Human Trabecular Meshwork Cells Research Articles

The effects of exosomes derived from trabecular meshwork cells on Schlemm\u2019s canal endothelial cells

Trabecular meshwork (TM) and Schlemm’s canal (SC) are the main structures within the conventional outflow pathway, and TM cells and SC endothelial (SCE) cells are essential for controlling intraocular pressure. To examine the interaction between TM cells and SCE cells, we investigated whether exosomes contribute to intercellular communication. Additionally, TM cells in glaucoma acquire mesenchymal characteristics in response to transforming growth factor (TGF)-β2 and extracellular matrix proteins such as collagen type 1 (Col-1); these changes result in increased resistance of aqueous outflow. In this study, we stimulated TM cells with TGF-β2 and Col-1 and characterized the exosomal miRNAs (exomiRs) released in response to each stimulus. Isolated exosomes were rich in miRNAs, with downregulated miR-23a-5p and upregulated miR-3942-5p and miR-7515 levels following Col-1 or TGF-β2 stimulation. Next, a miRNA-mRNA network under TGF-β2 stimulation was constructed. There were no connections among the 3 miRNAs and predicted genes under Col-1 stimulation. GO and KEGG analyses revealed that the identified miRNAs were associated with various signaling pathways, including the inflammatory response. Interestingly, SCE cells treated with miR-7515 mimic showed increased VEGFA, VEGFR2, PECAM, and Tie2 expression. Ultrastructures typical of exosomes and positive staining for exosomal markers were observed in human TM cells. Our data showed that TM cells may communicate with SCE cells via exomiRs and that miR-7515 may be important for SCE cell reprogramming.

Autotaxin May Have Lysophosphatidic Acid-Unrelated Effects on Three-Dimension (3D) Cultured Human Trabecular Meshwork (HTM) Cells.

Purpose: The objective of the current study was to evaluate the effects of the autotaxin (ATX)–lysophosphatidic acid (LPA) signaling axis on the human trabecular meshwork (HTM) in two-dimensional (2D) and three-dimensional (3D) cultures of HTM cells. Methods: The effects were characterized by transendothelial electrical resistance (TEER) and FITC-dextran permeability (2D), measurements of size and stiffness (3D), and the expression of several genes, including extracellular matrix (ECM) molecules, their modulators, and endoplasmic reticulum (ER) stress-related factors. Results: A one-day exposure to 200 nM LPA induced significant down-sizing effects of the 3D HTM spheroids, and these effects were enhanced slightly on longer exposure. The TEER and FITC-dextran permeability data indicate that LPA induced an increase in the barrier function of the 2D HTM monolayers. A one-day exposure to a 2 mg/L solution of ATX also resulted in a significant decrease in the sizes of the 3D HTM spheroids, and an increase in stiffness was also observed. The gene expression of several ECMs, their regulators and ER-stress related factors by the 3D HTM spheroids were altered by both ATX and LPA, but in different manners. Conclusions: The findings presented herein suggest that ATX may have additional roles in the human TM, in addition to the ATX–LPA signaling axis.

Pan-ROCK and ROCK2 Inhibitors Affect Dexamethasone-Treated 2D- and 3D-Cultured Human Trabecular Meshwork (HTM) Cells in Opposite Manners.

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.

TRPV4 is activated by mechanical stimulation to induce prostaglandins release in trabecular meshwork, lowering intraocular pressure.

Trabecular meshwork constitutes the conventional outflow pathway and controls intraocular pressure by regulating aqueous outflow. Mechanical stimulation has been studied as one of the triggers to regulate aqueous outflow in trabecular meshwork, but it is not well understood. We investigated that how transient receptor potential cation channel subfamily V member 4 (TRPV4) functions in human trabecular meshwork cells (HTMC) and affects intraocular pressure (IOP). HTMC were treated with TRPV4 siRNA, followed by incubation for 24 hours. We confirmed the suppression of TRPV4 mRNA expression and the reduction of Ca2+ influx by the TRPV4 agonist GSK1016790A in TRPV4 siRNA-treated HTMC. TRPV4 siRNA-treated HTMC exhibited a significant reduction in Ca2+ influx and production of arachidonic acid and prostaglandin (PG) E2 induced by mechanical stretch, and direct activation of TRPV4 by GSK1016790A increased production of arachidonic acid, PGE2, and PGD2 and inhibited gel contraction. Furthermore, TRPV4-deficient mice had higher IOP than wild-type mice, and GSK1016790A administration lowered IOP. These results suggest that TRPV4 mediates the cellular response induced by trabecular meshwork stretch, leading to IOP reduction through the production of prostaglandins and inhibition of cell contraction. Targeting TRPV4 may have therapeutic benefits that lead to lowering IOP in glaucoma patients.

Long Non-coding RNA GAS5 Knockdown Attenuates H2O2-Induced Human Trabecular Meshwork Cell Apoptosis and Promotes Extracellular Matrix Deposition by Suppressing miR-29b-3p and Upregulating STAT3.

The long non-coding RNA GAS5 (GAS5) is reportedly implicated in glaucoma. However, its significance in human trabecular meshwork cells (HTMCs) remains largely unclear. Here, we investigated the effect of GAS5 on the function of HTMCs and its interaction with miR-29b-3p in HTMCs. We established an H2O2-induced oxidative injury model using HTMCs. RT-qPCR or western blotting was performed to examine the expression of the indicated genes. Luciferase reporter assay was used to determine the interaction between GAS5, miR-29b-3p, miR-29b-3p, and STAT3. CCK8 assay was used to assess the proliferative rate of HTMCs. Exposure to H2O2 increased the expression of Bax, cleaved caspase-3, and extracellular matrix (ECM) proteins, accompanied by reduced Bcl-2 expression. These H2O2-induced changes were effectively alleviated by GAS5 knockdown with sh-GAS5. MiR-29b-3p was directly regulated by GAS5. The effect of sh-GAS5 on ECM protein expression was also observed with the miR-29b-3p mimic. STAT3 was directly regulated by miR-29b-3p. MiR-29b-3p silencing alleviated STAT3 inhibition, followed by the restoration of cell vitality, Bax, Bcl-2, and cleaved caspase-3 expression, and ECM deposition. Our study is the first experimental investigation to shed light on a novel molecular mechanism of the GAS5/miR-29b-3p/STAT3 axis in an H2O2-induced oxidative injury model using HTMCs, which may offer a promising therapeutic approach against glaucoma.

Establishment of appropriate glaucoma models using dexamethasone or TGF\u03b22 treated three-dimension (3D) cultured human trabecular meshwork (HTM) cells

To establish appropriate ex vivo models for a glaucomatous trabecular meshwork (TM), two-dimensional (2D) and three-dimensional (3D) cultures of human trabecular meshwork cells (HTM) were prepared in the presence of 250 nM dexamethasone (DEX) or 5 ng/mL TGFβ2, and characterized by the following analyses; transendothelial electrical resistance (TEER) measurements, FITC dextran permeability, scanning electron microscopy and the expression of the extracellular matrix (ECM) including collagen (COL)1, 4 and 6, and fibronectin (FN), α-smooth muscle actin (α-SMA), tissue inhibitor of metalloproteinase (TIMP)1–4, and matrix metalloproteinase (MMP)2, 9 and 14. DEX and TGFβ2 both caused a significant increase or decrease in the TEER values and FITC dextran permeability. During the 3D spheroid culture, DEX or TGFβ2 induced a mild and significant down-sizing and an increase in stiffness, respectively. TGFβ2 induced a significant up-regulation of COL1 and 4, FN, α-SMA, and MMP 2 and 14 (2D) or COL1 and 6, and TIMP2 and 3 (3D), and DEX induced a significant up-regulation of FN (3D) and TIMP4 (2D and 3D). The findings presented herein indicate that DEX or TGFβ2 resulted in mild and severe down-sized and stiff 3D HTM spheroids, respectively, thus making them viable in vitro HTM models for steroid-induced and primary open angle glaucoma.

Identification of Estrogen Signaling in a Prioritization Study of Intraocular Pressure-Associated Genes.

Elevated intraocular pressure (IOP) is the only modifiable risk factor for primary open-angle glaucoma (POAG). Herein we sought to prioritize a set of previously identified IOP-associated genes using novel and previously published datasets. We identified several genes for future study, including several involved in cytoskeletal/extracellular matrix reorganization, cell adhesion, angiogenesis, and TGF-β signaling. Our differential correlation analysis of IOP-associated genes identified 295 pairs of 201 genes with differential correlation. Pathway analysis identified β-estradiol as the top upstream regulator of these genes with ESR1 mediating 25 interactions. Several genes (i.e., EFEMP1, FOXC1, and SPTBN1) regulated by β-estradiol/ESR1 were highly expressed in non-glaucomatous human trabecular meshwork (TM) or Schlemm’s canal (SC) cells and specifically expressed in TM/SC cell clusters defined by single-cell RNA-sequencing. We confirmed ESR1 gene and protein expression in human TM cells and TM/SC tissue with quantitative real-time PCR and immunofluorescence, respectively. 17β-estradiol was identified in bovine, porcine, and human aqueous humor (AH) using ELISA. In conclusion, we have identified estrogen receptor signaling as a key modulator of several IOP-associated genes. The expression of ESR1 and these IOP-associated genes in TM/SC tissue and the presence of 17β-estradiol in AH supports a role for estrogen signaling in IOP regulation.

Sodium 4-Phenylbutyrate Reduces Ocular Hypertension by Degrading Extracellular Matrix Deposition via Activation of MMP9.

Ocular hypertension (OHT) is a serious adverse effect of the widely prescribed glucocorticoid (GC) therapy and, if left undiagnosed, it can lead to glaucoma and complete blindness. Previously, we have shown that the small chemical chaperone, sodium-4-phenylbutyrate (PBA), rescues GC-induced OHT by reducing ocular endoplasmic reticulum (ER) stress. However, the exact mechanism of how PBA rescues GC-induced OHT is not completely understood. The trabecular meshwork (TM) is a filter-like specialized contractile tissue consisting of TM cells embedded within extracellular matrix (ECM) that controls intraocular pressure (IOP) by constantly regulating aqueous humor (AH) outflow. Induction of abnormal ECM deposition in TM is a hallmark of GC-induced OHT. Here, we investigated whether PBA reduces GC-induced OHT by degrading abnormal ECM deposition in TM using mouse model of GC-induced OHT, ex vivo cultured human TM tissues and primary human TM cells. We show that topical ocular eye drops of PBA (1%) significantly lowers elevated IOP in mouse model of GC-induced OHT. Importantly, PBA prevents synthesis and deposition of GC-induced ECM in TM. We report for the first time that PBA can degrade existing abnormal ECM in normal human TM cells/tissues by inducing matrix metalloproteinase (MMP)9 expression and activity. Furthermore, inhibition of MMPs activity by chemical-inhibitor (minocycline) abrogated PBA’s effect on ECM reduction and its associated ER stress. Our study indicates a non-chaperone activity of PBA via activation of MMP9 that degrades abnormal ECM accumulation in TM.

Dexamethasone induced glaucoma associated features exhibited differentially in retinal ganglion cells and human trabecular meshwork cells

The Journal of the National Science Foundation of Sri Lanka publishes the results of research in all aspects of Science and Technology. The journal also has a website at http://www.nsf.gov.lk/. 2020 Impact Factor: 0.515The JNSF provides immediate open access to its content on the principle that making research freely available to the public supports a greater global exchange of knowledge.

Using CRISPR Interference as a Therapeutic Approach to Treat TGFβ2-Induced Ocular Hypertension and Glaucoma.

PurposePrimary open angle glaucoma (POAG) is a leading cause of blindness worldwide with elevated intraocular pressure (IOP) as the most important risk factor. POAG IOP elevation is due to pathological changes in the trabecular meshwork (TM). Elevated TGFβ2 contributes to these changes and increases IOP. We have shown that histone hyperacetylation is associated with TGFβ2 elevation in the TM. In this study, we determined if clustered regularly interspaced short palindromic repeats (CRISPR) interference could specifically deacetylate histones and decrease TGFβ2 in the TM.MethodsWe tested the efficiency of different promoters in driving KRAB-dCAS9 expression in human TM cells. We also screened and determined the optimal sgRNA sequence in the inhibition of TGFβ2. Chromatin immunoprecipitation-qPCR was used to determine the binding of KRAB-dCAS9. An adenovirus-mediated TGFβ2-induced ocular hypertension (OHT) mouse model was used to determine the effect of the CRISPR interference system in vivo.ResultsWe found that the CRISPR interference system inhibited TGFβ2 expression in human TM cells, and properly designed sgRNA targeted the promoter of the TGFβ2 gene. Using sgRNA targeting the CMV promoter of the Ad5-CMV-TGFβ2 viral vector, we found that lentivirus-mediated KRAB-dCAS9 and sgRNA expression was able to inhibit Ad5-CMV-TGFβ2-induced OHT in C57BL/6J female and male mice eyes. This inhibition of OHT was associated with decreased levels of TGFβ2 and extracellular matrix proteins in the mouse eye.ConclusionsOur results indicate that CRISPR interference is a useful tool for gene inhibition and may be a therapeutic approach to treat TGFβ2-induced OHT.

RhoA/ROCK-YAP/TAZ Axis Regulates the Fibrotic Activity in Dexamethasone-Treated Human Trabecular Meshwork Cells.

High intraocular pressure (IOP) is a major risk factor for glaucoma, a leading cause of irreversible blindness. Abnormal fibrotic activity in the human trabecular meshwork (HTM) cells is considered to be partly responsible for the increased resistance of aqueous humor outflow and IOP. This study aimed to identify the fibrotic pathways using integrated bioinformatics and further elucidate their mechanism of regulating fibrotic activity in dexamethasone (DEX)-treated HTM cells. Microarray datasets from the GEO database were obtained and analyzed by GEO2R. Bioinformatics analyses, including GO and KEGG analyses, were performed to explore biological functions and signaling pathways of differentially expressed genes (DEGs). The fibrotic pathways and targets were determined by western blot, RT-qPCR, or immunofluorescence staining. The cellular elastic modulus was measured using an atomic force microscope. A total of 204 DEGs, partly enriched in fibrotic activity (collagen-containing ECM, fibroblast activation) and Rap1, Ras, TGF-β, and Hippo pathways, were identified. Experimental results showed that DEX induced fibrotic activity and regulated the expression of RhoA/ROCK in HTM cells. Similarly, the constitutively active RhoA (RhoAG14V) also promoted the fibrotic activity of HTM cells. Mechanistically, RhoAG14V induced the expression and nuclear translocation of YAP/TAZ to produce CTGF. Moreover, inhibition of ROCK or YAP decreased the expression of Collagen I and α-SMA proteins induced by DEX or RhoAG14V in HTM cells. In conclusion, these results indicate that RhoA/ROCK-YAP/TAZ axis plays a crucial role in regulating the fibrotic activity of DEX-treated HTM cells.

The RNA-binding protein and stress granule component ATAXIN-2 is expressed in mouse and human tissues associated with glaucoma pathogenesis.

Polyglutamine repeat expansions in the Ataxin-2 (ATXN2) gene were first implicated in Spinocerebellar Ataxia Type 2, a disease associated with degeneration of motor neurons and Purkinje cells. Recent studies linked single nucleotide polymorphisms in the gene to elevated intraocular pressure in primary open angle glaucoma (POAG); yet, the localization of ATXN2 across glaucoma-relevant tissues of the vertebrate eye has not been thoroughly examined. This study characterizes ATXN2 expression in the mouse and human retina, and anterior eye, using an antibody validated in ATXN2-/- retinas. ATXN2-ir was localized to cytosolic sub compartments in retinal ganglion cell (RGC) somata and proximal dendrites in addition to GABAergic, glycinergic, and cholinergic amacrine cells in the inner plexiform layer (IPL) and displaced amacrine cells. Human, but not mouse retinas showed modest immunolabeling of bipolar cells. ATXN2 immunofluorescence was prominent in the trabecular meshwork and pigmented and nonpigmented cells of the ciliary body, with analyses of primary human trabecular meshwork cells confirming the finding. The expression of ATXN2 in key POAG-relevant ocular tissues supports the potential role in autophagy and stress granule formation in response to ocular hypertension.

Suberoylanilide hydroxamic acid (SAHA) inhibits transforming growth factor-beta 2-induced increases in aqueous humor outflow resistance

Transforming growth factor-beta 2 (TGF-β2) is highly concentrated in the aqueous humor of primary open-angle glaucoma patients. TGF-β2 causes fibrosis of outflow tissues, such as the trabecular meshwork (TM), and increases intraocular pressure by increasing resistance to aqueous humor outflow. Recently, histone deacetylase (HDAC) activity was investigated in fibrosis in various tissues, revealing that HDAC inhibitors suppress tissue fibrosis. However, the effect of HDAC inhibitors on fibrosis in the eye was not determined. Here, we investigated the effect of suberoylanilide hydroxamic acid (SAHA), an HDAC inhibitor, on TGF-β2-induced increased resistance to aqueous humor outflow. We found that SAHA suppressed TGF-β2-induced outflow resistance in perfused porcine eyes. Moreover, SAHA cotreatment suppressed TGF-β2-induced ocular hypertension in rabbits. The permeability of monkey TM (MTM) and Schlemm’s canal (MSC) cell monolayers was decreased by TGF-β2 treatment. SAHA inhibited the effects of TGF-β2 on the permeability of these cells. TGF-β2 also increased the expression of extracellular matrix proteins (fibronectin and collagen type I or IV) in MTM, MSC, and human TM (HTM) cells, while SAHA inhibited TGF-β2-induced extracellular matrix protein expression in these cells. SAHA also inhibited TGF-β2-induced phosphorylation of Akt and ERK, but did not inhibit Smad2/3 phosphorylation, the canonical pathway of TGF-β signaling. Moreover, SAHA induced the expression of phosphatase and tensin homolog, a PI3K/Akt signaling factor, as well as bone morphogenetic protein 7, an endogenous antagonist of TGF-β. These results imply that SAHA prevents TGF-β2-induced increases in outflow resistance and regulates the non-Smad pathway of TGF-β signaling in TM and MSC cells.

Effects of selective EP2 receptor agonist, omidenepag, on trabecular meshwork cells, Schlemm\u2019s canal endothelial cells and ciliary muscle contraction

This study investigated the effects of omidenepag (OMD), a novel selective EP2 receptor agonist, on human trabecular meshwork (HTM) cells, monkey Schlemm’s canal endothelial (SCE) cells, and porcine ciliary muscle (CM) to clarify the mechanism of intraocular pressure (IOP) reduction involving conventional outflow pathway. In HTM and SCE cells, the effects of OMD on transforming growth factor-β2 (TGF-β2)-induced changes were examined. The expression of actin cytoskeleton and extracellular matrix (ECM) proteins, myosin light chain (MLC) phosphorylation in HTM cells were evaluated using real-time quantitative PCR, immunocytochemistry, and western blotting. The expression of barrier-related proteins, ZO-1 and β-catenin, and permeability of SCE cells were evaluated using immunocytochemistry and transendothelial electrical resistance. The CM contraction was determined by contractibility assay. OMD significantly inhibited expression of TGF-β2 induced mRNA, protein, and MLC-phosphorylation on cytoskeletal and ECM remodeling in the HTM dose dependently. In SCE cells, OMD suppressed TGF-β2-induced expression of the barrier-related proteins and decreased SCE monolayer permeability. OMD at 3 µM significantly inhibited CM contraction, however, the effect was not significant at lower concentrations. IOP lowering effect of OMD through conventional outflow pathway is exerted by increasing outflow facilities with the modulation of TM cell fibrosis and SCE cell permeability.

Recombinant antibodies recognize conformation-dependent epitopes of the leucine zipper of misfolding-prone myocilin

Recombinant antibodies with well-characterized epitopes and known conformational specificities are critical reagents to support robust interpretation and reproducibility of immunoassays across biomedical research. For myocilin, a protein prone to misfolding that is associated with glaucoma and an emerging player in other human diseases, currently available antibodies are unable to differentiate among the numerous disease-associated protein states. This fundamentally constrains efforts to understand the connection between myocilin structure, function, and disease. To address this concern, we used protein engineering methods to develop new recombinant antibodies that detect the N-terminal leucine zipper structural domain of myocilin and that are cross-reactive for human and mouse myocilin. After harvesting spleens from immunized mice and in vitro library panning, we identified two antibodies, 2A4 and 1G12. 2A4 specifically recognizes a folded epitope while 1G12 recognizes a range of conformations. We matured antibody 2A4 for improved biophysical properties, resulting in variant 2H2. In a human IgG1 format, 2A4, 1G12, and 2H2 immunoprecipitate full-length folded myocilin present in the spent media of human trabecular meshwork (TM) cells, and 2H2 can visualize myocilin in fixed human TM cells using fluorescence microscopy. These new antibodies should find broad application in glaucoma and other research across multiple species platforms.

Downregulation of secreted protein acidic and rich in cysteine in human trabecular meshwork cells.

Secreted protein acidic and rich in cysteine (SPARC) may play a notable role in aqueous humor outflow through the trabecular meshwork (TM). SPARC is a potential therapeutic target in glaucoma, and the mechanism by which it regulates intraocular pressure remains unclear. The present study aimed to observe the effects of SPARC in human TM cells (HTMCs) in vitro. SPARC was downregulated by recombinant lentiviral vectors in HTMCs, and the subsequent levels of F-actin expression, zonula occludens-1 (ZO-1) expression and cellular phagocytosis were observed and calculated. It was revealed that after 48 h of culture, the expression levels of SPARC, F-actin and ZO-1 were significantly decreased in the lentivirus group compared with those in the blank control and empty vector control groups. The downregulation of SPARC promoted phagocytosis in HTMCs after 24 or 48 h of culture. This indicated that the downregulation of SPARC decreased the expression levels of the cytoskeleton-associated proteins F-actin and ZO-1, promoted phagocytosis in HTMCs and may affect the outflow of aqueous humor via the TM pathway.

Screening of the Drug-Induced Effects of Prostaglandin EP2 and FP Agonists on 3D Cultures of Dexamethasone-Treated Human Trabecular Meshwork Cells.

The objective of the current study was to perform a screening of the drug-induced effects of the prostaglandin F2α (PGF2α) and EP2 agonist, omidenepag (OMD), using two- and three-dimensional (2D and 3D) cultures of dexamethasone (DEX)-treated human trabecular meshwork (HTM) cells. The drug-induced effects on 2D monolayers were characterized by measuring the transendothelial electrical resistance (TEER) and fluorescein isothiocyanate (FITC)–dextran permeability, the physical properties of 3D spheroids, and the gene expression of extracellular matrix (ECM) molecules, including collagen (COL) 1, 4 and 6, and fibronectin (FN), α smooth muscle actin (αSMA), a tissue inhibitor of metalloproteinase (TIMP) 1–4, matrix metalloproteinase (MMP) 2, 9 and 14 and endoplasmic reticulum (ER) stress-related factors. DEX induced a significant increase in TEER values and a decrease in FITC–dextran permeability, respectively, in the 2D HTM monolayers, and these effects were substantially inhibited by PGF2α and OMD. Similarly, DEX also caused decreased sizes and an increased stiffness in the 3D HTM spheroids, but PGF2α or OMD had no effects on the stiffness of the spheroids. Upon exposure to DEX, the following changes were observed: the upregulation of COL4 (2D), αSMA (2D), and TIMP4 (2D and 3D) and the downregulation of TIMP1 and 2 (3D), MMP2 and 14 (3D), inositol-requiring enzyme 1 (IRE1), activating transcription factor 6 (ATF6) (2D), and glucose regulator protein (GRP)78 (3D). In the presence of PGF2α or OMD, the downregulation of COL4 (2D), FN (3D), αSMA (2D), TIMP3 (3D), MMP9 (3D) and the CCAAT/enhancer-binding protein homologous protein (CHOP) (2D), and the upregulation of TIMP4 (2D and 3D), MMP2, 9 and 14 (2D), respectively, were observed. The findings presented herein suggest that 2D and 3D cell cultures can be useful in screening for the drug-induced effects of PGF2α and OMD toward DEX-treated HTM cells.

Overexpression and Activation of αvβ3 Integrin Differentially Affects TGFβ2 Signaling in Human Trabecular Meshwork Cells.

Studies from our laboratory have suggested that activation of αvβ3 integrin-mediated signaling could contribute to the fibrotic-like changes observed in primary open angle glaucoma (POAG) and glucocorticoid-induced glaucoma. To determine how αvβ3 integrin signaling could be involved in this process, RNA-Seq analysis was used to analyze the transcriptomes of immortalized trabecular meshwork (TM) cell lines overexpressing either a control vector or a wild type (WT) or a constitutively active (CA) αvβ3 integrin. Compared to control cells, hierarchical clustering, PANTHER pathway and protein-protein interaction (PPI) analysis of cells overexpressing WT-αvβ3 integrin or CA-αvβ3 integrin resulted in a significant differential expression of genes encoding for transcription factors, adhesion and cytoskeleton proteins, extracellular matrix (ECM) proteins, cytokines and GTPases. Cells overexpressing a CA-αvβ3 integrin also demonstrated an enrichment for genes encoding proteins found in TGFβ2, Wnt and cadherin signaling pathways all of which have been implicated in POAG pathogenesis. These changes were not observed in cells overexpressing WT-αvβ3 integrin. Our results suggest that activation of αvβ3 integrin signaling in TM cells could have significant impacts on TM function and POAG pathogenesis.

Diverse effects of pan-ROCK and ROCK2 inhibitors on 2 D and 3D cultured human trabecular meshwork (HTM) cells treated with TGF\u03b22

A pan-ROCK-inhibitor, ripasudil (Rip), and a ROCK2 inhibitor, KD025, were used To study the effects of Rho-associated coiled-coil containing protein kinase (ROCK)1 and 2 on two-dimensional (2D) and three-dimensional (3D) cultures of a TGFβ2-treated human trabecular meshwork (HTM) cells. In the presence of 5 ng/mL TGFβ2, the effects of these inhibitors were characterized by transendothelial electrical resistance (TEER), FITC-dextran permeability, and the size and stiffness of 3D sphenoids, the expression of extracellular matrix (ECM) including collagen1, 4 and 6, and fibronectin, α-smooth muscle actin, a tissue inhibitor of metalloproteinase (TIMP)1–4, and matrix metalloproteinase (MMP)2, 9 and 14. TGFβ2 caused a significant increase in the TEER values, and decrease in FITC-dextran permeability, as well as a decrease in the sizes and stiffness of the 3D sphenoids. In the presence of ROCK inhibitors, the TGFβ2-induced effects of the TEER and FITC-dextran permeability were inhibited, especially by KD025. Rip induced a significant increase in sizes and a decrease in the stiffness of the TGFβ2-treated 3D sphenoids, although the effects of KD025 were weaker. Gene expressions of most of the ECMs, TIMP2 and MMP9 of 2D and 3D HTM cells were significantly up-regulated by TGFβ2. Those were significantly and differently modulated by Rip or KD025.

Role of MCP-1 and IL-8 in viral anterior uveitis, and contractility and fibrogenic activity of trabecular meshwork cells

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.