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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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The International Diabetes Federation predicts that by 2045 the number of individuals afflicted with diabetes will increase to 629 million. Furthermore, ∼352 million individuals with impaired glucose tolerance are at increased risk for developing diabetes. Several mechanisms have been proposed for the onset of metabolic dysfunction and demise of the islet β-cell leading to the pathogenesis of diabetes. It is widely accepted that the onset of type 2 diabetes is due to an intricate interplay between genetic expression of the disease and a multitude of factors including increased oxidative and endoplasmic reticulum stress consequential to glucolipotoxicity and inflammation. Compelling experimental evidence from in vitro and in vivo studies implicates intracellular generation of ceramide (CER), a biologically-active sphingolipid, as a trigger in the onset of β-cell demise under above pathological conditions. Recent pharmacological and molecular biological evidence affirms regulatory roles for Ras-related C3 botulinum toxin substrate 1 (Rac1), a small G protein, in the islet β-cell function in health and diabetes. In this Commentary, we overviewed the emerging evidence implicating potential cross-talk between Rac1 and ceramide signaling pathways in the onset of metabolic dysregulation of the islet β-cell culminating in impaired physiological insulin secretion, loss of β-cell mass and the onset of diabetes. Further, we propose a model depicting contributory roles of defective protein lipidation (prenylation) pathway in the induction of metabolic defects in the β-cell under metabolic stress conditions. Potential avenues for the identification of novel therapeutic targets for the prevention/treatment of diabetes and its associated complications are highlighted. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Background Diabetes is known to affect visual function before onset of retinopathy (diabetic retinopathy (DR)). Protection of visual function may signal disruption of mechanisms underlying DR. Methods This was a 6-month randomised, controlled clinical trial of patients with type 1 and type 2 diabetes with no retinopathy or mild to moderate non-proliferative retinopathy assigned to twice daily consumption of placebo or a novel, multi-component formula containing xanthophyll pigments, antioxidants and selected botanical extracts. Measurement of contrast sensitivity, macular pigment optical density, colour discrimination, 5-2 macular threshold perimetry, Diabetic Peripheral Neuropathy Symptoms, foveal and retinal nerve fibre layer thickness, glycohaemoglobin (HbA1c), serum lipids, 25-OH-vitamin D, tumour necrosis factor α (TNF-a) and high-sensitivity C reactive protein (hsCRP) were taken at baseline and 6 months. Outcomes were assessed by differences between and within groups at baseline and at study conclusion using meand ± SDs and t tests (p<0.05) for continuous variables. Results There were no significant intergroup differences at baseline. At 6 months, subjects on active supplement compared with placebo had significantly better visual function on all measures (p values ranging from 0.008 to <0.0001), significant improvements in most serum lipids (p values ranging from 0.01 to 0.0004), hsCRP (p=0.01) and diabetic peripheral neuropathy (Fisher's exact test, p=0.0024) No significant changes in retinal thickness, HbA1c, total cholesterol or TNF-α were found between the groups. Conclusions This study provides strong evidence of clinically meaningful improvements in visual function, hsCRP and peripheral neuropathy in patients with diabetes, both with and without retinopathy, and without affecting glycaemic control. Trial registration number www.ClinicalTrials.gov Identifier: NCT01646047 [ABSTRACT FROM AUTHOR]

Post-translational prenylation involves incorporation of 15-(farnesyl) or 20-(geranylgeranyl) carbon derivatives of mevalonic acid into highly conserved C-terminal cysteines of proteins. The farnesyl transferase (FTase) and the geranylgeranyl transferase (GGTase) mediate incorporation of farnesyl and geranylgeranyl groups, respectively. At least 300 proteins are prenylated in the human genome; the majority of these are implicated in cellular processes including growth, differentiation, cytoskeletal function and vesicle trafficking. From a functional standpoint, isoprenylation is requisite for targeting of modified proteins to relevant cellular compartments for regulation of effector proteins. Pharmacological and molecular biological studies have provided compelling evidence for key roles of this signaling pathway in physiological insulin secretion in normal rodent and human islets. Recent evidence indicates that inhibition of prenylation results in mislocalization of unprenylated proteins, and surprisingly, they remain in active (GTP-bound) conformation. Sustained activation of G proteins has been reported in mice lacking GGTase, suggesting alternate mechanisms for the activation of non-prenylated G proteins. These findings further raise an interesting question if mislocalized, non-prenylated and functionally active G proteins cause cellular pathology since aberrant protein prenylation has been implicated in the onset of cardiovascular disease and diabetes. Herein, we overview the existing evidence to implicate prenylation in islet function and potential defects in this signaling pathways in the diabetic β-cell. We will also identify critical knowledge gaps that need to be addressed for the development of therapeutics to halt defects in these signaling steps in β cells in models of impaired insulin secretion, metabolic stress and diabetes. [ABSTRACT FROM AUTHOR]

Abstract: Increased intracellular generation of reactive oxygen species [ROS] has been implicated in the pathology of metabolic [diabetes] and neurodegenerative [Alzheimer's] diseases. Accumulating evidence suggests NADPH oxidases [Noxs] as the principal source for cellular ROS in humans. Of this class of enzymes, the phagocyte-like Nox [Nox2] has come under intense scrutiny as one of the “culprits” for the induction of cellular damage culminating in the onset of diabetes and its complications. Functional regulation of Nox2 is fairly complex due to its membranous [gp91phox, p22phox] and cytosolic [p40phox, p47phox, p67phox and Rac1] cores, which require specific post-translational modification steps [phosphorylation and lipidation] for their membrane association. Therefore, optimal efficacy of Nox2 depends upon precise regulation of these signaling steps followed by translocation of the cytosolic components to the membrane. Interestingly, numerous recent studies have reported sustained activation of Nox2, ROS-derived oxidative stress, and cellular dysfunction in in vitro and in vivo models of glucolipotoxicity and diabetes. These investigations employed a variety of cell-permeable peptides and pharmacological inhibitors to impede Nox2 holoenzyme assembly and activation in pancreatic islet β-cells, cardiomyocytes and retinal endothelial cells under conditions of glucolipotoxicity and diabetes. Herein, we highlight the existing evidence to implicate Nox2 as the “trigger” of cellular damage, and identify critical gaps in our current understanding that need to be addressed to further affirm the roles of Nox2 as a potential therapeutic target for the treatment of diabetes and other metabolic disorders. [Copyright &y& Elsevier]

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. • Diabetes epidemic is an epidemic of its complications. • Epigenetic modifications can modulate the interplay between genes and environment. • Epigenetic modifications are implicated in diabetes and its complications. • Epigenetic changes are reprogrammable, and can be targeted with therapeutics. [ABSTRACT FROM AUTHOR]

OBJECTIVE--To determine the subunit expression and functional activation of phagocyte-like NADPH oxidase (Nox), reactive oxygen species (ROS) generation and caspase-3 activation in the Zucker diabetic fatty (ZDF) rat and diabetic human islets. RESEARCH DESIGN AND METHODS--Expression of core components of Nox was quantitated by Western blotting and densitometry. ROS levels were quantitated by the 2',7'-dichlorofluorescein diacetate method. Rac1 activation was quantitated using the gold-labeled immunosorbent assay kit. RESULTS--Levels of phosphorylated p47phox, active Rac1, Nox activity, ROS generation, Jun NH2-terminal kinase (JNK) 1/2 phosphorylation, and caspase-3 activity were significantly higher in the ZDF islets than the lean control rat islets. Chronic exposure of INS 832/13 cells to glucolipotoxic conditions resulted in increased JNK1/2 phosphorylation and caspase-3 activity; such effects were largely reversed by SP600125, a selective inhibitor of JNK. Incubation of normal human islets with high glucose also increased the activation of Rac1 and Nox. Lastly, in a manner akin to the ZDF diabetic rat islets, Rac1 expression, JNK1/2, and caspase-3 activation were also significantly increased in diabetic human islets. CONCLUSIONS--We provide the first in vitro and in vivo evidence in support of an accelerated Rac1--Nox--ROS--JNK1/2 signaling pathway in the islet β-cell leading to the onset of mitochondrial dysregulation in diabetes. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Abstract: H-Ras, a small molecular weight G-protein, undergoes post-translational modifications enabling its translocation from cytosol to the membrane. Hyperglycemia increases apoptosis of retinal capillary cells via activation of H-Ras, which can be ameliorated by farnesylation inhibitors. Our aim is to investigate the mechanism of retinal H-Ras activation in diabetes. H-Ras and Raf-1 were quantified in the retinal membrane and cytosol fractions obtained from streptozotocin-induced diabetes rats, and the role of post-translation modification was determined by investigating the effect of simvastatin on diabetes-induced alterations. The effect of H-Ras-siRNA on membrane translocation and apoptosis was also determined in bovine retinal endothelial cells (BRECs). Diabetes increased expressions of H-Ras and Raf-1 in the retinal membranes, and simvastatin prevented such translocation. Glucose-exposure of BRECs increased membrane H-Ras expression and H-Ras-siRNA prevented this translocation, and also decreased their apoptosis. Thus, membrane translocation of H-Ras is a plausible mechanism responsible for accelerated apoptosis of retinal capillary cells in diabetes. [Copyright &y& Elsevier]

Background: Oxidative stress and inflammation are implicated in the pathogenesis of retinopathy in diabetes. The aim of this study is to examine the effect of curcumin, a polyphenol with antioxidant and anti-inflammatory properties, on diabetes-induced oxidative stress and inflammation in the retina of rats. Methods: A group of streptozotocin-induced diabetic rats received powdered diet supplemented with 0.05% curcumin (w/w), and another group received diet without curcumin. The diets were initiated soon after induction of diabetes, and the rats were sacrificed 6 weeks after induction of diabetes. The retina was used to quantify oxidative stress and pro-inflammatory markers. Results: Antioxidant capacity and the levels of intracellular antioxidant, GSH (reduced form of glutathione) levels were decreased by about 30-35%, and oxidatively modified DNA (8-OHdG) and nitrotyrosine were increased by 60-70% in the retina of diabetic rats. The levels of interleukin-1β (IL-1β) and vascular endothelial growth factor (VEGF) were elevated by 30% and 110% respectively, and the nuclear transcription factor (NF-κB) was activated by 2 fold. Curcumin administration prevented diabetes-induced decrease in the antioxidant capacity, and increase in 8-OHdG and nitrotyrosine; however, it had only partial beneficial effect on retinal GSH. Curcumin also inhibited diabetes-induced elevation in the levels of IL-1β, VEGF and NF-κB. The effects of curcumin were achieved without amelioration of the severity of hyperglycemia. Conclusion: Thus, the beneficial effects of curcumin on the metabolic abnormalities postulated to be important in the development of diabetic retinopathy suggest that curcumin could have potential benefits in inhibiting the development of retinopathy in diabetic patients. [ABSTRACT FROM AUTHOR]

Oxidative stress is increased in the retina in diabetes, and it is considered to play an important role in the development of retinopathy. alpha-Lipoic acid, a thiol antioxidant, has been shown to have beneficial effects on polyneuropathy and on the parameters of oxidative stress in various tissues, including nerve, kidney, and retina. The purpose of this study was to examine the effect of alpha-lipoic acid on retinal capillary cell apoptosis and the development of pathology in diabetes. Retina was used from streptozotocin-induced diabetic rats receiving diets supplemented with or without alpha-lipoic acid (400 mg/kg) for 11 months of diabetes. Capillary cell apoptosis (by terminal transferase-mediated dUTP nick-end labeling) and formation of acellular capillaries were investigated in the trypsin-digested retinal microvessels. The effect of alpha-lipoic acid administration on retinal 8-hydroxy-2'deoxyguanosine (8-OHdG) and nitrotyrosine levels was determined by enzyme-linked immunosorbent assay. alpha-Lipoic acid administration for the entire duration of diabetes inhibited capillary cell apoptosis and the number of acellular capillaries in the retina, despite similar severity of hyperglycemia in the two diabetic groups (with and without alpha-lipoic acid). Retinal 8-OHdG and nitrotyrosine levels were increased by over twofold and 70%, respectively, in diabetes, and alpha-lipoic acid administration inhibited these increases. Our results demonstrate that the long-term administration of alpha-lipoic acid has beneficial effects on the development of diabetic retinopathy via inhibition of accumulation of oxidatively modified DNA and nitrotyrosine in the retina. alpha-Lipoic acid supplementation represents an achievable adjunct therapy to help prevent vision loss in diabetic patients. [ABSTRACT FROM AUTHOR]

Oxidative stress is increased in the retina in diabetes, and it is considered to play an important role in the development of retinopathy. α-Lipoic acid, a thiol antioxidant, has been shown to have beneficial effects on polyneuropathy and on the parameters of oxidative stress in various tissues, including nerve, kidney, and retina. The purpose of this study was to examine the effect of α-lipoic acid on retinal capillary cell apoptosis and the development of pathology in diabetes. Retina was used from streptozotocin-induced diabetic rats receiving diets supplemented with or without α-lipoic acid (400 mg/kg) for 11 months of diabetes. Capillary cell apoptosis (by terminal transferase-mediated dUTP nick-end labeling) and formation of acellular capillaries were investigated in the trypsin-digested retinal microvessels. The effect of α-lipoic acid administration on retinal 8-hydroxy-2'deoxyguanosine (8-OHdG) and nitrotyrosine levels was determined by enzyme-linked immunosorbent assay. α-Lipoic acid administration for the entire duration of diabetes inhibited capillary cell apoptosis and the number of acellular capillaries in the retina, despite similar severity of hyperglycemia in the two diabetic groups (with and without α-lipoic acid). Retinal 8-OHdG and nitrotyrosine levels were increased by over twofold and 70%, respectively, in diabetes, and α-lipoic acid administration inhibited these increases. Our results demonstrate that the long-term administration of α-lipoic acid has beneficial effects on the development of diabetic retinopathy via inhibition of accumulation of oxidatively modified DNA and nitrotyrosine in the retina. α-Lipoic acid supplementation represents an achievable adjunct therapy to help prevent vision loss in diabetic patients. Diabetes 53:3233--3238, 2004 [ABSTRACT FROM AUTHOR]

Studies have shown that good metabolic control (GC) is beneficial in slowing the progression of nephropathy in diabetes, and if the duration of poor metabolic control (PC) is prolonged before reinstitution of GC, nephropathy is not easily reversed. This study is to identify the biochemical abnormalities that could contribute to the resistance of nephropathy to reverse after establishment of GC in rats. The effect of reinstitution of GC and its duration is evaluated on oxidative stress and nitric oxide (NO) levels in the renal cortex and urine of diabetic rats. The rats were maintained in GC (5% glycated hemoglobin, GHb) soon after or 6 months after induction of hyperglycemia, and were sacrificed 13 months after induction of diabetes. For rats in which GC was initiated soon after induction of diabetes, oxidative stress [as measured by the levels of lipid peroxides (LPOs), 8-hydroxy-2′-deoxyguanosine (8-OHdG), and reduced glutathione (GSH)] and NO in urine and renal cortex were not different from that observed in normal control rats, but when reinstitution of GC was delayed for 6 months after induction of diabetes, oxidative stress and NO remain elevated in both urine and renal cortex. This suggests that hyperglycemia-induced oxidative stress and NO can be prevented if GC is initiated very early, but are not easily reversed if PC is maintained for longer durations. Understanding the mechanisms responsible for this phenomenon could reveal novel means to reverse nephropathy in diabetic patients. [Copyright &y& Elsevier]

Hyperglycemia is thought to be the underlying factor in the development of diabetic retinopathy, but the mechanisms involved remain partially understood. Diabetes increases oxidative stress, and reactive oxygen species affect the interactions between a small-molecular- weight G-protein, H-Ras, and several of its effector proteins. The purpose of this study was to examine the regulatory role of H-Ras in glucose-induced apoptosis of retinal endothelial cells. The expressions of H-Ras and its effector protein (Raf-1) were compared in the retina obtained from diabetic rats (2-8 months' duration) and age-matched normal rats and in retinal endothelial cells exposed to high-glucose medium. The effect of inhibition of H-Ras function on glucose-induced capillary cell death and the potential involvement of oxidative stress in diabetes-induced activation of H-Ras were also determined. The expressions of H-Ras and Raf-1 were increased in the retina in diabetes, and antioxidant therapy prevented diabetes-induced increased protein and mRNA expressions. The inhibitors of Ras farnesylation inhibited glucose-induced nitric oxides and apoptosis in isolated retinal endothelial cells. Thus, the results suggest that functional activation of H-Ras might be one of the signaling steps involved in glucose-induced capillary cell apoptosis and supports the role of H-Ras in the development of retinopathy in diabetes. [ABSTRACT FROM AUTHOR]

Clinical and experimental studies have shown that reinstitution of good glycemic control (GC) after a period of poor glycemic control (PC) does not produce immediate benefits on the progression of retinopathy, and hyperglycemia is sufficient to initiate the development of diabetic retinopathy. In this study, the effect of reinstitution of GC on hyperglycemia-induced increased oxidative stress and nitrative stress was evaluated in the retina of rats maintained in PC before initiation of GC. In diabetic rats, 2 or 6 months of PC (GHb >11.0%) was followed by 7 months of GC (GHb <5.5%). Reinstitution of GC after 2 months of PC inhibited elevations in retinal lipid peroxides and NO levels by approximately 50%, but failed to have any beneficial effects on nitrotyrosine formation. However, reversal of hyperglycemia after 6 months of PC had no significant effect on retinal oxidative stress and NO levels (P < 0.02 vs. normal). In the same rats, inducible nitric oxide synthase expression and nitrotyrosine levels remained elevated by >80% compared with normal rats or rats kept in GC for the duration. This suggests that oxidative and nitrative modifications in retina occur early in the course of development of retinopathy in diabetes. These abnormalities are not easily reversed by reinstitution of GC, and the duration of PC before initiation of GC influences the outcome of the reversal. Characterization of the abnormalities responsible for the resistance of retinopathy to arrest after reinstitution of GC will help identify potential future therapies to inhibit progression of diabetic retinopathy. [ABSTRACT FROM AUTHOR]

Apoptosis of retinal endothelial cells and pericytes is postulated to contribute to the development of retinopathy in diabetes. The goal of this study is to investigate diabetes-induced activation of retinal caspase-3, an apoptosis executer enzyme, in retina, and examine the effects of antioxidants on the activation. Caspase-3 activation was determined in the retina of alloxan diabetic rats (2-14 months duration) and in the isolated retinal capillary cells (endothelial cells and pericytes) by measuring cleavage of caspase-3 specific fluorescent substrate, and cleavage of caspase-3 holoenzyme and poly (ADP ribosyl) polymerase. Effect of antioxidants on the activation of caspase-3 was determined by feeding a group of diabetic rats diet supplemented with a comprehensive mixture of antioxidants, including Trolox, α-tocopherol, N -acetyl cysteine, ascorbic acid, β-carotene and selenium for 2-14 months, and also under in vitro conditions by incubating isolated retinal capillary cells with antioxidants with wide range of actions. Caspase-3 was activated in the rat retina at 14 months of diabetes ( P <0.05 vs. normal), but not at 2 months of diabetes, and administration of antioxidants for the entire duration inhibited this activation. In the isolated retinal capillary cells incubated in 25 mM glucose medium, caspase-3 activity was increased by 50% compared to the cells incubated in 5 mM glucose ( P <0.02), and antioxidants or caspase-3 inhibitor inhibited this increase. Our results suggest that increased oxidative stress in diabetes is involved in the activation of retinal caspase-3 and apoptosis of endothelial cells and pericytes. Antioxidants might be inhibiting the development of diabetic retinopathy by inhibiting microvascular apoptosis. [ABSTRACT FROM AUTHOR]

In the retinas of diabetic animals, protein kinase C (PKC) activity is elevated, and Na+-K+-ATPase and calcium ATPase activities are subnormal. These abnormalities are also present in another model of diabetic retinopathy, experimental galactosemia. We have investigated the relationship between hyperglycemia-induced abnormalities of PKC and ATPases using a selective inhibitor of beta isoform of PKC (LY333531). Diabetes or experimental galactosemia of 2 months' duration resulted in > 50% elevation of PKC activity in the retina, and administration of LY333531 prevented the elevation. In retinas of the same rats, the LY333531 prevented hyperglycemia-induced decreases of both Na+-K+-ATPase and calcium ATPase activities. Retinal microvessels, the main site of lesions in diabetic retinopathy, likewise showed elevated activity of PKC and inhibition of ATPases in diabetes and in experimental galactosemia, and administration of LY333531 to diabetic animals prevented these abnormalities. PKC activity in sciatic nerves, in contrast, became subnormal in diabetes and experimental galactosemia, and LY333531 had no effect on PKC activity in the sciatic nerve. PKC activity in the cerebral cortex was not affected by diabetes or experimental galactosemia. The results suggest that diabetes-induced reductions in Na+-K+-ATPase and calcium ATPase in the retina are mediated in large part by PKC-beta. The availability of an agent that can normalize the hyperglycemia-induced increase in PKC activity in the retina should facilitate investigation of the role of PKC in the development of diabetic retinopathy. [ABSTRACT FROM AUTHOR]

A correction is presented to the article "Abrogation of MMP-9 Gene Protects Against the Development of Retinopathy in Diabetic Mice by Preventing Mitochondrial Damage" which appeared in the previous issue.

Mitochondrial dysfunction is involved in the pathology of two major blinding retinal diseases, diabetic retinopathy (DR) and age-related macular degeneration (AMD). These diseases accumulate mitochondrial defects in distinct retinal subcellular structures, the vascular/neural network in DR and the retinal pigment epithelium (RPE) in AMD. These mitochondrial defects cause a metabolic crisis that drives disease. With no treatments to stop these diseases, coupled with an increasing population suffering from AMD and DR, there is an urgent need to develop new therapeutics targeting the mitochondria to prevent or reverse disease-specific pathology. In diabetic retinopathy (DR), cytosolic reactive oxygen species (ROS) accumulation in retinal vasculature occurs before mitochondrial damage and mitochondrial damage precedes capillary cell loss. In DR, mitochondrial fission is increased and mitochondrial DNA has increased damage and lower copy numbers and is hypermethylated. The retina and retinal pigment epithelium (RPE) are codependent tissues that exchange the energy substrates required for their survival. RPE mitochondrial damage occurs early in age-related macular degeneration (AMD) and leads to a bioenergetic crisis due to an imbalance in available energy substrates. Therapeutic strategies targeted towards maintaining mitochondrial homeostasis (structural, functional, and genomic) have the potential to prevent the development and progression of both DR and AMD. [ABSTRACT FROM AUTHOR]

Increased superoxide production is considered as a unifying mechanism for diabetic microvascular complications. Mitochondria are the major endogenous source of superoxide. Superoxide levels are elevated in the retina and its capillary cells in diabetes. The purpose of this study is to elucidate the mechanism involved in the oxidative damage of retinal mitochondria in diabetes. Mitochondria were prepared from the retina of streptozotocin- diabetic rats (4 months duration) and also from age-matched normal control rats by centrifugation method. Mitochondrial GSH was quantified by enzymatic recycling method; superoxide levels using lucigenin; oxidatively modified DNA (8-OHdG) by an ELISA; and H[sub 2]0[sub 2] levels were measured using a kit from Cell Technology. The membrane permeability transition of the mitochondria was determined by inducing transition with calcium chloride. In the same mitochondria the enzyme activities of complex I and complex III of the electron transport chain were quantified by spectrophotometric methods. Four months of diabetes decreased retinal mitochondrial GSH levels by 30%, and elevated superoxide and 8-OHdG levels by about 2 fold and H[sub 2]0[sub 2] levels by almost 70% compared to the normal control rats. The activities of complex I and complex III were decreased by about 15 and 30% respectively. In the same preparation swelling of retinal mitochondria was increased by 40%, suggesting an increase in membrane permeability transition. Thus, in diabetes the retinal mitochondria are swollen, its DNA is oxidatively modified and superoxide levels are elevated; and complex III appears to be one of the major sources of increased superoxide observed in the retina in diabetes. Understanding the mechanisms responsible for mitochondrial damage in diabetes may reveal novel means for inhibiting the development of diabetic retinopathy. [ABSTRACT FROM AUTHOR]

Abstract: Diabetic retinopathy resists arrest of its progression after reestablishment of good glycemic control that follows a profound period of poor glycemic control. The objective of this study was to elucidate the role of inflammation in the resistance of retinopathy to arrest after termination of hyperglycemia. Streptozotocin-diabetic rats were (a) maintained either in poor glycemic control [PC group; glycated hemoglobin (GHb)>11%] or in good glycemic control (GC group; GHb<7%) for 12 months or (b) allowed to be in poor glycemic control for 6 months followed by good glycemic control for 6 additional months. At 12 months, retina was analyzed for pro-inflammatory mediators. Twelve months of PC increased retinal interleukin 1β (IL-1β) mRNA by 2-fold and its protein expression by 25% compared with the values obtained from normal rat retina. Tumor necrosis factor α (TNF-α) was elevated approximately 3-fold (both mRNA and protein), and the receptors for IL-1β and TNF-α were increased by 40% each. The concentrations of intercellular cell adhesion molecule 1 and vascular cell adhesion molecule 1 were elevated by 40% and 150%, respectively, and inducible nitric oxide synthase transcripts were elevated by 6-fold. Six months of good glycemic control that followed 6 months of poor glycemic control failed to reverse the elevations in IL-1β, TNF receptor type I, and intercellular cell adhesion molecule 1 but had some beneficial effects on TNF-α, inducible nitric oxide synthase, and vascular cell adhesion molecule 1, however these mediators remained significantly elevated. However, the GC group showed no significant change in the retinal pro-inflammatory mediators compared with the normal rats. Failure to reverse retinal inflammatory mediators supports their important role in the resistance of retinopathy to arrest after cessation of hyperglycemia. [Copyright &y& Elsevier]

Highlights: [•] Activation of retinal gelatinase MMPs is an early event in diabetic retinopathy. [•] Activated cytosolic MMPs damage mitochondria and accumulate in them. [•] MMPs in the mitochondria damage them, activating the apoptotic machinery. [Copyright &y& Elsevier]

Abstract: Retinal mitochondria become dysfunctional and their DNA (mtDNA) is damaged in diabetes. The biogenesis of mitochondrial DNA is tightly controlled by nuclear–mitochondrial transcriptional factors, and translocation of transcription factor A (TFAM) to the mitochondria is essential for transcription and replication. Our aim is to investigate the effects of diabetes on nuclear–mitochondrial communication in the retina and its role in the development of retinopathy. Damage of mtDNA, copy number, and biogenesis (PGC1, NRF1, TFAM) were analyzed in the retinas from streptozotocin-diabetic wild-type (WT) and MnSOD transgenic (Tg) mice. Binding between TFAM and chaperone Hsp70 was quantified by coimmunoprecipitation. The key parameters were confirmed in isolated retinal endothelial cells and in the retinas from human donors with diabetic retinopathy. Diabetes in WT mice increased retinal mtDNA damage and decreased copy number. The gene transcripts of PGC1, NRF1, and TFAM were increased, but mitochondrial accumulation of TFAM was significantly decreased, and the binding of Hsp70 and TFAM was subnormal compared to WT nondiabetic mice. However, Tg diabetic mice were protected from retinal mtDNA damage and alterations in mitochondrial biogenesis. In retinal endothelial cells, high glucose decreased the number of mitochondria, as demonstrated by MitoTracker green staining and by electron microscopy, and impaired the transcriptional factors. Similar alterations in biogenesis were observed in the donors with diabetic retinopathy. Thus, retinal mitochondrial biogenesis is under the control of superoxide radicals and is impaired in diabetes, possibly by decreased transport of TFAM to the mitochondria. Modulation of biogenesis by pharmaceutical or molecular means may provide a potential strategy to retard the development/progression of diabetic retinopathy. [Copyright &y& Elsevier]