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At least 523 records · Page 29Linked to original sources
PubMed2005

Soluble CD44 is cytotoxic to trabecular meshwork and retinal ganglion cells in vitro.

PURPOSE: Current glaucoma research targets neuroprotective therapies for retinal ganglion cells (RGCs) in primary open-angle glaucoma (POAG). The purpose of this study was to determine whether the 32-kDa ectodomain fragment of CD44-soluble CD44 (sCD44)-which is increased in the aqueous of patients with POAG, affects RGC and trabecular meshwork (TM) cell survival in vitro. METHODS: sCD44 was isolated from human or fetal calf serum (FCS) by urea solubilization and immunoprecipitation. A transformed rat RGC-like cell line (RGC-5), human and bovine TM cells, and control cells were grown in Dulbecco's modified Eagle's medium containing 10% FCS until confluent and then were incubated in medium containing 0.1% FCS and treated with various doses of purified sCD44 and 17-alpha-methyl testosterone (17-alpha-MT). The cytotoxicity of sCD44 was verified by heat-inactivation, pretreatment with a pan-caspase inhibitor, and coadministration of anti-CD44 neutralizing antibody or hyaluronic acid (HA). Cell viability was assessed by trypan blue staining, cell counting, and phase-contrast microscopy. RESULTS: There was a statistically significant dose- and time-dependent decrease in the number of cells and viability in the RGC-5 and TM cells treated with sCD44. Within 12 hours of sCD44 treatment, RGC-5 and TM cells displayed cell rounding, detachment, and swelling. sCD44-induced cell death was cell specific. Smooth muscle cells were resistant to sCD44, whereas human cortical neuronal-like cells were susceptible to sCD44 after 24 hours, but recovered. The cytotoxicity of sCD44 was blocked by heat-inactivation, pretreatment with a pan-caspase inhibitor, or coadministration of anti-CD44 antibody or HA. 17-alpha-MT prevented sCD44 cytotoxicity in both RGC-5 and TM cells. CONCLUSIONS: The results indicate that exogenous sCD44 adversely affects RGC-5 and TM cell survival in vitro by activating proapoptotic pathways.

Animals↗
PubMed1996

Cultured human trabecular meshwork cells express functional alpha 2A adrenergic receptors.

PURPOSE: For the treatment of glaucoma, alpha-2 adrenergic receptor (alpha 2-AR) agonists are thought to lower intraocular pressure primarily by decreasing aqueous humor production. Effects on the outflow pathways, however, also may occur. To begin to examine this possibility, the authors characterized the alpha 2-AR subtypes present in cultures of human trabecular meshwork (HTM) cells using both immunofluorescence microscopy and functional measures of alpha 2-AR activation. METHODS: For immunofluorescence microscopy, subtype-specific polyclonal antibodies that recognize each of the human alpha 2-AR subtypes (alpha 2A, alpha 2B, alpha 2C) were used. Functional studies involved the inhibition of forskolin-stimulated cyclic adenosine monophosphate (cAMP) production, the stimulation of mitogen-activated protein (MAP) kinase activity, and the stimulation of mitotic activity as reflected by the expression of proliferating cell nuclear antigen (PCNA). RESULTS: From the immunofluorescence microscopy, there was evidence for the presence of the alpha 2A subtype, but not alpha 2B or alpha 2C subtype, on HTM cells. The administration of the alpha 2-agonist, dexmedetomidine, to HTM cells resulted in a 90% inhibition of forskolin-stimulated cAMP formation, a twofold stimulation of MAP kinase activity, and a threefold increase in the expression of PCNA. Additionally, preincubation of cells with either of the alpha 2-AR-selective antagonists, rauwolscine or atipamezole, reversed the functional effects of dexmedetomidine. CONCLUSIONS: Functional alpha 2A-ARs are present on HTM cells where they may affect the outflow pathway during the treatment of glaucoma with alpha 2-AR agonists.

Adrenergic alpha-Agonists↗
PubMed1999

Adenoviral reporter gene transfer to the human trabecular meshwork does not alter aqueous humor outflow. Relevance for potential gene therapy of glaucoma.

Obstruction of the aqueous humor outflow from the anterior chamber of the eye leads to an elevation of intraocular pressure in glaucoma, the second major cause of blindness worldwide. Our goal is to be able to modulate aqueous humor outflow resistance by gene transfer to the cells of the trabecular meshwork (TM). We have previously shown that adenoviral vectors are able to transfer a reporter gene to the TM of postmortem human donors. However, assessing gene therapy for glaucoma requires models that can monitor changes in aqueous humor outflow facility (C = flow/pressure). In this study we used four replication-deficient adenoviruses in two such perfusion models. In the first model, whole porcine eyes were infected, perfused at constant pressure and flow changes recorded for 5 h. In the second one, anterior segments from human eyes were infected, perfused at constant flow and pressure changes recorded for 3 days. A single dose of 10(8) adenovirus plaque forming units (pfu) causes a reduction in C while single doses of 10(7), 10(6) and 10(5) p.f.u. do not affect outflow facility and retain positive gene transfer. These findings indicate that adenovirus, at effective doses, could become useful vectors for gene therapy of glaucoma.

Adenoviridae↗
PubMed2001

Non-secretion of mutant proteins of the glaucoma gene myocilin in cultured trabecular meshwork cells and in aqueous humor.

Until recently, very little was known about the molecular mechanisms responsible for the development of glaucoma, a leading cause of blindness worldwide. Mutations in the glaucoma gene myocilin (MYOC, GLC1A) are associated with elevated intraocular pressure and the development of autosomal dominant juvenile glaucoma and a subset of adult-onset glaucoma. MYOC is expressed in the trabecular meshwork (TM), a tissue responsible for drainage of aqueous humor from the eye, and the tissue involved in elevated intraocular pressure associated with glaucoma. To better understand the role of MYOC in glaucoma pathogenesis, we examined the expression of normal and mutant myocilin in cultured ocular (TM) and non-ocular cells as well as in the aqueous humor of patients with and without MYOC glaucoma. Normal myocilin was secreted from cultured cells, but very little to no myocilin was secreted from cells expressing five different mutant forms of MYOC. In addition, no mutant myocilin was detected in the aqueous humor of patients harboring a nonsense MYOC mutation (Q368X). Co-transfection of cultured cells with normal and mutant myocilin led to suppression of normal myocilin secretion. These studies suggest that MYOC glaucoma is due either to insufficient levels of secreted myocilin or to compromised TM cell function caused by congestion of the TM secretory pathway.

Animals↗
PubMed2005

Effects of novel ethacrynic acid derivatives on human trabecular meshwork cell shape, actin cytoskeletal organization, and transcellular fluid flow.

To determine efficacy and therapeutic index in the context of ocular hypotensive activity of the new ethacrynic acid (ECA) derivatives of the series (SA8,248 and SA8,389), 9,000 series (SA9,000, SA9,622 and SA9,995) and ticrynafen, we undertook a comparative evaluation of the dose-dependent effects of these compounds on human trabecular meshwork (HTM) cell shape, actin cytoskeletal organization, focal adhesions and transcellular fluid flow. Responses were either scored using an arbitrary scale of 1-5 or quantified. Compounds of the 9000 series (SA9,995>SA9,000>SA9,622) were found to be 14- to 20-fold more potent than ECA, ticrynafen or analogs from the 8,000 series (SA8,389>SA8,248) in terms of ability to induce cell shape alterations in HTM cells. Similarly, compounds of the 9,000 series (SA9,995>SA9,622>SA9,000) were found to be much stronger (2 to 20 fold) than ECA, ticrynafen or analogs of the 8000 series in terms of affecting decreases in actin stress fiber content in HTM cells. Analogs of the 9000 series (SA9,622>SA9,995>SA9,000) were also observed to be 8 to 10 fold more potent than ECA (SA8,389>ECA>SA8,248>ticrynafen) at eliciting decreases in cellular focal adhesions. Interestingly, analogs of the 9000 series (SA9,000>SA9,622>SA9,995) and SA8,248 demonstrated a huge increase (by many folds) in transcellular fluid flow of HTM cell monolayers as compared to ECA and ticrynafen. Collectively, these analyses revealed that the structural modification of ECA improves its ocular hypotensive efficacy, indicating that the SA9,000 series compounds might be promising novel ocular hypotensive drugs.

Actins↗
PubMed2001

Mechanisms of action of unoprostone on trabecular meshwork contractility.

PURPOSE: This study was performed to clarify the possible mechanism behind the ocular hypotensive effect of unoprostone isopropyl (Rescula; Novartis Ophthalmics AG, Basel, Switzerland), a new docosanoid that has been shown to reduce intraocular pressure (IOP) in patients with ocular hypertension or primary open-angle glaucoma. To gain insight into the possible mode of action, the effects of unoprostone on ciliary muscle (CM) and trabecular meshwork (TM) contractility, intracellular calcium levels, and membrane channels were investigated. METHODS: The effects of unoprostone (M1 metabolite = free acid, 10(-5) M) and endothelin (ET)-1 (10(-9) M) on bovine TM (BTM) and ciliary muscle (CM) strips were investigated, by using a custom-made force-length transducer system. The effects of unoprostone and ET-1 (5 x 10(-8) M) on intracellular Ca(2+) mobilization in cultured human TM (HTM) were measured using fura-2AM as a fluorescent probe. Patch-clamp experiments were performed on HTM and BTM cells to investigate the unoprostone-dependent modulation of membrane currents. RESULTS: In isolated TM and CM strips, unoprostone almost completely inhibited ET-induced contractions (TM: 2.9% +/- 4.3% vs. 19.6% +/- 5.7%, P < 0.05, n = 6; CM: 1.4% +/- 1.6% vs. 30.1% +/- 5.3%, P < 0.01, n = 6; 100% = maximal carbachol-induced (10(-6) M) contraction). However, neither carbachol-induced contraction nor baseline tension was affected by unoprostone. Furthermore, unoprostone had no effect on baseline intracellular calcium levels (baseline: 126 +/- 45 nM versus unoprostone: 132 +/- 42 nM, n = 8) in HTM cells. The endothelin-induced increase (679 +/- 102 nM), however, was almost completely (P < 0.01) blocked by unoprostone (178 +/- 40 nM). In patch-clamp recordings, unoprostone could be shown to double the amplitude of outward current (HTM: 200% +/- 33%; n = 6; BTM: 179% +/- 20%; n = 8). This effect was blocked by the specific inhibitor of maxi-K channels, iberiotoxin. CONCLUSIONS: This study presents evidence for direct interaction of unoprostone with the contractility of the TM and CM. This compound may lower IOP by affecting aqueous outflow, most probably conventional outflow pathways (i.e., TM) through inhibition of ET-dependent mechanisms. In addition, unoprostone interacts with the maxi-K channel. Although primarily Ca(2+)-sensitive signal-transduction pathways seem to be involved, effects of unoprostone on Ca(2+)-independent pathways and uveoscleral outflow cannot be excluded.

Adult↗
PubMed2003

Common actions of adenosine receptor agonists in modulating human trabecular meshwork cell transport.

A(1) adenosine receptors (ARs) reduce, and A(2)ARs increase intraocular pressure, partly by differentially altering resistance to aqueous humor outflow. It is unknown whether the opposing effects of A(1)AR and A(2)AR agonists are mediated at different outflow-pathway cell targets or by opposing actions on a single cell target. We tested whether a major outflow-pathway cell, the trabecular meshwork (TM) cell might constitute the primary AR-agonist target and respond differentially to A(1), A(2A) and A(3)AR agonists. Receptor activation in human TM cells was identified by applying subtype-selective AR agonists: CPA and ADAC for A(1)ARs, CGS 21680 and DPMA for A(2A)ARs, and Cl-IB-MECA and IB-MECA for A(3)ARs. Stimulation of A(1), A(2A) and A(3)ARs elevated Ca(2+), measured with fura-2. Whole-cell patch clamping indicated that AR agonists activated ion channels non-uniformly, possibly reflecting variability in magnitude of agonist-triggered second-messenger responses. A(1), A(2A) and A(3)AR agonists all reduced volume, determined by calcein cell imaging. The endogenous source of adenosine delivery to the outflow pathway could be the TM cells since these cells were stimulated to release ATP by hypotonic perfusion. We conclude that: (1) TM cells express functional A(1), A(2A) and A(3)ARs; and (2) the reported differential effects of AR agonists on aqueous humor outflow are not mediated by differential actions on TM-cell Ca(2+) and volume, but likely by actions on separate cell targets.

Adenosine↗
PubMedDate not supplied

Ultrastructural changes in the trabecular meshwork of congenital glaucoma.

Trabeculectomy specimens from 10 patients with congenital glaucoma were studied by electron microscopy. In all cases proliferation of fibrous connective tissue was found from the inner wall of Schlemm's canal. Accumulation of collagen fibres, sometimes of varied thickness and irregular striation, and agglomerations of microfibrillar material with the structure of collagen microfibrils were observed. This material formed basement membrane-like structures and 'shaggy' or fingerprint-like patterns. The ultrastructural examination of the trabecular meshwork revealed no significant differences between the morphological picture of primary congenital glaucoma and that found in Axenfeld and Rieger anomalies. Ultrastructural pictures of glaucoma cases examined in the present study converged also with those of juvenile glaucoma cases reported in literature, which may indicate the existence of a pathogenic factor common to these forms of glaucoma.

Female↗
PubMed2006

First look at the effect of overexpression of TIGR/MYOC on the transcriptome of the human trabecular meshwork.

Wild-type TIGR/MYOC is a secreted protein implicated in the development of steroid glaucoma. Mutations in TIGR/MYOC have been linked to some patients who develop elevated intraocular pressure (IOP) and glaucoma. Because there is evidence of some other factors contributing to the TIGR/MYOC causative role in glaucoma, and because substantial increased levels of a particular cellular mRNA and protein might alter expression of other host genes, we began to investigate the effect of TIGR/MYOC overexpression on the transcriptome of human trabecular meshwork cells. We used a recombinant adenovirus carrying wild-type TIGR/MYOC cDNA, primary HTM cells, 300 viral particles per cell and U133 Affymetrix GeneChips. Our results indicate that 2361 out of the 22,284 genes (10.6%) were altered more than two-fold (p<or=0.005) by the overexpression of TIGR/MYOC. A higher proportion of the altered genes were downregulated (1412 vs. 949). Potentially relevant upregulated genes include angiopoietin 2, matrix metalloproteinase 1 (MMP1) and thrombomodulin; among those downregulated we observed growth arrest specific 1, proteins involved in the ubiquitination pathway and vascular cell adhesion molecule 1. In addition, collagen type 1, one of the MMP1 substrates, was also downregulated. Genes affected by wild-type TIGR/MYOC might prove to be candidate mediators for future studies of the mechanisms of glaucoma.

Angiopoietin-2↗
PubMed2005

Gene transfer to trabecular meshwork endothelium via direct injection into the Schlemm canal and in vivo toxicity study.

PURPOSE: Our aim was to investigate the efficiency of adenoviral gene transfer via direct injection into the Schlemm canal ex vivo in human donor eyes and to examine the effect of human MMP-3 transgene expression in a rat model in vivo. METHODS: A viscocanalostomy-like operation was performed and adenoviral vector encoding for MMP-3 and green fluorescent protein was injected into human Schlemm canal or rat anterior chamber. RESULTS: Transgene expression was high in trabecular meshwork endothelium in human donor eyes. In vivo, adenovirus caused dose-dependent inflammation. CONCLUSIONS: Direct injection of adenoviral vectors into the Schlemm canal has potential in glaucoma treatment.

Adenoviridae↗
PubMed1996

Inhibition of dexamethasone-induced cytoskeletal changes in cultured human trabecular meshwork cells by tetrahydrocortisol.

PURPOSE: To determine the cellular mechanism of action of the intraocular pressure (IOP) lowering steroid tetrahydrocortisol (THF). METHODS: Tetrahydrocortisol was evaluated for glucocorticoid antagonist activity using in vitro and in vivo assays. Systemically administered THF was evaluated for its ability to inhibit dexamethasone-induced body weight loss and systemic hypertension in rats. In vitro receptor antagonism was tested using the supernatant fraction of IM9 cells as the source of soluble glucocorticoid receptor in 3H-dexamethasone displacement binding assays. In addition, six different primary human trabecular meshwork (TM) cell lines were cultured for 0 to 14 days in the absence or presence of dexamethasone (10(-7) M) and/or THF (10(-6) to 10(-8) M). The effects of these steroids on the TM cytoskeleton were determined by epifluorescent microscopy and by transmission electron microscopy. RESULTS: Tetrahydrocortisol was unable to inhibit the dexamethasone (DEX)-induced systemic hypertension and decrease in body mass in rats and was unable to displace 3H-DEX from the soluble human glucocorticoid receptor. However, THF inhibited the DEX-induced formation of cross-linked actin networks in cultured human TM cells in a progressive and dose-dependent manner (IC50 = 5.7 x 10(-7) M). Dexamethasone caused changes in the TM cell microtubules that were reversed partially by concomitant treatment with THF. Tetrahydrocortisol alone appeared to increase microfilament bundling in TM cells. CONCLUSIONS: Tetrahydrocortisol was not a glucocorticoid antagonist at the level of the classical glucocorticoid receptor and did not appear to antagonize systemically mediated glucocorticoid activity in the rat. Tetrahydrocortisol inhibited DEX-induced changes in the TM microfilaments and microtubules. These results may explain partially the IOP lowering activity of THF because glucocorticoid-mediated changes in the TM cytoskeleton have been proposed to be involved in the generation of ocular hypertension.

Actins↗
PubMed1999

A trabecular meshwork glucocorticoid response (TIGR) gene mutation affects translocational processing.

PURPOSE: To examine possible effects of the E323K mutation in the trabecular meshwork glucocorticoid response (TIGR) gene (also known as myocilin [MYOC]), using assays of translocational processing through the endoplasmic reticulum (ER). The E323K mutation was of particular interest, since the mutation shows a strong association with early onset open-angle glaucoma, but has a minimal predicted effect on protein structure. METHODS: Normal and mutant TIGR cDNA constructs were used to generate protein products in the presence of endoplasmic reticulum (ER) membranes, using an assay previously developed to detect alterations in the ER translocation function. "Paused" regions for potential protein modifications were defined by proteinase K (PK) sensitivity in the presence of ER membranes, with the ability to restart translocation when treated with EDTA. The effects of the E323K mutation were evaluated, as well as mutations located on either side of E323K (G246R, G364V, P370L) as the other mutations had substantial predicted structural changes in addition to clear disease associations. RESULTS: The native TIGR molecule was observed to have a paused region that corresponds to the region of highest olfactomedin (OLF) homology. The E323K mutation, located near the beginning of this region, dramatically altered the normal pattern of nascent proteins observed in the translocational pausing assay. A prominent band appeared with the E323K mutation, which could represent a new product or a marked enhancement of a faint band normally seen, approximately 3 kDa higher than the major paused band. The other TIGR mutants examined did not show this effect. CONCLUSIONS: The major translocational pause that starts near the beginning of the region of high OLF homology may help to explain the high frequency of glaucoma-associated mutations in this area. The observed effect of the E323K mutation on the products of translocational processing suggests a delay in the normal pausing process of TIGR biogenesis. This delay points to a potentially distinct pathogenic mechanism for E323K as compared with the other TIGR mutations so far evaluated.

Cytoskeletal Proteins↗
PubMed1990

Quantitative analysis of collagen content and amino acids in trabecular meshwork.

The purpose of this study was to compare collagen content in the TM of normal and glaucomatous eyes, and to establish whether collagen levels change with age. Collagen content was measured in 30 normal and 27 age matched glaucoma trabeculectomy specimens by the sirius red dye binding technique, and in 14 normal and 15 age matched glaucoma specimens by amino acid analysis. Both dye binding data and amino acid analysis showed no statistical difference between normal and glaucoma samples. Age had no significant effect on mean optical densities or on the collagen-specific amino acids proline, hydroxyproline, and hydroxylysine. Amino acid variability, however, was statistically different between the two groups. These results indicate that mean collagen levels in the trabecular meshwork of glaucomatous eyes do not differ from those in normal eyes.

Adult↗
PubMed2005

Effects of dinucleoside polyphosphates on trabecular meshwork cells and aqueous humor outflow facility.

The most important risk factor for the development of glaucoma is elevated intraocular pressure (IOP). Hypotensive drugs decrease IOP, preventing optic nerve damage and further vision loss. The balance between aqueous humor (AH) production and drainage determines IOP, and problems in AH outflow pathways are associated with open-angle glaucoma development. Previous studies have shown the presence of diadenosine tetraphosphate (Ap(4)A) and pentaphosphate (Ap(5)A) in the AH. Topic application of Ap(4)A to the cornea decreased IOP, whereas Ap(5)A increased it. Because dinucleoside polyphosphates stimulate P2Y purinergic receptors, we studied their presence in trabecular meshwork (TM) cells. Additionally, the effects of diadenosine polyphosphates (Ap(n)As; n = 3-5) and Up(4)U (P(1),P(4)-(diuridine 5')-tetraphosphate; INS365) in outflow facility were tested. P2Y(1), P2Y(2), and P2Y(4) receptors were detected in TM cells by Western blot and immunocytochemistry. In TM cells, Ap(3)A, Ap(4)A, and Ap(5)A induced discrete intracellular calcium concentration ([Ca(2+)](i)) mobilizations compared with higher and more sustained [Ca(2+)](i) mobilizations after Up(4)U application. In bovine ocular anterior segments perfused at constant pressure, 1 microM Ap(3)A or Ap(4)A increased outflow facility, whereas Up(4)U or Ap(5)A did not modify it. 2-MeSADP, a selective P2Y(1) agonist, induced outflow facility increases similar to those obtained after Ap(3)A and Ap(4)A, and these were prevented by addition of the selective P2Y(1) receptor antagonist MRS-2179 (2'-deoxy-N(6)-methyladenosine-3',5'-diphosphate). Our results demonstrate that the hypotensive effect of Ap(4)A and other dinucleotides is mediated, at least in part, by increasing trabecular outflow facility through activation of P2Y(1) receptors. The latter would seem to be an interesting target in the development of antiglaucomatous drugs to selectively increase AH outflow.

Animals↗
PubMed1989

Obstruction of the trabecular meshwork by retinal rod outer segments.

Rod outer segments (ROS) have been described in the anterior chamber of patients with the Schwartz syndrome (retinal detachment, uveitis, and glaucoma). The effect of ROS on aqueous outflow was studied by intracameral injection of ROS in human eye bank eyes and in vivo in the cat. Injection of 10(8) ROS in eye bank eyes (n = 8) caused a 19.7% decrease in outflow facility. This was significantly different than in control eyes (n = 8), where the outflow facility decreased 5.2%. Injection of intracameral ROS in the cat resulted in a rise in intraocular pressure (IOP) from a baseline of 19.7 +/- 1.3 mm Hg to a peak of 30.0 +/- 0.7 mm Hg at 24 hours. By 48 hours, IOP returned to baseline. Control injection did not cause a change in IOP. Electron microscopy of the cat trabecular meshwork revealed occlusion of the intratrabecular spaces by ROS with little evidence of inflammatory activity.

Aged↗
PubMed1987

Collagen distribution in the lamina cribrosa and the trabecular meshwork of the human eye.

Tengroth and Ammitzböll found the collagen content of the optic disc in glaucoma to differ from that of normal eyes. A theory was advanced that a primary collagen disturbance might be involved in the pathogenesis of glaucoma. The connective tissue in the body has a supportive function in almost all the organs. The tensile strength and elasticity of connective tissue is mainly due to the presence of collagen fibres and elastic fibres, which also maintain the shape of the tissues. There are many different types of collagen, three of which are discussed in this paper. Type I collagen is found in tendons, skin, and numerous other organs, for example the eye. Type III is found mainly in the blood vessels but is also present in other tissues with a mesodermal origin, and type IV is found in the basement membranes. To elucidate the precise distribution of collagen types in the ocular structures an immunhistochemical study was undertaken in normal human eyes. The amino acids proline, hydroxyproline, and hydroxylysine, which are characteristic of collagen, were also analysed. Collagen types I, III, and IV were found in the lamina cribrosa, the trabecular meshwork, and the retrolaminary optic nerve. In contrast, only type I was found in the sclera.

Collagen↗
PubMed2004

Thrombospondin-1 in the trabecular meshwork: localization in normal and glaucomatous eyes, and induction by TGF-beta1 and dexamethasone in vitro.

Transforming growth factor-beta2 (TGF-beta2) is elevated in the aqueous humor of patients with primary open-angle glaucoma (POAG), and high levels of TGF-beta2 are thought to contribute to the pathogenesis of POAG. Most TGF-beta2 in the eye is present in a latent, inactive form and the mechanisms of its in vivo activation are unclear. Since thrombospondin-1 (TSP-1) is one of the most potent in vivo activating molecules of TGF-betas, we investigated the localization and expression of TSP-1 in the aqueous humor outflow pathways. TSP-1 immunohistochemistry was performed in the eyes of human donors (8 normal and 17 with glaucoma). In addition, the eyes of Tsp-1(-/-)-deficient mice and normal Tsp-1(+/+) mice were investigated. TSP-1 mRNA expression was assessed by reverse transcription-polymerase chain reaction and Northern blotting of RNA from fresh trabecular meshwork (TM), and human and mouse TM cells in vitro. In addition, Northern and Western blot analyses of TM cells after incubation with TGF-beta and dexamethasone were performed. In most of the eyes, TSP-1 immunolabeling was predominantly observed in extracellular areas of the juxtacanalicular (cribriform) part of the TM. Some focal staining was observed in the corneoscleral and uveal parts of the TM. In the eyes of six glaucoma patients (including one with steroid-induced glaucoma), TSP-1 immunoreactivity was considerably more intense and all regions of the TM were positively labeled. In double labeling experiments, staining for TSP-1 did not overlap with that of fibronectin or type VI collagen. mRNA for TSP-1 was detected in both fresh and cultured TM cells. Incubation of TM cells with TGF-beta1 and dexamethasone caused a marked increase in TSP-1 expression. TSP-1 in the TM might act as a potent local endogenous activator of TGF-betas in the aqueous humor and mediate any local effects of TGF-beta and/or dexamethasone on the outflow of aqueous humor.

Adult↗
PubMed2005

Regulation of glucocorticoid responsiveness in glaucomatous trabecular meshwork cells by glucocorticoid receptor-beta.

PURPOSE: Glucocorticoid administration can lead to increased intraocular pressure in greater than 90% of patients with primary open-angle glaucoma (POAG), compared with 30% to 40% of the general population. The molecular mechanisms for increased steroid responsiveness among patients with glaucoma are unknown. An alternative splicing variant of the human glucocorticoid receptor GRbeta has dominant negative activity and has been implicated in a variety of steroid-resistant diseases. GRbeta also may play a role in glucocorticoid hyperresponsiveness in glaucoma. METHODS: Western blot analysis was performed to detect the expression of GRalpha and GRbeta in TM cells and its regulation by dexamethasone (DEX). Immunocytochemistry was used to compare the subcellular expression of GRbeta between normal and glaucomatous TM cell lines. DEX transgene induction in a luciferase reporter was performed to investigate the differential glucocorticoid responsiveness between multiple normal and glaucomatous TM cell lines. Overexpression of GRbeta was conducted in glaucomatous TM cell lines, and the regulation of GRbeta in the Dex-induced reporter gene luciferase or endogenous myocilin and fibronectin expression were determined. RESULTS: Trabecular meshwork (TM) cell lines derived from normal individuals expressed higher levels of GRbeta than did glaucomatous TM cells. Glaucomatous TM cells were more susceptible to DEX induction of a luciferase reporter gene than were TM cells derived from normal donors. Overexpression of GRbeta in glaucomatous TM cells inhibited DEX induction of a luciferase reporter gene as well as the endogenous genes MYOC and fibronectin. CONCLUSIONS: The decreased amount of GRbeta in glaucomatous TM cells could result in enhanced glucocorticoid responsiveness and ocular hypertension.

Adolescent↗