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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. We expect that this set of standard practices will increase scientific rigor when using mouse models and will better enable researchers to replicate and build upon previous findings.

Elevated intraocular pressure arising from impaired aqueous humor drainage through the trabecular pathway is a major risk factor for glaucoma. To understand the molecular basis for Rho GTPase-mediated resistance to aqueous humor drainage, we investigated the possible interrelationship between actomyosin contractile properties and extracellular matrix (ECM) synthesis in human trabecular meshwork (TM) cells expressing a constitutively active form of RhoA (RhoAV14). TM cells expressing RhoAV14 exhibited significant increases in fibronectin, tenascin C, laminin, [alpha]-smooth muscle actin ([alpha]-SMA) levels, and matrix assembly in association with increased actin stress fibers and myosin light-chain phosphorylation. RhoAV14-induced changes in ECM synthesis and actin cytoskeletal reorganization were mimicked by lysophosphatidic acid and TGF-[[beta].sub.2], known to increase resistance to aqueous humor outflow and activate Rho/Rho kinase signaling. RhoAV14, lysophosphatidic acid, and TGF-[[beta].sub.2] stimulated significant increases in Erk1/2 phosphorylation, paralleled by profound increases in fibronectin, serum response factor (SRF), and [alpha]-SMA expression. Treatment of RhoA-activated TM cells with inhibitors of Rho kinase or Erk, on the other hand, decreased fibronectin and [alpha]-SMA levels. Although suppression of SRF expression (both endogenous and RhoA, TGF-[[beta].sub.2]-stimulated) via the use of short hairpin RNA decreased [[alpha]-SMA levels, fibronectin was unaffected. Conversely, fibronectin induced [alpha]-SMA expression in an SRF-dependent manner. Collectively, data on RhoA-induced changes in actomyosin contractile activity, ECM synthesis/assembly, and Erk activation, along with fibronectin-induced [alpha]-SMA expression in TM cells, reveal a potential molecular interplay between actomyosin cytoskeletal tension and ECM synthesis/assembly. This interaction could be significant for the homeostasis of aqueous humor drainage through the pressure-sensitive trabecular pathway. fibronectin; actomyosin; extracellular signal-regulated kinases; serum response factor doi:10.1152/ajpcell.00317.2009.

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 vectordriven 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 RhoAVI4 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. cytoskeleton; gene expression; extracellular matrix; aqueous humor drainage

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 RhoAVI4 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 RhoAVI4-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. [ABSTRACT FROM AUTHOR]

This study illustrates a vital role for ankyrin-B in lens architecture, growth and function through its involvement in membrane protein and spectrin-actin cytoskeletal organization and stability The transparent ocular lens is essential for vision by focusing light onto the retina. Despite recognizing the importance of its unique cellular architecture and mechanical properties, the molecular mechanisms governing these attributes remain elusive. This study aims to elucidate the role of ankyrin-B (AnkB), a membrane scaffolding protein, in lens cytoarchitecture, growth and function using a conditional knockout (cKO) mouse model. AnkB cKO mouse has no defects in lens morphogenesis, but exhibited changes that supported a global role for AnkB in maintenance of lens clarity, size, cytoarchitecture, and stiffness. Notably, absence of AnkB led to nuclear cataract formation, evident from P16. AnkB cKO lens fibers exhibit progressive disruption in membrane organization of the spectrin-actin cytoskeleton, channel proteins, cell-cell adhesion, shape change, loss and degradation of several membrane proteins (e.g., NrCAM. N-cadherin and aquaporin-0) along with a disorganized plasma membrane and impaired ball-and-socket membrane interdigitations. Furthermore, absence of AnkB led to decreased lens stiffness. Collectively, these results illustrate the essential role for AnkB in lens architecture, growth and function through its involvement in membrane protein and cytoskeletal organization.

Dysregulation of the mechanical properties and cell adhesive interactions of trabecular meshwork (TM) are known to impair aqueous humor drainage and elevate intraocular pressure in glaucoma patients. The identity of regulatory mechanisms underlying TM mechanotransduction, however, remains elusive. Here we analyzed the phosphotyrosine proteome of human TM cell-extracellular matrix (ECM) adhesion complexes, which play a key role in sensing and transducing extracellular chemical and mechanical cues into intracellular activities, using a two-level affinity pull-down (phosphotyrosine antibody and titanium dioxide beads) method and mass spectrometry. This analysis identified ~1,000 tyrosine-phosphorylated proteins of TM cell-ECM adhesion complexes. Many consensus adhesome proteins were found to be tyrosine phosphorylated. Interestingly, several of the phosphotyrosinylated proteins found in TM cell-ECM adhesion complexes are known to be required for podocyte glomerular filtration, indicating the existence of molecular parallels that are likely relevant to the shared fluid barrier and filtration functions of the two mechanosensitive cell types.

Intercellular adherens junctions and cell-extracellular matrix interactions are presumed to influence aqueous humor (AH) drainage via the conventional route, however, their direct role in modulation of intraocular pressure (IOP) is not well understood. Here, we investigated the role of Rac GTPase signaling in basal and growth factor-induced formation of adherens junctions in human trabecular meshwork (HTM) cells as compared to human umbilical vascular endothelial cells, and evaluated the effects of inhibition of Rac GTPase activity on IOP in rabbits. Expression of a constitutively active Rac1 GTPase or treatment with platelet derived growth factor (PDGF), a known activator of Rac GTPase, induced formation of β-catenin-based adherens junctions, actin cytoskeletal reorganization and membrane ruffle in HTM cells. In contrast, treatment of HTM cells with inhibitors of Rac GTPase caused cell-cell separation, a decrease in adherens junctions, and reorganization of actin stress fibers to the cell cortical regions and focal adhesion to the cell leading edges. Both, constitutively active Rac1 and PDGF stimulated generation of Reactive Oxygen Species (ROS) in HTM cells, and ROS were found to increase adherens junction formation and transendothelial electrical resistance (TEER) in HTM cells. Topical application of Rac GTPase inhibitors (EHT1864 and NSC23766), however, only marginally influenced IOP in rabbit eyes. Taken together, these data reveal that while Rac GTPase signaling plays a significant role in regulation of adherens junctions, ROS production and TEER in cells of the AH outflow pathway, Rac inhibitors showed only a marginal influence on IOP in live rabbits.

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 A(2) (iPLA(2)), which controls eicosanoid synthesis, in regulation of TM cell contraction and AH outflow using mechanism-based isoform specific inhibitors (R)-bromoenol lactone (R-BEL, iPLA(2)γ specific) and (S)-bromoenol lactone (S-BEL, iPLA(2)β specific). Immunohistochemical analysis revealed intense staining for both iPLA(2)β and γ isoforms throughout the TM, juxtacanalicular tissue, and Schlemm's canal of human eye. Inhibition of iPLA(2)γ by R-BEL or small interfering RNA-mediated silencing of iPLA(2)γ 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 iPLA(2)γ plays a significant and isoform-specific role in regulation of AH outflow facility by altering the contractile characteristics of the TM. The effects of iPLA(2)γ on TM contractile status appear to involve arachidonic acid and Rho GTPase signaling pathways.

Purpose: Based on pharmacological inhibition of activity, a role has been proposed for Rho-kinase in the modulation of aqueous outflow and intraocular pressure (IOP). This study employed a molecular approach to specifically address the role of Rho-kinase in the modulation of aqueous humor outflow., Methods: Adenoviral vectors expressing green fluorescent protein alone (Ad-GFP) or the dominant negative Rho-binding domain of Rho-kinase and GFP (Ad-DNRK-GFP) were utilized in these experiments. Human and porcine primary trabecular meshwork (TM) cells were infected with 30 MOI (multiplicity of infection) of Ad-GFP alone or with Ad-DNRK-GFP. Changes in cell shape, actomyosin cytoskeletal integrity, and the status of myosin light chain (MLC) phosphorylation were evaluated. The aqueous outflow facility was determined in organ cultured anterior segments of human cadaver eyes infected with 10(7) pfu adenoviral vectors (Ad-GFP or Ad-DNRK-GFP) using a constant flow perfusion system., Results: Expression of DNRK resulted in changes in cell shape (cell rounding, cell-cell detachment) and decreased actin stress fiber and focal adhesion staining in TM cells. These cellular changes were associated with substantially reduced myosin light chain phosphorylation. Additionally, organ cultured human eye anterior segments infected with Ad-DNRK-GFP exhibited a significant increase in the outflow facility (80%, n=9) compared to control anterior segments infected with Ad-GFP (5%)., Conclusions: This study demonstrated that specific inhibition of Rho-kinase activity in trabecular meshwork cells led to alterations in cell shape and presumed contractile properties, and we hypothesize that these morphological and contractile events underlie the observed effects of dominant negative Rho-kinase on the aqueous humor outflow facility. These data provide molecular evidence for the hypothesis of Rho-kinase being a potential cellular target involved in the regulation of aqueous humor outflow resistance, with implications for novel glaucoma therapy.

Purpose: The primary goal of this study was to characterize the Rac GTPase associated, NADPH oxidase-mediated Reactive Oxygen Species (ROS)-generating system in the lens tissue., Methods: NADPH oxidase activity in lens tissue was determined by quantifying superoxide-induced lucigenin photoemission. Immunological and PCR/RT-PCR techniques were utilized to determine expression of different components of the NADPH oxidase system in lens tissue. Growth factor stimulated ROS production was determined quantitatively in human lens epithelial cells using dichlorofluorescein diacetate., Results: Lens homogenates from different species showed generation of superoxide in a lucigenin-enhanced chemiluminescence assay in the presence of NADPH. This activity was found to be lens protein concentration dependent, heat sensitive, and inhibitable by superoxide dismutase and the flavoprotein inhibitor, diphenyleneiodonium (DPI). The distribution of superoxide generating activity in lens was confined predominantly to the lens epithelium, with very low levels in cortex and none in the nucleus. Immunological assays have demonstrated the presence of p67phox and p47phox in lens tissue, while PCR and RT-PCR reactions amplified DNA products corresponding to the p67phox, p40phox, p22phox, gp91phox, and Rac1 components of the NADPH oxidase complex from human and mouse lens cDNA libraries. Serum starved human lens epithelial cells stimulated with different growth factors including EGF, b-FGF, PDGF, TGF-beta, and LPA demonstrated increased production of ROS, a response which was blocked by inhibitors of NADPH oxidase, such as DPI and the antioxidant-N-acetyl cysteine (NAC). RT-PCR analysis of human lens RNA confirmed readily detectable levels of expression of low molecular weight protein tyrosine phosphatase (LMW-PTP), which is a well-characterized target of redox signaling pathway(s)., Conclusions: These data demonstrate the presence of a functional nonphagocytic NADPH oxidase system in lens that is predominantly localized to the lens epithelium. Several growth factors appear to stimulate the activity of lens NADPH oxidase, resulting in increased production of ROS in lens epithelial cells, indicating that redox signaling may have an important role in growth factor effects on lens growth and development.