MicroRNA-100 Mediates Hydrogen Peroxide-Induced Apoptosis of Human Retinal Pigment Epithelium ARPE-19 Cells.
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ABSTRACT:
SUBMITTER: Chang YS
PROVIDER: S-EPMC8067261 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Since melatonin production has been documented in extrapineal and extraneuronal tissues, we investigated the expression of molecular elements of the melatoninergic system in human RPE cells (ARPE-19). The expression of key enzymes for melatonin synthesis: tryptophan hydroxylases (TPH1 and TPH2); arylalkylamine N-acetyltransferase (AANAT) and hydroxyindole-O-methyltransferase (HIOMT) was detected in ARPE-19 cells using RT-PCR. TPH1 and AANAT proteins were detected in ARPE by Western blotting, while sequential metabolism of tryptophan, serotonin and N-acetylserotonin to melatonin was shown by RP-HPLC. We also demonstrated, by means of RT-PCR, that ARPE expressed mRNA encoding the melatonin receptors: MT2 (but not MT1), two isoforms of nuclear receptor (RORalpha1 and RORalpha4/RZR1), and quinone oxidoreductase (NQO2). By analogy with other peripheral tissues, for example the skin, the expression of these metabolic elements in RPE cells suggests that the RPE represents an additional source of melatonin in the eye, to regulate local homeostasis and prevent from oxidative damage in intra-, auto- and/or paracrine fashions.
| S-EPMC2728552 | biostudies-literature
Project description:BackgroundOxidative damage to retinal pigment epithelial (RPE) cells contributes to the development of age-related macular degeneration, which is among the leading causes of visual loss in elderly people. In the present study, we evaluated the protective role of triphenylphosphonium (TPP)-Niacin against hydrogen peroxide (H2O2)-induced oxidative stress in RPE cells.MethodsThe cellular viability, lactate dehydrogenase release, reactive oxygen species (ROS) generation, and mitochondrial function of retinal ARPE-19 cells were determined under treatment with H2O2 or pre-treatment with TPP-Niacin. The expression level of mitochondrial related genes and some transcription factors were assessed using real-time polymerase chain reaction (RT-qPCR).ResultsTPP-Niacin significantly improved cell viability, reduced ROS generation, and increased the antioxidant enzymes in H2O2-treated ARPE-19 cells. Mitochondrial dysfunction from the H2O2-induced oxidative stress was also considerably diminished by TPP-Niacin treatment, along with reduction of the mitochondrial membrane potential (MMP) and upregulation of the mitochondrial-associated gene. In addition, TPP-Niacin markedly enhanced the expression of transcription factors (PGC-1α and NRF2) and antioxidant-associated genes (especially HO-1 and NQO-1).ConclusionWe verified the protective effect of TPP-Niacin against H2O2-induced oxidative stress in RPE cells. TPP-Niacin is believed to protect against mitochondrial dysfunction by upregulating antioxidant-related genes, such as PGC-1α, NRF2, HO-1, and NQO-1, in RPE cells.
| S-EPMC7819191 | biostudies-literature
Project description:Age-related macular degeneration (AMD) is a leading cause of blindness and progressive loss of central vision in the elderly population. The important factor of AMD pathogenesis is the degeneration of retinal pigment epithelial (RPE) cells by oxidative stress. Inactivation of PTEN can disrupt intercellular adhesion in the RPE cells, but the mechanism of oxidative stress is less known. Here we presented evidence that UVB-mediated oxidative stress induced apoptosis in ARPE-19 cells. Downregulation of the expression of PTEN in UVB-irradiative RPE cells triggered DNA damage and increased the level of UVB-induced apoptosis by activating p53-dependent pathway. However, overexpression of PTEN increased cell survival by suppressing p-H2A in response to DNA damage and apoptosis. When using Pifithrin-α (one of p53 inhibitors), the level of p53-dependent apoptosis was significantly lower than untreated, which suggested that p53 was possibly involved in PTEN-dependent apoptosis. Thus, it elucidated the molecular mechanisms of UVB-induced damage in RPE cells and may offer an alternative therapeutic target in dry AMD.
| S-EPMC5340936 | biostudies-literature
Project description:PurposeMicroRNAs (miRNAs) are important regulators of many cellular functions due to their ability to target mRNAs for degradation or translational inhibition. Previous studies have reported that the expression of microRNA-9 (miR-9) is regulated by retinoic acid and reactive oxygen species (ROS). We have previously shown that N-(4-hydroxyphenyl)-retinamide (4HPR), a retinoic acid derivative, induces ROS generation and apoptosis in cultured human retinal pigment epithelial (RPE) cells, known as ARPE-19 cells. The aim of the present study was to investigate the expression of miR-9 in ARPE-19 cells in response to 4HPR treatment, and to identify other miRNAs normally expressed in these cells.MethodsARPE-19 cells in culture were treated with 4HPR, the total RNA fractions were isolated, and the expression of various miRNAs and mRNAs was analyzed using real-time PCR. The miRNA expression profile of ARPE-19 cells was analyzed using microarray hybridization.ResultsTreatment of ARPE-19 cells with 4HPR resulted in apoptosis characterized by the increased expression of HMOX1 and GADD153 genes. A twofold increase in the expression of miR-9 was also observed during this response. Potential binding sites for the transcription factors encoded by CEBPA and CEBPB genes were found to be present in the putative promoter regions of all three genes encoding miR-9. 4HPR-induced miR-9 expression was associated with parallel increases in the expression of these transcription factor genes. 5-Aza-2'-deoxycytidine, a methyl transferase inhibitor, also increased the expression of miR-9 in ARPE-19 cells. Microarray hybridization analysis identified let-7b, let-7a, miR-125b, miR-24, miR-320, miR-23b, let-7e, and let-7d as the most abundant miRNAs normally expressed in ARPE-19 cells. These miRNAs are known to regulate cell growth, differentiation or development. The 4HPR treatment increased the expression of miR-16, miR-26b, miR-23a, and miR-15b in ARPE-19 cells, although these increases were modest when compared to the increase in the expression of miR-9.ConclusionsOur studies demonstrate that miR-9 is expressed in the RPE cell line ARPE-19, and its expression is increased by a retinoic acid derivative and by an inhibitor of promoter hypermethylation. Several miRNAs with inherent ability to regulate cell growth, differentiation and development are also normally expressed in ARPE-19 cells. Thus, miR-9 and other miRNAs could be important in maintaining RPE cell function.
| S-EPMC2925906 | biostudies-literature
Project description:PURPOSE: The aim of this study was to characterize the arylsulfatase I (ARSI) gene that has been shown to be preferentially expressed in the human retinal pigment epithelium cell line ARPE-19 and to propose it as a candidate gene responsible for inherited eye diseases such as retinitis pigmentosa (RP). METHODS: Full-length cDNA clones encoding ARSI, arylsulfatase A (ARSA), and sulfatase modifying factor 1 (SUMF1) were isolated from ARPE-19 cDNA libraries constructed using the vector-capping method. The expression vectors for their FLAG-tagged proteins were transfected into ARPE-19 cells, and the expression products were characterized by western blot analysis and arylsulfatase assay. The entire region of the ARSI gene locus was sequenced using the genomic DNA samples of 68 RP patients. RESULTS: Transiently produced ARSI-FLAG was localized to the endoplasmic reticulum and was detected in the cellular fraction and the medium. When ARSI-FLAG and SUMF1-FLAG were coexpressed, the conditioned medium of the transfected cells showed arylsulfatase activity at a range of neutral pH. No mutation was found in the ARSI gene locus of the RP patients examined. CONCLUSIONS: ARSI may be a secreted sulfatase and may function in the extracellular space. Although ARSI may not be a causative gene for lysosomal storage diseases, preferentially expressed in the eye, ARSI would be a candidate gene causing inherited eye diseases for future mutation screening.
| S-EPMC2650720 | biostudies-literature
Project description:Age-related macular degeneration (AMD) is a major cause of severe and irreversible vision loss with limited effective therapies. Diminished autophagy and increased oxidative damage caused by ROS in the retinal pigment epithelium (RPE) have been implicated in the pathogenesis of AMD, and strategies aimed at enhancing autophagy are likely to protect these cells from oxidative damage. We have previously shown that berberine (BBR), an isoquinoline alkaloid isolated from Chinese herbs, was able to protect human RPE cells from H2O2-induced oxidative damage through AMPK activation. However, the precise mechanisms behind this protective effect remain unclear. Given the essential role of AMPK in autophagy activation, we postulated that BBR may confer protection against H2O2-induced oxidative damage by stimulating AMPK-dependent autophagy. Our results showed that BBR was able to induce autophagy in D407 cells, whereas autophagy inhibitor PIKIII or silencing of LC3B blocked the protective effect of BBR. Further analysis showed that BBR activated the AMPK/mTOR/ULK1 signaling pathways and that both pharmacological and genetic inhibitions of the AMPK pathway abolished the autophagy-stimulating effect of BBR. Similar results were obtained in primary cultured human RPE cells. Taken together, these results demonstrate that BBR is able to stimulate autophagy in D407 cells via the activation of AMPK pathway and that its protective effect against H2O2-induced oxidative damage relies on its autophagy-modulatory effect. Our findings also provide evidence to support the potential application of BBR in preventing and treating AMD.
| S-EPMC8378965 | biostudies-literature
Project description:PURPOSE: To study the effect of subtoxic levels of hydrogen peroxide (H(2)O(2)) on the expression and release of interleukin-6 (IL-6) by cultured retinal pigment epithelial (RPE) cells and to explore the relevant signal pathways. METHODS: Cultured human RPE cells were stimulated with various subtoxic concentrations of H(2)O(2) for different periods. Conditioned medium and cells were collected. IL-6 in the medium and IL-6 mRNA in the collected cells were measured using an IL-6 enzyme-linked immunosorbent assay kit and reverse transcriptase polymerase chain reaction, respectively. Nuclear factor-kappaB (NF-?B) in nuclear extracts and phosphorylated p38 mitogen-activated protein kinase (MAPK), extracellular signal-regulated kinase (ERK), and c-Jun N-terminal kinases (JNK) in cells cultured with and without H(2)O(2) were measured by NF-?B and MAPK enzyme-linked immunosorbent assay kits. Inhibitors of p38 (SB203580), ERK (UO1026), JNK (SP600125), and NF-?B (BAY11-7082) were added to the cultures before the addition of H(2)O(2) to test their effects(.) RESULTS: Subtoxic levels of H(2)O(2) (100 µM and less) increased the IL-6 mRNA level and the release of IL-6 protein by the cultured human RPE cells in a dose- and time-dependent manner. This was accompanied by an increase of NF-?B in nuclear extracts and phosphorylated p38 MAPK, ERK, and JNK in cell lysates, particularly in the p38 and NF-?B. The NF-?B inhibitor decreased the H(2)O(2)-induced expression of IL-6. The p38 inhibitor, but not the ERK or JNK inhibitor, completely abolished H(2)O(2)-induced expression of IL-6 by RPE cells. The p38 inhibitor also abolished the increase of NF-?B in nuclear extracts in cells treated with H(2)O(2). CONCLUSIONS: H(2)O(2) stimulated the production of IL-6, a key factor in the modulation of immune responses, inflammatory processes, and the occurrence of autoimmune diseases, which recently has been documented to be increased in age-related macular degeneration (AMD). This may be a molecular linkage for the oxidative stress and inflammatory/autoimmune reactions in AMD and may provide a novel target for the treatment of AMD.
| S-EPMC2956668 | biostudies-literature
Project description:Background:Proliferative vitreoretinopathy (PVR) is a severe blinding complication of retinal detachment surgery. Increasing evidence demonstrate that PVR is associated with oxidative stress. Scutellarin is a natural flavone compound that has been reported to have anti-oxidative activity. However, the effect of scutellarin on PVR remains unknown. In the current study, we assessed the effect of scutellarin on hydrogen peroxide (H2O2)-induced oxidative injury in human retinal pigment epithelium cells (ARPE-19). Methods:ARPE-19 cells were pretreated with different concentrations of scutellarin for 2 h, and then challenged with H2O2 (1 mM) for 24 h. The levels of reactive oxygen species (ROS), malondialdehyde (MDA) and superoxide dismutase (SOD) and glutathione (GSH) activity were measured to assess the level of oxidative stress. Flow cytometry was performed to detect the apoptosis rate of ARPE-19 cells. Expression levels of bcl-2, bax, cleaved-caspase-3, p-JAK2, JAK2, p-STAT3, and STAT3 were measured using western blot. Results:Our results revealed that scutellarin improved the cell viability of H2O2-induced ARPE-19 cells. Scutellarin alleviated the H2O2-induced oxidative stress in ARPE-19 cells, which was illustrated by reduced levels of ROS and MDA, accompanied by increased SOD activity and GSH level. The increased apoptosis rate of ARPE-19 cells caused by H2O2 induction was significantly decreased after scutellarin treatment. H2O2 treatment resulted in significant increase in bax expression and decrease in bcl-2 expression, while the changes in the expressions of bax and bcl-2 were reversed by scutellarin treatment. In addition, scutellarin promoted the activation of JAK2/STAT3 signaling pathway in H2O2-induced ARPE-19 cells. Suppression of JAK2/STAT3 signaling pathway abolished the protective effects of scutellarin on H2O2-induced ARPE-19 cells. Conclusion:These findings suggested that scutellarin was capable for alleviating H2O2-induced oxidative damage in ARPE-19 cells, which might be ascribed to the activation of JAK2/STAT3 signaling pathway.
| S-EPMC6341765 | biostudies-literature
Project description:Antibody-mediated rejection is characterized by donor-specific antibody produced by B cells. However, to our knowledge, B cell invasion and antibody in the inflamed retina after transplantation of retinal pigment epithelial (RPE) cells has not been reported. To determine if RPE transplantation could be performed using allografts, we established in vivo immune rejection models with induced pluripotent stem cell (iPSC)-RPE allografts and determined whether RPE-specific antibody could be detected in these models. We detected alloantibodies in the serum from recipient monkeys that had immune attacks in the retina in an immunofluorescent assay using the transplanted iPSC-RPE cells as the antigen. In addition to T cell and antigen-presenting cell immunity, peripheral blood cells and lymph nodes in animal models with allogeneic iPSC-RPE cells also had activated B cells, which were probably secreting alloantibodies. Using serum and transplanted cells, alloreactive antibody can be detected for the diagnosis of immune rejection after transplantation.
| S-EPMC5688231 | biostudies-literature
Project description:Lipofuscin accumulates with age in the retinal pigment epithelium (RPE) in discrete granular organelles and may contribute to age-related macular degeneration. Because previous studies suggest that lipofuscin contains protein that may impact pathogenic mechanisms, we pursued proteomics analysis of lipofuscin. The composition of RPE lipofuscin and its mechanisms of pathogenesis are poorly understood in part because of the heterogeneity of isolated preparations. We purified RPE lipofuscin granules by treatment with proteinase K or SDS and showed by light, confocal, and transmission electron microscopy that the purified granules are free of extragranular material and associated membranes. Crude and purified lipofuscin preparations were quantitatively compared by (i) LC MS/MS proteomics analyses, (ii) immunoanalyses of oxidative protein modifications, (iii) amino acid analysis, (iv) HPLC of bisretinoids, and (v) assaying phototoxicity to RPE cells. From crude lipofuscin preparations 186 proteins were identified, many of which appeared to be modified. In contrast, very little protein ( approximately 2% (w/w) by amino acid analysis) and no identifiable protein were found in the purified granules, which retained full phototoxicity to cultured RPE cells. Our analyses showed that granules in purified and crude lipofuscin preparations exhibit no statistically significant differences in diameter or circularity or in the content of the bisretinoids A2E, isoA2E, and all-trans-retinal dimer-phosphatidylethanolamine. The finding that the purified granules contain minimal protein yet retain phototoxic activity suggests that RPE lipofuscin pathogenesis is largely independent of associated protein. The purified granules also exhibited oxidative protein modifications, including nitrotyrosine generated from reactive nitrogen oxide species and carboxyethylpyrrole and iso[4]levuglandin E(2) adducts generated from reactive lipid fragments. This finding is consistent with previous studies demonstrating RPE lipofuscin to be a potent generator of reactive oxygen species and supports the hypothesis that such species, including reactive fragments from lipids and retinoids, contribute to the mechanisms of RPE lipofuscin pathogenesis.
| S-EPMC2493379 | biostudies-literature