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Expressional and functional involvement of gap junctions in aqueous humor outflow into the ocular trabecular meshwork of the anterior chamber.

ABSTRACT: Purpose:The ocular trabecular meshwork (TM) responsible for aqueous humor (AH) drainage is crucial for regulating intraocular pressure (IOP) of the eye. An IOP elevation that causes distended TM is involved in the pathogenesis of glaucoma, suggesting intercellular connections important for the TM pathophysiology. The goal of this study was to examine whether gap junction proteins between endothelial cells in the TM are expressional and functional. Methods:The expression levels of the gap junction channels in normal human TM cells were determined with real-time PCR and western blot assays. Immunohistochemistry (IHC) staining was performed to examine the localization of gap junction proteins in normal human TM cells and tissues. IOP and the outflow of AH were measured after intercameral injection of gap junction blockers in C57/BL6 mice. Results:Gap junction proteins GJA1, GJA8, GJB6, and GJC1 were robustly expressed in human TM cells from three individuals. Among the four gap junction channels, GJA1 and GJA8 exhibited the most abundance in the TM. The IHC analysis further confirmed that these proteins were expressed on the membrane between adjacent cells. In the human TM tissues, GJA1, GJA8, GJB6, and GJC1 were also observed along the trabecular beams. Inhibition of gap junctions with intracameral injection of blockers resulted in a statistically significant increase in aqueous humor outflow resistance and IOP elevation in mice. Conclusions:The GJA1 and GJA8 gap junction proteins, in particular, are robustly expressed in human TM cells and tissues. Pharmacological inhibition of gap junction channels causes an increased resistance of AH outflow and an elevation of IOP in mice. The present findings suggest the functional role of gap junction channels for regulation of AH outflow in the TM, and activation of gap junctions might represent a therapeutic strategy for treatment of glaucoma.

SUBMITTER: Yu H

PROVIDER: S-EPMC6545340 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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Expressional and functional involvement of gap junctions in aqueous humor outflow into the ocular trabecular meshwork of the anterior chamber.

Yu Hongxia H Miao Yongzhen Y Chen Wenshi W Qi Xia X Yang Xian X Pan Xiaojing X Wang KeWei K Zhu Wei W

Molecular vision 20190426

<h4>Purpose</h4>The ocular trabecular meshwork (TM) responsible for aqueous humor (AH) drainage is crucial for regulating intraocular pressure (IOP) of the eye. An IOP elevation that causes distended TM is involved in the pathogenesis of glaucoma, suggesting intercellular connections important for the TM pathophysiology. The goal of this study was to examine whether gap junction proteins between endothelial cells in the TM are expressional and functional.<h4>Methods</h4>The expression levels of ...[more]

PMID: 31205407

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Project description:Key pointsTrabecular meshwork (TM) is a highly mechanosensitive tissue in the eye that regulates intraocular pressure through the control of aqueous humour drainage. Its dysfunction underlies the progression of glaucoma but neither the mechanisms through which TM cells sense pressure nor their role in aqueous humour outflow are understood at the molecular level. We identified the Piezo1 channel as a key TM transducer of tensile stretch, shear flow and pressure. Its activation resulted in intracellular signals that altered organization of the cytoskeleton and cell-extracellular matrix contacts and modulated the trabecular component of aqueous outflow whereas another channel, TRPV4, mediated a delayed mechanoresponse. This study helps elucidate basic mechanotransduction properties that may contribute to intraocular pressure regulation in the vertebrate eye.AbstractChronic elevations in intraocular pressure (IOP) can cause blindness by compromising the function of trabecular meshwork (TM) cells in the anterior eye, but how these cells sense and transduce pressure stimuli is poorly understood. Here, we demonstrate functional expression of two mechanically activated channels in human TM cells. Pressure-induced cell stretch evoked a rapid increase in transmembrane current that was inhibited by antagonists of the mechanogated channel Piezo1, Ruthenium Red and GsMTx4, and attenuated in Piezo1-deficient cells. The majority of TM cells exhibited a delayed stretch-activated current that was mediated independently of Piezo1 by TRPV4 (transient receptor potential cation channel, subfamily V, member 4) channels. Piezo1 functions as the principal TM transducer of physiological levels of shear stress, with both shear and the Piezo1 agonist Yoda1 increasing the number of focal cell-matrix contacts. Analysis of TM-dependent fluid drainage from the anterior eye showed significant inhibition by GsMTx4. Collectively, these results suggest that TM mechanosensitivity utilizes kinetically, regulatory and functionally distinct pressure transducers to inform the cells about force-sensing contexts. Piezo1-dependent control of shear flow sensing, calcium homeostasis, cytoskeletal dynamics and pressure-dependent outflow suggests potential for a novel therapeutic target in treating glaucoma.

Project description: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. In this study, flow regions of the TM were characterized using fluorescent tracers and PCR arrays. Anterior segments from human donor eyes were perfused at physiological pressure in an ex vivo organ culture system. Fluorescently-labeled microspheres of various sizes were perfused into anterior segments to label flow regions. Actively perfused microspheres were segmentally distributed, whereas microspheres soaked passively into anterior segments uniformly labeled the TM and surrounding tissues with no apparent segmentation. Cell-tracker quantum dots (20 nm) were localized to the outer uveal and corneoscleral TM, whereas larger, modified microspheres (200 nm) localized throughout the TM layers and Schlemm's canal. Distribution of fluorescent tracers demonstrated a variable labeling pattern on both a macro- and micro-scale. Quantitative PCR arrays allowed identification of a variety of extracellular matrix genes differentially expressed in high and low flow regions of the TM. Several collagen genes (COL16A1, COL4A2, COL6A1 and 2) and MMPs (1, 2, 3) were enriched in high, whereas COL15A1, and MMP16 were enriched in low flow regions. Matrix metalloproteinase activity was similar in high and low regions using a quantitative FRET peptide assay, whereas protein levels in tissues showed modest regional differences. These gene and protein differences across regions of the TM provide further evidence for a molecular basis of segmental flow routes within the aqueous outflow pathway. New insight into the molecular mechanisms of segmental aqueous outflow may aid in the design and delivery of improved treatments for glaucoma patients.

Dataset Information

Expressional and functional involvement of gap junctions in aqueous humor outflow into the ocular trabecular meshwork of the anterior chamber.

Publications

Expressional and functional involvement of gap junctions in aqueous humor outflow into the ocular trabecular meshwork of the anterior chamber.

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