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^(-6)mol/L) group,HA1077(3×10^(-5)mol/L)group,and DEX(1×10^(-6)mol/L) and HA1077(3×10^(-5)mol/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.

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.

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.