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(Investigative Ophthalmology and Visual Science. 2007;48:2152-2161.)
© 2007 by The Association for Research in Vision and Ophthalmology, Inc.
DOI:  10.1167/iovs.06-1271
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The Rho-Kinase Inhibitor H-1152P Suppresses the Wound-Healing Activities of Human Tenon’s Capsule Fibroblasts In Vitro

Aysegül Tura, Salvatore Grisanti, Katrin Petermeier, and Sigrid Henke-Fahle

From the University Eye Hospital of the Center of Ophthalmology at the Eberhard-Karls University of Tübingen, Tübingen, Germany.


   Abstract
Top
 Abstract
Materials and Methods
 Results
 Discussion
 References
 
PURPOSE. To analyze the role of Rho-kinase signaling in the wound-healing activities of human Tenon’s capsule fibroblasts by using H-1152P, a potent inhibitor of this kinase, in vitro.

METHODS. The optimal concentration of H-1152P was determined by MTT test. Cell proliferation was measured by BrdU incorporation and Ki-67 immunostaining, whereas cell viability was investigated by ethidium homodimer-1 dye exclusion. The actin cytoskeleton organization was demonstrated by {alpha}-smooth muscle actin (SMA) immunostaining and Alexa 488-phalloidin staining. Cell migration was studied on restrained collagen gels and in a scratch-wound assay followed by Ki-67 and fibronectin immunostaining. The effect of H-1152P on contraction was analyzed in floating collagen gels populated with fibroblasts, which were subsequently processed for fibronectin immunostaining. The levels of adducin and the protein kinase A (PKA)–dependent phosphorylation of this protein were detected by immunoblot analysis, to rule out interference with PKA.

RESULTS. Incorporation of BrdU and upregulation of Ki-67 were reduced by 80% to 90% in cells incubated with 10 µM of this inhibitor for 4 days (P < 0.01). H-1152P caused the disassembly of stress fibers in a dose-dependent manner without exerting toxic effects and without a considerable interference with the PKA-pathway. H-1152P also significantly suppressed cell migration 3- to 3.5-fold and the contraction of collagen lattices fivefold with a dose-dependent impairment in the assembly of the fibronectin network.

CONCLUSIONS. These findings imply a role for Rho-kinase in the wound-healing activities of human Tenon’s capsule fibroblasts and show the potential of H-1152P as a safe and specific means to suppress these events.

Glaucoma filtration surgery is the most frequently used procedure for reducing intraocular pressure (IOP) in patients with glaucoma who failed to respond well to pharmacological therapy or laser treatment.1 2 Elevated IOP, the major risk factor that may be responsible for the optic nerve injury in glaucoma, arises when aqueous humor outflow through the natural pathway is impeded.3 The purpose of glaucoma filtration surgery is to create a scleral fistula that enables the drainage of aqueous humor from the anterior chamber to the subconjunctival space. Although this method has an immediate effect on lowering the IOP, the fibroproliferative response within the filtration area may prevent long-term success.4 5 6

A variety of pharmacologic agents such as corticosteroids and antimetabolites have been in clinical use to increase the success rate of filtration surgery.7 However, some antimetabolites such as fluorouracil and mitomycin C exert their effects by causing cell death,8 9 and diffusion into adjacent ocular tissues may result in impairment of cells other than the targeted ones,10 11 inducing vision threatening complications. This risk eventually necessitated the development of safer strategies, such as the administration of monoclonal anti-TGF-ß antibodies,12 photoablation at the site of surgery,13 and application of decorin, a naturally occurring proteoglycan inhibiting TGF-ß and PDGF.14

The wound-healing response of fibroblasts comprises dynamic events like proliferation, motility, and contractility, which require the continuous remodeling of the actin cytoskeleton. Identifying the intracellular signaling pathways that govern the cytoskeletal arrangement in Tenon’s capsule fibroblasts may therefore constitute the basis of alternative therapies specifically targeting the key mediators of these cascades. A central role in the organization of the actin cytoskeleton during various motile events is attributed to the RhoA protein from the Rho family of small GTPases.15 RhoA coordinates these events by transmitting the extracellular signals to downstream effectors such as the Rho-associated coiled-coil kinase (Rho kinase/ROCK). ROCK is a serine-threonine protein kinase that mainly promotes myosin II activity by inhibiting the myosin light-chain phosphatase and phosphorylating myosin light chain. This in turn favors the assembly of actin-myosin filaments that generate the tensile strength underlying the RhoA-ROCK–associated dynamic events in numerous cell types,16 such as the contraction of ciliary muscle and trabecular meshwork cells. Administration of the ROCK inhibitors HA-1077, Y-27632, and H-1152P was reported to induce the relaxation of these cells by suppressing myosin light-chain phosphorylation and promote the outflow of aqueous humor.17 18 19 Moreover, a very recent study reports the activation of RhoA in human Tenon’s capsule fibroblasts stimulated with TGF-ß and the reduction in TGF-ß-induced fibroblast contractility in response to the ROCK inhibitors, providing the first evidence of the involvement of the RhoA-ROCK pathway in an essential aspect of wound-healing in these cells.20

To acquire further insight into the role of ROCK-signaling in the wound-healing response of human Tenon’s capsule fibroblasts, we used H-1152P, the most specific of the currently available ROCK inhibitors,21 in the present in vitro study. The outcomes of H-1152P treatment on the proliferation, survival, and cytoskeletal organization of serum-stimulated fibroblasts were characterized by biochemical and morphologic analyses. Furthermore, the role of H-1152P on fibroblast motility and collagen gel contraction were investigated with particular emphasis on the extent of fibronectin network assembly. Last, the specificity of H-1152P action was analyzed to ensure that the observed effects reflected the outcomes of ROCK-inhibition.


   Materials and Methods
Top
 Abstract
 Materials and Methods
Results
 Discussion
 References
 
Cell Culture
Samples of human Tenon’s capsule were obtained from five patients undergoing glaucoma filtration surgery, according to the tenets of the Declaration of Helsinki for the use of human tissue, and informed consent was obtained from the subjects after explanation of the nature and possible consequences of the study. Strips of tissue were dissected into 1- to 2-mm cubes and maintained in DMEM/F-12 (1:1) medium supplemented with 10% fetal calf serum (FCS), 2 mM L-glutamine, 100 U/mL penicillin, and 100 µg/mL streptomycin (all from Invitrogen, Carlsbad, CA) at 37°C in a humidified atmosphere with 5% CO2. The fibroblasts migrating from these tissues were harvested after approximately 3 weeks. Cells for subculture or experimentation were detached from the culture flasks by incubation with 0.05% trypsin and 0.02% EDTA (Invitrogen), centrifuged, and suspended in fresh medium containing FCS unless otherwise stated. The cells between the third and seventh passages were used for the experiments. Incubations with H-1152P (Calbiochem, Darmstadt, Germany) were performed without replenishing the inhibitor, to detect whether this inhibitor can exert long-lasting effects.

Growing Fibroblasts on Collagen-Coated Coverslips
Type I collagen from rat tail (Roche, Penzberg, Germany) was dissolved in sterile 0.2% acetic acid at a final concentration of 2 mg/mL. A thin film of this solution was spread onto sterile coverslips with a diameter of 10 mm (~5 µg/cm2) and air-dried in the laminar flow hood for 1 hour. The coverslips were then rinsed with phosphate-buffered saline (PBS) and placed immediately in 24-well plates. Fibroblasts were added at a density of 3000 cells/coverslip, allowed to attach for 3 hours, and incubated with or without H-1152P in duplicates for 4 days.

Migration of Fibroblasts on Collagen Gels
Collagen gels with an approximate depth of 1 mm were prepared by mixing a cold type I collagen solution (Cellon, Strassen, Luxembourg) with 10x modified Eagle’s medium (MEM, Invitrogen), 0.2 M HEPES, and 0.2 N NaOH on ice at a ratio of 8:1:1 (vol/vol/vol) and casting 25 µL of this solution into each well (6.5 mm diameter) of a sterile 10-well slide placed in a 100-mm diameter Petri dish. To prevent the drying of gels, 1 to 2 mL of culture medium without serum was pipetted into the dish avoiding contact with the slides, and the gels were allowed to polymerize at 37°C. Fibroblasts were harvested by trypsinization and suspended in culture medium without serum at a concentration of 1 x 106 cells/mL. The polymerized gels were inoculated with 1 µL of this cell suspension at the center, and the cells were allowed to adhere for 2 to 3 hours at 37°C. The gels were then covered with culture medium containing 10% FCS and incubated with or without H-1152P. Images of the cells were acquired using a digital camera connected to an inverted microscope (Axiovert; Carl Zeiss Meditec GmbH, Göttingen, Germany) immediately after the attachment of the cells (T0) and at certain time points throughout a 14-day incubation. The area occupied by cells on each gel was calculated using an image analysis program (Axiovision; Carl Zeiss Meditec GmbH). At the end of the incubation, the cells were fixed for 10 minutes in 4% paraformaldehyde (PFA) and processed for immunofluorescence staining.

Scratch-Wound Assay
Fibroblasts harvested by trypsinization were seeded in 24-well plates at a concentration of 5000 cells/well and grown until reaching confluence. A wound was gently introduced in the center of the cell monolayers using a sterile 1000-µL pipette tip. To remove the cell debris, the wells were washed twice with PBS, and the cells were incubated with or without H-1152P, in duplicate for 48 hours. Phase-contrast images of marked regions along the wound area were obtained using an inverted microscope (Axiovert; Carl Zeiss Meditec GmbH) immediately after creating the wound and at the end of 48 hours. The initial wound area and the areas that remained unoccupied by cells after 48 hours were measured using the software (Axiovision; Carl Zeiss Meditec GmbH). After the incubation, the cells were fixed for 10 minutes in PFA and processed for immunostaining.

Contraction Assay
Before the collagen gels were prepared, fibroblasts were harvested by trypsinization, washed once with complete medium, and suspended at a concentration of 4 x 105 cells/mL. A cold solution of type I collagen (3 mg/mL in 0.2% acetic acid, Roche) was mixed with 10x MEM and 0.2 M HEPES and 0.2 N NaOH on ice at a ratio of 8:1:1 (vol/vol/vol). An equal volume of cell suspension was added into the neutralized collagen solution to give a final concentration of 1.2 mg/mL collagen and 2 x 105 cells/mL. The solution was cast into the wells of a 24-well tissue culture plate (500 µL/well) and allowed to polymerize at 37°C. The gels were then gently detached from the plastic surface using a sterile pipette tip and incubated with or without H-1152P for 7 days in duplicate. Images of the gels were acquired by scanning the culture plate with a flatbed scanner (GT-9600; Epson, Meerbusch, Germany) immediately after the detachment of the gels and at several time points throughout the incubation. The area of the gels was determined by using image-analysis software (Soft Imaging System, Münster, Germany).

Processing Three-Dimensional–Collagen Gels for Immunohistochemistry
Collagen gels in 24-well plates were washed with PBS, fixed in 4% PFA for 30 minutes, incubated in 4% sucrose-PBS overnight at 4°C, and kept in 20% sucrose-5% glycerol for 2 days at 4°C. The gels were embedded in OCT compound (Sakura Finetec, Torrance, CA) and 16 µm sagittal cryosections were prepared. The sections were fixed in ice-cold acetone for 10 minutes, air-dried, and stored at –20°C. To stain the nuclei in whole gels after fixation, they were permeabilized in 0.1% TritonX-100/PBS (PBST) for 15 minutes, incubated with 0.5 µg/mL DAPI (4',6'-diamino-2-phenylindole; Invitrogen-Molecular Probes, Eugene, OR) in PBS for 5 minutes, washed in PBS, and mounted in Mowiol (Sigma-Aldrich, Steinheim, Germany). The gels were analyzed by fluorescence microscopy (Carl Zeiss Meditec, GmbH) with commercial software (Openlab; Improvision, Tübingen, Germany).

Immunofluorescence Staining
After a 10-minute fixation in 4% PFA, the cells were washed with PBS, blocked with 3% BSA in PBST for 20 minutes, and incubated with monoclonal antibodies against Ki-67 (clone Ki-S5, 1:100 dilution in blocking buffer; Dianova, Hamburg, Germany) or {alpha}-SMA (1:75 dilution; Dianova) overnight at 4°C in a humidified chamber. After three washes of 5 minutes each in PBS, the cells were incubated for 1 hour with Cy3- or Alexa488-conjugated anti-mouse antibodies, to detect Ki-67 and {alpha}-SMA, respectively (diluted 1:400 in blocking buffer; Jackson ImmunoResearch, West Grove, PA), and counterstained with DAPI. The immunostaining against fibronectin was performed with the same protocol, omitting Triton X-100 from the blocking buffer and using a polyclonal antibody against fibronectin (1:400 dilution; Dianova) that was detected with a Cy3-conjugated goat anti-rabbit secondary antibody (1:400 dilution; Jackson ImmunoResearch). For Ki-67-fibronectin double immunostaining, the Ki-67 protein was detected with an Alexa488-conjugated goat anti-mouse antibody (1:400 dilution; Invitrogen-Molecular Probes).

Alexa488 Phalloidin Staining
Fibroblasts grown in uncoated 96-well plates were fixed for 10 minutes with 4% PFA, permeabilized with PBST for 5 minutes, preincubated with 1% bovine serum albumin (BSA)-PBS to prevent nonspecific binding, incubated with Alexa488-phalloidin (Invitrogen-Molecular Probes) diluted in 1% BSA-PBS at a final concentration of 5 U/mL for 30 minutes, and counterstained with DAPI.

MTT Test
Fibroblasts were seeded into 96-well cell culture plates at densities specified in the Results section, with 8 to 10 wells for each treatment group. The cells were allowed to settle for 3 hours and cultivated for 1 to 4 days in the presence or absence of H-1152P in a total volume of 200 µL per well. Twenty microliters of MTT stock solution (5 mg/mL in PBS, Sigma-Aldrich) was added to each well, and the cells were incubated at 37°C for 3 hours. The solution was discarded by gently inverting the plates, and the wells were filled with 200 µL of lysis solution (0.6% acetic acid and 10% SDS in dimethyl sulfoxide [DMSO]). After the plates were shaken vigorously for 20 minutes, the absorbances in each well were read with a spectrophotometric plate reader (SLT Spectra 400; ATX, Salzburg, Austria) at 570 nm with background subtraction at 690 nm.

BrdU Incorporation
Fibroblasts were seeded into 96-well plates and grown for 4 days as described earlier. Incorporation of BrdU into DNA was measured with a colorimetric detection kit (BrdU Cell Proliferation Assay; Calbiochem) according to the manufacturer’s instructions. The BrdU label was introduced into the culture medium at the end of day 3 and was made available for the cells during the final 24 hours of incubation. Cells incubated without the BrdU label served as the negative control. The absorbances were read at 450 nm with background subtraction at 570 nm with a spectrophotometric plate reader. The mean absorbance for each group was determined after subtracting the mean value of the negative control.

Ethidium Homodimer-1 Staining
Ethidium homodimer-1 is a fluorescent nucleic acid dye that cannot penetrate intact cell membranes. The dye is thus excluded from healthy cells and is commonly used as an indicator of cell damage. Fibroblasts grown on collagen-coated coverslips were incubated with 4 µM ethidium homodimer-1 (Invitrogen-Molecular Probes) in 0.1% glucose-PBS for 30 minutes. After washing with 0.1% glucose-PBS, the cells were fixed, permeabilized, and counterstained with DAPI, as described earlier. Fibroblasts incubated with 70% ethanol for 20 minutes served as the positive control.

Western Blot Analysis
Fibroblasts grown in 75-cm2 flasks for 4 days were washed twice with ice-cold PBS. Five hundred microliters of ice-cold lysis buffer (50 mM Tris-HCl [pH 7.4], 150 mM NaCl, 1% NP-40, 1 mM EDTA, and 1% protease inhibitor cocktail, added just before use; Sigma-Aldrich) was added to each flask, and the flasks were kept on ice for 5 minutes. The cells were transferred into a microfuge tube and maintained under gentle agitation at 4°C for 15 minutes. Cell lysates were cleared by centrifugation at 12,000g for 20 minutes. Protein concentration of the supernatants was determined using the BCA assay (Pierce, Rockford, IL), according to the manufacturer’s instructions. Twenty micrograms of protein was separated in 12% denaturing SDS-PAGE gels and transferred onto nitrocellulose membranes. Staining with PonceauS (Sigma-Aldrich) was performed to verify equal protein loading. The destained membranes were blocked in 5% nonfat milk in Tris-buffered saline (TBS) containing 0.1% Tween-20 for 30 minutes and incubated with polyclonal anti-adducin or anti-phospho(S726)-adducin antibody (diluted 1:500 in blocking buffer; Abcam, Cambridge, UK) overnight at 4°C. After washing with TBS, the membranes were incubated with a biotin-conjugated secondary donkey anti-rabbit antibody (diluted 1:1000 in blocking buffer; Jackson ImmunoResearch) and with horseradish peroxidase–conjugated streptavidin (diluted 1:1000 in blocking buffer; Dianova) for 1 hour at room temperature each. Signal detection was performed by incubating the membranes for 2 minutes with enhanced chemiluminescence solution (10 mL of 0.025% luminol in 0.1 M Tris-HCl [pH 8.6] mixed with 3 µL of 30% H2O2 and 1 mL of 0.11% para-hydroxycoumaric acid in DMSO) and exposing the membranes to autoradiograph film (X-Omat; Sigma-Aldrich) overnight. The signal intensity was quantified with commercial software (Quantity One, ver. 4.6.2; Bio-Rad, Munich, Germany).

Statistical Analysis
The data were analyzed by two tailed Student’s t-test, and P < 0.05 was accepted as significant.


   Results
Top
 Abstract
 Materials and Methods
 Results
Discussion
 References
 
Optimal Concentration of H-1152P
To analyze the cycling rate of Tenon’s capsule fibroblasts as well as the effect of various concentrations of H-1152P on this process, we initially performed MTT tests on cells seeded at densities decreasing serially from 5000 to 625 cells per well and incubated with or without H-1152P for 1, 2, and 4 days. After 24 hours, the addition of H-1152P induced a slight but nonsignificant decrease in the extinction values (Fig. 1a) . This pattern appeared essentially unchanged until the end of day 2 (Fig. 1b) . On day 4, the extinction values in untreated cells increased approximately twofold, and the administration of H-1152P at 1, 10, and 50 µM resulted in significant decreases (16%, 34%, and 51%, respectively; P < 0.0005). This H-1152P-dependent decrease became more pronounced at lower seeding densities, amounting to 27% (P < 0.03), 60% (P < 0.0005), and 67% (P < 0.005) reduction with 1, 10, and 50 µM H-1152P, respectively, in fibroblasts plated at a density of 625 cells/well. Because the decrease observed after incubation with 50 µM H-1152P did not significantly surpass the effect obtained with 10 µM, we deduced the latter concentration of this inhibitor as being optimal for causing a considerable reduction in the amount of viable fibroblasts (Fig. 1c) .


Figure 1
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  		FIGURE 1. Dose-dependent effect of H-1152P on the amount of viable fibroblasts. Cells seeded at densities varying between 5000 and 625 cells/well were incubated with or without H-1152P for (a) 1 day (n = 5 experiments), (b) 2 days (n = 3 experiments), and (c) 4 days (n = 4 experiments), after which they were subjected to an MTT test. *P < 0.03, **P < 0.001.

 
Antiproliferative Effect of H-1152P
To clarify whether the H-1152P-dependent reduction in the amount of viable fibroblasts results from an impairment of cell proliferation or the toxicity of this inhibitor, we measured the levels of BrdU incorporation into the DNA of proliferating fibroblasts that were plated at the densities mentioned earlier and incubated with or without H-1152P for 4 days. The extinction values of untreated cells revealed a gradual increase at lower seeding densities, which were reduced by H-1152P administration in a dose-dependent manner. Addition of 10 µM H-1152P was sufficient to keep the extinction values between 0.1 and 0.135, regardless of the initial cell number, accounting for significant decreases in cell proliferation by 80% to 90%, compared with that in control cultures (P < 0.01, Fig. 2 ).


Figure 2
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  		FIGURE 2. BrdU incorporation into fibroblasts during the last 24 hours of a 4-day incubation period. The mean absorbances were calculated from three independent experiments with the exception of the 5000 cells/well group (n = 2). *P < 0.04, **P < 0.01, ***P < 0.00005.

 
To further confirm the antiproliferative effect of H-1152P, immunostaining was performed with antibodies against Ki-67, a nuclear protein associated with cell proliferation,22 on fibroblasts that were grown for 4 days on collagen-coated coverslips. Our results revealed an upregulation of Ki-67 expression in approximately 50% of the nuclei in the untreated control cultures, whereas only approximately 5% of the cells stained positively for this marker after treatment with 10 µM H-1152P (P < 0.01, Figs. 3a 3b ).


Figure 3
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  		FIGURE 3. The antiproliferative effect of H-1152P on Tenon’s capsule fibroblasts. (a) Fibroblasts grown on collagen-coated coverslips were incubated for 4 days and used for Ki-67 immunostaining to analyze cell proliferation. DAPI counterstaining was performed to visualize the nuclei. (b) Quantification of (Ki-67)+ nuclei (n = 2 experiments); **P < 0.01. (c) Ethidium homodimer-1 (EthD-1) staining demonstrating the viability of fibroblasts after 4 days in contrast to the cells of the positive control which were treated with 70% ethanol. The images for Ki-67 and EthD-1 stainings are representative of two independent experiments. Bar, 25 µm.

 
The extent of cell damage at this time point was determined by performing ethidium homodimer-1 staining. Incubating the fibroblasts with 70% ethanol induced significant cellular toxicity, as demonstrated by the strong ethidium homodimer-1 staining in the positive control cells. In contrast, very weak staining was observed in both the H-1152P-treated and untreated fibroblasts grown on collagen-coated coverslips, indicating the viability of these cells. The number of DAPI-stained cells was reduced in a dose-dependent manner without an accompanying increase in the intensity of EthD-1 staining in H-1152P-treated cells, suggesting that H-1152P inhibits the proliferation of Tenon’s capsule fibroblasts without exerting toxic effects (Fig. 3c) .

Short-Term Application of H-1152P
The application of a growth inhibitor as a single dose might be desirable for in vivo use. This method may be used when the inhibitor does not have a very short half-life or is able to exert long-lasting effects. To investigate whether a short-term exposure to H-1152P could also lead to long-lasting effects on the proliferation of Tenon’s capsule fibroblasts, we performed an MTT test on cells that were incubated for 1 day, with or without H-1152P and for 3 days in fresh medium lacking this inhibitor. Compared with the results obtained after continuous exposure to H-1152P for 4 days (Fig. 1c) , the short-term treatment with H-1152P resulted in a milder decrease of ~20% in the extinction values. This modest effect was statistically significant (P < 0.04) in all the groups except for the cells seeded at the lowest density (Fig. 4) .


Figure 4
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  		FIGURE 4. Short-term application of H-1152P. Fibroblasts seeded into 96-well plates were incubated for 24 hours, with or without H-1152P. The medium was then replenished and the cells were incubated further for 3 days in the absence of H-1152P. Cell proliferation was quantified by performing MTT test at the end of this period (*P < 0.04).

 
Rearrangement of the Actin Cytoskeleton in Response to H-1152P
The H-1152P-induced changes in the organization of the actin cytoskeleton were visualized by performing Alexa-phalloidin staining on fibroblasts grown for 4 days. Untreated fibroblasts exhibited numerous stress fibers aligned as parallel bundles. Administration of H-1152P led to a dose-dependent decrease in the abundance and length of these bundles, resulting in a gradually weakening staining for filamentous actin and a gain in cell diameter, accompanied by the sprouting of randomly oriented protrusions, the lengths of which exhibited a dose-dependent increase in H-1152P-treated cells (Fig. 5a) .


Figure 5
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  		FIGURE 5. Changes in the organization of actin cytoskeleton in response to H-1152P. (a) Fibroblasts seeded in 96 well-plates at a density of 1250 cells/well were fixed after 4 days of incubation and stained with Alexa 488-phalloidin and DAPI to visualize the actin filaments and cell nuclei, respectively. The images shown are representative of three independent experiments. (b) {alpha}-SMA immunostaining in fibroblasts after 4 days of incubation to detect the extent of myofibroblast differentiation. The staining appeared very weak in both the treated and untreated groups and localized mainly to the perinuclear region. The images demonstrate the few cells in which {alpha}-SMA organized as bundles could be detected and are not representative of the entire well. The experiment was performed twice with duplicates for each treatment group. Bar: (a) 50 µm; (b) 25 µm.

 
A particular event observed in normal tissues under circumstances requiring mechanical force development and in pathologic tissues with hypertrophic scarring is the transition of fibroblasts into myofibroblasts, an intermediate cell type between fibroblasts and smooth muscle cells, with stress fibers rich in {alpha}-SMA.23 To analyze the extent of this differentiation in Tenon’s capsule fibroblasts after 4 days, we performed immunostaining for {alpha}-SMA on cells that were grown as described earlier. Only a few {alpha}-SMA-positive cells were detected, both among the untreated fibroblasts and the cells that received H-1152P for 4 days. Yet, the staining in the control cells appeared more intense, revealing prominent {alpha}-SMA bundles (Fig. 5b) .

Fibroblast Migration in Response to H-1152P
The effect of H-1152P on the motility of Tenon’s capsule fibroblasts was studied by inoculating restrained collagen gels with a small colony of fibroblasts and measuring the area occupied by cells over 14 days as well as performing an in vitro wound-healing assay. Untreated fibroblasts in the former assay acquired an elongated shape with a protruding edge and spread out radially on the gel, invading an area approximately eight times larger than the initial size and reaching confluence at the end of 14 days. In the presence of 1 µM H-1152P fibroblasts did not seem to be impaired in this respect. However, the cells acquired a less polarized morphology, and the increase in area covered by the cells was reduced by a factor of 3 with 10 µM H-1152P (P < 0.03, Figs. 6a 6b ). Migration appeared to be restricted to the surface of the gels, at least for the first 14-days after the attachment of fibroblasts to the gel, since no cells were detected inside the gels in any of the treatment groups. However, a dose-dependent decrease was observed in the amount of fibronectin deposited into the extracellular matrix (ECM) in response to H-1152P. In untreated control cells, strongly stained long fibrils aligned parallel to the longitudinal axis of cells were detected, both at the inoculation zone and along the tracks toward the farthermost points reached by the cells, with the staining intensity being stronger at the center, possibly due to the higher cell density at this location. The micrographs demonstrating the fibronectin immunostaining are therefore presented separately for the center and the periphery, to avoid misinterpretation while comparing the staining intensity in the treated and untreated groups. The fibroblasts treated with 10 µM H-1152P were loosely arranged, and their ECM exhibited almost no fibrils (Fig. 6c) .


Figure 6
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  		FIGURE 6. Effect of H-1152P on fibroblast migration on restrained collagen gels. (a) Collagen gels on multiwell glass slides (n = 5–8 gels per slide) were inoculated with an equal amount of fibroblasts. After the attachment of cells, the slides were incubated with or without H-1152P, and the changes in the area occupied by cells were monitored over 14 days. The mean increase in area was calculated from two independent experiments (*P < 0.05, **P < 0.03). (b) Phase-contrast images of the cells on collagen gels. Arrowhead: the leading edge of an untreated cell. (c) Representative images of fibronectin immunostaining (orange) at the inoculation zone (center) and the periphery of the collagen gels covered with fibroblasts (blue) after 14 days. Bar, 50 µm.

 
The migration of Tenon’s capsule fibroblasts into the wound area created at the center of confluent monolayers of these cells was also suppressed by the ROCK-inhibitor in a dose-dependent manner. Untreated fibroblasts rich in stress fibers were able to migrate into the wound area and organize a dense cellular network, leaving only 10% of the initial wound area unoccupied after 48 hours. Administration of 10 µM H-1152P significantly suppressed the recovery, with 35% of the initial wound region left unpopulated at the end of 48 hours (Figs. 7a 7b ; P < 0.005, n= 2 experiments). The expression of Ki-67 in fibroblasts at the wound’s center and the deposition of fibronectin in this area also exhibited a dose-dependent reduction in response to H-1152P (Fig. 7c) .


Figure 7
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  		FIGURE 7. In vitro wound-healing assay. (a) Phase-contrast images of the fibroblasts immediately after the induction of the wound into the confluent cell layer (0 hours) and Alexa 488-phalloidin staining exhibiting the cellular organization at the wound center after 48 hours. (b) Percentage of wound area after 48 hours. The mean values are calculated from two independent experiments with duplicates for each treatment group. **P < 0.005. (c) Double immunostaining for Ki-67 (green) and fibronectin (orange) at the wound area after 48 hours. Bar: (a) 100 µm; (b) 25 µm.

 
Effect of H-1152P on Collagen Gel Contraction
Wound contraction facilitates the healing process by drawing the wound margins closer and by reducing the amount of new tissue necessary to re-establish integrity. Floating collagen gels populated with fibroblasts are commonly used in in vitro models simulating this process, particularly at the final stage of wound healing, when the fibroblasts exhibit a more quiescent phenotype with low proliferative capacity.24 25 26 27 In this study, the impact of H-1152P on wound contraction was investigated by measuring the area of fibroblast-populated floating collagen gels, incubated with or without this inhibitor over a 7-day period. The area of the collagen gels decreased by 25% at the end of 7 days in untreated control cultures, whereas only a 5% contraction was observed in the presence of 10 µM H-1152P (P < 0.005; Fig. 8a ). Untreated cells were densely packed under these 3D-culturing conditions and exhibited a dendritic morphology, whereas the cells that received H-1152P treatment were more loosely arranged, with distorted protrusions (Fig. 8b) . In all the treatment groups, cell density gradually declined from the center toward the periphery of the gels (Fig. 8c) . The immunostaining performed on cross-sections of the gels using anti-fibronectin antibodies revealed a similar trend in the assembly pattern of this protein, with stronger staining intensity at the center, possibly due to the local differences in the cell density. The fibronectin network appeared nevertheless more prominent in untreated gels, compared with the H-1152P-treated groups (Fig. 8d) .


Figure 8
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  		FIGURE 8. Effect of H-1152P on fibroblasts in three-dimensional collagen gel culture. (a) Fibroblast-populated collagen gels were gently detached from the wells in which they were cast and incubated with or without H-1152P (n = 2 gels per treatment group). The area of the collagen gels was measured over 7 days with an image analyzer program and the percentage of contraction at each time point was calculated with respect to the initial gel size. Data shown are the mean results of three independent experiments; *P < 0.04, **P < 0.01, ***P < 0.005. (b) Phase contrast images of the fibroblasts in collagen gels. Arrows: the protrusions, which acquire a more distorted morphology in response to H-1152P, (c) DAPI staining of the gels viewed from above and (d) fibronectin immunostaining on the cross-sections of gels showing the organization of cells and the deposition of fibronectin into ECM, respectively, after 7 days’ incubation, with or without H-1152P. Bar, 50 µm.

 
Specificity of H-1152P Action
Like most kinase inhibitors, H-1152P suppresses the activity of ROCK mainly by occupying the ATP-binding domain of this enzyme and thus depriving the kinase of its source for the {gamma}-phosphoryl group required for the phosphorylation of its substrates.28 However, owing to the sequence homology between the ATP-binding domains of ROCK and PKA, H-1152P can also inactivate PKA at higher concentrations.29 30 To clarify whether H-1152P interferes with the PKA pathway in Tenon’s capsule fibroblasts at the concentrations tested, Western blot analyses were performed to analyze the levels of PKA-dependent adducin phosphorylation in these cells. Adducin is a cytoskeletal protein that recruits the actin filaments to the spectrin-based membrane skeleton. The PKA-mediated phosphorylation of {alpha}-and {gamma}-adducin occurs at serine (S)726 and S662, respectively,31 which could be detected using antibodies recognizing these phosphorylated residues in our blots. The signal intensity of the bands normalized to the protein levels in the corresponding lane did not appear to be considerably altered for phospho(p)-{alpha}-adducin. Control blots performed using a polyclonal antibody recognizing {alpha}-adducin displayed a similar trend in the total amount of this protein, suggesting that the PKA-dependent phosphorylation of {alpha}-adducin is not impaired by H-1152P. In contrast, the levels of phospho-{gamma}-adducin appeared slightly reduced in response to H-1152P and it was not possible to detect the changes in the total level of this isoform. However, increasing the concentration of H-1152P did not result in a further decrease in the intensity of p-{gamma}-adducin bands. H-1152P treatment therefore appeared less likely to have significantly interfered with the PKA-dependent phosphorylation of the {gamma}-isoform (Fig. 9) .


Figure 9
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  		FIGURE 9. H-1152P appears less likely to interfere with the PKA-dependent phosphorylation of adducin. Representative Western blot showing the levels of {alpha}-and {gamma}-adducin (Mr ~103 and ~85 kDa) phosphorylated at Serine 726 and 662, respectively, by PKA in fibroblasts incubated for 4 days with or without H-1152P (phospho-adducin). Right: the total level of {alpha}-adducin in response to H-1152P treatment. The lower-molecular-weight bands on this blot may represent degradation products of {alpha}-adducin or correspond to other proteins weakly recognized by the antibody that became detectable after extended film exposure. The signal intensity of each band was normalized to the levels of the total protein in the corresponding lane (visualized by PonceauS staining). The ratio of the normalized signal intensity in treated fibroblasts to the signal intensity in control fibroblasts (C) is given below the respective lane. M: molecular weight marker (in kilodaltons).

 

   Discussion
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 Abstract
 Materials and Methods
 Results
 Discussion
References
 
In the present study, we provide further insight into the role of ROCK-mediated signaling in human Tenon’s capsule fibroblasts in vitro. Incubation of these cells with H-1152P, a highly potent inhibitor of ROCK, resulted in a dose-dependent reduction in the assembly of stress fibers, the contractile bundles of actin and myosin II that generate the tension within the cytoskeleton required for dynamic events. This inhibitor also significantly impaired the wound-healing activities of Tenon’s capsule fibroblasts without exerting toxic effects.

The antiproliferative effect of H-1152P on Tenon’s capsule fibroblasts was demonstrated by measuring BrdU incorporation and analyzing Ki-67 expression, which both displayed an 80 to 90% decrease after 4 days of incubation with 10 µM of this inhibitor. Owing to the absence of significant toxic effects in any of the groups, the H-1152P associated decrease in the cleavage of MTT, as determined by the MTT test, can also be interpreted as a reduction of the mitotic activity in these cells. However, the maximum decrease under these conditions was estimated to be 60% by the latter method. This discrepancy may have arisen from the parameters quantified by each procedure. The first two methods measure the extent of events that are hallmarks of cell proliferation,22 32 whereas the MTT test allows the estimation of viable cell amount based on the activity of mitochondrial dehydrogenases.33 However, the mitochondrial activity may not proceed at the same level in all phases of the cell cycle, as suggested by a study showing the decrease in adenosine triphosphatase function in HeLa cells during the S-phase and mitosis particularly within the endoplasmic reticulum and mitochondria.34 A possible reduction in the activity of mitochondrial dehydrogenases during the proliferative stages might therefore have obscured the real amount of fibroblasts undergoing these phases in our assays. The MTT test nevertheless allowed us to screen large amount of samples efficiently for the overall trend in cell proliferation or survival.

Regardless of the detection method used, the decrease in cell proliferation after continuous exposure to H-1152P for 4 days gained more significance in cells plated at lower densities (Figs. 1 2) , possibly due to contact inhibition at high seeding densities, which may have withdrawn the cells from further division and accounted for a less prominent difference between the treated and untreated groups. However, a 1-day exposure to this inhibitor was not as effective in suppressing the proliferation occurring on the following 3 days (Fig. 4) , especially under low-density seeding conditions that favored cell division, indicating that the continuous exposure to H-1152P is necessary to cause a stronger inhibitory effect on proliferation. These findings also suggest that the H-1152P present in the medium for 4 days still exerts an antiproliferative effect. The significant reduction in BrdU incorporation between days 3 and 4 in cells incubated with H-1152P without replenishing this inhibitor provides further support for this view. The half-life of H-1152P under different cell culture conditions or in vivo is to our knowledge not determined yet. However, our findings on Tenon’s capsule fibroblasts provide evidence of the stability of H-1152P, even though it was not possible to predict the exact amount of the inhibitor that remained active after 4 days in culture.

Administration of H-1152P also suppressed the migration of Tenon’s capsule fibroblasts on a restrained collagen matrix and into a wound area created in cell monolayers. Though it was not possible to exclude the contribution of cell proliferation from the outcomes, most of the cells treated with the ROCK inhibitor exhibited features suggestive of an impairment in motility, such as the absence of a distinct leading or trailing edge. The ROCK-pathway indeed plays an essential role in the protrusion of the leading edge in fibroblasts via clustering integrins, the receptors for ECM components like collagen and fibronectin, to the end of stress fibers, and in forming focal adhesions that enable the cells to adhere to the substratum.35 36 37 Furthermore, ROCK activity coordinates the retraction of the cell rear by localizing integrin to the leading edge of migrating monocytes and promoting actomyosin-based contractility.38 The H-1152P induced decrease in the amount of fibronectin deposited into ECM was another remarkable event detected in these migration assays. Fibronectin fibrils constitute a considerable portion of the wound matrix, promoting cell adhesion and providing a substrate for migration.39 Assembly of the fibronectin network requires the interactions between this protein and integrin receptors as well as an intact actin cytoskeleton,40 which is mediated by RhoA in a variety of cell types.41 Our results suggest the involvement of ROCK signaling in this process in human Tenon’s capsule fibroblasts, the impairment of which may have contributed to the reduction in the motility of these cells.

Fibroblasts cultured in collagen matrices have served as useful models to study wound contraction in vitro. The contraction of these gels appears to rely on the tractional forces generated by motile cells as the cells try to migrate through the matrix by drawing the proximal collagen fibers,42 43 as well as on the adhesive interactions between cells and matrix.44 45 Untreated Tenon’s capsule fibroblasts within a floating 3D-matrix extended rigid protrusions in contrast to the H-1152P-treated cells. The distortion of these processes in the latter group of fibroblasts, possibly due to cytoskeletal instability, may have hindered the establishment of strong connections with the matrix and accounted for the H-1152P-dependent reduction in lattice contraction. H-1152P also induced a less dense cellular organization and impaired the assembly of the fibronectin network within the gels. Fibronectin was indeed reported to be one of the factors promoting collagen matrix contraction.46 However, cellular fibronectin, rather than the serum isoform was found to be required for this process.47 48 The H-1152P-induced decrease in the assembly of a fibronectin matrix may suggest an association between the expression of this protein and ROCK activity in Tenon’s capsule fibroblasts, which remains to be investigated.

H-1152P is a very potent and specific ROCK inhibitor with a Ki of 1.6 nM for Rho-kinase, 630 nM for protein kinase A, 9.270 µM for protein kinase C, and 10.1 µM for myosin light-chain kinase in cell-free assays. However, higher concentrations of this inhibitor ranging between 0.1 and 10 µM are necessary for the suppression of ROCK in NT-2 cells,29 possibly because of competition with intracellular ATP present at the micromolar range.49 Our observations on Tenon’s capsule fibroblasts support these findings and underscore the need for high doses of this molecule for ROCK-inhibition in cell-based assays. However, this increases the risk of unintentionally targeting other kinases, with PKA being the most likely candidate owing to the relatively lower Ki of H-1152P for this kinase. PKA is a ubiquitously expressed intracellular signaling molecule that regulates ion channel conductivity, gene transcription, cell metabolism, actin cytoskeletal dynamics, and migration.50 PKA can also directly phosphorylate and inactivate RhoA.51 52 This broad spectrum of functions, some of which antagonize the activity of ROCK, may therefore give rise to complications or weaken the effects when an inhibitor showing differential affinity for both kinases is applied at high concentrations. To ascertain that the PKA pathway was not influenced by H-1152P in Tenon’s capsule fibroblasts, we analyzed the phosphorylation level of adducin isoforms at S726/S662 as an indicator of PKA activity. The PKA mediated phosphorylation mainly at S726 causes its dissociation from the F-actin cytoskeleton in vitro. The supernatants of the cell lysates we collected after centrifugation at 12,000g are expected to contain both the cytoskeleton-bound and the dissociated forms of adducin.53 Our immunoblots revealed no significant change in the levels of p-{alpha}-adducin and only a slight decrease in the level of p-{gamma}-adducin, which did not become more pronounced at increasing concentrations of H-1152P. Though the reason for this decrease remains to be elucidated, these findings favor the view that H-1152P exerted its effects on Tenon’s capsule fibroblasts without considerably interfering with PKA activity at the concentrations tested.

Taken together, these data demonstrate that ROCK is involved in the execution of essential dynamic events in human Tenon’s capsule fibroblasts and that H-1152P specifically inhibits this multifunctional kinase without inducing toxic effects. This, in turn, highlights the potential of H-1152P as an effective and safe means of suppressing the undesirable wound-healing activities of these cells for improving the success rate of glaucoma filtration surgery.


   Footnotes
 
Supported by Herbert Funke-Stiftung.

Submitted for publication October 23, 2006; revised December 25, 2006; accepted February 21, 2007.

Disclosure: A. Tura, None; S. Grisanti, None; K. Petermeier, None; S. Henke-Fahle, None

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Corresponding author: Aysegül Tura, University Eye Hospital of the Center of Ophthalmology at the Eberhard-Karls University of Tübingen, Schleichstrasse 12, 72076 Tübingen, Germany; ayseguel.tura{at}med.uni-tuebingen.de.


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