scholarly journals The role of calcium-independent phospholipase A2γ in modulation of aqueous humor drainage and Ca2+ sensitization of trabecular meshwork contraction

2012 ◽  
Vol 302 (7) ◽  
pp. C979-C991 ◽  
Author(s):  
Padmanabhan P. Pattabiraman ◽  
Fred B. Lih ◽  
Kenneth B. Tomer ◽  
Ponugoti Vasantha Rao
Keyword(s):  
Arachidonic Acid ◽  
Aqueous Humor ◽  
Trabecular Meshwork ◽  
Rho Gtpase ◽  
Focal Adhesions ◽  
Intense Staining ◽  
Outflow Facility ◽  
Bromoenol Lactone ◽  
Mlc Phosphorylation

The contractile and relaxation characteristics of trabecular meshwork (TM) are presumed to influence aqueous humor (AH) drainage and intraocular pressure. The mechanisms underlying regulation of TM cell contractile properties, however, are not well understood. This study investigates the role of calcium-independent phospholipase A2 (iPLA2), which controls eicosanoid synthesis, in regulation of TM cell contraction and AH outflow using mechanism-based isoform specific inhibitors (R)-bromoenol lactone (R-BEL, iPLA2γ specific) and (S)-bromoenol lactone (S-BEL, iPLA2β specific). Immunohistochemical analysis revealed intense staining for both iPLA2β and γ isoforms throughout the TM, juxtacanalicular tissue, and Schlemm's canal of human eye. Inhibition of iPLA2γ by R-BEL or small interfering RNA-mediated silencing of iPLA2γ expression induced dramatic changes in TM cell morphology, and decreased actin stress fibers, focal adhesions, and myosin light-chain (MLC) phosphorylation. AH outflow facility increased progressively and significantly in enucleated porcine eyes perfused with R-BEL. This response was associated with a significant decrease in TM tissue MLC phosphorylation and alterations in the morphology of aqueous plexi in R-BEL-perfused eyes. In contrast, S-BEL did not affect either of these parameters. Additionally, R-BEL-induced cellular relaxation of the TM was associated with a significant decrease in the levels of active Rho GTPase, phospho-MLC phosphatase, phospho-CPI-17, and arachidonic acid. Taken together, these observations demonstrate that iPLA2γ plays a significant and isoform-specific role in regulation of AH outflow facility by altering the contractile characteristics of the TM. The effects of iPLA2γ on TM contractile status appear to involve arachidonic acid and Rho GTPase signaling pathways.

scholarly journals Role of MCP-1 and IL-8 in viral anterior uveitis, and contractility and fibrogenic activity of trabecular meshwork cells

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Jiyoung Lee ◽  
Jin A. Choi ◽  
Hyun-hee Ju ◽  
Ju-Eun Kim ◽  
Soon-Young Paik ◽  
...  
Keyword(s):  
Viral Infection ◽  
Aqueous Humor ◽  
Trabecular Meshwork ◽  
Anterior Uveitis ◽  
Rho Gtpase ◽  
Open Angle Glaucoma ◽  
Focal Adhesions ◽  
Aqueous Humor Outflow ◽  
Trabecular Meshwork Cells ◽  
Aqueous Outflow

AbstractThe inflammatory chemokines, monocyte chemoattractant protein (MCP)-1 and IL-8, are produced by normal trabecular meshwork cells (TM) and elevated in the aqueous humor of primary open angle glaucoma (POAG) and hypertensive anterior uveitis associated with viral infection. However, their role in TM cells and aqueous humor outflow remains unclear. Here, we explored the possible involvement of MCP-1 and IL-8 in the physiology of TM cells in the context of aqueous outflow, and the viral anterior uveitis. We found that the stimulation of human TM cells with MCP-1 and IL-8 induced significant increase in the formation of actin stress fibers and focal adhesions, myosin light chain phosphorylation, and the contraction of TM cells. MCP-1 and IL-8 also demonstrated elevation of extracellular matrix proteins, and the migration of TM cells. When TM cells were infected with HSV-1 and CMV virus, there was a significant increase in cytoskeletal contraction and Rho-GTPase activation. Viral infection of TM cells revealed significantly increased expression of MCP-1 and IL-8. Taken together, these results indicate that MCP-1 and IL-8 induce TM cell contractibility, fibrogenic activity, and plasticity, which are presumed to increase resistance to aqueous outflow in viral anterior uveitis and POAG.

scholarly journals Microtubule disassembly increases endothelial cell barrier dysfunction: role of MLC phosphorylation

2001 ◽  
Vol 281 (3) ◽  
pp. L565-L574 ◽  
Author(s):  
Alexander D. Verin ◽  
Anna Birukova ◽  
Peiyi Wang ◽  
Feng Liu ◽  
Patrice Becker ◽  
...  
Keyword(s):  
Endothelial Cell ◽  
Electrical Resistance ◽  
Rho Gtpase ◽  
Dependent Manner ◽  
Transendothelial Electrical Resistance ◽  
Barrier Dysfunction ◽  
Microtubule Disruption ◽  
Barrier Regulation ◽  
Mlc Phosphorylation

Endothelial cell (EC) barrier regulation is critically dependent on cytoskeletal components (microfilaments and microtubules). Because several edemagenic agents induce actomyosin-driven EC contraction tightly linked to myosin light chain (MLC) phosphorylation and microfilament reorganization, we examined the role of microtubule components in bovine EC barrier regulation. Nocodazole or vinblastine, inhibitors of microtubule polymerization, significantly decreased transendothelial electrical resistance in a dose-dependent manner, whereas pretreatment with the microtubule stabilizer paclitaxel significantly attenuated this effect. Decreases in transendothelial electrical resistance induced by microtubule disruption correlated with increases in lung permeability in isolated ferret lung preparations as well as with increases in EC stress fiber content and MLC phosphorylation. The increases in MLC phosphorylation were attributed to decreases in myosin-specific phosphatase activity without significant increases in MLC kinase activity and were attenuated by paclitaxel or by several strategies (C3 exotoxin, toxin B, Rho kinase inhibition) to inhibit Rho GTPase. Together, these results suggest that microtubule disruption initiates specific signaling pathways that cross talk with microfilament networks, resulting in Rho-mediated EC contractility and barrier dysfunction.

Role of Lysophospholipid Growth Factors in the Modulation of Aqueous Humor Outflow Facility

2004 ◽  
Vol 45 (7) ◽  
pp. 2263 ◽  
Author(s):  
Priyatham Sai Mettu ◽  
Pei-Feng Deng ◽  
Uma K. Misra ◽  
Govind Gawdi ◽  
David L. Epstein ◽  
...  
Keyword(s):  
Growth Factors ◽  
Aqueous Humor ◽  
Outflow Facility ◽  
Aqueous Humor Outflow

The role of protein kinase C in modulation of aqueous humor outflow facility

2003 ◽  
Vol 76 (1) ◽  
pp. 39-47 ◽  
Author(s):  
Rahul N Khurana ◽  
Pei-Feng Deng ◽  
David L Epstein ◽  
P Vasantha Rao
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Aqueous Humor ◽  
Outflow Facility ◽  
Aqueous Humor Outflow ◽  
Kinase C

scholarly journals Novel molecular insights into RhoA GTPase-induced resistance to aqueous humor outflow through the trabecular meshwork

2008 ◽  
Vol 295 (5) ◽  
pp. C1057-C1070 ◽  
Author(s):  
Min Zhang ◽  
Rupalatha Maddala ◽  
Ponugoti Vasantha Rao
Keyword(s):  
Aqueous Humor ◽  
Trabecular Meshwork ◽  
Rho Gtpase ◽  
Cytoskeletal Proteins ◽  
Fiber Formation ◽  
Aqueous Humor Outflow ◽  
Ecm Proteins ◽  
Rhoa Gtpase ◽  
Cell Adhesive ◽  
Adhesive Interactions

Impaired drainage of aqueous humor through the trabecular meshwork (TM) culminating in increased intraocular pressure is a major risk factor for glaucoma, a leading cause of blindness worldwide. Regulation of aqueous humor drainage through the TM, however, is poorly understood. The role of RhoA GTPase-mediated actomyosin organization, cell adhesive interactions, and gene expression in regulation of aqueous humor outflow was investigated using adenoviral vector-driven expression of constitutively active mutant of RhoA (RhoAV14). Organ-cultured anterior segments from porcine eyes expressing RhoAV14 exhibited significant reduction of aqueous humor outflow. Cultured TM cells expressing RhoAV14 exhibited a pronounced contractile morphology, increased actin stress fibers, and focal adhesions and increased levels of phosphorylated myosin light chain (MLC), collagen IV, fibronectin, and laminin. cDNA microarray analysis of RNA extracted from RhoAV14-expressing human TM cells revealed a significant increase in the expression of genes encoding extracellular matrix (ECM) proteins, cytokines, integrins, cytoskeletal proteins, and signaling proteins. Conversely, various ECM proteins stimulated robust increases in phosphorylation of MLC, paxillin, and focal adhesion kinase and activated Rho GTPase and actin stress fiber formation in TM cells, indicating a potential regulatory feedback interaction between ECM-induced mechanical strain and Rho GTPase-induced isometric tension in TM cells. Collectively, these data demonstrate that sustained activation of Rho GTPase signaling in the aqueous humor outflow pathway increases resistance to aqueous humor outflow through the trabecular pathway by influencing the actomyosin assembly, cell adhesive interactions, and the expression of ECM proteins and cytokines in TM cells.

scholarly journals Mechanotransduction and dynamic outflow regulation in trabecular meshwork requires Piezo1 channels

2020 ◽  
Author(s):  
Oleg Yarishkin ◽  
Tam T. T. Phuong ◽  
Jackson M. Baumann ◽  
Michael L. De Ieso ◽  
Felix Vazquez-Chona ◽  
...  
Keyword(s):  
Intraocular Pressure ◽  
Shear Flow ◽  
Aqueous Humor ◽  
Trabecular Meshwork ◽  
Molecular Mechanisms ◽  
Pressure Sensors ◽  
Focal Adhesions ◽  
Ruthenium Red ◽  
Pressure Transducers ◽  
Aqueous Humor Outflow

AbstractMechanosensitivity of the trabecular meshwork (TM) is a key determinant of intraocular pressure (IOP) yet our understanding of the molecular mechanisms that subserve it remains in its infancy. Here, we show that mechanosensitive Piezo1 channels modulate the TM pressure response via calcium signaling and dynamics of the conventional outflow pathway. Pressure steps evoked fast, inactivating cation currents and calcium signals that were inhibited by Ruthenium Red, GsMTx4 and Piezo1 shRNA. Piezo1 expression was confirmed by transcript and protein analysis, and by visualizing Yoda1-mediated currents and [Ca2+]i elevations in primary human TM cells. Piezo1 activation was obligatory for transduction of physiological shear stress and was coupled to reorganization of F-actin cytoskeleton and focal adhesions. The importance of Piezo1 channels as pressure sensors was shown by the GsMTx4 -dependence of the pressure-evoked current and conventional outflow function. We also demonstrate that Piezo1 collaborates with the stretch-activated TRPV4 channel, which mediated slow, delayed currents to pressure steps. 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 a novel potential therapeutic target for treating glaucoma.Significance StatementTrabecular meshwork (TM) is a highly mechanosensitive tissue in the eye that regulates intraocular pressure through the control of aqueous humor drainage. Its dysfunction underlies the progression of glaucoma but neither the mechanisms through which TM cells sense pressure nor their role in aqueous humor 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. These findings provide a new mechanistic framework for trabecular mechanotransduction and its role in the regulation of fast fluctuations in ocular pressure, as well as chronic remodeling of TM architecture that epitomizes glaucoma.

Intracellular Cl regulates Na-K-Cl cotransport activity in human trabecular meshwork cells

1999 ◽  
Vol 277 (3) ◽  
pp. C373-C383 ◽  
Author(s):  
Luanna K. Putney ◽  
Cecile Rose T. Vibat ◽  
Martha E. O’Donnell
Keyword(s):  
Cell Volume ◽  
Aqueous Humor ◽  
Trabecular Meshwork ◽  
Cell Types ◽  
Cellular Mechanisms ◽  
Outflow Facility ◽  
Aqueous Humor Outflow ◽  
Trabecular Meshwork Cells ◽  
Cotransport System ◽  
Stimulation Of

The trabecular meshwork (TM) of the eye plays a central role in modulating intraocular pressure by regulating aqueous humor outflow, although the mechanisms are largely unknown. We and others have shown previously that aqueous humor outflow facility is modulated by conditions that alter TM cell volume. We have also shown that the Na-K-Cl cotransport system is a primary regulator of TM cell volume and that its activity appears to be coordinated with net efflux pathways to maintain steady-state volume. However, the cellular mechanisms that regulate cotransport activity and cell volume in TM cells have yet to be elucidated. The present study was conducted to investigate the hypothesis that intracellular Cl concentration ([Cl]i) acts to regulate TM cell Na-K-Cl cotransport activity, as has been shown previously for some other cell types. We demonstrate here that the human TM cell Na-K-Cl cotransporter is highly sensitive to changes in [Cl]i. Our findings reveal a marked stimulation of Na-K-Cl cotransport activity, assessed as ouabain-insensitive, bumetanide-sensitive K influx, in TM cells following preincubation of cells with Cl-free medium as a means of reducing [Cl]i. In contrast, preincubation of cells with media containing elevated K concentrations as a means of increasing [Cl]i results in inhibition of Na-K-Cl cotransport activity. The effects of reducing [Cl]i, as well as elevating [Cl]i, on Na-K-Cl cotransport activity are concentration dependent. Furthermore, the stimulatory effect of reduced [Cl]i is additive with cell-shrinkage-induced stimulation of the cotransporter. Our studies also show that TM cell Na-K-Cl cotransport activity is altered by a variety of Cl channel modulators, presumably through changes in [Cl]i. These findings support the hypothesis that regulation of Na-K-Cl cotransport activity, and thus cell volume, by [Cl]i may participate in modulating outflow facility across the TM.

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