Human Schlemm's Canal Research Articles

Targeting YAP/TAZ mechanosignaling to ameliorate stiffness-induced Schlemm's canal cell pathobiology.

Pathological alterations in the biomechanical properties of the Schlemm's canal (SC) inner wall endothelium and its immediate vicinity are strongly associated with ocular hypertension in glaucoma due to decreased outflow facility. Specifically, the underlying trabecular meshwork is substantially stiffer in glaucomatous eyes compared with that from normal eyes. This raises the possibility of a critical involvement of mechanotransduction processes in driving SC cell dysfunction. Yes-associated protein (YAP) has emerged as a key contributor to glaucoma pathogenesis. However, the molecular underpinnings of SC cell mechanosignaling via YAP and transcriptional coactivator with PDZ-binding motif (TAZ) in response to glaucomatous extracellular matrix (ECM) stiffening are not well understood. Using a novel biopolymer hydrogel that facilitates dynamic and reversible stiffness tuning, we investigated how ECM stiffening modulates YAP/TAZ activity in primary human SC cells, and whether disruption of YAP/TAZ mechanosignaling attenuates SC cell pathobiology and increases ex vivo outflow facility. We demonstrated that ECM stiffening drives pathologic YAP/TAZ activation and cytoskeletal reorganization in SC cells, which was fully reversible by matrix softening in a distinct time-dependent manner. Furthermore, we showed that pharmacologic or genetic disruption of YAP/TAZ mechanosignaling abrogates stiffness-induced SC cell dysfunction involving altered cytoskeletal and ECM remodeling. Finally, we found that perfusion of the clinically used, small molecule YAP/TAZ inhibitor verteporfin (without light activation) increases ex vivo outflow facility in normal mouse eyes. Collectively, our data provide new evidence for a pathologic role of aberrant YAP/TAZ mechanosignaling in SC cell dysfunction and suggest that YAP/TAZ inhibition has therapeutic value for treating ocular hypertension in glaucoma.NEW & NOTEWORTHY Pathologically altered biomechanical properties of the Schlemm's canal (SC) inner wall microenvironment were recently validated as the cause for increased outflow resistance in ocular hypertensive glaucoma. However, the involvement of specific mechanotransduction pathways in these disease processes is largely unclear. Here, we demonstrate that Yes-associated protein (YAP)/transcriptional coactivator with PDZ-binding motif (TAZ) are central regulators of glaucoma-like SC cell dysfunction in response to extracellular matrix stiffening and that targeted disruption of YAP/TAZ mechanosignaling attenuates SC cell pathobiology and enhances outflow function.

Rare protective variants and glaucoma-relevant cell stressors modulate Angiopoietin-like 7 expression.

Rare missense and nonsense variants in the Angiopoietin-like 7 (ANGPTL7) gene confer protection from primary open-angle glaucoma (POAG), though the functional mechanism remains uncharacterized. Interestingly, a larger variant effect size strongly correlates with in silico predictions of increased protein instability (r = -0.98), suggesting that protective variants lower ANGPTL7 protein levels. Here, we show that missense and nonsense variants cause aggregation of mutant ANGPTL7 protein in the endoplasmic reticulum (ER) and decreased levels of secreted protein in human trabecular meshwork (TM) cells; a lower secreted:intracellular protein ratio strongly correlates with variant effects on intraocular pressure (r = 0.81). Importantly, accumulation of mutant protein in the ER does not increase expression of ER stress proteins in TM cells (P > 0.05 for all variants tested). Cyclic mechanical stress, a glaucoma-relevant physiologic stressor, also significantly lowers ANGPTL7 expression in primary cultures of human Schlemm's canal (SC) cells (-2.4-fold-change, P = 0.01). Collectively, these data suggest that the protective effects of ANGPTL7 variants in POAG stem from lower levels of secreted protein, which may modulate responses to physiologic and pathologic ocular cell stressors. Downregulation of ANGPTL7 expression may therefore serve as a viable preventative and therapeutic strategy for this common, blinding disease.

YAP/TAZ Mediate TGFβ2-Induced Schlemm's Canal Cell Dysfunction.

Elevated transforming growth factor beta2 (TGFβ2) levels in the aqueous humor have been linked to glaucomatous outflow tissue dysfunction. Potential mediators of dysfunction are the transcriptional coactivators, Yes-associated protein (YAP) and transcriptional coactivator with PDZ binding motif (TAZ). However, the molecular underpinnings of YAP/TAZ modulation in Schlemm's canal (SC) cells under glaucomatous conditions are not well understood. Here, we investigate how TGFβ2 regulates YAP/TAZ activity in human SC (HSC) cells using biomimetic extracellular matrix hydrogels, and examine whether pharmacological YAP/TAZ inhibition would attenuate TGFβ2-induced HSC cell dysfunction. Primary HSC cells were seeded atop photo-cross-linked extracellular matrix hydrogels, made of collagen type I, elastin-like polypeptide and hyaluronic acid, or encapsulated within the hydrogels. HSC cells were induced with TGFβ2 in the absence or presence of concurrent actin destabilization or pharmacological YAP/TAZ inhibition. Changes in actin cytoskeletal organization, YAP/TAZ activity, extracellular matrix production, phospho-myosin light chain levels, and hydrogel contraction were assessed. TGFβ2 significantly increased YAP/TAZ nuclear localization in HSC cells, which was prevented by either filamentous-actin relaxation or depolymerization. Pharmacological YAP/TAZ inhibition using verteporfin without light stimulation decreased fibronectin expression and actomyosin cytoskeletal rearrangement in HSC cells induced by TGFβ2. Similarly, verteporfin significantly attenuated TGFβ2-induced HSC cell-encapsulated hydrogel contraction. Our data provide evidence for a pathologic role of aberrant YAP/TAZ signaling in HSC cells under simulated glaucomatous conditions and suggest that pharmacological YAP/TAZ inhibition has promising potential to improve outflow tissue dysfunction.

Modeling the biomechanics of the conventional aqueous outflow pathway microstructure in the human eye

Background and objectiveIntraocular pressure (IOP) is determined by aqueous humor outflow resistance, which is a function of the combined resistance of Schlemm's canal (SC) endothelium and the trabecular meshwork (TM) and their interactions in the juxtacanalicular connective tissue (JCT) region. Aqueous outflow in the conventional outflow pathway results in pressure gradient across the TM, JCT, and SC inner wall, and induces mechanical stresses and strains that influence the geometry and homeostasis of the outflow system. The outflow resistance is affected by alteration in tissues’ geometry, so there is potential for active, two-way, fluid-structure interaction (FSI) coupling between the aqueous humor (fluid) and the TM, JCT, and SC inner wall (structure). However, our understanding of the biomechanical interactions of the aqueous humor with the outflow connective tissues and its contribution to the outflow resistance regulation is incomplete. MethodsIn this study, a microstructural finite element (FE) model of a human eye TM, JCT, and SC inner wall was constructed from a segmented, high-resolution histologic 3D reconstruction of the human outflow system. Three different elastic moduli (0.004, 0.128, and 51.5 MPa based on prior reports) were assigned to the TM/JCT complex while the elastic modulus of the SC inner wall was kept constant at 0.00748 MPa. The hydraulic conductivity was programmed separately for the TM, JCT, and SC inner wall using a custom subroutine. Cable elements were embedded into the TM and JCT extracellular matrix to represent the directional stiffness imparted by anisotropic collagen fibril orientation. The resultant stresses and strains in the outflow system were calculated using fluid-structure interaction method. ResultsThe higher TM/JCT stiffness resulted in larger stresses, but smaller strains in the outflow connective tissues, and resulted in a 4- and 5-fold larger pressure drop across the SC inner wall, respectively, compared to the most compliant model. Funneling through µm-sized SC endothelial pores was evident in the models at lower tissue stiffness, but aqueous flow was more turbulent in models with higher TM/JCT stiffness. ConclusionsThe mechanical properties of the outflow tissues play a crucial role in the hydrodynamics of the aqueous humor in the conventional outflow system.

Evaluation of different OCT systems in quantitative imaging of human Schlemm's canal

We examined the performance of human Schlemm’s canal (SC) imaging using different OCT devices: CIRRUS 5000 (840 nm, spectral-domain (SD)-OCT), PLEX Elite 9000 (1060 nm, swept-source (SS)-OCT) and CASIA SS-1000 (1310 nm, SS-OCT), and analyzed potential impact factors on visualization and the quantitative assessment of SC morphology in a pilot study. Ten healthy subjects were imaged using three OCT devices by a single experienced operator on the same day. Each eye underwent two cubic scans by each device, one on nasal and the other on temporal quadrant. The B-scan showing the largest SC was manually selected for processing. Four quantitative metrics, including one morphological metric as cross-sectional area (CSA), and three performance metrics as contrast, continuity, and coverage, were derived from the datasets. Repeated-measures ANOVA was used to investigate the difference between these parameters from the three devices (P < 0.05). We found the CSA measured from CIRRUS was significantly larger than PLEX, followed by CASIA. The contrast was highest in CIRRUS, followed by PLEX and CASIA. The coverage was also higher in CIRRUS as compared to PLEX and CASIA. No significant difference was seen in the continuity from the three devices. In summary, we showed the measurements from the three devices were not interchangeable.

An In Vitro Bovine Cellular Model for Human Schlemm's Canal Endothelial Cells and Their Response to TGFβ Treatment.

PurposeDue to the limited availability of primary human Schlemm's canal (SC) endothelial cells, we aimed to develop an in vitro cellular model using the angular aqueous plexus (AAP) cells from bovine eyes.MethodsWe harvested a mixture of cells from the trabecular meshwork region including AAP loops from multiple donors, followed by puromycin treatment and immunostaining of Von Willebrand factor and vascular endothelial (VE)-cadherin to confirm identity. Previously identified differentially expressed genes in glaucomatous SC cells were examined in non-glaucomatous SC cells (n = 3) under 0% or 15% equibiaxial strain for 24 hours using droplet digital polymerase chain reaction (ddPCR) and analyzed using the Ingenuity Pathway Analysis (IPA) software application to identify upstream regulators. To compare the cellular responses to candidate regulators of these mechanoresponsive genes, AAP and human SC cells (n = 3) were treated with 5 or 10 ng/mL transforming growth factor beta-2 (TGFβ2) for 24 or 48 hours, followed with expression profiling using real-time PCR or ddPCR.ResultsWe found that the isolated AAP cells displayed uniform cobblestone-like morphology and positive expression of two endothelial markers. In stretched SC cells, nine glaucoma-related genes were upregulated, and IPA implicated TGFβ as a potential upstream regulator. The effects of TGFβ2 treatment were similar for both AAP and SC cells in a dose- and time-dependent manner, activating TGFBR1 and SMAD2, inhibiting BMP4, and altering expression of three glaucoma-related genes (DCN, EZR, and CYP1B1).ConclusionsBovine AAP cells may serve as an alternative cellular model of human SC cells.Translational RelevanceThese AAP cells may be used to study the functional pathways related to the outflow facility.

ABCA1 Regulates IOP by Modulating Cav1/eNOS/NO Signaling Pathway

PurposeThis study aimed to investigate the role and pathophysiological mechanism of ATP binding cassette transporter A1 (ABCA1) in regulating the IOP and aqueous humor outflow.MethodsABCA1 expression was measured in trabecular meshwork samples obtained from patients with POAG and human donor eyes by Western blot. To further evaluate the functional significance of ABCA1, porcine angular aqueous plexus (AAP) cells, which are equivalent to human Schlemm's canal endothelial cells, were either treated with ABCA1 agonist GW3965 or transduced with lentivirus expressing ABCA1-shRNA. Transendothelial electrical resistance, protein expression, and nitric oxide (NO) concentration were measured. GW3965 was administered by intracameral injection. IOP and aqueous humor outflow facility were also measured.ResultsABCA1 expression was significantly higher in the trabecular meshwork tissue of patients with POAG compared with controls. ABCA1 upregulation in angular aqueous plexus cells decreased the transendothelial electrical resistance in the angular aqueous plexus monolayers accompanied by a 0.56-fold decrease in caveolin-1 expression and a 2.85-fold and 1.17-fold increase in endothelial NO synthase expression and NO concentration, respectively (n = 3, P < 0.05). Conversely, ABCA1 downregulation increased transendothelial electrical resistance and caveolin-1 expression and decreased endothelial NO synthase expression and NO production (n = 3, P < 0.05). GW3965 decreased IOP and significantly increased conventional outflow facility (P < 0.05).ConclusionsRegulation of aqueous humor outflow via the caveolin-1/endothelial NO synthase/NO pathway is a newly defined function of ABCA1 that is different from its traditional role in mediating cholesterol efflux. ABCA1 is a compelling, novel therapeutic candidate for the treatment of glaucoma and ocular hypertension.

The β4-Subunit of the Large-Conductance Potassium Ion Channel KCa1.1 Regulates Outflow Facility in Mice.

PurposeThe large-conductance calcium-activated potassium channel KCa1.1 (BKCa, maxi-K) influences aqueous humor outflow facility, but the contribution of auxiliary β-subunits to KCa1.1 activity in the outflow pathway is unknown.MethodsUsing quantitative polymerase chain reaction, we measured expression of β-subunit genes in anterior segments of C57BL/6J mice (Kcnmb1-4) and in cultured human trabecular meshwork (TM) and Schlemm's canal (SC) cells (KCNMB1-4). We also measured expression of Kcnma1/KCNMA1 that encodes the pore-forming α-subunit. Using confocal immunofluorescence, we visualized the distribution of β4 in the conventional outflow pathway of mice. Using iPerfusion, we measured outflow facility in enucleated mouse eyes in response to 100 or 500 nM iberiotoxin (IbTX; N = 9) or 100 nM martentoxin (MarTX; N = 12). MarTX selectively blocks β4-containing KCa1.1 channels, whereas IbTX blocks KCa1.1 channels that lack β4.Results Kcnmb4 was the most highly expressed β-subunit in mouse conventional outflow tissues, expressed at a level comparable to Kcnma1. β4 was present within the juxtacanalicular TM, appearing to label cellular processes connecting to SC cells. Accordingly, KCNMB4 was the most highly expressed β-subunit in human TM cells, and the sole β-subunit in human SC cells. To dissect functional contribution, MarTX decreased outflow facility by 35% (27%, 42%; mean, 95% confidence interval) relative to vehicle-treated contralateral eyes, whereas IbTX reduced outflow facility by 16% (6%, 25%).ConclusionsThe β4-subunit regulates KCa1.1 activity in the conventional outflow pathway, significantly influencing outflow function. Targeting β4-containing KCa1.1 channels may be a promising approach to lower intraocular pressure to treat glaucoma.

A bioengineering approach to Schlemm's canal-like stem cell differentiation for in vitro glaucoma drug screening

Human Schlemm's canal (HSC) cells are critical for understanding outflow physiology and glaucoma etiology. However, primary donor cells frequently used in research are difficult to isolate. HSC cells exhibit both vascular and lymphatic markers. Human adipose-derived stem cells (ADSCs) represent a potential source of HSC due to their capacity to differentiate into both vascular and lymphatic endothelial cells, via VEGF-A and VEGF-C. Shear stress plays a critical role in maintaining HSC integrity, function, and PROX1 expression. Additionally, the human trabecular meshwork (HTM) microenvironment could provide cues for HSC-like differentiation. We hypothesize that subjecting ADSCs to VEGF-A or VEGF-C, shear stress, and co-culture with HTM cells could provide biological, mechanical, and cellular cues necessary for HSC-like differentiation. To test this hypothesis, effects of VEGF-A, VEGF-C, and shear stress on ADSC differentiation were examined and compared to primary HSC cells in terms of cell morphology, and HSC marker expression using qPCR, immunoblotting, and immunocytochemistry analysis. Furthermore, the effect of co-culture with HTM cells on porous scaffolds on ADSC differentiation was studied. Treatment with VEGF-C under shear stress is effective in differentiating ADSCs into PROX1-expressing HSC-like cells. Co-culture with HTM cells on porous scaffolds leads to HTM/ADSC-derived HSC-like constructs that regulate through-flow and respond as expected to dexamethasone. Statement of significanceWe successfully generated human Schlemm's canal (HSC) like cells from adipocyte-derived stem cells induced by biochemical and biomechanical cues as well as bioengineered human trabecular meshwork (HTM) on micropatterned, porous SU8 scaffolds. These stem cell-derived HSC-like cells co-cultured with HTM cells on SU8 scaffolds can regulate through-flow, and in particular, are responsive to steroid treatment as expected. These findings show that ADSC-derived HSC-like cells have the potential to recreate the ocular outflow pathway for in vitro glaucoma drug screening.To the best of our knowledge, it is the very first time to demonstrate derivation of Schlemm's canal-like cells from stem cells. It provides an important alternative source to primary Schlemm's canal cells that are very difficult to be isolated and cultured from human donors.

Interaction Between Pilocarpine and Ripasudil on Intraocular Pressure, Pupil Diameter, and the Aqueous-Outflow Pathway.

To explore interactions between pilocarpine and the ROCK inhibitor, ripasudil, on IOP and pupil diameter in human eyes, and morphological and functional changes in outflow tissues in vitro. IOP and pupil diameter were measured after pilocarpine and/or ripasudil, which were topically applied in healthy subjects. Human trabecular meshwork (HTM) cells were used in a gel contraction assay, for the evaluation of phosphorylation of myosin light chain and cofilin, and immunostaining for cytoskeletal proteins. Porcine ciliary muscle (CM) was used in a CM contraction assay. The permeability of human Schlemm's canal endothelial (SCE) cells was evaluated by measuring transendothelial electrical resistance and fluorescein permeability. Both pilocarpine and ripasudil significantly reduced IOP in human eyes, but pilocarpine interfered with ripasudil-induced IOP reduction when concomitantly introduced. Ripasudil significantly inhibited gel contraction, TGFβ2-induced stress fiber formation, α-smooth muscle actin expression, and phosphorylation of both myosin light chain and cofilin in HTM cells. Pilocarpine reduced these effects, significantly inhibited the ripasudil-induced HTM cell responses to TGFβ2 stimulation, and increased the permeability in SCE cells. In CM, ripasudil inhibited pilocarpine-stimulated contraction, but ripasudil did not have significant effects on pilocarpine-induced miosis. Pilocarpine interfered with the direct effects of ROCK inhibitor on the conventional outflow pathway leading to IOP reduction and cytoskeletal changes in trabecular meshwork cells, but did not affect the relaxation effect of the ROCK inhibitor. It is therefore necessary to consider possible interference between these two drugs, which both affect the conventional outflow.

Co-expression of vasoactive intestinal peptide and protein gene product 9.5 surrounding the lumen of human Schlemm's canal

Previous studies of aqueous humor outflow have focused primarily on resistance at the trabecular meshwork (TM), and little is known about the function of Schlemm's canal (SC). Here, we aimed to investigate whether SC is innervated by the peripheral nervous system. Ten eye specimens from eight donors were processed for histological analysis. CD31 was used to identify SC, after which we used protein gene product (PGP) 9.5 as a marker to detect nerve fibers around SC. We then characterized the nerves by double staining for PGP9.5 and sympathetic nerve markers, such as tyrosine hydroxylase (TH) and dopamine beta-hydroxylase (DβH), or the parasympathetic marker vasoactive intestinal peptide (VIP), as well as sensory nerve marker calcitonin gene-related peptide (CGRP) and vesicular glutamate transporter 2 (VGLUT2). Immunohistochemistry and immunofluorescence were also performed to detect the expression of γ-epithelial Na+ channel (γ-ENaC) in SC. We found that different markers were expressed in the anterior chamber angle, with the luminal surface of SC were only positive stained for PGP9.5, VIP, and γ-ENaC. CGRP and VGLUT2 were expressed in TM and scleral spur (SS), whereas TH and DβH were absent in both TM and SC. Furthermore, PGP9.5 was co-expressed with VIP and γ-ENaC in the region surrounding the SC as well as in SS. Our findings indicate that the peripheral nerves anatomically spread in the tissues around the SC and the local nerve fibers may be parasympathetic or sensory rather than sympathetic.

Stanniocalcin-1 Is an Ocular Hypotensive Agent and a Downstream Effector Molecule That Is Necessary for the Intraocular Pressure-Lowering Effects of Latanoprost.

PurposeTo identify downstream signaling molecules through which intraocular pressure (IOP) is lowered following treatment with the prostaglandin analog latanoprost.MethodsTotal RNA and protein isolated from primary human Schlemm's canal cells (n = 3) treated with latanoprost (free acid; 100 nM) were processed for quantitative PCR and Western blot analysis. IOP was evaluated in stanniocalcin-1 (STC-1−/−) and wild-type mice following treatment with latanoprost or Rho kinase inhibitor Y27632. Human anterior segment pairs (n = 8) were treated with recombinant STC-1 (5, 50, or 500 ng/mL) and pressure was recorded using custom-designed software. The effect of recombinant STC-1 (0.5 mg/mL) on IOP was evaluated in wild-type mice. Tissue morphology was evaluated by light and transmission electron microscopy.ResultsIncreased STC-1 mRNA (4.0- to 25.2-fold) and protein expression (1.9- to 5.1-fold) was observed within 12 hours following latanoprost treatment. Latanoprost reduced IOP in wild-type mice (22.0% ± 1.9%), but had no effect on STC-1−/− mice (0.5% ± 0.7%). In contrast, Y27632 reduced IOP in both wild-type (12.5% ± 1.2%) and in STC-1−/− mice (13.1% ± 2.8%). Human anterior segments treated with STC-1 (500 ng/mL) showed an increase in outflow facility (0.15 ± 0.03 to 0.27 ± 0.09 μL/min/mm Hg) while no change was observed in paired vehicle-treated controls. Recombinant STC-1 reduced IOP in wild-type mice by 15.2% ± 3.0%. No observable morphologic changes were identified between treatment groups when evaluated by microscopy.ConclusionsLatanoprost-induced reduction of IOP is mediated through the downstream signaling molecule STC-1. When used by itself, STC-1 exhibits ocular hypotensive properties.

Expression Profiling of Human Schlemm's Canal Endothelial Cells From Eyes With and Without Glaucoma.

Ocular hypertension is a major risk factor for glaucoma and the inner wall of Schlemm's canal (SC) endothelia participates in the regulation of aqueous humor outflow resistance. This study aimed to identify differentially expressed genes in primary cultures of SC cells from glaucoma patients. This study examined SC samples from three glaucoma cases and four controls. Schlemm's canal cells were isolated from eight different postmortem human eyes. Total RNA was extracted, labeled, and hybridized to Illumina HumanWG-6 BeadChips containing probes for approximately 47,000 human transcripts. After extracting the data using Illumina GenomeStudio software, the data were normalized and analyzed using the R package limma in Bioconductor. Using Protein ANalysis THrough Evolutionary Relationships (PANTHER) software, gene ontology analysis of highly expressed genes was executed in controls and glaucoma groups separately. Pathway analysis was performed with differentially expressed genes using WebGestalt (WEB-based GEne SeT AnaLysis Toolkit). Selected genes were validated using droplet digital PCR (ddPCR). Gene ontology analysis indicated similar functional categories in cases and controls. Differential analysis identified a total of 113 genes with at least 2-fold expression changes in cases. Pathway analysis indicated significant enrichment of genes in cell adhesion, heparin binding, glycosaminoglycan binding, filopodium, and extracellular matrix remodeling. Eighteen selected genes with differential expression were successfully validated using ddPCR. This study represents the first genome-wide expression study of human primary SC cells from glaucoma patients and provides a potential list of targets regulating SC cell stiffness and pore formation, eventually the outflow resistance in glaucoma individuals.

Genetic variants and cellular stressors associated with exfoliation syndrome modulate promoter activity of a lncRNA within the LOXL1 locus.

Exfoliation syndrome (XFS) is a common, age-related, systemic fibrillinopathy. It greatly increases risk of exfoliation glaucoma (XFG), a major worldwide cause of irreversible blindness. Coding variants in the lysyl oxidase-like 1 (LOXL1) gene are strongly associated with XFS in all studied populations, but a functional role for these variants has not been established. To identify additional candidate functional variants, we sequenced the entire LOXL1 genomic locus (∼40 kb) in 50 indigenous, black South African XFS cases and 50 matched controls. The variants with the strongest evidence of association were located in a well-defined 7-kb region bounded by the 3'-end of exon 1 and the adjacent region of intron 1 of LOXL1. We replicated this finding in US Caucasian (91 cases/1031 controls), German (771 cases/1365 controls) and Japanese (1484 cases/1188 controls) populations. The region of peak association lies upstream of LOXL1-AS1, a long non-coding RNA (lncRNA) encoded on the opposite strand of LOXL1. We show that this region contains a promoter and, importantly, that the strongly associated XFS risk alleles in the South African population are functional variants that significantly modulate the activity of this promoter. LOXL1-AS1 expression is also significantly altered in response to oxidative stress in human lens epithelial cells and in response to cyclic mechanical stress in human Schlemm's canal endothelial cells. Taken together, these findings support a functional role for the LOXL1-AS1 lncRNA in cellular stress response and suggest that dysregulation of its expression by genetic risk variants plays a key role in XFS pathogenesis.

A biomimetic Schlemm's canal inner wall: A model to study outflow physiology, glaucoma pathology and high-throughput drug screening

Glaucoma is a disease that damages the optic nerve, frequently leading to blindness. Elevated intraocular pressure (IOP) is the only modifiable risk factor for glaucoma, which is expected to affect 80 million people by 2020, causing bilateral blindness in over 10 million individuals. Because pathological changes to Schlemm's canal (SC) may account for significant resistance to outflow, there is considerable interest in characterizing and evaluating the Schlemm's canal as a target for glaucoma therapeutics. In conventional, two-dimensional culture, human Schlemm's canal (HSC) cells lose spatial, mechanical and biochemical cues, resulting in altered gene expression and cell signaling than observed in vivo, compromising the clinical relevance of data obtained from such systems. Here, we report, for the first time, that 3D culture of HSC cells on microfabricated scaffolds with defined physical and biochemical cues, rescued expression of key HSC markers, VE-cadherin and PECAM1, and mediated pore formation, crucial for the Schlemm's canal regulation of IOP. We demonstrated that following treatment with the glaucopathogenic agent, TGF-β2, HSC cells undergo an endothelial–mesenchymal transition, which together with the increase in extracellular matrix (ECM) proteins might account for the decrease in outflow facility observed in patients with high TGF-β2 levels in their aqueous humor. We also demonstrated that unlike 2D cultures, 3D cultures of HSC cells are amenable to gene transfer. Thus, our data imply that 3D culture of HSC cells may be used as a platform to advance our understanding of HSC physiology and pathology and as a model for high-throughput drug and gene screening.

Shear stress-triggered nitric oxide release from Schlemm's canal cells.

Endothelial nitric oxide (NO) synthase is regulated by shear stress. At elevated intraocular pressures when the Schlemm's canal (SC) begins to collapse, shear stress is comparable with that in large arteries. We investigated the relationship between NO production and shear stress in cultured human SC cells. Schlemm's canal endothelial cells isolated from three normal and two glaucomatous human donors were seeded into Ibidi flow chambers at confluence, cultured for 7 days, and subjected to steady shear stress (0.1 or 10 dynes/cm(2)) for 6, 24, or 168 hours. Cell alignment with flow direction was monitored, and NO production was measured using 4-amino-5-methylamino-2',7'-difluorofluorescein (DAF-FM) and Griess reagents. Human trabecular meshwork (TM) and umbilical vein endothelial cells (HUVECs) were used as controls. Normal SC strains aligned with the direction of flow by 7 days. Comparing 0.1 vs. 10 dynes/cm(2), NO levels increased by 82% at 24 hours and 8-fold after 7 days by DAF-FM, and similar results were obtained with Griess reagent. Shear responses by SC cells at 24 hours were comparable with HUVECs, and greater than TM cells, which appeared shear-insensitive. Nitric oxide production by SC cells was detectable as early as 6 hours and was inhibited by 100 μM nitro-L-arginine methyl ester. Two glaucomatous SC cell strains were either unresponsive or lifted from the plate in the face of shear. Shear stress triggers NO production in human SC cells, similar to other vascular endothelia. Increased shear stress and NO production during SC collapse at elevated intraocular pressures may in part mediate IOP homeostasis.

Endothelial nitric oxide synthase-related mechanotransduction changes in aged porcine angular aqueous plexus cells.

To investigate effects of aging on endothelial nitric oxide synthase (eNOS) expression and signaling in angular aqueous plexus (AAP) (functional equivalent to human Schlemm's canal) cells subjected to shear stress. The AAP cells were isolated differentially from porcine outflow tissues using puromycin selection. Cell aging was induced by culturing cells in hyperoxia condition (40% oxygen and 5% carbon dioxide) for 14 days. The AAP cells grown in chamber slides were exposed to a shear stress of 8 dynes/cm(2) for 24 hours. Expression of eNOS, eNOS-phospho Thr495, eNOS-phospho Ser1177, and Akt-phospho was tested by Western blot analysis and immunofluorescence staining. Nitric oxide (NO) levels were measured using the Griess assay. Compared with control, eNOS levels in aged cells were significantly reduced by 60% (P < 0.05; n = 6). Phosphorylation of eNOS at Ser1177 and Akt at Ser473 was 63% and 80% lower in aged cells, respectively, whereas phosphorylation of the eNOS inhibition site (Thr495) increased by 6.1-fold (P < 0.05; n = 6). Shear stress (8 dynes/cm(2) for 24 hours) increased eNOS abundance (total protein and at cell borders) and phosphorylation at Ser1177 by 1.7-fold and 1.8-fold, respectively (P < 0.05; n = 6), whereas aged cells were unresponsive. In control cells exposed to shear stress, the NO concentration was 1.8-fold higher than in the static group (P < 0.05; n = 4); however, aged cells were unresponsive to shear stress (mean ± SD, 4.3 ± 1.3 vs. 4.1 ± 1.4 μM). Aged AAP cells appear compromised in their mechanotransduction machinery involving eNOS, the protein product of the gene, NOS3, polymorphisms of which impart a risk for the development of glaucoma.

Morphological changes in Schlemm's canal in treated and newly diagnosed untreated glaucomatous eyes.

A custom built 1310 nm center wavelength swept source optical coherence tomography instrument was used to measure morphological changes in treated and newly diagnosed untreated glaucomatous human Schlemm's canal (SC). Thirty-seven primary open-angle glaucoma patients were divided into two groups depending on the patients having been treated or not. The statistical results showed that there were significant differences between the treated and untreated groups' SC areas (treated, 7935.6875±680.003 μm(2); untreated, 3890.71875±871.49844 μm(2); P<0.001), the circumferences (treated, 580.37891±44.96529 μm; untreated, 381.9026±41.22123 μm; P<0.001), and the long diameters (treated, 272.87806±25.7254 μm; untreated, 185.24047±19.72786 μm; P<0.001). We hypothesize that, after drug treatment, the SC will expand and the morphometric values especially the areas will become larger, thus helping to reduce intraocular pressure.

Biomechanical strain as a trigger for pore formation in Schlemm's canal endothelial cells

The bulk of aqueous humor passing through the conventional outflow pathway must cross the inner wall endothelium of Schlemm's canal (SC), likely through micron-sized transendothelial pores. SC pore density is reduced in glaucoma, possibly contributing to obstructed aqueous humor outflow and elevated intraocular pressure (IOP). Little is known about the mechanisms of pore formation; however, pores are often observed near dome-like cellular outpouchings known as giant vacuoles (GVs) where significant biomechanical strain acts on SC cells. We hypothesize that biomechanical strain triggers pore formation in SC cells. To test this hypothesis, primary human SC cells were isolated from three non-glaucomatous donors (aged 34, 44 and 68), and seeded on collagen-coated elastic membranes held within a membrane stretching device. Membranes were then exposed to 0%, 10% or 20% equibiaxial strain, and the cells were aldehyde-fixed 5 min after the onset of strain. Each membrane contained 3-4 separate monolayers of SC cells as replicates (N = 34 total monolayers), and pores were assessed by scanning electron microscopy in 12 randomly selected regions (∼65,000 μm2 per monolayer). Pores were identified and counted by four independent masked observers. Pore density increased with strain in all three cell lines (p < 0.010), increasing from 87 ± 36 pores/mm2 at 0% strain to 342 ± 71 at 10% strain; two of the three cell lines showed no additional increase in pore density beyond 10% strain. Transcellular “I-pores” and paracellular “B-pores” both increased with strain (p < 0.038), however B-pores represented the majority (76%) of pores. Pore diameter, in contrast, appeared unaffected by strain (p = 0.25), having a mean diameter of 0.40 μm for I-pores (N = 79 pores) and 0.67 μm for B-pores (N = 350 pores). Pore formation appears to be a mechanosensitive process that is triggered by biomechanical strain, suggesting that SC cells have the ability to modulate local pore density and filtration characteristics of the inner wall endothelium based on local biomechanical cues. The molecular mechanisms of pore formation and how they become altered in glaucoma may be studied in vitro using stretched SC cells.

Cell Senescence Reduced the Mechanotransduction Sensitivity of Porcine Angular Aqueous Plexus Cells to Elevation of Pressure

To investigate the effect of pressure/flow on the barrier function and protein expression of normal and senescent porcine aqueous humor plexus (AAP) cells, which are the porcine equivalent of human Schlemm's canal endothelial cells. AAP cells were grown for 2 weeks in physiological (5% O2) or hyperoxic conditions (40% O2) to model cell senescence. Control and senescent AAP cells were subjected to control and elevated hydrostatic pressure gradient of 10 mm Hg for 72 hours. Hydraulic conductivity (HC) and transendothelial electric resistance (TEER) were measured. The expressions of senescence-associated β-galactosidase and DNA damage marker 8-hydroxy-2'-deoxyguanosine (8-OHdG) were monitored, and the protein expression profile was analyzed by Western blot. After 14 days of hyperoxia, AAP cells stained positive for 8-OHdG and β-galactosidase. Pressure elevation/flow resulted in significant increase of HC in control cells (from 1.37 ± 0.12 to 1.64 ± 0.18 μL/mm Hg/min/cm(2), P < 0.05), but not in senescent cells (1.15 ± 0.17 and 1.08 ± 0.10 μL μL/mm Hg/min/cm(2)). TEER changes were consistent with the HC results. Western blot analysis showed that the expression level of myosin light chain, claudin-5, and VE-cadherin significantly reduced under pressure elevation in control cells but not in senescent cells. AAP cells are mechano-sensitive; however, cell senescence rendered the cells less responsive to mechanical stimulus, which may have pathological consequences.