Julia M. Santos

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

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

Mitochondrial biogenesis and the development of diabetic retinopathy.

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.

Skeletal Muscle Pathways of Contraction-Enhanced Glucose Uptake

Muscle contraction acutely increases glucose transport in both healthy and type 2 diabetic individuals. Since glucose uptake during muscle contraction has been observed in the absence of insulin, the existence of an insulin-independent pathway has been suggested to explain this phenomenon. However, the exact mechanism behind the translocation of GLUT4 vesicles through the sarcolemma during muscle contraction is still unknown. Some substances, such as AMPK and calcium activated proteins, have been suggested as potential mediators but the exact mechanisms of their involvement remain to be elucidated. A hypothetical convergence point between the insulin cascade and the potential pathways triggered by muscle contraction has been suggested. Therefore, the earliest concept that two different routes exist in skeletal muscle has been progressively modified to the notion that glucose uptake is induced by muscle contraction via components of the insulin pathway. With further consideration, increased glucose uptake and enhanced insulin sensitivity observed during/after exercise might be explained by a metabolic- and calcium-dependent activation of several intermediate molecules of the insulin cascade. This paper aimed to review the literature in order to examine in detail these concepts behind muscle contraction-induced glucose uptake.

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.

Damaged Mitochondrial DNA Replication System and the Development of Diabetic Retinopathy

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.

Epigenetic Modifications and Diabetic Retinopathy

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.

Role of mitochondria biogenesis in the metabolic memory associated with the continued progression of diabetic retinopathy and its regulation by lipoic acid

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.

A compensatory mechanism protects retinal mitochondria from initial insult in diabetic retinopathy.

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 20mM glucose for 6-96h. 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.

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.

TIAM1-RAC1 signalling axis-mediated activation of NADPH oxidase-2 initiates mitochondrial damage in the development of diabetic retinopathy.

Aims/hypothesisIn diabetes, increased retinal oxidative stress is seen before the mitochondria are damaged. Phagocyte-like NADPH oxidase-2 (NOX2) is the predominant cytosolic source of reactive oxygen species (ROS). Activation of Ras-related C3 botulinum toxin substrate 1 (RAC1), a NOX2 holoenzyme member, is necessary for NOX2 activation and ROS generation. In this study we assessed the role of T cell lymphoma invasion and metastasis (TIAM1), a guanine nucleotide exchange factor for RAC1, in RAC1 and NOX2 activation and the onset of mitochondrial dysfunction in in vitro and in vivo models of glucotoxicity and diabetes.MethodsRAC1 and NOX2 activation, ROS generation, mitochondrial damage and cell apoptosis were quantified in bovine retinal endothelial cells exposed to high glucose concentrations, in the retina from normal and streptozotocin-induced diabetic rats and mice, and the retina from human donors with diabetic retinopathy.ResultsHigh glucose activated RAC1 and NOX2 (expression and activity) and increased ROS in endothelial cells before increasing mitochondrial ROS and mitochondrial DNA (mtDNA) damage. N6-[2-[[4-(diethylamino)-1-methylbutyl]amino]-6-methyl-4-pyrimidinyl]-2-methyl-4,6-quinolinediamine, trihydrochloride (NSC23766), a known inhibitor of TIAM1–RAC1, markedly attenuated RAC1 activation, total and mitochondrial ROS, mtDNA damage and cell apoptosis. An increase in NOX2 expression and membrane association of RAC1 and p47phox were also seen in diabetic rat retina. Administration of NSC23766 to diabetic mice attenuated retinal RAC1 activation and ROS generation. RAC1 activation and p47phox expression were also increased in the retinal microvasculature from human donors with diabetic retinopathy.Conclusions/interpretationThe TIAM1–RAC1–NOX2 signalling axis is activated in the initial stages of diabetes to increase intracellular ROS leading to mitochondrial damage and accelerated capillary cell apoptosis. Strategies targeting TIAM1–RAC1 signalling could have the potential to halt the progression of diabetic retinopathy in the early stages of the disease.