Dexamethasone alters F-actin architecture and promotes cross-linked actin network formation in human trabecular meshwork tissue

Elevated intraocular pressure is an important risk factor for the development of glaucoma, a leading cause of irreversible blindness. This ocular hypertension is due to increased hydrodynamic resistance to the drainage of aqueous humor through specialized outflow tissues, including the trabecular meshwork (TM) and the endothelial lining of Schlemm's canal. We know that glucocorticoid therapy can cause increased outflow resistance and glaucoma in susceptible individuals, that the cytoskeleton helps regulate aqueous outflow resistance, and that glucocorticoid treatment alters the actin cytoskeleton of cultured TM cells. Our purpose was to characterize the actin cytoskeleton of cells in outflow pathway tissues in situ, to characterize changes in the cytoskeleton due to dexamethasone treatment in situ, and to compare these with changes observed in cell culture. Human ocular anterior segments were perfused with or without 10(-7) M dexamethasone, and F-actin architecture was investigated by confocal laser scanning microscopy. We found that outflow pathway cells contained stress fibers, peripheral actin staining, and occasional actin "tangles." Dexamethasone treatment caused elevated IOP in several eyes and increased overall actin staining, with more actin tangles and the formation of cross-linked actin networks (CLANs). The actin architecture in TM tissues was remarkably similar to that seen in cultured TM cells. Although CLANs have been reported previously in cultured cells, this is the first report of CLANs in tissue. These cytoskeletal changes may be associated with increased aqueous humor outflow resistance after ocular glucocorticoid treatment.     

Novel molecular insights into RhoA GTPase-induced resistance to aqueous humor outflow through the trabecular meshwork

Impaired drainage of aqueous humor through the trabecular meshwork (TM) culminating in increased intraocular pressure is a major risk factor for glaucoma, a leading cause of blindness worldwide. Regulation of aqueous humor drainage through the TM, however, is poorly understood. The role of RhoA GTPase-mediated actomyosin organization, cell adhesive interactions, and gene expression in regulation of aqueous humor outflow was investigated using adenoviral vector-driven expression of constitutively active mutant of RhoA (RhoAV14). Organ-cultured anterior segments from porcine eyes expressing RhoAV14 exhibited significant reduction of aqueous humor outflow. Cultured TM cells expressing RhoAV14 exhibited a pronounced contractile morphology, increased actin stress fibers, and focal adhesions and increased levels of phosphorylated myosin light chain (MLC), collagen IV, fibronectin, and laminin. cDNA microarray analysis of RNA extracted from RhoAV14-expressing human TM cells revealed a significant increase in the expression of genes encoding extracellular matrix (ECM) proteins, cytokines, integrins, cytoskeletal proteins, and signaling proteins. Conversely, various ECM proteins stimulated robust increases in phosphorylation of MLC, paxillin, and focal adhesion kinase and activated Rho GTPase and actin stress fiber formation in TM cells, indicating a potential regulatory feedback interaction between ECM-induced mechanical strain and Rho GTPase-induced isometric tension in TM cells. Collectively, these data demonstrate that sustained activation of Rho GTPase signaling in the aqueous humor outflow pathway increases resistance to aqueous humor outflow through the trabecular pathway by influencing the actomyosin assembly, cell adhesive interactions, and the expression of ECM proteins and cytokines in TM cells.     

Myocilin is associated with mitochondria in human trabecular meshwork cells.

The trabecular meshwork (TM) is a specialized tissue located at the chamber angle of the eye next to the cornea. This tissue is believed to be responsible for regulation of the aqueous humor outflow and control of the intraocular pressure (IOP). Alterations in functions of the TM may lead to IOP elevation and development of glaucoma, a major cause of blindness. The myocilin gene has recently been directly linked to open-angle glaucomas. The gene product was originally identified as a protein inducible in TM cells by treatment with glucocorticoids such as dexamethasone (DEX) and termed TIGR (TM inducible-glucocorticoid response). The exact nature and function of the myocilin protein so far still remain elusive. In this study, myocilin was localized to the perinuclear region of both DEX-treated and control TM cells. Its distribution overlapped considerably with that of mitochondria. Subcellular fractionation and Western blot analyses suggested a rather extensive association of myocilin with mitochondria. The DEX-treated TM cells were found to undergo apoptosis, when exposed to anti-Fas antibody, to a significantly higher degree than the untreated control cells. It appears that the TM cell integrity remains intact after DEX treatment. However, the induced myocilin or myocilin-mitochondria association seems to render the cells more susceptible to a second stress or challenge. This vulnerability may be the basis that ultimately leads to pathological consequences.     

Senescent phenotype of trabecular meshwork cells displays biomarkers in primary open-angle glaucoma

Glaucoma is a major cause of irreversible blindness, affecting more than 70 million individuals worldwide. Elevated intraocular pressure (IOP) is a major risk factor in the development of glaucoma and in the progression of glaucomatous damage. High IOP usually occurs as a result of an increase in aqueous humor outflow resistance in trabecular meshwork (TM). Primary open angle glaucoma (POAG) is characterized by quantifiable parameters including the IOP, the aqueous outflow facility, and geometric measurements of the optic disc and visual defects. Morphological and biochemical analyses of the TM of POAG patients revealed loss of cells, increased accumulation of extracellular matrix (ECM), changes in the cytoskeleton, cellular senescence and the process of subclinical inflammation. Various biochemical and molecular biology biomarkers of TM cells senescence are considered in the article. Oxidative stress is becoming an important factor more likely to be involved in the pathogenesis of POAG. Treatment of TM cells with oxidative stress induced POAG-typical changes like ECM accumulation, cell death, disarrangement of the cytoskeleton, advanced senescence and the release of inflammatory markers. Oxidative stress is able to induce characteristic glaucomatous TM changes and these oxidative stress-induced TM changes can be minimized by the use of antioxidants, such as carnosine-related analogues and IOP-lowering substances. There is evidence demonstrating that carnosine related analogues may have antioxidative capacities, can prevent cellular senescence and the attrition of telomeres during the action of oxidative stress. Prevention of oxidative stress exposure to the TM with N-acetylcarnosine ophthalmic prodrug of carnosine and oral formulation of non-hydrolized carnosine may help to reduce the progression of POAG. The previous work has demonstrated that carnosine is able to reach the TM directly via the transcorneal and systemic pathways of administration with N-acetylcarnosine ophthalmic prodrug and oral formulation of non-hydrolized carnosine. We suggest in this article that dual therapy with N-acetylcarnosine lubricant eye drops, oral formulation of non-hydrolized carnosine combined with anti-glaucoma adrenergic drug may become the first-line therapy in glaucoma due to their efficiency in reducing IOP, prevention and reversal of oxidative stress-induced damages in TM and the low rate of severe side effects during combined treatment.     

Role of the autotaxin-lysophosphatidic acid axis in glaucoma,aqueous humor drainage and fibrogenic activity

Ocular hypertension due to impaired aqueous humor (AH) drainage through the trabecular meshwork (TM) is a major risk factor for glaucoma, a leading cause of irreversible blindness. However, the etiology of ocular hypertension remains unclear. Although autotaxin, a secreted lysophospholipase D and its catalytic product lysophosphatidic acid (LPA) have been shown to modulate AH drainage through TM, we do not have a complete understanding of their role and regulation in glaucoma patients, TM and AH outflow. This study reports a significant increase in the levels of autotaxin, lysophosphatidylcholine (LPC), LPA and connective tissue growth factor (CTGF) in the AH of Caucasian and African American open angle glaucoma patients relative to age-matched non-glaucoma patients. Treatment of human TM cells with dexamethasone, tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) increased the levels of autotaxin protein, a response that was mitigated by inhibitors of glucocorticoid receptor, NF-kB and SMAD3. Dexamethasone, TNF-α, IL-1β and LPC treatment of TM cells also led to an increase in the levels of CTGF, fibronectin and collagen type 1 in an autotaxin dependent manner. Additionally, in perfused enucleated mouse eyes, autotaxin and LPC were noted to decrease, while inhibition of autotaxin was increased aqueous outflow through the TM. Taken together, these results provide additional evidence for dysregulation of the autotaxin-LPA axis in the AH of glaucoma patients, reveal molecular insights into the regulation of autotaxin expression in TM cells and the consequences of autotaxin inhibitors in suppressing the fibrogenic response and resistance to AH outflow through the TM.     

Mechanisms of ATP release, the enabling step in purinergic dynamics

The only effective intervention to slow onset and progression of glaucomatous blindness is to lower intraocular pressure (IOP). Among other modulators, adenosine receptors (ARs) exert complex regulation of IOP. Agonists of A(3)ARs in the ciliary epithelium activate Cl(-) channels, favoring increased formation of aqueous humor and elevated IOP. In contrast, stimulating A(1)ARs in the trabecular outflow pathway enhances release of matrix metalloproteinases (MMPs) from trabecular meshwork (TM) cells, reducing resistance to outflow of aqueous humor to lower IOP. These opposing actions are thought to be initiated by cellular release of ATP and its ectoenzymatic conversion to adenosine. This view is now supported by our identification of six ectoATPases in trabecular meshwork (TM) cells and by our observation that external ATP enhances TM-cell secretion of MMPs through ectoenzymatic formation of adenosine. ATP release is enhanced by cell swelling and stretch. Also, enhanced ATP release and downstream MMP secretion is one mediator of the action of actin depolymerization to reduce outflow resistance. Inflow and outflow cells share pannexin-1 and connexin hemichannel pathways for ATP release. However, vesicular release and P2X(7) release pathways were functionally limited to inflow and outflow cells, respectively, suggesting that blocking exocytosis might selectively inhibit inflow, lowering IOP.     

The regulation of connective tissue growth factor expression influences the viability of human trabecular meshwork cells

Connective tissue growth factor (CTGF) induces extracellular matrix (ECM) synthesis and contractility in human trabecular meshwork (HTM) cells. Both processes are involved in the pathogenesis of primary open‐angle glaucoma. To date, little is known about regulation and function of CTGF expression in the trabecular meshwork (TM). Therefore, we analysed the effects of different aqueous humour proteins and stressors on CTGF expression in HTM cells. HTM cells from three different donors were treated with endothelin‐1, insulin‐like growth factor (IGF)‐1, angiotensin‐II, H₂O₂ and heat shock and were analysed by immunohistochemistry, real‐time RT‐PCR and Western blotting. Viability after H₂O₂ treatment was measured in CTGF silenced HTM‐N cells and their controls. Latrunculin A reduced expression of CTGF by about 50% compared to untreated HTM cells, whereas endothelin‐1, IGF‐1, angiotensin‐II, heat shock and oxidative stress led to a significant increase. Silencing of CTGF resulted in a delayed expression of αB‐crystallin and in reduced cell viability in comparison to the controls after oxidative stress. Conversely, CTGF treatment led to a higher cell viability rate after H₂O₂ treatment. CTGF expression is induced by factors that have been linked to glaucoma. An increased level of CTGF appears to protect TM cells against damage induced by stress. The beneficial effect of CTGF for viability of TM cells is likely associated with the effects on increased ECM synthesis and higher contractility of the TM, thereby contributing to reduced aqueous humour outflow facility causing increased intraocular pressure.     

Potential role of lysosomal dysfunction in the pathogenesis of primary open angle glaucoma

Primary open angle glaucoma (POAG) is a late onset disease usually accompanied by elevated intraocular pressure (IOP) that results from the failure of the trabecular meshwork (TM) to maintain normal levels of aqueous humor outflow resistance. Cells in the TM are subjected to chronic oxidative stress through reactive oxygen species (ROS) present in the aqueous humor (AH) and generated by normal metabolism. Exposure to ROS is thought to contribute to the morphological and physiological alterations of the outflow pathway in aging and POAG. Our results indicate that chronic exposure of TM cells to oxidative stress causes the accumulation of nondegradable material within the lysosomal compartment leading to diminished lysosomal activity and increased SA-β-Gal expression. Because the lysosomal compartment is responsible for maintaining general cellular turnover, such impaired activity may lead to a progressive cellular decline in the TM cell function and thus contribute to the progression of POAG.     

Galectin-8 Promotes Cytoskeletal Rearrangement in Trabecular Meshwork Cells through Activation of Rho Signaling

Purpose

The trabecular meshwork (TM) cell-matrix interactions and factors that influence Rho signaling in TM cells are thought to play a pivotal role in the regulation of aqueous outflow. The current study was designed to evaluate the role of a carbohydrate-binding protein, galectin-8 (Gal8), in TM cell adhesion and Rho signaling.

Methods

Normal human TM cells were assayed for Gal8 expression by immunohistochemistry and Western blot analysis. To assess the role of Gal8 in TM cell adhesion and Rho signaling, the cell adhesion and spreading assays were performed on Gal8-coated culture plates in the presence and the absence of anti-β₁ integrin antibody and Rho and Rho-kinase inhibitors. In addition, the effect of Gal8-mediated cell-matrix interactions on TM cell cytoskeleton arrangement and myosin light chain 2 (MLC2) phosphorylation was examined.

Principal Findings

We demonstrate here that Gal8 is expressed in the TM and a function-blocking anti-β₁ integrin antibody inhibits the adhesion and spreading of TM cells to Gal8-coated wells. Cell spreading on Gal8 substratum was associated with the accumulation of phosphorylated myosin light chain and the formation of stress fibers that was inhibited by the Rho inhibitor, C3 transferase, as well as by the Rho-kinase inhibitor, Y27632.

Conclusions/Significance

The above findings present a novel function for Gal8 in activating Rho signaling in TM cells. This function may allow Gal8 to participate in the regulation of aqueous outflow.     

Glypican-4 regulated actin cytoskeletal reorganization in glucocorticoid treated trabecular meshwork cells and involvement of Wnt/PCP signaling

A common adverse response to the clinical use of glucocorticoids (GCs) is elevated intraocular pressure (IOP) which is a major risk factor for glaucoma. Elevated IOP arises due to impaired outflow of aqueous humor (AH) through the trabecular meshwork (TM). Although GC-induced changes in actin cytoskeletal dynamics, contractile characteristics, and cell adhesive interactions of TM cells are believed to influence AH outflow and IOP, the molecular mechanisms mediating changes in these cellular characteristics are poorly understood. Our studies focused on evaluating changes in the cytoskeletal and cytoskeletal-associated protein (cytoskeletome) profile of human TM cells treated with dexamethasone (Dex) using label-free mass spectrometric quantification, identified elevated levels of specific proteins known to regulate actin stress fiber formation, contraction, actin networks crosslinking, cell adhesion, and Wnt signaling, including LIMCH1, ArgBP2, CNN3, ITGBL1, CTGF, palladin, FAT1, DIAPH2, EPHA4, SIPA1L1, and GPC4. Several of these proteins colocalized with the actin cytoskeleton and underwent alterations in distribution profile in TM cells treated with Dex, and an inhibitor of Abl/Src kinases. Wnt/Planar Cell Polarity (PCP) signaling agonists-Wnt5a and 5b were detected prominently in the cytoskeletome fraction of TM cells, and studies using siRNA to suppress expression of glypican-4 (GPC4), a known modulator of the Wnt/PCP pathway revealed that GPC4 deficiency impairs Dex induced actin stress fiber formation, and activation of c-Jun N-terminal Kinase (JNK) and Rho kinase. Additionally, while Dex augmented, GPC4 deficiency suppressed the formation of actin stress fibers in TM cells in the presence of Dex and Wnt5a. Taken together, these results identify the GPC4-dependent Wnt/PCP signaling pathway as one of the crucial upstream regulators of Dex induced actin cytoskeletal reorganization and cell adhesion in TM cells, opening an opportunity to target the GPC4/Wnt/PCP pathway for treatment of ocular hypertension in glaucoma.     

The role of oxidative stress in glaucoma

DNA damage is related to a variety of degenerative diseases such as cancer, atherosclerosis and neurodegenerative diseases, depending on the tissue affected. Increasing evidence indicates that reactive oxygen species (ROS) play a key role in the pathogenesis of primary open angle glaucoma (POAG), the main cause of irreversible blindness worldwide. Oxidative DNA damage is significantly increased in the ocular epithelium regulating aqueous humor outflow, i.e., the trabecular meshwork (TM), of glaucomatous patients compared to controls. The pathogenic role of ROS in glaucoma is supported by various experimental findings, including (a) resistance to aqueous humor outflow is increased by hydrogen peroxide by inducing TM degeneration; (b) TM possesses remarkable antioxidant activities, mainly related to superoxide dismutase-catalase and glutathione pathways that are altered in glaucoma patients; and (c) intraocular-pressure increase and severity of visual-field defects in glaucoma patients parallel the amount of oxidative DNA damage affecting TM. Vascular alterations, which are often associated with glaucoma, could contribute to the generation of oxidative damage. Oxidative stress, occurring not only in TM but also in retinal cells, appears to be involved in the neuronal cell death affecting the optic nerve in POAG. The highlighting of the pathogenic role of ROS in POAG has implications for the prevention of this disease as indicated by the growing number of studies using genetic analyses to identify susceptible individuals and of clinical trials testing the efficacy of antioxidant drugs for POAG management.     

Proteasome inhibition by chronic oxidative stress in human trabecular meshwork cells

The pathophysiologic mechanisms leading to the malfunction of the trabecular meshwork (TM)-Schlemm's canal (SC) outflow pathway in glaucoma are still unclear. We hypothesize that chronic oxidative stress may contribute to the malfunction of the outflow pathway by impairing the intracellular proteasome system of the cells, decreasing the ability of the tissue to modulate outflow resistance. To study the effects of chronic oxidative stress on proteasome function, primary cultures of human TM cells were incubated under 40% oxygen and proteasome activity was analyzed by measuring the accumulation of enhanced green fluorescent protein fused to a PEST motif. Changes in proteasome content, cellular senescence, and cell viability were also monitored. After 10 days of exposure to chronic oxidative stress, TM cells showed a marked decline in proteasome activity that was associated with premature senescence and decreased cell viability. These results suggest that proteasome failure may be involved in glaucoma pathophysiology.     

Intracellular Cl regulates Na-K-Cl cotransport activity in human trabecular meshwork cells

The trabecular meshwork (TM) of the eye plays a central role inmodulating intraocular pressure by regulating aqueous humor outflow,although the mechanisms are largely unknown. We and others have shownpreviously that aqueous humor outflow facility is modulated byconditions that alter TM cell volume. We have also shown that theNa-K-Cl cotransport system is a primary regulator of TM cell volume andthat its activity appears to be coordinated with net efflux pathways tomaintain steady-state volume. However, the cellular mechanisms thatregulate cotransport activity and cell volume in TM cells have yet tobe elucidated. The present study was conducted to investigate thehypothesis that intracellular Cl concentration([Cl]_i) acts toregulate TM cell Na-K-Cl cotransport activity, as has been shownpreviously for some other cell types. We demonstrate here that thehuman TM cell Na-K-Cl cotransporter is highly sensitive to changes in[Cl]_i. Our findingsreveal a marked stimulation of Na-K-Cl cotransport activity, assessedas ouabain-insensitive, bumetanide-sensitive K influx, in TM cells following preincubation of cells with Cl-free medium as a means ofreducing [Cl]_i. Incontrast, preincubation of cells with media containing elevated Kconcentrations as a means of increasing [Cl]_i results ininhibition of Na-K-Cl cotransport activity. The effects of reducing[Cl]_i, as well aselevating [Cl]_i, onNa-K-Cl cotransport activity are concentration dependent. Furthermore, the stimulatory effect of reduced[Cl]_i is additive withcell-shrinkage-induced stimulation of the cotransporter. Our studiesalso show that TM cell Na-K-Cl cotransport activity is altered by avariety of Cl channel modulators, presumably through changes in[Cl]_i. These findingssupport the hypothesis that regulation of Na-K-Cl cotransport activity,and thus cell volume, by[Cl]_i may participatein modulating outflow facility across the TM.

     

Mapping Molecular Differences and Extracellular Matrix Gene Expression in Segmental Outflow Pathways of the Human Ocular Trabecular Meshwork

Elevated intraocular pressure (IOP) is the primary risk factor for glaucoma, and lowering IOP remains the only effective treatment for glaucoma. The trabecular meshwork (TM) in the anterior chamber of the eye regulates IOP by generating resistance to aqueous humor outflow. Aqueous humor outflow is segmental, but molecular differences between high and low outflow regions of the TM are poorly understood. In this study, flow regions of the TM were characterized using fluorescent tracers and PCR arrays. Anterior segments from human donor eyes were perfused at physiological pressure in an ex vivo organ culture system. Fluorescently-labeled microspheres of various sizes were perfused into anterior segments to label flow regions. Actively perfused microspheres were segmentally distributed, whereas microspheres soaked passively into anterior segments uniformly labeled the TM and surrounding tissues with no apparent segmentation. Cell-tracker quantum dots (20 nm) were localized to the outer uveal and corneoscleral TM, whereas larger, modified microspheres (200 nm) localized throughout the TM layers and Schlemm’s canal. Distribution of fluorescent tracers demonstrated a variable labeling pattern on both a macro- and micro-scale. Quantitative PCR arrays allowed identification of a variety of extracellular matrix genes differentially expressed in high and low flow regions of the TM. Several collagen genes (COL16A1, COL4A2, COL6A1 and 2) and MMPs (1, 2, 3) were enriched in high, whereas COL15A1, and MMP16 were enriched in low flow regions. Matrix metalloproteinase activity was similar in high and low regions using a quantitative FRET peptide assay, whereas protein levels in tissues showed modest regional differences. These gene and protein differences across regions of the TM provide further evidence for a molecular basis of segmental flow routes within the aqueous outflow pathway. New insight into the molecular mechanisms of segmental aqueous outflow may aid in the design and delivery of improved treatments for glaucoma patients.     

Mechanism of the reconstruction of aqueous outflow drainage

Glaucoma is the leading cause of irreversible blindness worldwide. The reconstruction of aqueous outflow drainage (RAOD) has recently been proposed to aid in restoring aqueous outflow drainage in primary open-angle glaucoma. However, the mechanism of RAOD remains to be fully understood. Based on literature review and research studies, the potential mechanisms of RAOD are the following: (i) Circumferential dilation of the Schlemm’s canal (SC) and surrounding collector channels. (ii) Instant formation of microcracks through RAOD procedures. (iii) Formation of more pores, and local detachment between the SC endothelium (SCE) and basement membrane. (iv) Activation of stem cells by constant mechanical stress caused by the tensional suture placed at the anterior part of the SC. (v) Reversal of trabecular meshwork (TM) herniation. (vi) Mobilization of the reserve of the aqueous drainage. (vii) Change of SCE phenotype. (viii) Mechanosensing and mechanotransducing of TM.