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1.  Interrelationship between activation of matrix metalloproteinases and mitochondrial dysfunction in the development of diabetic retinopathy 
Biochemical and biophysical research communications  2013;438(4):10.1016/j.bbrc.2013.07.066.
Mitochondria dysfunction plays a significant role in the apoptosis of retinal cells. Diabetes activates retinal matrix metalloproteinases (MMP-9 and MMP-2), damages retinal mitochondria and activates the apoptotic machinery. This study is to investigate the temporal relationship between the activation of retinal MMPs and mitochondria damage in the development of diabetic retinopathy. Time course of activation of cytosolic MMP-9 and MMP-2 was investigated in the retinal endothelial cells incubated in high glucose for 6–96 h, and correlated with their mitochondrial accumulation and mitochondrial damage. This was confirmed in the retina from rats diabetic for 15 days to ~12 months (streptozotocin-induced). The results show that the activation of cytosolic MMP-9 and MMP-2 is an early event, which is followed by their accumulation in the mitochondria. Increased mitochondrial MMPs dysfunction them and begin to damage their DNA, which initiates a vicious cycle of reactive oxygen species. Thus, modulation of these gelatinase MMPs by pharmacological agents during the early stages of diabetes could provide a strategy to inhibit the development of diabetic retinopathy.
doi:10.1016/j.bbrc.2013.07.066
PMCID: PMC3864795  PMID: 23891690
Diabetes; Matrix metalloproteinase; Mitochondria; Retinopathy
2.  Regulation of Matrix Metalloproteinase-9 by Epigenetic Modifications and the Development of Diabetic Retinopathy 
Diabetes  2013;62(7):2559-2568.
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.
doi:10.2337/db12-1141
PMCID: PMC3712057  PMID: 23423566
3.  Impaired Transport of Mitochondrial Transcription Factor and the Metabolic Memory Phenomenon Associated with the Progression of Diabetic Retinopathy 
Diabetes damages retinal mitochondrial DNA (mtDNA), and compromises the mtDNA transcription. In the transcription and replication of mtDNA, nuclear-encoded transcription factor A (TFAM) is considered as a key activator, and we have shown that in diabetes while retinal TFAM gene expression is increased, its mitochondrial levels are decreased. This study investigates the role of mitochondrial outer and inner membrane transport systems in the transfer of TFAM into the mitochondria in diabetes, and how reversal of hyperglycemia affects the ability of TFAM to reach to the mitochondria. Components of membrane transport system, Tom70, Tom40, Tim23 and Tim44, were analyzed in the retina from streptozotocin-induced diabetic rats maintained in poor control (PC) or in good control (GC) for 8 months, or in PC for 4 months followed by in GC for 4 months (Rev). The binding of TFAM with Tom70 and Tim44 was determined by co-immunoprecipitation, and that with mtDNA by ChIP. Retinal expressions of Tom70, Tom40 and Tim44 were significantly decreased in diabetes, and the binding of TFAM with Tom70, Tim44 and mtDNA were impaired. Reversal of hyperglycemia had no beneficial effect on decreased binding of TFAM and Tom proteins and mtDNA. Thus, subnormal membrane transport system in diabetes impairs the transfer of TFAM into the mitochondria, and decreased TFAM-mtDNA binding results in subnormal mitochondria transcription. These processes continue to be dysfunctional even after the hyperglycemic insult is terminated. Strategies targeting mitochondrial membrane transport proteins could have potential in improving mitochondrial biogenesis and slowing/halting the progression of diabetic retinopathy.
doi:10.1002/dmrr.2384
PMCID: PMC3596457  PMID: 23255365
Diabetic retinopathy; Metabolic memory; Mitochondria; mtDNA biogenesis
4.  Beneficial effects of the nutritional supplements on the development of diabetic retinopathy 
Purpose
Increased oxidative stress and inflammatory mediators are implicated in the development of diabetic retinopathy, and in rats, its development can be prevented by antioxidants. Carotenoids are some of the powerful antioxidants, and diabetes decreases lutein and zeaxanthin levels in the serum and retina. The aim of this study is to investigate the effect of carotenoid containing nutritional supplements (Nutr), which is in clinical trials for ‘Diabetes Vision Function’, on diabetic retinopathy.
Methods
Streptozotocin-induced diabetic rats (Wistar, male) were fed Purina 5001 supplemented with nutritional supplements containing zeaxanthin, lutein, lipoic acid, omega-3 fatty acids and other nutrients, or without any supplementation. Retinal function was analyzed at ~4 months of diabetes by electroretinography. After 11 months of diabetes, capillary cell apoptosis (TUNEL-staining) and histopathology (degenerative capillaries) were quantified in trypsin-digested retinal vasculature. Retina was also analyzed for mitochondrial damage (by quantifying gene expressions of mtDNA-encoded proteins of the electron transport chain), VEGF and inflammatory mediators, interleukin-1β and NF-kB.
Results
Diabetes impaired retinal function decreasing the amplitudes of both a- and b-waves. In the same animals, retinal capillary cell apoptosis and degenerative capillaries were increased by 3–4 fold. Gene expressions of mtDNA encoded proteins were decreased, and VEGF, interleukin-1β and NF-kB levels were elevated. Supplementation with the nutrients prevented increased capillary cell apoptosis and vascular pathology, and ameliorated these diabetes-induced retinal abnormalities.
Conclusions
Nutritional supplementation prevents diabetic retinopathy, and also maintains normal retinal function, mitochondrial homeostasis and inflammatory mediators. Thus, this supplementation could represent an achievable and inexpensive adjunct therapy to also inhibit retinopathy, a slow progressing disease feared most by diabetic patients.
doi:10.1186/1743-7075-11-8
PMCID: PMC3937140  PMID: 24479616
Carotenoids; Diabetic retinopathy; Macular pigment; Mitochondria; Nutritional supplements; Zeaxanthin
5.  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.
Diabetes increases retinal Nrf2 levels, but decreases its DNA binding activity. Due to increased binding of Nrf2 with its inhibitor, the recruitment of Nrf2 at the promoter of GCLC, a rate-limiting enzyme in GSH biosynthesis, is decreased, resulting in subnormal antioxidant defense system.
doi:10.1167/iovs.13-11598
PMCID: PMC3676188  PMID: 23633659
antioxidant defense; diabetic retinopathy; Nrf2
6.  A compensatory mechanism protects retinal mitochondria from initial insult in diabetic retinopathy 
Free radical biology & medicine  2012;53(9):1729-1737.
In the pathogenesis of diabetic retinopathy, an increase in retinal oxidative stress precedes mitochondrial dysfunction and capillary cell apoptosis. This study is designed to understand the mechanism responsible for the protection of mitochondria damage in the early stages of diabetic retinopathy. After 15 days–12 months of streptozotocin-induced diabetes in rats, retina was analyzed for mitochondria DNA (mtDNA) damage by extended length PCR. DNA repair enzyme and replication machinery were quantified in the mitochondria, and the binding of mitochondrial transcriptional factor A (TFAM) with mtDNA was analyzed by ChIP. Key parameters were confirmed in the retinal endothelial cells incubated in 20 mM glucose for 6–96 h. Although reactive oxygen species (ROS) were increased within 15 days of diabetes, mtDNA damage was observed at 6 months of diabetes. After 15 days of diabetes DNA repair/replication enzymes were significantly increased in the mitochondria, but at 2 months, their mitochondrial accumulation started to come down, and mtDNA copy number and binding of TFAM with mtDNA became significantly elevated. However, at 6 months of diabetes, the repair/replication machinery became subnormal and mtDNA copy number significantly decreased. A similar temporal relationship was observed in endothelial cells exposed to high glucose. Thus, in the early stages of diabetes, increased mtDNA biogenesis and repair compensates for the ROS-induced damage, but, with sustained insult, this mechanism is overwhelmed, and mtDNA and electron transport chain (ETC) are damaged. The compromised ETC propagates a vicious cycle of ROS and the dysfunctional mitochondria fuels loss of capillary cells by initiating their apoptosis.
doi:10.1016/j.freeradbiomed.2012.08.588
PMCID: PMC3632051  PMID: 22982046
Diabetic retinopathy; Mitochondria; mtDNA biogenesis; mtDNA damage
7.  Epigenetic Modifications and Diabetic Retinopathy 
BioMed Research International  2013;2013:635284.
Diabetic retinopathy remains one of the most debilitating chronic complications, but despite extensive research in the field, the exact mechanism(s) responsible for how retina is damaged in diabetes remains ambiguous. Many metabolic pathways have been implicated in its development, and genes associated with these pathways are altered. Diabetic environment also facilitates epigenetics modifications, which can alter the gene expression without permanent changes in DNA sequence. The role of epigenetics in diabetic retinopathy is now an emerging area, and recent work has shown that genes encoding mitochondrial superoxide dismutase (Sod2) and matrix metalloproteinase-9 (MMP-9) are epigenetically modified, activates of epigenetic modification enzymes, histone lysine demethylase 1 (LSD1), and DNA methyltransferase are increased, and the micro RNAs responsible for regulating nuclear transcriptional factor and VEGF are upregulated. With the growing evidence of epigenetic modifications in diabetic retinopathy, better understanding of these modifications has potential to identify novel targets to inhibit this devastating disease. Fortunately, the inhibitors and mimics targeted towards histone modification, DNA methylation, and miRNAs are now being tried for cancer and other chronic diseases, and better understanding of the role of epigenetics in diabetic retinopathy will open the door for their possible use in combating this blinding disease.
doi:10.1155/2013/635284
PMCID: PMC3826295  PMID: 24286082
8.  Diabetic Retinopathy and Signaling Mechanism for Activation of Matrix Metalloproteinase-9 
Journal of cellular physiology  2012;227(3):1052-1061.
In the pathogenesis of diabetic retinopathy, H-Ras (a small molecular weight G-protein) and matrix metalloproteinase-9 (MMP9) act as pro-apoptotic, accelerating the apoptosis of retinal capillary cells, a phenomenon that predicts its development and the activation of MMP9 is under the control of H-Ras. The goal of this study is to elucidate the cellular mechanism by which H-Ras activates MMP9 culminating in the development of diabetic retinopathy. Using isolated retinal endothelial cells, the effect of regulation of H-Ras downstream signaling cascade, Raf-1, MEK, and ERK, was investigated on glucose-induced activation of MMP9. In vitro results were confirmed in the retina obtained from diabetic mice manipulated for MMP9 gene, and also in the retinal microvasculature obtained from human donors with diabetic retinopathy. Regulation of Raf-1/MEK/ERK by their specific siRNAs and pharmacologic inhibitors prevented glucose-induced activation of MMP9 in retinal endothelial cells. In MMP9-KO mice, diabetes had no effect on retinal MMP9 activation, and H-Ras/Raf-1/MEK signaling cascade remained normal. Similarly, donors with diabetic retinopathy had increased MMP9 activity in their retinal microvessels, the site of histopathology associated with diabetic retinopathy, and this was accompanied by activated H-Ras signaling pathway (Raf-1/ERK). Collectively, these results suggest that Ras/Raf-1/MEK/ERK cascade has an important role in the activation of retinal MMP9 resulting in the apoptosis of its capillary cells. Understanding the upstream mechanism responsible for the activation of MMP9 should help identify novel molecular targets for future pharmacological interventions to inhibit the development/progression of diabetic retinopathy.
doi:10.1002/jcp.22822
PMCID: PMC3801274  PMID: 21567393
9.  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.
doi:10.1517/13543784.2012.681043
PMCID: PMC3802521  PMID: 22519597
diabetes; diabetic retinopathy; matrix metalloproteinases; retina
10.  Damaged Mitochondrial DNA Replication System and the Development of Diabetic Retinopathy 
Antioxidants & Redox Signaling  2012;17(3):492-504.
Abstract
Aim
In the pathogenesis of diabetic retinopathy, retinal mitochondria are damaged, superoxide levels are elevated, and mitochondrial DNA (mtDNA) biogenesis is impaired. mtDNA has a noncoding region, displacement loop (D-loop), which has essential transcription and replication elements, and this region is highly vulnerable to oxidative damage. The aim of this study is to investigate the effect of diabetes on the D-loop damage and the mtDNA replication machinery. Results: Using retina from wild-type (WT) and mitochondrial superoxide dismutase transgenic (Tg) mice, we have investigated the effect of diabetes on retinal D-loop damage and on the replication system. The results were confirmed in the isolated retinal endothelial cells in which the DNA polymerase gamma 1 (POLG1) function was genetically manipulated. Diabetes damaged retinal mtDNA, and the damage was more at the D-loop region compared with the cytochrome B region. Gene transcripts and mitochondrial accumulation of POLG1, POLG2, and mtDNA helicase, the enzymes that form replisome to bind/unwind and extend mtDNA, were also decreased in WT-diabetic mice compared with WT-normal mice. Tg-diabetic mice were protected from diabetes-induced damage to the D-loop region. Overexpression of POLG1 prevented high glucose-induced D-loop damage. This was accompanied by a decrease in mitochondrial superoxide levels. Innovation and Conclusions: Integrity of the retinal D-loop region and the mtDNA replication play important roles in the mtDNA damage experienced by the retina in diabetes, and these are under the control of superoxide. Thus, the regulation of mtDNA replication/repair machinery has the potential to prevent mitochondrial dysfunction and the development of diabetic retinopathy. Antioxid. Redox Signal. 17, 492–504.
doi:10.1089/ars.2011.4333
PMCID: PMC3365359  PMID: 22229649
11.  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.
Diabetes alters the histone 3 lysine 4 (H3K4) methylation status of retinal Sod2 via activation of lysine-specific demethylase 1, and H3K4 remains hypomethylated even after hyperglycemic insult is terminated.
doi:10.1167/iovs.12-10854
PMCID: PMC3590072  PMID: 23221071
12.  Mitochondria DNA Replication and DNA Methylation in the Metabolic Memory Associated with Continued Progression of Diabetic Retinopathy 
Purpose.
Diabetic retinopathy fails to halt after cessation of hyperglycemic insult, and a vicious cycle of mitochondria damage continues. The aim of our study was to investigate the effect of termination of hyperglycemia on retinal mtDNA replication, and elucidate the mechanism responsible for the continued mtDNA damage.
Methods.
Polymerase gamma 1 (POLG1), the catalytic subunit of the mitochondrial DNA replication enzyme, and the damage to the displacement loop region of mtDNA (D-loop) were analyzed in the retina from streptozotocin-diabetic rats maintained in poor glycemic control (PC, glycated hemoglobin ∼11%) or in good glycemic control (GC, glycated hemoglobin ∼6%) for 6 months, or in PC for three months followed by GC for three months (Rev). To understand the mechanism DNA methylation status of POLG1 promoter was investigated by methylation-specific PCR. The key parameters were confirmed in the isolated retinal endothelial cells exposed to high glucose, followed by normal glucose.
Results.
POLG1 continued to be down-regulated, the D-loop region damaged, and the CpG islands at the regulatory region of POLG hyper-methylated even after three months of GC that had followed three months of PC (Rev group). Similar results were observed in the retinal endothelial cells exposed to normal glucose after being exposed to high glucose.
Conclusions.
Continued hypermethylation of the CpG sites at the regulatory region of POLG affects its binding to the mtDNA, compromising the transcriptional activity. Modulation of DNA methylation using pharmaceutic or molecular means could help maintain mitochondria homeostasis, and prevent further progression of diabetic retinopathy.
Diabetes-induced damage to the retinal mtDNA replication system compromises the transcriptional activity and the mitochondria remains dysfunctional even after hyperglycemic insult is terminated, resulting in the continued progression of diabetic retinopathy.
In diabetes, hypermethylation of the CpG sites at the regulatory region of DNA polymerase impairs its binding to the mtDNA, and this results in impaired DNA replication. Termination of hyperglycemic insult fails to provide any benefit to these abnormalities, suggesting their role in the development and in metabolic memory phenomenon associated with the continued progression of diabetic retinopathy.
doi:10.1167/iovs.12-9732
PMCID: PMC3415280  PMID: 22743328
13.  Interleukin-1β and mitochondria damage, and the development of diabetic retinopathy 
Mitochondrial dysfunction is considered to play an important role in the development of diabetic retinopathy. Recent evidence has also shown many similarities between diabetic retinopathy and a low grade chronic inflammatory disease. The aim of this study is to understand the interrelationship between proinflammtory mediator, IL-1β and mitochondrial dysfunction in the accelerated loss of capillary cells in the retina. Using IL-1β receptor gene knockout (IL-1R1−/−) diabetic mice, we have investigated the effect of regulation of IL-1β on mitochondrial dysfunction and mtDNA damage, and increased retinal capillary cell apoptosis and the development of retinopathy. Retinal mitochondrial dysfunction and mtDNA damage were significantly ameliorated in IL-1R1−/− mice, diabetic for ~10 months, compared to the wild-type diabetic mice. This was accompanied by protection of accelerated capillary cell apoptosis and the development of acellular capillaries, histopathology associated with diabetic retinopathy. Thus, mitochondrial damage could be one of the key events via which increased inflammation contributes to the activation of the apoptotic machinery resulting in the development of diabetic retinopathy, and the possible mechanism via which inflammation contributes to the development of diabetic retinopathy includes continuous fueling of the vicious cycle of mitochondrial damage, which could be disrupted by inhibitors of inflammatory mediators.
doi:10.1007/s12177-011-9074-6
PMCID: PMC3342404  PMID: 23272269
Diabetic retinopathy; Interleukin-1β Mitochondria
14.  Mitochondria biogenesis and the development of diabetic retinopathy 
Free radical biology & medicine  2011;51(10):1849-1860.
Retinal mitochondria become dysfunctional and their DNA (mtDNA) is damaged in diabetes. Biogenesis of mitochondria 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 effect 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 retina from streptozotocin-diabetic wildtype (WT) and MnSOD transgenic (Tg) mice. Binding between TFAM and chaperone Hsp70 was quantified by co-immunoprecipitation. The key parameters were confirmed in isolated retinal endothelial cells, and in the retina 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-non diabetic 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 mitochondria 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.
doi:10.1016/j.freeradbiomed.2011.08.017
PMCID: PMC3202722  PMID: 21911054
Diabetic Retinopathy; DNA damage; Mitochondria; Superoxide dismutase
15.  Abrogation of MMP-9 Gene Protects Against the Development of Retinopathy in Diabetic Mice by Preventing Mitochondrial Damage 
Diabetes  2011;60(11):3023-3033.
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.
doi:10.2337/db11-0816
PMCID: PMC3198054  PMID: 21933988
16.  Increased Phagocyte-Like NADPH Oxidase and ROS Generation in Type 2 Diabetic ZDF Rat and Human Islets 
Diabetes  2011;60(11):2843-2852.
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.
doi:10.2337/db11-0809
PMCID: PMC3198065  PMID: 21911753
17.  Beyond AREDS: Is There a Place for Antioxidant Therapy in the Prevention/Treatment of Eye Disease? 
This review provides a perspective about the possible use of AREDS-based micronutrients for the treatment of diabetic retinopathy.
Age-related macular degeneration (AMD), the major cause of blindness in adults (65 years of age and older), and diabetic retinopathy, the major cause of blindness in working adults, are chronic, progressive diseases with multifaceted etiologies that are not fully understood. Progression and lack of treatment of both diseases may lead to the advanced stage with neovascularization. Although the detailed cellular mechanisms leading to the development of AMD and diabetic retinopathy remain elusive, oxidative damage to the retina and its pigment epithelium are considered to be involved. Clinical studies have shown that the progression of AMD can be slowed down by nutritional antioxidants, but trials with antioxidants for diabetic retinopathy (very limited in number) have been inconclusive. Long-term administration of the AREDS antioxidants, the same nutritional antioxidants that have been demonstrated to slow the progression of AMD, have yielded exciting results in preventing the pathogenesis of retinopathy in diabetic rodents. These results suggest the merit of testing the AREDS antioxidants in a clinical trial to prevent the development and/or progression of diabetic retinopathy, with the possibility of reducing the impact of this common vision-threatening disease.
doi:10.1167/iovs.10-6768
PMCID: PMC3230310  PMID: 22065212
18.  Role of Mitochondria Biogenesis in the Metabolic Memory Associated with the Continued Progression of Diabetic Retinopathy and Its Regulation by Lipoic Acid 
Abnormalities in retinal mitochondria biogenesis continue to progress even after hyperglycemic insult is terminated, suggesting their major role in the metabolic memory phenomenon associated with the continued progression of diabetic retinopathy.
Purpose.
Termination of hyperglycemia does not arrest the progression of diabetic retinopathy, and retinal mitochondrial DNA (mtDNA) remains damaged, resulting in a continuous cycle of mitochondrial dysfunction. This study is to investigate the role of mitochondria biogenesis (regulated by nuclear mitochondrial signaling) in the metabolic memory phenomenon.
Methods.
Mitochondria DNA copy number, functional integrity, and biogenesis (peroxisome proliferator-activated receptor-γ coactivator-1α [PGC1], nuclear respiratory factor 1 [NRF1], mitochondrial transcriptional factor [TFAM]) were analyzed in the retina from streptozotocin-diabetic rats maintained in poor or good control for 12 months (PC and GC respectively), or in PC for 6 months followed by 6 months of GC (Rev). The effect of direct inhibition of superoxide on prior insult was investigated by supplementing lipoic acid (LA) during their 6 months of GC (R+LA). Binding of TFAM with chaperones (heat shock proteins 70 and 60, Hsp70 and Hsp60 respectively) was quantified by coimmunoprecipitation. The key parameters and the number of mitochondria (by transmission electron microscopy and fluorescence microscopy) were confirmed in isolated retinal endothelial cells.
Results.
Six months of GC in the rats in Rev group did not provide any benefit to diabetes-induced decreased mtDNA copy number, increased gene transcripts of PGC1, NRF1, and TFAM, and decreased mitochondrial TFAM. The binding of TFAM with the chaperones remained subnormal. Supplementation of LA (R+LA), however, had a significant beneficial effect on the impaired mitochondria biogenesis, and also on the continued progression of diabetic retinopathy. Similar results of reversal of high glucose insult were observed in isolated retinal endothelial cells.
Conclusions.
Dysregulated mitochondria biogenesis contributes to the metabolic memory, and supplementation of GC with therapies targeted in modulating mitochondria homeostasis has potential in helping diabetic patients retard progression of retinopathy.
doi:10.1167/iovs.11-8203
PMCID: PMC3230862  PMID: 22003111
19.  Diabetic Retinopathy and Damage to Mitochondrial Structure and Transport Machinery 
Retinal mitochondria fusionγÇôfission and protein import machinery are severely affected in diabetes, and reversal of hyperglycemia fails to provide any benefit to these abnormalities, suggesting their role in the development and in the continued progression of diabetic retinopathy.
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.
doi:10.1167/iovs.11-8045
PMCID: PMC3263781  PMID: 22003103
20.  Epigenetic Changes in Mitochondrial Superoxide Dismutase in the Retina and the Development of Diabetic Retinopathy 
Diabetes  2011;60(4):1304-1313.
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.
doi:10.2337/db10-0133
PMCID: PMC3064104  PMID: 21357467
21.  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.
PMCID: PMC3214730  PMID: 20939803
Antioxidants; apoptosis; diabetic retinopathy; metabolic memory; mitochondria; oxidative stress
22.  Novel Role of Mitochondrial Matrix Metalloproteinase-2 in the Development of Diabetic Retinopathy 
In diabetes, increased accumulation of MMP2 in the retinal mitochondria degrades their membranes by modulating Hsp60 and damaging the gap junction protein connexin 43. Cytochrome c leaks out of the mitochondria and activates the apoptotic machinery, resulting in accelerated loss of retinal capillary cells.
Purpose.
In the pathogenesis of diabetic retinopathy, retinal mitochondria become dysfunctional, their DNA is damaged, and capillary cells undergo accelerated apoptosis. Matrix metalloproteinase-2 (MMP2) becomes activated and proapoptotic, and the therapies that inhibit the development of diabetic retinopathy alleviate MMP2 activation. The authors sought to elucidate the possible mechanism by which activated MMP2 contributes to mitochondrial dysfunction.
Methods.
The effect of the regulation of MMP2 on mitochondrial dysfunction and the subcellular localization of the molecular chaperone important for mitochondrial integrity (Hsp60) and gap junction protein connexin 43 were investigated in retinal endothelial cells. The results were confirmed in retinal mitochondria isolated from diabetic mouse overexpressing MnSOD and in the retinas of normal rats that received intravitreal administration of MMP2.
Results.
High glucose increased MMP2 and decreased connexin 43 in the mitochondria of retinal endothelial cells. Although the Hsp60 gene transcript was increased, its abundance in the mitochondria was decreased, and its interaction with MMP2 was increased. In mice, the overexpression of MnSOD protected retinal mitochondria from diabetes-induced increases in MMP2 and decreases in Hsp60 and connexin 43. MMP2 administration in normal rats damaged the retinal mitochondria, decreased Hsp60 and connexin 43, and accelerated the apoptosis of retinal capillary cells.
Conclusions.
Elevated MMP2 in the mitochondria degrades its membranes by modulating Hsp60 and damaging connexin 43, and this activates the apoptotic machinery. Better understanding of MMP2-mediated mitochondrial damage could help identify new strategies for the treatment of this blinding disease.
doi:10.1167/iovs.10-6368
PMCID: PMC3109059  PMID: 21345984
23.  Role of Mitochondrial DNA Damage in the Development of Diabetic Retinopathy, and the Metabolic Memory Phenomenon Associated with Its Progression 
Antioxidants & Redox Signaling  2010;13(6):797-805.
Abstract
Diabetic retinopathy does not halt after hyperglycemia is terminated; the retina continues to experience increased oxidative stress, suggesting a memory phenomenon. Mitochondrial DNA (mtDNA) is highly sensitive to oxidative damage. The goal is to investigate the role of mtDNA damage in the development of diabetic retinopathy, and in the metabolic memory. mtDNA damage and its functional consequences on electron transport chain (ETC) were analyzed in the retina from streptozotocin-diabetic rats maintained in poor control (PC, glycated hemoglobin >11%) for 12 months or PC for 6 months followed by good control (GC, GHb < 6.5%) for 6 months. Diabetes damaged retinal mtDNA and elevated DNA repair enzymes (glycosylase). ETC proteins that were encoded by the mitochondrial genome and the glycosylases were compromised in the mitochondria. Re-institution of GC after 6 months of PC failed to protect mtDNA damage, and ETC proteins remained subnormal. Thus, mtDNA continues to be damaged even after PC is terminated. Although the retina tries to overcome mtDNA damage by inducing glycosylase, they remain deficient in the mitochondria with a compromised ETC system. The process is further exacerbated by subsequent increased mtDNA damage providing no relief to the retina from a continuous cycle of damage, and termination of hyperglycemia fails to arrest the progression of retinopathy. Antioxid. Redox Signal. 13, 797–805.
doi:10.1089/ars.2009.2932
PMCID: PMC2935337  PMID: 20088705
24.  Role of Histone Acetylation in the Development of Diabetic Retinopathy and the Metabolic Memory Phenomenon 
Journal of cellular biochemistry  2010;110(6):1306-1313.
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 5mM or 20mM 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.
doi:10.1002/jcb.22644
PMCID: PMC2907436  PMID: 20564224
Diabetic retinopathy; Histone acetylation; Histone deacetylase; Metabolic memory
25.  Metabolic Memory and Diabetic retinopathy: Role of Inflammatory Mediators in Retinal Pericytes 
Experimental eye research  2010;90(5):617-623.
Diabetic retinopathy shares many characteristics features of a low grade chronic inflammatory disease. Its progression resists arrest when good metabolic control is re-established after a period of poor metabolic control, suggesting a ‘metabolic memory’ phenomenon. The aim of this study is to investigate the effect of reversal of high glucose to normal glucose on the inflammatory mediators in pericytes, the site of histopathology in diabetic retinopathy. Bovine retinal pericytes were incubated in high glucose (20mM) for 2 days followed by normal glucose (5mM) for 4 days (2→4), or in high glucose for 4 days followed by normal glucose for 4 days (4→4) or 8 days (4→8). Pericytes incubated in continuous normal or high glucose for 2-12 days served as controls. Continuous high glucose exposure for 2-12 days significantly elevated gene expressions and protein concentrations of IL-1β, NF-kB, VEGF, TNF-α, TGF-β and ICAM-1 in retinal pericytes. Four days of normal glucose that followed 2 days of high glucose (2→4) had marginal, but significant, beneficial effect on the increases in these inflammatory mediators. Four days of normal glucose in 4→4 group failed to reverse increases in inflammatory mediators and cell apoptosis remained elevated, but addition of dexamethasone during normal glucose exposure ameliorated such increases. However, when normal glucose exposure, after 4 days of high glucose was extended to 8 days (4→8), increases in these mediators were significantly decreased. Hyperglycemia-induced elevations in inflammatory mediators in retinal microvascular cells resist reversal after re-institution of normal glucose conditions. Both, the duration of the initial exposure to high glucose, and normal glucose that follows high glucose, are critical in determining the outcome of the alterations in the inflammatory mediators.
doi:10.1016/j.exer.2010.02.006
PMCID: PMC2857535  PMID: 20170650
Diabetic retinopathy; Inflammation; Metabolic memory; Pericytes; Retina

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