ABSTRACT
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-ß 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.
Subject(s)
Cell Transdifferentiation , Dexamethasone , Glaucoma , Myofibroblasts , Rho Guanine Nucleotide Exchange Factors , Trabecular Meshwork , Dexamethasone/pharmacology , Trabecular Meshwork/drug effects , Trabecular Meshwork/metabolism , Trabecular Meshwork/cytology , Cell Transdifferentiation/drug effects , Animals , Humans , Myofibroblasts/drug effects , Myofibroblasts/metabolism , Myofibroblasts/cytology , Mice , Rho Guanine Nucleotide Exchange Factors/metabolism , Rho Guanine Nucleotide Exchange Factors/genetics , Glaucoma/pathology , Glaucoma/metabolism , Cells, Cultured , Glucocorticoids/pharmacology , Mice, Inbred C57BL , MaleABSTRACT
Members of the Rho family of small monomeric GTPases regulate a plethora of critical cellular functions including gene expression, cell cycle progression, and the dynamic modeling of the actin cytoskeleton. Diversity among Rho family members is derived, in part, from variations in their subcellular distribution. Localization of newly synthesized (naïve) Rho proteins to target subcellular compartments is largely governed by lipid modifications, including posttranslational prenylation. Here, using well-established and widely available contemporary methodologies, detailed protocols by which to semiquantitatively evaluate the functional consequence of posttranslational prenylation in human trabecular meshwork cells are described. We propose the novel concept that posttranslational prenylation itself is a key regulator of mammalian Rho GTPase protein expression and turnover.
Subject(s)
Trabecular Meshwork , Humans , Trabecular Meshwork/metabolism , Trabecular Meshwork/cytology , Cells, Cultured , Terpenes/metabolism , Protein Prenylation , Monomeric GTP-Binding Proteins/metabolism , Monomeric GTP-Binding Proteins/genetics , rho GTP-Binding Proteins/metabolism , rho GTP-Binding Proteins/genetics , Protein Processing, Post-TranslationalABSTRACT
The trabecular meshwork (TM) from primary open-angle glaucoma (POAG) cases has been found to contain decreased levels of intracellular plasmalogens. Plasmalogens are a subset of lipids involved in diverse cellular processes such as intracellular signaling, membrane asymmetry, and protein regulation. Proper plasmalogen biosynthesis is regulated by rate-limiting enzyme fatty acyl-CoA reductase (Far1). ATPase phospholipid transporting 8B2 (ATP8B2) is a type IV P-type ATPase responsible for the asymmetric distribution of plasmalogens between the intracellular and extracellular leaflets of the plasma membranes. Here we describe the methodology for extraction and culturing of TM cells from corneal tissue and subsequent downregulation of ATP8B2 using siRNA transfection. Further quantification and downstream effects of ATP8B2 gene knockdown will be analyzed utilizing immunoblotting techniques.
Subject(s)
Glaucoma, Open-Angle , Plasmalogens , Trabecular Meshwork , Trabecular Meshwork/metabolism , Trabecular Meshwork/cytology , Humans , Plasmalogens/metabolism , Glaucoma, Open-Angle/metabolism , Glaucoma, Open-Angle/genetics , Glaucoma, Open-Angle/pathology , Adenosine Triphosphatases/metabolism , Adenosine Triphosphatases/genetics , RNA, Small Interfering/genetics , Down-Regulation , Cells, Cultured , Gene Knockdown TechniquesABSTRACT
BACKGROUND: Various cell culture platforms that could display native environmental cue-mimicking stimuli were developed, and effects of environmental cues on cell behaviors were studied with the cell culture platforms. Likewise, various cell culture platforms mimicking native trabecular meshwork (TM) composed of juxtacanalicular, corneoscleral and uveal meshwork located in internal scleral sulcus were used to study effects of environmental cues and/or drug treatments on TM cells and glaucoma development. Glaucoma is a disease that could cause blindness, and cause of glaucoma is not clearly identified yet. It appears that aqueous humor (AH) outflow resistance increased by damages on pathway of AH outflow can elevate intraocular pressure (IOP). These overall possibly contribute to development of glaucoma. METHODS: For the study of glaucoma, static and dynamic cell culture platforms were developed. Particularly, the dynamic platforms exploiting AH outflow-mimicking perfusion or increased IOP-mimicking increased pressure were used to study how perfusion or increased pressure could affect TM cells. Overall, potential mechanisms of glaucoma development, TM structures and compositions, TM cell culture platform types and researches on TM cells and glaucoma development with the platforms were described in this review. RESULTS AND CONCLUSION: This will be useful to improve researches on TM cells and develop enhanced therapies targeting glaucoma.
Subject(s)
Cell Culture Techniques , Glaucoma , Trabecular Meshwork , Trabecular Meshwork/cytology , Humans , Cell Culture Techniques/methods , Intraocular Pressure , Aqueous Humor , AnimalsABSTRACT
The progressive degradation in the trabecular meshwork (TM) is related to age-related ocular diseases like primary open-angle glaucoma. However, the molecular basis and biological significance of the aging process in TM have not been fully elucidated. Here, we established a dynamic single-cell transcriptomic landscape of aged macaque TM, wherein we classified the outflow tissue into 12 cell subtypes and identified mitochondrial dysfunction as a prominent feature of TM aging. Furthermore, we divided TM cells into 13 clusters and performed an in-depth analysis on cluster 0, which had the highest aging score and the most significant changes in cell proportions between the two groups. Ultimately, we found that the APOE gene was an important differentially expressed gene in cluster 0 during the aging process, highlighting the close relationship between cell migration and extracellular matrix regulation, and TM function. Our work further demonstrated that silencing the APOE gene could increase migration and reduce apoptosis by releasing the inhibition on the PI3K-AKT pathway and downregulating the expression of extracellular matrix components, thereby increasing the aqueous outflow rate and maintaining intraocular pressure within the normal range. Our work provides valuable insights for future clinical diagnosis and treatment of glaucoma.
Subject(s)
Aging , Single-Cell Analysis , Trabecular Meshwork , Transcriptome , Animals , Aging/genetics , Trabecular Meshwork/metabolism , Trabecular Meshwork/cytology , Apolipoproteins E/genetics , Apolipoproteins E/metabolism , Cell Movement/genetics , Macaca , Apoptosis/geneticsABSTRACT
A major risk factor for glaucoma, the first leading cause of irreversible blindness worldwide, is the decellularisation of the trabecular meshwork (TM) in the conventional outflow pathway. Stem cell-based therapy, particularly the utilisation of induced pluripotent stem cells (iPSCs), presents an enticing potential for tissue regeneration and intraocular pressure (IOP) maintenance in glaucoma. We have previously observed that differentiated iPSCs can stimulate endogenous cell proliferation in the TM, a pivotal factor in TM regeneration and aqueous humour outflow restoration. In this study, we investigated the response of TM cells in vivo after interacting with iPSC-derived cells and identified two subpopulations responsible for this relatively long-term tissue regeneration: ATP Binding Cassette Subfamily G Member 2 (ABCG2)-positive cells and Nestin (NES)-positive cells. We further uncovered that alterations of these responsive cells are linked to ageing and different glaucoma etiologies, suggesting that ABCG2+ subpopulation decellularization could serve as a potential risk factor for TM decellularization in glaucoma. Taken together, our findings illustrated the proliferative subpopulations in the conventional outflow pathway when stimulated with iPSC-derived cells and defined them as TM precursors, which may be applied to develop novel therapeutic approaches for glaucoma.
Subject(s)
ATP Binding Cassette Transporter, Subfamily G, Member 2 , Cell Proliferation , Glaucoma , Induced Pluripotent Stem Cells , Regeneration , Trabecular Meshwork , Induced Pluripotent Stem Cells/metabolism , Induced Pluripotent Stem Cells/cytology , ATP Binding Cassette Transporter, Subfamily G, Member 2/metabolism , Humans , Trabecular Meshwork/metabolism , Trabecular Meshwork/cytology , Glaucoma/metabolism , Glaucoma/pathology , Glaucoma/therapy , Regeneration/physiology , Animals , Nestin/metabolism , Cell Differentiation , Cells, Cultured , Mice , Male , Female , Neoplasm ProteinsABSTRACT
A common adverse response to the clinical use of glucocorticoids (GCs) is elevated intraocular pressure (IOP) which is a major risk factor for glaucoma. Elevated IOP arises due to impaired outflow of aqueous humor (AH) through the trabecular meshwork (TM). Although GC-induced changes in actin cytoskeletal dynamics, contractile characteristics, and cell adhesive interactions of TM cells are believed to influence AH outflow and IOP, the molecular mechanisms mediating changes in these cellular characteristics are poorly understood. Our studies focused on evaluating changes in the cytoskeletal and cytoskeletal-associated protein (cytoskeletome) profile of human TM cells treated with dexamethasone (Dex) using label-free mass spectrometric quantification, identified elevated levels of specific proteins known to regulate actin stress fiber formation, contraction, actin networks crosslinking, cell adhesion, and Wnt signaling, including LIMCH1, ArgBP2, CNN3, ITGBL1, CTGF, palladin, FAT1, DIAPH2, EPHA4, SIPA1L1, and GPC4. Several of these proteins colocalized with the actin cytoskeleton and underwent alterations in distribution profile in TM cells treated with Dex, and an inhibitor of Abl/Src kinases. Wnt/Planar Cell Polarity (PCP) signaling agonists-Wnt5a and 5b were detected prominently in the cytoskeletome fraction of TM cells, and studies using siRNA to suppress expression of glypican-4 (GPC4), a known modulator of the Wnt/PCP pathway revealed that GPC4 deficiency impairs Dex induced actin stress fiber formation, and activation of c-Jun N-terminal Kinase (JNK) and Rho kinase. Additionally, while Dex augmented, GPC4 deficiency suppressed the formation of actin stress fibers in TM cells in the presence of Dex and Wnt5a. Taken together, these results identify the GPC4-dependent Wnt/PCP signaling pathway as one of the crucial upstream regulators of Dex induced actin cytoskeletal reorganization and cell adhesion in TM cells, opening an opportunity to target the GPC4/Wnt/PCP pathway for treatment of ocular hypertension in glaucoma.
Subject(s)
Actins , Cytoskeletal Proteins , Cytoskeleton , Dexamethasone , Glucocorticoids , Glypicans , Trabecular Meshwork , Humans , Actins/metabolism , Cells, Cultured , Cytoskeletal Proteins/deficiency , Cytoskeletal Proteins/genetics , Cytoskeletal Proteins/metabolism , Dexamethasone/pharmacology , Glaucoma/metabolism , Glaucoma/pathology , Glucocorticoids/pharmacology , Glypicans/deficiency , Glypicans/metabolism , Intraocular Pressure , Trabecular Meshwork/cytology , Trabecular Meshwork/drug effects , Trabecular Meshwork/metabolism , Wnt Signaling Pathway/drug effects , Cytoskeleton/metabolism , Cell Polarity/drug effects , rho-Associated Kinases/metabolism , Stress Fibers/drug effects , Cell Adhesion/drug effectsABSTRACT
The trabecular meshwork (TM) is the leading site of aqueous humor outflow in the eye and plays a critical role in maintaining normal intraocular pressure. When the TM fails to maintain normal intraocular pressure, glaucoma may develop. Mitochondrial damage has previously been found in glaucomatous TM cells; however, the precise metabolic activity of glaucomatous TM cells has yet to be quantitatively assessed. Using dexamethasone (Dex) treated primary human TM cells to model glaucomatous TM cells, we measure the respiratory and glycolytic activity of Dex-treated TM cells with an extracellular flux assay. We found that Dex-treated TM cells had quantifiably altered metabolic profiles, including increased spare respiratory capacity and ATP production rate from oxidative phosphorylation. Therefore, we propose that reversing or preventing these metabolic changes may represent an avenue for future research.
Subject(s)
Adenosine Triphosphate/metabolism , Cytoskeletal Proteins/metabolism , Dexamethasone/pharmacology , Eye Proteins/metabolism , Glucocorticoids/pharmacology , Glycoproteins/metabolism , Trabecular Meshwork/drug effects , Aged , Cells, Cultured , Female , Humans , Male , Middle Aged , Tissue Donors , Trabecular Meshwork/cytology , Trabecular Meshwork/metabolismABSTRACT
The human anterior segment perfusion culture model is a valuable tool for studying the trabecular meshwork (TM) and aqueous humor outflow in glaucoma. The traditional model relies on whole eye globes resulting in high cost and limited availability. Here, we developed a glue-based method which enabled us to use human corneal rims for perfusion culture experiments. Human corneal rim perfusion culture plates were 3D printed. Human corneal rims containing intact TM were attached and sealed to the plate using low viscosity and high viscosity glues, respectively. The human corneal rims were perfused using the constant flow mode, and the pressure changes were recorded using a computerized system. Outflow facility, TM stiffness, and TM morphology were evaluated. When perfused at rates from 1.2 to 3.6 µl/min, the outflow facility was 0.359 ± 0.216 µl/min/mmHg among 10 human corneal rims. The stiffness of the TM in naïve human corneal rim was similar to that of perfusion cultured human corneal rim. Also, the stiffness of TM of corneal rims perfused with dexamethasone was significantly higher than the control. Human corneal rims with glue contamination in the TM could be differentiated by high baseline intraocular pressure as well as high TM stiffness. Histology studies showed that the TM tissues perfused with plain medium appeared normal. We believed that our glued-based method is a useful tool and low-cost alternative to the traditional anterior segment perfusion culture model.
Subject(s)
Aqueous Humor/physiology , Cornea/cytology , Models, Biological , Organ Culture Techniques , Trabecular Meshwork/cytology , Elastic Modulus , Humans , Intraocular Pressure/physiology , Microscopy, Atomic Force , Tissue Adhesives , Tissue Donors , Trabecular Meshwork/physiologyABSTRACT
PURPOSE: Primary open-angle glaucoma (POAG) is a complex heterogeneous disease. While several POAG genes have been identified, a high proportion of estimated heritability remains unexplained. Elevated intraocular pressure (IOP) is a leading POAG risk factor and dysfunctional extracellular matrix (ECM) in the trabecular meshwork (TM) contributes to elevated IOP. In this study, we sought to identify missense variants in ECM genes that correlate with ocular hypertensive POAG. METHODS: Whole-genome sequencing was used to identify genetic variants in five members of a large POAG family (n = 68) with elevated IOP. The remaining family members were screened by Sanger sequencing. Unrelated normal (NTM) and glaucomatous (GTM) cells were sequenced for the identified variants. The ECM protein levels were determined by Western immunoblotting and confocal and electron microscopy investigated ECM ultrastructural organization. RESULTS: Three ECM gene variants were significantly associated with POAG or elevated IOP in a large POAG pedigree. These included rs2228262 (N700S; thrombospondin-1 (THBS1, TSP1)), rs112913396 (D563 G; collagen type VI, alpha 3 (COL6A3)) and rs34759087 (E987K; laminin subunit beta 2 (LAMB2)). Screening of unrelated TM cells (n = 27) showed higher prevalence of the THBS1 variant but not the LAMB2 variant, in GTM cells (39%) than NTM cells (11%). The rare COL6A3 variant was not detected. TSP1 protein was upregulated and COL6A3 was down-regulated in TM cells with N700S subject to mechanical stretch, an in vitro method that mimics elevated IOP. Immunofluorescence showed increased TSP1 immunostaining in cell strains with N700S compared to wild-type TM cells. Ultrastructural studies showed ECM disorganization and altered collagen type VI distribution in GTM versus NTM cells. CONCLUSIONS: Our results suggest that missense variants in ECM genes may not cause catastrophic changes to the TM, but over many years, subtle changes in ECM may accumulate and cause structural disorganization of the outflow resistance leading to elevated IOP in POAG patients.
Subject(s)
Aqueous Humor/metabolism , DNA/genetics , Extracellular Matrix Proteins/genetics , Glaucoma, Open-Angle/genetics , Mutation, Missense , Thrombospondin 1/genetics , Trabecular Meshwork/metabolism , Adult , Aged , Blotting, Western , Cells, Cultured , DNA Mutational Analysis , Extracellular Matrix Proteins/metabolism , Female , Glaucoma, Open-Angle/metabolism , Humans , Intraocular Pressure/physiology , Male , Middle Aged , Pedigree , Thrombospondin 1/metabolism , Trabecular Meshwork/cytologyABSTRACT
Effects of a pan-ROCK-inhibitor, ripasudil (Rip), and a ROCK2 inhibitor, KD025 on dexamethasone (DEX)-treated human trabecular meshwork (HTM) cells as a model of steroid-induced glaucoma were investigated. In the presence of Rip or KD025, DEX-treated HTM cells were subjected to permeability analysis of 2D monolayer by transendothelial electrical resistance (TEER) and FITC-dextran permeability, physical properties, size and stiffness analysis (3D), and qPCR of extracellular matrix (ECM), and their modulators. DEX resulted in a significant increase in the permeability, as well as a large and stiff 3D spheroid, and those effects were inhibited by Rip. In contrast, KD025 exerted opposite effects on the physical properties (down-sizing and softening). Furthermore, DEX induced several changes of gene expressions of ECM and their modulators were also modulated differently by Rip and KD025. The present findings indicate that Rip and KD025 induced opposite effects toward 2D and 3D cell cultures of DEX-treated HTM cells.
Subject(s)
Dexamethasone/pharmacology , Protein Kinase Inhibitors/pharmacology , Trabecular Meshwork/cytology , Trabecular Meshwork/drug effects , rho-Associated Kinases/antagonists & inhibitors , Biomarkers , Cell Culture Techniques , Cells, Cultured , Extracellular Matrix/metabolism , Extracellular Matrix Proteins/metabolism , HumansABSTRACT
Primary congenital glaucoma (PCG) is a severe disease characterized by developmental defects in the trabecular meshwork (TM) and Schlemm's canal (SC), comprising the conventional aqueous humor outflow pathway of the eye. Recently, heterozygous loss of function variants in TEK and ANGPT1 or compound variants in TEK/SVEP1 were identified in children with PCG. Moreover, common variants in ANGPT1and SVEP1 have been identified as risk alleles for primary open angle glaucoma (POAG) in GWAS studies. Here, we show tissue-specific deletion of Angpt1 or Svep1 from the TM causes PCG in mice with severe defects in the adjacent SC. Single-cell transcriptomic analysis of normal and glaucomatous Angpt1 deficient eyes allowed us to identify distinct TM and SC cell populations and discover additional TM-SC signaling pathways. Furthermore, confirming the importance of angiopoietin signaling in SC, delivery of a recombinant ANGPT1-mimetic promotes developmental SC expansion in healthy and Angpt1 deficient eyes, blunts intraocular pressure (IOP) elevation and RGC loss in a mouse model of PCG and lowers IOP in healthy adult mice. Our data highlight the central role of ANGPT1-TEK signaling and TM-SC crosstalk in IOP homeostasis and provide new candidates for SC-targeted glaucoma therapy.
Subject(s)
Aqueous Humor/metabolism , Cell Communication/physiology , Glaucoma, Open-Angle/pathology , Glaucoma, Open-Angle/therapy , Angiopoietin-1/administration & dosage , Angiopoietin-1/genetics , Angiopoietin-1/metabolism , Animals , Anterior Chamber/blood supply , Anterior Chamber/cytology , Anterior Chamber/metabolism , Calcium-Binding Proteins/genetics , Calcium-Binding Proteins/metabolism , Cell Adhesion Molecules/genetics , Cell Adhesion Molecules/metabolism , Cell Communication/drug effects , Disease Models, Animal , Endothelial Cells/cytology , Endothelial Cells/metabolism , Gene Expression Profiling , Glaucoma, Open-Angle/genetics , Glaucoma, Open-Angle/metabolism , Intraocular Pressure/drug effects , Intraocular Pressure/genetics , Mice , Mice, Knockout , Neural Crest/cytology , Neural Crest/metabolism , Proteins/genetics , Proteins/metabolism , Recombinant Proteins/administration & dosage , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Signal Transduction/drug effects , Single-Cell Analysis , Trabecular Meshwork/cytology , Trabecular Meshwork/metabolismABSTRACT
Glaucoma is one of the leading causes of vision loss worldwide, characterised with irreversible optic nerve damage and progressive vision loss. Primary open-angle glaucoma (POAG) is a subset of glaucoma, characterised by normal anterior chamber angle and raised intraocular pressure (IOP). Reducing IOP is the main modifiable factor in the treatment of POAG, and the trabecular meshwork (TM) is the primary site of aqueous humour outflow (AH) and the resistance to outflow. The structure and the composition of the TM are key to its function in regulating AH outflow. Dysfunction and loss of the TM cells found in the natural ageing process and more so in POAG can cause abnormal extracellular matrix (ECM) accumulation, increased TM stiffness, and increased IOP. Therefore, repair or regeneration of TM's structure and function is considered as a potential treatment for POAG. Cell transplantation is an attractive option to repopulate the TM cells in POAG, but to develop a cell replacement approach, various challenges are still to be addressed. The choice of cell replacement covers autologous or allogenic approaches, which led to investigations into TM progenitor cells, induced pluripotent stem cells (iPSCs), and mesenchymal stem cells (MSCs) as potential stem cell source candidates. However, the potential plasticity and the lack of definitive cell markers for the progenitor and the TM cell population compound the biological challenge. Morphological and differential gene expression of TM cells located within different regions of the TM may give rise to different cell replacement or regenerative approaches. As such, this review describes the different approaches taken to date investigating different cell sources and their differing cell isolation and differentiation methodologies. In addition, we highlighted how these approaches were evaluated in different animal and ex vivo model systems and the potential of these methods in future POAG treatment.
Subject(s)
Intraocular Pressure/physiology , Trabecular Meshwork/cytology , Animals , Biomarkers/metabolism , Humans , Stem Cells/cytology , Trabecular Meshwork/transplantationABSTRACT
Trabecular meshwork (TM) regulates the intraocular pressure (IOP) through the control of aqueous humor outflow. Previous reports show that TM cells express 11 types of mechanosensitive molecules, including Piezo 1, which sense mechanical stimuli. However, the role of Piezo 1 on TM remains unclear. Thus, in this study, we focused on the Piezo 1 and examined its role in TM cells. Immunostaining showed that Piezo 1 was expressed in mouse TM and human TM cells. Moreover, the eye drops containing Piezo 1 agonist Yoda 1 reduced the IOP in mice, and also reduced fibronectin expression level around the TM. In addition, Piezo 1 activation suppressed human TM cells migration/proliferation, and decreased fibronectin expression level. On the other hand, Piezo 1 activation increased matrix metalloproteinase (MMP)-2 expression responsible for fibronectin degradation. These findings could contribute to the development of new treatments for glaucoma.
Subject(s)
Gene Expression/genetics , Glaucoma/genetics , Glaucoma/therapy , Intraocular Pressure/genetics , Ion Channels/physiology , Animals , Cell Movement/genetics , Cell Proliferation/genetics , Cells, Cultured , Fibronectins/genetics , Fibronectins/metabolism , Humans , Ion Channels/genetics , Ion Channels/metabolism , Male , Matrix Metalloproteinase 2/genetics , Matrix Metalloproteinase 2/metabolism , Mice , Mice, Inbred Strains , Molecular Targeted Therapy , Trabecular Meshwork/cytology , Trabecular Meshwork/metabolismABSTRACT
This study aims to investigate the beneficial effects of exosomes derived from bone marrow mesenchymal stem cells (BMSCs) on trabecular meshwork cells under oxidative stress and predict candidate genes associated with this process. Trabecular meshwork cells were pretreated with BMSC-derived exosomes for 24 h, and exposed to 0.1 mM H2O2 for 6 h. Survival rate of trabecular meshwork cells was measured with CCK-8 assay. Production of intracellular reactive oxygen species (iROS) was measured using a flow cytometer. RT-PCR and ELISA were used to detect mRNA and protein levels of inflammatory cytokines and matrix metalloproteinases (MMPs). Sequencing of RNA and miRNA for trabecular meshwork cells from Exo and control groups was performed on BGISEQ500 platform. Phenotypically, pretreatment of BMSC-derived exosomes improves survival rate of trabecular meshwork cells exposed to H2O2, reduces production of iROS, and inhibits expression of inflammatory cytokines, whereas increases expression of MMPs. There were 23 miRNAs, 307 lncRNAs, and 367 mRNAs differentially expressed between Exo and control groups. Exosomes derived from BMSCs may protect trabecular meshwork cells from oxidative stress. Candidate genes responsible for beneficial effects, such as DIO2 and HMOX1, were predicted.
Subject(s)
Bone Marrow Cells/cytology , Exosomes/genetics , Exosomes/physiology , Mesenchymal Stem Cells/metabolism , Oxidative Stress/genetics , Trabecular Meshwork , Cell Survival , Cytokines/metabolism , Genetic Association Studies , Heme Oxygenase-1 , Humans , Hydrogen Peroxide/adverse effects , Inflammation Mediators/metabolism , Iodide Peroxidase , Matrix Metalloproteinases/metabolism , MicroRNAs/metabolism , Reactive Oxygen Species/metabolism , Trabecular Meshwork/cytology , Iodothyronine Deiodinase Type IIABSTRACT
The inflammatory chemokines, monocyte chemoattractant protein (MCP)-1 and IL-8, are produced by normal trabecular meshwork cells (TM) and elevated in the aqueous humor of primary open angle glaucoma (POAG) and hypertensive anterior uveitis associated with viral infection. However, their role in TM cells and aqueous humor outflow remains unclear. Here, we explored the possible involvement of MCP-1 and IL-8 in the physiology of TM cells in the context of aqueous outflow, and the viral anterior uveitis. We found that the stimulation of human TM cells with MCP-1 and IL-8 induced significant increase in the formation of actin stress fibers and focal adhesions, myosin light chain phosphorylation, and the contraction of TM cells. MCP-1 and IL-8 also demonstrated elevation of extracellular matrix proteins, and the migration of TM cells. When TM cells were infected with HSV-1 and CMV virus, there was a significant increase in cytoskeletal contraction and Rho-GTPase activation. Viral infection of TM cells revealed significantly increased expression of MCP-1 and IL-8. Taken together, these results indicate that MCP-1 and IL-8 induce TM cell contractibility, fibrogenic activity, and plasticity, which are presumed to increase resistance to aqueous outflow in viral anterior uveitis and POAG.
Subject(s)
Chemokine CCL2/metabolism , Eye Infections, Viral/immunology , Interleukin-8/metabolism , Trabecular Meshwork/cytology , Uveitis, Anterior/virology , Adult , Aqueous Humor/immunology , Cell Movement , Cells, Cultured , Cytomegalovirus/pathogenicity , Extracellular Matrix Proteins/metabolism , Eye Infections, Viral/pathology , Herpesvirus 1, Human/pathogenicity , Humans , Middle Aged , Primary Cell Culture , Receptors, CCR2/metabolism , Receptors, Interleukin-8A/metabolism , Trabecular Meshwork/immunology , Trabecular Meshwork/virology , Uveitis, Anterior/immunology , Uveitis, Anterior/pathologyABSTRACT
Activation of autophagy is one of the responses elicited by high intraocular pressure (IOP) and mechanical stretch in trabecular meshwork (TM) cells. However, the mechanosensor and the molecular mechanisms by which autophagy is induced by mechanical stretch in these or other cell types is largely unknown. Here, we have investigated the mechanosensor and downstream signaling pathway that regulate cyclic mechanical stretch (CMS)-induced autophagy in TM cells. We report that primary cilia act as a mechanosensor for CMS-induced autophagy and identified a cross-regulatory talk between AKT1 and noncanonical SMAD2/3 signaling as critical components of primary cilia-mediated activation of autophagy by mechanical stretch. Furthermore, we demonstrated the physiological significance of our findings in ex vivo perfused eyes. Removal of primary cilia disrupted the homeostatic IOP compensatory response and prevented the increase in LC3-II protein levels in response to elevated pressure challenge, strongly supporting a role of primary cilia-mediated autophagy in regulating IOP homeostasis.
Subject(s)
Cilia/metabolism , Proto-Oncogene Proteins c-akt/metabolism , Smad2 Protein/metabolism , Smad3 Protein/metabolism , Trabecular Meshwork/metabolism , Autophagy , Cells, Cultured , Cilia/pathology , Gene Knockdown Techniques , Humans , Intraocular Pressure/physiology , Intravital Microscopy , Mechanotransduction, Cellular/genetics , Ocular Hypertension/pathology , Ocular Hypertension/physiopathology , Primary Cell Culture , Proto-Oncogene Proteins c-akt/genetics , Smad2 Protein/genetics , Smad3 Protein/genetics , Stress, Mechanical , Time-Lapse Imaging , Trabecular Meshwork/cytology , Trabecular Meshwork/pathologyABSTRACT
We previously identified and characterized human trabecular meshwork stem cells (TMSCs) based on high expression of ABCG2/p75 positivity and high nucleus to cytoplasmic ratio. These TMSCs expressing high ABCG2 and p75 were located to the insert region of the human TM. Additionally, we demonstrated an age-related reduction in the TMSC content which was significantly associated with TM cell loss. In continuation, this study was aimed to determine the TMSC content in glaucomatous donor eyes wherein a drastic reduction in TM cellularity has already been reported. Anterior segments from known glaucomatous (n = 6) and age-matched normal (n = 8) donors were dissected into four quadrants. A minimum of three sections from each quadrant were used for histopathological analysis as well as immunostaining. Analysis of hematoxylin and eosin-stained sections from glaucomatous tissues revealed a decrease in total TM cellularity, thickening of trabecular beams, fusion of trabeculae, absence of patent Schlemm's canal compared to age-matched controls. In addition, the TM thickness at various positions of the meshwork and the coronal as well as the meridional diameters of the Schlemm's canal were observed to be significantly reduced in glaucomatous eyes. Further, sections from both the groups were immunostained for universal stem cell marker ABCG2 and neural crest derived stem cell marker p75. The images were acquired using Leica SP8 confocal microscope. Quantification of total TM cellularity based on nuclear counterstain (mean ± SD) using ImageJ identified 69.33 ± 12.77 cells/section in control eyes. In glaucomatous donors, the TM cellularity was found to be reduced significantly to 41.83 ± 9.0 (p = 0.0007). In addition, a reduction in the percentage of TMSCs (cells with high ABCG2 expression and p75 positivity) was evident in glaucomatous donors (0.14 ± 0.17%) compared to age-matched controls (4.73 ± 5.46%) (p = 0.064). Thus, the present study confirmed the significant decline in TM cellularity and a reducing trend in the TMSC content, though this reduction was non-significant in glaucomatous donor eyes. Further studies are essential to elucidate the role of TMSCs in the pathogenesis of primary open angle glaucoma.
Subject(s)
Glaucoma, Open-Angle/pathology , Stem Cells/cytology , Tissue Donors , Trabecular Meshwork/cytology , Adult Stem Cells/cytology , Adult Stem Cells/metabolism , Aged , Aged, 80 and over , Biomarkers , Cell Count , Female , Glaucoma, Open-Angle/metabolism , Humans , Immunohistochemistry , Male , Microscopy, Confocal , Middle Aged , Stem Cells/metabolism , Trabecular Meshwork/metabolismABSTRACT
To elucidate molecular pharmacology of Rho-associated coiled-coil containing protein kinase inhibitors (ROCK-i, Ripasudil and Y27632) on their efficiency for aqueous outflow, 2D or 3D cultures of a human trabecular meshwork (HTM) were prepared in the presence of TGFß2. Those were examined by transendothelial electrical resistance (TEER, 2D), electronic microscopy (EM, 2D and 3D), expression of the extracellular matrix (ECM) including collagen1 (COL1), COL4 and COL6, and fibronectin (FN) by immunolabeling and/or quantitative PCR (3D), and solidity of 3D organoids by a micro-squeezer. TGFß2 significantly increased the TEER values in 2D cultures, and the ECM expression indicated that the 3D organoids assumed a more densely packed shape. ROCK-i greatly reduced the TGFß2-induced enhancement of TEER and the immunolabeled ECM expression of the 3D organoids. In contrast, the mRNA expression of COL1 was increased, and those of COL4 and FN were unchanged. EM revealed that TGFß2 caused the HTM cells to become more compact and abundant ECM deposits within the 3D organoids were observed. These were significantly inhibited by ROCK-i. The dense solids caused by the presence of TGFß2 were significantly suppressed by ROCK-i. Current study indicates that ROCK-i cause beneficial effects toward the spatial configuration of TGFß2-induced HTM 3D organoids.
Subject(s)
Antihypertensive Agents/pharmacology , Glaucoma, Open-Angle/drug therapy , Optic Nerve Diseases/prevention & control , Trabecular Meshwork/drug effects , rho-Associated Kinases/antagonists & inhibitors , Amides/pharmacology , Amides/therapeutic use , Antihypertensive Agents/therapeutic use , Cell Culture Techniques , Cell Line , Glaucoma, Open-Angle/complications , Humans , Intraocular Pressure/drug effects , Isoquinolines/pharmacology , Isoquinolines/therapeutic use , Microscopy, Electron, Scanning , Optic Nerve Diseases/etiology , Organoids/drug effects , Organoids/metabolism , Organoids/ultrastructure , Pyridines/pharmacology , Pyridines/therapeutic use , Spheroids, Cellular , Sulfonamides/pharmacology , Sulfonamides/therapeutic use , Trabecular Meshwork/cytology , Trabecular Meshwork/metabolism , Trabecular Meshwork/ultrastructure , Transforming Growth Factor beta2/metabolism , rho-Associated Kinases/metabolismABSTRACT
Aberrant remodeling of trabecular meshwork (TM) extracellular matrix (ECM) may induce ocular hypertensive phenotypes in human TM (hTM) cells to cause ocular hypertension, via a yet unknown mechanism. Here, we show that, in the absence of exogenous transforming growth factor-beta2 (TGFß2), compared with control matrices (VehMs), glucocorticoid-induced cell-derived matrices (GIMs) trigger non-Smad TGFß2 signaling in hTM cells, correlated with overexpression/activity of structural ECM genes (fibronectin, collagen IV, collagen VI, myocilin), matricellular genes (connective tissue growth factor [CTGF], secreted protein, acidic and rich in cysteine), crosslinking genes/enzymes (lysyl oxidase, lysyl oxidase-like 2-4, tissue transglutaminase-2), and ECM turnover genes/enzymes (matrix metalloproteinases-MMP2,14 and their inhibitors-TIMP2). However, in the presence of exogenous TGFß2, VehMs and GIMs activate Smad and non-Smad TGFß2 signaling in hTM cells, associated with overexpression of α-smooth muscle actin (α-SMA), and differential upregulation of aforementioned ECM genes/proteins with new ones emerging (collagen-I, thrombospondin-I, plasminogen activator inhibitor, MMP1, 9, ADAMTS4, TIMP1); with GIM-TGFß2-induced changes being mostly more pronounced. This suggests dual glaucomatous insults potentiate profibrotic signaling/phenotypes. Lastly, we demonstrate type I TGFß receptor kinase inhibition abrogates VehM-/GIM- and/or TGFß2-induced upregulation of α-SMA and CTGF. Collectively, pathological TM microenvironments are sufficient to elicit adverse cellular responses that may be ameliorated by targeting TGFß2 pathway.