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- Karlsson, Jan-Olof, 1944, et al.
(författare)
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Acute Effects of the Sigma-2 Receptor Agonist Siramesine on Lens Epithelial Cells
- 2007
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Ingår i: Invest. Ophthalmol. Vis. Sci.. ; 48:5, s. 2432-
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Konferensbidrag (refereegranskat)abstract
- PurposeExperiments were carried out to study the effects of siramesine on markers for apoptosis, oxidative damage and mitochondrial function in primary cultures of human lens epithelial cells (HLEC). MethodsHLEC were incubated with 25 {micro}M siramesine for 1, 2, 3, 4, 6 and 8 hours. Caspase-3 was assayed in cell extracts with DEVD-AMC. Mitochondrial depolarization was assayed with JC-1. Peroxide production was studied with DCF-DA and superoxide levels with hydroethidium. Glutathione levels were assayed with monochlorobimane. ResultsSiramesine induced a significant increase of caspase-3 activity after 6h exposure to 25 {micro}M siramesine. Nuclear morphology examined with Hoechst 33342 showed signs of apoptosis after the same time intervals. A significant increase in the production of peroxide and superoxide were found up to 4 -8 hours after administration of siramesine. ConclusionsSiramesine, a piperidine analogue, was developed for the treatment of psychiatic disorders and is considered to be relatively nontoxic. This study, and others, indicate effects on cell growth, apoptosis and production of ROS. The sigma-2 receptor may be a regulator of HLEC growth and apoptosis.
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- Karlsson, Jan-Olof, 1944, et al.
(författare)
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Inhibition of Glycogen Synthase Kinase (GSK-3) Protects Against Oxidative Stress and Attenuates Apoptosis in Human Lens Epithelial Cells and the Mouse Lens in Organ Culure
- 2005
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Ingår i: Invest. Ophthalmol. Vis. Sci.. ; 46:5, s. 3867-
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Konferensbidrag (refereegranskat)abstract
- Purpose: GSK-3 may regulate Wnt signaling, gene expression, the cell cycle, cell differentiation and apoptosis. Inhibition of GSK-3 can be obtained via the structurally unrelated substances lithum or Kenpaullone. The lens and the lens epithelial cells are excellent models to study the role of this enzyme. Methods: Primary cultures of human lens epithelial cells (HLEC) or the mouse lens in organ culture were exposed to the GSK-3 inhibitors lithium (2 mM) or Kenpaullone (2 {micro}M) for times upp to 24h.The cells were, before or after treatment, placed in medium containing fluorogenic indicators of oxidative damage. DCFH-DA was used to assay peroxides, mitochondrial function was evaluated with Rhodamine 123 and JC-1, monochlorobimane was used to assay intracellular glutathione (GSH) levels, Proteolytic activities were assayed, on line, with cell-permeable fluorogenic substrates.Proteasome and calpain activities were assayed with LLVY-AMC, Cathepsin B with RR-AMC or FR-AMC. Metalloproteases were assayed with AAF-AMC. Caspase-3, 8 and 9 were assayed in cell extracts with DEVD-, IETD- or LEHD-AMC, respectively. Results: The mitochondrial membrane potential and the level of GSH increased by 10% after treatment of HLEC with Li or Kenpaullone for 24h. No change was observed for peroxide production. The basal (low) level of caspase-3 activity was decreased by at least 20%. No significant effects were found concerning caspase-8 or 9 activities. No effect was observed on the activity of calpain, the proteasome, metalloproteases and cathepsin D/E activity.The whole mouse lens in organ culture showed essentially the same elevated mitochondrial potential. The GSH increase was even more evident in the whole lens preparation. Conclusions: Inhibition of GSK-3 may protect against oxidative stress (and cataract) via prevention of MPT induction and attenuate apoptosis in HLEC.
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- Petersen, Anne, 1962, et al.
(författare)
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Effects Of Dexamethasone In The Lens
- 2006
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Ingår i: Invest. Ophthalmol. Vis. Sci.. ; 47:5, s. 4110-
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Konferensbidrag (refereegranskat)abstract
- Purpose: The aim of the study was to investigate effects of glucocorticoids in the lens. Methods: Lens epithelial cells (HLEC) were exposed to dexamethasone for 24 hours. Cells were assayed for changes in superoxide production using dihydroethidium (HET), for alterations in peroxide production using DCFH-DA or for GSH variations using monochlorobimane (MCB). Apoptosis was determined by Caspase-3 assay and by nuclear morphology of Hoechst stained cells. Mitochondria depolarisation was measured using the potential-sensitive colour JC-1. Morphology was examined by transmission electron microscopy (TEM). Results: Apoptosis were increased in HLEC exposed to 1, 10, 100 and 1000 {micro}M dexamethasone as revealed by nuclear morphology studies. Caspase-3 activity was increased at 100 and 1000 {micro}M dexamethasone. No effect on GSH, superoxide or peroxide production by dexamethasone was present. High concentrations of dexamethasone (1000 {micro}M) depolarised the mitochondria. TEM showed multilayering of cells, mitochondrial changes and accumulation of membrane delimited electron dense material. Conclusions: The mechanism underlying dexamethasone induced apoptosis and morphological changes in HLEC are probably not due to oxidative effects.
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- Petersen, Anne, 1962, et al.
(författare)
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Effects of dexamethasone on human lens epithelial cells in culture
- 2008
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Ingår i: Mol Vis. - 1090-0535. ; 14, s. 1344-52
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Tidskriftsartikel (refereegranskat)abstract
- PURPOSE: Treatment with glucocorticoids is a well known risk factor for cataract development, although the pathogenic mechanism has not been elucidated. The aim of the study was to investigate the effects of glucocorticoids in cultured human lens epithelial cells. METHODS: Human lens epithelial cells (HLECs) were exposed to dexamethasone for 24 h. The number of viable cells was determined using the 3-[4, 5-dimethylthiazolyl-2]-2, 5-diphenyltetrazolium bromide (MTT) assay, and proliferation was quantified using Ki-67. Apoptosis was investigated by measuring caspase-3 activity and by evaluating nuclear morphology of cells stained with Hoechst 33342. Mitochondria depolarization was measured using the potential-sensitive color, JC-1. Cells were assayed for changes in superoxide production using dihydroethidium (HET), for alterations in peroxide production using dichlorofluorescein diacetate (DCFH-DA), and for glutathione (GSH) variations using monochlorobimane (MCB). Caspase-3 activity was also measured in HLECs simultaneously exposed to dexamethasone and the glucocorticoid antagonist, RU486. RESULTS: Low doses of dexamethasone (0.1 microM) resulted in increased proliferation of HLECs. Apoptosis was increased in HLECs exposed to 1 microM, 10 microM, and 100 microM of dexamethasone as revealed by nuclear morphology studies. Apoptosis was also confirmed by measuring caspase-3 activation. No effect on superoxide production by dexamethasone was seen. There were no effects on GSH levels or mitochondrial depolarization either. Only the highest concentration of dexamethasone (100 microM) caused an increase in peroxide production. In HLECs incubated with the glucocorticoid antagonist, RU486, apoptosis was induced at a lower concentration of dexamethasone (0.1 microM) than with dexamethasone alone. CONCLUSIONS: Low doses of dexamethasone cause a moderate increase in proliferation of cultured HLECs. Slightly higher but still physiologically relevant concentrations of dexamethasone result in a dose-dependent increase in apoptosis. Dexamethasone-induced apoptosis in HLECs does not seem to involve oxidative mechanisms. The proapoptotic effect of dexamethasone does not appear to act through the glucocorticoid receptor. Effects on proliferation and/or dysregulation of apoptosis in lens epithelial cells may be an important factor in human steroid-induced posterior subcapsular cataract.
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- Petersen, Anne, 1962, et al.
(författare)
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Intracellular effects of NSAIDs/ASA in oxidatively stressed human lens epithelial cells in culture.
- 2008
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Ingår i: Ophthalmic research. - : S. Karger AG. - 1423-0259 .- 0030-3747. ; 40:2, s. 77-85
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Tidskriftsartikel (refereegranskat)abstract
- The aim of the study was to examine the effects of nonsteroidal anti-inflammatory drugs (NSAIDs)/acetylsalicylic acid (ASA) on human lens epithelial cells (HLECs) during oxidative stress. HLECs were exposed to H2O2 in the absence or presence of indomethacin, diclofenac, celecoxib (NSAIDs) or ASA for 24 h. HLECs were assayed for changes in superoxide and peroxide production and for variations in glutathione. Mitochondrial depolarization was measured using the membrane potential-sensitive dye JC-1. The results of the study include reduction in superoxide and peroxide production as well as reduction in glutathione depletion in oxidatively stressed HLECs incubated with low concentrations of NSAIDs/ASA. However, no protection against H2O2-induced mitochondrial depolarization by NSAIDs/ASA could be seen. In conclusion, NSAIDs/ASA display reactive oxygen species-scavenging properties in H2O2-exposed HLECs in culture.
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| 9. |
- Petersen, Anne, 1962, et al.
(författare)
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Potential Protective Effects of NSAIDs/ASA in Oxidatively Stressed Human Lens Epithelial Cells and Intact Mouse Lenses in Culture
- 2005
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Ingår i: Ophthalmic Res. - : S. Karger AG. - 0030-3747. ; 37:6, s. 318-327
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Tidskriftsartikel (refereegranskat)abstract
- Purpose: To study possible toxic effects of indomethacin, diclofenac, and celecoxib (NSAIDs) and acetylsalicylic acid (ASA) as well as potentially protective effects of these substances in oxidatively stressed human lens epithelial cells (HLEC) and in intact mouse lenses in culture. Methods: HLEC and mouse lenses were incubated with NSAIDs or ASA alone or in the presence of H(2)O(2). To study apoptosis the cells were then either stained with Hoechst 33342 or assayed for caspase-3 activity. Mouse lenses were studied with respect to lens transparency. Results: Low concentrations of NSAIDs/ASA caused a significant protection against H(2)O(2)-induced apoptosis in HLEC whereas higher concentrations were toxic. Conclusion: The protective effects of NSAIDs/ASA against oxidative damage are confined to a relatively small therapeutic window. Copyright (c) 2005 S. Karger AG, Basel.
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