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Glaucoma, a neurodegenerative disease characterized by progressive death of retinal ganglion cells (RGCs) and chronic axonal degeneration, is often associated with elevated intraocular pressure.Autophagy, which means self-eating, is a mechanism of cell degradation and recycling.Excessive autophagy or impaired autophagy may lead to cell dysfunction and even cell death.Recent studies have shown that autophagy is closely related to trabecular meshwork cell dysfunction, RGCs apoptosis and optic nerve degeneration caused by different factors, including oxidative stress, mechanical stimulation, high intraocular pressure, axon injury, genetic factors and so on.Regulating autophagy to protect RGCs may provide new ideas for glaucoma treatments.In this article, the definition and classification of autophagy, the regulation of autophagy, the role of autophagy in the process of oxidative stress and mechanical stimulation on the function of trabecular meshwork cells, and the impact of autophagy on RGCs under high intraocular pressure and RGCs axonal injury, the relationship between autophagy and apoptosis in RGCs, as well as the latest research results on autophagy and hereditary optic nerve degeneration, regulation of autophagy via gene and drug for the treatment of glaucoma were reviewed.
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@#<p>Glaucoma is a common irreversible blinding eye disease, pathological elevated intraocular pressure is the main clinical feature. The formation of intraocular pressure, related to aqueous circulation, will be pathologically elevated when the aqueous cycle is abnormal. Trabecular network, which plays a key role in maintaining normal intraocular pressure, is the main component of aqueous outflow channel. Imbalance of oxidative stress manifested as oxidation and antioxidant effects is a direct risk factor for elevated intraocular pressure in glaucoma. When it comes to the trabecular meshwork cells, a series of changes such as deposition and degeneration of extracellular matrix, autophagy and aging will eventually occur, and finally the dysfunction of trabecular meshwork cells and increased aqueous outflow resistance, causing intraocular pressure pathological elevated. In this paper, we reviewed the research progress on the relationship between oxidative stress in trabecular meshwork cells and the pathogenesis of glaucoma, in order to provide evidence for further research and reference for exploring the pathogenesis, prevention and treatment of glaucoma.
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Objective To explore the role of forkhead box F2 (FoxF2) in the extracellular matrix of trabecular meshwork.Methods The cultured human trabecular meshwork cells (HTMCs) were divided into Scramble control group and FoxF2 small hairpin RNA (shRNA) group,then FoxF2 shRNA,the FoxF2 restructuring interference carrier was built,HTMCs were infected with FoxF2 shRNA lentivirus.Western blot assay was used to detect the expression of FoxF2 protein and extracellular matrix.Furthermore,Transwell counting experiment was used to analyze the migration ability of HTMCs.Results The cultured HTMCs grew well and showed a long spindle shape.The growth status of HTMCs was well,and their morphological characteristics were consistent with the HTMCs in vivo.The relative expression level of FoxF2 protein in the FoxF2 shRNA group was lower than that in the Scramble control group,with a significant difference between them (0.72 ± 0.02 vs.1.27 ± 0.05;t =16.68,P ＜ 0.01).The relative expression level of fibronectin (FN),collagen type Ⅰ (COL Ⅰ) and α-smooth muscle actin (α-SMA) were 0.43±0.03,0.53 ±0.08 and O.86±0.15 in the FoxF2 shRNA group,and 0.87±0.04,1.66±0.06 and 1.73 ±0.13 in the Scramble control group,respectively,the relative expression levels of FN,COL Ⅰ and α-SMA in the FoxF2 shRNA group were significantly lower than those in the Scramble control group (t =15.08,18.81,7.50,all at P＜0.01).The migration number of HTMCs in the FoxF2 shRNA group was significantly lower than that in the Scramble control group (117.30±11.41 vs.251.00±10.37;t =8.72,P＜0.01).Conclusions The FoxF2 shRNA lentivirus are successfully constructed,which can decrease the expression of FoxF2 in HTMCs.Low expression of FoxF2 can reduce the expression level of extracellular matrix protein in HTMCs and inhibit the migration ability of HTMCs.
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@#Primary open angle glaucoma is a kind of chronic disease characterized by progressive damage to the optic disc as a result of persistent elevation of intraocular pressure. The complicated and unidentified mechanism of primary open angle glaucoma makes its clinical treatment relatively difficult nowadays. In primary open angle glaucoma, IOP elevation is often a result of reduced aqueous humor flow through the trabecular meshwork, which plays an important regulating role in drainage process. The morphology, quantity, structure and function of trabecular meshwork cell can increase the outflow resistance of aqueous humor, leading to elevation of IOP. Research is proving that induced pluripotent stem cells(iPSCs), bone mesenchymal stem cells(BMSCs)and adipose-derived stem cells(ADSCs)have been used for trabecular meshwork cell differentiation and regeneration, providing a reliable cell source for trabecular meshwork stem cell replacement therapy in primary open angle glaucoma. Recent studies have showed that trabecular meshwork stem cells have absolute superiority in differentiating into trabecular meshwork cells, which provides new target for cell transplantation to treat glaucoma. This marks a new era of stem cell therapy for primary open angle glaucoma and also brings new hope to the treatment of glaucoma. This article reviews different types of stem cells for trabecular meshwork transplantation and may provide novel development of therapeutic strategies for primary open angle glaucoma with cell transplantation in the future.
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@#AIM: To investigate the changes of fibronectin expression in cultured bovine trabecular meshwork cells when cells were stimulated by different concentrations of IL-6 to provide evidence for early diagnosis and new therapy of POAG.<p>METHODS: We identify third-generation bovine trabecular meshwork cells which were got from tissue mass culture method. Then the relative expression of FN gene and protein in cells were detected by Real-Time PCR and Western-blot after 24h stimulation with 0ng/mL, 0.1ng/mL, 0.5ng/mL IL-6.<p>RESULTS: The cultured bovine trabecular cells are coincident with what recorded in the book. Real-time PCR and Western blot showed that the amount of FN mRNA produced by cells was 1.000±0.000, 0.213±0.004, 0.056±0.001, 0.019±0.002 respectively, and the protein expression was 1.167±0.012, 0.662±0.009, 0.238±0.011, 0.061±0.011 respectively. There was a significant difference among four groups(<i>P</i><0.05).<p>CONCLUSION: Cultured bovine trabecular meshwork cells have a negative correlation with the expression of FN protein after being stimulated by exogenous IL-6, and the results are consistent with the actual state of the disease. We speculate that the IL-6 participate in the pathogenesis and progression of POAG by affecting the expression of FN gene and protein and changing the structure of trabecular meshwork.
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Objective To investigate the effects of transient receptor potential melastatin (TRPM) 7 on dexamethasone (Dex)-mediated cytoskeleton remodeling in human trabecular meshwork.Methods Human trabecular meshwork cells (HTMs) were primarily cultured and the cells of generation 3 to 6 were used in this study.The expression of TRPM7 protein in the cells was located using immunofluorescence technology.Dex at the dose of 0.2 mg was added into culture medium for 4 days with the final concentration of 1×10-5,1×10-6 and 1×10-7 mol/L,respectively.Western blot assay was employed to detect the relative expression level of TRPM7 protein.Cultured cells were divided into non-transfected group,siRNA transfected group,TRPM7-siRNA1 transfected group and TRPM7-siRNA2 transfected group,and the expressions of TRPM7 protein and p-cofilin protein in the cells were assayed by Western blot method.Cultured cells were divided into normal control group,Dex-treated group,siRNA transfected group and TRPM7-siRNA transfected group,and the expression of phalloidin (a cytoskeletal protein) and Vinculin (focal adhesion protein) was detected by immunofluorescence staining.In addition,cultured cells were divided into normal control group,Dex-treated group,2-APB (a Ca2+ inhibitor) treated group,ethylene glycol tetraacetic acid (EGTA) (a calcium chelator)-treated group,TRPM7-siRNA transfected group and TRPM7-siRNA+Dex group,and the [Ca2+] i in the cells was observed by Fluo-3AM immunofluorescence staining.Western blot assay was used to detect the expression of p-cofilin in the cells.Results TRPM7 was positively expressed on the cell membrane.The relative expression of TRPM7 was gradually reduced with an increase of Dex dose (F=4.210,P＜0.05),and the expression of TRPM7 was significantly decreased in 1 × 10-5 mol/L Dex group compared with the normal control group (P＜ 0.05).Western blot assay revealed that the relative expression levels of TRPM7 in the TRPM7-siRNA1 and TRPM7-siRNA2 group were significantly lower than those of non-siRNA transfected group and siRNA transfected group (all at P＜0.05).In the Dex-treated group and TRPM7-siRNA transfected group,the cells were enlarged in size with the lessened processes in comparison with the normal control group.Immunofluorescence staining showed that the actin fiber and vinculin increased in the Dex-treated group,and more spread but depolymerized actin fiber was seen in the TRPM7-siRNA transfected group.Compared with the normal control group,the fluorescence intensity of [Ca2×] i was weak in the Dex-treated group and TRPM7-siRNA transfected group.The relative expression levels of p-confilin protein was lower in the TRPM7-siRNA transfected group than that in the siRNA transfected group (0.317 ±0.031 vs.0.092±0.071) (t =5.030,P =0.007).Conclusions Dex induces the downregulation of TRPM7 expression in HTMs.The downregulation of TRPM7 probably participates in Dex-induced cytoskeletal remodeling by causing the depolymerization of actin cytoskeleton and reduction of [Ca2+] i in HTMs.
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Objective To investigate the protection and mechanism of procyanidins (PC) against H2O2 induced oxidative damage of human trabecular meshwork cells (HTMC) in order to provide an experimental foundation for glaucoma clinical treatment.Methods HTMC were cultured and then divided randomly into 5 groups.As untreated group:Normal cultured HTMC;Control group:Normal cultured HTMC + H2O2 (500 μmol · L-1 for 1 hour);Treated group:Normal cultured HTMC + H2O2 (500 μmol ·L-1 for 1 hour) + PC (PC fmal concentrations were 0.02 g · L-1,0.05 g · L-1,0.10 g· L-1).Real-time fluorescence quantitative polymerase chain reaction (PCR) was used to investigate the expression of mitochondrial complex Ⅰ mRNA.Results Compared with untreated group (1.000 0 ± 0.000 0),the differences of mitochondrial complexⅠ mRNA expression in 0.02 g · L-1 PC (0.401 3 ±0.010 3),0.05 g · L-1 PC (0.791 5 ± 0.008 5) groups were statistically significant (all P ＜ 0.01),but the 0.10 g ·L-1 PC group (1.043 0 ± 0.062 2) had no significant differences (P ＞ 0.05).The differences between PC treated groups and control group were statistically significant (P ＜0.01),which showed HTMC treated with PC could increase the expression of mitochondrial complex Ⅰ mRNA.The differences in each PC treated groups were statistically significant (P ＜ 0.01),which showed the expression of mitochondrial complex Ⅰ mRNA were increased along with the concentration of PC gradually increased.Conclusion Exogenetic PC can increase the expression of mitochondrial complex Ⅰ mRNA in the oxidative damaged HTMC,and in a certain range of concentration,the protective effects of PC have the positive relationship of dose-effect,which suggest that PC may be a good candidate for further study of the clinical treatment of glaucoma.
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Objective To investigate the effect of procyanidins (PC) on cell apoptosis and cytochrome C releasing in human trabecular meshwork cells (HTMC) under oxidative stress induced by H2 O2.Methods HTMC were cultured and then divided randomly into 5 groups.Normal cultured HTMC served as normal group without treatment,and normal cultured HTMC in control group were treated with 500 μrnol · L-1 H2O2 for 1 h,as well as normal cells in treatment groups were treated with 500 imol · L-1 H2 O2 for 1 h combined with different concentrations of PC (0.02 g · L-1,0.05 g · L-1,0.10 g · L-1).Then flow cytometry and Western blot were applied to detect the cell apoptosis rate and the release of cytochrome C (CytC) respectively.Results Compared with the normal group,the apoptotic rates of the control group and the PC treatment groups were significantly increased,and the difference was statistically significant (all P ＜ 0.01).Compared with the control group,the apoptotic rates of the 3 PC treatment groups were decreased,and the differences were statistically significant (all P ＜ 0.01).Moreover,the apoptotic rate in 0.02 g · L-1,0.05 g · L-1,0.10 g · L-1 PC treatment groups was decreased as PC concentration increased,and the differences were statistically significant (all P ＜ 0.01).Furthermore,compared with the normal group,the release of cytochrome C in the control group,0.02 g · L-1 and 0.05 g · L-1 PC treatment group enhanced,and the differences were statistically significant (all P ＜ 0.01),but there was no significant difference between 0.01 g · L-1 PC treatment group and the normal group (P ＞ 0.05).Compared with the control group,the release of cytochrome C in the 3 PC treatment group was attenuated with significant differences (all P ＜0.01).Meanwhile,the release of cytochrome C was decreased in the 3 PC treatment groups with a concentration-dependent manner,and the pairwise differences were statistically significant (all P ＜ 0.01).Conclusion Exogenous PC can decrease the apoptotic rate of HTMC under oxidative stress and reduce the release of cytochrome C,as well as it also has an antioxidant effect in a certain concentration-dependent manner.
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Objective To investigate the effect of procyanidins (PC) on cell apoptosis and cytochrome C releasing in human trabecular meshwork cells (HTMC) under oxidative stress induced by H2 O2.Methods HTMC were cultured and then divided randomly into 5 groups.Normal cultured HTMC served as normal group without treatment,and normal cultured HTMC in control group were treated with 500 μrnol · L-1 H2O2 for 1 h,as well as normal cells in treatment groups were treated with 500 imol · L-1 H2 O2 for 1 h combined with different concentrations of PC (0.02 g · L-1,0.05 g · L-1,0.10 g · L-1).Then flow cytometry and Western blot were applied to detect the cell apoptosis rate and the release of cytochrome C (CytC) respectively.Results Compared with the normal group,the apoptotic rates of the control group and the PC treatment groups were significantly increased,and the difference was statistically significant (all P ＜ 0.01).Compared with the control group,the apoptotic rates of the 3 PC treatment groups were decreased,and the differences were statistically significant (all P ＜ 0.01).Moreover,the apoptotic rate in 0.02 g · L-1,0.05 g · L-1,0.10 g · L-1 PC treatment groups was decreased as PC concentration increased,and the differences were statistically significant (all P ＜ 0.01).Furthermore,compared with the normal group,the release of cytochrome C in the control group,0.02 g · L-1 and 0.05 g · L-1 PC treatment group enhanced,and the differences were statistically significant (all P ＜ 0.01),but there was no significant difference between 0.01 g · L-1 PC treatment group and the normal group (P ＞ 0.05).Compared with the control group,the release of cytochrome C in the 3 PC treatment group was attenuated with significant differences (all P ＜0.01).Meanwhile,the release of cytochrome C was decreased in the 3 PC treatment groups with a concentration-dependent manner,and the pairwise differences were statistically significant (all P ＜ 0.01).Conclusion Exogenous PC can decrease the apoptotic rate of HTMC under oxidative stress and reduce the release of cytochrome C,as well as it also has an antioxidant effect in a certain concentration-dependent manner.
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PURPOSE: To compare the effects of anti-inflammatory agents, specifically bromfenac, loteprednol, and prednisolone, on the permeability of cultured human trabecular meshwork cell (HTMC) monolayers. METHODS: HTMCs were cultured until confluency in the inner chamber of Transwell, then exposed to 1/1,000 or 1/500 diluted commercial 0.1% bromfenac, 0.5% loteprednol, and 1% prednisolone for 24 hours. The permeabilities of carboxyfluorescein through the HTMC monolayer were measured with a spectrofluorometer after 2 hours in the outer chamber. Cellular viabilities were assessed with an 3-[4,5–dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay. RESULTS: Bromfenac and loteprednol diluted at 1/1,000 or 1/500 did not significantly affect the cellular survival (p > 0.05). Bromfenac did not affect the permeability via the HTMC monolayer (p > 0.05) and loteprednol decreased the permeability (p < 0.05). In addition, 1/2,000 prednisolone also decreased the permeability (p < 0.05). CONCLUSIONS: Among the anti-inflammatory agents, the non-steroidal anti-inflammatory agent bromfenac did not affect the permeability, while loteprednol and prednisolone decreased the permeability through the HTMC monolayer. Thus, loteprednol and prednisolone may decrease the trabecular outflow.
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Humans , Anti-Inflammatory Agents , Loteprednol Etabonate , Permeability , Prednisolone , Trabecular MeshworkABSTRACT
PURPOSE: To compare the effects of anti-inflammatory agents, specifically bromfenac, loteprednol, and prednisolone, on the permeability of cultured human trabecular meshwork cell (HTMC) monolayers. METHODS: HTMCs were cultured until confluency in the inner chamber of Transwell, then exposed to 1/1,000 or 1/500 diluted commercial 0.1% bromfenac, 0.5% loteprednol, and 1% prednisolone for 24 hours. The permeabilities of carboxyfluorescein through the HTMC monolayer were measured with a spectrofluorometer after 2 hours in the outer chamber. Cellular viabilities were assessed with an 3-[4,5–dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay. RESULTS: Bromfenac and loteprednol diluted at 1/1,000 or 1/500 did not significantly affect the cellular survival (p > 0.05). Bromfenac did not affect the permeability via the HTMC monolayer (p > 0.05) and loteprednol decreased the permeability (p < 0.05). In addition, 1/2,000 prednisolone also decreased the permeability (p < 0.05). CONCLUSIONS: Among the anti-inflammatory agents, the non-steroidal anti-inflammatory agent bromfenac did not affect the permeability, while loteprednol and prednisolone decreased the permeability through the HTMC monolayer. Thus, loteprednol and prednisolone may decrease the trabecular outflow.
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Humans , Anti-Inflammatory Agents , Loteprednol Etabonate , Permeability , Prednisolone , Trabecular MeshworkABSTRACT
PURPOSE: To investigate the effects of tetrahydrozoline (THZ) on the survival of cultured human trabecular meshwork cells (HTMC) and the permeability of HTMC monolayer. METHODS: Primary cultured HTMC were exposed to an adrenergic agonist (0.01, 0.1, 1.0 or 10 µM THZ) for 1 day and 3 days. Carboxyfluorescein permeability through the HTMC monolayer was measured using Transwell. Cellular viability and nitric oxide (NO) production were assessed using MTT and Griess assays, respectively. RESULTS: THZ did not affect the cellular survival (p > 0.05) or NO production (p > 0.05). THZ significantly increased the carboxyfluorescein permeability through the HTMC monolayer in a dose-dependent manner compared with non-exposed control (p < 0.05) after exposure for 1 and 3 days. CONCLUSIONS: THZ does not affect the survival of HTMC but decreases the permeability of HTMC monolayer in a dose-dependent manner. Thus, THZ may possibly decrease trabecular outflow.
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Humans , Adrenergic Agonists , Nitric Oxide , Permeability , Trabecular MeshworkABSTRACT
PURPOSE: To determine the effect of exogenous nitric oxide (NO) on the migration of trabecular meshwork (TM) cells and its association with expression of matrix metalloproteinases (MMPs). METHODS: Primary human TM cells treated with 1 or 10 microM S-nitroso-N-acetyl-penicillamine (SNAP) and examined for changes in adherence. TM cells were seeded onto transwell culture inserts, and changes in their migratory activity were quantified. Reverse transcription polymerase chain reaction was performed to determine the relative changes in mRNA expression of MMPs and tissue inhibitor of metalloproteinases (TIMPs). RESULTS: Treatment with SNAP did not significantly suppress TM cell adhesion or migration (p > 0.05). Treatment of TM cells with 10 microM SNAP decreased expression of MMP-2 and increased expression of membrane type MMP-1 and TIMP-2. Treatment with interleukin-1alpha triggered MMP-3 expression but did not exert significant effects on MMP-3 activation in response to SNAP. CONCLUSIONS: These data suggest that NO revealed no significant effect on the migration of TM cells because NO decreased MMP-2 and increased TIMP-2 expression. Although expression of certain MMPs and TIMPs change in response to NO donors, NO may modulate trabecular outflow by changing the cellular production of extracellular matrix without having a significant effect on the migration of TM cells.
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Humans , Cell Movement/drug effects , Cell Survival/drug effects , Cells, Cultured , DNA Primers/chemistry , Gene Expression Regulation, Enzymologic/physiology , Matrix Metalloproteinases/genetics , Nitric Oxide Donors/pharmacology , RNA, Messenger/genetics , Real-Time Polymerase Chain Reaction , S-Nitroso-N-Acetylpenicillamine/pharmacology , Tissue Inhibitor of Metalloproteinase-2/genetics , Trabecular Meshwork/cytologyABSTRACT
Background Evidences indicated that oxidative stress damage is an essential pathological process in primary open angle glaucoma.Sulforaphane (SFN) can play an antioxidative stress role to many tissues and cells by activating Nrf2/ARE single pathway.However,whether SFN has a protective role to oxidative stress induced damage of trabecular meshwork cells is still unclear.Objective This study was to investigate the antioxidant effect of SFN against H2O2-induced oxidative damage in bovine trabecular meshwork cells.Methods Trabecular cells were isolated from fresh black bovine eyeballs and primarily cultured and passaged.The third generation of cells were incubated to 96-well dish at a density of 1 ×103/well for 24 hours and divided into 4 groups.The cells were incubated using 100 μl serum-free medium in the blank control group.Oxidative damage models were established by adding 100 μmol/L H2O2(100 μl) in medium in the H2O2 group.The cells were cultured with the medium containing 10 μmol/L SFN (100 μl) in the SFN group,and 100 μl H2O2 at the final concentration of 100 μmol/L was added in the SFN-treated cell medium in the SFN +H2O2 group.The cell vitality in various groups was assayed by using cell counting kit-8 (CCK-8).The apoptosis rate of the cells was detected by Annexin V-FITC/PI double-staining with flow cytometry.Results Cultured cells showed a spindle shape with uniform size,abundant cytoplasm,numberous pigmented particles and big nucleolus.The relative cell vitality reduced to (67.00± 1.27)％ and (80.00±6.25)％ in the H2O2 group and SFN+H2O2 group in comparison with 100％ in the blank control group,and the cell vitality in the SFN+ H2O2 group was lower than that in the SFN group but higher than that in the H2 O2 group (both at P＜0.01).The mean apoptosis rate was (11.33 ±0.77) ％,(32.31 ± 1.03) ％,(10.44 ±0.68) ％ and (17.68 ±0.21) ％ in the blank control group,H2 O2 group,SFN group and SFN+H2O2 group,respectively,showing a significant difference among the groups (F=539.96,P＜0.01),and the apoptosis rate in the SFN+H2O2 group was significantly lower than that in the H2O2 group but higher than that in the blank control group and SFN group (all at P＜0.01).Conclusions SFN can improve the antioxidative stress ability of trabecular meshwork cells and alleviate the damage induced by oxidative stress.
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PURPOSE: To investigate the effects of nitric oxide (NO) on the permeability of cultured human trabecular meshwork cell (HTMC) monolayer. METHODS: HTMCs were cultured until confluency in the Transwell inner chamber and then exposed to 0, 10 or 100 microm S-nitroso-N-acetyl-DL-penicillamine (SNAP) and 0.5 mm L-NG-Nitroarginine methyl ester (L-NAME) for 24 hours. Permeabilities of carboxyfluorescein through the HTMC monolayer were measured using a spectrofluorometer after 2 hours in the outer chamber. Cellular viabilities and production of NO were assessed using 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) and Griess assay, respectively. RESULTS: The cellular survival was not affected by 10 or 100 microm SNAP (p > 0.05) but NO production increased in a dose-dependent manner (p 0.05). CONCLUSIONS: NO increased the permeability of carboxyfluorescein through the HTMC monolayer in a dose-dependent manner. Thus, NO could increase trabecular outflow by increasing the permeability of trabecular cell layer in addition to trabeular messwork (TM) relaxation.
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Humans , NG-Nitroarginine Methyl Ester , Nitric Oxide Synthase , Nitric Oxide , Permeability , Relaxation , Trabecular MeshworkABSTRACT
PURPOSE: To investigate the effects of benzalkonium chloride (BAC), mitomycin C (MMC) and dexamethasone (DEX) on cellular stress in cultured human trabecular meshwork cell (HTMC) monolayers. METHODS: HTMCs were cultured in the inner Transwell chamber until confluence and then were exposed to BAC, MMC or DEX for 6 hours. The carboxyfluorescein permeability through the HTMC monolayer was measured using a spectrofluorometer at 532 nm after 2 hours in the outer chamber. The 3-[4, 5 -dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide (MTT) assay was used to evaluate cellular viabilities. RESULTS: The carboxyfluorescein permeability through the HTMC monolayer increased and cell survival decreased with 0.002% BAC (p 0.05). CONCLUSIONS: BAC and MMC induced cellular toxicity and stress at lower concentrations but did not affect survival of cultured HTMCs.
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Humans , Benzalkonium Compounds , Cell Survival , Dexamethasone , Mitomycin , Permeability , Trabecular MeshworkABSTRACT
PURPOSE: To investigate the effects of bimatoprost on the permeability of cultured human trabecular meshwork cells (HTMC) monolayer. METHODS: HTMCs were cultured until confluency in the inner Transwell chamber and then exposed to benzalkonium chloride, brimonidine, latanoprost or bimatoprost for 1 week. Carboxyfluorescein permeability through the HTMC monolayer was measured using a spectrofluorometer after 2 hours in the outer chamber. Cellular viability was assessed using the MTT assay. RESULTS: Each drug diluted at 1/1000X did not affect the cellular survival (p > 0.05). Brimonidine, latanoprost and bimatoprost did not affect the carboxyfluorescein permeability through the HTMC monolayer (p > 0.05). The carboxyfluorescein permeability was not different between latanoptost and bimatoprost after 1 week of exposure (p > 0.05). CONCLUSIONS: Bimatoprost, a drug known to increase trabecular outflow, does not affect the carboxyfluorescein permeability through the HTMC monolayer. Thus, the effect on the trabecular outflow of bimatoprost may not be significant.
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Humans , Benzalkonium Compounds , Permeability , Trabecular Meshwork , Bimatoprost , Brimonidine TartrateABSTRACT
Aim: To study the effects of triamcinolone acetonide (TA) on cultured human trabecular meshwork (HTM) cells. Materials and Methods: HTM cells were cultured and treated with 125, 250, 500 and 1000 μg/mL concentration of TA for 24 h. The cells were treated with both crystalline TA (TA‑C) (commercial preparation) and solubilized TA (TA‑S). Cell viability was measured by a trypan blue dye exclusion test. The activity of caspse‑3/7 was measured by a fluorescence caspase kit and DNA laddering was evaluated by electrophoresis on 3% agarose gel. Levels of lactate dehydrogenase (LDH) were assessed with LDH cytotoxicity assay kit‑II. Results: Mean cell viabilities of HTM cells after 24 h exposure to TA‑C 125, 250, 500, and 1000 μg/mL were 75.4 ±2.45% (P < 0.0001), 49.43 ± 1.85% (P < 0.0001), 17.07 ± 2.39% (P < 0.0001), and 3.7 ± 0.9% (P < 0.0001), respectively, compared with the untreated HTM cells 92.49 ± 1.21%. The mean cell viabilities with 125, 250, 500, and 1000 μg/mL of TA‑S were 94.47 ± 1.60% (P > 0.05), 90.13 ± 0.40% (P < 0.01), 85.57 ± 0.47% (P < 0.001), and 71.67 ± 3.30% (P < 0.0001), respectively, compared to DMSO‑equivalent cultures. Untreated HTM control had a cell viability of 96.57 ± 1.98%. DMSO‑treated controls of 125, 250, 500, and 1000 μg/mL had a cell viability of 94.73 ± 0.57%, 96.97 ± 1.08%, 93.97 ± 1.85%, and 97.27 ± 1.15%, respectively. There was no increase of caspase‑3/7 activity in cultures treated with either TA‑C or TA‑S. DNA laddering showed no bands in the TA‑C or TA‑S treated cultures. There were significantly higher LDH release rates at all concentrations of TA‑C compared to TA‑S. Conclusions: Results show that the effect of TA‑C and TA‑S on HTM cells is due to cell death by necrosis at all concentrations except 125 μg/mL of TA‑S. Elevated levels of LDH confirmed necrotic cell death. Our study also infers the relative safety of TA‑S over TA‑C.
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Objective To analyze the influence of dexamethasone in the hypotonic-induced volume-sensitive chloride currents in human trabecular meshwork cells,and to investigate the possible mechanism of volume-sensitive chloride channels(VACC)in the glucocorticoid-induced glaucoma(GIG)cases.Methods The human trabecular meshwork cells were seeded in 35 mm diameter plastic petri dishes,so that they could grow in monolayer.The cultured cells were divided into normal cell culture medium group and dexamethasone 1 d,3 d,7 d groups.The chlorine current density values of the cells in four groups were recorded respectively by the whole-cell patch-clamp technique. The differences among groups were compared.Results In normal group,after hypotonic stimulation,under+100 and-100 mV voltage clamp,the outward and inward current density values of the trabecular cells were (19.94±0.87) and (-6.53±0.41)pA/pF.In dexamethasone 1 d,3 d,and 7 d groups,under the same condition,the outward and inward current density values of the trabecular cells were (19.39 ± 1.40)and (-6.42 ± 0.28)pA/pF, (17.97±2.35)and (-5.82±0.94)pA/pF,(17.16±1.16)and (-5.65±0.43)pA/pF.The trabecular cells cultured with dexamethasone for 1 d had lower outward and inward current density values under hypotonic stimulation compared with normal group,but there was no significant difference (P>0.05).The trabecular cells cultured with dexamethasone for 3 d and 7 d,when compared with normal group,had significantly lower outward and inward current density values under hypotonic stimulation (P<0.05 ). Conclusion Dexamethasone could reduce the volume-sensitive chloride current in trabecular meshwork cells,which would affect trabecular meshwork cell volume adjustment.This would possibly cause the increase of the aqueous humor outflow resistance among GIG cases.
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PURPOSE: To investigate and compare the effects of topical carbonic anhydrase inhibitors on the production and expression of nitric oxide in cultured human trabecular meshwork cells (HTMC). METHODS: Primarily cultured HTMC were exposed to 0, 10, and 100 microM dorzolamide and brinzolamide using serum-deprived media for 3 days. Production of nitric oxide was assessed with Griess assay. Expressions of eNOS mRNA were assessed with RT-PCR. RESULTS: Both dorzolamide and brinzolamide increased the production of nitric oxide eNOS activity (p < 0.05). Dorzolamide had a more potent effect than brinzolamide on the production of nitric oxide and the expression of eNOS mRNA. CONCLUSIONS: Topical carbonic anhydrase inhibitors increased the production of nitric oxide, which was accompanied by increased eNOS activity. Dorzolamide had a more potent effect than brinzolamide on the production of nitric oxide and expression of eNOS mRNA in HTMC. The increased production of nitric oxide by topical carbonic anhydrase inhibitors involves mechanisms other than carbonic anhydrase inhibition.