cytochrome-c-t and Retinal-Diseases

Retinal ischemia plays a central role in several retinal diseases. The pathogenesis of retinal ischemia involves changes in gene expression. Valproic acid (VPA), a broad-spectrum histone deacetylase inhibitor, is an anticonvulsant and mood-stabilizing drug with neuroprotective effects. Here, we investigated whether VPA protects the retina and optic nerve axon from ischemic damage in a rat model and determined a possible protective mechanism. Adult male Wistar rats were randomized into sham, ischemia/reperfusion (I/R)-plus-vehicle, and I/R-plus-VPA groups. Rats received subcutaneous injections of 300 mg/kg VPA or phosphate-buffered saline twice a day after retinal ischemia induced by acute high intraocular pressure. Twenty-four hours after I/R, retinal neuron apoptosis was evaluated using the TUNEL assay. The expression of heat-shock protein 70 (Hsp70), activated-caspase-3, and apoptotic-protease-activating factor-1 (apaf-1), acetylation levels of histone H3, release of cytochrome c, and interaction between Hsp70 and apaf-1 were analyzed by immunoblotting analysis in all groups; the transcriptional activation of the Hsp70 gene and interaction between the Hsp70 promoter with p300 or HDAC1 were analyzed using chromatin immunoprecipitation assay. Seven days after I/R, the histological changes in the retina were evaluated using hematoxylin and eosin staining, and optic nerve axon damage was evaluated using toluidine blue staining and transmission electron microscopy. The density of retinal ganglion cells (RGCs) was analyzed using Fluoro-Gold retrograde labeling at 7, 14, 21 days after I/R. VPA markedly attenuated I/R-induced retinal neuron apoptosis, damage to RGCs, and morphological injury to the retina and optic nerve axons. VPA resulted in the upregulation of Hsp70 and hyperacetylation of histone H3, accompanied by Hsp70 promoter hyperacetylation, which may result from increased p300 recruitment to the Hsp70 promoter. Furthermore, VPA increased the binding between Hsp70 and apaf-1 to block apoptosome formation and reduced the release of cytochrome c and activation of caspase-3 in the retina after I/R. Therefore, VPA-mediated neuroprotection against I/R injury in the retina may involve cytoprotective Hsp70 induction via transcriptional activation and inhibition of the mitochondria-mediated apoptosis pathway.

Topics: Acetylation; Animals; Apoptosis; Apoptotic Protease-Activating Factor 1; Axons; Caspase 3; Cytochromes c; Histone Deacetylase Inhibitors; Histone Deacetylases; Histones; HSP70 Heat-Shock Proteins; In Situ Nick-End Labeling; Injections, Subcutaneous; Male; Neuroprotective Agents; Rats; Rats, Wistar; Reperfusion Injury; Retinal Diseases; Retinal Ganglion Cells; Transcriptional Activation; Valproic Acid

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

To determine whether intraocular pressure (IOP) elevation alters OPA1 expression and triggers OPA1 release, as well as whether the uncompetitive N-methyl-d-aspartate (NMDA) glutamate receptor antagonist memantine blocks OPA1 release and subsequent apoptotic cell death in glaucomatous DBA/2J mouse retina.. Preglaucomatous DBA/2J mice received memantine (5 mg/kg, intraperitoneal injection, twice daily for 3 months) and IOP in the eyes was measured monthly. RGC loss was counted after FluoroGold labeling. OPA1, Dnm1, Bcl-2, and Bax mRNA were measured by qPCR. OPA1 protein was assessed by immunohistochemistry and Western blot. Apoptotic cell death was assessed by TUNEL staining.. Memantine treatment significantly increased RGC survival in glaucomatous DBA/2J mice and increased the 75-kDa OPA1 isoform, but did not alter the 80- and 90-kDa isoforms. The isoforms of OPA1 were significantly increased in the cytosol of the vehicle-treated glaucomatous retinas but were significantly decreased in memantine-treated glaucomatous retinas. OPA1 immunoreactivity was decreased in the photoreceptors of both vehicle- and memantine-treated glaucomatous retinas, but was increased in the outer plexiform layer of only the memantine-treated glaucomatous retinas. Memantine blocked apoptotic cell death in the GCL, increased Bcl-2 gene expression, and decreased Bax gene expression.. OPA1 release from mitochondria in glaucomatous mouse retina is inhibited by blockade of glutamate receptor activation. Because this OPA1 effect was accompanied by increased Bcl-2 expression, decreased Bax expression, and apoptosis blockade, glutamate receptor activation in the glaucomatous retina may involve a distinct mitochondria-mediated cell death pathway.

Topics: Animals; Apoptosis; bcl-2-Associated X Protein; Blotting, Western; Cell Survival; Cytochromes c; Dynamin I; Excitatory Amino Acid Antagonists; Female; Gene Expression; Glaucoma; GTP Phosphohydrolases; Immunohistochemistry; In Situ Nick-End Labeling; Injections, Intraperitoneal; Intraocular Pressure; Memantine; Mice; Mice, Inbred C57BL; Mice, Inbred DBA; Mitochondria; Polymerase Chain Reaction; Proto-Oncogene Proteins c-bcl-2; Receptors, N-Methyl-D-Aspartate; Retinal Diseases; Retinal Ganglion Cells; RNA, Messenger

Autoantibodies against recoverin, a Ca2+-binding protein found in patients with cancer-associated retinopathy (CAR syndrome), penetrate retinal cells and induce their apoptosis via a mitochondrial pathway. The goal of this study was to investigate whether the entry of anti-recoverin antibody into E1A.NR3 retinal cells causes a change in intracellular Ca2+. Intracellular Ca2+ was measured using the Ca2+-sensitive fluorescent dye Fura-2 AM in living E1A.NR3 retinal cells treated with anti-recoverin antibody Rec-1, patients' autoantibodies, and control rat and human IgG. The exposure of retinal cells to Rec-1 antibody and to the CAR patients' autoantibodies in vitro caused a significant increase in intracellular Ca2+, while non-specific antibodies did not induce such an effect. Co-treatment of the E1A.NR3 cells with Rec-1 in the presence of nifedipine, a L-type Ca2+ channel blocker, significantly suppressed the increase of Ca2+. Treatment with nifedipine also blocked changes in the anti-apoptotic protein bcl-xL and in expressions of the pro-apoptotic protein bax. Nifedipine-treated cells also showed a decrease in cytosolic cytochrome c release and a decrease in caspase 3 activation, compared to cells treated only with Rec-1 antibody. The increase in the antibody-induced Ca2+ is at least in part dependent on extracellular Ca2+. Nifedipine was found to inhibit the entry of Ca2+ into the cells and to protect them from Rec-1-induced apoptosis. Increased levels of intracellular Ca2+ may lead to retinal dysfunction and degeneration in the CAR syndrome. Our results provide a molecular basis for the use of Ca2+ blockers in the treatment of the CAR syndrome.

Topics: Animals; Antibodies, Monoclonal; Apoptosis; Autoantibodies; bcl-2-Associated X Protein; bcl-X Protein; Calcium; Calcium Channel Blockers; Calcium-Binding Proteins; Cell Line; Cytochromes c; Humans; Neoplasms; Nifedipine; Rats; Recoverin; Retina; Retinal Diseases; Syndrome

Oxidative stress is increased in the retina in diabetes, and antioxidants inhibit activation of caspase-3 and the development of retinopathy. The purpose of this study was to investigate the effect of diabetes on the release of cytochrome c from mitochondria and translocation of Bax into mitochondria in the rat retina and in the isolated retinal capillary cells.. Mitochondria and cytosol fractions were prepared from retina of rats with streptozotocin-induced diabetes and from the isolated retinal endothelial cells and pericytes incubated in 5 or 20 mM glucose medium for up to 10 days in the presence of superoxide dismutase (SOD) or a synthetic mimetic of SOD (MnTBAP). The release of cytochrome c into the cytosol and translocation of the proapoptotic protein Bax into the mitochondria were determined by the Western blot technique and cell death by caspase-3 activity and ELISA assay.. Diabetes of 8 months' duration in rats increased the release of cytochrome c into the cytosol and Bax into the mitochondria prepared from the retina, and this phenomenon was not observed at 2 months of diabetes. Incubation of isolated retinal capillary cells with 20 mM glucose increased cytochrome c content in the cytosol and Bax in the mitochondria, and these abnormalities were accompanied by increased cell apoptosis. Inclusion of SOD or its mimetic inhibited glucose-induced release of cytochrome c, translocation of Bax, and apoptosis.. Retinal mitochondria become leaky when the duration of diabetes is such that capillary cell apoptosis can be observed; cytochrome c starts to accumulate in the cytosol and Bax into the mitochondria. Inhibition of superoxides inhibits glucose-induced release of cytochrome c and Bax and inhibits apoptosis in both endothelial cells and pericytes. Identifying the mechanism by which retinal capillary cells undergo apoptosis may reveal novel therapies to inhibit the development of retinopathy in diabetes.

Topics: Animals; bcl-2-Associated X Protein; Blotting, Western; Capillaries; Cell Death; Cell Membrane Permeability; Cytochromes c; Cytosol; Diabetes Mellitus, Experimental; Endothelium, Vascular; Enzyme-Linked Immunosorbent Assay; Male; Mitochondrial Diseases; Pericytes; Protein Transport; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Rats; Rats, Wistar; Retinal Diseases; Retinal Vessels