Morphological changes in the trabecular meshwork and Schlemm's canal after treatment with topical intraocular pressure-lowering agents.
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ABSTRACT:
SUBMITTER: Park JH
PROVIDER: S-EPMC8437975 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Ocular hypertension in glaucoma develops due to age-related cellular dysfunction in the conventional outflow tract, resulting in increased resistance to aqueous humor outflow. Two cell types, trabecular meshwork (TM) and Schlemm's canal (SC) endothelia, interact in the juxtacanalicular tissue (JCT) region of the conventional outflow tract to regulate outflow resistance. Unlike endothelial cells lining the systemic vasculature, endothelial cells lining the inner wall of SC support a transcellular pressure gradient in the basal to apical direction, thus acting to push the cells off their basal lamina. The resulting biomechanical strain in SC cells is quite large and is likely to be an important determinant of endothelial barrier function, outflow resistance and intraocular pressure. This review summarizes recent work demonstrating how biomechanical properties of SC cells impact glaucoma. SC cells are highly contractile, and such contraction greatly increases cell stiffness. Elevated cell stiffness in glaucoma may reduce the strain experienced by SC cells, decrease the propensity of SC cells to form pores, and thus impair the egress of aqueous humor from the eye. Furthermore, SC cells are sensitive to the stiffness of their local mechanical microenvironment, altering their own cell stiffness and modulating gene expression in response. Significantly, glaucomatous SC cells appear to be hyper-responsive to substrate stiffness. Thus, evidence suggests that targeting the material properties of SC cells will have therapeutic benefits for lowering intraocular pressure in glaucoma.
| S-EPMC4268318 | biostudies-literature
Project description:Previously, we have shown that Tfap2b, the gene encoding transcription factor AP-2β, is needed for normal mouse eye development. Specifically, targeted loss of Tfap2b in neural crest cells (NCCs)1 and their derivatives, particularly the periocular mesenchyme (POM), resulted in anterior segment defects affecting the cornea and angle tissue. These defects were further associated with an increase in intraocular pressure (IOP). The present study investigates the underlying changes in embryonic and postnatal POM cell development and differentiation caused by loss of AP-2β in the NCCs, particularly in the structures that control aqueous outflow, using Wnt1Cre+/-; Tfap2b-/lox; tdTomatolox/+ mice (AP-2β neural crest cell knockout or AP-2β NCC KO). Toluidine blue-stained sections and ultrathin sections stained with uranyl acetate and lead citrate were used to assess morphology and ultrastructure, respectively. Immunohistochemistry of KO and control eyes was performed at embryonic day (E) 15.5, E18.5, postnatal day (P) 1, P7 and P14 using phospho-histone H3 (PH3), α-smooth muscle actin (α-SMA), myocilin and endomucin antibodies, as well as a TUNEL assay. Conditional deletion of AP-2β in the NCC-derived POM resulted in defects that appeared during both embryogenesis and postnatal stages. Fate mapping of the knockout cells in the mutants revealed that the POM migrated appropriately into the eye during embryogenesis. However, during postnatal stages a significant reduction in POM proliferation in the angle region was observed in the mutants compared to controls. This was accompanied by a lack of expression of appropriate trabecular meshwork and Schlemm's canal markers. This is the first study to show that AP-2β is required for development and differentiation of the trabecular meshwork and Schlemm's canal. Together, these defects likely contributed to the elevated intraocular pressure (IOP) previously reported in the AP-2β NCC KO mice.
| S-EPMC8005257 | biostudies-literature
Project description: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.
| S-EPMC8578291 | biostudies-literature
Project description:PurposeThe purpose of this study was to distinguish differences in gene expression between cells cultured from the juxtacanalicular trabecular meshwork (JCTM) and those from Schlemm's canal (SC), to gain clues to differences between those cell types, and to add to our baseline knowledge of gene expression differences in these cell types for later comparison between cells from nonprimary open-angle glaucoma (POAG) and POAG outflow tissues.MethodsA set of JCTM and SC cells was cultured from each of 2 donor eyes by an explant method, grown to passage 3, and frozen in liquid nitrogen. The cells were thawed, total RNA was extracted, and the probes made from total RNAs were hybridized to MICROMAX human cDNA microarray slides in 2 separate trials. Differentially expressed genes were analyzed using PubMed, Prosite, and IPA software, and the expression of several of the genes including intercellular adhesion molecule-1 (ICAM-1), tenascin, and β-spectrin was assessed by immunofluorescence.ResultsSchlemm's canal cells differentially expressed ICAM-1, spectrin, complement, fibulin-1, and several genes consistent with an endothelial origin in both arrays, while the JCTM cells more often overexpressed genes consistent with contractile, matrix function, and neural character. At the same time, many genes highly expressed in the first array were not highly overexpressed in the second. One highly overexpressed gene in the JCTM in both arrays, that for heparan sulfate 3-O-sulfotransferase-1 precursor, is thought to be somewhat unique, and could affect the glycosaminoglycan functionality in the extracellular matrix (ECM).ConclusionsWe found generally good agreement between the 2 array trials, but some contradictions as well. Many of the genes overexpressed in each cell type had been described in earlier work, but several were new. Tables of genes, grouped by cellular function, and the complete datasets are provided for the development of new hypotheses.
| S-EPMC3991976 | biostudies-literature
Project description:Elevated intraocular pressure (IOP) narrows Schlemm's canal (SC), theoretically increasing luminal shear stress. Using engineered adenoviruses containing a functional fragment of the shear-responsive endothelial nitric oxide synthase (eNOS) promoter, we tested effects of shear stress and elevated flow rate on reporter expression in vitro and ex vivo. Cultured human umbilical vein endothelial cells (HUVECs) and SC cells were transduced with adenovirus containing eNOS promoter driving secreted alkaline phosphatase (SEAP) or green fluorescent protein (GFP) and subjected to shear stress. In parallel, human anterior segments were perfused under controlled flow. After delivering adenoviruses to the SC lumen by retroperfusion, the flow rate in one anterior segment of pair was increased to double pressure. In response to high shear stress, HUVECs and SC cells expressed more SEAP and GFP than control. Similarly, human anterior segments perfused at higher flow rates released significantly more nitrites and SEAP into perfusion effluent, and SC cells expressed increased GFP near collector channel ostia compared to control. These data establish that engineered adenoviruses have the capacity to quantify and localize shear stress experienced by endothelial cells. This is the first in situ demonstration of shear-mediated SC mechanobiology as a key IOP-sensing mechanism necessary for IOP homeostasis.
| S-EPMC7118084 | biostudies-literature
Project description:Ripasudil hydrochloride hydrate (K-115), a specific Rho-associated coiled-coil containing protein kinase (ROCK) inhibitor, was the first ophthalmic solution developed for the treatment of glaucoma and ocular hypertension in Japan. Topical administration of K-115 decreased intraocular pressure (IOP) and increased outflow facility in rabbits. This study evaluated the effect of K-115 on monkey trabecular meshwork (TM) cells and Schlemm's canal endothelial (SCE) cells. K-115 induced retraction and rounding of cell bodies as well as disruption of actin bundles in TM cells. In SCE-cell monolayer permeability studies, K-115 significantly decreased transendothelial electrical resistance (TEER) and increased the transendothelial flux of FITC-dextran. Further, K-115 disrupted cellular localization of ZO-1 expression in SCE-cell monolayers. These results indicate that K-115 decreases IOP by increasing outflow facility in association with the modulation of TM cell behavior and SCE cell permeability in association with disruption of tight junction.
| S-EPMC4725980 | biostudies-other
Project description:Lowering intraocular pressure (IOP) delays or prevents the loss of vision in primary open-angle glaucoma (POAG) patients with high IOP and in those with normal tension glaucoma showing progression. Abundant evidence demonstrates that inhibition of contractile machinery of the trabecular meshwork cells is an effective method to lower IOP. However, the mechanisms involved in the regulation of trabecular contraction are not well understood. Although microRNAs have been shown to play important roles in the regulation of multiple cellular functions, little is known about their potential involvement in the regulation of IOP. Here, we showed that miR-200c is a direct postranscriptional inhibitor of genes relevant to the physiologic regulation of TM cell contraction including the validated targets Zinc finger E-box binding homeobox 1 and 2 (ZEB1 and ZEB2), and formin homology 2 domain containing 1 (FHOD1), as well as three novel targets: lysophosphatidic acid receptor 1 (LPAR1/EDG2), endothelin A receptor (ETAR), and RhoA kinase (RHOA). Consistently, transfection of TM cells with miR-200c resulted in strong inhibition of contraction in collagen populated gels as well as decreased cell traction forces exerted by individual TM cells. Finally, delivery of miR-200c to the anterior chamber of living rat eyes resulted in a significant decrease in IOP, while inhibition of miR-200c using an adenoviral vector expressing a molecular sponge led to a significant increase in IOP. These results demonstrate for the first time the ability of a miRNA to regulate trabecular contraction and modulate IOP in vivo, making miR-200c a worthy candidate for exploring ways to alter trabecular contractility with therapeutic purposes in glaucoma.
| S-EPMC3522713 | biostudies-literature
Project description: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.
| S-EPMC8355290 | biostudies-literature
Project description:We investigated in vivo changes in Schlemm's canal and the trabecular meshwork in eyes with primary open angle glaucoma (POAG). Relationships between Schlemm's canal diameter, trabecular meshwork thickness, and intraocular pressure (IOP) were examined. Forty POAG patients and 40 normal individuals underwent 80-MHz Ultrasound Biomicroscopy examinations. The Schlemm's canal and trabecular meshwork were imaged in superior, inferior, nasal and temporal regions. Normal individuals had an observable Schlemm's canal in 80.3% of sections, a meridional canal diameter of 233.0±34.5 ?m, a coronal diameter of 44.5±12.6 ?m and a trabecular meshwork thickness of 103.9±11.1 ?m, in POAG patients, Schlemm's canal was observable in 53.1% of sections, a meridional canal diameter of 195.6±31.3 ?m, a coronal diameter of 35.7±8.0 ?m, and a trabecular meshwork thickness of 88.3±13.2 ?m, which significantly differed from normal (both p <0.001). Coronal canal diameter (r = -0.623, p < 0.001) and trabecular meshwork thickness (r = -0.663, p < 0.001) were negatively correlated with IOP, but meridional canal diameter was not (r = -0.160, p = 0.156). Schlemm's canal was observable in 50.5% and 56.6% of POAG patients with normal (<21 mmHg) and elevated (>21 mmHg) IOP, respectively (? = 1.159, p = 0.282). Coronal canal diameter was significantly lower in the elevated IOP group (32.6±4.9 ?m) than in the normal IOP group (35.7±8.0 ?m, p < 0.001). This was also true of trabecular meshwork thickness (81.9±10.0 ?m vs. 97.1±12.0 ?m, p < 0.001). In conclusion, eyes with POAG had fewer sections with an observable Schlemm's canal. Canal diameter and trabecular meshwork thickness were also lower than normal in POAG patients. Schlemm's canal coronal diameter and trabecular meshwork thickness were negatively correlated with IOP.
| S-EPMC4699705 | biostudies-literature
Project description:PurposeIn this study, we investigate how adenosine monophosphate-activated protein kinase (AMPK) affects extracellular matrix (ECM) and cellular tone in the trabecular meshwork (TM), and examine how deletion of its catalytic α2 subunit affects IOP and aqueous humor clearance in mice.MethodsHuman TM tissue was examined for expression of AMPKα1 and AMPKα2, genomically distinct isoforms of the AMPK catalytic subunit. Primary cultured human TM cells were treated for 24 hours with the AMPK activator 5-amino-1-β-Dffff-ribofuranosyl-imidazole-4-carboxamide (AICAR), under basal or TGF-β2 stimulatory conditions. Conditioned media (CM) was probed for secreted protein acidic and rich in cysteine (SPARC), thrombospondin-1 (TSP-1), and ECM proteins, and cells were stained for F-actin. Cells underwent adenoviral infection with a dominant negative AMPKα subunit (ad.DN.AMPKα) and were similarly analyzed. Intraocular pressure, central corneal thickness (CCT), and aqueous clearance were measured in AMPKα2-null and wild-type (WT) mice.ResultsBoth AMPKα1 and AMPKα2 are expressed in TM. AICAR activated AMPKα and suppressed the expression of various ECM proteins under basal and TGF-β2 stimulatory conditions. AICAR decreased F-actin staining and increased the phospho-total RhoA ratio (Ser188). Transforming growth factor-β2 transiently dephosphorylated AMPKα. Infection with ad.DN.AMPKα upregulated various ECM proteins, decreased the phospho-total RhoA ratio, and increased F-actin staining. AMPKα2-null mice exhibited 6% higher IOP and decreased aqueous clearance compared with WT mice, without significant differences in CCT or angle morphology.ConclusionsCollectively, our data identify AMPK as a critical regulator of ECM homeostasis and cytoskeletal arrangement in the TM. Mice that are AMPKα2-null exhibit higher IOPs and decreased aqueous clearance than their WT counterparts.
| S-EPMC4023495 | biostudies-literature