Human Trabecular Meshwork Cells Research Articles

Dynamic traction force in trabecular meshwork cells: A 2D culture model for normal and glaucomatous states

Glaucoma, which is associated with intraocular pressure (IOP) elevation, results in trabecular meshwork (TM) cellular dysfunction, leading to increased rigidity of the extracellular matrix (ECM), larger adhesion forces between the TM cells and ECM, and higher resistance to aqueous humor drainage. TM cells sense the mechanical forces due to IOP dynamic and apply multidimensional forces on the ECM. Recognizing the importance of cellular forces in modulating various cellular activities and development, this study is aimed to develop a 2D in vitro cell culture model to calculate the 3D, depth-dependent, dynamic traction forces, tensile/compressive/shear strain of the normal and glaucomatous human TM cells within a deformable polyacrylamide (PAM) gel substrate. Normal and glaucomatous human TM cells were isolated, cultured, and seeded on top of the PAM gel substrate with embedded FluoSpheres, spanning elastic moduli of 1.5 to 80 kPa. Sixteen-hour post-seeding live confocal microscopy in an incubator was conducted to Z-stack image the 3D displacement map of the FluoSpheres within the PAM gels. Combined with the known PAM gel stiffness, we ascertained the 3D traction forces in the gel. Our results revealed meaningfully larger traction forces in the glaucomatous TM cells compared to the normal TM cells, reaching depths greater than 10-µm in the PAM gel substrate. Stress fibers in TM cells increased with gel rigidity, but diminished when stiffness rose from 20 to 80 kPa. The developed 2D cell culture model aids in understanding how altered mechanical properties in glaucoma impact TM cell behavior and aqueous humor outflow resistance. Statement of significanceGlaucoma, a leading cause of irreversible blindness, is intricately linked to elevated intraocular pressures and their subsequent cellular effects. The trabecular meshwork plays a pivotal role in this mechanism, particularly its interaction with the extracellular matrix. This research unveils an advanced 2D in vitro cell culture model that intricately maps the complex 3D forces exerted by trabecular meshwork cells on the extracellular matrix, offering unparalleled insights into the cellular biomechanics at play in both healthy and glaucomatous eyes. By discerning the changes in these forces across varying substrate stiffness levels, we bridge the gap in understanding between cellular mechanobiology and the onset of glaucoma. The findings stand as a beacon for potential therapeutic avenues, emphasizing the gravity of cellular/extracellular matrix interactions in glaucoma's pathogenesis and setting the stage for targeted interventions in its early stages.

Knockdown of lncRNA PVT1 protects human trabecular meshwork cells against H2O2-induced injury via the regulation of the miR-29a-3p/VEGF/MMP-2 axis

PurposeHuman trabecular meshwork cell (HTMC) dysfunction results in imbalanced aqueous humor inflow and outflow, leading to an increase in intraocular pressure (IOP). Uncontrolled high IOP can promote the occurrence of glaucoma, an irreversible optic neuropathy. Here, we explored whether the long non-coding RNA plasmacytoma variant translocation 1 (lncRNA PVT1)/microRNA-29a-3p (miR-29a-3p) axis could ameliorate HTMC dysfunction under oxidative stress by modulating the expression of the proangiogenic factor vascular endothelial growth factor (VEGFA) and the profibrotic factor metalloproteinase-2 (MMP-2). MethodsHTMCs were cultured under H2O2-induced oxidative stress for 48 h. The expression of lncRNA PVT1, miR-29a-3p, VEGFA, MMP-2, intracellular adhesion molecule-1 (ICAM-1), and alpha-smooth muscle actin (α-SMA) was detected by reverse transcription quantitative real-time polymerase chain reaction, western blotting, and immunofluorescence. Interference experiments were conducted via the transfection of HTMCs with small interfering RNA (siRNA) targeting lncRNA PVT1 or miR-29a-3p mimics. A luciferase reporter assay was undertaken to identify the presence of a miR-29a-3p binding site in lncRNA PVT1. Flow cytometry and Transwell and Cell Counting Kit-8 assays were employed to evaluate HTMC functions under oxidative stress with different treatments. ResultsIn HTMCs, the expression of lncRNA PVT1 was induced by H2O2 treatment, whereas that of miR-29a-3p was inhibited. The levels of angiogenic factors (VEGFA, ICAM-1) and fibrosis-associated mediators (MMP-2, α-SMA) were upregulated in HTMCs under oxidative stress. The siRNA-mediated suppression of lncRNA PVT1 or the upregulation of miR-29a-3p significantly suppressed the expression of VEGFA, MMP-2, ICAM-1, and α-SMA. A luciferase reporter assay confirmed that lncRNA PVT1 directly targeted miR-29a-3p and acted as a miR-29a-3p sponge. The knockdown of lncRNA PVT1 restored the level of miR-29a-3p in H2O2-treated HTMCs, thereby inhibiting VEGFA and MMP-2, its target mRNAs. HTMC dysfunction, including increased apoptosis and decreased cell mobility and viability, could be effectively ameliorated by lncRNA PVT1 downregulation or miR-29a-3p overexpression under oxidative stress. ConclusionLncRNA PVT1 has potential as a therapeutic target for inhibiting VEGFA and MMP-2, thus protecting HTMCs, suppressing the progression of fibrosis, and, consequently, improving the outcome of glaucoma filtration surgery.

Effects of hyperglycemia on the TGF-β pathway in trabecular meshwork cells

BackgroundHyperglycemia, which can lead to apoptosis, hypertrophy, fibrosis, and induces hyperinflammation in diabetic vascular complications due to oxidative stress. In order to elucidate the potential dual roles and regulatory signal transduction of TGF-β1 and TGF-β2 in human trabecular meshwork cells (HTMCs), we established an oxidative cell model in HTMCs using 5.5, 25, 50, and 100 mM d-glucose-supplemented media and characterized the TGF-β-related oxidative stress pathway. MethodsFurther analysis was conducted to investigate oxidative damage and protein alterations in the HTMC caused by the signal transduction. This was done through a series of qualitative cell function studies, such as cell viability/apoptosis analysis, intracellular reactive oxygen species (ROS) detection, analysis of calcium release concentration, immunoblot analysis to detect the related protein expression alteration, and analysis of cell fibrosis to study the effect of different severities of hyperglycemia. Also, we illustrated the role of TGF-β1/2 in oxidative stress-induced injury by shRNA-mediated knockdown or stimulation with recombinant human TGF-β1 protein (rhTGF-β1). ResultsResults from the protein expression analysis showed that p-JNK, p-p38, p-AKT, and related SMAD family members were upregulated in HTMCs under hyperglycemia. In the cell functional assays, HTMCs treated with rhTGFβ-1 (1 ng/mL) under hyperglycemic conditions showed higher proliferation rates and lower ROS and calcium levels. ConclusionsTo summarize, mechanistic analyses in HTMCs showed that hyperglycemia-induced oxidative stress activated TGF-β1 along with its associated pathway. General Significance: While at low concentrations, TGF-β1 protects cells from antioxidation, whereas at high concentrations, it accumulates in the extracellular matrix, causing further HTMC dysfunction.

The Effects of ROCK Inhibitor on Prevention of Dexamethasone-Induced Glaucoma Phenotype in Human Trabecular Meshwork Cells.

This study investigated the effects of dexamethasone (Dex) on human trabecular meshwork (TM) cells, a model of glucocorticoid-induced glaucoma, and evaluated the impact of ripasudil (Rip) as a co-delivery or sequential dosing strategy. In vitro experiments were conducted to assess the effects of Dex and Rip on TM cells. Confocal microscopy was used to evaluate the impact of Dex and Rip on F-actin staining signals. Contractility of the TM cells upon Dex and Rip treatment mimicking co-delivery and sequential delivery was quantified using collagen gel contraction assay. Transepithelial electrical resistance (TEER) values and fluorescein isothiocyanate (FITC)-dextran permeability were also measured to assess the impact of Dex and Rip on TM cells. Dex and Rip did not exhibit cytotoxicity at the maximum tested concentration (20 µM). Dex-treated TM cells exhibited higher F-actin staining signals compared to controls, which were reduced when co-treated with Rip. Rip inhibited Dex-induced collagen gel contraction activity in both co-delivery and sequential treatments. Dex resulted in increased TEER values as the dose increased, whereas TEER values were maintained when co-treated with Rip. Co-delivery of Rip has the potential to prevent glaucoma symptoms when patients are treated with Dex. This study highlights the importance of identifying strategies to reduce the side effects of prolonged use of glucocorticoids, such as Dex, in the treatment of various diseases. This study demonstrates the potential of co-delivering ripasudil with dexamethasone to mitigate glucocorticoid-induced ocular hypertension and a secondary glaucoma that resembles primary open-angle glaucoma, providing insights for the development of novel preventive strategies in clinical care.

Inhibition of TGF-β2-Induced Trabecular Meshwork Fibrosis by Pirfenidone.

Trabecular meshwork (TM) fibrosis is a crucial pathophysiological process in the development of primary open-angle glaucoma. Pirfenidone (PFD) is a new, broad-spectrum antifibrotic agent approved for the treatment of idiopathic pulmonary fibrosis. This study investigated the inhibitory effect of PFD on TM fibrosis and evaluated its efficacy in lowering intraocular pressure (IOP). Human TM cells were isolated, cultured, and characterized. Cell Counting Kit-8 was used to evaluate the proliferation and toxicity of different concentrations of PFD on normal or fibrotic TM cells. TM cells were treated with transforming growth factor beta-2 (TGF-β2) in the absence or presence of PFD. Western blotting and immunofluorescence analyses were used to analyze changes in the TM cell cytoskeleton and extracellular matrix (ECM) proteins, including alpha-smooth muscle actin (α-SMA), F-actin, collagen IV (COL IV), and fibronectin (FN). An ocular hypertension (OHT) mouse model was induced with Ad-TGF-β2C226/228S and then treated with PFD or latanoprost (LT) eye drops to confirm the efficacy of PFD in lowering IOP. PFD inhibited the proliferation of fibrotic TM cells in a dose-dependent manner and inhibited TGF-β2-induced overexpression of α-SMA, COL IV, and FN in TM cells. PFD stabilized F-actin. In vivo, PFD eye drops reduced the IOP of the OHT models and showed no significant difference compared with LT eye drops. PFD inhibited TGF-β2-induced TM cell fibrosis by rearranging the disordered cytoskeleton and decreasing ECM deposition, thereby enhancing the aqueous outflow from the TM outflow pathway and lowering IOP, which provides a potential new approach to treating glaucoma. Our work with pirfenidone provides a new approach to treat glaucoma.

Small RNA Sequencing Reveals a Distinct MicroRNA Signature between Glucocorticoid Responder and Glucocorticoid Non-Responder Primary Human Trabecular Meshwork Cells after Dexamethasone Treatment.

Glucocorticoids (GCs) are known to regulate several physiological processes and are the mainstay in the management of inflammatory eye diseases. The long-term use of GC causes raised intraocular pressure (IOP) or ocular hypertension (OHT) in about 30-50% of the susceptible individuals depending on the route of administration, and can lead to steroid-induced secondary glaucoma. The present study aims to understand the role of microRNAs (miRNAs) in differential glucocorticoid (GC) responsiveness in human trabecular meshwork (HTM) cells using small RNA sequencing. The human organ-cultured anterior segment (HOCAS) model was used to identify whether donor eyes were from GC-responders (GC-R; n = 4) or GC-non-responders (GC-NR; n = 4) following treatment with either 100 nM dexamethasone (DEX) or ethanol (ETH) for 7 days. The total RNA was extracted from cultured HTM cells with known GC responsiveness, and the differentially expressed miRNAs (DEMIRs) were compared among the following five groups: Group #1: ETH vs. DEX-treated GC-R; #2: ETH vs. DEX-treated GC-NR; #3: overlapping DEGs between Group #1 and #2; #4: Unique DEMIRs of GC-R; #5: Unique DEMIRs of GC-NR; and validated by RT-qPCR. There were 13 and 21 DEMIRs identified in Group #1 and Group #2, respectively. Seven miRNAs were common miRNAs dysregulated in both GC-R and GC-NR (Group #3). This analysis allowed the identification of DEMIRs that were unique to GC-R (6 miRNAs) and GC-NR (14 miRNAs) HTM cells, respectively. Ingenuity Pathway Analysis identified enriched pathways and biological processes associated with differential GC responsiveness in HTM cells. This is the first study to reveal a unique miRNA signature between GC-R and GC-NR HTM cells, which raises the possibility of developing new molecular targets for the management of steroid-OHT/glaucoma.

Identification of the novel role of sterol regulatory element binding proteins (SREBPs) in mechanotransduction and intraocular pressure regulation.

Trabecular meshwork (TM) cells are contractile and mechanosensitive, and they aid in maintaining intraocular pressure (IOP) homeostasis. Lipids are attributed to modulating TM contractility, with poor mechanistic understanding. In this study using human TM cells, we identify the mechanosensing role of the transcription factors sterol regulatory element binding proteins (SREBPs) involved in lipogenesis. By constitutively activating SREBPs and pharmacologically inactivating SREBPs, we have mechanistically deciphered the attributes of SREBPs in regulating the contractile properties of TM. The pharmacological inhibition of SREBPs by fatostatin and molecular inactivation of SREBPs exvivo and invivo, respectively, results in significant IOP lowering. As a proof of concept, fatostatin significantly decreased the SREBPs responsive genes and enzymes involved in lipogenic pathways as well as the levels of the phospholipid, cholesterol, and triglyceride. Further, we show that fatostatin mitigated actin polymerization machinery and stabilization, and decreased ECM synthesis and secretion. We thus postulate that lowering lipogenesis in the TM outflow pathway can hold the key to lowering IOP by modifying the TM biomechanics.

Antioxidative effects of ghrelin on human trabecular meshwork cells

Glaucoma is a group of neurodegenerative diseases characterized by loss of retinal ganglion cells and visual field defects and is one of the major causes of irreversible blindness worldwide. Primary open-angle glaucoma (POAG) is one of the classifications of glaucoma. Oxidative stress in trabecular reticulated cells is one of the possible mechanisms of the development of glaucoma. At present, there is still a lack of effective methods to treat glaucoma. Ghrelin is characterized by its wide distribution and high potency and has anti-inflammatory, antioxidant, and anti-apoptotic effects, which may be beneficial in the treatment of glaucoma. In this study, we investigated whether ghrelin can protect human trabecular meshwork cells (HTMCs) from oxidative damage induced by hydrogen peroxide (H2O2), as well as the possible mechanism of action. CCK8 and flow cytometry results revealed that treatment of HTMCs with ghrelin showed a dose-dependent protective effect against H2O2-induced damage. Ghrelin significantly decreased the rate of apoptosis and levels of reactive oxygen species (ROS) and malondialdehyde (MDA) and increased the level of superoxide dismutase (SOD) and catalase (CAT) in HTMCs. The difference was statistically significant compared with the H2O2 group. Ghrelin activated Nrf2/HO-1/NQO-1 signaling pathways and decreased HIF-1α level in H2O2-injured HTMCs as shown on qPCR and Western blot. In conclusion, ghrelin can protect HTMCs from oxidative damage induced by H2O2 and reduce apoptosis in HTMCs, which can be a new approach to treating POAG. The underlying therapeutic mechanism may be related to Nrf2/HO-1/NQO-1 signaling pathways and HIF-1α.

Cryo-Electrospinning Generates Highly Porous Fiber Scaffolds Which Improves Trabecular Meshwork Cell Infiltration.

Human trabecular meshwork is a sieve-like tissue with large pores, which plays a vital role in aqueous humor outflow. Dysfunction of this tissue can occur, which leads to glaucoma and permanent vision loss. Replacement of trabecular meshwork with a tissue-engineered device is the ultimate objective. This study aimed to create a biomimetic structure of trabecular meshwork using electrospinning. Conventional electrospinning was compared to cryogenic electrospinning, the latter being an adaptation of conventional electrospinning whereby dry ice is incorporated in the fiber collector system. The dry ice causes ice crystals to form in-between the fibers, increasing the inter-fiber spacing, which is retained following sublimation. Structural characterization demonstrated cryo-scaffolds to have closer recapitulation of the trabecular meshwork, in terms of pore size, porosity, and thickness. The attachment of a healthy, human trabecular meshwork cell line (NTM5) to the scaffold was not influenced by the fabrication method. The main objective was to assess cell infiltration. Cryo-scaffolds supported cell penetration deep within their structure after seven days, whereas cells remained on the outer surface for conventional scaffolds. This study demonstrates the suitability of cryogenic electrospinning for the close recapitulation of trabecular meshwork and its potential as a 3D in vitro model and, in time, a tissue-engineered device.

Nanofibrous PCL-Based Human Trabecular Meshwork for Aqueous Humor Outflow Studies.

Primary open-angle glaucoma is characterized by the progressive degeneration of the optic nerve, with the high intraocular pressure (IOP) being one of the main risk factors. The human trabecular meshwork (HTM), specifically the juxtacanalicular tissue (JCT), is responsible for placing resistance to the aqueous humor (AH) outflow and the resulting IOP control. Currently, the lack of a proper in vitro JCT model and the complexity of three-dimensional models impede advances in understanding the relationship between AH outflow and HTM degeneration. Therefore, we design an in vitro JCT model using a polycaprolactone (PCL) nanofibrous scaffold, which supports cells to recapitulate the functional JCT morphology and allow the study of outflow physiology. Mechanical and morphological characterizations of the electrospun membranes were performed, and human trabecular meshwork cells were seeded over the scaffolds. The engineered JCT was characterized by scanning electron microscopy, quantitative real-time polymerase chain reaction, and immunochemistry assays staining HTM cell markers and proteins. A pressure-sensitive perfusion system was constructed and used for the investigation of the outflow facility of the polymeric scaffold treated with dexamethasone (a glucocorticoid) and netarsudil (a novel IOP lowering the rho inhibitor). Cells in the in vitro model exhibited an HTM-like morphology, expression of myocilin, fibronectin, and collagen IV, genetic expression, outflow characteristics, and drug responsiveness. Altogether, the present work develops an in vitro JCT model to better understand HTM cell biology and the relationship between the AH outflow and the HTM and allow further drug screening of pharmacological agents that affect the trabecular outflow facility.

In silico study about β-amyloid’s role in Alzheimer’s disease and glaucoma and prediction of its interactions with glaucoma related proteins

Aim: The significance of β-amyloid protein as a key player in neuro-degenerative disorders viz. Alzheimer’s disease (AD), Parkinson’s disease (PD) has been extensively researched and reported. Glaucoma being another prominent form of neuro-degeneration involving the loss of retinal ganglion cells (RGCs) and human trabecular meshwork (HTM) cells, is also found to be similar to AD in many aspects, but its relation with β-amyloid has not been studied too far up to understanding its causation and pathogenesis where β-amyloid is expected to play important role. This study is an attempt to evaluate the chances of β-amyloid’s role in pathogenesis of retinal neurodegenerative disorder called glaucoma, in silico. Methods: The study involved determination of feasibility of interaction between β-amyloid and well known glaucoma related proteins namely, myocilin and optineurin. The computational tool called Hex 8.0.0 has been used in this work. Results: The docking score for β-amyloid and myocilin was found to be –724.1 kJ mol–1 while that for β-amyloid and wild-type optineurin pair was found to be –296.9 kJ mol–1 and that for β-amyloid and mutated optineurin was –607.1 kJ mol–1. Conclusions: Interaction of β-amyloid with myocilin and optineurin in both forms (wild-type and mutated) is quite energetically favorable. The binding between β-amyloid and mutated optineurin is higher in comparison to that between β-amyloid and wild-type optineurin. Thus, functional significance of β-amyloid in glaucoma pathogenesis is fairly possible which should be studied and proved through in vitro and in vivo studies.

Apoptosis inhibitor of macrophages/CD5L enhances phagocytosis in the trabecular meshwork cells and regulates ocular hypertension.

The trabecular meshwork (TM) cells of the eye are important for controlling intraocular pressure (IOP) and regulating outflow resistance in the aqueous humor. TM cells can remove particles and cellular debris by phagocytosis, decreasing both outflow resistance and IOP. However, the underlying mechanisms remain unclear. Here, we investigate whether apoptosis inhibitor of macrophages (AIM), which mediates the removal of dead cells and debris in renal tubular epithelial cells, regulates the phagocytic capacity of TM cells. In vitro experiments revealed that CD36, the main receptor for AIM, colocalized with AIM in human TM cells; additionally, phagocytosis was stimulated when AIM was provided. Furthermore, in a mouse model with transient IOP elevation induced by laser iridotomy (LI), removal of accumulated iris pigment epithelial cells or debris in the TM and recovery of IOP to baseline levels were delayed in AIM-/- mice, compared with control mice. However, treatment with AIM eyedrops rescued AIM-/- mice from the elevated IOP after LI. Since AIM is a protein known to inhibit macrophage apoptosis, we additionally verified its involvement in macrophage removal of cellular debris and IOP. There were no statistically significant differences in the number of macrophagesbetween control mice and AIM-/- mice in the TM. Additionally, we confirmed the rescue effect of the rAIM eyedrops after macrophageshad been removed by clodronate liposomes. Therefore, AIM plays an important role in regulating the phagocytic capacity of TM cells, thereby affecting outflow resistance. Our results suggest that drugs targeting the phagocytic capacity of TM cells via the AIM-CD36 pathway may be used to treat glaucoma.

The correlation between primary open-angle glaucoma (POAG) and gut microbiota: a pilot study towards predictive, preventive, and personalized medicine.

Glaucoma is the leading cause of irreversible blindness worldwide. Emerged evidence has shown that glaucoma is considered an immune system related disorder. The gut is the largest immune organ in the human body and the gut microbiota (GM) plays an irreversible role in maintaining immune homeostasis. But, how the GM influences glaucoma remains unrevealed. This study aimed at investigating the key molecules/pathways mediating the GM and the glaucoma to provide new biomarkers for future predictive, preventive, and personalized medicine. Datasets from the primary open-angle glaucoma (POAG) patients (GSE138125) and datasets for target genes of GM/GM metabolites were downloaded from a public database. For GSE138125, the differentially expressed genes (DEGs) between healthy and POAG samples were identified. And the online Venn diagram tool was used to obtain the DEGs from POAG related to GM. After which GM-related DEGs were analyzed by correlation analysis, pathway enrichment analysis, and protein-protein interaction (PPI) network analysis. Human trabecular meshwork cells were used for validation, and the mRNA level of hub genes was verified by quantitative real-time polymerase chain reaction (RT-qPCR) in the in vitro glaucoma model. A total of 16 GM-related DEGs in POAG were identified from the above 2 datasets (9 upregulated genes and 7 downregulated genes). Pathway enrichment analysis indicated that these genes are mostly enriched in immune regulation especially macrophages-related pathways. Then 6 hub genes were identified by PPI network analysis and construction of key modules. Finally, RT-qPCR confirmed that the expression of the hub genes in the in vitro glaucoma model was consistent with the results of bioinformatics analysis of the mRNA chip. This bioinformatic study elucidates NFKB1, IL18, KITLG, TLR9, FKBP2, and HDAC4 as hub genes for POAG and GM regulation. Immune response modulated by macrophages plays an important role in POAG and may be potential targets for future predictive, preventive, and personalized diagnosis and treatment. The online version contains supplementary material available at 10.1007/s13167-023-00336-2.

Sigma-1 Receptor Activation Is Protective against TGFβ2-Induced Extracellular Matrix Changes in Human Trabecular Meshwork Cells.

The trabecular meshwork (TM) route is the principal outflow egress of the aqueous humor. Actin cytoskeletal remodeling in the TM and extracellular matrix (ECM) deposition increase TM stiffness, outflow resistance, and elevate intraocular pressure (IOP). These alterations are strongly linked to transforming growth factor-β2 (TGFβ2), a known profibrotic cytokine that is markedly elevated in the aqueous humor of glaucomatous eyes. Sigma-1 receptor (S1R) has been shown to have neuroprotective effects in the retina, but data are lacking about its role in the TM. In this study, we identified the presence of S1R in mouse TM tissue and investigated the effect of an S1R agonist fluvoxamine (FLU) on TGFβ2-induced human TM cells regarding cell proliferation; ECM-related functions, including F-actin reorganization; and the accumulation of ECM elements. TGFβ2 increased the proliferation, cytoskeletal remodeling, and protein levels of fibronectin, collagen type IV, and connective tissue growth factor, and decreased the level of matrix metalloproteinase-2. Most importantly, FLU reversed all these effects of TGFβ2, suggesting that S1R agonists could be potential candidates for preserving TM function and thus maintaining normal IOP.

Sigma-1 Receptor Agonist Fluvoxamine Ameliorates Fibrotic Response of Trabecular Meshwork Cells.

Primary open-angle glaucoma remains a global issue, lacking a definitive treatment. Increased intraocular pressure (IOP) is considered the primary risk factor of the disease and it can be caused by fibrotic-like changes in the trabecular meshwork (TM) such as increased tissue stiffness and outflow resistance. Previously, we demonstrated that the sigma-1 receptor (S1R) agonist fluvoxamine (FLU) has anti-fibrotic properties in the kidney and lung. In this study, the localization of the S1R in TM cells was determined, and the anti-fibrotic efficacy of FLU was examined in both mouse and human TM cells. Treatment with FLU reduced the F-actin rearrangement, inhibited cell proliferation and migration induced by the platelet-derived growth factor and decreased the levels of fibrotic proteins. The protective role of the S1R in fibrosis was confirmed by a more pronounced increase in alpha smooth muscle actin and F-actin bundle and clump formation in primary mouse S1R knockout TM cells. Furthermore, FLU demonstrated its protective effects by increasing the production of nitric oxide and facilitating the degradation of the extracellular matrix through the elevation of cathepsin K. These findings suggest that the S1R could be a novel target for the development of anti-fibrotic drugs and offer a new therapeutic approach for glaucoma.

Effects of Brimonidine, Omidenepag Isopropyl, and Ripasudil Ophthalmic Solutions to Protect against H2O2-Induced Oxidative Stress in Human Trabecular Meshwork Cells

Purpose We investigated whether hydrogen peroxide (H2O2)-induced oxidative stress causes human trabecular meshwork (HTM) cell dysfunction observed in open angle glaucoma (OAG) in vitro, and the effects of topical glaucoma medications on oxidative stress in HTM cells. Methods We used commercially available ophthalmic solutions of brimonidine, omidenepag isopropyl, and ripasudil in the study. HTM cells were exposed to H2O2 for 1 h, with or without glaucoma medications. We assessed cell viability and senescence via WST-1 and senescence-associated-β-galactosidase (SA-β-Gal) activity assays. After exposure to H2O2 and glaucoma medications, we evaluated changes in markers of fibrosis and stress by using real-time quantitative polymerase chain reaction (qPCR) to measure the mRNA levels of collagen type I alpha 1 chain (COL1A1), fibronectin, alpha-smooth muscle actin (α-SMA), matrix metalloproteinase-2 (MMP-2), endoplasmic reticulum stress markers of C/EBP homologous protein (CHOP), 78-kDa glucose-regulated protein (GRP78), and splicing X-box binding protein-1 (sXBP-1). Results HTM cell viability decreased and SA-β-Gal activity increased significantly after exposure to H2O2. Treatment with three ophthalmic solutions attenuated these changes. Real-time qPCR revealed that H2O2 upregulated the mRNA levels of COL1A1, fibronectin, α-SMA, CHOP, GRP78, and sXBP-1, whereas it downregulated MMP-2 mRNA expression significantly. Brimonidine suppressed the upregulation of stress markers CHOP and GRP78. Additionally, omidenepag isopropyl and ripasudil decreased the upregulation of COL1A1 and sXBP-1. Furthermore, ripasudil significantly suppressed fibrotic markers fibronectin and α-SMA, compared with the other two medications. Conclusion In vitro, H2O2 treatment of HTM cells induced characteristic changes of OAG, such as fibrosis changes and the upregulation of stress markers. These glaucomatous changes were attenuated by additional treatments with brimonidine, omidenepag isopropyl, and ripasudil ophthalmic solutions.

Modulation of Mitochondrial Metabolic Parameters and Antioxidant Enzymes in Healthy and Glaucomatous Trabecular Meshwork Cells with Hybrid Small Molecule SA-2.

Oxidative stress (OS)-induced mitochondrial damage is a risk factor for primary open-angle glaucoma (POAG). Mitochondria-targeted novel antioxidant therapies could unearth promising drug candidates for the management of POAG. Previously, our dual-acting hybrid molecule SA-2 with nitric oxide-donating and antioxidant activity reduced intraocular pressure and improved aqueous humor outflow in rodent eyes. Here, we examined the mechanistic role of SA-2 in trabecular meshwork (TM) cells in vitro and measured the activity of intracellular antioxidant enzymes during OS. Primary human TM cells isolated from normal (hNTM) or glaucomatous (hGTM) post-mortem donors and transformed glaucomatous TM cells (GTM-3) were used for in vitro assays. We examined the effect of SA-2 on oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) in vitro using Seahorse Analyzer with or without the oxidant, tert-butyl hydroperoxide (TBHP) treatment. Concentrations of total antioxidant enzymes, catalase (CAT), malondialdehyde (MDA), and glutathione peroxidase (GPx) were measured. We observed significant protection of both hNTM and hGTM cells from TBHP-induced cell death by SA-2. Antioxidant enzymes were elevated in SA-2-treated cells compared to TBHP-treated cells. In addition, SA-2 demonstrated an increase in mitochondrial metabolic parameters. Altogether, SA-2 protected both normal and glaucomatous TM cells from OS via increasing mitochondrial energy parameters and the activity of antioxidant enzymes.

Effects of Vascular Endothelial Growth Factor on YAP/TAZ Signaling in Trabecular Meshwork Cells

Purpose: To investigate the effects of vascular endothelial growth factor (VEGF) on yes-associated protein (YAP)/transcriptional coactivator with a PDZ-binding motif (TAZ), a Hippo pathway-related transcription factor, and the role of YAP/TAZ induced by trabecular meshwork stimulation.Methods: Human trabecular meshwork cells were cultured and treated with various VEGF concentrations to verify cell cytotoxicity using the CCK-8 solution. Transforming growth factor β-2 (TGFβ2; 5 ng/mL) and VEGF (30 ng/mL) were applied and YAP/TAZ expression was assessed by western blotting, reverse transcription quantitative polymerase chain reaction and immunocytochemistry. Fibronectin, collagen 1, and myocilin expression were also assessed by western blotting. The cells were stained using Alexa Fluor 488-phalloidin to observe F-actin changes.Results: YAP and TAZ expression increased following TGFβ2 and VEGF treatment for 24 hours. Fibronectin and collagen 1 increased significantly in all three treatment groups, while myocilin increased in the TGFβ2 and TGFβ2+VEGF groups. The F-actin staining showed increased cross-linking in the trabecular meshwork cells.Conclusions: VEGF induced YAP/TAZ signaling and increased trabecular meshwork cell fibrosis. Based on the functional changes caused by VEGF, it is suggested that VEGF and YAP/TAZ may increase aqueous humor outflow resistance in trabecular meshwork cells.

Activation of Sonic Hedgehog Signaling Pathway Regulates Human Trabecular Meshwork Cell Function.

Purpose: To investigate the effects of Sonic hedgehog (Shh) signaling on primary human trabecular meshwork (HTM) cells. Methods: Primary HTM cells were isolated from healthy donors and cultured. Recombinant Shh (rShh) protein and cyclopamine were used to activate and inhibit the Shh signaling pathway, respectively. A cell viability assay was performed to assess the effects of rShh on the activity of primary HTM cells. Functional assessment of cell adhesion and phagocytosis was also performed. The proportion of apoptotic cells was examined using flow cytometry. Fibronectin (FN) and transforming growth factor beta2 (TGF-β2) protein were detected to assess the influence of rShh on the metabolism of the extracellular matrix (ECM). Real-time polymerase chain reaction (RT-PCR) and western blot analyses were used to examine mRNA and protein expression of Shh signaling pathway-associated factors GLI Family Zinc Finger 1 (GLI1) and Suppressor of Fused (SUFU). Results: rShh significantly enhanced primary HTM cell viability at a concentration of 0.5 μg/mL. rShh increased the adhesion and phagocytic abilities of primary HTM cells, and decreased cell apoptosis. FN and TGF-β2 protein expression increased in primary HTM cells treated with rShh. rShh upregulated the transcriptional activity and protein levels of GLI1, and downregulated those of SUFU. Correspondingly, the rShh-induced GLI1 upexpression was partially blocked by pretreatment with the Shh pathway inhibitor cyclopamine at a concentration of 10 μM. Conclusions: Activation of Shh signaling can regulate the function of primary HTM cells through GLI1. Regulation of Shh signaling may be a potential target for attenuating cell damage in glaucoma.

Recreating the Trabecular Outflow Tissue on Implantable, Micropatterned, Ultrathin, Porous Polycaprolactone Scaffolds.

Glaucoma, where increased intraocular pressure (IOP) leads to damage to the optic nerve and loss of sight, is amongst the foremost causes of irreversible blindness worldwide. In primary open angle glaucoma, the increased IOP is a result of the malfunctioning human trabecular meshwork (HTM) cells' inability to properly regulate the outflow of aqueous humor from the eye. A potential future treatment for glaucoma is to replace damaged HTM cells with a tissue-engineered substitute, thus restoring proper fluid outflow. Polycaprolactone (PCL) is a versatile, biodegradable, and implantable material that is widely used for cell culture and tissue engineering. In this work, PCL scaffolds were lithographically fabricated using a sacrificial process to produce submicron-thick scaffolds with openings of specific sizes and shapes (e.g., grid, hexagonal pattern). The HTM cell growth on gelatin-coated PCL scaffolds was assessed by scanning electron microscopy, tetrazolium metabolic activity assay, and cytoskeletal organization of F-actin. Expression of HTM-specific markers and ECM deposition were assessed by immunocytochemistry and qPCR analysis. Gelatin-coated, micropatterned, ultrathin, porous PCL scaffolds with a grid pattern supported proper HTM cell growth, cytoskeleton organization, HTM-marker expression, and ECM deposition, demonstrating the feasibility of using these PCL scaffolds to tissue-engineer implantable, healthy ocular outflow tissue.