cytochrome-c-t and Diabetic-Retinopathy

Diabetic retinopathy is thought to be trigger by glucose- induced oxidative stress which leads to an increase of the mitochondrial permeability through opening the permeability transition pore (MTP). In several cell types, hexokinases interact with the mitochondria regulating MTP opening, avoiding cytochrome c release. We studied HK I mitochondrial proportion in control and streptozotocin-induced diabetic rat retinas. In the normal retina, 50% of HK I was linked to mitochondria, proportion that did not change up to 60 days of diabetes. Mitochondria from normal and diabetic rat retinas showed a limited swelling, and similar cytochrome c levels. G-6-P and glycogen content increased 3-6-fold in diabetic rat retinas, while lactate content did not vary. Results suggest that mitochondrial bound HK produce G-6-P and drove it to glycogen synthesis, controlling ROS production and lactate toxicity.

Topics: Animals; Cytochromes c; Diabetes Mellitus; Diabetic Retinopathy; Disease Models, Animal; Female; Glucose-6-Phosphate; Hexokinase; Mitochondria; Rats; Retina; Streptozocin

To determine whether high glucose (HG) or diabetes alters mitochondrial morphology and promotes mitochondrial fragmentation in retinal vascular cells and thereby triggers apoptosis associated with diabetic retinopathy.. To assess whether diabetes promotes mitochondrial fragmentation and thereby triggers apoptosis, retinas from nondiabetic and diabetic rats were analyzed using electron microscopy (EM) and in parallel, wild-type, diabetic, and OPA1+/- mice were analyzed for optic atrophy gene 1 (OPA1) and cytochrome c levels using Western blot (WB) analysis. To assess the relationship between mitochondrial fragmentation and OPA1 levels, rat retinal endothelial cells (RRECs) were grown in normal (N; 5 mmol/L) medium, HG (30 mmol/L) medium, or in N medium transfected with OPA1 siRNA for seven days. Cells were examined for OPA1 expression and cytochrome c release by WB. In parallel, cells were stained with MitoTracker Red and assessed for mitochondrial fragmentation in live cells using confocal microscopy.. EM images revealed significant mitochondrial fragmentation in vascular cells of retinal capillaries of diabetic rats compared with that of nondiabetic rats. WB analysis showed significant OPA1 downregulation concomitant with increased levels of proapoptotic cytochrome c levels in cells grown in HG and in cells transfected with OPA1 siRNA alone. Similarly, OPA1 level was significantly reduced in diabetic retinas compared with that of nondiabetic retinas. Interestingly, OPA1+/- animals exhibited elevated cytochrome c release similar to those of diabetic mice.. Findings indicate that diabetes promotes mitochondrial fragmentation in retinal vascular cells, which are driven, at least in part, by decreased OPA1 levels leading to apoptosis in diabetic retinopathy.

Topics: Animals; Apoptosis; Blotting, Western; Cells, Cultured; Cytochromes c; Diabetes Mellitus, Experimental; Diabetic Retinopathy; Endothelium, Vascular; Glucose; GTP Phosphohydrolases; Mice; Mice, Inbred C57BL; Microscopy, Confocal; Microscopy, Electron; Mitochondria; Mitochondrial Diseases; Rats; Rats, Sprague-Dawley; Retinal Vessels; RNA, Small Interfering; Transfection

The aim of this study was to investigate the protective effects and mechanisms of hesperidin, a plant based active flavanone found in citrus fruits, under the oxidative stress and apoptosis induced by high levels of glucose in retinal ganglial cells (RGCs). RGC-5 cells were pretreated with hesperidin (12.5, 25, or 50 μmol/L) for 6 h followed by exposure to high (33.3 mmol/L) d-glucose for 48 h. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was adopted to evaluate cell viability. Mitochondrial function was estimated by measuring the mitochondrial membrane potential (ΔΨm). A fluorescent probe was employed to evaluate the intercellular production of reactive oxygen species (ROS). Colorimetric assay kits were used to evaluate lipid peroxidation, antioxidant enzyme activities, and protein carbonyls formation. The expression of apoptosis-related proteins and mitogen-activated protein kinase (MAPK) were measured with Western blotting. Hesperidin inhibited high glucose-mediated cell loss and restored mitochondrial function including a reversion of ΔΨm loss and cytochrome c release. Treated with hesperidin, high glucose-induced increase in ROS, malondialdehyde, and protein carbonyl levels were blocked in RGC-5 cells. Hesperidin was found to elevate the activities of superoxide dismutase, catalase, glutathione peroxidase, and to recover glutathione levels. Hesperidin inhibited high glucose-induced cell apoptosis by attenuating the downregulation of caspase-9, caspase-3, and Bax/Bcl-2. Furthermore, the phosphorylation of c-Jun N-terminal kinases (JNK) and p38 MAPK triggered by high glucose were attenuated in RGC-5 cells after their incubation with hesperdin. We concluded that hesperidin may protect RGC-5 cells from high glucose-induced injury since it owns the properties of antioxidant action and blocks mitochondria-mediated apoptosis.

Topics: Animals; Antioxidants; Apoptosis; Cell Survival; Cytochromes c; Diabetic Retinopathy; Dose-Response Relationship, Drug; Gene Expression Regulation, Enzymologic; Glucose; Hesperidin; Lipid Peroxidation; MAP Kinase Kinase 4; Membrane Potential, Mitochondrial; Mice; Oxidative Stress; p38 Mitogen-Activated Protein Kinases; Protein Carbonylation; Reactive Oxygen Species; Retinal Ganglion Cells

Blood-retinal barrier (BRB) breakdown, the early hallmark of diabetic retinopathy (DR), is thought to depend on retinal inflammation and cell damage. The proinflammatory factor interleukin-1β (IL-1β) was demonstrated to cause inflammation as well as cell apoptosis during the process of BRB breakdown. This study extensively evaluated the protective effect of puerarin, a major active component extracted from the traditional herb Radix puerariae, against IL-1β-induced cell dysfunction in TR-iBRB2 cells, a retinal capillary endothelial cell line.. TR-iBRB2 cells were pretreated with IL-1β (10 ng/ml) for 24 h and then exposed to puerarin (0, 10, 25, and 50 μM) for another 24 h. Leukocyte endothelial adhesion was assessed through a cell-based assay using lymphoblastoid cells. Cell apoptosis was evaluated with flow cytometry, and the expression of adhesion molecules and apoptosis-related molecules was assessed with western blot analysis.. Our data showed that puerarin attenuated IL-1β-mediated leukostasis and cell apoptosis in TR-iBRB2 cells. Furthermore, puerarin strikingly prevented IL-1β-induced molecular events of the upstream and downstream signaling pathways involved in this cellular process.. These findings may significantly contribute to better understanding of the protective effect of puerarin, in particular for DR, as well as provide novel insights into the potential application of this compound in DR therapy.

Topics: Animals; Apoptosis; Cell Adhesion; Cell Line; Cytochromes c; Diabetic Retinopathy; Drugs, Chinese Herbal; Endothelial Cells; Humans; Intercellular Adhesion Molecule-1; Interleukin-1beta; Isoflavones; Leukostasis; Phytotherapy; Rats; Retinal Vessels; Vascular Cell Adhesion Molecule-1

To determine connexin 43 (Cx43) localization in mitochondria and investigate the effects of high glucose (HG) on mitochondrial Cx43 (mtCx43) expression and whether altered mtCx43 channel activity is involved in promoting apoptosis in retinal endothelial cells.. MtCx43 localization was determined using immunostaining, green fluorescent protein (GFP)-tagged Cx43 followed by confocal imaging, and Western blot analysis using protein isolated from mitochondria of rat retinal endothelial cells (RRECs). To assess HG effects on mtCx43 expression, RRECs were grown in normal (5 mM) or HG (30 mM) medium for 7 days, and mtCx43 protein level assessed by Western blot analysis. To determine if mtCx43 channel inhibition affected mitochondrial morphology, RRECs grown sparsely were left untreated or treated with β-glycerrhetinic acid (β-GA), an inhibitor of connexin channels, and imaged using confocal microscopy. Additionally, mitochondria isolated from RRECs were treated with β-GA, and cytochrome c release assessed by Western blot.. Cx43 localization on the mitochondria of RRECs was confirmed with immunofluorescence staining using Cx43 antibody and GFP-tagged Cx43 imaged in live cells. Western blot analysis indicated that Cx43 was located primarily on the inner mitochondrial membrane, and mtCx43 protein level was significantly reduced in RRECs grown in HG condition. Treatment of RRECs with β-GA significantly decreased mtCx43 phosphorylation, induced mitochondrial fragmentation, and isolated mitochondria treated with β-GA showed increased cytochrome c release.. HG-induced downregulation of mtCx43 protein resulting in decreased channel activity may promote mitochondrial morphology changes and cytochrome c release, suggesting a novel mechanism for hyperglycemia-induced apoptosis in diabetic retinopathy.

Topics: Animals; Apoptosis; Blotting, Western; Cells, Cultured; Connexin 43; Cytochromes c; Diabetic Retinopathy; Disease Models, Animal; Down-Regulation; Endothelial Cells; Glucose; Microscopy, Confocal; Mitochondria; Rats; Retinal Vessels; Sweetening Agents

To investigate the effect of mitochondria-targeted antioxidant peptide SS31 on prevention of high glucose-induced injury on human retinal endothelial cells (HRECs).. Cultured P3-P5 HRECs were divided into three groups: 5 mM glucose group, 30 mM glucose group and 30 mM glucose co-treated with 100 nM SS31 group. 24 and 48 h after treatment, Annexin V-FITC/PI staining was used to evaluate the survival of HRECs. Overproduction of ROS was assessed by MitoSOX staining under confocal microscope. Change of mitochondrial potential (ΔΨ(m)) of HRECs was measured by flow cytometry after JC-1 fluorescent probe staining. Release of cytochrome c was assessed by confocal microscopy and western blot. Expression of caspase-3 and thioredoxin-2 (Trx-2) were measured by western blot and real-time PCR.. Compared to the high glucose group, co-treatment with 100 nM SS31 significantly protected HRECs from high glucose-induced injury, reduced the production of ROS in mitochondria, stabilized ΔΨ(m), decreased the release of cytochrome c from mitochondria to cytoplasm, decreased the expression of caspase-3 and increased the expression of Trx-2 in high glucose-treated HRECs.. SS31 attenuates the high glucose-induced injuries on HRECs by stabilizing ΔΨ(m), decreasing ROS production, preventing the release of cytochrome c from mitochondria, decreasing the expression of caspase-3 and increasing the expression of Trx-2. Our study suggests that SS31 may be as a potential new treatment for diabetic retinopathy and other oxidative stress-related diseases.

Topics: Antioxidants; Caspase 3; Caspase Inhibitors; Cells, Cultured; Cytochromes c; Diabetic Retinopathy; Endothelial Cells; Glucose; Humans; Hyperglycemia; Membrane Potential, Mitochondrial; Mitochondria; Mitochondrial Proteins; Oligopeptides; Retina; Thioredoxins

In the pathogenesis of diabetic retinopathy, retinal mitochondria become dysfunctional resulting in accelerated apoptosis of its capillary cells. Matrix metalloproteinase-2 (MMP2) is considered critical in cell integrity and cell survival, and diabetes activates MMP2 in the retina and its capillary cells. This study aims at elucidating the mechanism by which MMP2 contributes to the development of diabetic retinopathy. Using isolated bovine retinal endothelial cells, the effect of regulation of MMP2 (by its siRNA and pharmacological inhibitor) on superoxide accumulation and mitochondrial dysfunction was evaluated. The effect of inhibiting diabetes-induced retinal superoxide accumulation on MMP2 and its regulators was investigated in diabetic mice overexpressing mitochondrial superoxide dismutase (MnSOD). Inhibition of MMP2 ameliorated glucose-induced increase in mitochondrial superoxide and membrane permeability, prevented cytochrome c leakage from the mitochondria, and inhibited capillary cell apoptosis. Overexpression of MnSOD protected the retina from diabetes-induced increase in MMP2 and its membrane activator (MT1-MMP), and decrease in its tissue inhibitor (TIMP-2). These results implicate that, in diabetes, MMP2 activates apoptosis of retinal capillary cells by mitochondrial dysfunction increasing their membrane permeability. Understanding the role of MMP2 in the pathogenesis of diabetic retinopathy should help lay ground for MMP2-targeted therapy to retard the development of retinopathy in diabetic patients.

Topics: Animals; Apoptosis; Capillaries; Cattle; Cytochromes c; Diabetic Retinopathy; Endothelial Cells; Glucose; Matrix Metalloproteinase 14; Matrix Metalloproteinase 2; Mice; Mitochondria; Retina; Retinal Diseases; Superoxide Dismutase; Superoxides; Tissue Inhibitor of Metalloproteinase-2

Mitochondrial dysfunction has been implicated in diabetic complications; however, it is unknown whether hyperglycemia affects mitochondrial morphology and metabolic capacity during development of diabetic retinopathy. We investigated high glucose (HG) effects on mitochondrial morphology, membrane potential heterogeneity, cellular oxygen consumption, extracellular acidification, cytochrome c release, and apoptosis in retinal endothelial cells. Rat retinal endothelial cells grown in normal (5 mmol/L) or HG (30 mmol/L) medium and double-stained with MitoTracker Green and tetramethylrhodamine-ethyl-ester-perchlorate were examined live with confocal microscopy. Images were analyzed for mitochondrial shape change using Form Factor and Aspect Ratio values, and membrane potential heterogeneity, using deviation of fluorescence intensity values. Rat retinal endothelial cells grown in normal or HG medium were analyzed for transient changes in oxygen consumption and extracellular acidification using an XF-24 flux analyzer, cytochrome c release by Western blot, and apoptosis by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assay. Rat retinal endothelial cells grown in HG medium exhibited increased mitochondrial fragmentation concurrent with membrane potential heterogeneity. Metabolic analysis showed increased extracellular acidification in HG with reduced steady state/maximal oxygen consumption. Cytochrome c and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling-positive cells were also increased in HG. Thus, HG-induced mitochondrial fragmentation with concomitant increase in membrane potential heterogeneity, reduced oxygen consumption, and cytochrome c release may underlie apoptosis of retinal endothelial cells as seen in diabetic retinopathy.

Topics: Animals; Cells, Cultured; Cytochromes c; Diabetic Retinopathy; Endothelial Cells; Fluorescent Dyes; Glucose; Humans; In Situ Nick-End Labeling; Membrane Potentials; Mitochondria; Rats; Retina

To investigate the expression of the antiapoptotic and proapoptotic markers in diabetic retinas.. In total, 12 donor eyes from six subjects with diabetes mellitus, and 10 eyes from five nondiabetic subjects without known ocular disease serving as control subjects were examined. Immunohistochemical techniques were used with antibodies directed against cyclooxygenase-2 (Cox-2), Akt (protein kinase B), Mcl-1, Bad, cytochrome c, apoptosis-inducing factor (AIF), tumour necrosis factor receptor-1-associated death domain protein (TRADD), and Fas-associated death domain protein (FADD).. In retinas from all subjects without diabetes, cytoplasmic immunoreactivity for the antiapoptotic molecules Cox-2, Akt, and Mcl-1 was noted in ganglion cells. Cytoplasmic immunostaining for Cox-2 was also noted in the retinal pigment epithelial cells. Weak immunoreactivity for the mitochondrial apoptogenic proteins cytochrome c, and AIF was noted in the inner segments of photoreceptors, in the inner one-third of the outer plexiform layer, in cells in the inner nuclear layer, in the inner plexiform layer, and in ganglion cells. There was no immunoreactivity for the other antibodies tested. All diabetic retinas showed de novocytoplasmic immunoreactivity for Bad in ganglion cells, and in occasional cells in the inner nuclear layer. Upregulation of cytochrome cand AIF immunoreactivity was noted. Cox-2, Akt, and Mcl-1 immunoreactivity was not altered in the diabetic retinas. There was no immunoreactivity for TRADD, and FADD.. Ganglion cells in diabetic and nondiabetic retinas express the antiapoptotic molecules Cox-2, Akt, and Mcl-1. Retinal ganglion cells express the proapoptotic molecule Bad in response to diabetes-induced neuronal injury. Diabetic retinas show upregulation of the mitochondrial proteins cytochrome c, and AIF.

Topics: Aged; Apoptosis; Apoptosis Inducing Factor; bcl-Associated Death Protein; Biomarkers; Cyclooxygenase 2; Cytochromes c; Diabetes Mellitus, Type 1; Diabetes Mellitus, Type 2; Diabetic Retinopathy; Fas-Associated Death Domain Protein; Female; Humans; Immunohistochemistry; Male; Middle Aged; Myeloid Cell Leukemia Sequence 1 Protein; Neoplasm Proteins; Proto-Oncogene Proteins c-akt; Proto-Oncogene Proteins c-bcl-2; Retina; TNF Receptor-Associated Death Domain Protein