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PubMed1999

Expression of alternatively spliced growth factor receptor isoforms in the human trabecular meshwork.

PURPOSE: Growth factors act through high-affinity cell surface receptors expressed by target cells and are critical modulators of cell function. Because aqueous humor is known to contain growth factors, these molecules may play a key role in maintaining the normal function of the human trabecular meshwork (HTM). Alternate mRNA splicing is an important mechanism used by cells to generate diverse isoforms of growth factor receptors. Although previous investigators have suggested that HTM cells may express alternative isoforms of several growth factor receptors, there have been no studies to verify these preliminary findings. The objective of this study was to determine whether cultured and ex vivo HTM cells express alternate isoforms of hepatocyte, keratinocyte, and transforming growth factor beta (TGFbeta)-II receptors and to characterize the isoform molecular sequences. METHODS: To determine whether cells within the HTM express mRNA for alternate isoforms of growth factor receptors, total RNA was isolated from several well-characterized HTM cell lines that were established from donors of various ages and from fresh ex vivo HTM tissues from healthy donors. After cDNA synthesis, polymerase chain reaction was initiated using specific primers for alternate forms of the following receptors: hepatocyte growth factor (HGFR), keratinocyte growth factor (KGFR), and transforming growth factor beta receptor II (TGFbetaR-II). Specificity and characterization of the polymerase chain reaction amplification products were determined by nucleic acid sequencing. RESULTS: Amplification products of the expected size for the growth factor isoforms were expressed in cell lines and in ex vivo tissues. Nucleic acid sequencing showed that cultured HTM cells and fresh ex vivo trabecular meshwork tissues expressed specific mRNA for alternatively spliced isoforms of HGFR, KGFR, and TGFbetaR-II The HGFR alternate isoform contained a 96-bp insert in the C-terminal coding region of the cytoplasmic tyrosine kinase domain. The KGFR alternate isoform is a soluble, truncated form, because it has no transmembrane or cytoplasmic domain as does the normal membrane-associated form. The TGFbetaR-II alternate isoform contained a 75-bp insert in the N-terminal coding region of the extracellular domain. CONCLUSIONS: In vitro and ex vivo HTM cells express mRNA for alternatively spliced isoforms of HGFR, KGFR, and TGFbetaR-II. These alternatively spliced receptor isoforms may be functional within the HTM and may play a critical role in maintaining the normal microenvironment of this important tissue.

Aged↗

PubMed2000

Regulation of human myocilin/TIGR gene transcription in trabecular meshwork cells and astrocytes: role of upstream stimulatory factor.

BACKGROUND: Mutations in the myocilin (MYOC)/TIGR gene are responsible for autosomal-dominant juvenile primary open-angle glaucoma (POAG). In patients with non-autosomal-dominant POAG, such mutations are rare, but the expression of MYOC/TIGR in the trabecular meshwork (TM) of the eye is considerably higher than in normals. We performed transfection, DNAse I footprinting, mutagenesis and electrophoretic mobility shift assays (EMSA) to identify elements responsible for the basal transcription of MYOC/TIGR in TM cells and astrocytes. RESULTS: DNAse I footprinting experiments of the human MYOC/TIGR promoter showed a major protected area between nt -106 to -77, which was not conserved in the homologous region of the mouse myoc/tigr promoter. In addition, the TATA-box was protected, as well as at least three downstream sites, including an AP-1-like sequence. Deletion of the -106 to -77 region caused a substantial loss of functional promotor activity in all cell types. Site-directed mutagenesis and EMSA experiments revealed the presence of two regulatory elements in the -106 to -77 region. Each of these cis-elements is essential for minimal promoter activity. The 5'-half of the region contains a sequence with similarities to NF-kappaB-related sites, however, binding of NF-kappaB could not be confirmed by EMSA. The 3'-half contains a canonical E-box sequence. EMSA experiments showed that the upstream regulatory factor (USF) was binding to the E-box sequence and that the binding can be supershifted by specific antibodies. CONCLUSIONS: Several DNA-protein binding elements contribute to a transcription of MYOC/TIGR, and USF is critically required for its basal transcription in trabecular meshwork cells and astrocytes.

Animals↗

PubMed1984

Aqueous outflow in the beagle. I. Postnatal morphologic development of the iridocorneal angle: pectinate ligament and uveal trabecular meshwork.

The present study examined the postnatal development of the pectinate ligaments and uveal trabecular meshwork of the normal canine chamber angle of the Beagle breed at one day, one through ten weeks, three, four and six months, and one and two years of age. Angle tissue was prepared and examined by standard techniques for light microscopy and scanning and transmission electron microscopy. Developmentally, pectinate ligaments matured by the third week although areas continued to develop through the eighth week. Each primary pectinate ligament possessed a large, posterior extracellular space at its iridal attachment. The uveal meshwork, which developed by rarefaction, was not formed at birth and did not appear adult-like until the end of the second month. Adult trabecular cells were ultrastructurally identical throughout the angle, having been characterized by having cilia and "special" rough endoplasmic reticulum.

Aging↗

PubMed1976

The sympathetic innervation of the ciliary body and trabecular meshwork of the cat. Fluorescence histochemistry and electron microscopy.

The ciliary body of the cat was investigated by fluorescence histochemistry and electron microscopy in an attempt to clarify its sympathetic innervation. Subconjunctival doses of 5-hydroxydopamine (5-OHDA) or 6-hydroxydopamine (6-OHDA) were given to establish the precise location of the sympathetic nerve terminals. The distribution of noradrenergic fibers and terminals was shown by fluorescence histochemistry to be sparse in the trabecular meshwork and the anterior portion of the ciliary muscle, but dense in the subepithelial tissue. The small and large dense core vesicles which occur in many nerve endings of the subepithelial tissue adjacent to the pigmented epithelial layer increased in electron density following the administration of 5-OHDA. Many degenerating nerve endings were found in the same region of animals treated with 6-OHDA. In contrast, there were few noradrenergic terminals in the ciliary muscle except for a portion of the smooth muscle which was shown to be dually innervated. The noradrenergic fibers in the subepithelial region and the trabecular meshwork may play an important role in aqueous secretion and outflow.

Adrenergic Fibers↗

PubMed1992

Glycosaminoglycans of trabecular meshwork and adjacent tissues of the rabbit eye.

Glycosaminoglycans (GAGs) extracted from the trabecular meshwork (TM) of rabbit eyes were analyzed by cellulose acetate electrophoresis and sequential enzymatic degradation, and compared with GAGs extracted from the adjacent tissues including the cornea, sclera, iris and ciliary body. The TM contained hyaluronic acid (HA), chondroitin 4- and/or 6-sulfate (CS), dermatan sulfate (DS), keratan sulfate (KS), heparan sulfate (HS) and an unidentified band of Alcian-blue-stained material which was resistant to degradation by the enzymes and chemicals used. The GAGs of the rabbit TM included: 13.9% HA, 31.6% CS, 47.1% DS, 4.6% KS and 1.2% HS, and were distinct from those of the cornea and the sclera.

Animals↗

PubMed1996

Glycans of the trabecular meshwork in primary open angle glaucoma.

AIMS: Glycan expression was compared in glaucomatous trabecular meshwork (TM) and normal TM in order to determine any differences which may reflect pathological changes underlying primary open angle glaucoma (POAG). METHODS: Resin embedded TM from trabeculectomy specimens from 15 eyes with POAG and from 12 eyes with normal anterior segments were probed with a panel of biotinylated lectins and an avidin-peroxidase revealing system at the light microscope level. Statistical analyses were performed on the comparative staining results. RESULTS: The lectins ConA and ePHA showed strong staining in all areas of both glaucomatous and normal TM; ePHA staining of Schlemm's canal (SC) from POAG TM was significantly less than that from normal TM (ePHA-SC p = 0.04). The lectins PSA, LCA, and SNA bound moderately strongly to SC endothelium and weakly to the endothelium of the corneoscleral meshwork (CSM); glaucomatous SC endothelial binding was significantly less than that of normal SC endothelium for PSA and LCA (PSA-SC p = 0.002, LCA-SC p = 0.002). STA and DSA showed moderately strong binding while WGA, ECA, AHA, and MPA bound weakly throughout the TM; for DSA and MPA this staining was significantly greater in POAG than in normal TM (DSA-SC p = 0.001, DSA-CSM p = 0.002, MPA-SC p = 0.01, MPA-CSM p = 0.02). Jac stained strongly throughout the TM and showed no significant difference in POAG compared with normal TM (Jac-SC p = 0.6, Jac-CSM p = 1). 1PHA, SBA, DBA, CTA, UEA-1 and LTA did not bind to glaucomatous TM or normal TM. There were no age-related changes seen. CONCLUSIONS: The expression of some complex and hybrid, bisected and non-bisected N-linked glycans is significantly diminished in glaucomatous TM compared with normal TM. Some glycans with multiple N-acetylglucosamine residues and O-linked glycans with terminal and subterminal galactosyl groups are significantly increased in POAG TM. Glycan expression does not change significantly with age in POAG or normal TM.

Adult↗

PubMed2002

In vitro localization of TIGR/MYOC in trabecular meshwork extracellular matrix and binding to fibronectin.

PURPOSE: To determine whether trabecular meshwork-inducible glucocorticoid response/myocilin (TIGR/MYOC) protein associates with the extracellular matrix (ECM) of human trabecular meshwork (HTM) cells. METHODS: The extracellular localization of TIGR/MYOC was examined by immunofluorescence microscopy in HTM cultures treated with and without dexamethasone and ascorbate and in a transformed HTM cell line, TM-1, transiently transfected with TIGR/MYOC cDNA. Antibodies to TIGR/MYOC, fibronectin, laminin, type IV collagen, or thrombospondin were used to determine the extracellular localization of TIGR/MYOC. Solid phase binding assays using 125I-recombinant TIGR/MYOC and types I and IV collagens, fibronectin, and laminin were done to examine the association of TIGR/MYOC with these proteins and to identify a specific TIGR/MYOC binding site within fibronectin. The domains of fibronectin tested were the fibrin/collagen binding domain, the RGD domain, and the Heparin II (Hep II) domain. RESULTS: TIGR/MYOC colocalized with fibronectin, laminin, and type IV collagen, but not thrombospondin in both dexamethasone and dexamethasone/ascorbate-treated HTM cultures and in TM-1 cultures transfected with TIGR/MYOC cDNA. In solid phase binding assays, 125I-TIGR/MYOC bound fibronectin but not laminin or type IV collagen. Binding to fibronectin could be competed with excess TIGR/MYOC or fibronectin. Specific binding was found for the Hep II domain of fibronectin. CONCLUSIONS: TIGR/MYOC can associate with components of the ECM via interactions with the Hep II domain of fibronectin. The interactions with the Hep II domain of fibronectin could alter cell-matrix interactions in the TM and provides an interesting lead to explore the role(s) of TIGR/MYOC in both steroid-induced and primary open angle glaucoma.

Adult↗

PubMed2000

Molecular cloning of the bovine MYOC and induction of its expression in trabecular meshwork cells.

PURPOSE: Myocilin gene (MYOC) was identified as one of the disease-causing genes of primary open-angle glaucoma. This study was conducted to establish a system for the investigation of the biological role of MYOC in vitro by using bovine eyes, which are easy to obtain and have been widely used to examine the aqueous outflow system. The cDNA sequence of the bovine MYOC was determined and its expression in bovine eyes was examined with a quantitative polymerase chain reaction (PCR) assay. METHODS: Bovine MYOC cDNA was obtained from cultured bovine trabecular meshwork cells, and part of its sequence was determined using a primer pair designed based on the known sequence of the human MYOC gene. The 3' and 5' ends of this sequence were determined using the method of 3' and 5' rapid amplification of cDNA ends. The induction of the MYOC gene in cultured bovine trabecular meshwork cells after exposure to dexamethasone was quantitatively examined with real-time quantitative PCR using a probe designed according to the sequence of the determined bovine MYOC gene. RESULTS: Bovine MYOC protein was composed of 490 amino acids, which was 81.6% identical with that of human MYOC protein. Most of the amino acid residues of which mutation was reported to cause glaucoma were conserved in the bovine MYOC protein. After 2 weeks of treatment with 500 nM dexamethasone, expression of bovine MYOC mRNA was amplified 14-fold (14.1+/-5.1-fold, mean +/- SEM) measured by real-time quantitative PCR. CONCLUSIONS: The cDNA sequence of the bovine MYOC gene had a high degree of similarity to that of the human MYOC gene. Investigation of the function of bovine MYOC may contribute to identifying the role of MYOC protein in the aqueous outflow system.

Amino Acid Sequence↗

PubMed1979

The trabecular meshwork of a non-glaucomatous eye with the exfoliation syndrome. Electronmicroscopic study.

The trabecular meshwork of a non-glaucomatous eye with the exfoliation syndrome was investigated. The exfoliation material was found mainly in massive deposits in the subendothelial region of the outer and inner wall of Schlemm's canal, in the cribriform area and the uveal meshwork. The other alterations of the trabeculum corneosclerale corresponded to the age of the patient. The concept that the accumulation of exfoliation material is an important pathogenetic factor in the development of glaucoma was not confirmed by this study.

Aged↗

PubMed1993

[Porcine trabecular meshwork organ culture].

In order to investigate whether porcine eyes could be useful for trabecular meshwork (TM) organ culture, changes in ultrastructure and synthesis of glycosaminoglycans (GAGs) of organ-cultured porcine TM were studied. The presence of thrombospondin was also detected in porcine TM, using an immunohistochemical method. Explants were cultured for periods of 7 to 21 days and subsequently some of them were labeled with 3H-glucosamine and 35S-sulfate for 48 hours. There was very little change in the ultrastructure of the explants during our culture periods. The total incorporation of 3H-glucosamine and 35S-sulfate into trabecular GAGs for up to 21 days showed similar patterns. Immunoreactivity for thrombospondin was found in the trabecular sheets. This organ culture system suggests a model for studying the various drug and hormone induced alterations in this tissue.

Animals↗

PubMed1993

[Transplantation of functional trabecular meshwork in 9 cases].

On the basis of experiments on rabbits, transplantation of functional trabecular meshwork was performed in 9 cases of absolute angle-closure glaucoma. The surgical reactions were not serious, and the postoperative intraocular pressure decreased significantly by 50%-80% on discharge from the hospital, and in 4 cases followed up for 3 months, by 20%-60%, with 1 case of ocular hypotension. This modality of trabeculoplasty opens a new prospect of ophthalmological organ transplantation for probation.

Aged↗

PubMed1991

Fibronectin in the trabecular meshwork: immunofluorescent and immunoelectron microscopic findings.

We examined the fibronectin (FN) secretion of cultured trabecular meshwork (TM) cells in a normal human eye by indirect immunofluorescent technique using mouse anti-human FN monoclonal antibody and FITC-conjugated goat anti-mouse IgG. To localize FN on frozen sections of normal TM, which were obtained from 7 enucleated eyes owing to traumatic eyeball rupture, the same indirect immunofluorescent method was used. Immunoelectron microscopy was applied to demonstrate the distribution pattern of FN in the normal TM of 2 human eyes using an avidin-biotin-peroxidase complex method. In the tissue culture of TM, the TM cell walls and extracellular matrices showed an intense staining with antibody to FN. Indirect immunofluorescent staining of FN on frozen sections of TM showed strong positive reactions in the subendothelial region. There was no reaction in the central core of the trabecular beam. Immunoelectron microscopy revealed the reaction products to FN in the areas lining the trabecular endothelial cells.

Adolescent↗

PubMed1985

Trabecular meshwork glycosaminoglycans in human and cynomolgus monkey eye.

The glycosaminoglycans (GAGs) extractable from the trabecular meshworks (TM) of human and non-human primate eye have been analyzed by sequential enzymatic degradation and cellulose acetate electrophoresis. For comparison, similar extracts of the cornea, sclera, iris, and ciliary body have also been analyzed. The distribution of glycosaminoglycans in human and in cynomolgus monkey TMs are similar, although not identical. The human TM contains hyaluronic acid (HA), chondroitin-4-sulfate and/or 6-sulfate (CS), dermatan sulfate (DS), keratan sulfate (KS), heparan sulfate (HS), and an unidentified band of Alcian Blue staining material, which is resistant to the enzymes that we used. Based upon quantitation of the Alcian Blue staining intensities of extracted GAGs, which have been corrected by a relative dye-binding factor, the GAGs of the human TM include: 29.0% HA, 14.1% CS, 21.5% DS, 20.3% KS, and 15.0% HS. The cynomolgus monkey trabecular GAGs include: 12.8% HA, 14.3% CS, 15.2% DS, 42.1% KS, and 15.6% HS.

Animals↗

PubMed1983

Altered cortisol metabolism in cells cultured from trabecular meshwork specimens obtained from patients with primary open-angle glaucoma.

Cells cultured from trabecular meshwork specimens obtained from patients with primary open angle glaucoma (TMPOAG cells) exhibited two major differences in cortisol-metabolizing enzymes when compared with similar cells from nonglaucomatous patients. One is a marked increase (greater than 100-fold) in delta 4-reductase activity and the other is a decrease (4-fold) in 3-oxidoreductase activity. Peripheral lymphocytes from one of these patients as well as from five additional patients with POAG, did not show these abnormalities, indicating that the defects are not found in all cortisol-metabolizing cells. The abnormal metabolism of cortisol by TMPOAG cells may be of significance in the pathogenesis of POAG.

Adult↗

PubMed2003

Effect of nitric oxide on the proliferation of cultured porcine trabecular meshwork cells.

To investigate the effect of nitric oxide (NO) on the proliferation of trabecular meshwork (TM) cells, primarily cultured porcine TM cells were exposed to NO donor (SNAP, S-nitroso-N-acetyl-D,L-penicillamine) with and without its inhibitor (L-NAME, Nomega-Nitro-L-arginine methyl ester). The proliferation of TM cells was quantified by a rapid colorimetric assay. Acridine orange/Hoechest 33342 staining and flow cytometry with annexin-PI were done. As a result, NO inhibited the proliferation of TM cells significantly in a dose-dependent manner and this inhibitory effect was abolished by L-NAME. Fluorescent microscopy and flow cytometric analysis revealed that NO induced apoptotic cell death. The current results suggest that NO inhibit the proliferation of TM cells and apoptosis may be involved in some degree.

Acridine Orange↗

PubMed1994

Immunohistochemistry study of the glaucomatous and normal human trabecular meshwork.

Although class II-expressing cells have been reported previously in the human trabecular meshwork (HTM), the exact identity of these cells and their possible role in the normal and the pathological state remains unclear. Preliminary results suggest increased human leucocyte antigen (HLA)-DR expression in the glaucomatous TM. To investigate this possibility, 20 trabeculectomy specimens obtained from patients with primary open-angle glaucoma (POAG) were examined immunohistochemically for HLA-DR, leucocyte common antigen (LCA; CD45) and a macrophage marker (CD68). The patients varied in age from 42 to 86 years (average, 69 years). The degree of antibody staining in the TM was compared with that of 20 human control eyes obtained from an eye bank less than 24 h after death. The donors varied in age from 47 to 91 years (average, 69 years); there was no known history of eye disease amongst the control group. Evaluation of specimen was performed by two observers using a grading system ranging from negative (0) to strongly positive (3) and by computer-aided optic densitometry. HLA-DR-positive cells were present in both the normal and the glaucomatous TM. Occasional cells stained for LCA and CD68 in the TM of both groups. Subpopulations of the HLA-DR-staining cells were apparent on serial sectioning. An age effect on antigen staining was not observed; postmortem delay of greater than 48 h resulted in decreased specimen antigenicity. Both evaluation techniques failed to reveal a significant difference in HLA-DR, LCA or CD68 staining between the glaucomatous and the normal TM.

Adult↗

PubMed2005

Induction of TGF-beta1 in the trabecular meshwork under cyclic mechanical stress.

The pathophysiological mechanisms involved in the failure of the trabecular meshwork (TM) to maintain normal levels of aqueous outflow in glaucoma are not yet understood. Aberrant activation of the transforming growth factor beta-1 (TGF-beta1) pathway has been implicated in several degenerative diseases. We investigated the possibility that chronic cyclic mechanical stress that affects the TM might result in increased production of TGF-beta1. Primary cultures of TM cells subjected to cyclic mechanical stress (5% stretching, 1 cycle/sec) demonstrate a significant increase in total and biologically active secreted TGF-beta1 that was associated with activation of the TGF-beta1 promoter, measured using a recombinant adenovirus expressing the secreted reporter gene secreted alkaline phosphatase protein (SEAP) under the TGF-beta1 gene promoter (AdTGFbeta1-SEAP). Associated changes in the transcription of MMP-2, TIMP-2, and CTGF were assessed by semiquantitative PCR. Immunohistochemical analysis of TGF-beta1 in organ culture of human eyes revealed a generalized accumulation of this protein in the extracellular matrix (ECM) of the TM, while expression of the TGF-beta1 promoter, analyzed using the LacZ reporter gene, was localized in some specific cells within the outflow pathway. Induction of the TGF-beta1 promoter in organ culture was demonstrated using a novel model for cyclic mechanical stress in human perfused anterior segments infected with AdTGFbeta1-SEAP. Given the relevant physiological and pathophysiological roles of TGF-beta1, its induction after cyclic mechanical stress in the TM supports the hypothesis that this cytokine might play a significant role in the physiology of the TM, and contribute to the pathological changes of this tissue in certain forms of glaucoma.

Anterior Eye Segment↗

PubMed2002

cDNA microarray analysis of gene expression changes induced by dexamethasone in cultured human trabecular meshwork cells.

PURPOSE: To profile gene expression changes induced by dexamethasone in cultured human trabecular meshwork (TM) cells and identify genes related to the occurrence of steroid-induced glaucoma. METHODS: At confluence, dexamethasone (final concentration 10(-7) M in 0.1% ethanol) or vehicle alone (control, 0.1% ethanol) was applied to cultured human TM cells from eyes of four normal subjects. After 7 days of application, a labeled cDNA probe was synthesized from extracted total RNA and hybridized to a human cDNA microarray containing 2400 genes. After hybridization, the tyramide signal was amplified, and the fluorescent signals on each microarray were scanned and analyzed. RESULTS: In dexamethasone-treated TM cells, simultaneous analysis of 2400 human genes indicated a more than twofold increase in 30 genes. Five of them, myocilin (MYOC), decorin, insulin-like growth factor binding protein 2, ferritin L chain, and fibulin-1C, were the most upregulated genes with higher-than-control expression levels in all four experiments. Their upregulation was further confirmed by semiquantitative RT-PCR. Downregulation, with fluorescent signals decreased to less than a half, was found in 34 genes. The dexamethasone-induced expression changes in most of these TM cell genes have not been reported previously. CONCLUSIONS: cDNA microarray is a useful tool for gene expression analysis that confirms previous reports of upregulated mRNA expression of MYOC after treatment with dexamethasone in human TM cells. Changes in other genes subsequent to the treatment with dexamethasone may also reduce outflow facility, providing new insights into the pathogenesis of steroid-induced glaucoma.

Adolescent↗