The Involvement of the Rho-Kinase Pathway and Its Regulation in Cytokine-Induced Collagen Gel Contraction by Hyalocytes

Abstract

purpose. To investigate the involvement of the Rho-kinase pathway in collagen gel contraction by hyalocytes.

methods. An in vitro type I collagen gel contraction assay using cultured bovine hyalocytes was performed to evaluate the effect of PDGF-BB and TGF-β2. The effect of both cytokines on the phosphorylation of myosin light chain (MLC) was analyzed by Western blot analysis. To confirm the involvement of the Rho-kinase pathway in the collagen gel contraction, the effects of Y27632, a specific Rho-kinase inhibitor were examined. The effect of hydroxyfasudil, another potent Rho-kinase inhibitor, was also evaluated. The expression of α-smooth muscle actin (αSMA) was analyzed by Western blot analysis to examine the myofibroblast-like transdifferentiation of the hyalocytes.

results. Maximum collagen gel contraction was observed within 24 hours after treatment with PDGF-BB and much stronger contraction with TGF-β2, whose effect was time dependent, at least up to 5 days. Although transient and maximum MLC phosphorylation by PDGF-BB was observed at ∼4 hours after stimulation (180.8%, P < 0.01), TGF-β2–elicited MLC phosphorylation occurred in a time-dependent manner at least up to 24 hours (220.0%, P < 0.01) and was maintained up to 5 days. Y27632 demonstrated significant inhibition of collagen gel contraction induced by both cytokines. Hydroxyfasudil dose-dependently (0.03–20.00 μM) prohibited the phosphorylation of MLC, and inhibited collagen gel contraction at a concentration corresponding to that which inhibited MLC phosphorylation. TGF-β2, but not PDGF-BB, also caused myofibroblast-like transdifferentiation with αSMA overexpression, which was downregulated by hydroxyfasudil in part (P < 0.01).

conclusions. The hyalocytes have a contractile property in the presence of PDGF-BB and TGF-β2. Whereas PDGF-BB initiates collagen gel contraction by transient activation of the Rho-kinase pathway, sustained activation of the Rho-kinase pathway and myofibroblast-like transdifferentiation appears to be involved in the TGF-β2–dependent contractile properties of hyalocytes.

Accumulating studies using both light and electron microscopes have indicated that hyalocytes are morphologically very similar to macrophages, and the most popular concept about their origin proposes that the hyalocytes originate from the blood monocytes. ^{1 2} The hyalocytes are therefore assumed to be resident macrophages in the vitreous cavity under physiological conditions and to be actively associated with the maintenance of vitreous transparency. ^{3 4 5 6}  

In contrast, it is thought that the hyalocytes themselves may be involved in vitreoretinal interface diseases such as idiopathic epiretinal membrane formation, macular hole, and diabetic macular edema. ^{7 8} Faulborn et al. ⁹ reported that human hyalocytes in diabetic eyes had a shape different from those in normal eyes, and their number seemed to increase. In addition, proliferative vitreoretinopathy (PVR) is a serious problem in patients with retinal detachments leading to severe vision loss or blindness despite the major advances in understanding their pathogenesis and the development of vitreoretinal surgeries. The pathologic course of PVR is characterized by the migration and proliferation of several cell types, including retinal pigment epithelial cells, glial cells, macrophages, and myofibroblast-like cells of unknown origin, which organize into a proliferative membrane. ^{7 10 11 12} It has been reported that the mononuclear phagocytes participate in the pathogenesis of PVR through production of cytokines that stimulate the migration and/or proliferation of other type of cells. ¹³ We hypothesized that the hyalocytes may contribute to the pathogenesis of PVR, not only by the production of cytokines to regulate the neighboring cells, but also by being one of the effector cell types exerting contractile force on the membrane as transformed myofibroblast-like cells. 

Platelet-derived growth factor (PDGF) and transforming growth factor (TGF)-β are multifunctional cytokines, and high levels of these cytokines have been found in the vitreous humor or epiretinal membrane of patients with PVR, ^{14 15 16 17} suggesting a possible association with the development of vitreoretinal disease. Both PDGF and TGF-β are known to modulate the proliferation and differentiation of many cell types and are considered to bring about the increase of extracellular matrix production, resulting in the formation and contraction of proliferative membranes. ^{18 19 20} Contraction of the proliferative membrane results in the progression of the diseased state. ¹⁸  

Myosin light chain (MLC) phosphorylation plays pivotal roles in smooth muscle contraction and in the actin-myosin interaction to form stress fibers and contractile rings in nonmuscle cells. ^{21 22} The phosphorylation state of MLC is dependent on at least two pathways. ²³ It has been reported that MLC phosphorylation is regulated by MLC kinase (MLCK) and MLC phosphatase ^{21 24 25} and is supposed to facilitate cell contractility and cell motility. ^{25 26} Recent studies have shown, however, that the phosphorylation state of MLC can also be regulated by Rho-kinase, which is the target protein of a small guanosine triphosphatase (GTPase) Rho. ^{23 27} Rho and Rho-kinase have been implicated in cell adhesion, migration, proliferation, and cytokinesis of vascular smooth muscle cells, ^{28 29 30 31 32 33} but their roles within the hyalocytes has yet to be clarified. 

Because cell-mediated contraction of tissues containing fibrillar collagens can be biologically advantageous during the contraction phase of wound healing and can lead to loss of organ function, pharmacologic treatment is needed in addition to the improvement of vitreoretinal surgery. Although the precise pharmacokinetics are still unclear, hydroxyfasudil, a metabolite of fasudil after oral administration, is already in clinical use for cerebral vasospasm and is a potent and selective inhibitor of Rho-kinase with minimum effect on other kinases, including MLCK and protein kinase C (PKC) as previously demonstrated by in vitro kinase assay. ³⁴  

We hypothesized that the hyalocytes, which mainly exist in the vitreal cortex, ³⁵ and the Rho/Rho-kinase pathway may be associated with the vitreoretinal interface diseases. In the present study herein, we demonstrated the contractile properties of hyalocytes in the presence of PDGF-BB and TGF-β2. The possible involvement of the Rho-kinase–mediated pathway in cytokine-induced collagen gel contraction by hyalocytes was also demonstrated, suggesting that the hyalocytes and Rho-kinase pathway might be therapeutic targets for the treatment of vitreoretinal interface diseases. 

Materials and Methods

Recombinant human PDGF-BB and TGF-β2 were purchased from Sigma-Aldrich, Tokyo, Japan). TGF-β2 was used in an active form by reconstitution of the lyophilized powder with phosphate-buffered saline (PBS) containing 4 mM HCl. Goat polyclonal antibodies against MLC and phosphorylated (p)MLC were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). A mouse monoclonal antibody against αSMA was obtained from Sigma-Aldrich. Y27632, a specific Rho-kinase inhibitor, was purchased from CalBiochem (La Jolla, CA). Hydroxyfasudil, a potent and selective Rho-kinase inhibitor, was generously provided by Asahi Kasei Pharma Corp. (Tokyo, Japan). 

Cell Isolation and Identification

Bovine hyalocytes were isolated as we previously reported, ³⁶ but with some modification. The posterior part of the vitreous body was extracted and washed once in Dulbecco’s modified Eagle’s medium (DMEM; Invitrogen-Gibco, San Diego, CA). The vitreous was chopped into several pieces and incubated in type I collagen–coated dishes in DMEM containing 10% fetal bovine serum (FBS; Invitrogen-Gibco) for 1 week. The cells that proliferated on the dishes were then subcultured in type I collagen–coated dishes in DMEM supplemented with 10% FBS as well. Cultured hyalocytes obtained between passages 2 and 5, which had shown no obvious morphologic change in the meantime, were used for the following experiments. Isolated cells were immunocytochemically identified as hyalocytes expressing S-100 protein, but neither expressed glial fibrillary acidic protein (GFAP) nor cytokeratin, as previously described. ³⁶  

Collagen Gel Contraction Assay

The contraction assay was performed as previously described. ³⁶ The hyalocytes were collected by the treatment of cultures with trypsin-EDTA for 3 minutes, washed with unsupplemented DMEM, and resuspended in DMEM at a density of 2.2 × 10⁶ cells/mL. Type I collagen (Koken Co., Ltd., Tokyo, Japan), two kinds of reconstitution buffer, hyalocyte suspension, and distilled water were mixed on ice at a ratio of 7:1:1:1:1 (final concentration of type I collagen gel, 1.9 mg/mL; final cell density, 2 × 10⁵ cells/mL). The resultant mixture (0.5 mL) was added to a 24-multiwell plate (Nunc, Roskilde, Denmark), and the formation of collagen gel was induced by incubation at 37°C under 5% CO₂ for 60 minutes. After the gels formed, 0.5 mL of DMEM containing 1% FBS was added to each well. The gels were freed from the walls of the culture wells with a microspatula 24 hours after gelling and used for the experiments. The diameter of the collagen gel was measured with a ruler at indicated time points after stimulation. For quantitative purposes, contraction data are presented as the reduction in diameter of the collagen gels. 

Phosphorylation of MLC

Subconfluent hyalocytes were starved in DMEM containing 1% FBS for 24 hours. The cells were then stimulated with 10 ng/mL of PDGF-BB (0.3 nM) or 9 ng/mL of TGF-β2 (0.3 nM) for the indicated time. The hyalocytes were washed once in cold 1× PBS and lysed in 1× Laemmli buffer (50 mM Tris [pH 6.8], 2% SDS, and 10% glycerol) containing protease inhibitors (1 mM phenylmethylsulfonyl fluoride [PMSF], 2 μg/mL aprotinin, 10 μg/mL leupeptin, 1 mM NaF, and 0.5 mM Na₃VO₄). ³⁷ The extraction was subjected to 15% SDS-PAGE and transferred to nitrocellulose filters (New England BioLabs, Beverly, MA). After the cells were blocked with 3% skim milk, the blots were incubated overnight at 4°C with an antibody against p-MLC (1:1000). After another wash, the membranes were incubated with horseradish-peroxidase–labeled rabbit anti-goat IgG (Bio-Rad, Richmond, CA; 1:4000) for 1 hour at room temperature. Visualization was performed with an enhanced chemiluminescence (ECL; Amersham, Arlington Heights, IL) detection system, according to the manufacturer’s instructions. Lane-loading differences were normalized by reblotting the membranes with an antibody against MLC (1:1000). The signal density was measured and analyzed on computer (NIH image, ver. 1.55; available by ftp at zippy.nimh.nih.gov/ or at http://rsb.info.nih.gov/nih-image; developed by Wayne Rasband, National Institutes of Health, Bethesda, MD), and quantified by the ratio of p-MLC to MLC. 

Determination of Hydroxyfasudil Concentration and Pretreatment Time

Subconfluent hyalocytes in regular culture conditions (DMEM containing 10% FBS) were treated with hydroxyfasudil for 30 minutes at indicated concentrations or for the indicated time (0, 2, 5, 15, 30, 60 min) at a concentration of 20 μM. Western blot analysis for p-MLC and MLC was performed as just described. 

Expression of αSMA by Hyalocytes Embedded in the Collagen Gels

Five days after the treatment, the medium was removed and 0.5 mL of serum-free DMEM containing 3 mg/mL collagenase (collagenase type II; Worthington Biochemicals, Freehold, NJ) was added to each well. They were incubated at 37°C until the collagen gels were dissolved. The hyalocytes were sedimented, and the supernatant was removed. The cells were lysed in 1× Laemmli buffer, and the same amount of protein from the extractions was subjected to Western blot analysis for the detection of αSMA (1:2000). 

Counting Viable Cells in Collagen Gels

The viable cells in the collagen gels were counted, to exclude the effect of cell growth or cytotoxicity on the collagen gel contraction or its inhibition. After 5 days’ treatment, the collagen gels were dissolved as described earlier, and the cell suspension was collected. The viable cells were counted with a hemocytometer after trypan blue staining. 

Statistical Analysis

The experimental data are expressed as the mean ± SD. Statistical significance was assumed at P < 0.05, determined with Student’s t-test in normally distributed populations. 

Results

Effect of PDGF-BB and TGF-β2 on Collagen Gel Contraction by Hyalocytes

We hypothesized that the hyalocytes might be involved in vitreoretinal diseases at least in part because of their contractile property. We thus confirmed the contractile property of hyalocytes in the presence of PDGF-BB or TGF-β2 in three-dimensional collagen gels. In this study, we used an established in vitro wound-contraction model involving a type I collagen gel with hyalocytes embedded in it. The control gels showed no apparent contraction up to 5 days, whereas PDGF-BB and TGF-β2 significantly decreased (P < 0.01) the collagen gel’s diameter (Fig. 1) . Collagen gels stimulated by PDGF-BB revealed a maximum contraction within 24 hours (13.8% contraction), and no further contraction was observed. In contrast, TGF-β2–induced collagen gel contraction occurred in a time-dependent manner, at least up to 5 days (51.8% contraction). 

MLC Phosphorylation by PDGF-BB or TGF-β2

To examine whether the hyalocytes’ contraction is associated with the phosphorylation of MLC, we investigated the effects of PDGF-BB and TGF-β2 on the phosphorylation state of MLC. PDGF-BB dephosphorylated MLC first, showing significant dephosphorylation 5 minutes after stimulation. Maximum phosphorylation of MLC was achieved at ∼4 hours after stimulation (180.8%) and declined thereafter. In contrast, the TGF-β2–dependent phosphorylation of MLC was gradually increased in a time-dependent manner up to at least 24 hours (220.0%, Fig. 2 ), and the phosphorylation state of MLC was maintained up to 5 days (data not shown). 

Involvement of the Rho-Kinase Pathway in Collagen Gel Contraction by Hyalocytes

We confirmed the involvement of the Rho-kinase pathway in cytokine-induced collagen gel contraction by hyalocytes by using the specific Rho-kinase inhibitor Y27632 (Fig. 3) . Five days after the addition of 10 ng/mL PDGF-BB or 9 ng/mL TGF-β2, the collagen gels were photographed and the diameters measured. Whereas PDGF-BB and TGF-β2 stimulated collagen gel contraction by 14.0% (P < 0.01) and 34.1% (P < 0.01), Y27632 significantly prohibited the contraction by up to 1.2% (P < 0.01 versus control plus PDGF-BB) and 6.0% (P < 0.01 versus control plus TGF-β2), respectively. 

These results indicate that both PDGF-BB–and TGF-β2–dependent collagen gel contraction substantially involve the Rho/Rho-kinase pathway. 

Inhibition of MLC Phosphorylation by Hydroxyfasudil

Hydroxyfasudil, an active metabolite of fasudil, preferentially inhibits Rho-kinase. ³⁴ To examine whether hydroxyfasudil inhibits the phosphorylation of MLC in hyalocytes, we first investigated the dose- and time-dependent inhibitory effect of hydroxyfasudil on MLC phosphorylation in regular culture conditions (DMEM containing 10% FBS). As shown in Figures 4A and 4B , hydroxyfasudil inhibited the phosphorylation state of MLC in a concentration-dependent manner, first showing significant inhibition at a concentration of 0.3 μM and almost complete inhibition at a concentration of 20 μM. Next, we examined the time-dependent inhibition of MLC by hydroxyfasudil at the concentration of 20 μM. As shown in Figures 4C and 4D , abrupt inhibition of MLC phosphorylation (or possible dephosphorylation) was observed within 2 minutes, with almost complete inhibition after 30 minutes, and this inhibitory effect was maintained at least up to 24 hours (data not shown). 

Inhibitory Effect of Hydroxyfasudil on PDGF-BB–and TGF-β2–Dependent MLC Phosphorylation

We next examined whether PDGF-BB–and TGF-β2–dependent MLC phosphorylation was also mediated through the Rho/Rho-kinase pathway, by using hydroxyfasudil at a concentration of 20 μM with pretreatment for 30 minutes. In accordance with the time points at which the cytokines significantly stimulated MLC phosphorylation, as described earlier, the stimulation periods were 4 hours for PDGF-BB and 24 hours for TGF-β2. As shown in Figures 5A and 5B , whereas PDGF-BB stimulated MLC phosphorylation 180.2% compared with the vehicle, hydroxyfasudil strongly attenuated the phosphorylation to the extent that it was 30.7% of the vehicle alone. TGF-β2–dependent MLC phosphorylation was dramatically decreased in the presence of hydroxyfasudil, as well (Figs. 5C 5D) . These results indicate that PDGF-BB–and TGF-β–dependent MLC phosphorylation is mainly mediated through the Rho/Rho-kinase pathway. 

Inhibition of Collagen Gel Contraction by Hydroxyfasudil

In this study, we demonstrated that the Rho/Rho-kinase pathway is substantially involved in collagen gel contraction by hyalocytes, theoretically suggesting that hydroxyfasudil has therapeutic benefits for the treatment of tractional vitreoretinal diseases. We thus confirmed the inhibitory effect of hydroxyfasudil on collagen gel contraction induced by PDGF-BB or TGF-β2 (Fig. 6) . As expected, whereas the collagen gel diameter was decreased by these cytokines by 13.8% and 51.8%, respectively, after 5 days, 20 μM hydroxyfasudil significantly diminished these effects by up to 0.7% (P < 0.01 versus PDGF-BB alone) and 14.4% (P < 0.01 versus TGF-β2 alone), respectively. 

Expression of αSMA and Cell Viability

The expression of αSMA was examined to confirm the myofibroblast-like transdifferentiation of the hyalocytes. Whereas vehicle or PDGF-BB–treated cells showed scanty expression of αSMA, TGF- β2–treated hyalocytes showed prominent overexpression of αSMA. The pretreatment of hyalocytes with hydroxyfasudil resulted in partial downregulation of αSMA expression by 20% (P < 0.01; Fig. 7A ). We also confirmed whether cytokine and/or hydroxyfasudil treatment affects cell growth and/or cell viability by counting the viable cells after the treatment of collagen gels with collagenase. As shown in Figure 7B , both cytokines and hydroxyfasudil showed no significant effect on the number of cells in three-dimensional collagen gels, suggesting that collagen gel contraction and its inhibition are independent of cell proliferation and viability. 

Discussion

Hyalocytes are morphologically very similar to macrophages and are assumed to be resident macrophages in the vitreous cavity under physiological conditions. ^{1 2} However, recent studies have indicated that the hyalocytes may also be involved in vitreoretinal diseases. ^{3 4 5 6 36} In the present study, we demonstrated the contractile property of hyalocytes in the presence of PDGF-BB and TGF-β2. We also confirmed that the myofibroblast-like transdifferentiation of hyalocytes and the Rho-kinase–mediated MLC phosphorylation are associated with the cells’ contractile property. Finally, our data indicated that the contractile property of hyalocytes is a possible contributor to vitreoretinal interface diseases and also is a possible therapeutic target for their treatment with Rho-kinase inhibitors such as hydroxyfasudil. 

Both PDGF-BB and TGF-β2 are the multifunctional cytokines thought to be one of the key regulators of various vitreoretinal diseases. ^{15 16 17} The main cause of failure after retinal reattachment surgery is PVR, in which contractile fibrocellular membranes are formed on the retinal surface and vitreous base. Recently, elevated levels of PDGF-BB and TGF-β2 were immunohistochemically confirmed in the PVR membrane and measured in the vitreous of patients with PVR, suggesting a possible association of these cytokines with the disease. ^{38 39 40 41} PVR is characterized by the migration and proliferation of various kinds of cells, including retinal glial cells, macrophages, and retinal pigment epithelial cells, which organize into an epiretinal membrane. ^{7 10 11 12} Although inflammatory cells and macrophages including hyalocytes have also been described as contributors to the formation of epiretinal membranes, ⁷ little is known about the role of hyalocytes in membrane contraction. Although myofibroblast-like cells are observed in epiretinal membranes and are thought to be the main contributor to the membrane contraction, their origin is unclear. ^{7 42}  

We hypothesized that the hyalocytes might contribute to the pathogenesis of vitreoretinal interface diseases, not only by the production of cytokines and/or chemokines that regulate the neighboring cells, but also by contributing to the contractile force of the membranes as transformed myofibroblast-like cells. Our data demonstrate the possibility that hyalocytes are capable of transforming into myofibroblast-like cells in the presence of TGF-β2. 

The phosphorylation of MLC is a critical step in the formation of actin stress fibers, and the phosphorylation state of MLC is dependent on at least two pathways. ²³ First, the Rho/Rho-kinase pathway directly enhances the phosphorylation state of MLC or indirectly affects it by inactivating the MLC phosphatase through the phosphorylation of the myosin-binding subunit. Second, the MLCK pathway directly phosphorylates MLC at the same site as Rho-kinase does. ^{25 43 44}  

To confirm the involvement of the Rho/Rho-kinase pathway in collagen gel contraction by hyalocytes, we examined the inhibitory effect of Y27632, a specific Rho-kinase inhibitor, in a collagen gel contraction assay. Both PDGF-BB–and TGF-β2–dependent collagen gel contraction was significantly inhibited by Y27632, suggesting that the Rho/Rho-kinase pathway is substantially involved in the mechanisms of collagen gel contraction by hyalocytes in response to PDGF-BB and TGF-β2. These Rho-kinase inhibitors, nevertheless, did not completely prohibit gel contraction, even though a concentration that could completely inhibit MLC phosphorylation was used (data not shown), indicating that other pathways, including the MLCK pathway, may also be involved in gel contraction, at least in part. 

Hydroxyfasudil, a potent and selective inhibitor of Rho-kinase, is already in clinical use as a treatment for cerebral vasospasm. ⁴³ If hydroxyfasudil has pharmacologic therapeutic potential for certain vitreoretinal diseases, it may also be of great benefit to patients confronted by blindness due to disease. Hydroxyfasudil dose dependently (0.03–20 μM) suppressed MLC phosphorylation in hyalocytes, consistent with previous reports demonstrating the potent inhibition of Rho-kinase by hydroxyfasudil in vitro. ³⁴ Although the precise pharmacokinetics of hydroxyfasudil inhibition of Rho-kinase activity are still uncertain, it was clearly demonstrated that hydroxyfasudil is capable of inhibiting the activity of recombinant Rho-kinase in a dose-dependent manner, with little effect on other kinases such as MLCK and PKC at the concentration (20 μM) we used in this study. ³⁴  

Many other cytokines and chemokines, such as basic fibroblast growth factor, hepatocyte growth factor, tumor necrosis factor, interleukin-1β, and monocyte chemoattractant protein-1 are also reported to be possible contributors to vitreoretinal diseases, ^{45 46 47 48} but the effects of these factors on MLC phosphorylation and collagen gel contraction by hyalocytes were much less dominant than those of PDGF-BB and TGF-β2 (data not shown). Nevertheless, vitreoretinal disease, of itself, may be caused by the various effects of multiple factors, such as cell migration, cell proliferation, and membrane contraction. 

In this study, we demonstrated the promotional effect of PDGF-BB and TGF-β2 on the contraction of collagen gels containing hyalocytes. PDGF-BB caused phasic collagen gel contraction by transient phosphorylation of MLC. In contrast, TGF-β2 caused tonic collagen gel contraction by sustained phosphorylation of MLC and overexpression of αSMA, promoting transdifferentiation into the myofibroblast-like cells. The Rho/Rho-kinase pathway seems to be substantially involved in the mechanisms of PDGF-BB–and TGF-β2–dependent collagen gel contraction by hyalocytes. Although little is known about the contribution of hyalocytes to the occurrence of vitreoretinal diseases to date, our findings suggest the association of hyalocytes with vitreoretinal interface diseases (i.e., PVR, macular pucker, diabetic macular edema, proliferative diabetic retinopathy, and idiopathic macular hole). Hydroxyfasudil, a potent Rho-kinase inhibitor, may be a beneficial pharmacologic treatment of various diseases. Further examination in vivo, is necessary to evaluate its therapeutic potential for clinical use in treatment of intraocular diseases. 

 Supported in part by Grants-in-Aid 13307024, 13557068, and 15591861; a grant for the 21st Century Center of Excellence Program from the Japanese Ministry of Education, Culture, Sports, Science and Technology; and a grant from the Program for Promotion of Fundamental Studies in Health Sciences of the Organization for Pharmaceutical Safety and Research of Japan.

 Submitted for publication December 10, 2003; revised May 24 and July 13, 2004; accepted July 20, 2004.

 Disclosure: K. Hirayama, None; Y. Hata, None; Y. Noda, None; M. Miura, None; I. Yamanaka, None; H. Shimokawa, None; T. Ishibashi, 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.

 

Figure 1.

 

Collagen gel contraction by PDGF-BB and TGF-β2. Three-dimensional collagen gels containing hyalocytes were treated with vehicle, PDGF-BB at a concentration of 10 ng/mL (0.3 nM), or TGF-β2 at a concentration of 9 ng/mL (0.3 nM) in a 24-well plate (n = 4). The gels were photographed and the gel diameter was measured. The change in the collagen gel diameter was measured at the indicated time points, and recorded as a percentage of the diameter at time 0. (A) Dotted line: control; dashed line: PDGF-BB; solid line: TGF-β2. (B) A representative image of the gels at day 5. **P < 0.01 versus control at each time point. NS, no significant difference between PDGF-BB and TGF-β2 at 24 hours.

Figure 2.

 

MLC phosphorylation by PDGF-BB and TGF-β2. Subconfluent hyalocytes were starved in DMEM containing 1% FBS for 24 hours. The cells were then stimulated with 10 ng/mL PDGF-BB (0.3 nM) or 9 ng/mL TGF-β2 (0.3 nM) for the indicated time. Total cell lysates were subjected to Western blot analysis using an antibody against phosphorylated MLC (p-MLC). Lane-loading differences were normalized by reblotting the membranes with an antibody against MLC. (A, C) Representative Western blots. (B, D) The signal density was quantified and is demonstrated by the mean ratio of p-MLC to MLC in three independent experiments. *P < 0.05; **P < 0.01.

Figure 3.

 

Involvement of the Rho-kinase pathway in collagen gel contraction by hyalocytes. The collagen gels containing hyalocytes were pretreated with a control buffer or 10 μM Y27632 for 30 minutes and then stimulated with PDGF-BB (0.3 nM) or TGF-β2 (0.3 nM). The collagen gel diameter was measured 5 days after stimulation. Data are the percentage of the diameter at time 0. n = 4, *P < 0.01 versus control/vehicle; **P < 0.01 versus each control.

Figure 4.

 

The effect of hydroxyfasudil on MLC phosphorylation. Hyalocytes in regular culture conditions (DMEM supplemented with 10% FBS) were treated with hydroxyfasudil at the indicated concentrations for 30 minutes or with 20 μM hydroxyfasudil for the indicated times. Total cell lysates were subjected to Western blot analysis (A, C) to determine the appropriate pretreatment concentration and time for the inhibition of MLC phosphorylation. The signal density was quantified and is demonstrated (B, D) by the ratio of p-MLC to MLC compared with the ratio at time 0, in three independent experiments. *P < 0.05, **P < 0.01.

Figure 5.

 

Inhibitory effect of hydroxyfasudil on PDGF-BB–and TGF-β2–dependent MLC phosphorylation. Starved hyalocytes were stimulated with PDGF-BB for 4 hours or TGF-β2 for 24 hours in the absence or presence of 20 μM hydroxyfasudil. The total cell lysates were subjected to Western blot analysis for p-MLC and MLC. (A, C) Representative blots from three independent experiments. (B, D) Signal intensities were quantified and are expressed a percentage of the ratio of p-MLC to MLC at time 0. *P < 0.05, **P < 0.01. HF, hydroxyfasudil.

Figure 6.

 

The effect of hydroxyfasudil on cytokine-induced collagen gel contraction. The collagen gels containing hyalocytes were pretreated with a control buffer or 20 μM hydroxyfasudil for 30 minutes and then stimulated with PDGF-BB (0.3 nM) or TGF-β2 (0.3 nM). Left: five days after stimulation, the gels were photographed and their diameters measured. Right: data are expressed as a percentage of the gel diameter at time 0. *P < 0.01 compared with control/vehicle, **P < 0.01 compared with each control (n = 4). HF, hydroxyfasudil.

Figure 7.

 

Expression of αSMA by hyalocytes embedded in collagen gels and determination of cell viability. (A) Five days after treatment, the gels were dissolved by collagenase, and sedimented hyalocytes were lysed in 1× Laemmli buffer. The same amount of protein from the extractions was subjected to Western blot analysis for the detection of αSMA (1:2000). *P < 0.01 compared with control/vehicle, **P < 0.01 compared with control/TGF-β2 (n = 4). (B) The viable cells in the collagen gels were counted, to exclude the effect of cell growth or cytotoxicity on the collagen gel contraction or its inhibition. After 5 days of treatment, the collagen gels were dissolved and the cell suspension was collected. Viable cells were counted with a hemocytometer after trypan blue staining. NS, not significant (n = 4).

The authors thank Tadahisa Kagimoto (Department of Ophthalmology, Kyushu University Graduate School of Medical Sciences), Eve E. Sockett, and Yuka Nakamura for excellent help.