Immunohistochemical localization of MYOC/TIGR protein in the trabecular tissue of normal and glaucomatous eyes

PURPOSE: To examine immunohistochemically the localization of myocilin/trabecular meshwork inducible glucocorticoid response (MYOC/TIGR) protein in the glaucomatous and normal trabecular meshworks. METHODS: Trabecular tissues were used from one eye with late-onset goniodysgenetic glaucoma, three with primary open angle glaucoma (one of which had the MYOC/TIGR gene mutation), two with exfoliation glaucoma and one without glaucoma. For light microscopic immunohistochemistry, frozen sections were stained by the avidin-biotin complex method using anti-MYOC/TIGR polyclonal antibody. For electron microscopic immunohistochemistry, the pre-embedding method using the same antibody was performed. Double immunostaining using both anti-MYOC/TIGR and anti-type VI collagen antibodies was done by the immunofluorescence method. RESULTS: With light microscopy, immunoreactivity was seen in the whole trabecular meshwork of each of the specimens. No notable differences were detected in staining among the types of glaucoma, or between the eyes with and those without the gene mutation. Under electron microscopy, immunoreaction products were observed not only in the cytoplasm of the trabecular cells but also in the extracellular matrix, where staining was associated with the long-spacing collagen, fine granular materials and possibly microfibrils. With double immunohistochemistry, MYOC/TIGR was colocalized with type VI collagen in the trabecular meshwork. CONCLUSIONS: In glaucomatous and normal trabecular meshworks, the MYOC/TIGR protein is distributed in the extracellular matrix colocalizing with type VI collagen.     

Modulation of myocilin/TIGR expression in human trabecular meshwork.

PURPOSE: To study factors that modulate myocilin/trabecular meshwork inducible glucocorticoid response protein (TIGR) mRNA expression in human trabecular meshwork (TM). METHODS: mRNA from fresh TM of four human donors, from perfused anterior segment organ cultured TM of three donors, and from four primary TM cell lines of different donors was isolated. The full length cDNA of myocilin/TIGR was cloned from TM mRNA using a polymerase chain reaction approach and used as probe for northern blot analysis hybridization. Trabecular meshwork cell cultures were treated with transforming growth factor (TGF)-beta1 (1 ng/ml), dexamethasone (10(-7) M), and mechanical stretch (10%). RESULTS: mRNA for myocilin/TIGR could be readily detected by northern blot analysis hybridization in 2 to 3 microg of total RNA from all fresh and all organ-cultured TM samples. In contrast, no mRNA for myocilin/TIGR could be detected in 20 microg of total RNA isolated from three different primary TM cell lines. Only one TM cell line had a baseline expression of myocilin/TIGR, which was 35- to 55-fold lower than that of fresh or organ-cultured TM samples. Treatment of TM cell cultures with dexamethasone for 1 day markedly increased expression of myocilin/TIGR mRNA, an effect that was even more pronounced after 3 days of treatment. Treatment with TGF-beta1 for 24 hours had no effect; however, after 3 and 12 days of treatment a 3.8- and 4-fold increase in myocilin/TIGR mRNA expression was observed. Expression of myocilin/TIGR mRNA was also increased after 10% mechanical stretch; however, in contrast to the effects of TGF-beta-1, this effect was observed much earlier (8-24 hours) after treatment. CONCLUSIONS: Dynamic mechanical stimuli maintain myocilin/TIGR expression in TM in situ and lack of these stimuli in monolayer cell cultures might be involved in downregulation of myocilin/TIGR expression.     

Modulation of extracellular matrix turnover in the trabecular meshwork

Intraocular pressure (IOP) is the most critical risk factor for primary open angle glaucoma (POAG). In most cases of POAG, IOP is increased because of an abnormally high aqueous humor outflow resistance in the juxtacanalicular region of the trabecular meshwork. A distinct structural change in the trabecular meshwork of patients with POAG is the increase in fibrillar extracellular matrix in the juxtacanalicular region of the trabecular meshwork. Our knowledge on the molecular factors that govern turnover of the extracellular matrix in the trabecular meshwork has increased considerably in recent years. It has become clear that quality and quantity of the extracellular matrix in the trabecular meshwork are regulated by several signaling molecules that interact with each other to promote its synthesis, degradation, or extracellular modification. Transforming growth factor-β1 and β2 (TGF-β1 and TGF-β2) which derive from the aqueous humor or may be locally expressed induce in cultured trabecular meshwork cells the expression of a variety of extracellular matrix molecules. The action of TGF-βs very likely requires local activation by thrombospondin-1 and is partly mediated by its downstream mediator connective tissue growth factor, both of which are constitutively expressed in the trabecular meshwork. Bone morphogenetic proteins (BMP)-7 and -4 effectively antagonize the effects of TGF-β2 on matrix deposition. The antagonizing effects of BMP-7 are mediated in trabecular meshwork cells through Smad7. Smad7 is a key molecular switch to inhibit TGF-β2 signaling in the trabecular meshwork.     

Immunofluorescence method for quantifying the trabecular meshwork glucocorticoid response (TIGR) protein in trabecular meshwork and Schlemm's canal cells.

PURPOSE. Decreased flow of aqueous humor through the trabecular meshwork (TM) and into Schlemm's canal (SC) is believed to be a predominant factor in the development of the elevated intraocular pressure found in primary open-angle and steroid glaucoma. Recent biochemical and genetic evidence has suggested that alterations in the expression of the TM glucocorticoid response (TIGR) protein within the chamber angle may play a role in the development of these glaucomas. To understand the process of TIGR induction in outflow pathway cells we developed an assay for TIGR expression that could distinguish both individual cell responses and also provide a semi-quantitative comparison between cell cultures. The present study demonstrates this approach, using digital image capture of immunofluorescently stained human TM and SC cells after treatment with dexamethasone (Dex). METHODS. Confluent cultures of human TM and SC cells were treated with 1 M Dex or vehicle control for 10 days. The cells were then fixed, permeabilized, and stained using polyclonal antibodies produced against recombinant TIGR. Digital images of fluorescently stained cells, using the same exposure time within an experiment, were evaluated by tabulating the staining intensity of all cells on each image. Between 10 and 40 cells were evaluated per image, 8-10 frames/ sample, 2-3 samples per treatment. Each cell was ranked as either 0 (background), 1+ (minor), 2+ (moderate) or 3+ (very bright staining). RESULTS. TM cells showed a significant basal level of TIGR staining. About 20% of control cells showed appreciable levels of TIGR staining, with intensity levels evenly distributed between 1, 2 and 3+. Dex treatment increased the number of TM cells expressing TIGR to 60-80%, with the majority of cells showing 2+ to 3+ staining throughout the cytoplasm. SC cells showed no basal TIGR staining, but Dex-treated cells exhibited TIGR staining in 6-15% of cells. SC cell TIGR staining varied between 1+ to 2+ in intensity, and showed a distribution different from TM cells. Staining in SC cells was localized to a ribbon-like compartment adjacent to the nucleus. Such perinuclear localization was rarely seen in TM cells. CONCLUSIONS. The low standard errors of the mean TIGR responses within each experiment, and the reproducibility between experiments for each cell type, suggests good reliability for this method. At the same time, the consistent, marked contrast between TM and SC cells in their response to glucocorticoids demonstrates that the assay can distinguish significant differences between cell types. The data support the view that Dex has a cell type specific effect on TIGR induction. The different extent, pattern and localization of TIGR staining between cell types suggests that TIGR expression in SC cells could play a functional role in the outflow pathway, but one that may be distinct from that played in TM cells.     

Human trabecular meshwork cells in culture: morphology and extracellular matrix components

This study demonstrates the presence of laminin and collagen type IV in the extracellular space of human trabecular meshwork (HTM) cells in culture and its absence in cultures of fibroblasts from sclera adjacent to the outflow pathway. These basement membrane components can be detected by standard immunohistochemical techniques. Positive staining for these macromolecules is found only after the HTM cells have reached confluence. The presence of laminin and human collagen type IV in the early passages can serve as additional criteria for identification of HTM cells in culture. These cells provide a useful experimental system for studying the effects of drugs and other factors on the synthesis of components of the extracellular matrix.     

Immunohistochemical evaluation of the extracellular matrix in trabecular meshwork in steroid-induced glaucoma

BACKGROUND: To immunohistochemically examine the localization of the extracellular matrix (ECM) in the trabecular meshwork (TM) in steroid-induced glaucoma (SG) specimens. METHODS: We morphologically and immunohistochemically examined six trabecular tissues from three eyes with SG, two with primary open angle glaucoma (POAG), and one without glaucoma. For the morphological study, the ultra-microtome sections were observed using an electron microscope. For the light microscopic immunohistochemical analyses, frozen sections were stained by the avidin-biotin complex method using anti-type IV collagen, anti-type VI collagen, anti-heparan sulfate proteoglycan (HSPG), anti-fibronectin or anti-myocilin (MYOC) antibody. RESULTS: The morphological examinations revealed accumulations of basement membrane-like and fine fibrillar-like materials in the outer TM of SG specimen. Type IV collagen, HSPG and fibronectin antibodies in SG specimens showed a greater degree of staining in the outer TM in comparison to the POAG and non-glaucomatous specimens; in contrast, the other antibodies including the type VI collagen and MYOC, did not. CONCLUSIONS: The localization of ECMs in the TM is different in SG in comparison to that in POAG patients.     

Signal transduction mediated by adhesion of human trabecular meshwork cells to extracellular matrix

In this study we investigated the signaling event induced by adhesion of human trabecular meshwork (TM) cells to extracellular matrix (ECM) elements such as fibronectin. The role of tyrosine phosphorylation in adhesion was evaluated. A number of intracellular entities involved in the adhesion-mediated pathways were identified. For the experiments, human TM cells were seeded onto fibronectin- or polylysine (negative control)-coated plates. Fifteen, 30, 90 and 240 min after the seeding, cell lysates were collected. Immunoblotting analysis revealed that tyrosine phosphorylation occurred within 15 min of adhesion of TM cells to fibronectin and the level increased with time. The phosphotyrosyl proteins had molecular masses 25-220 kDa. A much lower level of tyrosine phosphorylation was observed when cells were plated on polylysine. Immunoprecipitation experiments indicated that the phosphotyrosine-containing proteins included focal adhesion kinase, paxillin, phosphatidylinositol 3-kinase and mitogen activated protein kinase. Within 30 min of adherence to fibronectin, human TM cells immunostained for paxillin and phosphotyrosine and exhibited prominent focal contacts. When treated with tyrosine kinase inhibitors genistein and herbimycin A and a protein kinase C (PKC) pseudosubstrate peptide inhibitor, cell adhesion to fibronectin was compromised and focal contact formation was limited. These results demonstrated that in human TM cells, tyrosine kinase was activated upon their adherence to fibronectin. PKC also appeared to play a role in modulation of the cell-matrix adhesion process. The current study provides insight into the signaling pathways that are linked to the ECM-induced events in TM cells. Elucidation of the hierarchy of signal responses may help develop strategies manipulating the cell-matrix interactions in the TM system.     

SPARC modulates expression of extracellular matrix genes in human trabecular meshwork cells

Purpose: To investigate the effects of secreted protein acidic and rich in cysteine (SPARC) on the expression of components of the extracellular matrix (ECM) in cultured human trabecular meshwork (TM) cells. Methods: Cultured human trabecular cells were transfected with small interfering RNAs (siRNAs) specific for the human SPARC gene. Protein and mRNA expressions of fibronectin (FN) and the α1chains of collagen I and collagen III were quantified. Results: After silencing of the SPARC gene by transfection of cells with SPARC siRNA, the expression of COL1A1 and COL3A1 mRNAs and proteins was significantly enhanced, as compared to that in the control group (all, p < 0.001). In contrast, SPARC siRNA significantly reduced the expression of FN and SPARC mRNAs and FN protein, as compared to that in the control group (all, p < 0.001.). Conclusions: SPARC modulates the expression of several ECM genes in cultured human TM cells.     

Functional properties of fibronectin in the trabecular meshwork

Fibronectin plays a number of important roles in the extracellular matrix (ECM) including providing structural support and signaling cues for cell survival, migration, differentiation, gene expression, growth factor signaling, and cell contractility. In this review, we examine recent findings about the biological and structural properties of fibronectin and discuss how these properties could contribute to the regulation of aqueous humor (AH) outflow in the trabecular meshwork (TM).     

Activation of extracellular signal-regulated kinase in trabecular meshwork cells.

A number of different agents, such as growth factors, cytokines and phorbol esters have been shown to modulate trabecular meshwork cell function. These studies were designed to evaluate the role extracellular signal-regulated kinase (ERK) pathway plays in mediating the responses to platelet-derived growth factor-BB (PDGF-BB) and phorbol 12-myristate 13-acetate (PMA) in trabecular meshwork cells. The human trabecular meshwork cell line, HTM-3, and the bovine trabecular meshwork (BTM) cells were treated with either PDGF-BB or PMA and the activation of ERK 1/2 evaluated. The effects of the MAP kinase kinase (MEK) inhibitor U0126, and the PKC inhibitor chelerythrine on ERK 1/2 were also determined. In a separate group of experiments, cells were treated with PDGF-BB or PMA and the secretion of matrix metalloproteinase-2 (MMP-2) evaluated. The addition of PDGF-BB or PMA produced time- and dose-dependent activation of ERK 1/2. Pretreatment with U0126 or chelerythrine significantly reduced ERK 1/2 activation induced by PDGF-BB or PMA. The addition of PDGF-BB or PMA stimulated the secretion of MMP-2. This secretory response was inhibited by pretreatment with the MEK inhibitor U0126. In trabecular meshwork cells, PDGF-BB and PMA activate ERK 1/2 by a PKC-dependent mechanism. Activation of ERK 1/2 by these agents in trabecular meshwork cells leads to the secretion of MMP-2. These studies provide evidence that ERK pathway is an important mechanism for integrating various signals that regulate trabecular function.     

Connective tissue growth factor induces extracellular matrix deposition in human trabecular meshwork cells

The major structural change in the human trabecular meshwork (TM) of eyes with primary open-angle glaucoma (POAG) is an increase in extracellular matrix (ECM) in the juxtacanalicular region of the TM. There is evidence that treatment with TGF-β2 causes an induction of ECM deposition in cultured human TM cells and that TGF-β2 is causatively involved in the JCT ECM increase in POAG. In the present study, we investigated the effects of connective tissue growth factor (CTGF) on the biology of cultured human TM cells. CTGF is a downstream mediator of TGF-β2-signaling, which is expressed at high amounts in the human TM in situ. HEK293 cells were transfected with an eukaryotic expression plasmid containing the coding sequences of human CTGF. Secreted CTGF was isolated and purified by chromatography. Primary human TM cells were incubated for 24 h with CTGF at concentrations of 2.5-100 ng/ml. Following treatment with CTGF, the expression of various ECM components that are expressed in the JCT, matrix metalloproteinases (MMPs) and integrins was investigated by real-time RT-PCR and western blot analyses. In addition, the activity of MMPs was investigated by gelatine zymography. The effect of CTGF silencing on TGF-β2-induced gene expression was investigated by transfection of immortalized HTM cells with CTGF-specific small interfering (si)RNA before TGF-β2 treatment. CTGF-treated human TM cells showed an increase in the expression of fibronectin, collagen types I, III, IV and VI, as well as in the integrin subunits aV and β1. Lower concentrations of CTGF caused an autoinduction of CTGF expression. No effects were observed on the expression and activity of MMP-2, MMP-9 and plasminogen activator inhibitor-1 (PAI-1). Transfection with CTGF-specific siRNA inhibited the TGF-β2-induced upregulation of CTGF and fibronectin. Our results indicate that treatment of human TM cells with recombinant CTGF causes distinct changes in gene expression and that CTGF is a critical mediator of the effects of TGF-β2 on ECM synthesis in human TM cells. An intriguing aspect supported by the data of the present work is that the pharmacologic modulation of CTGF might be a useful approach to develop novel therapeutic strategies to prevent or to reverse the structural changes that occur in the TM of eyes with POAG.     

In situ localization of cytoskeletal elements in the human trabecular meshwork and cornea

The authors compared cytoskeletal elements of the in situ human trabecular-meshwork cell with in situ human corneal cells using indirect immunofluorescence staining for tubulin and intermediate filaments (vimentin, cytokeratin, and desmin) and NBD-phallacidin staining for f-actin using both fixed frozen and unfixed frozen sections from postmortem eyes. Both f-actin and tubulin were found throughout the cell body of trabecular-meshwork cells, keratocytes, corneal endothelium, and corneal epithelium. The f-actin staining pattern was concentrated at the cell periphery of these four cell types. Vimentin stain was intensely localized in focal areas of the trabecular-meshwork cell, keratocytes, and throughout the corneal endothelium. A general anticytokeratin antibody was intensely localized in corneal epithelium and endothelium. However, PKK-1 anticytokeratin antibody was seen only in superficial layers of corneal epithelium and not in corneal endothelium. The 4.62 anticytokeratin antibody was not observed in either corneal epithelium or endothelium. None of these three cytokeratin antibodies were seen in trabecular-meshwork cells or keratocytes. Desmin stain was not noted in any of these cell types. In general, cytoskeletal staining of unfixed frozen sections showed a similar staining pattern for f-actin and tubulin but a more uniform and intense staining pattern for vimentin and cytokeratin compared with fixed frozen material. The authors conclude that these cytoskeletal stains can differentiate human trabecular-meshwork cells from cells of the cornea in situ.     

Glucocorticoid induction of the glaucoma gene MYOC in human and monkey trabecular meshwork cells and tissues.

PURPOSE. To examine the intracellular and extracellular expression of myocilin in the human and primate trabecular meshwork (TM) in the presence and absence of glucocorticoids. METHODS. Myocilin expression was examined in cultured human TM cells by Northern blot analysis and myocilin antibody-mediated immunoprecipitation. Myocilin expression was quantified using high-resolution two-dimensional polyacrylamide gel electrophoresis of radiolabeled proteins from human TM cells, TM tissue explants, and perfused human anterior segments cultured with and without dexamethasone (DEX) for 14 to 21 days, as well as TM tissue from pigtailed monkeys treated orally for 1 year with cortisone acetate. Immunofluorescence with anti-myocilin antibodies was used to localize cellular and extracellular expression of myocilin in cultured human TM cells. RESULTS. Glucocorticoid treatment caused a significant induction of myocilin mRNA, a tetrad of cell-associated proteins, and 8 to 20 secreted proteins (molecular mass [M(r)] 56 and 59 kDa and isoelectric point [pI] 5.2 and 5.3) in some, but not all the cultured human TM cells and explanted tissues. Western immunoblot analysis using anti-myocilin peptide antibodies identified these proteins as encoded by the MYOC gene. There was significant induction of the myocilin proteins in three perfusion-cultured human eyes, in which DEX-induced elevated intraocular pressure developed. Monkeys treated 1 year with cortisol acetate showed steroid glaucoma-like morphologic changes in the TM that correlated with the induction of myocilin in the TM. Immunofluorescence analysis of cultured TM cells localized myocilin intracellularly in discrete perinuclear and cytoplasmic vesicular deposits as well as extracellularly on the cell surface associated with the extracellular matrix. In several DEX-treated TM cell lines, there were significant levels of myocilin secreted into the media. Enzymatic deglycosylation of proteins in the TM media converted the higher molecular weight isoforms of myocilin (approximately 57 kDa) to the lower molecular weight isoforms ( approximately 55 kDa). CONCLUSIONS. Although the function of myocilin is unknown, induction of these TM proteins was found in eyes in which glucocorticoid-induced ocular hypertension developed. Therefore, myocilin may play an important pathogenic role in ocular hypertension in addition to its role in certain forms of POAG.     

维拉帕米对牛眼小梁细胞合成细胞外基质的影响

目的 探讨维拉帕米(Verapamil)对牛眼小梁细胞(BTMC)合成Ⅰ、Ⅲ、Ⅳ型胶原、纤维连接蛋白、透明质酸的影响。方法 采用免疫组化结合计算机图像分析和放免技术检测等于和低于0.01 mg/ml维拉帕米对BTMC合成胶原、纤维连接蛋白、透明质酸的影响。结果 0.001、0.005、0.01mg/ml维拉帕米可质量浓度依赖性抑制Ⅰ、Ⅲ型胶原、纤维连接蛋白的合成(P<0.05),而促进透明质酸的合成(P<0.05)。0.005、0.01mg/ml维拉帕米可质量浓度依赖性抑制Ⅳ型胶原(P<0.05),而0.001mg/ml维拉帕米无此作用(P>0.05)。结论 维拉帕米可通过抑制细胞外基质在小梁网异常沉积,减少房水外流阻力,有望在发病学环节上治疗原发性开角型青光眼。