Endothelial cell stiffness and type drive the formation of biomechanically-induced transcellular pores.

Formation of transcellular pores facilitates the transport of materials across endothelial barriers. In Schlemm's canal (SC) endothelium, impaired pore formation is associated with glaucoma. However, our understanding of the cellular processes responsible for pore formation is limited by lack of assays.

Lysyl oxidase-like 1-antisense 1 (LOXL1-AS1) lncRNA differentially regulates gene and protein expression, signaling and morphology of human ocular cells.

Pseudoexfoliation glaucoma (PEXG) is characterized by dysregulated extracellular matrix (ECM) homeostasis that disrupts conventional outflow function and increases intraocular pressure (IOP). Prolonged IOP elevation results in optic nerve head damage and vision loss. Uniquely, PEXG is a form of open angle glaucoma that has variable penetrance, is difficult to treat and does not respond well to common IOP-lowering pharmaceuticals.

Proposed Mechanism of Long-Term Intraocular Pressure Lowering With the Bimatoprost Implant.

Purpose: The purpose of this study was to evaluate the effects of pharmacologically relevant bimatoprost and bimatoprost free acid (BFA) concentrations on matrix metalloproteinase (MMP) gene expression in cells from human aqueous outflow tissues.

Methods: MMP gene expression by human trabecular meshwork (TM), scleral fibroblast (SF), and ciliary muscle (CM) cells exposed to 10 to 1000 µM bimatoprost or 0.1 to 10 µM BFA (intraocular concentrations after intracameral bimatoprost implant and topical bimatoprost dosing, respectively) was measured by polymerase chain reaction array.

YAP/TAZ Mediate TGFβ2-Induced Schlemm's Canal Cell Dysfunction.

Purpose: Elevated transforming growth factor beta2 (TGFβ2) levels in the aqueous humor have been linked to glaucomatous outflow tissue dysfunction. Potential mediators of dysfunction are the transcriptional coactivators, Yes-associated protein (YAP) and transcriptional coactivator with PDZ binding motif (TAZ). However, the molecular underpinnings of YAP/TAZ modulation in Schlemm's canal (SC) cells under glaucomatous conditions are not well understood.

Anterior Segment Anatomy and Conventional Outflow Physiology of the Tree Shrew (Tupaia belangeri).

Purpose: Rodent and primate models are commonly used in glaucoma research; however, both have their limitations. The tree shrew (Tupaia belangeri) is an emerging animal model for glaucoma research owing in part to having a human-like optic nerve head anatomy, specifically a collagenous load-bearing lamina. However, the anterior segment anatomy and function have not been extensively studied in the tree shrew.

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.

Fibrotic Changes to Schlemm's Canal Endothelial Cells in Glaucoma.

Previous studies have shown that glaucomatous Schlemm's canal endothelial cells (gSCECs) are stiffer and associated with reduced porosity and increased extracellular matrix (ECM) material compared to SCECs from healthy individuals. We hypothesised that Schlemm's canal (SC) cell stiffening was a function of fibrotic changes occurring at the inner wall of SC in glaucoma. This study was performed in primary cell cultures isolated from the SC lumen of human donor eyes.

Surface Engineering of FLT4-Targeted Nanocarriers Enhances Cell-Softening Glaucoma Therapy.

Primary open-angle glaucoma is associated with elevated intraocular pressure (IOP) that damages the optic nerve and leads to gradual vision loss. Several agents that reduce the stiffness of pressure-regulating Schlemm's canal (SC) endothelial cells, in the conventional outflow pathway of the eye, lower IOP in glaucoma patients and are approved for clinical use. However, poor drug penetration and uncontrolled biodistribution limit their efficacy and produce local adverse effects.

Targeted Delivery of Cell Softening Micelles to Schlemm's Canal Endothelial Cells for Treatment of Glaucoma.

Increased stiffness of the Schlemm's canal (SC) endothelium in the aqueous humor outflow pathways has been associated with elevated intraocular pressure (IOP) in glaucoma. Novel treatments that relax this endothelium, such as actin depolymerizers and rho kinase inhibitors, are in development. Unfortunately, these treatments have undesirable off-target effects and a lower than desired potency.

An In Vitro Bovine Cellular Model for Human Schlemm's Canal Endothelial Cells and Their Response to TGFβ Treatment.

Purpose: Due to the limited availability of primary human Schlemm's canal (SC) endothelial cells, we aimed to develop an in vitro cellular model using the angular aqueous plexus (AAP) cells from bovine eyes.

Methods: We harvested a mixture of cells from the trabecular meshwork region including AAP loops from multiple donors, followed by puromycin treatment and immunostaining of Von Willebrand factor and vascular endothelial (VE)-cadherin to confirm identity. Previously identified differentially expressed genes in glaucomatous SC cells were examined in non-glaucomatous SC cells (n = 3) under 0% or 15% equibiaxial strain for 24 hours using droplet digital polymerase chain reaction (ddPCR) and analyzed using the Ingenuity Pathway Analysis (IPA) software application to identify upstream regulators.

Differential DNA methylation patterns in human Schlemm's canal endothelial cells with glaucoma.

Purpose: Schlemm's canal (SC) endothelial cells derived from donors with or without glaucoma showed different mechanical properties and gene expression. As an important contributor to the regulation of intraocular pressure (IOP) and pathogenesis of primary open-angle glaucoma (POAG), the heritable key epigenetic changes, methylation may play an important role in the physiologic function of SC cells. This study aims to identify differentially methylated CpG sites (DMSs) in primary cultures of human SC cells with or without glaucoma.

Expression of mRNAs, miRNAs, and lncRNAs in Human Trabecular Meshwork Cells Upon Mechanical Stretch.

Purpose: Intraocular pressure (IOP), the primary risk factor for primary open-angle glaucoma, is determined by resistance to aqueous outflow through the trabecular meshwork (TM). IOP homeostasis relies on TM responses to mechanical stretch. To model the effects of elevated IOP on the TM, this study sought to identify coding and non-coding RNAs differentially expressed in response to mechanical stretch.

Shear Stress in Schlemm's Canal as a Sensor of Intraocular Pressure.

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.

Increased stiffness and flow resistance of the inner wall of Schlemm's canal in glaucomatous human eyes.

The cause of the elevated outflow resistance and consequent ocular hypertension characteristic of glaucoma is unknown. To investigate possible causes for this flow resistance, we used atomic force microscopy (AFM) with 10-µm spherical tips to probe the stiffness of the inner wall of Schlemm's canal as a function of distance from the tissue surface in normal and glaucomatous postmortem human eyes, and 1-µm spherical AFM tips to probe the region immediately below the tissue surface. To localize flow resistance, perfusion and imaging methods were used to characterize the pressure drop in the immediate vicinity of the inner wall using giant vacuoles that form in Schlemm's canal cells as micropressure sensors.

Probe Sensitivity to Cortical versus Intracellular Cytoskeletal Network Stiffness.

In development, wound healing, and pathology, cell biomechanical properties are increasingly recognized as being of central importance. To measure these properties, experimental probes of various types have been developed, but how each probe reflects the properties of heterogeneous cell regions has remained obscure. To better understand differences attributable to the probe technology, as well as to define the relative sensitivity of each probe to different cellular structures, here we took a comprehensive approach.

Intracameral Delivery of Layer-by-Layer Coated siRNA Nanoparticles for Glaucoma Therapy.

Glaucoma is the second leading cause of blindness worldwide, often associated with elevated intraocular pressure. Connective tissue growth factor (CTGF) is a mediator of pathological effects in the trabecular meshwork (TM) and Schlemm's canal (SC). A novel, causative therapeutic concept which involves the intracameral delivery of small interfering RNA against CTGF is proposed.

Pentablock copolymer dexamethasone nanoformulations elevate MYOC: in vitro liberation, activity and safety in human trabecular meshwork cells.

Aim: The aim of this study is to examine the elevation of MYOC in long-term treatment of human trabecular meshwork (HTM) cells using dexamethasone (DEX) encapsulated pentablock (PB) copolymer-based nanoparticles (NPs) (DEX-PB-NPs).

Materials & Methods: PB copolymers and DEX-PB-NPs were synthesized and characterized using nuclear magnetic resonance, gel permeation chromatography, and X-ray diffraction analyses. MYOC levels secreted from HTM cells were measured by western blot (WB) analysis.

Enhancement of Outflow Facility in the Murine Eye by Targeting Selected Tight-Junctions of Schlemm's Canal Endothelia.

The juxtacanalicular connective tissue of the trabecular meshwork together with inner wall endothelium of Schlemm's canal (SC) provide the bulk of resistance to aqueous outflow from the anterior chamber. Endothelial cells lining SC elaborate tight junctions (TJs), down-regulation of which may widen paracellular spaces between cells, allowing greater fluid outflow. We observed significant increase in paracellular permeability following siRNA-mediated suppression of TJ transcripts, claudin-11, zonula-occludens-1 (ZO-1) and tricellulin in human SC endothelial monolayers.

Differential response and withdrawal profile of glucocorticoid-treated human trabecular meshwork cells.

The goal of the study was to examine secreted protein response and withdrawal profiles from cultured human trabecular meshwork (HTM) cells following short- and long-term glucocorticoid treatment. Primary cultures of five human HTM cell strains isolated from 5 different individual donor eyes were tested. Confluent HTM cells were differentiated in culture media containing 1% FBS for at least one week, and then treated with Dexamethasone (Dex, 100 nM) 3 times/week for 1 or 4 weeks.

Successful Implementation of a Program for Increasing Donor Eyes for Research: The Duke-Miracles In Sight Program.

Purpose: Although essential for development of ocular therapeutics, the quality and quantity of human donor eyes for research have been on the decline. To streamline procurement protocols, provide better medical documentation of tissue, and improve freshness and number of eyes, a pilot cooperative program was undertaken between the Duke University Eye Center and Miracles In Sight Eye Bank.

Methods: For research eye donors who expire at Duke University Hospital, age restrictions to procurement were lifted, access to donors' electronic medical records was granted to researchers, and eye tissue was delivered directly to scientists.

Expression Profiling of Human Schlemm's Canal Endothelial Cells From Eyes With and Without Glaucoma.

Purpose: Ocular hypertension is a major risk factor for glaucoma and the inner wall of Schlemm's canal (SC) endothelia participates in the regulation of aqueous humor outflow resistance. This study aimed to identify differentially expressed genes in primary cultures of SC cells from glaucoma patients.

Methods: This study examined SC samples from three glaucoma cases and four controls.

Altered mechanobiology of Schlemm's canal endothelial cells in glaucoma.

Increased flow resistance is responsible for the elevated intraocular pressure characteristic of glaucoma, but the cause of this resistance increase is not known. We tested the hypothesis that altered biomechanical behavior of Schlemm's canal (SC) cells contributes to this dysfunction. We used atomic force microscopy, optical magnetic twisting cytometry, and a unique cell perfusion apparatus to examine cultured endothelial cells isolated from the inner wall of SC of healthy and glaucomatous human eyes.

Pigment epithelium-derived factor decreases outflow facility.

Purpose: Pigment epithelium-derived factor (PEDF) regulates blood-retinal barrier function. As a constituent of aqueous humor, the role of PEDF in conventional outflow function is unknown. The goals of the study were to examine the effects of PEDF on barrier function of cultured Schlemm's canal (SC) endothelia and outflow facility in mouse eyes in situ.

Structural basement membrane components and corresponding integrins in Schlemm's canal endothelia.

Purpose: The conventional outflow pathway provides the primary source of resistance to aqueous humor drainage, regulating intraocular pressure. Despite large pressure gradients across the inner wall of Schlemm's canal (SC), cells remain attached to their basement membrane. The goal of this study was to examine integrin-extracellular matrix binding partners of the inner wall basement membrane that facilitate attachment.