cytochrome-c-t and Macular-Degeneration

Topics: Antioxidants; Apoptosis Regulatory Proteins; Catalase; Cell Line; Cytochromes c; Drug Evaluation, Preclinical; Epithelial Cells; Gene Expression Regulation; Glutathione; Glutathione Peroxidase; Humans; Hydrogen Peroxide; Lutein; Macular Degeneration; MAP Kinase Signaling System; Molecular Structure; Oxidative Stress; Prodrugs; Reactive Oxygen Species; Retinal Pigment Epithelium

Mitochondria maintain their function by the process of mitochondrial dynamics, which involves repeated fusion and fission. It is thought that the failure of mitochondrial dynamics, especially excessive fission, is related to the progression of several diseases. A previous study demonstrated that mitochondrial fragmentation occurs in the retinal pigmented epithelial (RPE) cells of patients with non-exudative age-related macular degeneration (AMD). We predicted that the suppression of mitochondrial fragmentation offers a novel therapeutic strategy for non-exudative AMD. We investigated whether the inhibition of mitochondrial fission was effective against the oxidative stress-induced damage of ARPE-19 cells. The treatment of ARPE-19 cells with H

Topics: Cells, Cultured; Cytochromes c; Humans; Macular Degeneration; Mitochondria; Mitochondrial Dynamics; Oxidative Stress; Quinazolinones; Reactive Oxygen Species; Retinal Pigment Epithelium

Age-related macular degeneration (AMD) is the primary cause of blindness and severe vision loss in developed countries and is responsible for 8.7% of blindness globally. Ultraviolet radiation can induce DNA breakdown, produce reactive oxygen species, and has been implicated as a risk factor for AMD. This study investigated the effects of UVA radiation on Human retinal pigment epithelial cell (ARPE-19) growth and protein expression.. We detected 29 differentially expressed proteins (9 up-regulated and 20 down-regulated) in the exposed cells. Some of these proteins such as CALR, GRP78, NPM, Hsp27, PDI, ATP synthase subunit alpha, PRDX1, and GAPDH are associated with anti-proliferation, induction of apoptosis, and oxidative-stress protection. We also detected altered protein expression levels among caspases (caspase 3 and 9) and in the mitochondrial (cytosolic cytochrome C, AIF, Mcl-1, Bcl-2, Bcl-xl, Bax, Bad, and p-Bad) and ER stress-related (p-PERK, p-eIF2α, ATF4 and CHOP) apoptotic pathways.. UVA irradiation suppressed the proliferation of ARPE-19 cells in a dose-dependent manner, caused quantitative loses in transmembrane potential (ΔΨm), and induced both early and late apoptosis.

Topics: Apoptosis; Cell Survival; Cells, Cultured; Chromatography, Liquid; Cytochromes c; Endoplasmic Reticulum Chaperone BiP; Humans; Macular Degeneration; Oxidative Stress; Proteomics; Reactive Oxygen Species; Retinal Pigment Epithelium; Signal Transduction; Tandem Mass Spectrometry; Ultraviolet Rays

Age-related macular degeneration (AMD) causes the dysfunction of the retinal pigment epithelial (RPE) cells. In this study, we examined the effects of riluzole, a sustained activator of the TRAAK potassium channel, on human RPE (ARPE-19) cells in an oxidant-induced cell-injury model and elucidate the mechanism of riluzole on RPE cell apoptosis.. The follow four groups of ARPE-19 cells were treated with riluzole and/or tert-butyl hydroperoxide (t-BHP) for 24.0 h: control, t-BHP, riluzole, and t-BHP + riluzole. Cell apoptosis was measured by flow cytometry, and Western blotting was performed to analyze the expression of the weakly inward rectifying potassium (TRAAK) channel. Finally, the mitochondrial membrane potential (Δψm) was detected by flow cytometry, and cytochrome C (Cyt-c) release was assessed by Western blotting.. The viability of the cells in the cotreated group was significantly higher (85.6 ± 3.1%) than that in the t-BHP group (66.2 ± 2.5%). In addition, the cells in the cotreated group had a higher effect on increasing the expression of TRAAK than the t-BHP group. The results also showed that Cyt-c translocation significantly decreased and Δψm increased in the cotreated group.. These results demonstrate that riluzole protects RPE cells from apoptosis. The protection mechanism of riluzole could be from stabilizing mitochondrial Δψm and preventing the release of Cyt-c. Changes in TRAAK expression might also contribute to the protection of RPE cells.

Topics: Antioxidants; Apoptosis; Cell Survival; Cytochromes c; Epithelial Cells; Excitatory Amino Acid Antagonists; Humans; Macular Degeneration; Membrane Potential, Mitochondrial; Potassium Channels; Retinal Pigment Epithelium; Riluzole

Micro(mi)RNAs negatively regulate a wide variety of genes through degradation or posttranslational inhibition of their target genes. The purpose of this study was to investigate the role of miR-23a in modulating RPE cell survival and gene expression in response to oxidative damage.. The expression level of miR-23a was measured in macular retinal pigment epithelial (RPE) cells of donor eyes with aged-related macular degeneration (AMD) and age-matched normal eyes by using qRT-PCR. Cultured human ARPE-19 cells were transfected with miR-23a mimic or inhibitor. Cell viability was assessed by the MTT assay. Apoptosis was determined by incubating cells with hydrogen peroxide (H(2)O(2)) or t-butylhydroperoxide (tBH). Caspase-3 activity and DNA fragmentation were measured by enzyme-linked immunosorbent assays. The protein relevant to apoptosis, such as Fas expression level, was analyzed by Western blot analysis.. miR-23a expression was significantly downregulated in macular RPE cells from AMD eyes. H(2)O(2)-induced ARPE-19 cell death and apoptosis were increased by an miR-23a inhibitor and decreased by an miR-23a mimic. Computational analysis found a putative target site of miR-23a in the 3'UTR of Fas mRNA, which was verified by a luciferase reporter assay. Forced overexpression of miR-23a decreased H(2)O(2) or tBH-induced Fas upregulation, and this effect was blocked by downregulation of miR-23a.. The protection of RPE cells against oxidative damage is afforded by miR-23a through regulation of Fas, which may be a novel therapeutic target in retinal degenerative diseases.

Topics: Aged; Aged, 80 and over; Apoptosis; Apoptosis Inducing Factor; Blotting, Northern; Blotting, Western; Caspase 3; Cell Survival; Cells, Cultured; Cytochromes c; Enzyme-Linked Immunosorbent Assay; fas Receptor; Gene Expression Regulation; Humans; Hydrogen Peroxide; Macular Degeneration; MicroRNAs; Middle Aged; Mitochondria; Oxidants; Oxidative Stress; Retinal Pigment Epithelium; Reverse Transcriptase Polymerase Chain Reaction; tert-Butylhydroperoxide; Tissue Donors; Transfection

Oxidative stress from reactive oxygen species (ROS) has been implicated in many diseases, including age-related macular degeneration (AMD), in which the retinal pigment epithelium (RPE) is considered a primary target. Because manganese superoxide dismutase (SOD2), localized in mitochondria, is known to be a key enzyme that protects the cells against oxidative stress, this study was undertaken to examine oxidation-induced apoptosis in cultured RPE cells with various levels of SOD2.. Primary cultures of RPE cells were established from wild-type (WT), heterozygous Sod2-knockout mouse (HET) and hemizygous Sod2 mice with overexpression of the enzyme (HEMI). Purity of the RPE cell cultures was verified by immunostaining with antibody to RPE65 and quantified by flow cytometry. Oxidative stress was induced in RPE cells by exposing them to H(2)O(2) (0-500 muM) for 1 hour and reculturing them in normal medium for various times (0-24 hours). Apoptosis in the RPE was examined by TUNEL staining and quantified by cell-death-detection ELISA. Mitochondrial transmembrane potential (MTP) was measured by a cationic dye, and cytochrome c leakage from mitochondria was analyzed by Western blot analysis.. More than 95% of the cells in each culture were RPE65 positive, and the relative SOD2 levels in HET, WT, and HEMI cells were 0.6, 1.0, and 3.4, respectively. H(2)O(2)-induced apoptotic cell death was both dose and time dependent, and apoptosis in these cells was related to the cellular SOD2 level. Disruption of MTP and release of cytochrome c were observed to occur before apoptotic cell death, and they correlated with cellular SOD2.. The results demonstrate a critical role of SOD2 in protection against oxidative challenge. Cells from HET mice showed greater apoptotic cell death, whereas in those from HEMI mice, cell death induced by oxidative injury was suppressed.

Topics: Animals; Apoptosis; Blotting, Western; Carrier Proteins; Cell Survival; Cells, Cultured; cis-trans-Isomerases; Cytochromes c; Enzyme-Linked Immunosorbent Assay; Eye Proteins; Female; Flow Cytometry; Hydrogen Peroxide; Immunohistochemistry; In Situ Nick-End Labeling; Macular Degeneration; Male; Membrane Potentials; Mice; Mice, Inbred C3H; Mice, Inbred C57BL; Mice, Knockout; Mitochondria; Oxidants; Oxidative Stress; Pigment Epithelium of Eye; Superoxide Dismutase