Primary open angle glaucoma is an important type of glaucoma as it is one of the most common causes of blindness. Previous studies have demonstrated that in glaucomatous patients, the human trabecular meshwork (HTM) is markedly stiffened. The purpose of the present study was to determine the regulatory role of Yes-associated protein (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ) in HTM cells. Primary HTM cells were cultured with different concentrations of dexamethasone (DEX), and the expression levels of YAP and TAZ were evaluated using reverse transcription-quantitative polymerase chain reaction and western blotting. The results revealed that DEX increased the expression of YAP and TAZ in a dose-dependent manner. In addition, the western blot analysis of cytoskeleton-associated proteins revealed that the inhibition of YAP and/or TAZ using small interfering RNA resulted in the increased expression of collagen I, and decreased expression of fibronectin, laminin and collagen IV. The expression of β-catenin, a key protein in the Wnt pathway, was also observed to be regulated by YAP and TAZ. A 5-ethynyl-2'-deoxyuridine staining assay indicated that YAP and TAZ induced the proliferation of HTM cells. The investigation of cross-linked actin network formation by the HTM cells demonstrated that the knockdown of YAP and TAZ genes rescued HTM cells from cytoskeletal reorganization. Furthermore, functional evaluation of a HTM cell monolayer using a permeability assay demonstrated that the inhibition of YAP and TAZ attenuated the DEX-induced impairment of permeability. These findings suggest that YAP and TAZ play pivotal roles in the DEX-induced cytoskeletal changes of HTM cells, and reveal novel potential mechanisms for the development and progression of glaucoma.

This study aimed to investigate the differential responses of trabecular meshwork stem cells (TMSCs) and trabecular meshwork (TM) cells to endoplasmic reticulum (ER) stress inducers.

The aim of this study was to examine the expression of genes related to the Wnt signaling pathway, such as beta-catenin (CTNNB1) and secreted frizzled-related protein-1 (sFRP1), in human trabecular meshwork (TM) cells. In addition, the effect of oxidative stress on Wnt signaling was evaluated.

Trabecular meshwork and Schlemm's canal are the tissues appointed to modulate the aqueous humour outflow from the anterior chamber. The impairment of their functions drives to an intraocular pressure increase. The selective laser trabeculoplasty is a laser therapy of the trabecular meshwork able to decrease intraocular pressure. The exact response mechanism to this treatment has not been clearly delineated yet. The herein presented study is aimed at studying the gene expression changes induced in trabecular meshwork cells by selective laser trabeculoplasty (SLT) in order to better understand the mechanisms subtending its efficacy.

Hyaluronan (HA) in the ocular trabecular meshwork (TM) is a critical modulator of aqueous humor outflow. Individual HA strands in the pericellular matrix can coalesce to form cable-like structures, which have different functional properties. Here, we investigated HA structural configuration by TM cells in response to various stimuli known to stimulate extracellular matrix (ECM) remodeling. In addition, the effects of HA cable induction on aqueous outflow resistance was determined. Primary TM cell cultures grown on tissue culture-treated plastic were treated for 12-48 h with TNFα, IL-1α, or TGFβ2. TM cells grown on silicone membranes were subject to mechanical stretch, which induces synthesis and activation of ECM proteolytic enzymes. HA structural configuration was investigated by HA binding protein (HAbp) staining and confocal microscopy. HAbp-labeled cables were induced by TNFα, TGFβ2 and mechanical stretch, but not by IL-1α. HA synthase (HAS) gene expression was quantitated by quantitative RT-PCR and HA concentration was measured by ELISA assay. By quantitative RT-PCR, HAS-1, -2, and -3 genes were differentially up-regulated and showed temporal differences in response to each treatment. HA concentration was increased in the media by TNFα, TGFβ2 and IL-1α, but mechanical stretch decreased pericellular HA concentrations. Immunofluorescence and Western immunoblotting were used to investigate the distribution and protein levels of the HA-binding proteins, tumor necrosis factor-stimulated gene-6 (TSG-6) and inter-α-inhibitor (IαI). Western immunoblotting showed that TSG-6 and IαI were increased by TNFα, TGFβ2 and IL-1α, but mechanical stretch reduced their levels. The underlying substrate appears to affect the identity of IαI·TSG-6·HA complexes since different complexes were detected when TM cells were grown on a silicone substrate compared to a rigid plastic surface. Porcine anterior segments were perfused with 10 μg/ml polyinosinic:polycytidylic acid (polyI:C), a potent inducer of HA cables, and outflow rates were monitored for 72 h. PolyI:C had no significant effect on outflow resistance in porcine anterior segments perfused at physiological pressure. Collectively, HAS gene expression, HA concentration and configuration are differentially modified in response to several treatments that induce ECM remodeling in TM cells. In ocular TM cells, our data suggests that the most important determinant of HA cable formation appears to be the ratio of HA chains produced by the different HAS genes. However, the act of rearranging pericellular HA into cable-like structures does not appear to influence aqueous outflow resistance.

To better understand the role of exosomes in the trabecular meshwork (TM), the site of intraocular pressure control, the exosome proteome from primary cultures of human TM cell monolayers was analyzed. Exosomes were purified from urine and conditioned media from primary cultures of human TM cell monolayers and subjected to a two dimensional HPLC separation and MS/MS analyses using the MudPIT strategy. Spectra were searched against a human protein database using Sequest. Protein profiles were compared to each other and the Exocarta database and the presence of specific protein markers confirmed by Western blot analyses of exosomes from aqueous humor and human TM cell strains (n=5) that were untreated, or exposed to dexamethasone and/or ionomycin. TM cell exosomes contained 108 of the 143 most represented exosome proteins in ExoCarta, including previously characterized markers such as membrane organizing and tetraspanin proteins. Several cell-specific proteins in TM exosomes were identified including myocilin, emilin-1 and neuropilin-1. All TM exosome proteins had flotation densities on sucrose gradients and release responses to ionomycin typical for exosomes. Taken together, TM exosomes have a characteristic exosome protein profile plus contain unique proteins, including the glaucoma-causing protein, myocilin; suggesting a role for exosomes in the control of intraocular pressure.

Although the extracellular matrix (ECM) in trabecular meshwork (TM) cells is known to be important in intraocular pressure (IOP) regulation, the molecular mechanisms involved in generating a glaucomatous environment in the TM are not completely understood. Recently we identified a molecular pathway, transforming growth factor beta 2 (TGFβ2)-toll-like receptor 4 (TLR4) signaling crosstalk, as an important regulator of glaucomatous damage in the TM, which contributes to fibrosis. Here we evaluate a novel molecular target, A20, also known as tumor necrosis factor alpha-induced protein 3 (TNFAIP3), which may help to block pathological TGFβ2-TLR4 signaling. Primary human TM cells were analyzed for A20 message and for A20 and fibronectin protein expression after treatment with TGFβ2. A20 message increased when the TLR4 pathway was inhibited in TM cells. In addition, TGFβ2, a known inducer of fibrosis, increased fibronectin expression, while at the same time decreasing the expression of A20. We then overexpressed A20 in TM cells in order to test the effect on treatment with TGFβ2, lipopolysaccharide (LPS), or cellular fibronectin extra domain A (cFN-EDA). Importantly, overexpression of A20 rescued the fibrotic response when TM cells were treated with TGFβ2, LPS, or cFN-EDA. In situ hybridization was used to probe for A20 RNA expression in age-matched control (C57BL/6J) mice and mice that constitutively express the EDA isoform of fibronectin (B6.EDA+/+). In this novel mouse model of glaucoma, A20 RNA was increased versus age-matched control mice in a cyclic manner at 6 weeks and 1 year of age, but not at 8 months. Overall, these data suggest that A20 may work through a negative feedback mechanism attenuating the ability of TGFβ2-TLR4 signaling to induce fibrosis.

Primary open-angle glaucoma (POAG) is a leading cause of blindness due to chronic degeneration of retinal ganglion cells and their optic nerve axons. It is associated with disturbed regulation of intraocular pressure, elevated intraocular levels of TGF-β2, aberrant extracellular matrix (ECM) deposition and increased outflow resistance in the trabecular meshwork (TM). The mechanisms underlying these changes are not fully understood. Cell-matrix interactions have a decisive role in TM maintenance and it has been suggested that TGF-β-induced inhibition of matrix metalloproteases may drive aberrant ECM deposition in POAG. Invadopodia and podosomes (invadosomes) are distinct sites of cell-matrix interaction and localized matrix-metalloprotease (MMP) activity. Here, we report on the effects of TGF-β2 on invadosomes in human trabecular meshwork cells. Human TM (HTM) cells were derived from donor tissue and pretreated with vehicle or TGF-β2 (2 ng/ml) for 3d. Invadosomes were studied in ECM degradation assays, protein expression and MMP-2 activity were assessed by western blot and zymography and ECM protein transcription was detected by RT-qPCR. HTM cells spontaneously formed podosomes and invadopodia as detected by colocalization of Grb2 or Nck1 to sites of gelatinolysis. Pretreatment with TGF-β2 enhanced invadosomal proteolysis and zymographic MMP-2 activity as well as MMP-2, TIMP-2 and PAI-1 levels in HTM cell culture supernatants. Rho-kinase inhibition by H1152 blocked the effects of TGF-β2. Concomitant transcription of fibronectin and collagens-1, -4 and -6 was increased by TGF-β2 and fibrillar fibronectin deposits were observed in areas of invadosomal ECM remodelling. In contrast to a current hypothesis, our data indicate that TGF-β2 induces an active ECM remodelling process in TM cells, characterized by concurrent increases in localized ECM digestion and ECM expression, rather than a mere buildup of material due to a lack of degradation. Invadosomal cell adhesion and signaling may thus have a role in POAG pathophysiology.

To determine whether activity of carbohydrate metabolism enzymes (aldolase, pyruvate kinase, isocitrate dehydrogenase, and malate dehydrogenase) are altered in the glaucomatous trabecular meshwork (TM) compared to controls.

Trabecular meshwork (TM) fibrosis is a crucial pathophysiological process in the development of primary open-angle glaucoma. Pirfenidone (PFD) is a new, broad-spectrum antifibrotic agent approved for the treatment of idiopathic pulmonary fibrosis. This study investigated the inhibitory effect of PFD on TM fibrosis and evaluated its efficacy in lowering intraocular pressure (IOP).

Trabecular meshwork (TM) cell volume is a determinant of aqueous humor outflow resistance, and thereby IOP. Regulation of TM cell volume depends on chloride ion (Cl-) release through swelling-activated channels (ICl,Swell), whose pore is formed by LRRC8 proteins. Chloride ion release through swelling-activated channels has been reported to be regulated by calcium-activated anoctamins, but this finding is controversial. Particularly uncertain has been the effect of anoctamin Ano6, reported as a Ca2+-activated Cl- (CaCC) or cation channel in other cells. The current study tested whether anoctamin activity modifies volume regulation of primary TM cell cultures and cell lines.

To expand multi-potent progenitors from human trabecular meshwork (TM), we have created a new optimized method on two-dimensional (2D) followed by three-dimensional (3D) Matrigel in modified embryonic stem cell medium supplemented with 5% fetal bovine serum (MESCM + 5% FBS). The expanded TM cells were small cuboidal cells expressing TM markers such as AQP1, MGP, CHI3L1, and AnkG, embryonic stem cell (ESC) markers such as Oct4, Sox2, Nanog, and ABCG2, and neural crest (NC) markers such as p75NTR, FOXD3, Sox9, Sox10, and MSX1. Although expanded cells lost expression of these markers after passage, the cells regained the markers when Passage 2 cells were seeded on 3D Matrigel through activation of canonical BMP signaling. Such restored progenitors could differentiate into corneal endothelial cells, adipocytes, and chondrocytes but not keratocytes or osteocytes. Therefore, we have concluded that human TM harbors multipotent progenitors that can be effectively isolated and expanded using 2D Matrigel in MESCM + 5% FBS. This unique in vitro model system can be used to understand how TM is altered in glaucoma and whether such TM progenitor cells might one day be used for treating glaucoma or corneal endothelial dysfunction.

Aqueous humour does not drain uniformly through the trabecular meshwork (TM), but rather follows non-uniform or "segmental" routes. In this study, we examined whether segmental outflow patterns in the TM change over time in living mice and whether such changes are affected by age. Segmental outflow patterns were labelled by constant-pressure infusion of fluorescent tracer microparticles into the anterior chamber of anesthetised C57BL/6J mice at 3 or 8 months of age. Two different tracer colours were infused at separate time points with an interval of Δt = 0, 2, 7 or 14 days. In a separate experiment, one tracer was infused in vivo while the second tracer was infused ex vivo after 2 days. The spatial relationship between the two tracer patterns was analysed using the Pearson's correlation coefficient, r. In 3-month-old mice, there was a time-dependent decay in r, which was near unity at Δt = 0 and near zero at Δt = 14 days. In 8-month-old mice, r remained elevated for 14 days. Segmental outflow patterns measured in young mice ex vivo were not significantly different from those measured in vivo after accounting for the expected changes over 2 days. Therefore, segmental outflow patterns are not static in the TM but redistribute over time, achieving near complete loss of correlation by 2 weeks in young healthy mice. There is an age-related decline in the rate at which segmental outflow patterns redistribute in the TM. Further research is needed to understand the dynamic factors controlling segmental outflow.

Biophysical and biochemical attributes of the extracellular matrix are major determinants of cell fate in homeostasis and disease. Ocular hypertension and glaucoma are diseases where the trabecular meshwork tissue responsible for aqueous humor egress becomes stiffer accompanied by changes in its matrisome in a segmental manner with regions of high or low flow. Prior studies demonstrate these alterations in the matrix are dynamic in response to age and pressure changes. The underlying reason for segmentation or differential response to pressure and stiffening are unknown. This is largely due to a lack of appropriate models ( in vitro or ex vivo ) to study this phenomena. In this study, we characterize the biomechanical attributes, matrisome, and incidence of crosslinks in the matrix deposited by primary cells isolated from segmental flow regions and when treated with glucocorticosteroid. Data demonstrate that matrix deposited by cells from low flow regions are stiffer and exhibit a greater number of immature and mature crosslinks, and that these are exacerbated in the presence of steroid. We also show a differential response of high or low flow cells to steroid via changes observed in the matrix composition. We conclude that although a mechanistic basis for matrix stiffness was undetermined in this study, it is a viable tool to study cell-matrix interactions and further our understanding of trabecular meshwork pathobiology.

The house swine (Sus scrofa domestica Linnaeus 1758) is an important model organism regarding the study of neurodegenerative diseases, especially ocular neuropathies such as glaucoma. This is due to the high comparability of the porcine and human eye regarding anatomy and molecular features. In the pathogenesis of glaucoma, the trabecular meshwork (TM) forms a key ocular component in terms of intraocular pressure (IOP) elevation. Thereby, functional TM abnormalities are correlated with distinct proteomic alterations. However, a detailed analysis of the TM proteome has not been realized so far. Since the porcine eye has high potential as a model system to study ocular diseases such as glaucoma, the present study focuses on the in-depth analysis of the porcine TM proteome. By use of a bottom-up (BU) mass spectrometric (MS) platform utilizing electrospray ionization liquid chromatography tandem MS (LC-ESI-MS/MS) considering database-dependent and peptide de novo sequencing, more than 3000 TM proteins were documented with high confidence (FDR < 1%). A distinct number of proteins with neuronal association were revealed. To the best to our knowledge, many of these protein species have not been reported for TM tissue before such as reelin, centlein and high abundant neuroblast differentiation-associated protein AHNAK (AHNAK). Thereby, AHNAK might play a superordinate role in the TM regarding proposed tissue involvement in barrier function. Also, a high number of secretory proteins could be identified. The generated TM proteomic landscape underlines a multifunctional character of the TM beyond representing a simple drainage system. Finally, the protein catalogue of the porcine TM provides an in-depth view of the TM molecular landscape and will serve as an important reference map in terms of glaucoma research utilizing porcine animal models, porcine TM tissues and/or cultured TM cells.

To identify non-housekeeping genes definitively expressed in the human trabecular meshwork (TM).

To study the effects of triamcinolone acetonide (TA) on cultured human trabecular meshwork (HTM) cells.

Myocilin, a protein associated with the development of glaucoma, is expressed in most eye tissues with highest expression observed in trabecular meshwork cells. In culture, primary human trabecular meshwork cells incubated in 10% fetal bovine serum have reduced myocilin expression compared to in vivo, but incubation in human aqueous humor, their normal in vivo nutrient source, restores myocilin expression to near in vivo levels. To investigate the mechanism by which human aqueous humor stimulates myocilin accumulation in conditioned media from normal human trabecular meshwork cells, three independent trabecular meshwork cell lines were cultured in Dulbecco's Modified Eagle's Medium (DMEM) containing various supplements: fetal bovine serum (10%), human serum (0.2%), porcine aqueous humor (50%), bovine serum albumin (0.1%), dexamethasone (10(-7)M), human aqueous humor (50%) or heat-inactivated human aqueous humor (50%). Conditioned media from cultured primary trabecular meshwork cells following incubation in human aqueous humor showed significant accumulation of myocilin in a time- (15 min) and dose-dependent manner (half maximal effective concentration ∼ 30%) while intracellular myocilin levels decreased. Minimal myocilin accumulation was observed in conditioned media isolated from trabecular meshwork cells cultured in DMEM containing fetal bovine or human serum, bovine serum albumin, porcine aqueous humor, dexamethasone or DMEM alone. Heat inactivation of human aqueous humor nearly eliminated human aqueous humor-stimulated myocilin secretion. Inhibitors of new protein synthesis, gene transcription, the endoplasmic reticulum/Golgi system and endocytic/exocytic secretory pathways failed to inhibit human aqueous humor-stimulated myocilin secretion. Using immunolabeling and transmission electron microscopy, myocilin was found associated with 70-90 nm vesicle-like structures within the cytoplasm of human aqueous humor treated trabecular meshwork cells. These studies suggest that myocilin secretion from trabecular meshwork cells occurs in a Golgi-independent manner following human aqueous humor treatment. Heat-labile factors in human aqueous humor are responsible for the time- and dose-dependent release of myocilin from vesicle-like structures within the cytoplasm of trabecular meshwork cells.

Canonical Wnt signaling has emerged as a critical regulator of aqueous outflow facility and intraocular pressure (IOP). In this study, we examine the role of canonical Wnt signaling on extracellular matrix (ECM) expression in the trabecular meshwork (TM) and explore the molecular mechanisms involved.

It has been suggested that common mechanisms may underlie the pathogenesis of primary open-angle glaucoma (POAG) and steroid-induced glaucoma (SIG). The biomechanical properties (stiffness) of the trabecular meshwork (TM) have been shown to differ between POAG patients and unaffected individuals. While features such as ocular hypertension and increased outflow resistance in POAG and SIG have been replicated in mouse models, whether changes of TM stiffness contributes to altered IOP homeostasis remains unknown. We found that outer TM was stiffer than the inner TM and, there was a significant positive correlation between outflow resistance and TM stiffness in mice where conditions are well controlled. This suggests that TM stiffness is intimately involved in establishing outflow resistance, motivating further studies to investigate factors underlying TM biomechanical property regulation. Such factors may play a role in the pathophysiology of ocular hypertension. Additionally, this finding may imply that manipulating TM may be a promising approach to restore normal outflow dynamics in glaucoma. Further, novel technologies are being developed to measure ocular tissue stiffness in situ. Thus, the changes of TM stiffness might be a surrogate marker to help in diagnosing altered conventional outflow pathway function if those technologies could be adapted to TM.