Trabecular Meshwork Cells Research Articles

3D printed gelatin methacryloyl hydrogels for perfusion culture of human trabecular meshwork cells and glaucoma studies.

Glaucoma, a progressive eye disease leading to irreversible blindness, currently affects over 70 million people globally. Elevated intraocular pressure (IOP) is implicated in its development. IOP is carefully regulated by the trabecular meshwork (TM). However, studying TM behavior has been limited to traditional tissue culture studies or costly ex vivo cultures of animal and donor eyes. Developing novel functional TM models could enhance cell/tissue behavior understanding and aid therapeutic development for glaucoma. In this study, we 3D printed a simplified and reproducible model of the human TM (hTM) and studied hTM cell behavior under static and dynamic cultures. Gelatin Methacryloyl bioinks proved suitable for printing with viable and proliferative hTM cells expressing crucial marker genes in response to glucocorticoid induction. This, to our knowledge, is the first functional 3D printed hTM model and aims to facilitate TM research. Moreover, this easily reproducible model could also be applicable in the study of numerous other cell types throughout the body.

In Vivo Models of Steroid-Induced Intraocular Hypertension

Corticosteroids are widely utilized for their anti-inflammatory and immunosuppressive properties but often lead to ocular complications, including ocular hypertension. If untreated, ocular hypertension can progress to optic nerve atrophy and eventually result in steroid-induced glaucoma, which poses a risk of irreversible visual damage. Approximately 40% of individuals experience increased intraocular pressure after steroid use, and around 6% develop glaucoma. Although steroid-induced glaucoma is usually temporary and reversible if the treatment duration is under a year, prolonged exposure can cause permanent vision impairment. The pathogenesis of steroid-induced glaucoma is suggested to arise from increased outflow resistance of aqueous humor, primarily due to decreased expression of matrix metalloproteinases. This deficiency promotes the deposition of extracellular matrix and the dysfunction of trabecular meshwork cells. Additionally, modifications in the actin cytoskeleton increase the stiffness and alter the morphology of trabecular meshwork, further impeding aqueous humor outflow. Molecular changes, such as elevated expression of the MYOC gene, have also been implicated in restricting aqueous outflow. Various animal models, including rats, mice, primates, rabbits, cattle, sheep, cats, and dogs, have been developed to study steroid-induced glaucoma. These models exhibit pathological, pathophysiological, and molecular similarities to human disease, making them valuable for research. This review aims to summarize common animal models of steroid-induced ocular hypertension, discussing their advantages and limitations. The goal is to help researchers select appropriate models for future studies, thereby advancing the understanding of disease mechanisms and developing preventive strategies.

The Delayed Turnover of Proteasome Processing of Myocilin upon Dexamethasone Stimulation Introduces the Profiling of Trabecular Meshwork Cells' Ubiquitylome.

Glaucoma is chronic optic neuropathy whose pathogenesis has been associated with the altered metabolism of Trabecular Meshwork Cells, which is a cell type involved in the synthesis and remodeling of the trabecular meshwork, the main drainage pathway of the aqueous humor. Starting from previous findings supporting altered ubiquitin signaling, in this study, we investigated the ubiquitin-mediated turnover of myocilin (MYOC/TIGR gene), which is a glycoprotein with a recognized role in glaucoma pathogenesis, in a human Trabecular Meshwork strain cultivated in vitro in the presence of dexamethasone. This is a validated experimental model of steroid-induced glaucoma, and myocilin upregulation by glucocorticoids is a phenotypic marker of Trabecular Meshwork strains. Western blotting and native-gel electrophoresis first uncovered that, in the presence of dexamethasone, myocilin turnover by proteasome particles was slower than in the absence of the drug. Thereafter, co-immunoprecipitation, RT-PCR and gene-silencing studies identified STUB1/CHIP as a candidate E3-ligase of myocilin. In this regard, dexamethasone treatment was found to downregulate STUB1/CHIP levels by likely promoting its proteasome-mediated turnover. Hence, to strengthen the working hypothesis about global alterations of ubiquitin-signaling, the first profiling of TMCs ubiquitylome, in the presence and absence of dexamethasone, was here undertaken by diGLY proteomics. Application of this workflow effectively highlighted a robust dysregulation of key pathways (e.g., phospholipid signaling, β-catenin, cell cycle regulation) in dexamethasone-treated Trabecular Meshwork Cells, providing an ubiquitin-centered perspective around the effect of glucocorticoids on metabolism and glaucoma pathogenesis.

Dexamethasone Impairs ATP Production and Mitochondrial Performance in Human Trabecular Meshwork Cells.

Mitochondrial damage occurs in human trabecular meshwork (HTM) cells as a result of normal aging and in open angle glaucoma. Using an HTM cell model, we quantified mitochondrial function and ATP generation rates after dexamethasone (Dex) and TGF-β2 treatments, frequently used as in vitro models of glaucoma. Primary HTM cells were assayed for metabolic function using a Seahorse XFp Analyzer. We additionally assessed the mitochondrial copy number and the expression of transcripts associated with mitochondrial biogenesis and oxidative stress regulation. Cells treated with Dex, but not TGF-β2, exhibited a significant decrease in total ATP production and ATP from oxidative phosphorylation relative to that of the control. Dex treatment also resulted in significant decreases in maximal respiration, ATP-linked O2 consumption, and non-mitochondrial O2 consumption. We did not observe significant changes in the level of mitochondrial genomes or mRNA transcripts of genes involved in mitochondrial biogenesis and oxidative stress regulation. Decreased mitochondrial performance and ATP production are consistent with the results of prior studies identifying the effects of Dex on multiple cell types, including HTM cells. Our results are also consistent with in vivo evidence of mitochondrial damage in open-angle glaucoma. Overall, these results demonstrate a decrease in mitochondrial performance in Dex-induced glaucomatous models in vitro, meriting further investigation.

The S341P mutant MYOC renders the trabecular meshwork sensitive to cyclic mechanical stretch

The trabecular meshwork (TM) plays an essential role in the circulation of aqueous humor by sensing mechanical stretch. The balance between the outflow and inflow of aqueous humor is critical in regulating intraocular pressure (IOP). A dysfunctional TM leads to resistance to the outflow of aqueous humor, resulting in an elevated IOP, a major risk factor for glaucoma. It is widely accepted that mutant myocilin (MYOC) can cause damage to the TM. However, few studies have investigated how TM cells carrying mutant MYOC respond to cyclic mechanical stretch (CMS) and whether these cells are more sensitive to CMS under this genetic background. In this study, we applied mechanical stretch to TM cells using the Flexcell system to mimic physiological stress. In addition, we performed genome-wide transcriptome analysis and oxidized lipidomics to systematically compare the gene expression and oxylipin profiles of non-stretched control human primary TM cells, human primary TM cells under CMS (TM-CMS), and human primary TM cells overexpressing MYOCS341P under CMS (S341P-CMS). We found that TM cells that overexpressed MYOCS341P were more sensitive to mechanical stress. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis revealed that downregulated genes were most enriched in oxidative phosphorylation, indicating mitochondria dysfunction and the likelihood of oxidative stress. Oxidized lipidomics analysis revealed significant changes in oxylipin profiles between the S341P-CMS and TM-CMS groups. Through further genome-wide transcriptomic analysis, we identified several genes that may be involved in the sensitivity of TM cells that overexpressed MYOCS341P to mechanical stress, including SARM1, AHNAK2, NT5C, and SOX8. The importance of these genes was validated by quantitative real-time PCR. Collectively, our findings indicate that mitochondrial dysfunction may contribute to the damage that occurs to TM cells with a MYOCS341P background under mechanical stretch.

Tert-butyl hydroperoxide induces trabecular meshwork cells injury through ferroptotic cell death.

Trabecular meshwork (TM) tissue has a crucial role in regulating aqueous humor circulation in the eye, thus maintaining normal intraocular pressure (IOP). TM dysfunction causes IOP elevation, which leads to glaucoma. To investigate biological changes in TM tissue in patients with glaucoma, we analyzed the mRNA expression microarray dataset, GSE27276. Gene ontology analysis indicated that redox microenvironment imbalance is among the main changes of TM tissue in patients with glaucoma. Subsequently, we induced oxidative stress in TM cells using the tert-butyl hydroperoxide (tBHP) treatment, to generate in vivo and in vitro models, and conducted mRNA sequencing to identify genes with critical roles in maintaining the redox microenvironment balance. We found that the tBHP caused TM dysfunction in vivo, characterized by aqueous humor circulation resistance, IOP elevation, and TM cell death. Further, Kyoto Encyclopedia of Genes and Genomes pathway analysis showed that ferroptosis signaling was enriched in tBHP-treated TMcells. Consistently, in vitro analyses showed that levels of reactive oxygen species, ferric ion, and malondialdehyde were increased after the tBHP treatment, indicating TM cell ferroptosis. Furthermore, inhibiting ferroptosis alleviated tBHP-induced TM cell injury. This study provides new insights suggesting that inhibition of ferroptosis has potential as a treatment for glaucoma.

Resting human trabecular meshwork cells experience tonic cation influx.

The trabecular meshwork (TM) regulates intraocular pressure (IOP) by converting biochemical and biomechanical stimuli into intracellular signals. Recent electrophysiological studies demonstrated that this process is mediated by pressure sensing ion channels in the TM plasma membrane while the molecular and functional properties of channels that underpin ionic homeostasis in resting cells remain largely unknown. Here, we demonstrate that the TM resting potential is subserved by a powerful cationic conductance that disappears following Na+ removal and substitution with choline or NMDG+. Its insensitivity to TTX, verapamil, phenamil methanesulfonate and amiloride indicates it does not involve voltage-operated Na+, Ca2+ and epithelial Na+ (ENaC) channels or Na+/H+ exchange while a modest hyperpolarization induced by SEA-0440 indicates residual contribution from reversed Na+/Ca2+ exchange. Tonic cationic influx was inhibited by Gd3+ and Ruthenium Red but not GsMTx4, indicating involvement of TRP-like but not Piezo channels. Transcriptional analysis detected expression of most TRP genes, with the canonical transcriptome pool dominated by TRPC1 followed by the expression ofTRPV1, TRPC3 and TRPC5. TRPC3 antagonist Pyr3 and TRPC1,4,5 antagonist Pico1,4,5 did not affect the standing current, whereas the TRPC blocker SKF96365 promoted rather than suppressed, Na+ influx. TM cells thus maintain the resting membrane potential, control Na+ homeostasis, and balance K+ efflux through a novel constitutive monovalent cation leak current with properties not unlike those of TRP channels. Yet to be identified at the molecular level, this novel channel sets the homeostatic steady-state and controls the magnitude of pressure-induced transmembrane signals.

Engineered sensor actuator modulator as aqueous humor outflow actuator for gene therapy of primary open-angle glaucoma

Glaucoma, a blinding eye disease with optic neuropathy, is usually associated with elevated intraocular pressure (IOP). The currently available pharmacological and surgical treatments for glaucoma have significant limitations and side effects, which include systemic reactions to medications, patient non-compliance, eye infections, surgical device failure, and damage to the eye. Here, we present Sensor-Actuator-Modulator (SAM), an engineered double mutant version of the bacterial stretch-activated mechanosensitive channel of large conductance (MscL) that directly senses tension in the membrane lipid bilayer of cells and in response, transiently opens its large nonspecific pore to release cytoplasmic fluid. The heterologously expressed mechanosensitive SAM channel acts as a tension-activated pressure release valve in trabeculocytes. In the trabecular meshwork (TM), SAM is activated by membrane stretch caused by elevated IOP. We have identified several SAM variants that are activated at physiologically relevant pressures. Using this barogenetic technology, we have demonstrated that SAM is functional in cultured TM cells, and successfully transduced in vivo in TM cells by use of AAV2/8. Further, it is effective in enhancing aqueous humor outflow facility leading to lowering the IOP in a mouse model of ocular hypertension.

Characterization, enrichment, and computational modeling of cross-linked actin networks in trabecular meshwork cells.

Cross-linked actin networks (CLANs) are prevalent in the glaucomatous trabecular meshwork (TM), yet their role in ocular hypertension remains unclear. We used a human TM cell line that spontaneously forms fluorescently-labeled CLANs (GTM3L) to explore the origin of CLANs, developed techniques to increase CLAN incidence in GMT3L cells, and computationally studied the biomechanical properties of CLAN-containing cells. GTM3L cells were fluorescently sorted for viral copy number analysis. CLAN incidence was increased by (i) differential sorting of cells by adhesion, (ii) cell deswelling, and (iii) cell selection based on cell stiffness. GTM3L cells were also cultured on glass or soft hydrogel to determine substrate stiffness effects on CLAN incidence. Computational models were constructed to mimic and study the biomechanical properties of CLANs. All GTM3L cells had an average of 1 viral copy per cell. LifeAct-GFP expression level did not affect CLAN incidence rate, but CLAN rate was increased from ∼0.28% to ∼50% by a combination of adhesion selection, cell deswelling, and cell stiffness-based sorting. Further, GTM3L cells formed more CLANs on a stiff vs. a soft substrate. Computational modeling predicted that CLANs contribute to higher cell stiffness, including increased resistance of the nucleus to tensile stress when CLANs are physically linked to the nucleus. It is possible to greatly enhance CLAN incidence in GTM3L cells. CLANs are mechanosensitive structures that affect cell biomechanical properties. Further research is needed to determine the effect of CLANs on TM biomechanics and mechanobiology as well as the etiology of CLAN formation in the TM.

Dexamethasone induces trabecular meshwork cell myofibroblast transdifferentiation through ARHGEF26.

Glucocorticoid use may cause elevated intraocular pressure, leading to the development of glucocorticoid-induced glaucoma (GIG). However, the mechanism of GIG development remains incompletely understood. In this study, we subjected primary human trabecular meshwork cells (TMCs) and mice to dexamethasone treatment to mimic glucocorticoid exposure. The myofibroblast transdifferentiation of TMCs was observed in cellular and mouse models, as well as in human trabecular mesh specimens. This was demonstrated by the cytoskeletal reorganization, alterations in cell morphology, heightened transdifferentiation markers, increased extracellular matrix deposition, and cellular dysfunction. Knockdown of Rho guanine nucleotide exchange factor 26 (ARHGEF26) expression ameliorated dexamethasone-induced changes in cell morphology and upregulation of myofibroblast markers, reversed dysfunction and extracellular matrix deposition in TMCs, and prevented the development of dexamethasone-induced intraocular hypertension. And, this process may be related to the TGF-β pathway. In conclusion, glucocorticoids induced the myofibroblast transdifferentiation in TMCs, which played a crucial role in the pathogenesis of GIG. Inhibition of ARHGEF26 expression protected TMCs by reversing myofibroblast transdifferentiation. This study demonstrated the potential of reversing the myofibroblast transdifferentiation of TMCs as a new target for treating GIG.

Transcriptomic profiling of Schlemm's canal cells reveals a lymphatic-biased identity and three major cell states.

Schlemm's canal (SC) is central in intraocular pressure regulation but requires much characterization. It has distinct inner and outer walls, each composed of Schlemm's canal endothelial cells (SECs) with different morphologies and functions. Recent transcriptomic studies of the anterior segment added important knowledge, but were limited in power by SEC numbers or did not focus on SC. To gain a more comprehensive understanding of SC biology, we performed bulk RNA sequencing on C57BL/6J SC, blood vessel, and lymphatic endothelial cells from limbal tissue (~4500 SECs). We also analyzed mouse limbal tissues by single-cell and single-nucleus RNA sequencing (C57BL/6J and 129/Sj strains), successfully sequencing 903 individual SECs. Together, these datasets confirm that SC has molecular characteristics of both blood and lymphatic endothelia with a lymphatic phenotype predominating. SECs are enriched in pathways that regulate cell-cell junction formation pointing to the importance of junctions in determining SC fluid permeability. Importantly, and for the first time, our analyses characterize 3 molecular classes of SECs, molecularly distinguishing inner wall from outer wall SECs and discovering two inner wall cell states that likely result from local environmental differences. Further, and based on ligand and receptor expression patterns, we document key interactions between SECs and cells of the adjacent trabecular meshwork (TM) drainage tissue. Also, we present cell type expression for a collection of human glaucoma genes. These data provide a new molecular foundation that will enable the functional dissection of key homeostatic processes mediated by SECs as well as the development of new glaucoma therapeutics.

Trabecular meshwork cell differentiation in response to collagen and TGFβ-2 spatial interactions

Primary open-angle glaucoma (POAG) is currently the most prevalent cause of irreversible blindness globally. To date, few in vitro models that can faithfully recapitulate the complex architecture of the trabecular meshwork (TM) and the specialised trabecular meshwork cell (TMC) characteristics that are local to the structurally opposing regions. This study aimed to investigate the parameters that govern TMC phenotype by adapting the extracellular matrix structure to mimic the juxtacanalicular tissue (JCT) region of the TM. Initially, TMC phenotypic characteristics were investigated within type I collagen matrices of controlled fiber density and anisotropy, generated through confined plastic compression (PC). Notably, PC-collagen presented biophysical cues that induced JCT cellular characteristics (elastin, α-β-Crystallin protein expression, cytoskeletal remodelling, increased mesenchymal markers and JCT-specific genetic markers). In parallel, a pathological mesenchymal phenotype associated with POAG was induced through localised transforming growth factor -beta 2 (TGFβ-2) exposure. This resulted in a profile of alternative mesenchymal states (fibroblast/smooth muscle or myofibroblast) displayed by the TMC in vitro. Overall, the study provides an advanced insight into the biophysical cues that modulate TMC fate, inducing a JCT-specific phenotype and transient mesenchymal characteristics that reflect healthy and pathological scenarios. Statement of significanceGlaucoma is a leading cause of blindness, with a lack of long-term efficacy within current drug candidates. Reliable trabecular meshwork (TM) in vitro models will be critical for enhancing the fields understanding of healthy and disease states for pre-clinical testing.Trabecular meshwork cells (TMCs) display heterogeneity throughout the hierarchical TM, however our understanding into recapitulating these phenotypes in vitro, remains elusive. This study hypothesizes the importance of specific matrix/growth factor spatial stimuli in governing TMCs phenotype. By emulating certain biophysical/biochemical in vivo parameters, we introduce an advanced profile of distinct TMC phenotypic states, reflecting healthy and disease scenarios. A notion that has not be stated prior and a fundamental consideration for future 3D TM in vitro modelling.

TRPV4 and chloride channels mediate volume sensing in trabecular meshwork cells.

Aqueous humor drainage from the anterior eye determines intraocular pressure (IOP) under homeostatic and pathological conditions. Swelling of the trabecular meshwork (TM) alters its flow resistance but the mechanisms that sense and transduce osmotic gradients remain poorly understood. We investigated TM osmotransduction and its role in calcium and chloride homeostasis using molecular analyses, optical imaging, and electrophysiology. Anisosmotic conditions elicited proportional changes in TM cell volume, with swelling, but not shrinking, evoking elevations in intracellular calcium concentration [Ca2+]TM. Hypotonicity-evoked calcium signals were sensitive to HC067047, a selective blocker of TRPV4 channels, whereas the agonist GSK1016790A promoted swelling under isotonic conditions. TRPV4 inhibition partially suppressed hypotonicity-induced volume increases and reduced the magnitude of the swelling-induced membrane current, with a substantial fraction of the swelling-evoked current abrogated by Cl- channel antagonists 4,4'-diisothiocyanato-2,2'-stilbenedisulfonic acid (DIDS) and niflumic acid. The transcriptome of volume-sensing chloride channel candidates in primary human was dominated by ANO6 transcripts, with moderate expression of ANO3, ANO7, and ANO10 transcripts and low expression of LTTRC genes that encode constituents of the volume-activated anion channel. Imposition of 190 mosM but not 285 mosM hypotonic gradients increased conventional outflow in mouse eyes. TRPV4-mediated cation influx thus works with Cl- efflux to sense and respond to osmotic stress, potentially contributing to pathological swelling, calcium overload, and intracellular signaling that could exacerbate functional disturbances in inflammatory disease and glaucoma.NEW & NOTEWORTHY Intraocular pressure is dynamically regulated by the flow of aqueous humor through paracellular passages within the trabecular meshwork (TM). This study shows hypotonic gradients that expand the TM cell volume and reduce the outflow facility in mouse eyes. The swelling-induced current consists of TRPV4 and chloride components, with TRPV4 as a driver of swelling-induced calcium signaling. TRPV4 inhibition reduced swelling, suggesting a novel treatment for trabeculitis and glaucoma.

GSK3β Inhibitors Inhibit TGFβ Signaling in the Human Trabecular Meshwork.

Primary open-angle glaucoma (POAG) is a leading cause of blindness, and its primary risk factor is elevated intraocular pressure (IOP) due to pathologic changes in the trabecular meshwork (TM). We previously showed that there is a cross-inhibition between TGFβ and Wnt signaling pathways in the TM. In this study, we determined if activation of the Wnt signaling pathway using small-molecule Wnt activators can inhibit TGFβ2-induced TM changes and ocular hypertension (OHT). Primary human TM (pHTM) cells and transduced SBE-GTM3 cells were treated with or without Wnt and/or TGFβ signaling activators and used for luciferase assays; for the extraction of whole-cell lysate, conditioned medium, cytosolic proteins, and nuclear proteins for Western immunoblotting (WB); or for immunofluorescent staining. Human donor eyes were perfusion cultured to study the effect of Wnt activators on IOP. We found that the small-molecule Wnt activators (GSK3β inhibitors) (BIO, SB216763, and CHIR99021) activated canonical Wnt signaling in pHTM cells without toxicity at tested concentrations. This activation inhibited TGFβ signaling as well as TGFβ2-induced extracellular matrix deposition and formation of cross-linked actin networks in pHTM cells or SBE-GTM3 cells. We also observed nuclear translocation of both Smad4 and β-catenin in pHTM cells, which suggested that the cross-inhibition between the TGFβ and Wnt signaling pathways may occur in the nucleus. Using our ex vivo model, we found that CHIR99021 inhibited TGFβ2-induced OHT in perfusion-cultured human eyes. Our results showed that small-molecule Wnt activators have the potential for treating TGFβ signaling-induced OHT in patients with POAG.

Artificial Trabecular Meshwork Structure Combining Melt Electrowriting and Solution Electrospinning.

The human trabecular meshwork (HTM) is responsible for regulating intraocular pressure (IOP) by means of gradient porosity. Changes in its physical properties, like increases in stiffness or alterations in the extracellular matrix (ECM), are associated with increases in the IOP, which is the primary cause of glaucoma. The complexity of its structure limits the engineered models to one-layered and simple approaches, which do not accurately replicate the biological and physiological cues related to glaucoma. Here, a combination of melt electrowriting (MEW) and solution electrospinning (SE) is explored as a biofabrication technique used to produce a gradient porous scaffold that mimics the multi-layered structure of the native HTM. Polycaprolactone (PCL) constructs with a height of 20-710 µm and fiber diameters of 0.7-37.5 µm were fabricated. After mechanical characterization, primary human trabecular meshwork cells (HTMCs) were seeded over the scaffolds within the subsequent 14-21 days. In order to validate the system's responsiveness, cells were treated with dexamethasone (Dex) and the rho inhibitor Netarsudil (Net). Scanning electron microscopy and immunochemistry staining were performed to evaluate the expected morphological changes caused by the drugs. Cells in the engineered membranes exhibited an HTMC-like morphology and a correct drug response. Although this work demonstrates the utility of combining MEW and SE in reconstructing complex morphological features like the HTM, new geometries and dimensions should be tested, and future works need to be directed towards perfusion studies.

Role of TGF-β1/SMADs signalling pathway in resveratrol-induced reduction of extracellular matrix deposition by dexamethasone-treated human trabecular meshwork cells.

Deposition of extracellular matrix (ECM) in the trabecular meshwork (TM) increases aqueous humour outflow resistance leading to elevation of intraocular pressure (IOP) in primary open-angle glaucoma, which remains the only modifiable risk factor. Resveratrol has been shown to counteract the steroid-induced increase in IOP and increase the TM expression of ECM proteolytic enzymes; however, its effects on the deposition of ECM components by TM and its associated pathways, such as TGF-β-SMAD signalling remain uncertain. This study, therefore, explored the effects of trans-resveratrol on the expression of ECM components, SMAD signalling molecules, plasminogen activator inhibitor-1 and tissue plasminogen activator in dexamethasone-treated human TM cells (HTMCs). We also studied the nature of molecular interaction of trans -resveratrol with SMAD4 domains using ensemble docking. Treatment of HTMCs with 12.5 µM trans-resveratrol downregulated the dexamethasone-induced increase in collagen, fibronectin and α-smooth muscle actin at gene and protein levels through downregulation of TGF-β1, SMAD4, and upregulation of SMAD7. Downregulation of TGF-β1 signalling by trans-resveratrol could be attributed to its effect on the transcriptional activity due to high affinity for the MH2 domain of SMAD4. These effects may contribute to resveratrol's IOP-lowering properties by reducing ECM deposition and enhancing aqueous humour outflow in the TM.

Protective Effect of Nicotinamide Riboside on Glucocorticoid-Induced Glaucoma: Mitigating Mitochondrial Damage and Extracellular Matrix Deposition.

Glucocorticoid-induced glaucoma (GIG) is a prevalent complication associated with glucocorticoids (GCs), resulting in irreversible blindness. GIG is characterized by the abnormal deposition of extracellular matrix (ECM) in the trabecular meshwork (TM), elevation of intraocular pressure (IOP), and loss of retinal ganglion cells (RGCs). The objective of this study is to investigate the effects of nicotinamide riboside (NR) on TM in GIG. Primary human TM cells (pHTMs) and C57BL/6J mice responsive to GCs were utilized to establish in vitro and in vivo GIG models, respectively. The study assessed the expression of ECM-related proteins in TM and the functions of pHTMs to reflect the effects of NR. Mitochondrial morphology and function were also examined in the GIG cell model. GIG progression was monitored through IOP, RGCs, and mitochondrial morphology. Intracellular nicotinamide adenine dinucleotide (NAD+) levels of pHTMs were enzymatically assayed. NR significantly prevented the expression of ECM-related proteins and alleviated dysfunction in pHTMs after dexamethasone treatment. Importantly, NR protected damaged ATP synthesis, preventing overexpression of mitochondrial reactive oxygen species (ROS), and also protect against decreased mitochondrial membrane potential induced by GCs in vitro. In the GIG mouse model, NR partially prevented the elevation of IOP and the loss of RGCs. Furthermore, NR effectively suppressed the excessive expression of ECM-associated proteins and mitigated mitochondrial damage in vivo. Based on the results, NR effectively enhances intracellular levels of NAD+, thereby mitigating abnormal ECM deposition and TM dysfunction in GIG by attenuating mitochondrial damage induced by GCs. Thus, NR has promising potential as a therapeutic candidate for GIG treatment.

The TGFβ Induced MicroRNAome of the Trabecular Meshwork.

Primary open-angle glaucoma (POAG) is a progressive optic neuropathy with a complex, multifactorial aetiology. Raised intraocular pressure (IOP) is the most important clinically modifiable risk factor for POAG. All current pharmacological agents target aqueous humour dynamics to lower IOP. Newer therapeutic agents are required as some patients with POAG show a limited therapeutic response or develop ocular and systemic side effects to topical medication. Elevated IOP in POAG results from cellular and molecular changes in the trabecular meshwork driven by increased levels of transforming growth factor β (TGFβ) in the anterior segment of the eye. Understanding how TGFβ affects both the structural and functional changes in the outflow pathway and IOP is required to develop new glaucoma therapies that target the molecular pathology in the trabecular meshwork. In this study, we evaluated the effects of TGF-β1 and -β2 treatment on miRNA expression in cultured human primary trabecular meshwork cells. Our findings are presented in terms of specific miRNAs (miRNA-centric), but given miRNAs work in networks to control cellular pathways and processes, a pathway-centric view of miRNA action is also reported. Evaluating TGFβ-responsive miRNA expression in trabecular meshwork cells will further our understanding of the important pathways and changes involved in the pathogenesis of glaucoma and could lead to the development of miRNAs as new therapeutic modalities in glaucoma.

The Factors Affecting the Stability of IOP Homeostasis.

Shear-induced nitric oxide (NO) production by Schlemm's canal (SC) endothelial cells provides a fast, IOP-sensitive feedback signal that normally contributes to IOP homeostasis. Our goal was to analyze the response of this homeostatic system under constant flow perfusion (as occurs in vivo) vs. constant pressure perfusion (as typical for laboratory perfusions). A mathematical model of aqueous humor dynamics, including shear-mediated NO signaling, was formulated and analyzed for stability. The model includes Goldmann's equation, accounting for proximal and distal outflow resistance, and describes how elevated IOP causes narrowing of SC lumen that increases the shear stress on SC cells. Elevated shear stress stimulates NO production, which acts to reduce outflow resistance and relax trabecular meshwork cells to decrease trabecular meshwork stiffness, affecting the SC luminal caliber. During constant flow perfusion, the outflow system is typically stable, returning to baseline IOP after a perturbation. In contrast, during constant pressure perfusion, the outflow system can become unstable and exhibit a time-dependent change in outflow resistance that diverges from baseline. The stability of shear mediated IOP homeostasis is predicted to differ critically between constant flow vs. constant pressure perfusion. Because outflow facility is typically measured at a constant pressure in the laboratory, this instability may contribute to the characteristic time-dependent increase in outflow facility, known as washout, observed in many nonhuman species. Studies of IOP homeostasis should consider how the outflow system may respond differently under constant pressure vs. constant flow perfusion.

Paeoniflorin alleviates TGF-β2-mediated extracellular matrix remodeling and oxidative stress in human trabecular meshwork cells.

The multifunctional profibrotic cytokine transforming growth factor-beta2 (TGF-β2) is implicated in the pathophysiology of primary open angle glaucoma. Paeoniflorin (PAE) is a monoterpene glycoside with multiple pharmacological efficacies, such as antioxidant, anti-fibrotic, and anti-inflammatory properties. Studies have demonstrated that paeoniflorin protects human corneal epithelial cells, retinal pigment epithelial cells, and retinal microglia from damage. Here, the biological role of PAE in TGF-β2-dependent remodeling of the extracellular matrix (ECM) within the trabecular meshwork (TM) microenvironment. Primary or transformed (GTM3) human TM (HTM) cells conditioned in serum-free media were incubated with TGF-β2 (5ng/mL). PAE (300μM) was added to serum-starved confluent cultures of HTM cells for 2h, followed by incubation with TGF-β2 for 22h. SB-431542, a TGF-β receptor inhibitor (10μM), was used as a positive control. The levels of intracellular ROS were evaluated by CellROX green dye. Western blotting was used to measure the levels of TGF-β2/Smad2/3 signaling-related molecules. Collagen 1α1, collagen 4α1, and connective tissue growth factor (CTGF) expression was evaluated by RT-qPCR. Immunofluorescence assay was conducted to measure collagen I/IV expression in HTM cells. Phalloidin staining assay was conducted for evaluating F-actin stress fiber formation in the cells. PAE attenuated TGF-β2-induced oxidative stress and suppressed TGF-β2-induced Smad2/3 signaling in primary or transformed HTM cells. Additionally, PAE repressed TGF-β2-induced upregulation of collagen 1α1, collagen 4α1, and CTGF expression and reduced TGF-β2-mediated collagen I/IV expression and of F-actin stress fiber formation in primary or transformed HTM cells. PAE alleviates TGF-β2-induced ECM deposition and oxidative stress in HTM cells through inactivation of Smad2/3 signaling.