and hyperhomocysteinemia is shown to exacerbate diabetes-induced mitochondrial damage and worsen their dynamics. This study aims to investigate the temporal relationship between hyperhomocysteinemia and retinal mitochondrial quality control in diabetic retinopathy. METHODS: Human retinal endothelial cells incubated in 20 mM D-glucose for 24 to 96 h, in the absence or presence of 100 µM homocysteine, with/without a hydrogen sulfide donor GYY4137, were analyzed for mitochondrial ROS (MitoSox fluorescence), DNA damage (transcripts of mtDNA-encoded ND6 and CytB), copy numbers, oxygen consumption rate (Seahorse XF analyzer) and mitophagy (mitophagosomes immunofluorescence labeling and flow cytometry). Results were confirmed in the retina from mice genetically manipulated for hyperhomocysteinemia (cystathionine ß-synthase deficient mice, Cbs+/-), streptozotocin-induced diabetic for 8 to 24 weeks. At 24 weeks of diabetes, vascular health was evaluated by counting acellular capillaries in the trypsin digested retinal vasculature and by fluorescein angiography. RESULTS: Homocysteine, in high glucose medium, exacerbated mitochondrial ROS production, mtDNA damage and impaired mitochondrial respiration within 24 h, and slowed down/worsened mitochondrial biogenesis and mitophagy, as compared to 48 to 96 h in high glucose alone. GYY4137 supplementation ameliorated homocysteine + high glucose-induced mitochondrial damage and impairment in biogenesis and mitophagy. Similar results were obtained from Cbs+/- mice-mitochondrial ROS, mtDNA damage and decline in biogenesis and mitophagy were observed within eight weeks of diabetes vs. 16 to 24 weeks of diabetes in Cbs+/+ mice, and at 24 weeks of diabetes, Cbs+/- mice had significantly higher acellular capillaries and vascular leakage. CONCLUSIONS: Hyperhomocysteinemia, in a hyperglycemic environment, overwhelms the mitochondria, accelerating and exacerbating their dysfunction, and also delays/worsens their removal, augmenting the development of diabetic retinopathy. Thus, our results strengthen the importance of maintaining homocysteine-hydrogen sulfide balance during the early stages of diabetes for a patient to prevent/retard vision loss.

Impaired Removal of the Damaged Mitochondria in the Metabolic Memory Phenomenon Associated with Continued Progression of Diabetic Retinopathy.

Retinopathy fails to halt even after diabetic patients in poor glycemic control try to institute tight glycemic control, suggesting a "metabolic memory" phenomenon, and the experimental models have demonstrated that mitochondria continue to be damaged/dysfunctional, fueling into the vicious cycle of free radicals. Our aim was to investigate the role of removal of the damaged mitochondria in the metabolic memory. Using human retinal endothelial cells (HRECs), incubated in 20 mM D-glucose for 4 days, followed by 5 mM D-glucose for 4 additional days, mitochondrial turnover, formation of mitophagosome, and mitophagy flux were evaluated. Mitophagy was confirmed in a rat model of metabolic memory where the rats were kept in poor glycemic control (blood glucose ~ 400 mg/dl) for 3 months soon after induction of streptozotocin-induced diabetes, followed by 3 additional months of good control (BG < 150 mg/dl). Reversal of high glucose by normal glucose had no effect on mitochondrial turnover and mitophagosome formation, and mitophagy flux remained compromised. Similarly, 3 months of good glycemic control in rats, which had followed 3 months of poor glycemic control, had no effect on mitophagy flux. Thus, poor turnover/removal of the damaged mitochondria, initiated during poor glycemic control, does not benefit from the termination of hyperglycemic insult, and the damaged mitochondria continue to produce free radicals, suggesting the importance of mitophagy in the metabolic memory phenomenon associated with the continued progression of diabetic retinopathy.

Mitochondrial Genome-Encoded Long Noncoding RNA Cytochrome B and Mitochondrial Dysfunction in Diabetic Retinopathy.

Aims: Mitochondrial dysfunction is closely associated with the development of diabetic complications. In diabetic retinopathy, electron transport chain is compromised and mitochondrial DNA (mtDNA) is damaged, downregulating transcription of mtDNA-encoded cytochrome B (CYTB) and its antisense long noncoding RNA, long noncoding RNA cytochrome B (LncCytB). Our goal was to investigate the role of LncCytB in the regulation of CYTB and mitochondrial function in diabetic retinopathy. Methods: Using human retinal endothelial cells, genetically manipulated for LncCytB (overexpression or silencing), the effect of high glucose (20 mM d-glucose) on LncCytB-CYTB interactions (by chromatin isolation by RNA purification), CYTB gene expression (by real-time quantitative polymerase chain reaction), complex III activity, mitochondrial free radicals, and oxygen consumption rate (OCR, by Seahorse XF analyzer) was investigated. Key results were confirmed in the retinal microvessels from streptozotocin-induced diabetic mice. Results: High glucose decreased LncCytB-CYTB interactions, and while LncCytB overexpression ameliorated glucose-induced decrease in CYTB gene transcripts, complex III activity and OCR and increase in mitochondrial reactive oxygen species, LncCytB-siRNA further attenuated CYTB gene transcription, complex III activity, and OCR. Similar decrease in LncCytB-CYTB interactions and CYTB transcription was observed in diabetic mice. Furthermore, maintenance of mitochondrial homeostasis by overexpressing superoxide dismutase or sirtuin 1 in mice ameliorated diabetes-induced decrease in LncCytB-CYTB interactions and CYTB gene transcripts, and also improved complex III activity and mitochondrial respiration. Innovation and Conclusion: LncCytB downregulation in hyperglycemic milieu downregulates CYTB transcription, which inhibits complex III activity and compromises mitochondrial stability and OCR. Thus, preventing LncCytB downregulation in diabetes has potential of inhibiting the development of diabetic retinopathy, possibly via maintaining mitochondrial respiration. Antioxid. Redox Signal. 39, 817-828.

Impaired mitochondrial dynamics and removal of the damaged mitochondria in diabetic retinopathy.

Introduction: Mitochondrial dynamic plays a major role in their quality control, and the damaged mitochondrial components are removed by autophagy. In diabetic retinopathy, mitochondrial fusion enzyme, mitofusin 2 (Mfn2), is downregulated and mitochondrial dynamic is disturbed resulting in depolarized and dysfunctional mitochondria. Our aim was to investigate the mechanism of inhibition of Mfn2, and its role in the removal of the damaged mitochondria, in diabetic retinopathy. Methods: Using human retinal endothelial cells, effect of high glucose (20mM) on the GTPase activity of Mfn2 and its acetylation were determined. Role of Mfn2 in the removal of the damaged mitochondria was confirmed by regulating its acetylation, or by Mfn2 overexpression, on autophagosomes- autolysosomes formation and the mitophagy flux. Results: High glucose inhibited GTPase activity and increased acetylation of Mfn2. Inhibition of acetylation, or Mfn2 overexpression, attenuated decrease in GTPase activity and mitochondrial fragmentation, and increased the removal of the damaged mitochondria. Similar phenomenon was observed in diabetic mice; overexpression of sirtuin 1 (a deacetylase) ameliorated diabetes-induced inhibition of retinal Mfn2 and facilitated the removal of the damaged mitochondria. Conclusions: Acetylation of Mfn2 has dual roles in mitochondrial homeostasis in diabetic retinopathy, it inhibits GTPase activity of Mfn2 and increases mitochondrial fragmentation, and also impairs removal of the damaged mitochondria. Thus, protecting Mfn2 activity should maintain mitochondrial homeostasis and inhibit the development/progression of diabetic retinopathy.

Mitochondrial Quality Control and Metabolic Memory Phenomenon Associated with Continued Progression of Diabetic Retinopathy.

Diabetic retinopathy continues to progress even when hyperglycemia is terminated, suggesting a 'metabolic memory' phenomenon. Mitochondrial dysfunction is closely associated with the development of diabetic retinopathy, and mitochondria remain dysfunctional. Quality control of mitochondria requires a fine balance between mitochondrial fission-fusion, removal of the damaged mitochondria (mitophagy) and formation of new mitochondria (biogenesis). In diabetes, while mitochondrial fusion protein (Mfn2) is decreased, fission protein (Drp1) is increased, resulting in fragmented mitochondria. Re-institution of normal glycemia fails to reverse mitochondrial fragmentation, and dysfunctional mitochondria continue to accumulate. Our aim was to investigate the direct effect of regulation of the mitochondrial fusion process during normal glycemia that follows a high glucose insult on mitochondrial quality control in the 'metabolic memory' phenomenon. Human retinal endothelial cells, incubated in 20 mM glucose for four days, followed by 5 mM glucose for four additional days, with or without the Mfn2 activator leflunomide, were analyzed for mitochondrial fission (live cell imaging), mitophagy (flow cytometry and immunofluorescence microscopy), and mitochondrial mass (mitochondrial copy numbers and MitoTracker labeling). Mitochondrial health was determined by quantifying mitochondrial reactive oxygen species (ROS), respiration rate (Seahorse XF96) and mitochondrial DNA (mtDNA) damage. Addition of leflunomide during normal glucose exposure that followed high glucose prevented mitochondrial fission, facilitated mitophagy and increased mitochondrial mass. Glucose-induced decrease in mitochondrial respiration and increase in ROS and mtDNA damage were also prevented. Thus, direct regulation of mitochondrial dynamics can help maintain mitochondrial quality control and interfere with the metabolic memory phenomenon, preventing further progression of diabetic retinopathy.

Cross Talks between Oxidative Stress, Inflammation and Epigenetics in Diabetic Retinopathy.

Diabetic retinopathy, one of the most devastating complications of diabetes, is a multifactorial progressing disease with a very complex etiology. Although many metabolic, molecular, functional and structural changes have been identified in the retina and its vasculature, the exact molecular mechanism of its pathogenesis still remains elusive. Sustained high-circulating glucose increases oxidative stress in the retina and also activates the inflammatory cascade. Free radicals increase inflammatory mediators, and inflammation can increase production of free radicals, suggesting a positive loop between them. In addition, diabetes also facilitates many epigenetic modifications that can influence transcription of a gene without changing the DNA sequence. Several genes associated with oxidative stress and inflammation in the pathogenesis of diabetic retinopathy are also influenced by epigenetic modifications. This review discusses cross-talks between oxidative stress, inflammation and epigenetics in diabetic retinopathy. Since epigenetic changes are influenced by external factors such as environment and lifestyle, and they can also be reversed, this opens up possibilities for new strategies to inhibit the development/progression of this sight-threatening disease.

Mitochondrial Genome-Encoded Long Noncoding RNA and Mitochondrial Stability in Diabetic Retinopathy.

Mitochondria experience genomic and functional instability in diabetes, and mitochondrial dysfunction has a critical role in the development of diabetic retinopathy. Diabetes also alters expressions of many long noncoding RNAs (LncRNAs), the RNAs with >200 nucleotides and no open reading frame. LncRNAs are mainly encoded by the nuclear genome, but mtDNA also encodes three LncRNAs. Our goal was to investigate the effect of hyperglycemia on mtDNA-encoded LncRNA cytochrome B (LncCytB) in mtDNA stability in diabetic retinopathy. Retinal endothelial cells, transfected with LncCytB-overexpressing plasmids or siRNA, incubated in 5 mmol/L d-glucose (normal glucose [NG]) or 20 mmol/L d-glucose (high glucose [HG]) for 4 days, were analyzed for LncCytB expression by strand-specific PCR and its mitochondrial localization by RNA fluorescence in situ hybridization. Damage-sensitive mtDNA regions were examined by micrococcal nuclease (MNase) digestion sequencing and LncCytB occupancy at mtDNA by chromatin isolation by RNA purification. Protective nucleoids in mtDNA were analyzed by SYBR Green-MitoTracker Red staining and confirmed in isolated mitochondria by flow cytometry. Compared with NG, HG downregulated LncCytB by >50% but had no significant effect on the other mtDNA-encoded LncRNAs. mtDNA packaging was impaired, MNase sensitivity was increased, and LncCytB occupancy at mtDNA was decreased. While LncCytB overexpression ameliorated mtDNA damage and decrease in nucleoids and copy numbers, LncCytB-siRNA exacerbated damage and further reduced nucleoids. Retinal microvessels from streptozotocin-induced diabetic mice and human donors with diabetic retinopathy presented a similar decrease in LncCytB and mtDNA nucleoids. Thus, LncCytB has a major role in maintaining mitochondrial genomic stability, and its downregulation in the hyperglycemic milieu contributes to increased vulnerability of mtDNA to damage.

Regulation of serine palmitoyl-transferase and Rac1-Nox2 signaling in diabetic retinopathy.

Hyperlipidemia is considered as one of the major systemic factors associated with the development of diabetic retinopathy, and animal models have documented that its presence in a hyperglycemic environment exacerbates cytosolic ROS production (via activation of the Rac1-Nox2 axis) and mitochondrial damage. Hyperglycemia also accelerates Rac1 transcription via dynamic DNA methylation-hydroxymethylation of its promoter. In diabetes, ceramide metabolism in the retina is impaired and its accumulation is increased. Our aim was to investigate the effect of inhibition of the rate limiting enzyme of the de novo ceramide biosynthesis, serine palmitoyl-transferase (SPT), on Rac1 activation in diabetic retinopathy. Using human retinal endothelial cells, transfected with SPT-siRNA, and incubated in 20 mM D-glucose in the presence or absence of 50 µM palmitate (glucolipotoxic and glucotoxic, respectively), activities of Rac1 and Nox2, and ROS levels were quantified. For Rac1 transcriptional activation, 5 hydroxymethyl cytosine (5hmC) levels at its promoter were quantified. Key parameters were confirmed in retinal microvessels from streptozotocin-induced diabetic mice on a normal diet (type 1 diabetic model) or on a high-fat diet (45% kcal, type 2 diabetic model), injected intravitreally with SPT-siRNA. Compared to normal glucose, cells in high glucose, with or without palmitic acid, had increased Rac1-Nox2-ROS signaling, Rac1 transcripts and 5hmC levels at its promoter. Inhibition of SPT by SPT-siRNA or myriocin prevented glucotoxic- and glucolipotoxic-induced increase in Rac1-Nox2-ROS signaling and 5hmC at the Rac1 promoter. Similarly, in both type 1 and type 2 diabetic mouse models, SPT-siRNA attenuated the increase in the Rac1-Nox2-ROS axis and 5hmC at the Rac1 promoter. Thus, inhibition of the rate limiting enzyme of ceramide de novo biosynthesis, SPT, regulates activation of DNA methylation-hydroxymethylation machinery and prevents increased Rac1 transcription. This ameliorates the activation of Rac1-Nox2 signaling and protects the mitochondria from damaging cytosolic ROS, which prevents accelerated capillary cell loss. These results further raise the importance of regulating lipid levels in diabetic patients with dyslipidemia.

Long Noncoding RNAs and Mitochondrial Homeostasis in the Development of Diabetic Retinopathy.

Retinopathy is one of the most devastating complications of diabetes, which a patient fears the most. Hyperglycemic environment results in many structural, functional, molecular and biochemical abnormalities in the retina, and overproduction of mitochondrial superoxide, induced by hyperglycemic milieu, is considered to play a central role in the development of diabetic retinopathy. Expression of many genes associated with maintaining mitochondrial homeostasis is also altered. Recent research has shown that several long noncoding RNAs, RNAs with more than 200 nucleotides but without any reading frames, are aberrantly expressed in diabetes, and altered expression of these long noncoding RNAs is now being implicated in the development of diabetes and its complications including retinopathy. This review focuses the role of long noncoding RNAs in the development of diabetic retinopathy, with a special emphasis on the maintenance of mitochondrial homeostasis.

Involvement of High Mobility Group Box 1 Protein in Optic Nerve Damage in Diabetes.

Introduction: Diabetic patients routinely have high levels of high mobility group box 1 (HMGB1) protein in their plasma, vitreous and ocular membranes, which is strongly correlated with subclinical chronic inflammation in the eye. Our previous work has suggested that high HMGB1 in diabetes plays a role in retinal inflammation and angiogenesis, but its role in the optic nerve damage is unclear. Therefore, our goal is to examine the role of HMGB1 in optic nerve damage in diabetes. Methods: Gene expression of HMGB1 was quantified in the optic nerve from streptozotocin-induced diabetic mice by qRT-PCR, and their protein expressions by Western blot analysis and immunofluorescence staining. Using immunohistochemical technique, expression of reactive astrogliosis (indicator of neuroinflammation) and nerve demyelination/damage were determined by quantifying glial fibrillary acid protein (GFAP) and myelin basic protein (MBP), respectively. The role of HMGB1 in the optic nerve damage and alteration visual pathways was confirmed in mice receiving glycyrrhizin, a HMGB1 inhibitor. Similar parameters were measured in the optic nerve from human donors with diabetes. Results: Compared to normal mice, diabetic mice exhibited increased levels of HMGB1, higher GFAP expression, and decreased MBP in the optic nerve. Double immunofluorescence microscopy revealed that diabetes induced increased HMGB1 immunoreactivities were significantly colocalized with GFAP in the optic nerve. Glycyrrhizin supplementation effectively reduced HMGB1 and maintained normal axonal myelination and visual conduction. Results from mice optic nerve confirmed the results obtained from human donors with diabetes. Discussions: Thus, diabetes-induced HMGB1 upregulation promotes optic nerve demyelination and inflammation. The regulation of HMGB1 activation has potential to protect optic nerve damage and the abnormalities of visual pathways in diabetic patients.

Mitochondrial Dynamics in the Metabolic Memory of Diabetic Retinopathy.

Mitochondria play a central role in the development of diabetic retinopathy and in the metabolic memory associated with its continued progression. Mitochondria have a regulated fusion fission process, which is essential for their homeostasis. One of the major fission proteins, dynamin-related protein 1 (Drp1), is recruited to the mitochondria by fission protein 1 (Fis1) to initiate fragmentation. Our aim is to investigate the role of Drp1 in the altered mitochondrial dynamics in the continued progression of diabetic retinopathy. Methods. Drp1 activation, mitochondrial transport, and Drp1-Fis1 interactions were analyzed in retinal endothelial cells incubated in 20 mM glucose (HG), followed by 5 mM glucose (NG), for four days each (HG-NG group). The results were confirmed in retinal microvessels from streptozotocin-induced diabetic rats with poor glycemia (>350 mg/dl blood glucose, PC group), followed by normal glycemia (~100 mg/dl), for four months each (PC-GC group). Results. GTPase activity of Drp1, Fis1-Drp1 interactions, mitochondrial levels of Drp1, and fragmentation of the mitochondria were elevated in HG group. Mitochondrial Division Inhibitor 1 (Mdiv) or Drp1-siRNA attenuated Drp1 activation, mitochondrial fragmentation, and DNA damage. In HG-NG group, NG failed to ameliorate Drp1 activation and Drp1-Fis1 interactions, and the mitochondria remained fragmented. However, Mdiv supplementation in normal glucose, which had followed four days of high glucose (HG-NG/Mdiv group), inhibited Drp1 activation, mitochondrial fragmentation, and increase in ROS and prevented mitochondrial damage. Retinal microvessels from the rats in PC and PC-GC groups had similar Drp1 activation. Conclusion. Thus, Drp1 plays a major role in mitochondrial homeostasis in diabetic retinopathy and in the metabolic memory phenomenon associated with its continued progression. Supplementation of normal glycemia with a Drp1 inhibitor could retard development and further progression of diabetic retinopathy.

Mitochondrial Fragmentation in a High Homocysteine Environment in Diabetic Retinopathy.

Diabetic patients routinely have elevated homocysteine levels, and due to increase in oxidative stress, hyperhomocysteinemia is associated with increased mitochondrial damage. Mitochondrial homeostasis is directly related to the balance between their fission and fusion, and in diabetes this balance is disturbed. The aim of this study was to investigate the role of homocysteine in mitochondrial fission in diabetic retinopathy. Human retinal endothelial cells, either untransfected or transfected with siRNA of a fission protein (dynamin-related protein 1, Drp1) and incubated in the presence of 100 µM homocysteine, were analyzed for mitochondrial fragmentation by live-cell microscopy and GTPase activity of Drp1. Protective nucleoids and mtDNA damage were evaluated by SYBR DNA stain and by transcripts of mtDNA-encoded ND6 and cytochrome b. The role of nitrosylation of Drp1 in homocysteine-mediated exacerbation of mitochondrial fragmentation was determined by supplementing incubation medium with nitric-oxide inhibitor. Homocysteine exacerbated glucose-induced Drp1 activation and its nitrosylation, mitochondrial fragmentation and cell apoptosis, and further decreased nucleoids and mtDNA transcription. Drp1-siRNA or nitric-oxide inhibitor prevented glucose- and homocysteine-induced mitochondrial fission, damage and cell apoptosis. Thus, elevated homocysteine in a hyperglycemic environment increases Drp1 activity via increasing its nitrosylation, and this further fragments the mitochondria and increases apoptosis, ultimately leading to the development of diabetic retinopathy.

Epigenetic modifications in diabetes.

Diabetes is now considered as a 'silent epidemic' that claims over four million lives every year, and the disease knows no socioeconomic boundaries. Despite extensive efforts by the National and International organizations, and cutting-edge research, about 11% world's population is expected to suffer from diabetes (and its complications) by year 2045. This life-long disease damages both the microvasculature and the macrovasculature of the body, and affects many metabolic and molecular pathways, altering the expression of many genes. Recent research has shown that external factors, such as environmental factors, lifestyle and pollutants can also regulate gene expression, and contribute in the disease development and progression. Many epigenetic modifications are implicated in the development of micro- and macro- vascular complications including DNA methylation and histone modifications of several genes implicated in their development. Furthermore, several noncoding RNAs, such as micro RNAs and long noncoding RNAs, are also altered, affecting many biochemical pathways. Epigenetic modifications, however, have the advantage that they could be passed to the next generation, or can be erased. They are now being explored as therapeutical target(s) in the cancer field, which opens up the possibility to use them for treating diabetes and preventing/slowing down its complications.

Nuclear Genome-Encoded Long Noncoding RNAs and Mitochondrial Damage in Diabetic Retinopathy.

Retinal mitochondria are damaged in diabetes-accelerating apoptosis of capillary cells, and ultimately, leading to degenerative capillaries. Diabetes also upregulates many long noncoding RNAs (LncRNAs), including LncMALAT1 and LncNEAT1. These RNAs have more than 200 nucleotides and no open reading frame for translation. LncMALAT1 and LncNEAT1 are encoded by nuclear genome, but nuclear-encoded LncRNAs can also translocate in the mitochondria. Our aim was to investigate the role of LncMALAT1 and LncNEAT1 in mitochondrial homeostasis. Using human retinal endothelial cells, the effect of high glucose on LncMALAT1 and LncNEAT1 mitochondrial localization was examined by RNA fluorescence in situ hybridization. The role of these LncRNAs in mitochondrial membrane potential (by JC-I staining), mtDNA integrity (by extended length PCR) and in protective mtDNA nucleoids (by SYBR green staining) was examined in MALAT1- or NEAT1-siRNA transfected cells. High glucose increased LncMALAT1 and LncNEAT1 mitochondrial expression, and MALAT1-siRNA or NEAT1-siRNA ameliorated glucose-induced damage to mitochondrial membrane potential and mtDNA, and prevented decrease in mtDNA nucleoids. Thus, increased mitochondrial translocation of LncMALAT1 or LncNEAT1 in a hyperglycemic milieu plays a major role in damaging the mitochondrial structural and genomic integrity. Regulation of these LncRNAs can protect mitochondrial homeostasis, and ameliorate formation of degenerative capillaries in diabetic retinopathy.

Regulation of Rac1 transcription by histone and DNA methylation in diabetic retinopathy.

Cytosolic ROS, generated by NADPH oxidase 2 (Nox2) in diabetes, damage retinal mitochondria, which leads to the development of retinopathy. A small molecular weight G-protein essential for Nox2 activation, Rac1, is also transcriptionally activated via active DNA methylation-hydroxymethylation. DNA methylation is a dynamic process, and can also be regulated by histone modifications; diabetes alters retinal histone methylation machinery. Our aim is to investigate the role of histone methylation (H3K9me3) of Rac1 promoter in dynamic DNA methylation- transcriptional activation. Using human retinal endothelial cells in 20 mM D-glucose, H3K9me3 at Rac1 promoter was quantified by chromatin-Immunoprecipitation technique. Crosstalk between H3K9me3 and DNA methylation was examined in cells transfected with siRNA of histone trimethyl-transferase, Suv39H1, or Dnmt1, exposed to high glucose. Key parameters were confirmed in retinal microvessels from streptozotocin-induced diabetic mice, with intravitreally administered Suv39H1-siRNA or Dnmt1-siRNA. Compared to cells in normal glucose, high glucose increased H3K9me3 and Suv39H1 binding at Rac1 promoter, and Suv39H1-siRNA prevented glucose-induced increase 5 hydroxy methyl cytosine (5hmC) and Rac1 mRNA. Similarly, in diabetic mice, Suv39H1-siRNA attenuated increase in 5hmC and Rac1 mRNA. Thus, H3K9me3 at Rac1 promoter assists in active DNA methylation-hydroxymethylation, activating Rac1 transcription. Regulation of Suv39H1-H3K9 trimethylation could prevent further epigenetic modifications, and prevent diabetic retinopathy.

Diabetic Retinopathy and NADPH Oxidase-2: A Sweet Slippery Road.

Diabetic retinopathy remains the leading cause of vision loss in working-age adults. The multi-factorial nature of the disease, along with the complex structure of the retina, have hindered in elucidating the exact molecular mechanism(s) of this blinding disease. Oxidative stress appears to play a significant role in its development and experimental models have shown that an increase in cytosolic Reacttive Oxygen Speies (ROS) due to the activation of NADPH oxidase 2 (Nox2), is an early event, which damages the mitochondria, accelerating loss of capillary cells. One of the integral proteins in the assembly of Nox2 holoenzyme, Rac1, is also activated in diabetes, and due to epigenetic modifications its gene transcripts are upregulated. Moreover, addition of hyperlipidemia in a hyperglycemic milieu (type 2 diabetes) further exacerbates Rac1-Nox2-ROS activation, and with time, this accelerates and worsens the mitochondrial damage, ultimately leading to the accelerated capillary cell loss and the development of diabetic retinopathy. Nox2, a multicomponent enzyme, is a good candidate to target for therapeutic interventions, and the inhibitors of Nox2 and Rac1 (and its regulators) are in experimental or clinical trials for other diseases; their possible use to prevent/halt retinopathy will be a welcoming sign for diabetic patients.

Long Noncoding RNA MALAT1 and Regulation of the Antioxidant Defense System in Diabetic Retinopathy.

The retina experiences increased oxidative stress in diabetes, and the transcriptional activity of Nrf2, which is critical in regulating many antioxidant genes, is decreased. The nuclear movement/transcriptional activity of Nrf2 is mediated by its intracellular inhibitor Keap1, and retinal Keap1 levels are increased in diabetes. Gene expression is also regulated by long noncoding RNAs (LncRNAs). Our aim was to investigate the role of LncRNA MALAT1 in the regulation of Keap1-Nrf2-antioxidant defense in diabetic retinopathy. LncRNA MALAT1 expression (quantitative real-time PCR, immunofluorescence, and RNA sequencing), its interactions with Keap1 (FACS), Keap1-Nrf2 interactions, and transcription of the antioxidant response genes (immunofluorescence and nuclear RNA sequencing) were investigated in retinal endothelial cells exposed to high glucose. Glucose increased LncRNA MALAT1 levels by increasing Sp1 transcription factor binding at its promoter. Downregulation of LncRNA MALAT1 by its siRNA prevented glucose-induced increase in Keap1 and facilitated Nrf2 nuclear translocation and antioxidant gene transcription. Retinal microvessels from streptozotocin-induced diabetic mice and human donors with diabetic retinopathy also presented similar increases in LncRNA MALAT1 and its interactions with Keap1 and decreases in Nrf2-mediated antioxidant defense genes. Thus, LncRNA MALAT1, via Keap1-Nrf2, regulates antioxidant defense in diabetic retinopathy. Inhibition of LncRNA MALAT1 has potential to protect the retina from oxidative damage and to prevent or slow down diabetic retinopathy.