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At least 307 records · Page 17Linked to original sources
PubMed2002

Transformation of human trabecular meshwork cells with SV40 TAg alters promoter utilization.

PURPOSE: To compare promoter usage in primary differentiated and SV40 TAg transformed human trabecular meshwork cells (HTM and TM1 cells). METHODS: Cultured HTM and TM1 cells were transfected with vectors expressing MYOC/TIGR from the CMV-IE, IE4/5 (HSV immediate early 4/5), ICP6 (early gene ICP6 of HSV), EF-1 alpha (human elongation factor 1 alpha-subunit), or the UB6 (human ubiquitin) promoters, respectively. Immunoblotting was used to measure MYOC/TIGR protein expression. MYOC/TIGR expression at the RNA level was detected by Northern blotting. RESULTS: In primary HTM cells, CMV was the only promoter displaying substantial activity. In TM1 cells, several promoters were functional with the order in decreasing activity being EF-1 alpha > or = CMV > or = UB6 >> IE4/5. CONCLUSIONS: The difference between the normal and transformed HTM cells suggests that the latter cell type has alterations that influence cellular promoter function. The type of cell used is likely to be a crucial factor in evaluating the functions of promoter elements for genes expressed in the trabecular meshwork and in screening promoters for use in gene delivery studies, especially for evaluations of the MYOC/TIGR gene in relation to glaucoma mechanisms.

Adult↗
PubMed1985

Histoautoradiographic and electron microscopic studies on short-term explant cultures of the glaucomatous trabecular meshwork.

Explant cultures of 15 trabeculectomy specimens derived from glaucomatous eyes were studied, using electron microscopy and histoautoradiography. The morphology of the trabecular meshwork was monitored for 3-14 days after explanation. In 4 cases the trabeculectomy specimens were cut in 2-3 pieces each and then incubated with 14C-glucosamine at different times during the observation period (2-10 days). The cells of the cribriform layer and the outer corneoscleral trabeculae become "activated" as soon as 3-4 days after explantation, developing a large amount of endoplasmic reticulum, Golgi material, and numerous mitochondria. Histoautoradiographically, intense labeling of the trabecular meshwork, especially the cribriform layer, was found after incubation with 14C-glucosamine. We assume that under tissue culture conditions the "activated" cells of the glaucomatous meshwork produce various types of glycosaminoglycans (GAGs) but, based on the experiments alone, no final conclusions can be made in this respect.

Autoradiography↗
PubMed2005

Alterations in human trabecular meshwork cell homeostasis by selenium.

Epidemiological evidence indicates that selenium supplementation may increase risk for glaucoma and ocular hypertension. The purpose of this study was to determine the effects of selenium on trabecular meshwork cells, a likely site of pathology for glaucoma. Human trabecular meshwork (HTM) cells and human umbilical vein endothelial cells (HUVECs) were treated with selenium (MSeA) at or near physiologically relevant concentrations. Selenium uptake by cells was monitored using mass spectrometry. Alterations in protein secretion, intracellular signaling, and cell morphology were monitored; and the role of integrin signaling in MSeA-induced morphological alterations was investigated using divalent cation treatments. Radiolabeling was used to assess protein synthesis and secretion, while luciferase and MTT assays monitored total cellular ATP and cell viability, respectively. Whereas detectible changes in intracellular selenium were observed after exposure to 1-10 microM MSeA for 24hr, the majority remained in the conditioned medium. Selenium-induced morphological changes (< or =3 hr) occurred before alterations in protein secretion and intracellular signaling (3-6 hr). Zinc treatment prevented selenium-mediated alterations in protein secretion and changes in cell-matrix adhesion. MSeA treatment (5 microM) led to a 60% decrease in protein synthesis after 3 hr and a 30% reduction in secretion, although significant alterations in cell viability and total ATP were not observed after MSeA treatment. Selenium altered several indicators of HTM cell homeostasis, but did not affect viability at physiologically relevant doses. Similar results with HUVECs have implications for understanding selenium's mechanisms of action as an anti-angiogenic agent.

Adult↗
PubMed1987

Exogenous glycosaminoglycans stimulate hyaluronic acid synthesis by cultured human trabecular-meshwork cells.

Addition of hyaluronic acid (50-200 micrograms ml-1) to the defined, serum-free media of cultured human trabecular-meshwork cells resulted in an increase of glycosaminoglycan (GAG) synthesis as measured by the incorporation of [14C]glucosamine. Lesser stimulatory effects were exerted by dermatan sulfate and chondroitin-4- or -6-sulfate. Nearly 90% of the labeled GAGs were found to be exerted into the medium and ea. 10% were associated with the cell layer. Mainly hyaluronic acid synthesis was stimulated by the exogenous GAGs. Analysis of the GAG-pattern revealed that exogenous hyaluronic acid stimulated hyaluronic acid synthesis (positive feedback), while exogenous dermatan sulfate and chondroitin sulfate had additional effects on chondroitin sulfate synthesis. Cell growth of these cultures, which exhibited a limited proliferative capacity (ca. 18 population doublings during their life span) was not affected by the GAG treatment. Thus, exogenous hyaluronic acid and to a lower degree dermatan sulfate or chondroitin sulfate appeared to interfere with the GAG-metabolism of these human trabecular-meshwork cells in culture.

Cells, Cultured↗
PubMed1985

Glutathione reductase of calf trabecular meshwork.

Hydrogen peroxide has been found in both calf and human aqueous humor at a level of 25 microM. It is likely, therefore, that trabecular meshwork possesses mechanisms for detoxifying H2O2, both to protect itself and other more distal structures of the outflow pathway from oxidative damage. We have recently demonstrated an active glutathione peroxidase in calf trabecular meshwork. In this study, we have characterized the complementary enzyme, glutathione reductase. The activity was present at a level of 0.120 units/min/g wet of tissue (0.005 units/min/mg soluble protein). The enzyme quickly lost activity in crude extracts but could be stabilized by heating at 60 degrees C for 30 min. Denatured protein was removed by centrifuging at 43,000 X g. Heating at 80 degrees C for 10 min destroyed all enzyme activity. Addition of 1 mM GSSG protected the enzyme completely from heat denaturation; NADP+ and GSH offered some protection but NADPH provided none. The supernatant from the 60 degrees C heat treatment was further purified by affinity chromatography on 2',5'-ADP-agarose. Overall purification was 200-fold with a yield of 80%. The pH optimum of the purified enzyme was 7.0. The KmS for NADPH and GSSG were 19 microM and 78 microM, respectively. The heat inactivation properties of the purified enzyme were identical to those in the crude extract. An enzyme activity stain on disc gel electrophoresis showed that the enzyme exists in only one form.

Animals↗
PubMed1996

alpha B-crystallin in the primate ciliary muscle and trabecular meshwork.

The presence of alpha B-crystallin, a protein with heat-shock protein-like properties, has been demonstrated in ciliary muscle and trabecular meshwork derived from human and monkey eyes using immunohistochemical and polymerase chain reaction methods. Both frozen sections and cultured cells have been analyzed. In the ciliary muscle, alpha B-crystallin staining is localized in the region of the dense bands, in the cytoplasm of the muscle cells and in the Schwann cells of the nerves supplying the muscle. In the trabecular meshwork, two cell types could be distinguished on the basis of alpha B-crystallin occurrence. Whereas the trabecular cells covering the lamellae were virtually devoid of the protein, the subendothelial or cribriform cells contained relatively large amounts in parallel with a higher alpha B-crystallin mRNA level.

Adult↗
PubMed1996

Expression of mesenger RNA for transforming growth factor-beta 1 in bovine trabecular meshwork.

PURPOSE: To investigate the relationship between transforming growth factor-beta 1 (TGF-beta 1) and primary open-angle glaucoma, we have determined whether trabecular tissues have the expression of messenger RNA for TGF-beta 1. METHODS: Total RNA of 24 newborn bovine trabecular tissue were extracted by Guanidine isothiocyanate method. The TGF-beta 33 plasmid was brought into E. colibacillius HB101 and amplificated. After Bam HI endolase degradation and label with alpha-32p-dATP the RNA was hybridized with the cDNA (complementary DNA) probe and examined by autoradiography. RESULTS: The presence of mRNA for TGF-beta 1 in bovine trabecular meshwork was confirmed. CONCLUSIONS: The TGF-beta 1 present in normal aqueous humor must be at least partly derived from the trabecular meshwork. It offered a basis for understanding the relationship between abnormal synthesis, activation and clearance of TGF-beta 1 and the pathogenesis of primary open-angle glaucoma (POAG) in molecular biology.

Animals↗
PubMed2004

Fibronectin overexpression inhibits trabecular meshwork cell monolayer permeability.

PURPOSE: To study whether excess synthesis of an extracellular matrix (ECM) component, fibronectin (FN), underlying the monolayer of human trabecular meshwork (HTM) cells, influences permeability. METHODS: To upregulate FN expression, HTM cells were grown in high glucose (30 mM) medium for 10 days. In parallel, cells were grown in normal (5 mM) medium as control, and two separate groups of HTM cells were grown in high glucose medium for transfection with FN antisense phosphorothioate oligonucleotides (AS-FN oligos) to modulate high glucose induced FN overexpression, or random phosphorothioate oligonucleotides (Ran oligos) as control. FN protein expression and distribution was assessed by western blot analysis and immunofluorescence microscopy. In parallel, HTM cells were grown in transwell plates in normal or high glucose medium to perform in vitro permeability (IVP) assays and to assess transelectrical resistance (TER). RESULTS: Western blot analysis showed FN expression was upregulated by 27% (p=0.018) in HTM cells grown in high glucose medium compared to cells grown in normal medium. Immunofluorescence microscopy showed intense FN immunostaining, and IVP results showed a consistent reduction in monolayer permeability (13% reduction, p=0.004) in cells grown in high glucose medium compared to cells grown in normal medium. When cells grown in high glucose medium were transfected with AS-FN oligos FN expression was reduced by 33% (p=0.009) and resulted in increased permeability to near normal levels (98+/-7% of control, p=0.01), whereas random oligos had no effect on either FN expression or IVP. TER was significantly increased across TM cell monolayers grown in high glucose compared to those grown in normal medium (143+/-11% of control, p=0.001), which was reduced when cells were transfected with AS-FN oligos (109+/-7% of control, p=0.02) whereas cells transfected with random oligos showed no change. CONCLUSIONS: Excess FN synthesis by trabecular meshwork cells may contribute to blockage in aqueous outflow associated with the development of primary angle open glaucoma (POAG).

Blotting, Western↗
PubMed2001

[Muscarinic receptor subtype-3 expression in trabecular meshwork of normal human eye and primary open-angle glaucoma].

OBJECTIVE: To identify the muscarinic receptor subtype-3 (M3-receptor) expression in trabecular meshworks (TM) of normal human eyes and eyes with primary open-angle glaucoma (POAG) and discuss the pathological changes of TM in POAG. METHOD: TM specimens were taken from 5 normal persons and 10 patients with POAG at late stage. Then LSAB immunohistochemical technique was used to identify M3-receptors. The results were observed under microscope and compared with computer-image-analysis system. RESULTS: (1) The trabecular meshwork cells in all the 5 normal persons expressed M3-receptors. The positive cells mainly existed in the uveal part of TM, from Schwalbe's line to scleral spur. (2) In TM from eyes with POAG, the amount of both TM cells and M3-receptor positive cells were reduced obviously, even some specimens had no such positive expression. The positive cells were correspondingly decreased in number and scattered in distribution. CONCLUSION: The TM cells in normal human eyes express M3-receptors. The TM cells and M3-positive cells in eyes with late POAG are reduced in number and their distribution is irregular.

Adolescent↗
PubMed2004

New ethacrynic acid derivatives as potent cytoskeletal modulators in trabecular meshwork cells.

A series of ethacrynic acid (ECA) derivatives were synthesized and examined for ocular hypotensive activity. Efficacy was evaluated in a cell-shape assay, using human trabecular meshwork cells, and cytotoxicity in a (3-(4,5-dimethylthiazole-2-yl)-5-(3-carboxymethoxy phenyl)-2-(4-sulfophenyl)-2H-tetrazolium) assay, using cultured bovine trabecular meshwork cells. Many of the derivatives demonstrated efficacy equal to or greater than that of ECA. SA9000 was selected as the most promising candidate for a novel ocular hypotensive drug with few side effects.

Animals↗
PubMed1997

[Expression of messenger RNA for transforming growth factor-beta 1 in bovine trabecular meshwork].

OBJECTIVE: To investigate the relationship between transforming growth factor-beta 1 (TGF-beta 1) and primary open-angle glaucoma. METHODS: Total RNA of 24 new born bovine trabecular tissue was extracted by guanidine isothiocyanate method. The TGF-beta 33 plasmid was brought into colibacillus HB101 and amplified. After Bam HI enzyme degradation and label with alpha-32P-dATP, the RNA was hybridized with the complementary DNA (cDNA) probe and examined by autoradiography to determine the expression of messenger RNA (mRNA) for TGF-beta 1. RESULTS: The presence of mRNA for TGF-beta 1 in bovine trabecular meshwork was confirmed. CONCLUSION: The TGF-beta 1 present in normal aqueous humor at least partly is derived from the trabecular meshwork. The result offers a basis for understanding the relationship between abnormal synthesis, activation and clearance of TGF-beta 1 and the pathogenesis of primary open-angle glaucoma in molecular biology.

Animals↗
PubMed2006

Effects of latrunculin-B on outflow facility and trabecular meshwork structure in human eyes.

PURPOSE: To determine the effect of the F-actin-disrupting agent latrunculin-B on aqueous outflow facility and trabecular meshwork architecture in human eyes. METHODS: After baseline facility measurement in human eye bank eyes (n = 9 pairs), one eye of each pair received anterior chamber exchange and continued perfusion with medium containing 1 microM latrunculin-B. Contralateral eyes were treated in a similar manner with vehicle. Eyes were fixed by anterior chamber exchange and perfusion with universal fixative at 8 mm Hg (corresponding to a physiologic pressure of 15 mm Hg in vivo), and outflow pathway tissues were examined by transmission and scanning electron microscopy. RESULTS: Perfusion of eyes with 1 microM latrunculin-B caused a continuous and ongoing increase in outflow facility, resulting in a net facility difference of 64% 2 hours after drug administration (P < 0.006). Transmission electron microscopy showed subtle and focal detachment of the inner wall of Schlemm's canal, rarefaction of the juxtacanalicular tissue (JCT), and cell-cell and cell-matrix detachment. Scanning electron microscopy showed collapsed vacuoles in the inner wall of Schlemm's canal and a marked increase in the number and size of border (paracellular) pores in the inner wall. CONCLUSIONS: Latrunculin-B increases outflow facility in postmortem human eyes. The mechanism of facility increase is most likely due to loss of mechanical integrity of the trabecular meshwork as a consequence of reduction in cell-cell and cell-matrix adhesion. The facility increase and the extent of inner wall separation from the JCT that we observed were both qualitatively similar to that reported in living monkey eyes, but the magnitude of the facility increase and morphologic changes were much less than in the living monkey. This supports the idea that inner wall separation from the JCT may modulate outflow facility.

Aged↗
PubMed1998

Microtubule disruption leads to cellular contraction in human trabecular meshwork cells.

PURPOSE: To determine whether microtubule- and actin-altering drugs, which have been shown to increase aqueous humor outflow, cause cellular contraction in human trabecular meshwork (HTM) cells. METHODS: HTM cells were plated in culture dishes containing a polymerized deformable silicone substrate. After 48 hours, the dishes were placed on an inverted microscope and treated with ethacrynic acid, colchicine, vinblastine, cytochalasin B, or 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7) and then recorded on videotape for 15 minutes. An increase in silicone substrate wrinkle size and/or number indicated a contraction. Sham controls were used. RESULTS: Cellular contraction was observed with ethacrynic acid, colchicine, and vinblastine in the 10(-5) to 10(-4) M dosage range. Pretreatment with H-7 blocked these effects. Cytochalasin B did not produce cellular contraction. CONCLUSIONS: Microtubule disruption causes cellular contraction in HTM cells, and this effect depends on an intact actin cytoskeleton network. Contraction of trabecular meshwork cells in response to various stimuli is an attractive hypothesis for possible homeostatic mechanisms in the outflow pathway, and this may serve as a focus for novel glaucoma drug development.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1983

Laser energy levels for trabecular meshwork damage in the primate eye.

The authors treated the trabecular meshwork of 17 primate eyes with the argon laser by a variety of protocols in an attempt to cause moderate, consistent intraocular pressure elevation. This was achieved most satisfactorily with deliveries of 0.5 to 1.0 seconds and a total energy of at least 50 joules. The method allows production of a satisfactory laser-induced model of chronic glaucoma with one treatment session in most eyes. This experience provides a measure of the upper limit of acceptable total laser energy for therapeutic procedures in humans with this instrument. Delivery durations longer than 0.1 sec should be used with caution in trabeculoplasty.

Animals↗
PubMed1997

Experimental erbium: YAG laser photoablation of trabecular meshwork in rabbits: an in-vivo study.

Photoablative laser trabecular surgery has been proposed as an outflow-enhancing treatment for open-angle glaucoma. The aim of the study was to investigate the time course of repair response following low-thermal Erbium: YAG laser trabecular ablation. In 20 anaesthetized rabbits gonioscopically controlled ab-interno photoablation of the ligamenta pectinata and underlying trabecular meshwork (TM) was performed with a single-pulsed (200 microseconds) Erbium: YAG (2.94 microns) laser. The right eye received 12-15 single laser pulses (2 mJ) delivered through an articulated zirconium fluoride fiberoptic and a 200 microns (core diameter) quartz fiber tip, the left unoperated eye served as control. At time intervals of 30 minutes, 2, 10, 30, and 60 days after laser treatment, eyes were processed for light- and scanning electron microscopy. The applied energy density of 6-4 J cm-2 resulted in visible dissection of the ligamenta pectinata and reproducible microperforations of the TM exposing scleral tissue accompanied by blood reflux from the aqueous plexus. The initial ablation zones measured 154 +/- 36 microns in depth and 45 +/- 6 microns in width. Collateral thermal damage zones were 22 +/- 8 microns. At two days post-operative, ablation craters were still blood- and fibrin-filled. The inner surface of the craters were covered with granulocytes. No cellular infiltration of the collateral thermal damage zone was observed. At 10 days post-operative, progressive fibroblastic proliferation was observed, resulting in dense scar tissue formation with anterior synechiae, proliferating capillaries and loss of intertrabecular spaces inside the range of former laser treatment at 60 days post-operative. Trabecular microperforations were closed 60 days after laser treatment in all rabbits. IOP in treated and contralateral eyes did not significantly change its level during whole period of observation. Low-thermal infrared laser energy with minimal thermal damage to collateral structures could not effectively prevent early scarring of trabecular surgery in rabbits.

Animals↗
PubMed1993

Increased hydrolase activities in the human trabecular meshwork of glaucomatous eyes.

Alterations in the metabolic functions of trabecular meshwork (TM) cells are thought to be involved in the pathogenesis of primary open-angle glaucoma (POAG). In an investigation of this possibility, 30 trabeculectomy specimens from patients with POAG were examined histochemically for 11 lysosomal and membrane-bound enzymes. The patients ranged from 48 to 87 years in age. The degree of enzyme staining was compared with that of 15 age-matched controls obtained from an eye bank at less than 24 h after death. There was no history of eye disease in the controls. The enzymes examined were: dipeptidylpeptidases II and IV (DPPII and IV); beta-glucuronidase (beta-GLUC); acid-beta-galactosidase (s beta-GAL); N-acetyl-beta-D-glucosaminidase (NAG); nonspecific esterase (UE); acid phosphatase (SP); alkaline phosphatase (ALP); gamma-glutamyltransferase (GGT); and aminopeptidase A and M (APA and APM). Evaluation of the specimens was performed by two observers and by computer-aided optic densitometry. Results showed increased staining of SP, UE, GGT and APM in the pathological specimens as compared with the controls. SP and UE indicate phagocytic activity, APM is involved in collagen turnover and GGT participates in both drug detoxification and the breakdown of glutathione in the gamma-glutamyl cycle. Our observations show different hydrolase activities in the TM cells of human glaucomatous eyes as compared with normal values, suggesting that such metabolic differences may be related to the pathogenesis of POAG.

Aged↗
PubMed2002

Human trabecular meshwork cell volume regulation.

The volume of certain subpopulations of trabecular meshwork (TM) cells may modify outflow resistance of aqueous humor, thereby altering intraocular pressure. This study examines the contribution that Na+/H+, Cl-/HCO exchange, and K+-Cl- efflux mechanisms have on the volume of TM cells. Volume, Cl- currents, and intracellular Ca2+ activity of cultured human TM cells were studied with calcein fluorescence, whole cell patch clamping, and fura 2 fluorescence, respectively. At physiological bicarbonate concentration, the selective Na+/H+ antiport inhibitor dimethylamiloride reduced isotonic cell volume. Hypotonicity triggered a regulatory volume decrease (RVD), which could be inhibited by the Cl- channel blocker 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB), the K+ channel blockers Ba2+ and tetraethylammonium, and the K+-Cl- symport blocker [(dihydroindenyl)oxy]alkanoic acid. The fluid uptake mechanism in isotonic conditions was dependent on bicarbonate; at physiological levels, the Na+/H+ exchange inhibitor dimethylamiloride reduced cell volume, whereas at low levels the Na+-K+-2Cl- symport inhibitor bumetanide had the predominant effect. Patch-clamp measurements showed that hypotonicity activated an outwardly rectifying, NPPB-sensitive Cl- channel displaying the permeability ranking Cl- > methylsulfonate > aspartate. 2,3-Butanedione 2-monoxime antagonized actomyosin activity and both increased baseline [Ca2+] and abolished swelling-activated increase in [Ca2+], but it did not affect RVD. Results indicate that human TM cells display a Ca2+-independent RVD and that volume is regulated by swelling-activated K+ and Cl- channels, Na+/H+ antiports, and possibly K+-Cl- symports in addition to Na+-K+-2Cl- symports.

Bicarbonates↗
PubMed2006

Protein expression in a transformed trabecular meshwork cell line: proteome analysis.

PURPOSE: Characterization of the human trabecular meshwork (TM) proteome is hindered by the small mass of intact tissue and the slow growth of cultured cell strains. We have previously characterized a transformed TM cell strain (GTM3) that demonstrates many of the same protein expression and cell signaling systems of nontransformed cell strains. The aim of this study was to initiate a proteomic survey of GTM3 cells as the initial step toward characterization of the complete human TM proteome. METHODS: GTM3 cells were cultured to confluence, harvested and solubilized in urea/Nonidet. The protein extract (600 mug) was focused in immobilized isoelectric focusing (IEF) strips, separated by 10% SDS PAGE, and visualized with colloidal Coomassie Blue. Spots of interest were excised, destained, and the contained proteins subjected to in-gel reduction, derivatization, and tryptic digestion. Tryptic peptides were extracted and analyzed by electrospray LC/MS/MS. Protein identification was made using the TurboSequest search algorithm and a recent version of the nonredundant human protein database downloaded from the National Center for Biotechnology Information (NCBI). RESULTS: Eighty-seven (87) primary proteins and 93 variants of these proteins were identified. A website was created (TM proteome) that combines data such as graphic spot location within the gel, peptide sequence, apparent and calculated pI, apparent and calculated mass, percentage of coverage, and protein informatic website links. CONCLUSIONS: Proteomic analysis of a transformed human TM cell line has been initiated combining preparative two-dimensional PAGE separation, LC/MS/MS analysis of major proteins, and bioinformatic cataloging of the data. Further investigation of data from the transformed cell strain will be used in a comparative fashion for spot identification of analytical proteomic gels of human TM tissue and cultured normal cells. These initial data will form the base from which the characterization of protein expression in the normal and glaucomatous TM can be accomplished.

Cell Line, Transformed↗