Human trabecular meshwork cell behavior is influenced by collagen scaffold pore architecture and glycosaminoglycan composition.
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ABSTRACT:
SUBMITTER: Osmond MJ
PROVIDER: S-EPMC8360228 | biostudies-literature |
REPOSITORIES: biostudies-literature
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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:PurposeHomeostatic turnover of the trabecular meshwork extracellular matrix (ECM) is essential to regulate aqueous humor outflow and to maintain intraocular pressure homeostasis. In this study, we evaluated aqueous humor turnover, intraocular pressure, and trabecular meshwork organization in MMP-9 null mice.MethodsIntraocular pressure and aqueous humor turnover were measured in MMP-9 null versus wild-type mice. Morphology of the anterior segment of the eye, with special attention to the structural organization of the trabecular meshwork, was investigated by means of optical coherence tomography, light microscopy, and transmission electron microscopy. Furthermore, using quantitative real-time polymerase chain reaction and immunostainings, we evaluated the ECM composition of the trabecular meshwork. Finally, the integrity and function of the retina and optic nerve were assessed, via optical coherence tomography, histologic techniques, and optomotor testing.ResultsMMP-9 null mice displayed early-onset ocular hypertension and reduced aqueous humor turnover. While transmission electron microscopic analysis did not reveal any abnormalities in the cellular organization of the trabecular meshwork, detailed investigation of collagen expression indicated that there is an aberrant trabecular meshwork ECM composition in MMP-9 null mice. Notably, at the age of 13 months, no glaucomatous neurodegeneration was seen in MMP-9 null mice.ConclusionsOur observations corroborate MMP-9 as an important remodeler of the collagenous composition of the trabecular meshwork and provide evidence for a causal link between MMP-9 deficiency, trabecular meshwork ultrastructure, and ocular hypertension.
| S-EPMC5102567 | biostudies-literature
Project description:Ocular hypertension is a causal risk-factor to developing glaucoma. This is associated with stiffening of the trabecular meshwork (TM), the primary site of resistance to aqueous-humor-outflow. The mechanisms underlying this stiffening or how pathologic extracellular matrix (ECM) affects cell function are poorly understood. It is recognized that mechanotransduction systems allow cells to sense and translate the intrinsic biophysical properties of ECM into intracellular signals to control gene transcription, protein expression, and cell behavior. Using an anterior segment perfusion model, we document that there are significantly more low flow regions that are much stiffer, and fewer high flow regions that are less stiff in glaucomatous TM (GTM) when compared to non-glaucomatous TMs (NTM). GTM tissue also has fewer cells overall when compared with NTM tissue. In order to study the role of pathologic ECM in glaucoma disease progression, we conducted studies using cell derived matrices (CDM). First, we characterized the mechanics, composition and organization of fibronectin in ECM deposited by GTM and NTM cells treated with glucocorticosteroids. Then, we determined that these GTM-derived ECM are able to induce stiffening of normal NTM cells, and alter their gene/protein expression to resemble that of a glaucomatous phenotype. Further, we demonstrate that GTM-derived ECM causes endoplasmic reticular stress in NTM. They also became resistant to being reorganized by these NTM cells. These phenomena were exacerbated by ECMs obtained from steroid treated glaucoma model groups. Collectively, our data demonstrates that CDMs represent a novel tool for the study of bidirectional interactions between TM cells and their immediate microenvironment.Statement of significanceExtracellular matrix (ECM) changes are prevalent in a number of diseases. The precise mechanisms by which changes in the ECM contribute to disease progression is unclear, primarily due to absence of appropriate models. Here, using glaucoma as a disease model, we document changes in cell derived matrix (CDM) and tissue mechanics that contribute to the pathology. Subsequently, we determine the effect that ECMs from diseased and healthy individuals have on healthy cell behaviors. Data emanating from this study demonstrate that CDMs are a potent tool for the study of cell-ECM interactions.
| S-EPMC5899949 | biostudies-literature
Project description:The use of spinal fusion surgery as a treatment for degenerative spinal conditions and chronic back pain is increasing. However, this technique requires use of a bone grafting material to fuse the vertebrae, traditionally autologous bone, which consists of an optimal combination of osteogenic cell precursors, extracellular matrix proteins and mineral components. To date, this remains the 'gold standard' material but its supply is limited and is associated with a number of clinical and ethical difficulties; consequently, various combinations of cells with biological scaffold materials have been tested but have failed to achieve fusion rates even comparable to autologous bone. We successfully fabricated a novel collagen-based scaffold using self-organising atelocollagen combined with nano-hydroxyapatite and chondroitin sulphate, cross-linked by microbial transglutaminase. The scaffold was characterised using a range of imaging, chemical composition and thermal analysis techniques. It was found to exhibit appropriate stiffness and suitable pore size for the adhesion, growth and differentiation of MSCs. The low toxicity makes it suitable for clinical application, and its slow degradation profile would enable the scaffold to promote bone growth over an extended period. This material therefore shows promise for clinical use in spinal fusion and other procedures requiring the use of bone grafts.
| S-EPMC6029624 | 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:Multi-material polymer scaffolds with multiscale pore architectures were characterized and tested with vascular and heart cells as part of a platform for replacing damaged heart muscle. Vascular and muscle scaffolds were constructed from a new material, poly(limonene thioether) (PLT32i), which met the design criteria of slow biodegradability, elastomeric mechanical properties, and facile processing. The vascular-parenchymal interface was a poly(glycerol sebacate) (PGS) porous membrane that met different criteria of rapid biodegradability, high oxygen permeance, and high porosity. A hierarchical architecture of primary (macroscale) and secondary (microscale) pores was created by casting the PLT32i prepolymer onto sintered spheres of poly(methyl methacrylate) (PMMA) within precisely patterned molds followed by photocuring, de-molding, and leaching out the PMMA. Pre-fabricated polymer templates were cellularized, assembled, and perfused in order to engineer spatially organized, contractile heart tissue. Structural and functional analyses showed that the primary pores guided heart cell alignment and enabled robust perfusion while the secondary pores increased heart cell retention and reduced polymer volume fraction.
| S-EPMC5142531 | biostudies-literature
Project description:To determine the difference in protein glycosylation and glycosylation enzyme levels between glaucomatous and control trabecular meshwork (TM).Glaucomatous and normal donor (n = 12 each) TM tissues, lectin fluorescence, fluorophore-assisted carbohydrate analyses, and quantitative MS were used to determine the glycosylation levels. Primary TM cells and glycosylation inhibitors were used to determine their effect on cell shape and motility.In contrast to elevated levels of glycoproteins determined by lectin fluorescence, simultaneous hyper- and hypo-glycosylation in glaucomatous TM was revealed by fluorophore-assisted carbohydrate analyses. Analyses of enzymes showed elevation of beta-glycosidase 1 and decrease in galactosyltransferase family 6 domain containing protein 1 in the glaucomatous TM. Quantitative MS identified select protein level changes between glaucomatous and normal TM. Primary TM cells were treated with inhibitors to elicit hypo-glycosylation, which affected cell shape, motility, and fluorescent tracer transport across a layer of TM cells.Global protein glycosylation is aberrant in glaucomatous TM compared to controls. The results presented here suggest that the alteration in global TM protein glycosylation encompassing cellular and extracellular matrix proteins contributes to glaucoma pathology likely mediated through changes in properties of TM cells.
| S-EPMC4015974 | biostudies-literature
Project description:PurposeTo image the trabecular meshwork (TM) in its native unfixed state using a non-invasive, non-destructive technique.MethodsTwo-photon microscopy (2PM), including two-photon excitation fluorescence (2PEF) and second harmonic generation (SHG), was used to image flat-mounted trabecular meshwork samples from human cadaver eyes. Multiple images were analyzed along the tissue axis (z-axis) to generate a three-dimensional (3D) model of the region.ResultsA lattice of large collagen fibers (approximately 10 microm in diameter) were detected by inherent fluorescence (2PEF) and SHG. There are regions of both tightly overlapping bundles as well as fluid-filled regions visible from the surface of the TM. 3D analysis of multiple images reveals that the open regions deep in the TM penetrate the juxtacanalicular TM (JTM) and connect to the inner wall of Schlemm's canal (IWSC). These open regions may represent low-resistance fluid pathways between the anterior chamber and Schlemm's canal (SC).Conclusions2PM imaging of the outflow system of the human eye documented collagenous structures solely from inherent optical properties, without addition of an exogenous fluorescent label. 2PM successfully imaged into the TM without the need for fixation, embedding, or histological processing. Deep penetration using advanced optical techniques revealed regions likely representing pores in the IWSC that have been documented by multiple electron microscope studies. Our work reveals that 2PM imaging has potential as a new metric for evaluating the aqueous outflow region of the human eye and is worthy of further exploration.
| S-EPMC2890557 | biostudies-literature
Project description:PurposeTo evaluate the normal aging effects on trabecular meshwork (TM) parameters using Fourier domain anterior segment optical coherence tomography (ASOCT) images.Patients and methodsOne eye from 45 participants with open angles was imaged. Two independent readers measured TM area, TM length, and area and length of the TM interface shadow from 3 age groups (18-40, 41-60, and 61-80). Measurements were compared using stepwise regression analysis.ResultsThe average TM parameters were 0.0487 (± 0.0092) mm(2) for TM area, 0.5502 (± 0.1033) mm for TM length, 0.1623 (± 0.341) mm(2) for TM interface shadow area, and 0.7755 (± 0.1574) mm for TM interface shadow length. Interobserver reproducibility coefficients ranged from 0.45 (TM length) to 0.82 (TM area). TM area and length were not correlated with age. While the TM interface shadow length did not correlate with age, the TM interface shadow area increased with age. Race, sex, intraocular pressure, and gonioscopy score were not correlated with any TM parameters.ConclusionAlthough the TM measurements were not correlated with age, the TM interface shadow area increased with age. Further study is required to determine whether there is any relationship between the age-related ASOCT findings of the TM interface shadow area and physiologic function.
| S-EPMC3791583 | biostudies-literature
Project description:The lack of an animal model or an in vitro model limits experimental options for studying temporal molecular events in pseudoexfoliation syndrome (PXF), an age related fibrillopathy causing trabecular meshwork damage and glaucoma. Our goal was to create a workable in vitro model of PXF using primary human TM (HTM) cell lines simulating human disease. Primary HTM cells harvested from healthy donors (n = 3), were exposed to various concentrations (5 ng/mL, 10 ng/mL, 15 ng/mL) of transforming growth factor-beta1 (TGF-β1) for different time points. Morphological change of epithelial-mesenchymal transition (EMT) was analyzed by direct microscopic visualization and immunoblotting for EMT markers. Expression of pro-fibrotic markers were analyzed by quantitative RT-PCR and immunoblotting. Cell viability and death in treated cells was analyzed using FACS and MTT assay. Protein complex and amyloid aggregate formation was analyzed by Immunofluorescence of oligomer11 and amyloid beta fibrils. Effect of these changes with pharmacological inhibitors of canonical and non-canonical TGF pathway was done to analyze the pathway involved. The expression of pro-fibrotic markers was markedly upregulated at 10 ng/mL of TGF-β1 exposure at 48-72 h of exposure with associated EMT changes at the same time point. Protein aggregates were seen maximally at these time points that were found to be localized around the nucleus and in the extracellular matrix (ECM). EMT and pro-fibrotic expression was differentially regulated by different canonical and non-canonical pathways suggesting complex regulatory mechanisms. This in vitro model using HTM cells simulated the main characteristics of human disease in PXF like pro-fibrotic gene expression, EMT, and aggregate formation.
| S-EPMC8700223 | biostudies-literature