Molecular Vision 2001; 7:145-153 <http://www.molvis.org/molvis/v7/a21/> Received 3 September 2000 | Accepted 13 May 2001 | Published 27 June 2001

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Effect of H-7 on cultured human trabecular meshwork cells

Xuyang Liu,¹ Suping Cai,¹ Adrian Glasser,¹ Tova Volberg,² Jon R. Polansky,³ Donald J. Fauss,³ Curtis R. Brandt,¹ Benjamin Geiger,² Paul L. Kaufman¹
 
 

¹Department of Ophthalmology & Visual Sciences, University of Wisconsin, Madison, WI; ²Department of Molecular Cell Biology, Weizmann Institute of Science, Rehovot, Israel; ³Department of Ophthalmology, University of California at San Francisco, San Francisco, CA

Correspondence to: Paul L. Kaufman, M.D., Department of Ophthalmology and Visual Sciences, F4/328 Clinical Science Center, 600 Highland Avenue, Madison, WI, 53792; Phone: (608) 263-6074; FAX: (608) 263-1466; email: kaufmanp@mhub.ophth.wisc.edu

Abstract

Purpose: To determine the effect of the serine-threonine kinase inhibitor H-7, which blocks actomyosin contractility and increases outflow facility in live monkeys, on morphology, cytoskeleton, and cellular adhesions of human trabecular meshwork (HTM) cells in culture.

Methods: Cultured HTM cells were videographically recorded and evaluated before and after exposure to H-7 at different concentrations. The subcellular distribution of the actin-based cytoskeleton and associated anchor proteins including vinculin, paxillin, and b-catenin, as well as phosphotyrosine-containing proteins were evaluated by fluorescence immunocytochemistry and digital fluorescence microscopy.

Results: H-7 induced pronounced but reversible HTM cell thickening toward the cell center and deterioration of the actin cytoskeletal network. Cell-extracellular matrix (ECM) and cell-cell adhesions were also affected, but the b-catenin-rich, vinculin-containing adherens junctions were clearly more resistant than focal contacts. Phosphotyrosine labeling in focal contacts was highly sensitive to H-7.

Conclusions: H-7 induces alterations in cell shape, actin cytoskeleton, and associated focal adhesions in cultured HTM cells, which may be responsible for the effects of H-7 on outflow facility in live monkey eyes.

Introduction

Intracameral and topical administration of H-7 (1-(5-isoquinolinyl-sulfonyl)-2-methylpiperazine), a broad-spectrum serine-threonine kinase inhibitor, reversibly increases outflow facility across the trabecular meshwork, and reduces intraocular pressure (IOP), in live monkey eyes [1,2], and thus this drug has potential as an anti-glaucoma agent. To date, the effects of H-7 on the contractility, morphology, and adhesion molecules of cultured cells, including human trabecular meshwork (HTM) cells, have been studied [1,3,4]. In cultured bovine aortic endothelial cells (BAEC), exposure to H-7 results in a reversible disruption of actin microfilaments and a major alteration in the organization of cell-ECM adhesions [1]. In cultured HTM cells, H-7 blocks cellular contraction induced by ethacrynic acid, colchicine, and vinblastine, and disrupts actin filaments of HTM and Schlemm's canal (SC) cells without altering cell-cell contacts [3,4]. However, changes in morphology of living cells and changes in cellular adhesion characteristics of cultured HTM cells, such as measures of vinculin, b-catenin, paxillin, and phosphotyrosine, upon H-7 treatment have not been extensively examined previously. Such information might help to better understand the cellular and molecular basis for H-7's effect on outflow facility in vivo. In the present study, we specifically evaluated the effects of H-7 on morphology of living HTM cells in culture, and on dynamics of cellular adhesions of HTM cells. For the latter, particularly, a novel fluorescence microscopic approach (fluorescence ratio imaging; FRI) was used, and the labeling intensities and dynamics of the adhesion molecules were analyzed. The novel element in this analysis is the combination of image superimposition and FRI [5], which creates a unique immunofluorescent forum for the rapid quantitative investigation of the diversity in the fine distribution of cellular proteins and cellular changes. The unique feature of FRI is that it is highly sensitive to local differences in the relative fluorescent staining intensities irrespective of their absolute intensities, and can provide valuable information for even faintly labeled structures [5]. The HTM cells used in this study were cultured from an undifferentiated stage with fibroblast-like morphology to a highly confluent monolayer showing stable endothelial-like morphology, to resemble and behave as close to the in vivo situation as possible.

This study reports and discusses the effects of H-7 on HTM cells with emphasis on (a) shape changes of living cells, (b) molecular dynamics of cellular adhesions, and (c) comparison with our in vivo studies including H-7 induced changes in outflow facility, TM ultrastructure, and potential side effects of the drug. Most of these issues and relationships have not been addressed in previous studies with H-7.

Methods

HTM cell culture and H-7 treatment

HTM cells [6] were grown on Petri dishes or glass cover slips pre-coated with poly-L-lysine (Sigma Chemical Co., St. Louis, MO) and maintained in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum, 25 mg/ml gentamycin and 2.5 mg/ml amphotericin B, at 37°C, 8% CO₂. Cells of 3^rd-5^th passage were cultured to high confluence for 1 week, at which time they exhibited a stable monolayer endothelial-like morphology, and were then treated with H-7 (Sigma Chemical Co., St. Louis, MO) and analyzed.

Image analysis

For time lapse videography, a high resolution black & white charge coupled device (CCD) camera (COHU Inc., Electronics Division, San Diego, CA) was attached to the phototube of a Leitz DMIL inverted phase microscope (Leica, Wetzlar, Germany) and the output was fed to a video recorder and a TV screen. The identical field of cells was recorded at each time interval before and during exposure to H-7 and images were subsequently captured from the videotape. In addition, cells were recorded and then fixed for actin staining as described below after exposure to 20, 100, and 300 mM H-7 for 2 h, as well as after 30 min and 2 h recovery following drug washout. To evaluate the potential toxicity of H-7 for HTM cells, some cells were recorded while exposed to 600 mM H-7 (twice the generally accepted maximal dose for affecting the cytoskeleton in several types of cultured cells [1,7,8], and twice the maximally effective intracameral dose for increasing outflow facility in monkeys [1,9]) for 2 h and during recovery for 30 min and 2 h after drug washout.

Immunocytochemistry

Cells were cultured on glass cover slips pre-coated with poly-L-lysine, incubated with or without H-7 for the indicated time, and then fixed and fluorescently labeled for actin, vinculin, and b-catenin. The cells were washed with 50 mM MES (2-(N-morpholine) ethanesulfonic acid) buffer, and simultaneously permeabilized and fixed with 0.5% Triton X-100 and 3% paraformaldehyde. The cells were then exposed to rhodamine (TRITC) phalloidin (Sigma) for fluorescent labeling of actin, or exposed to monoclonal or polyclonal anti-human vinculin, and b-catenin primary antibodies followed by Texas Red (TR)-conjugated goat anti-mouse second antibody (Jackson Laboratories, West Grove, PA). The cells were examined and images generated using a Zeiss Axiophot fluorescent microscope with a 100x oil immersion lens (Zeiss, Thornwood, NY).

Digital fluorescence microscopy

To quantitatively compare the local labeling intensities of different adhesion molecules including actin, vinculin, paxillin, b-catenin, and phosphotyrosine-containing proteins, digital florescence microscopy was used in separate experiments. Cells were double-labeled for vinculin (cyanine dyes, CY3, Jackson Laboratories) and actin (fluorescein isothiocyanate, FITC, Sigma), paxillin (CY3) and phosphotyrosine (Alexa, Molecular Probes, Inc., Eugene, OR) or b-catenin (CY3) and actin (FITC) respectively. Cells were then washed, fixed and stained as described. Images were acquired using the DeltaVision system (Applied Precision, Issaquah, WA), equipped with a Zeiss Axiovert 100 microscope with a 100x 1.3 NA Plan-Neofluar objective (Oberkochen, Germany) and Photometrics 300 series scientific-grade cooled camera (Tucson, AZ) reading 12 bit images. Images were presented either by true color, or by color spectrum representing fluorescence intensities. FRI between two different components in double-labeled cells was used to determine the dynamics of cellular adherens junctions as described previously [5,10].

Results

HTM cell topography

Untreated HTM cells formed a monolayer of closely attached cells with "endothelial-like" morphology, and remained alike, albeit not identical, in appearance at each time point (Figure 1). Treatment with H-7 induced a dose- and time-dependent cell rounding and apparent thickening toward the center of the cells. A mild effect was observed within 60 min after treatment with 100 mM H-7 and increased with H-7 concentration or exposure time. After exposure to 300 mM H-7 for 1 h, cells became more refractile toward the cell center, yet their intercellular contacts were maintained and no cellular separation was observed. After 24 h exposure to H-7, about 50% of the cells treated with 20 mM H-7 appeared to be affected, whereas almost all the cells treated with 100 or 300 mM H-7 were involved (Figure 1). These changes were reversible, and no obviously detached cells were observed at any dose or time of treatment. For the 20 and 100 mM doses (24 h treatment), recovery seemed complete after 48 h in H-7-free medium (Figure 1). Essentially full recovery of the cells after 48 h was also observed for the 300 mM dose, as shown by a typical endothelial morphology. For each dose and time point, a confluent monolayer of cells very similar to the pretreatment appearance was observed and there was no obvious cell loss. This indicates that the cells observed were the original ones, since there was not enough time for new cells to grow to a highly confluent and differentiated appearance. These data suggest that H-7 does not appear to be irreversibly cytotoxic at the highest in vivo intraocular concentration studied [1,9]. Within 30 min after drug removal following 2 h exposure to different doses of H-7, including 600 mM (data not shown), morphological recovery of HTM cells appeared complete (Figure 2). For all doses, 2 h recovery (data not shown) appeared as complete as 30 min recovery, and no obviously detached cells were seen.

Effect of H-7 on the distribution of actin, vinculin, and b-catenin in HTM cells

H-7 induced disruption of the stress fiber network and focal adhesions. Actin-containing bundles deteriorated markedly after 30 min exposure to H-7, especially at the higher doses. With longer exposures (2 h), the stress fiber network was obliterated, especially at higher doses, leaving only a diffuse mesh of actin filaments mostly at the cell periphery (Figure 3). The H-7-treated cells also showed dramatic dose- and time-dependent deterioration of vinculin-containing focal adhesions, with mild alterations in vinculin-containing cell-cell junctions (Figure 4). The distribution of b-catenin was slightly altered, but in general cell-cell junctions were resistant to H-7, especially at lower doses (Figure 5). Staining for b-catenin was weak and cells were occasionally partially separated upon exposure to 300 mM H-7 (Figure 5).

Recovery of the actin-based cytoskeleton and vinculin-containing focal adhesions began as early as 30 min after 2 h treatment with H-7. Actin recovery occurred rapidly in cells treated with any dose, redistributing into both radial and circumferential bundles consistent with the recovery of cell morphology (Figure 2, Figure 3). There was not much difference in b-catenin labeling between H-7 treatment and recovery at the 20 and 100 mM doses; that is, because there was little effect, as described above, there was little recovery to be seen. However, at the 300 mM H-7 dose, which did affect b-catenin, recovery of b-catenin at the cell border was obvious within 30 min after drug removal, exhibiting a continuous zigzag appearance (Figure 5). In contrast, vinculin recovery in cell-matrix and cell-cell adhesions lagged behind that of actin microfilaments (Figure 3, Figure 4).

FRI analysis of H-7 induced changes in the actin cytoskeleton and the associated cellular adhesions

As described above, immunofluorescence analysis of HTM cells treated with H-7 demonstrated a striking, dose-dependent, and transient breakdown in the structure and organization of actin, vinculin and, to a limited extend, b-catenin. However, information regarding relative changes and dynamics in the local fluorescence intensities of the molecules associated with cellular adhesions, which is important for understanding the mechanisms underlying the drug's effects, was not defined by the conventional immunocytochemistry. Digital microscopy coupled with FRI analysis of cells double labeled for two proteins was utilized in order to examine the molecular dynamics of these changes in more detail.

Double staining for vinculin (red channel) and actin (green channel) in untreated cells revealed numerous stress fibers oriented primarily parallel to the long axis of the cells, and terminating at vinculin-containing focal adhesions and adherens junctions (Figure 6A-D). FRI analysis (Figure 6D) showed that within most individual adhesions there was extensive overlap between vinculin and actin labeling (i.e., abundance of sites where an intensity ratio of 1:1 was observed). After exposure to 300 mM H-7 for as little as 15 min (Figure 6E-H), HTM cells showed fewer and smaller focal contacts and stress fibers. FRI showed that the actin:vinculin ratio within the vast majority of residual adhesions remained unchanged, indicating a rapid but proportional loss of vinculin and actin from these structures (Figure 6H). This is consistent with the findings at 1 h of exposure to 300 mM H-7, which confirmed a breakdown of both vinculin and actin as well as an unchanged actin:vinculin ratio. Removal of the drug for as little as 5 min resulted in a rapid reorganization of actin filaments and an increase in the size, number, and intensity of vinculin-positive focal adhesions (Figure 6M-P). By 20 min after H-7 removal, actin and vinculin appeared essentially normal (Figure 6Q-T).

Double staining for paxillin (red channel) and phosphotyrosine (green channel) in untreated cells revealed that paxillin, unlike vinculin, was associated only with focal contacts and not with cell-cell junctions. A substantial portion of the phosphotyrosine, but not all, co-localized with paxillin at focal contacts, and was also associated with cell-cell junctions, indicating a positive correlation with cellular adhesions (Figure 7A). HTM cells treated with 300mM H-7 for 15 and 60 min showed a rapid and time-dependent reduction in phosphphotyrosine levels, followed by a slower loss of paxillin, in focal contacts (Figure 7E-L). FRI again indicated a more significant reduction in the number and intensity of sites positive for phosphphotyrosine than for paxillin and, as a result, a high ratio of paxillin to phosphotyrosine (Figure 7H). The number, size, and labeling intensity of paxillin-positive focal contacts increased dramatically upon removal of H-7 (Figure 7M-T). At 5 min the more prominent focal contacts already contained phosphotyrosine, and their number and size increased with time (Figure 7M-T), but the recovery was not complete, especially for phosphotyrosine, as shown by a fewer number of and less intense labelings, and by the relatively low ratio of the two components, compared to untreated cells (Figure 7Q-T).

Double staining for b-catenin (red channel) and actin (green channel) in untreated cells revealed that intercellular junctions were delineated by b-catenin staining appearing as continuous lines at cell-cell borders. Weaker, but significant actin labeling was also noted along the b-catenin-containing cell-cell adherens junctions, and cell-cell junctions were flanked by prominent actin bundles (Figure 8A-D). Upon H-7 treatment for 15 min, stress fibers and the actin bundles flanking the cell-cell adherens junctions were disrupted, whereas the b-catenin-rich cell-cell adhesions were maintained (Figure 8E-L). FRI analysis showed that the residual actin filaments were not associated with these b-catenin containing adhesions (Figure 8H). By 60 min of H-7 exposure (Figure 8I-L), b-catenin-containing cell-cell junctions were still present but less well organized. FRI indicated that the actin:b-catenin ratio at cell-cell junctions dropped to below 0.1, indicating a relative loss of actin from these structures (Figure 8H). Removal of H-7 resulted in a rapid re-formation of actin filaments and b-catenin-positive structures (Figure 8M-T). Five min after H-7 withdrawal, new stress fibers were formed throughout the ventral aspects of the cells and were also associated with b-catenin-containing cell-cell junctions. By 20 min cell-cell b-catenin-containing adherens junctions flanked by actin bundles were apparent (Figure 8M-T).

Discussion

In this study, the 2-dimensional topographic view of HTM cells showed that H-7-treated cells appeared to round or thicken toward the center in a dose- and time-dependent manner, based on increased refractility of the cells. By inhibiting actomyosin contractility, H-7 secondarily alters actin cytoskeleton organization and the associated focal adhesion complexes [7,11,12], thereby altering cell shape. The changes in cell morphology were consistent with those in the actin cytoskeleton and associated cellular adhesions, indicating that the increased cell refractility appeared subsequent to the H-7-induced disruption of the actin cytoskeleton and cellular adhesions, especially focal adhesions. It was suggested by Bill, Svedbergh, and Mäepea [13,14] that the proper attachment of SC inner wall endothelial cells to the subendothelial tissue is important for the formation of invaginations and pores, the vacuole-like structures in the inner wall cells through which aqueous humor reaches the canal lumen. If the attachment is too strong, the invagination process may not take place; if it is too weak, the inner wall cells might detach [14]. H-7 may loosen focal adhesions but not detach the cells, thus facilitating the formation of invaginations of the inner wall cells and enhancing outflow. Based on H-7's cytoskeletal effects, we may also hypothesize that H-7 can destabilize the overall architecture of the trabecular meshwork by inducing changes in cell shape and cellular attachments, opening the flow pathways and forming new flow routes [9], and thereby reducing flow resistance and IOP [1]. The data suggest that the cytoskeletal system is involved in the normal regulation of aqueous humor outflow resistance and the pathophysiology of certain forms of glaucoma [3,15], and may be targets for H-7 and other compounds capable of increasing outflow facility in vivo. The consistency of our data with studies of H-7-induced changes in outflow facility and TM ultrastructure supports this hypothesis. In monkeys, the maximal H-7-induced IOP reduction occurred at 2 h [1], when considerable disruption of actin and focal contacts was observed in cultured HTM cells. In addition, the H-7-induced outflow facility increase and cellular relaxation in the TM were reversible after the drug was removed from the anterior chamber (AC) for 2.5 h [16], while after 2 h in H-7 free medium, recoveries of actin and associated adhesion molecules of HTM cells were almost complete. However, the cellular mechanisms underlying the outflow increasing effects of various cytoskeletal compounds may not be the same. For example, latrunculin (LAT)-A, which increases facility about as much as H-7, disassembles actin filaments by sequestering monomeric actin, and preferentially affects intercellular adherens junctions [10,17,18]. Cytochalasin B results in an increase in the hydraulic conductivity of cultured monolayers of HTM cells, accompanied by a retraction of the cells and widening of the intercellular spaces [19], and a disruption of endothelial lining and a washout of extracellular material in TM tissue [20,21]. Calcium chelators, which primarily affect the intercellular junctions, produce a similar cell separation and outflow facility increase in living monkeys [22-24].

The role of intercellular junctions in H-7's ability to enhance outflow across the trabecular meshwork is not clear. Consistent with previous findings [3], our study showed that most cells still remained attached to their neighbors, even at high doses and long duration of H-7 treatment. Studies with other non-ocular cell types [1,7,25] also demonstrated dose- and time-dependent disruption of the actin cytoskeleton and cell-matrix adhesions, with only a relatively modest effect on intercellular junctions following H-7 treatment. Sabanay et al [9] determined the effects of H-7 on TM structure and fluid conductance of live monkeys using colloidal gold as a flow tracer. The data showed that H-7 expanded the intercellular spaces in the juxtacanalicular meshwork accompanied by removal of ECM material, and extended the SC inner wall cells, but had little effect on cell-cell junctions. As a result, gold tracer was widely seen along the inner canal wall but failed to pass between SC cells. It is possible, however, that the intercellular spaces may become slightly "leakier" to aqueous humor but not to gold particles upon H-7 treatment. This hypothesis is consistent with other studies [8,25,26]. In cultured Madin Darby Canine Kidney epithelial cells, H-7 prevented tight junction assembly through its effect on the cytoskeleton, associated with a decrease in transepithelial resistance but without interfering with adherens junction assembly. Taken together, these observations indicate that, as previously proposed [3], even trivial changes in intercellular junctions might also contribute to the effects of H-7 on outflow facility.

In this study, we employed digital microscopy and multiple image processing to characterize the molecular dynamics of various components of the actin cytoskeleton and the associated cellular junctions in cultured HTM cells in response to H-7 treatment. Vinculin-actin FRI revealed largely comparable distribution of the two proteins in focal contacts and in cell-cell junctions. Phosphotyrosine-paxillin FRI pointed to a highly similar labeling (i.e., uniform FRI) of focal contacts, suggesting an active involvement of tyrosine phosphorylation in assembly of focal contacts [5]. b-catenin-actin FRI showed that the cell-cell junctions were flanked by prominent actin bundles as expected from a typical zonula adherens [5]. FRI analysis of the cells at different time points indicated that phosphotyrosine was indeed more sensitive to H-7 than was paxillin. The loss of phosphotyrosine from focal contacts early after addition of H-7 suggests that tyrosine phosphorylation may be preferentially affected as an immediate result of H-7-induced cellular relaxation, leading to the suppression of substrate adhesion formation. However, the dynamics of phosphotyrosine in actin-derived cellular tension as well as intercellular interaction in HTM cells with or without H-7 treatment remains unknown. Clearly more work, including FRI analysis of phosphotyrosine vs. actin and/or intercellular adhesion and other focal adhesion molecules, is needed to elucidate the role of phosphotyrosine in the cellular and molecular basis for the effects of H-7 and other compounds acting via the cytoskeleton on outflow facility.

In previous studies in living monkeys [1], slit lamp examination revealed transient and mild punctate corneal epithelial defects and slight epithelial cloudiness following a topical dose of 150 or 400 mM H-7. The maximal facility-effective dose of topical H-7 (400 mM) also transiently increased the protein concentration in the AC, the rate of entry of i.v. fluorescein into the AC aqueous humor and cornea, and the corneal thickness, and caused the corneal endothelial cell borders to become indistinct on specular microscopy. However, no changes were found in the corneal endothelium and ciliary epithelium by light and electron microscopy, or in the lens and AC by slit lamp biomicroscopy, following a maximal facility-effective dose of intracameral H-7 (300 mM) [1]. This difference is likely due to different drug concentrations in the AC and cornea after different routes of drug administration. The high concentration (400 mM) and small volume (5ml x 4) formulations used in the topical drug protocols place the central cornea, onto which the drops are placed with blinking prevented, at a disadvantage, so that the cornea "sees" a much higher dose with topical than with intracameral administration [27]. Additionally, when penetrating into the cornea after topical administration, H-7 would reach the corneal endothelial cells and their focal contacts from the basement membrane (Descemet's membrane) side. However, when H-7 is delivered intracamerally, the presence of apical tight junctional complexes (maculae occludents) between adjacent cells, may prevent or inhibit the passage of H-7 from the aqueous humor to the lateral and basal aspects of the corneal endothelial cells [28,29].

It is not entirely clear why 300 mM intracameral H-7 induces cytoskeletal changes in the TM/SC but not corneal endothelium and ciliary epithelium. One possible explanation could be that H-7 might affect the TM at lower concentrations than the cornea because of their different tissue architecture and physiologic milieu. The TM is a suspended multilayered tissue, in which juxtacanalicular cells have no real basement membrane and the SC inner wall cells have only a thin, diaphanous, discontinuous basement membrane. When cellular contractility is inhibited by H-7, the TM can be readily distorted and distended by fluid flow down the pressure gradient between the AC and SC. However, the corneal endothelium is a single cell layer on a well-defined basement membrane/ECM structure (Descemet's membrane and stroma) with much less fluid flow across it than the TM and is thus less easily distended or distorted even when the contractile apparatus, and consequently cellular adhesions, are weakened. Similarly, the ciliary body geometry is less likely to be perturbed than the TM. In addition, the juxtacanalicular cells will be continuously bathed on all sides by drug-containing aqueous humor, while the other AC tissues will have at least one aspect facing away from the aqueous. Especially for the posteriorly located ciliary body, the continuous anteriorly directed aqueous flow may result in the ciliary epithelium being exposed to a much lower drug concentration compared to the TM [27].

Nonetheless, potential H-7-induced cytotoxicity is of concern. Our previous and current studies showed that H-7's outflow facility-increasing effect in live monkeys and the effects of H-7 on cell shape, actin cytoskeleton and cellular adhesions were all reversible after drug removal, and no apparent cell death was observed [1]. Particularly, rapid and almost full morphological recovery of HTM cells treated with H-7 occurred even at a 600 mM dose, which is twice the highest in vivo intraocular concentration studied [1]. Those data, plus the absence, or at most, the mild and transitory nature of any ocular side effects following H-7 treatment in vivo, indicate that the reversible changes we observed in cell shape and adhesions induced by H-7 at a 300 mM or lower dose may reflect a pharmacological induction of normal physiologic responses of cells in vivo and in vitro to ambient conditions. Other cytoskeleton-disrupting compounds such as LAT-A and B [17,18] and cytochalasins B and D [30,31] also induce large but reversible increases in outflow facility, again suggesting that the outflow facility increasing effects of such cytoskeletal agents can be relatively gentle and reflect a near-physiological modulation of a basic flow control mechanism rather than irreversible cytotoxicity. Such treatments, if targeted specifically to cells of the outflow pathway, augur well for potential clinical applicability.

Acknowledgements

Mary Ann Croft, MS assisted with statistical analysis of cellular topography and Baohe Tian, MD provided valuable editorial contributions. This work was supported by grants EY02698 (PLK), EY02477 (JRP) and EY07336 (CRB) from the National Eye Institute, Bethesda, Maryland; Research to Prevent Blindness, New York, New York (PLK); the Glaucoma Research Foundation, San Francisco, California (PLK and BG); American Health Assistance Foundation (CRB); Yeda Research and Development, the E. Neter Chair in Cell and Tumor Biology, the Minerva Foundation, and the Israel Science Foundation, Rehovot, Israel (BG).

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