Porcine Trabecular Meshwork Cells Research Articles

Cell senescence alters responses of porcine trabecular meshwork cells to shear stress.

Mechanical microenvironment and cellular senescence of trabecular meshwork cells (TMCs) are suspected to play a vital role in primary open-angle glaucoma pathogenesis. However, central questions remain about the effect of shear stress on TMCs and how aging affects this process. We have investigated the effect of shear stress on the biomechanical properties and extracellular matrix regulation of normal and senescent TMCs. We found a more significant promotion of Fctin formation, a more obvious realignment of F-actin fibers, and a more remarkable increase in the stiffness of normal cells in response to the shear stress, in comparison with that of senescent cells. Further, as compared to normal cells, senescent cells show a reduced extracellular matrix turnover after shear stress stimulation, which might be attributed to the different phosphorylation levels of the extracellular signal-regulated kinase. Our results suggest that TMCs are able to sense and respond to the shear stress and cellular senescence undermines the mechanobiological response, which may lead to progressive failure of cellular TM function with age.

The Effects of Cannabidiol on Aqueous Humor Outflow and Trabecular Meshwork Cell Signaling.

Intraocular pressure (IOP) is regulated primarily through aqueous humor production by ciliary body and drainage through uveoscleral and trabecular meshwork (TM) tissues. The goal of this study was to measure the effect of non-psychotropic cannabidiol (CBD) on aqueous humor outflow through TM and assess the effect of CBD on the TM cell signaling pathways that are important for regulating outflow. Perfused porcine eye anterior segment explants were used to investigate the effects of CBD on aqueous humor outflow. Cultured porcine TM cells were used to study the effects of CBD on TM cell contractility, myosin light chain (MLC) and myosin phosphatase targeting subunit 1 (MYPT1) phosphorylation, and RhoA activation. In the anterior segment perfusion experiments, aqueous humor outflow was increased significantly within 1 h after adding 1 µM CBD and the effect was sustained over the 5 h of measurement. Treatment of TM cells with 1 µM CBD significantly decreased TM cell-mediated collagen contraction, inhibited phosphorylation of MLC and MYPT1, and reduced RhoA activation. Our data demonstrate, for the first time, that as a potential therapeutic agent for lowering intraocular pressure, CBD can enhance aqueous humor outflow and modify TM cell signaling.

A Porcine Organ-Culture Glaucoma Model Mimicking Trabecular Meshwork Damage Using Oxidative Stress.

PurposeRe-cellularization of the trabecular meshwork (TM) using stem cells is a potential novel treatment for ocular hypertension associated with glaucoma. To assess the therapeutic efficacy of this approach, improved in vivo and ex vivo models of TM pathophysiology are needed. Here, we investigate whether oxidative stress, induced by hydrogen peroxide (H2O2), can model glaucomatous ocular hypertension in the readily available porcine anterior segment organ culture model.MethodsThe impact of H2O2 on TM cell viability and function was first evaluated in vitro using primary porcine TM cells. Oxidative stress was then induced by H2O2 infusion into perfused porcine anterior segments. Trabecular meshwork function was assessed by tracking matrix metalloproteinase (MMP) activity and the ability of the preparation to maintain intraocular pressure (IOP) homeostasis after a flow challenge (doubled fluid infusion rate). Finally, the TM was evaluated histologically.ResultsH2O2 treatment resulted in a titratable reduction in cellularity across multiple primary TM cell donor strains. In organ culture preparations, H2O2-treated eyes showed impaired IOP homeostasis (i.e., IOPs stabilized at higher levels after a flow challenge vs. control eyes). This result was consistent with reduced MMP activity and TM cellularity; however, damage to the TM microstructure was not histologically evident in anterior segments receiving H2O2.ConclusionsTitrated H2O2 infusion resulted in TM cellular dysfunction without destruction of TM structure. Thus, this porcine organ culture model offers a useful platform for assessing trabecular meshwork therapies to treat ocular hypertension associated with glaucoma.

Impact of pigment dispersion on trabecular meshwork cells.

Dysfunction of the trabecular meshwork (TM) in pigmentary glaucoma contributes to increased aqueous humor outflow resistance and intraocular pressure. In this study, we investigated the effect of pigment dispersion on trabecular meshwork cells. Porcine TM cells from ab interno trabeculectomy specimens were exposed to pigment dispersion, then, analyzed for changes in morphology, immunostaining, and ultrastructure. Their abilities to phagocytose migrate, and contraction was quantified. An expression microarray, using 23,937 probes, and a pathway analysis were performed. Stress fiber formation was increased in the pigment dispersion group (P) (60.1 ± 0.3%, n = 10) compared to control (C) (38.4 ± 2.5%, n = 11, p < 0.001). Phagocytosis declined (number of cells with microspheres in P = 37.0 ± 1.1% and in C= 68.7 ± 1.3%, n = 3, p < 0.001) and migration was reduced after 6h (cells within the visual field over 6h in P = 28.0.1 ± 2.3 (n = 12) and in C = 40.6 ± 3.3 (n = 13), p < 0.01). Pigment induced contraction at 24h onwards (p < 0.01). Microarray analysis revealed that Rho signaling was central to these responses. Exposure of TM cells to pigment dispersion resulted in reduced phagocytosis and migration, as well as increased stress fiber formation and cell contraction. The Rho signaling pathway played a central and early role, suggesting that its inhibitors could be used as a specific intervention in treatment of pigmentary glaucoma.

Discovery and Preclinical Development of Netarsudil, a Novel Ocular Hypotensive Agent for the Treatment of Glaucoma.

Purpose: Rho-associated protein kinase (ROCK) inhibitors lower intraocular pressure (IOP) by increasing aqueous outflow through the trabecular meshwork (TM). The preclinical characterization of netarsudil, a new ROCK/norepinephrine transporter (NET) inhibitor currently in clinical development, is presented herein.Methods: The kinase inhibitory activity of netarsudil was compared to its esterase metabolite, netarsudil-M1, and 3 other ROCK inhibitors using a commercially available kinase assay kit. Disruption of actin stress fibers was measured in primary porcine TM cells and disruption of focal adhesions in transformed human TM (HTM) cells. Induction of fibrosis markers after exposure to transforming growth factor-β2 (TGF-β2) was conducted in primary HTM cells. Ocular hypotensive activity and tolerability of topical formulations were evaluated in normotensive Dutch Belted rabbits and Formosan Rock monkeys. In vitro corneal metabolism assays were conducted using dog, pig, rabbit, monkey, and human corneas. In vivo ocular pharmacokinetics was studied in Dutch Belted rabbits.Results: Netarsudil inhibited kinases ROCK1 and ROCK2 with a Ki of 1 nM each, disrupted actin stress fibers and focal adhesions in TM cells with IC50s of 79 and 16 nM, respectively, and blocked the profibrotic effects of TGF-β2 in HTM cells. Netarsudil produced large reductions in IOP in rabbits and monkeys that were sustained for at least 24 h after once daily dosing, with transient, mild hyperemia observed as the only adverse effect.Conclusion: Netarsudil is a novel ROCK/NET inhibitor with high potency in biochemical and cell-based assays, an ability to produce large and durable IOP reductions in animal models, and favorable pharmacokinetic and ocular tolerability profiles.

Live cell imaging of actin dynamics in dexamethasone-treated porcine trabecular meshwork cells

The regulation of the actin cytoskeleton in trabecular meshwork (TM) cells is important for controlling outflow of the aqueous humor. In some reports, dexamethasone (DEX) increased the aqueous humor outflow resistance and induced unusual actin structures, such as cross-linked actin networks (CLAN), in TM cells. However, the functions and dynamics of CLAN in TM cells are not completely known, partly because actin stress fibers have been observed only in fixed cells. We conducted live-cell imaging of the actin dynamics in TM cells with or without DEX treatment. An actin-green fluorescent protein (GFP) fusion construct with a modified insect virus was transfected into porcine TM cells. Time-lapse imaging of live TM cells treated with 25 μM Y-27632 and 100 nM DEX was performed using an inverted fluorescence microscope. Fluorescent images were recorded every 15 s for 30 min after Y-27632 treatment or every 30 min for 72 h after DEX treatment. The GFP-actin was expressed in 22.7 ± 10.9% of the transfected TM cells. In live TM cells, many actin stress fibers were observed before the Y-27632 treatment. Y-27632 changed the cell shape and decreased stress fibers in a time-dependent manner. In fixed cells, CLAN-like structures were seen in 26.5 ± 1.7% of the actin-GFP expressed PTM cells treated with DEX for 72 h. In live imaging, there was 28% CLAN-like structure formation at 72 h after DEX treatment, and the lifetime of CLAN-like structures increased after DEX treatment. The DEX-treated cells with CLAN-like structures showed less migration than DEX-treated cells without CLAN-like structures. Furthermore, the control cells (without DEX treatment) with CLAN-like structures also showed less migration than the control cells without CLAN-like structures. These results suggested that CLAN-like structure formation was correlated with cell migration in TM cells. Live cell imaging of the actin cytoskeleton provides valuable information on the actin dynamics in TM cells.

Ascorbic Acid Modulation of Iron Homeostasis and Lysosomal Function in Trabecular Meshwork Cells

To investigate the antioxidant properties and biological functions of ascorbic acid (AA) on trabecular meshwork (TM) cells. Primary cultures of porcine TM cells were supplemented for 10 days with increasing concentrations of AA. Antioxidant properties against cytotoxic effect of H2O2 were evaluated by monitoring cell viability. Redox-active iron was quantified using calcein-AM. Intracellular reactive oxygen species (iROS) production was quantified using H2DCFDA. Ferritin and cathepsin protein levels were analyzed by Western blot. Autophagy was evaluated by monitoring lipidation of LC3-I to LC3-II. Lysosomal proteolysis and cathepsins activities were quantified using specific fluorogenic substrates. AA exerts a dual effect against oxidative stress in TM cells, acting as an anti-oxidant or a pro-oxidant, depending on the concentration used. The pro-oxidant effect of AA was mediated by free intracellular iron and correlated with increased protein levels of ferritin and elevated iROS. In contrast, antioxidant properties correlated with lower ferritin and basal iROS content. Ascorbic acid supplementation also caused induction of autophagy, as well as increased lysosomal proteolysis, with the latter resulting from higher proteolytic activation of lysosomal cathepsins in treated cultures. Our results suggest that the reported decrease of AA levels in plasma and aqueous humor can compromise lysosomal degradation in the outflow pathway cells with aging and contribute to the pathogenesis of glaucoma. Restoration of physiological levels of vitamin C inside the cells might improve their ability to degrade proteins within the lysosomal compartment and recover tissue function.

Controllable Urokinase Gene Expression in Trabecular Meshwork Cells

Purpose: Glaucoma is a type of progressive optic neuropathy that finally leads to blindness related to elevated intraocular pressure (IOP). Accumulation of extracellular matrix in trabecular meshwork (TM) and juxta-canalicular connective tissues, which form an aqueous outflow pathway, may be a major cause of increased IOP, thus the fibrinolytic system may be associated with regulation of IOP. We examined the possibility of controllable urokinase plasminogen activator (uPA) gene transfer into TM cells.Methods: TM cells were freshly isolated from porcine eyes and human TM cells obtained during trabeculectomy procedures and cultured. Total RNA was extracted from human TM cells and reverse transcribed into cDNA. A reverse-transcribed polymerase chain reaction (RT-PCR) method was then performed to detect the gene expression of uPA. The cDNA of human uPA was sub-cloned into an expression vector (pEYFP-N1 vector) and controllable expression vector (TRE-Tight vector), then each vector was independently transfected into cultured porcine TM cells. Doxycycline was added to the culture medium to activate the pTet-ON and TRE-Tight vectors. Finally, the expression of uPA was examined using an enzyme linked immunosorbent assay (ELISA).Results: ELISA findings revealed the expression of uPA (3.5 ng/ml) in medium from cultured porcine TM cells that had been transfected with the human uPA gene using the pEYFP-N1 vector. Doxycycline induced human uPA in the pTet-On and TRE-Tight vectors with human uPA gene co-transfected TM cells in a dose-dependent manner.Conclusions: Controllable gene transfer of uPA, which may degrade the extracellular matrix in juxta-canalicular connective tissue, into TM cells was achieved using a Tet-On system. The present method may be useful as a novel therapy for glaucoma.

Effects of roscovitine, a cell cycling-dependent kinase inhibitor, on intraocular pressure of rabbit and retinal ganglion cell damage

Glaucoma is characterized by increased intraocular pressure (IOP) and the death of retinal ganglion cells. Previously, we reported that roscovitine, a cell cyclin-dependent kinase (CDK) inhibitor, strongly induced relaxation of porcine trabecular meshwork cells, implicating an interaction with lowered IOP. In addition, the activity of CDKs is known to increase in response to high IOP, which is linked to retinal ganglion cell damage. However, the effects of roscovitine on IOP and retinal damage have not been investigated. Roscovitine has racemic isomers that differ in their inhibition of CDKs. Therefore, we investigated the effects of both the R-isomer and the S-isomer on the IOP of rabbits and on the death of cultured retinal ganglion cells. In the in vivo rabbit experiment, instillation of both isomers significantly lowered the IOP. In the in vitro cell experiment, the R-isomer amplified the effects of tunicamycin, an endoplasmic reticulum stress inducer, and increased oxygen–glucose deprivation-induced cell death, whereas S-isomer significantly inhibited this cell death. Therefore, both isomers of roscovitine can lower the IOP, but from the perspective of neuroprotective effects, the S-isomer was superior to the R-isomer. The S-isomer of roscovitine may be useful as an agent for lowering IOP and its neuroprotective effects.

Endogenous Production of Extracellular Adenosine by Trabecular Meshwork Cells: Potential Role in Outflow Regulation

To investigate the mechanisms for endogenous production of extracellular adenosine in trabecular meshwork (TM) cells and evaluate its physiological relevance to the regulation of aqueous humor outflow facility. Extra-cellular levels of adenosine monophosphate (AMP) and adenosine in porcine trabecular meshwork (PTM) cells treated with adenosine triphosphate (ATP), AMP, cAMP or forskolin with or without specific inhibitors of phosphodiesterases (IBMX) and CD73 (AMPCP) were determined by high-pressure liquid chromatography fluorometry. Extracellular adenosine was also evaluated in cell cultures subjected to cyclic mechanical stress (CMS) (20% stretching; 1 Hz) and after disruption of lipid rafts with methyl-β-cyclodextrin. Expression of CD39 and CD73 in porcine TM cells and tissue were examined by Q-PCR and Western blot. The effect of inhibition of CD73 on outflow facility was evaluated in perfused living mouse eyes. PTM cells generated extracellular adenosine from extracellular ATP and AMP but not from extracellular cAMP. Increased intracellular cAMP mediated by forskolin led to a significant increase in extracellular adenosine production that was not prevented by IBMX. Inhibition of CD73 resulted, in all cases, in a significant decrease in extracellular adenosine. CMS induced a significant activation of extracellular adenosine production. Inhibition of CD73 activity with AMPCP in living mouse eyes resulted in a significant decrease in outflow facility. These results support the concept that the extracellular adenosine pathway might play an important role in the homeostatic regulation of outflow resistance in the TM, and suggest a novel mechanism by which pathologic alteration of the TM, such as increased tissue rigidity, could lead to abnormal elevation of IOP in glaucoma.

Antioxidants Protect Trabecular Meshwork Cells From Hydrogen Peroxide-Induced Cell Death

To examine the protective effects of antioxidants in cultured trabecular meshwork (TM) cells exposed to oxidative stress. Primary porcine TM cells were pretreated with 50 μM resveratrol, 0.2 mM urate, 1 mM ascorbate, 1 mM reduced glutathione (rGSH), or 1 mM ρ-coumarate followed by exposure to hydrogen peroxide (0.5-4 mM). Cell metabolism was determined by mitochondrial enzyme activity and cell viability by uptake of the vital dye calcein, a fluorescent calcium binding dye. Reactive oxygen species (ROS), which may reflex oxidative damage, were determined by 2'7'-dichlorofluorescein diacetate. Trabecular meshwork cell metabolism was reduced to 72 ± 5% of control levels with 1 mM hydrogen peroxide (H2O2) treatment. TM cells that co-incubated with ascorbate (85% ± 5%), ρ-coumarate (98 ± 11%) or rGSH (103 ± 17%) had significantly increased metabolism compared to 1 mM H2O2 treatment. Resveratrol significantly increased TM cell metabolism at both 2 mM (102 ± 14% live) and 4 mM H2O2 (27 ± 12% live), with H2O2-treated cultures containing mostly metabolically inactive cells (3% at 2 mM; 2% at 4 mM). Similar results were obtained in cell viability assays. Ascorbate and resveratrol, but not ρ-coumarate or rGSH, decreased ROS levels in TM cells exposed to a sublethal dose of H2O2 (0.5 mM). Urate had no protective effect against H2O2 damage in any of the assays. Increased oxidative damage was demonstrated in the TM of patients with primary open angle glaucoma. The antioxidants (resveratrol, ascorbate, ρ-coumarate) and the antioxidant enzyme cofactor (rGSH) protected TM cells from H2O2-induced damage. Future experiments are needed to determine whether addition of antioxidants may maintain TM cell viability in vivo. Antioxidants could be applied either topically or coupled with extended-release vehicles for intraocular injection to reduce free radical formation leading to enhanced therapeutic outcomes. Ultimately, studies using animal models could determine whether application of antioxidants can ameliorate progression in diseases such as glaucoma and macular degeneration.

Up-Regulated Expression of Extracellular Matrix Remodeling Genes in Phagocytically Challenged Trabecular Meshwork Cells

BackgroundCells in the trabecular meshwork (TM), the tissue responsible for draining aqueous humor out of the eye, are known to be highly phagocytic. Phagocytic function in TM cells is thought to play an important role in the normal functioning of the outflow pathway. Dysfunction of phagocytosis could lead to abnormalities of outflow resistance and increased intraocular pressure (IOP). However, the molecular mechanisms triggered by phagocytosis in TM cells are completely unknown.Methodology/Principal FindingsGene expression profile analysis of human TM cells phagocytically challenged to E. coli or pigment under physiological and oxidative stress environment were performed using Affymetrix U133 plus 2.0 array and analyzed with Genespring GX. Despite the differential biological response elicited by E. coli and pigment particles, a number of genes, including MMP1, MMP3, TNFSF11, DIO2, KYNU, and KCCN2 showed differential expression with both phagocytic ligands in all conditions. Data was confirmed by qPCR in both human and porcine TM cells. Metacore pathway analysis and the usage of recombinant adenovirus encoding the dominant negative mutant of IkB identified NF-κB as a transcription factor mediating the up-regulation of at least MMP1 and MMP3 in TM cells with phagocytosis. In-gel zymography demonstrated increased collagenolytic and caseinolytic activities in the culture media of TM cells challenge to E. coli. In addition, collagenolytic I activity was further confirmed using the self-quenched fluorescent substrate DQ-Collagen I.Conclusions/SignificanceHere we report for the first time the differential gene expression profile of TM cells phagocytically challenged with either E. coli or pigment. Our data indicate a potential role of phagocytosis in outflow pathway tissue homeostasis through the up-regulation and/or proteolytic activation of extracellular matrix remodeling genes.

Effects of cardiotonic steroids on trabecular meshwork cells: Search for mediator of ouabain-enhanced outflow facility

Lowering intraocular pressure (IOP) is currently the only strategy documented to slow the onset and progression of glaucomatous blindness. Ouabain, a cardiotonic glycoside inhibitor of Na+, K+-activated ATPase, was recently reported to enhance outflow facility in porcine anterior segments at concentrations as low as 30 nM for ≥4 h, suggesting a novel approach to lowering IOP. The underlying mechanism is unknown, but associated cytoskeletal changes were observed in porcine trabecular meshwork cells. We have previously found that changes in ATP release and subsequent ectoenzymatic conversion to adenosine may play a role in linking cytoskeletal remodeling with modulation of outflow resistance. We now tested whether altered ATP release might also be a mediator of ouabain's effect on outflow facility. ATP release from transformed human TM5 and explant-derived human trabecular meshwork cells was measured by the luciferin–luciferase reaction. Matrix metalloproteinases (MMPs) were studied by zymography, cell Na+ concentration by SBFI fluorometry, gene expression of ATP-release pathways by real-time PCR, cell volume by electronic cell sorting and cell viability by the LDH and MTT methods. Actin was examined by confocal microscopy of phalloidin-stained cells. Contrary to expectation, ouabain at concentrations ≥10 nM inhibited swelling-triggered ATP release from TM5 cells after ≥4 h of exposure. Inhibition was enhanced by increasing ouabain concentration and exposure time. Similar effects were produced by the reversible cardiac aglycone strophanthidin. Ouabain also inhibited swelling-activated ATP release from explant-derived native human TM cells. Ouabain (4 h, 30 nM and 100 nM) did not alter gene expression of the ATP-release pathways, and cell viability was unchanged by exposure to ouabain (30 nM–1 μM). Preincubation with 30 nM ouabain for 4 h did not detectably change Na+ level, the regulatory volume decrease (RVD) or the actin cytoskeleton of TM5 cells, but did inhibit hypotonicity-elicited ATP release. Moreover, even when N-methyl-d-glucosamine replaced Na+ in the extracellular fluid, ouabain still inhibited swelling-initiated ATP release at 100 nM. In the absence of ouabain, extracellular ATP stimulated MMP secretion, which was largely blocked by inhibiting conversion of ATP to adenosine, as expected. In contrast, ouabain reduced ATP release, but did not alter secretion of MMP-2 and MMP-9 from cells pretreated for ≤4 h. The results suggest that: (1) ouabain can trigger enhancement of outflow facility independent of its transport and actin-restructuring effects exerted at higher concentration and longer duration; (2) ouabain exerts parallel independent effects on ATP release and outflow facility; and (3) these effects likely reflect ouabain-induced changes in the scaffolding and/or signaling functions of Na+, K+-activated ATPase.

Effect of Genistein on the Survival and Production of Nitric Oxide in Trabecular Meshwork Cells

Purpose: To investigate the effect of hypoxia on the survival and nitric oxide (NO) production of cultured trabecular meshwork (TM) cells. Methods: After inducing chemical hypoxia with sodium cyanide, the survival and nitrite production of the primarily cultured porcine TM cells were assessed with MTT and Griess assays. The effect of NOS inhibitor, -Nitro-L-arginine methyl ester (L-NAME), was also assessed. Flow cytometry using annexin/PI was done to evaluate apoptosis. Results: Chemical hypoxia decreased TM cell survival significantly (p

Characterization of Soluble Guanylate Cyclase in NO-Induced Increases in Aqueous Humor Outflow Facility and in the Trabecular Meshwork

Nitric oxide (NO) increases the rate at which aqueous humor exits the eye; however, the involvement of soluble guanylate cyclase (sGC) is unknown. This study investigated the role of sGC in mediating the NO-induced increases in outflow facility. Outflow facility was measured in porcine eyes using the anterior segment organ culture perfusion system. sGC activity was assessed by cGMP production in low-passage porcine and human trabecular meshwork (TM) cells and transformed human TM cells, as measured by enzyme immunoassay. sGCalpha and sGCbeta isoform expression were determined using Western blot analysis. Activation of sGC is necessary for the NO-induced increases in outflow facility (0.3215 microL/min per mm Hg [baseline outflow facility]+/-0.0837 [SEM]). NO resulted in increased sGC activity that was abolished by 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-1 (ODQ). Western blot analysis of total protein demonstrated an equivalent ratio of sGCalpha and sGCbeta subunit expression. In transformed cell fractions, however, the level of cytoplasmic sGCalpha subunit expression was decreased compared with low-passage human TM cells. Activation of sGC is involved in the NO-induced increases in outflow facility. The expression of alpha and beta sGC subunits in an equivalent ratio would suggest a functional sGC heterodimer because DETA-NO increased cGMP levels in low-passage human and porcine TM cells. However, the inability of DETA-NO to cause increases in cGMP levels in transformed TM cells suggests that though the sGC heterodimer is necessary, it is not sufficient and may require other factors not present in transformed cells.

Ouabain-induced changes in aqueous humour outflow facility and trabecular meshwork cytoskeleton

Background:Inhibition of the Na,K-ATPase by ouabain results in decreased intraocular pressure (IOP), by mechanisms that are not completely understood. Cellular mechanisms that regulate aqueous humour outflow through the trabecular meshwork...

Cultured Porcine Trabecular Meshwork Cells Display Altered Lysosomal Function When Subjected to Chronic Oxidative Stress

To investigate the effects of chronic oxidative stress on lysosomal function in trabecular meshwork (TM) cells. Confluent cultures of porcine TM cells were grown for 2 weeks in physiological (5% O(2)) or hyperoxic conditions (40% O(2)) in the presence or absence of the protease inhibitor leupeptin (10 microM). The following parameters were quantified using the fluorogenic probes indicated within parentheses: autofluorescence, intracellular reactive oxygen species (ROS; H(2)DCFDA), mitochondrial membrane potential (JC-1), mitochondrial content (Mitotracker Red; Invitrogen-Molecular Probes, Eugene, OR), lysosomal content (acridine orange and Lysotracker Red [Invitrogen-Molecular Probes]), autophagic vacuole content (MDC), SA-beta-galactosidase (FDG), and cathepsin activities (z-FR-AMC). Cathepsin levels were quantified by qPCR and Western blot analysis. Ultrastructural analysis was performed by transmission electron microscopy. Prolonged exposure of porcine TM cells to a hyperoxic environment led to an increase in ROS production and oxidized material. Electron micrographs revealed the cytoplasmic accumulation of lipofuscin-loaded lysosomes. Augmented lysosomal and autophagic vacuole content was confirmed with specific fluorophores. The mRNA and protein levels of several cathepsins were upregulated with oxidative stress. This upregulated expression did not correlate with increased lysosomal activity. The 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. Since the lysosomal system is responsible for the continuous turnover of cellular organelles, impaired lysosomal activity may lead to progressive failure of cellular TM function with age.

P38 MAP Kinase Pathway and Stromelysin Regulation in Trabecular Meshwork Cells

Increased expression of stromelysin-1 (matrix metalloproteinase [MMP]-3) by the trabecular meshwork (TM) initiates extracellular matrix turnover and increases aqueous humor outflow facility. Tumor necrosis factor (TNF)alpha and interleukin (IL)-1alpha are efficacious inducers of MMP-3 in TM. To facilitate understanding of the regulation of MMP-3, the authors investigated the involvement of p38 MAP kinase pathway proteins in this process. Western immunoblots were used to determine the effects of these cytokines and p38 MAP kinase pathway inhibitors on MMP-3 protein levels, p38 MAP kinase isoforms, and phosphorylation levels in human and porcine TM cells. The effects of a dominant-negative p38 MAP kinase construct on MMP-3 expression were evaluated. Morphologic changes in the cells were also examined. Both cytokines increased MMP-3 levels. The p38 MAP kinase inhibitor SB202190 diminished MMP-3 induction by TNFalpha at all times and at 24 hours by IL-1alpha but potentiated the IL-1alpha-induced increase in MMP-3 at later times. MMP-3 induction by both cytokines was blocked by dominant-negative p38 MAP kinase constructs. Each cytokine increased phosphorylation of the p38 MAP kinase pathway components and altered TM cell morphology. The p38 inhibitor blocked only the morphologic changes produced by TNFalpha. Human and porcine TM cells expressed p38 alpha, beta, delta, and gamma isoforms, which migrate coincident with bands of specific phosphorylation. The effects of p38 inhibitors and the dominant-negative construct on TNFalpha and IL-1alpha induction of MMP-3 demonstrate an essential role for p38 in this signaling process. Differences between p38 inhibitor effects on TNFalpha and IL-1alpha induction of MMP-3 suggest divergent p38 isoform use, as do the morphologic responses. The anomalous p38 inhibitor effect on IL-1alpha induction of MMP-3 and phosphorylation of p38 delta/gamma suggests complex interactions between p38 MAP kinase isoforms and their differential uses by TNFalpha and IL-1alpha in TM.

Noladin Ether Acts on Trabecular Meshwork Cannabinoid (CB1) Receptors to Enhance Aqueous Humor Outflow Facility

To study the effects of 2-arachidonyl glyceryl ether (noladin ether), an endocannabinoid ligand selective for cannabinoid (CB)1 receptor, on aqueous humor outflow facility, to investigate the involvement of trabecular meshwork CB1 receptors and the p42/44 MAP kinase signaling pathway and to explore the cellular mechanisms of noladin ether-induced changes of outflow facility. The effects of noladin ether on aqueous humor outflow facility were measured in a porcine anterior-segment-perfused organ culture model. The expression of CB1 receptors on cultured porcine trabecular meshwork cells and the coupling of these receptors to p42/44 MAP kinase was determined by immunofluorescence microscopy and Western blot analysis. Both Western blot and zymography were used to monitor the effects of noladin ether on matrix metalloproteinase (MMP)-2. In morphologic studies, AlexaFluor 488-labeled phalloidin staining was used to examine actin filament, and immunohistochemistry with anti-paxillin antibodies was used to detect focal adhesions. Within 1 hour after adding 3, 30, or 300 nM of noladin ether, the aqueous humor outflow facility increased concentration dependently. The effect of 30 nM of noladin ether was completely blocked by SR141716A, a selective CB1 antagonist. Positive signals were detected on cultured porcine trabecular meshwork cells with an anti-CB1 antibody in immunofluorescence microscopy and Western blot studies. Treatment of trabecular meshwork cells with 30 nM of noladin ether activated p42/44 MAP kinase, whereas pretreatment with SR141716A blocked the p42/44 MAP kinase-activating effects of noladin ether. In addition, the enhancement of outflow facility induced by noladin ether was blocked by pretreatment of porcine anterior segments with PD98059, an inhibitor of p42/44 MAP kinase pathway. Furthermore, noladin ether treatment caused rounding of trabecular meshwork cells, and there was a decrease of actin stress fibers, as well as a decrease in focal adhesions. These noladin ether-induced morphologic changes were also blocked by SR141716A and PD98059. The results demonstrate for the first time that administration of noladin ether, an endocannabinoid agonist selective for the CB1 receptor, increases aqueous humor outflow facility. The data also show that noladin ether-induced enhancement of outflow facility is mediated through the trabecular meshwork CB1 receptor, with an involvement of p42/44 MAP kinase signaling pathway and changes in actin cytoskeletons.

Blebbistatin, a Novel Inhibitor of Myosin II ATPase Activity, Increases Aqueous Humor Outflow Facility in Perfused Enucleated Porcine Eyes

To investigate the specific role of myosin II, a critical biochemical determinant of cellular contraction, in modulation of aqueous humor outflow facility through the trabecular meshwork (TM) pathway. Expression of the nonmuscle myosin II heavy chains (IIA, IIB, and IIC) in human TM and ciliary body (CB) cells was determined by RT-PCR analyses. The effects of inhibition of myosin II on cell morphology, actomyosin organization, and cell adhesions were evaluated in porcine TM and CB cells treated with blebbistatin, a cell-permeable, specific inhibitor of myosin II adenosine triphosphatase (ATPase) activity. Changes in aqueous humor outflow facility were determined in enucleated porcine eyes by using a constant-pressure Grant perfusion model system. Ultrastructural integrity of the outflow pathway in drug-perfused eyes was analyzed by transmission electron microscopy. Expression of nonmuscle myosin IIA and IIB was confirmed in both human TM and CB cells. Confluent cultures of primary porcine TM and CB cells treated with blebbistatin in the presence of serum revealed dose (10-200 microM)-dependent changes in cell morphology, decreases in actin stress fiber content and in focal adhesions and adherens junctions. These changes were found to be reversible within 24 hours of drug withdrawal from the cell culture media. Blebbistatin did not affect the status of myosin light chain phosphorylation in TM cells. Perfusion of enucleated porcine eyes for 5 hours with 100 and 200 microM blebbistatin produced a significant increase (P < 0.01, n = 7) in aqueous outflow facility (53% and 64%, respectively) from the baseline facility, compared with a 21% facility increase in sham control specimens. The integrity of the inner wall of aqueous plexi in drug-perfused porcine eyes was found to be intact, and TM cell morphology appeared to be similar to that noted in sham-treated eyes. These data demonstrate that selective inhibition of myosin II in the aqueous humor outflow pathway leads to increased aqueous outflow facility, suggesting a critical role for myosin II in the regulation of aqueous humor outflow facility. This study also suggests myosin II as a potential therapeutic target for lowering intraocular pressure in patients with glaucoma.