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

Purpose: Re-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. Methods: The 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. Results: H2O2 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. Conclusions: Titrated 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.

Targeting the vascular-specific phosphatase PTPRB protects against retinal ganglion cell loss in a pre-clinical model of glaucoma.

Elevated intraocular pressure (IOP) due to insufficient aqueous humor outflow through the trabecular meshwork and Schlemm's canal (SC) is the most important risk factor for glaucoma, a leading cause of blindness worldwide. We previously reported loss of function mutations in the receptor tyrosine kinase TEK or its ligand ANGPT1 cause primary congenital glaucoma in humans and mice due to failure of SC development. Here, we describe a novel approach to enhance canal formation in these animals by deleting a single allele of the gene encoding the phosphatase PTPRB during development. Compared to Tek haploinsufficient mice, which exhibit elevated IOP and loss of retinal ganglion cells, Tek+/-;Ptprb+/- mice have elevated TEK phosphorylation, which allows normal SC development and prevents ocular hypertension and RGC loss. These studies provide evidence that PTPRB is an important regulator of TEK signaling in the aqueous humor outflow pathway and identify a new therapeutic target for treatment of glaucoma.

Src mediates TGF-ß-induced intraocular pressure elevation in glaucoma.

Glaucoma, a progressive and irreversible optic neuropathy, is one of the leading causes of vision impairment worldwide. Elevation of intraocular pressure (IOP) due to transforming growth factor-ß (TGF-ß)-induced dysfunction of the trabecular meshwork is a risk factor for glaucoma, but the underlying molecular mechanisms remain elusive. Here, we show that Src kinase is involved in TGF-ß-induced IOP elevation. We observed that dasatinib, a potent Src inhibitor, suppressed TGF-ß2-induced IOP in rat eyes. Mechanistic analyses in human trabecular meshwork cells showed that TGF-ß2 activated Src signaling and concomitantly increased cytoskeletal remodeling, cell adhesion, and extracellular matrix (ECM) accumulation. Src was activated via TGF-ß2-induced upregulation of the Src scaffolding protein CasL, which mediates the assembly of focal adhesions, cytoskeletal remodeling, and ECM deposition. Activation of Src suppressed the expression of tissue plasminogen activator, thereby attenuating ECM degradation. Furthermore, the Src inhibitor ameliorated TGF-ß2-induced changes in the contractile and adhesive characteristics of trabecular meshwork cells, and ECM deposition. These findings underscore the crucial role of Src activity in TGF-ß-induced IOP elevation and identify Src signaling as a potential therapeutic target in glaucoma.

Effects of Lentivirus-Mediated C3 Expression on Trabecular Meshwork Cells and Intraocular Pressure.

Purpose: We evaluated the effects of lentivirus-mediated exoenzyme C3 transferase (C3) expression on cultured primary human trabecular meshwork (HTM) cells in vitro, and on rat intraocular pressure (IOP). Methods: HTM cells were cultured and treated with lentivirus vectors expressing either green fluorescent protein (GFP) only (LV-GFP) or GFP and C3 together (LV-C3-GFP). Changes in cell morphology and actin stress fibers were assessed. The vectors were also injected into the anterior chamber of rats, and GFP expression was visualized by a Micron III Retinal Imaging Microscope in vivo and a fluorescence microscope ex vivo. Changes in rat IOP were monitored by using a rebound tonometer and the eyes were evaluated by slit lamp. Results: LV-mediated C3 expression induced morphologic changes in HTM cells. The cells became retracted and rounded. GFP expression in the anterior chamber angle of rats was observed in vivo from 8 days to 48 days after injection of LV-C3-GFP or LV-GFP. IOP was significantly decreased in the LV-C3-GFP group starting 3 days post injection, and lasting for at least 40 days, when compared to either the contralateral control eyes (the LV-GFP group) or the ipsilateral baseline before injection (P < 0.05). No obvious inflammatory signs were observed in either the LV-C3-GFP or LV-GFP groups. Conclusions: LV-mediated C3 expression induced changes in morphology of cultured HTM cells. Intracameral injection of LV-C3-GFP lowered rat IOP for at least 40 days. No significant inflammatory reactions were observed in either the LV-C3-GFP or LV-GFP groups. This study supports the possible use of C3 gene therapy for the treatment of glaucoma.

Knockout of tissue transglutaminase ameliorates TGFß2-induced ocular hypertension: A novel therapeutic target for glaucoma?

Glaucoma is a vision threatening optic neuropathy that affects millions of people worldwide. In primary open angle, increased intraocular pressure (IOP) is the main risk factor for the development of this disease. Studies investigating the causes and mechanisms of increased IOP show fibrotic changes in the trabecular meshwork (TM) that are different from those of age-matched controls. Tissue transglutaminase (TGM2), an extracellular matrix (ECM) crosslinking enzyme, covalently crosslinks ECM proteins and causes excessive ECM protein deposition in the TM that could cause increased IOP. Previous literature reports increased expression of TGM2 in glaucomatous eyes compared to controls. We recently have shown that overexpression of TGM2 causes increased ECM crosslinking in the TM, increases IOP, and decreases aqueous humor (AH) outflow facility in mouse eyes. Therefore, we wanted to study the effect of TGM2 knockout (KO) on IOP in TGM2 floxed mice. Ad5.Cre transduction caused partial KO of TGM2, which decreased TGM2 expression in the TM region of mouse eyes. TGM2 KO significantly decreased IOP by itself and also in TGFß2 induced ocular hypertensive mice. TGM2 KO also restores the outflow facility in TGFß2 transduced eyes. Overall, TGM2 KO rescued the TGFß2-induced ocular hypertensive phenotype. Thus, TGM2 may offer potential as a new therapeutic target for glaucoma.

Role of the Autotaxin-LPA Pathway in Dexamethasone-Induced Fibrotic Responses and Extracellular Matrix Production in Human Trabecular Meshwork Cells.

Purpose: Dexamethasone (Dex) regulates aqueous humor outflow by inducing reorganization of the cytoskeleton and extracellular matrix (ECM) production. Rho kinase (ROCK) has an important role in this process, but the upstream pathway leading to its activation remains elusive. The purpose of the study was to determine the role of autotaxin (ATX), an enzyme involved in the generation of lysophosphatidic acid (LPA), in the Dex-induced fibrotic response and ECM production in human trabecular meshwork (HTM) cells. Methods: The expression of ATX in specimens from glaucoma patients was investigated by immunohistochemistry. Regulation of ATX expression and the changes in actin cytoskeleton, ECM production, myosin light chain (MLC) and cofilin phosphorylation, ATX secretion, and lysophospholipase D (lysoPLD) activity induced by Dex treatment in HTM cells were determined by immunofluorescence, real-time quantitative PCR, immunoblot, and the two-site immunoenzymetric and lysoPLD assays. Results: Significant ATX expression was found in conventional outflow pathway specimens from glaucoma patients. Dex treatment induced increases in ATX mRNA levels, protein expression, and secretion in HTM cells in association with reorganization of cytoskeleton and ECM accumulation. Significant suppression of these aforementioned changes was observed after ATX/LPA-receptor/ROCK inhibition as well as suppression of fibrotic changes and MLC and cofilin phosphorylation in HTM cells. Conclusions: The results of this study, including the robust induction of ATX by Dex treatment, in association with fibrotic changes and ECM production in HTM cells, collectively suggest a potential role for ATX-LPA pathway in the regulation of aqueous humor outflow and IOP in glaucomatous eyes.

Tissue Transglutaminase Elevates Intraocular Pressure in Mice.

Purpose: Tissue transglutaminase (TGM2) is elevated in glaucomatous trabecular meshwork (TM) tissues. We investigated whether increased expression of TGM2 increases extracellular matrix crosslinking in the TM, thereby increasing aqueous humor outflow resistance and elevating intraocular pressure (IOP) in mouse eyes. Methods: GTM3, primary human GTM 125-05, and cultured mouse TM cells were transduced with adenovirus serotype 5 expressing human transglutaminase 2 (Ad5.TGM2; multiplicity of infection [MOI]-75) and fixed for immunocytochemistry. To test the effect on IOP in living eyes, Ad5.TGM2 was injected intravitreally into one eye of BALB/cJ (n = 18) or C57BL/6J mice (n = 9). The uninjected contralateral eye and Ad5.GFP served as controls. Daytime conscious IOPs were measured twice per week. Aqueous outflow facility (C) was measured by constant flow infusion on completion of IOP measurements. Immunohistochemistry was performed on BALB/cJ mouse eyes to study TGM2 expression and activity. Results: The treatment of cultured TM cells with Ad5.TGM2 increased immunostaining of N-ε(γ-glutamyl) lysine crosslinks. Ad5.TGM2 injection significantly increased IOP in BALB/cJ (15.86 mm Hg [injected] vs. 10.70 mm Hg [control]) and in C57BL/6J mice (17.09 mm Hg [injected] vs. 12.01 mm Hg [control]). Mean aqueous outflow facility in the injected eyes of BALB/cJ (0.013 µL/min/mm Hg) and C57BL/6J mice (0.012 µL/min/mm Hg) was significantly lower than in the uninjected control eyes (BALB/cJ, 0.021 µL/min/mm Hg; C57BL/6J, 0.019 µL/min/mm Hg). The Ad5.TGM2 transduction of mouse eyes increased TGM2 expression in the TM region and increased N-ε(γ-glutamyl) lysine crosslinks. Conclusions: The increased expression of TGM2 in the TM increases N-ε(γ-glutamyl) lysine crosslinking in the TM, increases aqueous outflow resistance, and elevates IOP in mice. TGM2 may be at least partially responsible for ocular hypertension in POAG.

Functional Activity of Matrix Metalloproteinases 2 and 9 in Tears of Patients With Glaucoma.

Purpose: To evaluate the differential expression of tear matrix metalloproteinases (MMP) 2 and 9 in of patients with various forms of glaucoma. Methods: Tear samples were collected with a Schirmer's strip from 148 eyes of 113 patients (medically naïve patients with primary open-angle [POAG] or angle closure glaucoma [PACG] and those with pseudoexfoliation syndrome [PXF] or glaucoma [PXG]). These were compared to patients undergoing cataract surgery (controls) for this cross-sectional study. Functional activities of tear MMP-9 and MMP-2 were analyzed by gelatin zymography. Tenon's capsules (n = 15) were harvested from the inferior quadrant in those undergoing cataract surgery and protein expression of MMP-9 was analyzed by immunohistochemistry (IHC). Hydrogen peroxide (H2O2) stress-induced effects on in vitro activities of MMP-9 in human trabecular meshwork (HTM) cells were analyzed. Results: The MMP-9 activity in tears was increased significantly in POAG, (n = 27), PACG (n = 24), and PXF (n = 40) eyes compared to controls (n = 35), and was increased significantly in eyes with glaucoma compared to moderate/severe glaucoma (P < 0.001). The MMP-9 expression was significantly lower in PXG (n = 22) eyes. Immunohistochemistry of Tenon's capsule revealed increased expression of MMP-9 in primary glaucoma eyes. Increased MMP-9 activity was seen in in vitro by gelatin zymography and was confirmed by Western and immunofluorescent assay on HTM upon 800 and 1000 µM H2O2-induced stress for 2 to 3 hours with approximately 80% cell death. Conclusions: Increased tear MMP-9 activity in early glaucoma and pseudoexfoliation syndrome suggesting activation of extracellular matrix (ECM) degradation can be used as a tear-based predictive biomarker. Decreased expression in advanced stages suggests exhaustion of the degradation response.

Vertebrate Lonesome Kinase Regulated Extracellular Matrix Protein Phosphorylation, Cell Shape, and Adhesion in Trabecular Meshwork Cells.

Glaucoma, a leading cause of irreversible blindness, is commonly associated with elevated intraocular pressure (IOP) due to impaired aqueous humor (AH) drainage through the trabecular meshwork (TM). Although dysregulated production and organization of extracellular matrix (ECM) is presumed to increase resistance to AH outflow and elevate IOP by altering TM cell contractile and adhesive properties, it is not known whether regulation of ECM protein phosphorylation via the secretory vertebrate lonesome kinase (VLK) influences TM cellular characteristics. Here, we tested this possibility. Experiments carried out in this study reveal that the 32 kDa protein is a prominent VLK isoform detectable in lysates and conditioned media (CM) of human TM cells. Increased levels of VLK were observed in CM of TM cells subjected to cyclic mechanical stretch, or treated with dexamethasone, TGF-ß2, and TM cells expressing constitutively active RhoA GTPase. Downregulation of VLK expression in TM cells using siRNA decreased tyrosine phosphorylation (TyrP) of ECM proteins and focal adhesions, and induced changes in cell shape in association with reduced levels of actin stress fibers and phospho-paxillin. VLK was also demonstrated to regulate TGF-ß2-induced TyrP of ECM proteins. Taken together, these results suggest that VLK secretion can be regulated by external cues, intracellular signal proteins, and mechanical stretch, and VLK can in turn regulate TyrP of ECM proteins secreted by TM cells and control cell shape, actin stress fibers, and focal adhesions. These observations indicate a potential role for VLK in homeostasis of AH outflow and IOP, and in the pathobiology of glaucoma. J. Cell. Physiol. 232: 2447-2460, 2017. © 2016 Wiley Periodicals, Inc.

Effect of Nitric Oxide on the Expression of Matrix Metalloproteinase and Its Association with Migration of Cultured Trabecular Meshwork Cells.

PURPOSE: To determine the effect of exogenous nitric oxide (NO) on the migration of trabecular meshwork (TM) cells and its association with expression of matrix metalloproteinases (MMPs). METHODS: Primary human TM cells treated with 1 or 10 µM S-nitroso-N-acetyl-penicillamine (SNAP) and examined for changes in adherence. TM cells were seeded onto transwell culture inserts, and changes in their migratory activity were quantified. Reverse transcription polymerase chain reaction was performed to determine the relative changes in mRNA expression of MMPs and tissue inhibitor of metalloproteinases (TIMPs). RESULTS: Treatment with SNAP did not significantly suppress TM cell adhesion or migration (p > 0.05). Treatment of TM cells with 10 µM SNAP decreased expression of MMP-2 and increased expression of membrane type MMP-1 and TIMP-2. Treatment with interleukin-1α triggered MMP-3 expression but did not exert significant effects on MMP-3 activation in response to SNAP. CONCLUSIONS: These data suggest that NO revealed no significant effect on the migration of TM cells because NO decreased MMP-2 and increased TIMP-2 expression. Although expression of certain MMPs and TIMPs change in response to NO donors, NO may modulate trabecular outflow by changing the cellular production of extracellular matrix without having a significant effect on the migration of TM cells.

Secretion of matrix metalloproteinase-3 by co-cultured pigmented and non-pigmented human trabecular meshwork cells following selective laser trabeculoplasty.

BACKGROUND: Matrix metalloproteinases (MMPs) are reported to contribute to the mechanism of argon laser trabeculoplasty, but it is unknown whether they are also secreted after selective laser trabeculoplasty (SLT). The aim of this study was to investigate whether human primary trabecular meshwork (HTM) cells secrete MMP-3 after stimulation by SLT. The relationship between SLT and the two principal forms of cell death, apoptosis and necrosis, was also examined. METHODS: Non-pigmented primary HTM cells were challenged with melanin granules to artificially introduce pigmentation. Isolated non-pigmented HTM cells and 1:1 co-cultures of pigmented and non-pigmented cells were treated with 0.5-1.5 mJ SLT. Cellular metabolic activity (CMA) was quantified using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Apoptosis and necrosis were measured using a cell death ELISA and a lactate dehydrogenase assay, respectively. MMP-3 secretion was measured by ELISA. RESULTS: Co-cultures exhibited a dose-dependent decline in CMA and a dose-dependent increase in necrosis 4 and 24 h after SLT. Non-pigmented cells did not undergo necrosis and displayed a trend towards increased CMA. Apoptosis was reduced in non-pigmented cells but elevated in co-cultures. Increased MMP-3 secretion was observed from co-cultures but not isolated non-pigmented cells. CONCLUSIONS: Pigmentation is necessary for both post-SLT cell death and MMP-3 secretion. SLT appears to have a hormetic effect on non-pigmented HTM cells.

Receptor Protein Tyrosine Phosphatase Sigma (RPTP-σ) Increases pro-MMP Activity in a Trabecular Meshwork Cell Line Following Oxidative Stress Conditions.

PURPOSE: To elucidate the role of phosphatases in the eye drainage system by overexpressing the receptor tyrosine phosphatase sigma (RPTP-σ) in a human normal trabecular meshwork (NTM) cell line. METHODS: The efficacy, expression, and location of RPTP-σ were evaluated following its transfection in NTM cells (NTM(T)) and in NTM control cells. The cells were also analyzed for viability, matrix metalloproteinase (MMP) activity, and phosphatase activity following oxidative stress conditions. Assays were conducted in the presence or absence of a specific RPTP-σ inhibitor. RESULTS: Transfection efficacy measurements revealed that RPTP-σ expression measured via GFP fluorescence was significantly higher (×3.8) in NTM(T) cells than in control cells. Western blot analyses showed that RPTP-σ expression was significantly higher (×2.25) in NTM(T) cells than in control cells. No significant differences were observed in cell viability between NTM(T) and control cells after oxidative stress. We found that pro-MMP-2 and pro-MMP-9 showed a significantly higher activity (×2.18 and ×1.9; respectively) in NTM(T) cells than in control cells. Serine/threonine phosphatase activity in NTM(T) cells was significantly increased following oxidative stress. The specific phosphatase inhibitor PTP-IV inhibited 15% of the RPTP-σ expression in NTM cells and 31% in NTMT cells. The activity of pro-MMP-9, pro-MMP-2, and MMP-9 was significantly inhibited (48%, 35%, and 78% respectively). CONCLUSIONS: The findings indicate that RPTP-σ is expressed constituently in NTM cells and that oxidative stress changes the general phosphatase balance in NTM cells. In addition, the results show that expression levels of RPTP-σ affect the activity of various forms of MMP.

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.

Temperature oscillations drive cycles in the activity of MMP-2,9 secreted by a human trabecular meshwork cell line.

PURPOSE: Aqueous humor inflow falls 50% during sleeping hours without proportional fall in IOP, partly reflecting reduced outflow facility. The mechanisms underlying outflow facility cycling are unknown. One outflow facility regulator is matrix metalloproteinase (MMP) release from trabecular meshwork (TM) cells. Because anterior segment temperature must oscillate due to core temperature cycling and eyelid closure during sleep, we tested whether physiologically relevant temperature oscillations drive cycles in the activity of secreted MMP. METHODS: Temperature of transformed normal human TM cells (hTM5 line) was fixed or alternated 12 hours/12 hours between 33°C and 37°C. Activity of secreted MMP-2 and MMP-9 was measured by zymography, and gene expression by RT-PCR and quantitative PCR. RESULTS: Raising temperature to 37°C increased, and lowering to 33°C reduced, activity of secreted MMP. Switching between 37°C and 33°C altered MMP-9 by 40% ± 3% and MMP-2 by 22% ± 2%. Peripheral circadian clocks did not mediate temperature-driven cycling of MMP secretion because MMP-release oscillations did not persist at constant temperature after 3 to 6 days of alternating temperatures, and temperature cycles did not entrain clock-gene expression in these cells. Furthermore, inhibiting heat shock transcription factor 1, which links temperature and peripheral clock-gene oscillations, inhibited MMP-9 but not MMP-2 temperature-driven MMP cycling. Inhibition of heat-sensitive TRPV1 channels altered total MMP secretion but not temperature-induced modulations. Inhibiting cold-sensitive TRPM-8 channels had no effect. CONCLUSIONS: Physiologically relevant temperature oscillations drive fluctuations of secreted MMP-2 and MMP-9 activity in hTM5 cells independent of peripheral clock genes and temperature-sensitive TRP channels.

RhoA GTPase-induced ocular hypertension in a rodent model is associated with increased fibrogenic activity in the trabecular meshwork.

Ocular hypertension arising from increased resistance to aqueous humor (AH) outflow through the trabecular meshwork is a primary risk factor for open-angle glaucoma, a leading cause of blindness. Ongoing efforts have found little about the molecular and cellular bases of increased resistance to AH outflow through the trabecular meshwork in ocular hypertension patients. To test the hypothesis that dysregulated Rho GTPase signaling and a resulting fibrotic activity within the trabecular meshwork may result in ocular hypertension, we investigated the effects of expressing a constitutively active RhoA GTPase (RhoAV14) in the AH outflow pathway in Sprague-Dawley rats by using lentiviral vector-based gene delivery. Rats expressing RhoAV14 in the iridocorneal angle exhibited a significantly elevated intraocular pressure. Elevated intraocular pressure in the RhoAV14-expressing rats was associated with fibrotic trabecular meshwork and increased levels of F-actin, phosphorylated myosin light chain, α-smooth muscle actin, collagen-1A, and total collagen in the trabecular AH outflow pathway. Most of these changes were ameliorated by topical application of Rho kinase inhibitor. Human autopsy eyes from patients with glaucoma exhibited significant increases in levels of collagen-1A and total collagen in the trabecular AH outflow pathway. Collectively, these observations indicate that increased fibrogenic activity because of dysregulated RhoA GTPase activity in the trabecular AH outflow pathway increases intraocular pressure in a Rho kinase-dependent manner.

Lysyl oxidases in the trabecular meshwork.

The mechanical properties of the extracellular matrix (ECM) play an important role in maintaining cellular function and overall tissue homeostasis. Emerging evidence suggests that biomechanical modifications of the ECM may be initiators and/or drivers of disease, exemplified by increased tissue stiffness. Specific ECM cross-linking enzymes (tissue transglutaminase, lysyl oxidase, and lysyl oxidase-like 1) are expressed in the trabecular meshwork and are regulated by transforming growth factor beta (TGF-ß) isoforms. As TGF-ß isoforms are elevated in the aqueous humor of glaucoma patients, trabecular meshwork stiffness mediated by ECM cross-linking may be responsible for increased aqueous humor outflow resistance and elevated intraocular pressure.

DNA methyl transferases are differentially expressed in the human anterior eye segment.

PURPOSE: DNA methylation is an epigenetic mark involved in the control of genes expression. Abnormal epigenetic events have been reported in human pathologies but weakly documented in eye diseases. The purpose of this study was to establish DNMT mRNA and protein expression levels in the anterior eye segment tissues and their related (primary or immortalized) cell cultures as a first step towards future in vivo and in vitro methylomic studies. METHODS: Total mRNA was extracted from human cornea, conjunctiva, anterior lens capsule, trabeculum and related cell cultures (cornea epithelial, trabecular meshwork, keratocytes for primary cells; and HCE, Chang, B-3 for immortalized cells). cDNA was quantified by real-time PCR using specific primers for DNMT1, 2, 3A, 3B and 3L. Immunolocalization assays were carried out on human cornea using specific primary antibodies for DNMT1, 2 and 3A, 3B and 3L. RESULTS: All DNMT transcripts were detected in human cornea, conjunctiva, anterior lens capsule, trabeculum and related cells but showed statistically different expression patterns between tissues and cells. DNMT2 protein presented a specific and singular expression pattern in corneal endothelium. CONCLUSIONS: This study produced the first inventory of the expression patterns of DNMTs in human adult anterior eye segment. Our research highlights that DNA methylation cannot be ruled out as a way to bring new insights into well-known ocular diseases. In addition, future DNA methylation studies using various cells as experimental models need to be conducted with attention to approach the results analysis from a global tissue perspective.

Aberrant glycosylation in the human trabecular meshwork.

PURPOSE: To determine the difference in protein glycosylation and glycosylation enzyme levels between glaucomatous and control trabecular meshwork (TM). EXPERIMENTAL DESIGN: Glaucomatous and normal donor (n = 12 each) TM tissues, lectin fluorescence, fluorophore-assisted carbohydrate analyses, and quantitative MS were used to determine the glycosylation levels. Primary TM cells and glycosylation inhibitors were used to determine their effect on cell shape and motility. RESULTS: In contrast to elevated levels of glycoproteins determined by lectin fluorescence, simultaneous hyper- and hypo-glycosylation in glaucomatous TM was revealed by fluorophore-assisted carbohydrate analyses. Analyses of enzymes showed elevation of beta-glycosidase 1 and decrease in galactosyltransferase family 6 domain containing protein 1 in the glaucomatous TM. Quantitative MS identified select protein level changes between glaucomatous and normal TM. Primary TM cells were treated with inhibitors to elicit hypo-glycosylation, which affected cell shape, motility, and fluorescent tracer transport across a layer of TM cells. CONCLUSIONS AND CLINICAL RELEVANCE: Global protein glycosylation is aberrant in glaucomatous TM compared to controls. The results presented here suggest that the alteration in global TM protein glycosylation encompassing cellular and extracellular matrix proteins contributes to glaucoma pathology likely mediated through changes in properties of TM cells.

Ocular hypotensive efficacy of Src-family tyrosine kinase inhibitors via different cellular actions from Rock inhibitors.

We investigated the effects of Src-family tyrosine kinase (SFK) inhibitors on intraocular pressure (IOP) and trabecular meshwork (TM) cells. The SFK inhibitors, PP2, PP1, and damnacanthal, significantly lowered IOP from baseline following intracameral injection in ocular normotensive rabbits, and PP2 decreased trans-epithelial electrical resistance (TEER) of TM cell layers in a dose-dependent manner ranging from 0.1 µM to 100 µM. The maximal efficacy of PP2 on TEER was a reduction to 71.7% relative to the vehicle-treated group at 100 µM. PP2 decreased the adhesion of TM cells to culture surfaces either uncoated with specific ECM proteins dose-dependently or coated with extracellular matrix proteins such as laminin I, fibronectin, collagen type I and basement membrane extraction. Tyrosine phosphorylation of focal adhesion kinase and p130(cas) was decreased by PP2. On the other hand, major changes in actin staining of TM cells were not able to be detected after PP2 treatment, although quantitative analysis showed that PP2 induced some morphological changes which were in the different direction to those caused by Y-27632, a Rock inhibitor. Y-27632 at 10 µM increased the permeability of TM cell layers, but did not induce changes in the adhesion of TM cells. These results suggest that SFK inhibitors lower IOP, at least partly, by acting on TM cells in a manner that is distinct from Rock inhibitors.

MMPs in the trabecular meshwork: promising targets for future glaucoma therapies?

Glaucoma is one of the world's most common blinding diseases, affecting more than 60 million people worldwide. Although the disease presents as a neurodegenerative disorder affecting retinal ganglion cell axons in the optic nerve and their somata in the retina, the elicitors of this optic neuropathy are often located outside the neuroretina. Disturbances in aqueous humor outflow, leading to ocular hypertension, are considered to be the major risk factor for the development of glaucoma. Although an amplitude of pharmacological and surgical measures is available to lower IOP in glaucoma patients, these are not always sufficient to halt the disease. Multiple surveys in glaucoma patients, as well as in vitro studies in anterior segment explant or cell cultures, reported changes in the expression and activity of several matrix metalloproteinases (MMPs) in the aqueous humor and trabecular meshwork, in response to elevated IOP. In this review, we describe MMPs as important modulators of aqueous humor outflow, functioning in a feedback mechanism that continuously remodels the trabecular meshwork extracellular matrix composition in order to maintain a stable outflow resistance and IOP. We review the evidence for the involvement of MMPs in glaucoma disease onset and investigate their potential as therapeutic targets for the development of future glaucoma therapies.