Characterization of extracellular matrix deposited by segmental trabecular meshwork cells.

Purpose: Biophysical and biochemical attributes of the extracellular matrix are major determinants of cell fate in homeostasis and disease. Ocular hypertension and glaucoma are diseases where the trabecular meshwork tissue responsible for aqueous humor egress becomes stiffer accompanied by changes in its matrisome in a segmental manner with regions of high or low flow. Prior studies demonstrate these alterations in the matrix are dynamic in response to age and pressure changes.

Characterization of extracellular matrix deposited by segmental trabecular meshwork cells.

Biophysical and biochemical attributes of the extracellular matrix are major determinants of cell fate in homeostasis and disease. Ocular hypertension and glaucoma are diseases where the trabecular meshwork tissue responsible for aqueous humor egress becomes stiffer accompanied by changes in its matrisome in a segmental manner with regions of high or low flow. Prior studies demonstrate these alterations in the matrix are dynamic in response to age and pressure changes.

Valve-Like Outflow System Behavior With Motion Slowing in Glaucoma Eyes: Findings Using a Minimally Invasive Glaucoma Surgery-MIGS-Like Platform and Optical Coherence Tomography Imaging.

Purpose: This study aimed to investigate anatomic relationships and biomechanics of pressure-dependent trabecular meshwork and distal valve-like structure deformation in normal and glaucoma eyes using high-resolution optical coherence tomography (HR-OCT).

Methods: We controlled Schlemm's canal (SC) pressure during imaging with HR-OCT in segments of three normal (NL) and five glaucomatous (GL) eyes. The dissected limbal wedges were studied from 15 locations (5 NL and 10 GL).

Consensus Recommendation for Mouse Models of Ocular Hypertension to Study Aqueous Humor Outflow and Its Mechanisms.

Due to their similarities in anatomy, physiology, and pharmacology to humans, mice are a valuable model system to study the generation and mechanisms modulating conventional outflow resistance and thus intraocular pressure. In addition, mouse models are critical for understanding the complex nature of conventional outflow homeostasis and dysfunction that results in ocular hypertension. In this review, we describe a set of minimum acceptable standards for developing, characterizing, and utilizing mouse models of open-angle ocular hypertension.

Endogenous expression of Notch pathway molecules in human trabecular meshwork cells.

Purpose: Cells in the trabecular meshwork sense and respond to a myriad of physical forces through a process known as mechanotransduction. Whilst the effect of substratum stiffness or stretch on TM cells have been investigated in the context of transforming growth factor (TGF-β), Wnt and YAP/TAZ pathways, the role of Notch signaling, an evolutionarily conserved pathway, recently implicated in mechanotransduction, has not been investigated in trabecular meshwork (TM) cells. Here, we compare the endogenous expression of Notch pathway molecules in TM cells from glaucomatous and non-glaucomatous donors, segmental flow regions, and when subjected to cyclical strain, or grown on hydrogels of varying rigidity.

Circumferential trabecular meshwork cell density in the human eye.

The cells residing in the trabecular meshwork (TM) fulfill important roles in the maintenance of the tissue and the regulation of intraocular pressure (IOP). Here we examine (i) TM cell distribution along the circumference of the human eye, (ii) differences in TM cell density between regions of high and low outflow, and (iii) whether TM cell distribution in eyes from donors with primary open angle glaucoma (POAG) differs from that of normal eyes. Toward this end, the TM cell density from 12 radial segments around the circumference of the TM of human donor eyes (n = 6) with and without POAG was determined using histochemical methods.

Glucocorticoid-induced cell-derived matrix modulates transforming growth factor β2 signaling in human trabecular meshwork cells.

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 (TGFβ2), compared with control matrices (VehMs), glucocorticoid-induced cell-derived matrices (GIMs) trigger non-Smad TGFβ2 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 TGFβ2, VehMs and GIMs activate Smad and non-Smad TGFβ2 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-TGFβ2-induced changes being mostly more pronounced.

Crosslinked Extracellular Matrix Stiffens Human Trabecular Meshwork Cells Via Dysregulating β-catenin and YAP/TAZ Signaling Pathways.

Purpose: The purpose of this study was to determine whether genipin-induced crosslinked cell-derived matrix (XCDM) precipitates fibrotic phenotypes in human trabecular meshwork (hTM) cells by dysregulating β-catenin and Yes-associated protein (YAP)/ transcriptional coactivator with PDZ-binding motif (TAZ) signaling pathways.

Methods: Cell-derived matrices were treated with control or genipin for 5 hours to obtain respective uncrosslinked (CDM) and XCDMs and characterized. hTM cells were seeded on these matrices with/without Wnt pathway modulators in serum-free media for 24 hours.

Normal and glaucomatous outflow regulation.

Glaucoma remains only partially understood, particularly at the level of intraocular pressure (IOP) regulation. Trabecular meshwork (TM) and Schlemm's canal inner wall endothelium (SCE) are key to IOP regulation and their characteristics and behavior are the focus of much investigation. This is becoming more apparent with time.

Isolation and Characterization of primary human trabecular meshwork cells from segmental flow regions: New tools for understanding segmental flow.

Segmental flow in the human trabecular meshwork is a well-documented phenomenon but in depth mechanistic investigations of high flow (HF) and low flow (LF) regions are restricted due to the small amount of tissue available from a single donor. To address this issue we have generated and characterized multiple paired HF and LF cell strains. Here paired HF and LF cell strains were generated from single donors.

Generating cell-derived matrices from human trabecular meshwork cell cultures for mechanistic studies.

Ocular hypertension has been attributed to increased resistance to aqueous outflow often as a result of changes in trabecular meshwork (TM) extracellular matrix (ECM) using in vivo animal models (for example, by genetic manipulation) and ex vivo anterior segment perfusion organ cultures. These are, however, complex and difficult in dissecting molecular mechanisms and interactions. In vitro approaches to mimic the underlying substrate exist by manipulating either ECM topography, mechanics, or chemistry.

Elevated pressure influences relative distribution of segmental regions of the trabecular meshwork.

Elevated intraocular pressure (IOP) is the primary risk factor for glaucoma and is the only treatable feature of the disease. There is a correlation between elevated pressure and homeostatic reductions in the aqueous humor outflow resistance via changes in the extracellular matrix of the trabecular meshwork. It is unclear how these extracellular matrix changes affect segmental patterns of aqueous humor outflow, nor do we understand their causal relationship.

GPR158 in the Visual System: Homeostatic Role in Regulation of Intraocular Pressure.

GPR158 is a newly characterized family C G-protein-coupled receptor, previously identified in functional screens linked with biological stress, including one for susceptibility to ocular hypertension/glaucoma induced by glucocorticoid stress hormones. In this study, we investigated GPR158 function in the visual system. Gene expression and protein immunolocalization analyses were performed in mouse and human brain and eye to identify tissues where GPR158 might function.

Consensus recommendations for trabecular meshwork cell isolation, characterization and culture.

Cultured trabecular meshwork (TM) cells are a valuable model system to study the cellular mechanisms involved in the regulation of conventional outflow resistance and thus intraocular pressure; and their dysfunction resulting in ocular hypertension. In this review, we describe the standard procedures used for the isolation of TM cells from several animal species including humans, and the methods used to validate their identity. Having a set of standard practices for TM cells will increase the scientific rigor when used as a model, and enable other researchers to replicate and build upon previous findings.

Glaucomatous cell derived matrices differentially modulate non-glaucomatous trabecular meshwork cellular behavior.

Unlabelled: 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.

Biomechanical Rigidity and Quantitative Proteomics Analysis of Segmental Regions of the Trabecular Meshwork at Physiologic and Elevated Pressures.

Purpose: The extracellular matrix (ECM) of the trabecular meshwork (TM) modulates resistance to aqueous humor outflow, thereby regulating IOP. Glaucoma, a leading cause of irreversible blindness worldwide, is associated with changes in the ECM of the TM. The elastic modulus of glaucomatous TM is larger than age-matched normal TM; however, the biomechanical properties of segmental low (LF) and high flow (HF) TM regions and their response to elevated pressure, are unknown.

Estimating Human Trabecular Meshwork Stiffness by Numerical Modeling and Advanced OCT Imaging.

Purpose: The purpose of this study was to estimate human trabecular meshwork (hTM) stiffness, thought to be elevated in glaucoma, using a novel indirect approach, and to compare results with direct en face atomic force microscopy (AFM) measurements.

Methods: Postmortem human eyes were perfused to measure outflow facility and identify high- and low-flow regions (HF, LF) by tracer. Optical coherence tomography (OCT) images were obtained as Schlemm's canal luminal pressure was directly manipulated.

Pressure-induced expression changes in segmental flow regions of the human trabecular meshwork.

Elevated intraocular pressure (IOP) is thought to create distortion or stretching of the juxtacanalicular and Schlemm's canal cells and their extracellular matrix (ECM) leading to a cascade of events that restore IOP to normal levels, a process termed IOP homeostasis. The ECM of the trabecular meshwork (TM) is intricately involved in the regulation of outflow resistance and IOP homeostasis, as matrix metalloproteinase (MMP)-initiated ECM turnover in the TM is necessary to maintain outflow facility. Previous studies have shown ECM gene expression and mRNA splice form differences in TM cells in response to sustained stretch, implicating their involvement in the dynamic process of IOP homeostasis.

Bone matrix hypermineralization in prolyl-3 hydroxylase 1 deficient mice.

Lack of prolyl 3-hydroxylase 1 (P3H1) due to mutations in P3H1 results in severe forms of recessive osteogenesis imperfecta. In the present study, we investigated the bone tissue characteristics of P3H1 null mice. Histomorphometric analyses of cancellous bone in the proximal tibia and lumbar vertebra in 1-month and 3-month old mice demonstrated that P3H1 deficient mice had low trabecular bone volume and low mineral apposition rate, but normal osteoid maturation time and normal osteoblast and osteoclast surfaces.

Mapping molecular differences and extracellular matrix gene expression in segmental outflow pathways of the human ocular trabecular meshwork.

Elevated intraocular pressure (IOP) is the primary risk factor for glaucoma, and lowering IOP remains the only effective treatment for glaucoma. The trabecular meshwork (TM) in the anterior chamber of the eye regulates IOP by generating resistance to aqueous humor outflow. Aqueous humor outflow is segmental, but molecular differences between high and low outflow regions of the TM are poorly understood.

Extracellular matrix in the trabecular meshwork: intraocular pressure regulation and dysregulation in glaucoma.

The trabecular meshwork (TM) is located in the anterior segment of the eye and is responsible for regulating the outflow of aqueous humor. Increased resistance to aqueous outflow causes intraocular pressure to increase, which is the primary risk factor for glaucoma. TM cells reside on a series of fenestrated beams and sheets through which the aqueous humor flows to exit the anterior chamber via Schlemm's canal.

Intraocular pressure homeostasis: maintaining balance in a high-pressure environment.

Although glaucoma is a relatively common blinding disease, most people do not develop glaucoma. A robust intraocular pressure (IOP) homeostatic mechanism keeps ocular pressures within relatively narrow acceptable bounds throughout most peoples' lives. The trabecular meshwork and/or Schlemm's canal inner wall cells respond to sustained IOP elevation and adjust the aqueous humor outflow resistance to restore IOP to acceptable levels.

Biological role of prolyl 3-hydroxylation in type IV collagen.

Collagens constitute nearly 30% of all proteins in our body. Type IV collagen is a major and crucial component of basement membranes. Collagen chains undergo several posttranslational modifications that are indispensable for proper collagen function.

The effects of tenascin C knockdown on trabecular meshwork outflow resistance.

Purpose: Tenascin C (TNC) is a matricellular glycoprotein whose expression in adult tissue is indicative of tissue remodeling. The purpose of the current study was to determine the localization of TNC in trabecular meshwork (TM) tissue and to analyze the effects of TNC on intraocular pressure (IOP).

Methods: Human TM frontal sections were immunostained with anti-TNC and imaged by confocal microscopy.