Establishment of appropriate glaucoma models using dexamethasone or TGFβ2 treated three-dimension (3D) cultured human trabecular meshwork (HTM) cells.
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ABSTRACT:
SUBMITTER: Watanabe M
PROVIDER: S-EPMC8481525 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description: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. 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. 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. The findings presented herein suggest that ATX may have additional roles in the human TM, in addition to the ATX-LPA signaling axis.
| S-EPMC8584821 | biostudies-literature
Project description: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.
| S-EPMC8316578 | biostudies-literature
Project description: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.
| S-EPMC8587022 | biostudies-literature
Project description: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 TGFβ2. 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. TGFβ2 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 TGFβ2-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 TGFβ2 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 TGFβ2 were significantly suppressed by ROCK-i. Current study indicates that ROCK-i cause beneficial effects toward the spatial configuration of TGFβ2-induced HTM 3D organoids.
| S-EPMC7680137 | biostudies-literature
Project description:PurposeTreatment with corticosteroids can result in ocular hypertension and may lead to the development of steroid-induced glaucoma. The extent to which biomechanical changes in trabecular meshwork (TM) cells and extracellular matrix (ECM) contribute toward this dysfunction is poorly understood.MethodsPrimary human TM (HTM) cells were cultured for either 3 days or 4 weeks in the presence or absence of dexamethasone (DEX), and cell mechanics, matrix mechanics and proteomics were determined, respectively. Adult rabbits were treated topically with either 0.1% DEX or vehicle over 3 weeks, and mechanics of the TM were determined.ResultsTreatment with DEX for 3 days resulted in a 2-fold increase in HTM cell stiffness, and this correlated with activation of extracellular signal-related kinase 1/2 (ERK1/2) and overexpression of ?-smooth muscle actin (?SMA). Further, the matrix deposited by HTM cells chronically treated with DEX is approximately 4-fold stiffer, more organized, and has elevated expression of matrix proteins commonly implicated in glaucoma (decorin, myocilin, fibrillin, secreted frizzle-related protein [SFRP1], matrix-gla). Also, DEX treatment resulted in a 3.5-fold increase in stiffness of the rabbit TM.DiscussionThis integrated approach clearly demonstrates that DEX treatment increases TM cell stiffness concurrent with elevated ?SMA expression and activation of the mitogen-activated protein kinase (MAPK) pathway, stiffens the ECM in vitro along with upregulation of Wnt antagonists and fibrotic markers embedded in a more organized matrix, and increases the stiffness of TM tissues in vivo. These results demonstrate glucocorticoid treatment can initiate the biophysical alteration associated with increased resistance to aqueous humor outflow and the resultant increase in IOP.
| S-EPMC4509060 | biostudies-literature
Project description:Alterations in stiffness of the trabecular meshwork (TM) may play an important role in primary open-angle glaucoma (POAG), the second leading cause of blindness. Specifically, certain data suggest an association between elevated intraocular pressure (IOP) and increased TM stiffness; however, the underlying link between TM stiffness and IOP remains unclear and requires further study. We here first review the literature on TM stiffness measurements, encompassing various species and based on a number of measurement techniques, including direct approaches such as atomic force microscopy (AFM) and uniaxial tension tests, and indirect methods based on a beam deflection model. We also briefly review the effects of several factors that affect TM stiffness, including lysophospholipids, rho-kinase inhibitors, cytoskeletal disrupting agents, dexamethasone (DEX), transforming growth factor-?2 (TGF-?2), nitric oxide (NO) and cellular senescence. We then describe a method we have developed for determining TM stiffness measurement in mice using a cryosection/AFM-based approach, and present preliminary data on TM stiffness in C57BL/6J and CBA/J mouse strains. Finally, we investigate the relationship between TM stiffness and outflow facility between these two strains. The method we have developed shows promise for further direct measurements of mouse TM stiffness, which may be of value in understanding mechanistic relations between outflow facility and TM biomechanical properties.
| S-EPMC6501821 | biostudies-literature
Project description:It has been observed in several tissues that direct isolation of cells in serum-free media and on nonadhesive substrates results in the formation of spherical clusters of cells known as free-floating spheres. Such free-floating spheres have been hypothesized to contain undifferentiated multipotent progenitor cells. Our goal was to isolate and characterize such free-floating spheres from HTM cell primary cultures. For this purpose, HTM cells were incubated in serum-free media and on a nonadhesive substrate. Individual free-floating spheres generated in these conditions were isolated in 96-well plates, and their proliferative capacity was evaluated by monitoring their size increase over time. The expression of the TM markers, MGP and CHI3L1, was examined using recombinant adenoviruses containing the respective promoters. Morphology of the free-floating spheres was analysed in semithin sections, and the gene expression profile was obtained using Human Genome U133 Plus 2.0 Affymetrix microarrays. HTM cells incubated in serum-free media and on nonadhesive substrate generated free-floating spheres that could be grown for more than 3 months. Addition of serum to the culture media promoted the attachment of the spheres to the substrate, migration of cells from the spheres, and differentiation into cells phenotypically similar to normal TM cells. Gene profiling analysis demonstrated strong similarities between the gene expression profiles of the spheres and HTM cell monolayers. Both infection with the recombinant adenoviruses and gene array analysis demonstrated the expression of CHI3L1 and MGP, indicating that free-floating spheres likely originate from HTM cells. Gene array analysis also showed expression of the marker for neural precursor cells nestin, as well as leukemia inhibitory factor, a gene involved in the maintenance of the undifferentiated state of progenitor cells. Analysis of semithin sections indicated that these TM free-floating spheres were highly dynamic structures demonstrating a distinct radial gradient of cell proliferation, survival, and apoptosis. Extensive up- and down-regulation of gene expression was associated with the processes of sphere attachment and cell migration after the addition of serum. These results suggest that HTM primary cultures might contain relatively undifferentiated or progenitor cells. The availability of TM progenitor cell cultures could constitute a useful tool to investigate cell therapy approaches targeting the TM in glaucoma.
| S-EPMC3143838 | biostudies-literature
Project description:To contrast genome-wide gene expression profiles of cultured human trabecular meshwork (HTM) cells to that of control and primary open angle glaucoma (POAG) HTM tissues.Cultured HTM cells, HTM tissue dissected from control donors, and HTM tissue from POAG donors receiving medication for glaucoma were fixed in RNA latertrade mark. Total RNA extracted from these samples was linearly amplified with the Ovation Biotin RNA Amplification and Labeling System and individually hybridized to Affymetrix Human Genome U133 Plus 2.0 high density microarrays. Data analysis was performed using GeneSpring Software 7.0. Selected genes showing significant differential expression were validated by quantitative real-time PCR in nonamplified RNA.Cultured HTM cells retained the expression of some genes characteristic of HTM tissue, including chitinase 3-like 1 and matrix Gla protein, but demonstrated downregulation of physiologically important genes such as myocilin. POAG HTM tissue showed relatively small changes compared to that of control donors. These changes included the statistically significant upregulation of several genes associated with inflammation and acute-phase response, including selectin-E (ELAM-I), as well as the downregulation of the antioxidants paraoxonase 3 and ceruloplasmin.Downregulation in cultured HTM cells of genes potentially relevant for outflow pathway function highlights the importance of developing new conditions for the culture of TM cells capable of preserving the characteristics of TM cells in vivo. Comparative analysis between control and POAG tissues suggests that the upregulation of inflammation-associated genes might be involved in the progression of glaucoma.
| S-EPMC3152462 | biostudies-literature
Project description:Glaucoma is the second leading cause of irreversible blindness worldwide. Primary open angle glaucoma (POAG), the most common form of glaucoma, is often associated with elevation of intraocular pressure (IOP) due to the dysfunction of trabecular meshwork (TM) tissues. Currently, an ex vivo human anterior segment perfusion cultured system is widely used to study the effects of glaucoma factors and disease modifying drugs on physiological parameters like aqueous humor (AH) dynamics and IOP homeostasis. This system requires the use of freshly enucleated intact human eyes, which are sparsely available at very high cost. In this study, we explored the feasibility of using human donor corneoscleral segments for modeling morphological and biochemical changes associated with POAG. Among the number of corneas donated each year, many are deemed ineligible for transplantation due to stringent acceptance criteria. These ineligible corneoscleral segments were obtained from the Lions Eye Bank, Tampa, Florida. Each human donor anterior corneoscleral segment was dissected into four equal quadrants and cultured for 7 days by treating with the glaucoma factors dexamethasone (Dex) or recombinant transforming growth factor (TGF) β2 or transduced with lentiviral expression vectors containing wild type (WT) and mutant myocilin. Hematoxylin and Eosin (H&E) staining analysis revealed that the TM structural integrity is maintained after 7 days in culture. Increased TUNEL positive TM cells were observed in corneoscleral quadrants treated with glaucoma factors compared to their respective controls. However, these TUNEL positive cells were mainly confined to the scleral region adjacent to the TM. Treatment of corneoscleral quadrants with Dex or TGFβ2 resulted in glaucomatous changes at the TM, which included increased extracellular matrix (ECM) proteins and induction of endoplasmic reticulum (ER) stress. Western blot analysis of the conditioned medium showed an increase in ECM (fibronectin and collagen IV) levels in Dex- or TGFβ2-treated samples compared to control. Lentiviral transduction of quadrants resulted in expression of WT and mutant myocilin in TM tissues. Western blot analysis of conditioned medium revealed decreased secretion of mutant myocilin compared to WT myocilin. Moreover, increased ECM deposition and ER stress induction was observed in the TM of mutant myocilin transduced quadrants. Our findings suggest that the ex-vivo cultured human corneoscleral segment model is cost-effective and can be used as a pre-screening tool to study the effects of glaucoma factors and anti-glaucoma therapeutics on the TM.
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Project description:Primary open-angle glaucoma progression is associated with increased human trabecular meshwork (HTM) stiffness and elevated transforming growth factor beta 2 (TGFβ2) levels in the aqueous humor. Increased transcriptional activity of Yes-associated protein (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ), central players in mechanotransduction, are implicated in glaucomatous HTM cell dysfunction. Yet, the detailed mechanisms underlying YAP/TAZ modulation in HTM cells in response to alterations in extracellular matrix (ECM) stiffness and TGFβ2 levels are not well understood. Using biomimetic ECM hydrogels with tunable stiffness, here we show that increased ECM stiffness elevates YAP/TAZ nuclear localization potentially through modulating focal adhesions and cytoskeletal rearrangement. Furthermore, TGFβ2 increased nuclear YAP/TAZ in both normal and glaucomatous HTM cells, which was prevented by inhibiting extracellular-signal-regulated kinase and Rho-associated kinase signaling pathways. Filamentous (F)-actin depolymerization reversed TGFβ2-induced YAP/TAZ nuclear localization. YAP/TAZ depletion using siRNA or verteporfin decreased focal adhesions, ECM remodeling and cell contractile properties. Similarly, YAP/TAZ inactivation with verteporfin partially blocked TGFβ2-induced hydrogel contraction and stiffening. Collectively, our data provide evidence for a pathologic role of aberrant YAP/TAZ signaling in glaucomatous HTM cell dysfunction, and may help inform strategies for the development of novel multifactorial approaches to prevent progressive ocular hypertension in glaucoma.
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