Effect of Dexamethasone and Aquaporin-1 Antisense Oligonucleotides on the Aquaporin-1 Expression in Cultured Human Trabecular Meshwork Cells

The changes in the expression of aquaporin-1 （AQP1） mRNA and protein in cultured human trabecular meshwork （HTM） cells treated with dexamethasone and transfected with antisense oligonucleotides （AS-ODN） were studied, and the implication of AQP1 regulation in corticosteroid-glaucoma and the possibility of AS-ODN inhibiting the AQP1 expression were evaluated. The cultured HTM cells in vitro were treated with different concentrations of dexamethasone and transfected with oligonucleotides for 5 days respectively. Then, total RNA and protein of HTM cells were extracted. The changes of AQP1 mRNA and protein were demonstrated qualitatively and quantitatively by RT-PCR and Western blot. Band intensities were detected by imaging analysis. There was a parallel relationship between the results of RT-PCR and those of Western blot. The expression levels of AQP1 mRNA and protein in dexamethasone-treated groups were increased initially and decreased later as dexamethasone concentration was stepped up. In the 0.04 μg/mL and 0.4 μg/mL groups, the levels of AQP1 were higher than in control group （0 μg/mL）. In the 4 μg/ mL and 40 μg/mL groups, the AQP1 expression levels were lower than in control group. AS-ODN could down-regulate the expression of AQP1 mRNA and protein in a dose-dependent manner. At 5 μg/mL, down-regulation efficiency reached the maximum. There was no statistically significant difference in the expression of AQP1 mRNA and protein between all sense oligonucleotides groups and control group. It was suggested that dexamethasone may induce the changes of the AQP1 expression in HTM cells to be involved in the occurrence of corticosteroid-glaucoma. AS-ODN can down-regulate the AQP1 expression in HTM cells to some extent.

Inhibition of latrunculin-A on dexamethasone-induced fibronectin production in cultured human trabecular meshwork cells

AIM: To determine the effects of a low dose latrunculin (LAT)-A on dexamethasone (Dex)-induced upregulation of extracellular matrix proteins fibronectin (FN) in cultured human trabecular meshwork (HTM) cells. METHODS: HTM cells were cultured to confluent and incubated with 0.4 mu mol/L Dex and/or 0.05 mu mol/L LAT-A. FN expression in HTM cells was evaluated by Western blot and immunofluorescence microscopy. RESULTS: Dex up-regulated FN production in HTM cells, failed to do so when co-incubated with LAT-A. LAT-A decreased production of FN in cultured HTM cells. CONCLUSION: This study indicated that LAT-A may modulate the expression of fibronectin in trabecular meshwork to achieve treatment for steroids and other types of glaucoma. It has an important prospect as an intraocular pressure-lowering drug.

Effects of Nitric Oxide on Proliferation and Apoptosis of Cultured Bovine Trabecular Meshwork Cells

The effects of different doses of nitric oxide (NO) on the proliferation and apoptosis of the cultured bovine trabecular meshwork (TM) cells were studied. L arginine and N G nitro L arginine methyl (L NAME) were incubated with TM cells for 48 h. In the control group, no medicine was given. In the experimental groups, concentrations of L arginine and L NAME were 1×10 -7 mol/L, 1×10 -6 mol/L, 1×10 -5 mol/L, 1×10 -4 mol/L, 1×10 -3 mol/L and 1×10 -2 mol/L, respectively. NO 2 - in supernate, the proliferation and apoptosis of TM cells and mRNA expression of bcl 2 and bax were measured by Griess reagent, terminal deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL), MTT assay and in situ hybridization,respectively. The results showed that L arginine with concentration ≥1×10 -4 mol/L could induce apoptosis of the TM cells and inhibit the proliferation of TM cells through increasing the NO levels, down regulating bcl 2 mRNA expression and up regulating bax mRNA expression; L NAME with concentration ≥1×10 -5 mol/L could induce the proliferation of the TM cells through suppressing the production of NO. It was concluded that NO in high level could induce apoptosis of the TM cells and suppress the proliferation of the TM cells.

Effects of Hydrostatic Pressure on Cultured Bovine Trabecular Meshwork Cells

In order to explore the effects of pressure on trabecular meshwork cells, bovine trabecular meshwork cells were cultured in vitro and subjected to different levels of hydrostatic pressure. The cellular morphology, ultrastructure and phagocytosis were studied with inverted phase-contrast microscopy, light microscopy and transmission electron microscopy, etc. It was found that the cells subjected to 2. 0 kPa or 2. 67 kPa for 48 h had no remarkable difference as compared with the controls in terms of parameters observed. Those under 4. 0 kPa for 24 h showed slight changes in structure and a mild decrease in phagocytic function. The damage appeared more severe if the pressure was higher or lasted longer. From the above we conclude that trabecular meshwork cells can only bear pressure below a certain level. They may be destroyed structurally or impaired functionally by pressure over this level.

Effect of CD44 Suppression by Antisense Oligonucleotide on Attachment of Human Trabecular Meshwork Cells to HA

The effects of suppression of CD44 by CD44-specific antisense oligonucleotide on attachment of human trabecular meshwork cells to hyaluronic acid (HA) were observed and the possible relationship between CD44 and primary open-angle glaucoma (POAG) investigated. CD44-specific antisense oligonucleotide was delivered with cationic lipid to cultured human trabecular meshwork cells. The expression of CD44 suppressed by CD44-specific antisense oligonucleotide was detected by RT-PCR and Western blotting. The effect of CD44 suppression by specific antisense oligonucleotide on attachment of trabecular meshwork cells to HA was measured by MTT assay. Results showed that expression of CD44 was suppressed by CD44-specific antisense oligonucleotide. Antisense oligonucleotide also suppressed the adhesion of human trabecular meshwork cells to HA in a concentration dependent manner. It was concluded that attachment of human trabecular meshwork cells to HA was decreased when CD44 was suppressed by specific antisense oligonucleotide. CD44 might play a role in pathogenesis of POAG by affecting the adhesion of trabecular meshwork cells to HA.

Inhibitory Effect of Tissue Transglutaminase (tTG) Antisense Oligodeoxynucleotides on tTG Expression in Cultured Bovine Trabecular Meshwork Cells

To study the effect of tTG fully phosphorothioated antisense oligodeoxynucleotides （tTG-ASDON） on tTG expression in cultured bovine trabecular meshwork cells （BTMCs） in vitro and explore a new treatment alternative for primary open angle glaucoma （POAG）, the ASDON1 and ASDON2 complementary to the protein codogram region of tTG were designed, synthesized and phosphorothioated according to the secondary structure of tTG. The ASDON1 and ASDON2 were embedded in Lipofectamine and transfected into BTMCs. The untreated group served as negative controls. The expression of tTG in the mRNA and protein level were measured by semi-quantitative RT-PCR and immunohistochemical technique-Supervision method respectively. Our results showed that both the mRNA and the protein of tTG with tTG-ASDON1 and tTCr-ASDON2 were significantly decreased as compared with that of the controls （P〈0.05）. On the other hand, no significant difference was found between the ASDON1 group and the ASDON2 group. It is concluded that the expression of tTG mRNA and protein in cultured BTMC are down-regulated by tTG- ASDON. As a result, tTG-ASDON may be used for the treatment of POAG through the inhibitory effect on the expression of tTG.

Effects of dexamethasone and HA1077 on actin cytoskeleton and β-catenin in cultured human trabecular meshwork cells

AIM：To investigate the effects of dexamethasone（DEX） and 1-（5-isoquinolinesulfonyl）-homopiperazine（HA1077） on actin cytoskeleton and β-catenin in cultured human trabecular meshwork（HTM） cells.METHODS： The HTM cells were separated from human eyeball and cultured in vitro.They were divided into control group,DEX（1×10^-6mol/L） group,HA1077（3×10^-5mol/L）group,and DEX（1×10^-6mol/L） and HA1077（3×10^-5mol/L）group.Actin cytoskeleton and β-catenin in HTM cells of the four groups were examined by immunofluorescence and Western blot analyses.RESULTS： In DEX group,there were reorganization of actin cytoskeleton and formation of cross linked actin networks（CLANs）,which were partially reversed in DEX and HA1077 group.DEX treatment also induced an increased expression of β-catenin,which was obviously reduced in DEX and HA1077 group.Meanwhile,the cultured HTM cells in HA1077 group had lower expression of β-catenin than that in the control group. CONCLUSION： Our results show that HA1077 can reverse the changes of actin organization and expression of β-catenin induced by DEX in cultured HTM cells,suggesting that HA1077 may play an important role in increasing outflow and reducing intraocular pressure.

A Study of Toxicity of 5-Fluorouracil on Bovine Trabecular Meshwork Cells in Vitro

In order to explore whether the conventional use of 5 fluorouracil (5 Fu) had any toxic effects on trabecular meshwork cells, bovine trabecular meshwork cells were cultured in vitro and exposed to 5 Fu at different concentrations. The cellular morphology, ultrastructure, mortality and phagocytosis were studied under light microscopy, transmission electron microscopy and methods of Wright's stain. It was found that the toxic effects of 5 Fu on the cells were in a dose dependent mode. 1×10 -1 mg/ml of 5 Fu caused a large part of cells rounded up, while 1×10 -3 mg/ml of the drug only a rough appearance of the cell surface. Exposure to 1×10 -2 mg/ml of 5 Fu made mitochrone swollen and rough endoplasmic reticulum enlarged, with the cell mortality being 50.5 %. The latex microspheres engulfed in cytoplasm in cells receiving 1×10 -1 and 1×10 -2 mg/ml of 5 Fu were significantly decreased as compared with those in the control group ( P <0.01). It was concluded that the safe concentration of 5 Fu on bovine trabecular meshwork cells was 1×10 -3 mg/ml and the conventional dosage of 5 Fu in clinical practice would not cause injury to trabecular meshwork cells.

Regulatory Effect of Dexamethasone on Aquaporin-1 Expression in Cultured Bovine Trabecular Meshwork Cells

To evaluate the effect of dexamethasone on the expression of aquaporin-1 （AQP-1） in cultured bovine trabeeular meshwork cells, bovine trabeeular meshwork cells were cultured in vitro and reproduced to the third and the fourth generation, then treated with dexamethasone at the concentrations of 5, 25, 50, 250μg/L respectively for 7 days. Immunohistochemical technique-supervision method was employed to measure, and image analysis system to analyze the expression of AQP-1 in normal cultured bovine trahecular meshwork cells and those treated with dexamethasone. In normal bovine trabeeular meshwork cells, the grayseale of AQP-1 positive staining was 167.94± 1.18, while it was 168.92±0.91, 176.72±1.80, 180. 64±1.31, 185.64±1.58 in cells treated with 5, 25, 50, 250μg/L concentrations of dexamethasone. When the concentration of dexamethasone was higher than 25 μg/L, the expression of AQP-1 was significantly inhibited （P〈0.05）. The regulation of AQP-1 expression by dexamethasone in cultured bovine trahecular meshwork cells in vitro may be one of causes that retard the aqueous outflow in glueoeorticoid-induced glaucoma.

Expression profile analysis to identify potential gene changes induced by dexamethasone in the trabecular meshwork

AIM:To investigate potential gene changes in trabecular meshwork(TM)induced by dexamethasone(DEX)in steroidinduced glaucoma(SIG).METHODS:The expression data of 24 cases from a public functional genomics data were sorted to identify the mechanisms of action of DEX on the TM.The relationships of the differentially expressed genes(DEGs)were enriched using Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)analysis.In addition,the hub genes were screened by the Search Tool for the Retrieval of Interacting Genes Database(STRING)and Cytoscape tools.Finally,human TM cells(HTMCs)were treated with DEX to preliminarily explore the function of hub genes.RESULTS:Totally 47 DEGs,including 21 downregulated and 26 upregulated genes were identified.The primary enriched results of the DEGs consisted of inflammatory response,extracellular matrix(ECM),negative regulation of cell proliferation,TNF signalling pathway and the regulation of tr yptophan channels by inflammator y mediators.Subsequently,pro-melanin-enriched hormone(PMCH)and Bradykinin B1 receptor(BDKRB1)were screened as hub genes.It is verified in GSE37474 data set.Western blot and quantitative real-time polymerase chain reaction(q PCR)results showed that protein and RNA expression levels of BDKRB1 were significantly decreased after DEX treatment,while PMCH was not significantly changed.CONCLUSION:BDKRB1 may be a key gene involved in SIG onset,providing a suitable therapeutic target for improving the prognosis of SIG patients.

Expression of Tissue Transglutaminase in Cultured Bovine Trabecluar Meshwork Cells

Summary: To study whether cultured bovine trabecluar meshwork cells (BTMC) are capable of expressing tTG in protein and at mRNA level, BTMC were cultured in vitro and passaged three times, then the cells were transferred onto or cultured on sterile cover or submitted to isolation of RNA with Trizol, and the expression of tTG was detected by immunohistochemical technique and reverse transcription polymerase chain reaction (RT-PCR) respectively. Our results showed that tTG immunostaining was positive in the cytoplasm and rarely in the nucleus of cultured BTMC. No immunostaining was seen in the negative control. Moreover, a single RT-PCR amplified product whose sequence and size were in accordance with our known parameters was obtained. The expression of tTG in cultured BTMC was confirmed in protein and at mRNA level. BMTC is available more readily for the investigation of the relationship between tTG and primary open-angle glaucoma.

Identification, quantification and agerelated changes of human trabecular meshwork stem cells

Background:Loss of cells in the human trabecular meshwork(TM)has been reported with ageing and in glaucoma.This study aims to identify,quantify and determine the age-related changes of human TM stem cells(TMSCs).Methods:Isolation of TM cells/paraffin sectioning was carried out using human corneoscleral rings and whole globes.The TM cells/sections were immunostained for the stem cell markers ATP-binding cassette protein G2(ABCG2),nerve growth factor receptor p75 and AnkyrinG(AnkG).Images were acquired using Leica SP8 confocal microscope.The isolated cells were analyzed for two parameters-ABCG2 expression and nucleus to cytoplasmic ratio(N/C ratio).The total number of TM cells and those positive for ABCG2 and p75 in each section were quantified.Spearman rank order correlation was used to determine the association between age and the cell counts.Results:The TMSCs were identified based on two parameters-high ABCG2 expression and high N/C ratio>0.7.These stem cells were also positive for p75 and AnkG.The TMSC content based on the two parameters was 21.0±1.4%in<30 years age group,12.6±6.6%in 30–60 years and 4.0±3.5%in>60 years.The stem cells with high ABCG2 and p75 expression were restricted to the Schwalbe’s line region of the TM.A significant correlation was observed between the reduction in TMSC content and TM cell count during ageing.Conclusion:The human TMSCs were identified and quantified based on two parameter analysis.This study established a significant association between age-related reduction in TMSC content and TM cell loss.

Experimental study of growth of trabecular cells on the filters and hydraulic conductivity

Objectives To search the method of culturing human trabecular cells (HTC) on a filter support so as to provide a model to study the hydraulic conductivity of HTC in vivo.Methods The third passage of HTC was cultured on a nylon filter; after that we measured the rate of different irrigations through the filter with HTC [Lp, μl/(min· mm Hg·cm 2)]. Results HTC could continuously grow on the filters. The normal Lp was 10.45 μl/(min·mm Hg·cm 2). Irrigated by the solution of epinephrine (EPI) or dexamethasone (DEX), Lp of HTC were higher than that in controls of the same cultural time, while after being exposed to DEX for a few days, Lp was significantly decreased.Conclusions (1) More information of hydraulic conductivity and effects of pharmacologic agents on HTC could be got from the dynamic filtery model; (2) EPI could improve the conductivity of HTC while DEX could have the same effect in early period.

Role of mitochondria in the pathogenesis and treatment of glaucoma

Objective To gain insight into the potential mechanism of mitochondria dysfunction in pathogenesis, progression and therapeutic management of glaucoma. Data sources The data used in this review were mainly published in English from 2000 to present obtained from PubMed. The search terms were ＂mitochondria＂, ＂glaucoma＂ and ＂trabecular meshwork＂ or ＂retinal ganglion cells＂. Study selection Articles studying the mitochondria-related pathologic mechanism and treatment of glaucoma were selected and reviewed. Results Mitochondrial dysfunction or injury was demonstrated in different eye tissue of glaucoma. A variety of potential injuries （light, toxic materials, oxidative injury, mechanical stress, aging, etc.） and the inherent DNA defects are deemed to cause mitochondrial structural and functional destruction in trabecular meshwork cells, retinal ganglion cells, etc. of glaucoma. In addition, various new experimental and therapeutic interventions were used to preserve mitochondrial function, which may be useful for protecting against optic nerve degeneration or reducing the death of retinal ganglion cells in glaucoma. Conclusions Mitochondria play an important role in the pathogenesis of glaucoma, various strategies targeting mitochondrial protection might provide a promising way to delay the onset of glaucoma or protect RGCs against glaucomatous damage.