SDPR expression in human trabecular meshwork and its potential role in racial disparities of glaucoma.

In order to identify how differential gene expression in the trabecular meshwork (TM) contributes to racial disparities of caveolar protein expression, TM dysfunction and development of primary open angle glaucoma (POAG), RNA sequencing was performed to compare TM tissue obtained from White and Black POAG surgical (trabeculectomy) specimens. Healthy donor TM tissue from White and Black donors was analyzed by PCR, qPCR, immunohistochemistry staining, and Western blot to evaluate SDPR (serum deprivation protein response; Cavin 2) and CAV1/CAV2 (Caveolin 1/Caveolin 2). Standard transmission electron microscopy (TEM) and immunogold labeled studies were performed. RNA sequencing demonstrated reduced SDPR expression in TM from Black vs White POAG patients' surgical specimens, with no significant expression differences in other caveolae-associated genes, confirmed by qPCR analysis. No racial differences in SDPR gene expression were noted in healthy donor tissue by PCR analysis, but there was greater expression as compared to specimens from patients with glaucoma. Analysis of SDPR protein expression confirmed specific expression in the TM regions, but not in adjacent tissues. TEM studies of TM specimens from healthy donors did not demonstrate any racial differences in caveolar morphology, but a significant reduction of caveolae with normal morphology and immuno-gold staining of SDPR were noted in glaucomatous TM as compared to TM from healthy donors. Linkage of SDPR expression levels in TM, POAG development, and caveolar ultrastructural morphology may provide the basis for a novel pathway of exploration of the pathologic mechanisms of glaucoma. Differential gene expression of SDPR in TM from Black vs White subjects with glaucoma may further our understanding of the important public health implications of the racial disparities of this blinding disease.

Paeoniflorin alleviates TGF-ß2-mediated extracellular matrix remodeling and oxidative stress in human trabecular meshwork cells.

BACKGROUND: The multifunctional profibrotic cytokine transforming growth factor-beta2 (TGF-ß2) 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-ß2-dependent remodeling of the extracellular matrix (ECM) within the trabecular meshwork (TM) microenvironment. METHODS: Primary or transformed (GTM3) human TM (HTM) cells conditioned in serum-free media were incubated with TGF-ß2 (5 ng/mL). PAE (300 µM) was added to serum-starved confluent cultures of HTM cells for 2 h, followed by incubation with TGF-ß2 for 22 h. SB-431542, a TGF-ß receptor inhibitor (10 µM), 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-ß2/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. RESULTS: PAE attenuated TGF-ß2-induced oxidative stress and suppressed TGF-ß2-induced Smad2/3 signaling in primary or transformed HTM cells. Additionally, PAE repressed TGF-ß2-induced upregulation of collagen 1α1, collagen 4α1, and CTGF expression and reduced TGF-ß2-mediated collagen I/IV expression and of F-actin stress fiber formation in primary or transformed HTM cells. CONCLUSION: PAE alleviates TGF-ß2-induced ECM deposition and oxidative stress in HTM cells through inactivation of Smad2/3 signaling.

Comparative analysis of traction forces in normal and glaucomatous trabecular meshwork cells within a 3D, active fluid-structure interaction culture environment.

This study presents a 3D in vitro cell culture model, meticulously 3D printed to replicate the conventional aqueous outflow pathway anatomical structure, facilitating the study of trabecular meshwork (TM) cellular responses under glaucomatous conditions. Glaucoma affects TM cell functionality, leading to extracellular matrix (ECM) stiffening, enhanced cell-ECM adhesion, and obstructed aqueous humor outflow. Our model, reconstructed from polyacrylamide gel with elastic moduli of 1.5 and 21.7 kPa, is based on serial block-face scanning electron microscopy images of the outflow pathway. It allows for quantifying 3D, depth-dependent, dynamic traction forces exerted by both normal and glaucomatous TM cells within an active fluid-structure interaction (FSI) environment. In our experimental design, we designed two scenarios: a control group with TM cells observed over 20 hours without flow (static setting), focusing on intrinsic cellular contractile forces, and a second scenario incorporating active FSI to evaluate its impact on traction forces (dynamic setting). Our observations revealed that active FSI results in higher traction forces (normal: 1.83-fold and glaucoma: 2.24-fold) and shear strains (normal: 1.81-fold and glaucoma: 2.41-fold), with stiffer substrates amplifying this effect. Glaucomatous cells consistently exhibited larger forces than normal cells. Increasing gel stiffness led to enhanced stress fiber formation in TM cells, particularly in glaucomatous cells. Exposure to active FSI dramatically altered actin organization in both normal and glaucomatous TM cells, particularly affecting cortical actin stress fiber arrangement. This model while preliminary offers a new method in understanding TM cell biomechanics and ECM stiffening in glaucoma, highlighting the importance of FSI in these processes. STATEMENT OF SIGNIFICANCE: This pioneering project presents an advanced 3D in vitro model, meticulously replicating the human trabecular meshwork's anatomy for glaucoma research. It enables precise quantification of cellular forces in a dynamic fluid-structure interaction, a leap forward from existing 2D models. This advancement promises significant insights into trabecular meshwork cell biomechanics and the stiffening of the extracellular matrix in glaucoma, offering potential pathways for innovative treatments. This research is positioned at the forefront of ocular disease study, with implications that extend to broader biomedical applications.

The Role and Mechanism of Nicotinamide Riboside in Oxidative Damage and a Fibrosis Model of Trabecular Meshwork Cells.

Purpose: To investigate the potential effects and mechanism of nicotinamide riboside (NR) on the oxidative stress and fibrosis model of human trabecular meshwork (HTM) cell line cells. Methods: HTM cells were pretreated with NR, followed by the induction of oxidative injury and fibrosis by hydrogen peroxide (H2O2) and TGF-ß2, respectively. Cell viability was tested using Hoechst staining and MTT assays, cell proliferation was assessed by EdU assay, and cell apoptosis was detected by flow cytometry and western blotting. DCFH-DA and DHE probes were used to measure the level of reactive oxygen species (ROS), and MitoTracker staining was used to measure the mitochondrial membrane potential (MMP). Fibrotic responses, including cell migration and deposition of extracellular matrix (ECM) proteins, were detected via Transwell assays, qRT-PCR, and immunoblotting. Results: NR pretreatment improved the viability, proliferation, and MMP of H2O2-treated HTM cells. Compared to cells treated solely with H2O2, HTM cells treated with both NR and H2O2, exhibited a reduced rate of apoptosis and generation of ROS. Compared with H2O2 pretreatment, NR pretreatment upregulated expression of the JAK2/Stat3 pathway but inhibited mitogen-activated protein kinase (MAPK) pathway expression. Moreover, 10-ng/mL TGF-ß2 promoted cell proliferation and migration, which were inhibited by NR pretreatment. Both qRT-PCR and immunoblotting showed that NR inhibited the expression of fibronectin in a TGF-ß2-induced fibrosis model. Conclusions: NR has a protective effect on oxidative stress and fibrosis in HTM cells, which may be related to the JAK2/Stat3 pathway and MAPK pathway. Translational Relevance: Our research provides the ongoing data for potential therapy of NAD+ precursors in glaucoma.

Genetic Basis of Pigment Dispersion Syndrome and Pigmentary Glaucoma: An Update and Functional Insights.

Pigment Dispersion Syndrome (PDS) and Pigmentary Glaucoma (PG) comprise a spectrum of ocular disorders characterized by iris pigment dispersion and trabecular meshwork changes, resulting in increased intraocular pressure and potential glaucomatous optic neuropathy. This review summarizes recent progress in PDS/PG genetics including rare pathogenic protein coding alterations (PMEL) and susceptibility loci identified from genome-wide association studies (GSAP and GRM5/TYR). Areas for future research are also identified, especially the development of efficient model systems. While substantial strides have been made in understanding the genetics of PDS/PG, our review identifies key gaps and outlines the future directions necessary for further advancing this important field of ocular genetics.

Phacoemulsification combined with goniosynechialysis versus phacoemulsification alone for patients with primary angle­closure disease: A meta-analysis.

This meta-analysis aims to systematically compare the efficacy between phacoemulsification (PE) combined with goniosynechialysis (GSL) and PE alone for primary angle-closure disease (PACD) patients. All the data were searched from the PubMed, EMBASE and the Cochrane Library. The Cochrane Handbook was used to evaluate the quality of the included studies. Additionally, this meta-analysis was performed by using the Revman 5.4 software. Nine randomized controlled trials (RCTs) were included in this study. Compared with PE alone group, PE+GSL could result significant reduction in the IOP (MD, 1.81; p = 0.002). In the instrumental subgroup, also more reduction of IOP was shown in the PE+GSL group (MD, 2.11; p = 0.02). In the viscogonioplasty (VGP) subgroup, there was not no statistical difference between PE alone group and PE+GSL group (MD, 1.53; p = 0.11). Also, more reduction of peripheral anterior synechiae (PAS) was shown in the PE+GSL group (MD,59.15; p<0.00001). For the change in angle open distance (AOD)500, AOD 750, trabecular-iris space (TISA)500, number of glaucoma medications and best corrected visual acuity (BCVA), there was no difference between two groups (p = 0.25, 0.35, 0.17, 0.56, 0.08). For TISA 750, more improvement was shown in the PE+GSL group (p<0.00001). Instrumental separation had better effect on lowering IOP when it combined with PE. Both instrumental separation and VGP could reduce postoperative PAS. The operation of GSL has no obvious effect on postoperative vision.

Influence of Growth Differentiation Factor 15 on Intraocular Pressure in Mice.

Growth differentiation factor 15 (GDF15), a stress-sensitive cytokine, and a distant member of the transforming growth factor ß superfamily, has been shown to exhibit increased levels with aging, and in various age-related pathologies. Although GDF15 levels are elevated in the aqueous humor (AH) of glaucoma (optic nerve atrophy) patients, the possible role of this cytokine in the modulation of intraocular pressure (IOP) or AH outflow is unknown. The current study addresses this question using transgenic mice expressing human GDF15 and GDF15 null mice, and by perfusing enucleated mouse eyes with recombinant human GDF15 (rhGDF15). Treatment of primary cultures of human trabecular meshwork cells with a telomerase inhibitor, an endoplasmic reticulum stress-inducing agent, hydrogen peroxide, or an autophagy inhibitor resulted in significant elevation in GDF15 levels relative to the respective control cells. rhGDF15 stimulated modest but significant increases in the expression of genes encoding the extracellular matrix, cell adhesion proteins, and chemokine receptors (C-C chemokine receptor type 2) in human trabecular meshwork cells compared with controls, as deduced from the differential transcriptional profiles using RNA-sequencing analysis. There was a significant increase in IOP in transgenic mice expressing human GDF15, but not in GDF15 null mice, compared with the respective wild-type control mice. The AH outflow facility was decreased in enucleated wild-type mouse eyes perfused with rhGDF15. Light microcopy-based histologic examination of the conventional AH outflow pathway tissues did not reveal identifiable differences between the GDF15-targeted and control mice. Taken together, these results reveal the modest elevation of IOP in mice expressing human GDF15 possibly stemming from decreased AH outflow through the trabecular pathway.

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 SIGNIFICANCE: Glaucoma, 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.

Histopathologic correlates of trabecular meshwork in microincisional trabeculectomy.

PURPOSE: To report the histopathologic correlates of trabecular meshwork (TM) specimens procured by microincisional trabeculectomy (MIT) for different severities of glaucoma (early glaucoma: visual field mean deviation [MD] <-6 dB, moderate glaucoma: MD from - 6 to - 12 dB, and advanced glaucoma: MD <-12 dB). METHODS: TM specimens from four patients undergoing MIT with or without cataract surgery were analyzed by routine histopathology for structural changes. The number of cells, the number of cells with spindle-shaped nuclei suggestive of epithelial-mesenchymal transformation (EMT), and the distance between the trabecular beams were calculated using different tools on freely available ImageJ software using the line or pint/count tool. RESULTS: The TM specimens procured from two early and two advanced glaucoma cases showed decreasing cellularity and decreased compact arrangement of the trabecular beams in severe disease stages. The number of cells and preserved architecture in all four specimens were evident, with > 50 cells being present per section in all four cases despite the glaucoma being of advanced disease stage in two patients. CONCLUSION: The TM specimens obtained from MIT can be utilized for downstream analysis using different molecular methods for studying the molecular events in the tissue from early to severe glaucoma.

Inhibition of TGF-ß2-Induced Trabecular Meshwork Fibrosis by Pirfenidone.

Purpose: 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). Methods: 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-ß2) 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-ß2C226/228S and then treated with PFD or latanoprost (LT) eye drops to confirm the efficacy of PFD in lowering IOP. Results: PFD inhibited the proliferation of fibrotic TM cells in a dose-dependent manner and inhibited TGF-ß2-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. Conclusions: PFD inhibited TGF-ß2-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. Translational Relevance: Our work with pirfenidone provides a new approach to treat glaucoma.

Connective tissue growth factor: Role in trabecular meshwork remodeling and intraocular pressure lowering.

Connective tissue growth factor (CTGF) is a distinct signaling molecule modulating many physiological and pathophysiological processes. This protein is upregulated in numerous fibrotic diseases that involve extracellular matrix (ECM) remodeling. It mediates the downstream effects of transforming growth factor beta (TGF-ß) and is regulated via TGF-ß SMAD-dependent and SMAD-independent signaling routes. Targeting CTGF instead of its upstream regulator TGF-ß avoids the consequences of interfering with the pleotropic effects of TGF-ß. Both CTGF and its upstream mediator, TGF-ß, have been linked with the pathophysiology of glaucomatous optic neuropathy due to their involvement in the regulation of ECM homeostasis. The excessive expression of these growth factors is associated with glaucoma pathogenesis via elevation of the intraocular pressure (IOP), the most important risk factor for glaucoma. The raised in the IOP is due to dysregulation of ECM turnover resulting in excessive ECM deposition at the site of aqueous humor outflow. It is therefore believed that CTGF could be a potential therapeutic target in glaucoma therapy. This review highlights the CTGF biology and structure, its regulation and signaling, its association with the pathophysiology of glaucoma, and its potential role as a therapeutic target in glaucoma management.

iPSCs-Based Therapy for Trabecular Meshwork.

The trabecular meshwork (TM) of the eye serves as an essential tissue in controlling aqueous humor (AH) outflow and intraocular pressure (IOP) homeostasis. However, dysfunctional TM cells and/or decreased TM cellularity is become a critical pathogenic cause for primary open-angle glaucoma (POAG). Consequently, it is particularly valuable to investigate TM characteristics, which, in turn, facilitates the development of new treatments for POAG. Since 2006, the advancement in induced pluripotent stem cells (iPSCs) provides a new tool to (1) model the TM in vitro and (2) regenerate degenerative TM in POAG. In this context, we first summarize the current approaches to induce the differentiation of TM-like cells from iPSCs and compare iPSC-derived TM models to the conventional in vitro TM models. The efficacy of iPSC-derived TM cells for TM regeneration in POAG models is also discussed. Through these approaches, iPSCs are becoming essential tools in glaucoma modeling and for developing personalized treatments for TM regeneration.

Differential roles of FOXC2 in the trabecular meshwork and Schlemm's canal in glaucomatous pathology.

Impaired development and maintenance of Schlemm's canal (SC) are associated with perturbed aqueous humor outflow and intraocular pressure. The angiopoietin (ANGPT)/TIE2 signaling pathway regulates SC development and maintenance, whereas the molecular mechanisms of crosstalk between SC and the neural crest (NC)-derived neighboring tissue, the trabecular meshwork (TM), are poorly understood. Here, we show NC-specific forkhead box (Fox)c2 deletion in mice results in impaired SC morphogenesis, loss of SC identity, and elevated intraocular pressure. Visible-light optical coherence tomography analysis further demonstrated functional impairment of the SC in response to changes in intraocular pressure in NC-Foxc2 -/- mice, suggesting altered TM biomechanics. Single-cell RNA-sequencing analysis identified that this phenotype is predominately characterized by transcriptional changes associated with extracellular matrix organization and stiffness in TM cell clusters, including increased matrix metalloproteinase expression, which can cleave the TIE2 ectodomain to produce soluble TIE2. Moreover, endothelial-specific Foxc2 deletion impaired SC morphogenesis because of reduced TIE2 expression, which was rescued by deleting the TIE2 phosphatase VE-PTP. Thus, Foxc2 is critical in maintaining SC identity and morphogenesis via TM-SC crosstalk.

Improved magnetic delivery of cells to the trabecular meshwork in mice.

Glaucoma is the leading cause of irreversible blindness worldwide and its most prevalent subtype is primary open angle glaucoma (POAG). One pathological change in POAG is loss of cells in the trabecular meshwork (TM), which is thought to contribute to ocular hypertension and has thus motivated development of cell-based therapies to refunctionalize the TM. TM cell therapy has shown promise in intraocular pressure (IOP) control, but existing cell delivery techniques suffer from poor delivery efficiency. We employed a novel magnetic delivery technique to reduce the unwanted side effects of off-target cell delivery. Mesenchymal stem cells (MSCs) were labeled with superparamagnetic iron oxide nanoparticles (SPIONs) and after intracameral injection were magnetically steered towards the TM using a focused magnetic apparatus ("point magnet"). This technique delivered the cells significantly closer to the TM at higher quantities and with more circumferential uniformity compared to either unlabeled cells or those delivered using a "ring magnet" technique. We conclude that our point magnet cell delivery technique can improve the efficiency of TM cell therapy and in doing so, potentially increase the therapeutic benefits and lower the risk of complications such as tumorigenicity and immunogenicity.

Glaucoma: Novel antifibrotic therapeutics for the trabecular meshwork.

Glaucoma is a chronic and progressive neurodegenerative disease characterized by the loss of retinal ganglion cells and visual field defects, and currently affects around 1% of the world's population. Elevated intraocular pressure (IOP) is the best-known modifiable risk factor and a key therapeutic target in hypertensive glaucoma. The trabecular meshwork (TM) is the main site of aqueous humor outflow resistance and therefore a critical regulator of IOP. Fibrosis, a reparative process characterized by the excessive deposition of extracellular matrix components and contractile myofibroblasts, can impair TM function and contribute to the pathogenesis of primary open-angle glaucoma (POAG) as well as the failure of minimally invasive glaucoma surgery (MIGS) devices. This paper provides a detailed overview of the current anti-fibrotic therapeutics targeting the TM in glaucoma, along with their anti-fibrotic mechanisms, efficacy as well as the current research progress from pre-clinical to clinical studies.

Elevated Angiotensin-II Levels Contribute to the Pathogenesis of Open-Angle Glaucoma Via Inducing the Expression of Fibrosis-Related Genes in Trabecular Meshwork Cells Through a ROS/NOX4/SMAD3 Axis.

Glaucoma including primary open-angle glaucoma (POAG) results from elevations in intraocular pressure (IOP). An eye-localized renin-angiotensin system (RAS) has been implicated in IOP regulation, although its mechanism of action and contribution to glaucoma is poorly understood. Here, we detected significant increases in the levels of angiotensin II (ANGII) in aqueous humor samples from POAG patients. Moreover, we determined that the concentrations of ANGII were positively correlated with IOP, suggesting a role for elevated ANGII levels in eye pathogenesis. Functional investigations demonstrated that ANGII induces the expression of fibrosis-related genes of transformed and primary human trabecular meshwork cells (HTMCs) through the transcriptional upregulation of key fibrotic genes. Parallel experiments using a murine periocular conjunctival fornix injection model confirmed that ANGII induces the expression of fibrosis-related genes in trabecular meshwork (TM) cells in vivo along with increasing IOP. ANGII was revealed to function through increasing the levels of reactive oxygen species (ROS) via selectively upregulating NOX4, with NOX4 knockdown or inhibition with GLX351322 alleviating fibrotic changes induced by ANGII. We further show that ANGII activates Smad3, with both GLX351322 and an inhibitor of Smad3 (SIS3) decreasing the phosphorylation of Smad3 and dampening the ANGII-induced increases in fibrotic proteins. Moreover, NOX4 and Smad3 inhibitors also partially rescued the elevated IOP levels induced by ANGII. Our collective results therefore highlight ANGII as a biomarker and treatment target in POAG together with establishing a causal relationship between ANGII and up-regulation of the expression of fibrosis-related genes of TM cells via a NOX4/ROS axis in cooperation with TGFß/Smad3 signaling.

Tissue-engineered anterior segment eye cultures demonstrate hallmarks of conventional organ culture.

BACKGROUND: Glaucoma is a blinding disease largely caused by dysregulation of outflow through the trabecular meshwork (TM), resulting in elevated intraocular pressure (IOP). We hypothesized that transplanting TM cells into a decellularized, tissue-engineered anterior segment eye culture could restore the outflow structure and function. METHODS: Porcine eyes were decellularized with freeze-thaw cycles and perfusion of surfactant. We seeded control scaffolds with CrFK cells transduced with lentiviral vectors to stably express eGFP and compared them to scaffolds seeded with primary TM cells as well as to normal, unaltered eyes. We tracked the repopulation behavior, performed IOP maintenance challenges, and analyzed the histology. RESULTS: Transplanted cells localized to the TM and progressively infiltrated the extracellular matrix, reaching a distribution comparable to normal, unaltered eyes. After a perfusion rate challenge to mimic a glaucomatous pressure elevation, transplanted and normal eyes reestablished a normal intraocular pressure (transplanted = 16.5 ± 0.9 mmHg, normal = 16.9 ± 0.9). However, eyes reseeded with eGFP-expressing CrFK cells could not regulate IOP, remaining high and unstable (27.0 ± 6.2 mmHg) instead. CONCLUSION: Tissue-engineered anterior segment scaffolds can serve as readily available, scalable ocular perfusion cultures. This could reduce dependency on scarce donor globes in outflow research and may allow engineering perfusion cultures with specific geno- and phenotypes.

Human Pro370Leu Mutant Myocilin Induces the Phenotype of Open-Angle Glaucoma in Transgenic Mice.

To investigate the characteristics of mutation myocilin proteins and glaucoma pathological phenotype in transgenic mice with full-length human Pro370Leu mutant myocilin gene (Tg-MYOCP370L). Tg-MYOCP370L mice were established using the CRISPR/Cas9 system. Long-term intraocular pressure (IOP) was measured, myocilin protein expressions in anterior chamber angle, retina, optic nerve tissues and aqueous humor were detected by western blot. RBPMS, myocilin, Iba-1 and GFAP expression were visualized by immunofluorescence. H&E staining was applied to assess the ocular angle and retinal morphology. Aqueous humor dynamics were visualized by Gadolinium magnetic resonance imaging (Gd-MRI). TUNEL assay was used to evaluate the specific cell apoptosis in trabecular meshwork and retina. Optomotor and electroretinography tests were employed to evaluate the visual function in Tg-MYOCP370L and wild-type (WT) mice. Homozygous myocilin mutation at position 503 (C > T) was identified by PCR and sequencing in Tg-MYOCP370L mice. Myocilin protein expression was overexpressed in eye tissues of Tg-MYOCP370L mice with reduced myocilin secretion in aqueous humor. H&E staining showed normal histological morphology of anterior chamber angle whereas decreased thickness and nuclei in ganglion cell layer were found (P < 0.05). Gd signals were significantly increased in the anterior chamber of Tg-MYOCP370L compared with WT eyes (P < 0.05). IOP was elevated in Tg-MYOCP370L mice starting at 5 months of age, with significant RGC loss (P < 0.05). Upregulation of caspase-3 and caspase-9 expressions and increased TUNEL-positive cells were found in eyes of Tg-MYOCP370L mice. Excessive activation of retinal glial cells and impaired visual function were detected in Tg-MYOCP370L mice. Tg-MYOCP370L mice can induce the phenotype of open-angle glaucoma, featured as IOP elevation, activated retinal glial cells, loss of RGCs and impaired visual function. These pathologic changes may arise from the abnormal mutant myocilin protein accumulation in the trabecular meshwork and injured aqueous humor drainage. Therefore, Tg-MYOCP370L mice model can serve as an effective animal model for glaucoma research, especially for glaucoma-associated myocilin mutation studies.

Glycosaminoglycans Influence Extracellular Matrix of Human Trabecular Meshwork Cells Cultured on 3D Scaffolds.

Glaucoma is a multifactorial progressive optic neuropathy characterized by the loss of retinal ganglion cells leading to irreversible blindness. It is the leading cause of global irreversible blindness and is currently affecting over 70 million people. Elevated intraocular pressure (IOP) is considered the only modifiable risk factor and is a target of numerous treatment modalities. Researchers have assigned this elevation of IOP to accumulation of extracellular matrix (ECM) components in the aqueous humor (AH) outflow pathway. The major drainage structure for AH outflow is the trabecular meshwork (TM). The ECM of the TM is important in regulating IOP in both normal and glaucomatous eyes. In this work, we have studied the role of exogeneous glycosaminoglycans (GAGs), glucocorticoids, and culture conditions on the expression of the ECM gene and proteins by human TM (hTM) cells cultured on biomaterial scaffolds. Gene and protein expression levels of elastin, laminin, and matrix metalloproteinase-2 (MMP-2) were evaluated using quantitative PCR and immunohistochemistry. Pressure gradient changes in cell-laden scaffolds in perfusion cultures were also monitored. Our findings show that GAGs and dexamethasone play an influencing role in hTM ECM turnover at both transcriptional and translational levels by altering expression levels of elastin, laminin, and MMP-2. Understanding the role of exogeneous factors on hTM cell behavior is helpful in gaining insights on glaucoma pathogenesis and ultimately pivotal in development of novel therapeutics against the disease.

PBX1 attenuates H2O2-induced oxidant stress in human trabecular meshwork cells via promoting NANOG-mediated PI3K/AKT signaling pathway.

Oxidative stress-induced excessive extracellular matrix (ECM) deposition in trabecular meshwork (TM) tissue is considered the major pathological procedure of glaucoma. This study aimed to explore the role and regulatory mechanism of pre-B-cell leukemia transcription factor 1 (PBX1) in H2O2-induced human trabecular meshwork cells (HTMCs). Expressions of PBX1, NANOG, ECM, and pathway-related factors were detected by qRT-PCR and western blot. Cell viability and apoptosis of HTMCs were measured using CCK-8 and flow cytometry assays. Reactive oxygen species (ROS), superoxide dismutase (SOD), and L-glutathione (GSH) levels were detected to evaluate oxidative stress. Through luciferase reporter assay, the association between PBX1 and NANOG was verified. Results presented that PBX1 was significantly upregulated in H2O2-induced HTMCs. Functionally, PBX1 and NANOG promoted cell viability, inhibited cell apoptosis and ECM deposition, suppressed ROS accumulation, and enhanced the productions of SOD and GSH in H2O2-stimulated HTMCs, while PBX1 inhibition showed the opposite effects. In addition, PBX1 promoted the transcription of NANOG by upregulating the promoter activity of NANOG which activated the PI3K-AKT signaling pathway. What's more, the inhibitions of PI3K-AKT signaling pathway or NANOG reversed the protective effect of PBX1 on H2O2-stimulated HTMCs. In summary, our study firstly revealed that PBX1 attenuated the oxidative damage in HTMCs via regulating NANOG-mediated PI3K/AKT signaling, suggesting that PBX1 might be a potential treatment target for glaucoma patients.