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Br J Pharmacol. Jan 2010; 159(2): 304–315.
Published online Dec 18, 2009. doi:  10.1111/j.1476-5381.2009.00477.x
PMCID: PMC2825352

Blebbistatin inhibits contraction and accelerates migration in mouse hepatic stellate cells

Abstract

Background and purpose:

Blebbistatin, an inhibitor of myosin-II-specific ATPase, has been used to inhibit contraction of invertebrate and mammalian muscle preparations containing non-muscle myosin. Activated hepatic stellate cells have contractile properties and play an important role in the pathophysiology of liver fibrosis and portal hypertension. Therefore, hepatic stellate cells are considered as therapeutic target cells. In the present study, we studied the effect of blebbistatin during the transition of mouse hepatic stellate cells into contractile myofibroblasts.

Experimental approach:

Effects of blebbistatin on cell morphology were evaluated by phase contrast microscopy. Cell stress fibres and focal adhesions were investigated by dual immunofluorescence staining and visualized using fluorescence microscopy. Contractile force generation was examined by silicone wrinkle formation assays and collagen gel contraction assays. Intracellular Ca2+ release in response to endothelin-1 was measured by using Fluo-4. Cell migration was measured by wound healing experiments.

Key results:

In culture-activated hepatic stellate cells, blebbistatin was found to change both cell morphology and function. In the presence of blebbistatin, stellate cells became smaller, acquired a dendritic morphology and had less myosin IIA-containing stress fibres and vinculin-containing focal adhesions. Moreover, blebbistatin impaired silicone wrinkle formation, reduced collagen gel contraction and blocked endothelin-1-induced intracellular Ca2+ release. Finally, it promoted wound-induced cell migration.

Conclusions and implications:

By inhibiting myosin II ATPase, blebbistatin has profound effects on the morphology and function of activated hepatic stellate cells. Our data suggest that myosin II could be a therapeutic target in the treatment of liver fibrosis and portal hypertension.

Keywords: blebbistatin, myosin, hepatic stellate cells, contraction, migration, non-muscle myosin, α-SMA, stress fibres

Introduction

Hepatic stellate cells (HSCs) are located in the perisinusoidal space of Disse and represent 5–8% of cells in the normal liver. They play a pivotal role in vitamin A metabolism, in synthesis and degradation of extracellular matrix (ECM) and in regulation of sinusoidal blood flow (Geerts, 2001). Upon liver injury, they undergo transition into α-smooth muscle actin (α-SMA)-expressing myofibroblasts, which are characterized by loss of stored vitamin A, increased synthesis of collagen and α-SMA, reorganization of the actin cytoskeleton and increased deposition of ECM components. HSCs migrate to sites of inflammation and produce collagen and ECM constituents during the wound healing process, ultimately resulting in hepatic fibrosis and cirrhosis (Friedman, 2000; Geerts, 2001). They play an important role in the pathophysiology of fibrosis and portal hypertension and the paradigm of HSC activation provides an important framework for defining therapeutic targets (Gressner, 1991; Rockey and Weisiger, 1996; Geerts, 2001; Rockey, 2001; Reynaert et al., 2002; Friedman, 2008). Myosins, which are actin-based motor proteins, play multiple vital roles in many different cell types, such as cardiomyocytes, kidney, neuronal and astrocytoma cells (Sellers, 2000; Guha et al., 2005; Dou et al., 2007; Haviv et al., 2008; Salhia et al., 2008). Specific inhibitors of myosin–actin interaction may provide insight into potential novel therapeutic approaches.

Blebbistatin was identified as a non-muscle myosin II-specific inhibitor (Straight et al., 2003), although smooth muscle myosin and some non-conventional myosins (I, V and X) have been reported to be minimally influenced as well (Limouze et al., 2004). Blebbistatin is cell-permeable and inhibits myosin function during cell division and motility in different cell types, such as neuronal cells, astrocytoma cells and vascular smooth muscle cells (Guha et al., 2005; Shu et al., 2005; Zhang and Rao, 2005; Calaminus et al., 2007; Rosner et al., 2007; Salhia et al., 2008; Wang et al., 2008). It further inhibits contraction of invertebrate and mammalian muscle preparations containing non-muscle myosin (Katayama et al., 2006; Dou et al., 2007; Fedorov et al., 2007; Rosner et al., 2007; Farman et al., 2008; Salhia et al., 2008). Little is known about the effects of blebbistatin on mouse HSCs, which are considered therapeutic target cells for liver fibrosis and portal hypertension (Reynaert et al., 2002; Friedman, 2008). Migration and contraction are characteristic of activated HSCs, which are important for the formation of fibrous septa and the development and severity of portal hypertension (Friedman, 2003). Using blebbistatin we investigated whether non-muscle myosin II plays a role during the in vitro transdifferentiation of HSCs and in the contractility and migration of activated HSCs.

Methods

Isolation and culture of mouse HSCs

All animal care and experimental procedures were according to the institution's guidelines for the care and use of laboratory animals in research and this study was approved by the local ethical committee. All procedures were performed with animals under nembutal anaesthesia. HSCs used in this study were isolated by a modified collagenase-pronase digestion method as previously described (Reynaert et al., 2001; Uyama et al., 2006). About 1.5 × 106 freshly isolated HSCs were plated on 10 cm petri-dishes. To analyse activated HSCs, cells were trypsinized and replated when they had reached confluency after 5–6 days.

Western blot analysis

Cells were homogenized in sample buffer (62.5 mM Tris-HCl, pH 6.8, 2% SDS, 10% glycerol and proteinase inhibitor added before use), boiled for 10 min, sonicated and boiled for 15 s. Protein concentration was determined using the BCA protein assay kit (Pierce, Rockford, Illinois, USA). 10 µg samples in loading buffer with β-mercaptoethanol were loaded and separated by SDS-PAGE, then electroblotted onto polyvinylidene difluoride membrane (Millpore, Bedford, MA, USA). After blocking with 5% skim milk, 0.05% Tween 20 in PBS at room temperature for 1.5 h, the upper part of the blot was probed with primary antibodies to NMMHC-II isoforms, the lower part of the blot was incubated with primary antibodies to actin isoforms for 2 h at room temperature. The blots were washed and incubated with secondary antibodies for 1 h at room temperature. Proteins were detected by enhanced chemiluminescence using ECL substrate (Amersham, Rosendaal, the Netherlands).

Phase contrast and fluorescence microscopy

Cells were cultured in absence or presence of blebbistatin (50 µM) for 2 h at the indicated days and then photographed. For immunodetection, cells were plated on non-coated coverslips in 24-well plates and fixed for 10 min in acetone/ethanol (3:1) at −20°C at day 11, washed three times with PBS and blocked with 2% BSA/PBS for 30 min at room temperature. Following three washes with PBS, cells were incubated with primary antibodies for 90 min. After three washes with PBS, cells were incubated with fluorochrome-labelled secondary antibodies for 1 h under light-protected conditions. Cells were visualized by fluorescence microscopy (Axioskop, ZEISS, Jena, Germany).

Silicone substrate wrinkle assay

A thin layer of silicone fluid 200 (viscosity, 30 000 centistokes) was spread on the surface of a glass-bottom tissue culture dish (dish type; ‘The optics of glass in a petri dish’, MatTek, Ashland, USA). This surface was then exposed to a low flame from a Bunsen burner, allowing a film of tiny wrinkles to form on the fluid surface. The silicone took 2 s to polymerize, and was sterilized by UV exposure. Cells were added to the dishes in DMEM supplemented with 10% FCS + antibiotics and were incubated at 37°C for 1 or 2 days. Cells were then starved for 3 h with serum-free medium to exclude the well-known contractile effect of serum, followed by treatment with blebbistatin (25 µM) for 2 h. Wrinkle formation was evaluated by microscopy. Cells were then treated with endothelin-1 (ET-1; 4 × 10−8M) for 10 min. Wrinkle formation was assessed before and after treatment with blebbistatin or ET-1.

Collagen gel contraction assay

Contraction of HSCs on collagen gel lattices was examined in six-well flat-bottom tissue culture plates (Becton Dickinson, Bedford, MA). Briefly, culture plates were pre-incubated with 2% BSA/PBS for 2 h at 37°C, washed and air-dried. Rat-tail collagen (BD Biosciences) was adjusted to physiological strength and pH with PBS and 1 M NaOH. Two millilitres of this solution (final collagen concentration: 1.5 mg·mL−1) was added to each well of six-well plates. The plate was then incubated at 37°C for 1 h to allow gelatinizing. HSCs at day 13 were trypsinized, re-suspended (~0.5 × 106 cells·mL−1), and plated on top of the gels (2 mL per well). After incubation overnight to allow cell attachment, serum-free conditions were introduced for 3 h. Gels were then detached from the plate by gentle circumferential dislodgment using the tip of a 200 µl pipette. Then, medium with or without blebbistatin (50 µM) was added. After 15 min, ET-1 (2 × 10−8M) (Sigma-Aldrich) was added to elicit contraction, medium was used as a control. Twenty-four hours post treatment, gels were photographed, the collagen gel area was measured and the ‘post treatment collagen gel area’ was calculated, relative to the original gel area (set to 100%).

Measurement of intracellular Ca2+ release

Mouse HSCs were plated on 35 mm poly-D-lysine-coated glass-bottom microwell dishes (MatTek, Ashland, MA). Measurement was carried out at day 11 on cells treated with or without blebbistatin (50 µM, 2 h) following 24 h of starvation. Cells were subsequently washed in incubation buffer, and then loaded with Fluo-4 AM (1 µM, Invitrogen) in 0.005% Pluronic F-127 (Sigma) by incubation for 25 min at 37°C. Pluronic® F-127 is a non-ionic, surfactant polyol that facilitates the solubilization of water-insoluble dyes like Fluo-4 AM. After the loading incubation, unincorporated Fluo-4 was removed by washing the coverslips. Next, ET-1 (final concentration: 4 × 10−8M) was applied and cytosolic free Ca2+ was assessed by confocal laser scanning microscopy (Zeiss; LSM-510). Image data were analysed off-line using the Zeiss LSM510 analysis software V2.53.

Wound healing assay

To investigate the effect of blebbistatin on wound-induced migration, 24-well plates were coated with 5% collagen for 1.5 h at room temperature, washed three times with PBS, and blocked by 2% BSA/PBS. The 4- and 10-day-old cells were seeded at ~1 × 105 and ~5 × 104 cells per well respectively. Once the cells had attached properly, medium without FBS was added to starve the cells for 3 h. Then a ‘wound’ was made with a tip and medium with or without blebbistatin (0, 12.5, 25 and 50 µM) was added. Pictures were taken at the start and 24 h after ‘wound induction’. Cell migration was analysed by counting the number of cells in the ‘wound’.

Proliferation

Freshly isolated HSCs were replated on 96-well plate (1.6 × 104 cells per well). At day 3, medium was replaced by serum-free medium and cells were starved overnight, treated with or without blebbistatin (25 µM) for 2 h followed by stimulation with platelet-derived growth factor-BB (20 ng·mL−1). After an overnight incubation, the WST-1 cell proliferation assay (Cayman, Michigan, USA) were performed according to the manufacturer's protocols.

Cell adhesion assay

24-well plates were coated with 5% collagen type I at room temperature for 2 h and blocked with 0.2% BSA/PBS at room temperature for 1 h. After trypsinization, 4- and 10-day-old cells were seeded at ~1 × 105 and ~5 × 104 cells per well respectively, and incubated for 1 h at 37°C in the presence or absence of blebbistatin (0, 12.5, 25 and 50 µM). Then, plates were washed three times with PBS to remove unattached cells. Adherent cells were stained by 0.1% crystal violet, followed by 0.2% Triton X-100 in PBS. Absorbance was measured using a microplate spectrophotometer (Bio-Rad, Hercules, USA) at a wavelength of 450 nm corrected to 595 nm.

Statistical analysis

All experiments were repeated with mouse HSCs from at least three isolations. Data were expressed as means ± a standard deviation. All statistical tests were performed using GraphPad Prism version 4.0 for Windows (GraphPad Software, San Diego, CA). One-way anova (Tukey's Multiple Comparison Test) was used for collagen gel contraction and wound healing assays.

Materials

Anti-myosin IIA, anti-myosin IIB anti-α-SMA, anti-β-actin, endothelin-1 and blebbistatin were purchased from Sigma (Saint Louis, USA). 10 mM Blebbistatin stock solution was prepared in ethanol; in all experiments the control condition was medium containing vehicle control. Rat tail collagen type I was obtained from BD Biosciences (San Jose, CA, USA). Anti-rabbit IgG Alexa Fluor 488 and anti-mouse IgG Alexa Fluor 633 were purchased from Molecular Probe. Swine anti-rabbit immunoglobulins/HRP and goat anti-mouse immunoglobulins/HRP were from DAKO (Glostrup, Denmark).

Results

Effect of blebbistatin on the morphology of mouse HSCs

Culture-activated HSCs developed spreading lamellipodia and formed stress fibres. When treated with blebbistatin (0, 12.5, 25 and 50 µM) for 2 h, cells became smaller, acquired a more dendritic morphology and had less stress fibres and lamellipodia (Figure 1A). Western blot analysis showed that these changes were not due to changes in the protein expression of myosin IIA, IIB or α-SMA (Figure 1B). Myosin IIC was undetectable in mouse HSCs (data not shown). This treatment was reversible since washing the blebbistatin-treated cells with normal medium resulted in a normal HSC morphology after 24 h (Figure 1A).

Figure 1
Effects of blebbistatin on mouse hepatic stellate cells (HSCs). (A) Phase contrast images showing that blebbistatin induced morphological changes in HSCs. Cells at day 10 were treated with or without blebbistatin (0, 12.5, 25 and 50 µM, 2 h), ...

Myosin IIA–α-SMA-containing stress fibres were disrupted in blebbistatin-treated HSCs

Considering that protein levels of actin and myosin II were not influenced, we studied whether the organization of myosin–actin stress fibres was altered in blebbistatin-treated cells by dual immunofluorescence staining for myosin IIA and α-SMA. Figure 2 suggests that myosin IIA and α-SMA filaments were localized in stress fibres that sometimes overlapped in control cells. When cells were treated with blebbistatin, the stress fibres disappeared. Although myosin IIA and α-SMA were still present, myosin IIA and α-SMA filaments were disrupted (Figure 2).

Figure 2
Blebbistatin dissociates myosin IIA –α-smooth muscle actin (α-SMA)-containing stress fibres. Cells at day 11 were treated with or without blebbistatin (50 µM) for 2 h, and then fixed in acetone/ethanol (1:3) at −20 ...

Silicone wrinkles do not form under blebbistatin-treated HSCs

As myosin is known to play a role in the contractility of HSCs (Saab et al., 2002), we investigated the effect of blebbistatin in a silicone wrinkle assay as a measure of contractility of a deformable matrix. Due to the very thin silicone film, only minimal power is needed to form wrinkles. In contrast to other techniques, such as collagen gel contraction, this assay permits the assessment of single-cell contractions. Moreover, the formation of wrinkles is reversible, allowing the analysis of the reversibility of drug effects. In untreated conditions, HSCs induced wrinkling of the silicone substrate, which was significantly enhanced by ET-1, a potent contractile peptide for HSCs. In contrast, cells treated with blebbistatin did not form any wrinkles in the silicone coating, not even after ET-1 stimulation (Figure 3A). Removal of blebbistatin and continued culturing of the HSCs in the presence of normal medium resulted in cells recovering and regaining their ability to form wrinkles. Approximately 3 days after withdrawal of blebbistatin, cells had returned to control values (Figure 3B).

Figure 3
Blebbistatin inhibits silicone wrinkle formation, both in the presence and in the absence of the potent constrictor ET-1. (A) Mouse hepatic stellate cells (HSCs) were cultured on silicone substrate. At day 7 wrinkle formation was assessed before and after ...

Blebbistatin inhibits ET-1-induced collagen gel contraction

To further investigate the inhibitory effect of blebbistatin on HSC contraction, we performed a hydrated collagen gel assay to evaluate ET-1-induced gel contraction in the presence and absence of blebbistatin. This assay allowed us to measure the effect of blebbistatin on the contraction of a large number of HSCs. We observed in control plates that collagen fibril bundles formed around groups of cells. In the presence of blebbistatin, no or only minimal collagen fibril bundle formation was seen (Figure 4A). HSCs growing on collagen-gel resulted in a basal level of gel surface decrease of 20% (Figure 4B and C). After ET-1 stimulation, we observed a significant decrease in the gel surface (Figure 4B), the reduction being 46.5% of the original gel size (2.3 times basal level) (Figure 4C). The ET-1-induced decrease was blocked by pre-treatment of the HSCs with blebbistatin (Figure 4B), resulting in a 6.9% reduction of original gel surface (Figure 4C). This shows that blebbistatin can inhibit both ET-1-induced and basal-level gel contraction by HSCs.

Figure 4
Blebbistatin blocks collagen gel contraction by activated hepatic stellate cells (HSCs). (A) Morphology of 14-day-cultured HSCs on collagen gel, 24 h after stimulation with 2 × 10−8 M ET-1 in the absence or presence of 50 µM blebbistatin. ...

Intracellular Ca2+ release is blocked in blebbistatin-treated mouse HSCs

Because Ca2+ transients are involved in contraction in many cell types including activated stellate cells (Pinzani et al., 1992a; Bataller et al., 1998), and the cytoskeleton may be an integral component of Ca2+ regulation (Tseng et al., 1997; Cotrina et al., 1998), we measured [Ca2+]i in our experimental system. HSCs were loaded with Fluo-4 and the intracellular Ca2+ levels were evaluated using fluorescent microscopy. Control cells showed a clear increase in [Ca2+]i upon ET-1 stimulation (Figure 5A and C), while most blebbistatin-treated cells did not respond upon exposure to ET-1 (Figure 5B and C). A few cells did show a positive response to ET-1, but expressed the morphology of control cells, suggesting that these cells were not or only partially inhibited by blebbistatin (Figure 5B and C).

Figure 5
Blebbistatin blocks ET-1-induced intracellular Ca2+ release. Original fluorescence images of [Ca2+]i measurements following addition of ET-1 (4 × 10−8 M) in hepatic stellate cells (HSCs) in the absence (A) or presence (B) of blebbistatin ...

Blebbistatin promotes wound-induced mouse HSC migration

Following acute or chronic liver injury, HSCs are activated and migrate to sites of injury (Friedman, 2000). In vitro, HSCs undergo a culture-induced transdifferentiation process during which an increasing number of α-SMA containing stress fibres are formed. By cell morphological analysis and immunostaining, we rarely observed bundles of stress fibres at day 4 in culture (data not shown) while well-formed α-SMA-containing stress fibres can be easily detected in cells at day 10 in culture (Figure 2). To determine the effect of blebbistatin on HSC migration, wound healing assays were performed using both day 4 and day 10 cultured HSCs. Migration of cells during wound closure was studied 24 h after scraping cells from an area in a confluent monolayer of cells. In comparison with untreated HSCs, cells treated with blebbistatin migrated faster (Figure 6A and B) and in higher numbers (Figure 6C) into the wound. This increased number was not due to proliferation in blebbistatin-treated HSC cultures (Figure 6D).

Figure 6
Wound healing assay showing that blebbistatin promotes wound-induced hepatic stellate cell (HSC) migration. (A) At day 4 after seeding, a ‘wound’ was made and cells were treated with blebbistatin (concentration: 0, 12.5, 25, 50 µM) ...

Blebbistatin decreases adhesion of activated but not quiescent HSCs to collagen type I

In view of the results of cell contraction and migration, we presumed that a reduction in stress fibres would decrease cell adhesion to collagen type I. This, in turn, would block the formation of contractile force and facilitate cell migration. Therefore, cell adhesion assays were performed with HSCs at day 4 (few stress fibres) and day 10 (fully developed stress fibres) in culture. Figure 7 clearly demonstrates that blebbistatin significantly decreased HSC adhesion at day 10 (Figure 7B), but did not influence cell adhesion at day 4 (Figure 7A).

Figure 7
Cell adhesion assay. (A) Blebbistatin has no significant effect on the adhesion of quiescent hepatic stellate cells (HSCs). Images show cell adhesion after crystal violet staining of cells at day 4, treated with different concentrations of blebbistatin ...

Vinculin-containing focal adhesions disappear in blebbistatin-treated HSCs

Both classical and supermature focal adhesions have been reported in myofibroblasts and the formation and stability of supermature focal adhesions depend on high SMA-mediated contractile activity of myofibroblast stress fibres (Goffin et al., 2006). As we showed that blebbistatin induced a disruption of stress fibres, it might be possible that it also affects focal adhesions in activated HSCs. To this end, we stained HSCs with antibodies to vinculin, which is a major component of focal adhesions. In control cells, vinculin was distributed to the cell membrane and cytoplasm to various degrees, including vinculin-containing focal adhesions organized into supermature focal adhesions (Figure 7C). Treatment of the cells with blebbistatin (50 µM) for 2 h led to the disappearance of the normal distribution pattern of vinculin at the cell membrane and in most of the cytoplasm. In these blebbistatin-treated cells, vinculin showed mainly a perinuclear localization, suggesting that vinculin-containing supermature focal adhesions cannot form in the presence of blebbistatin (Figure 7C).

Discussion and conclusions

In the present study, we demonstrate that blebbistatin has profound effects on multiple aspects of the transdifferentiation of mouse HSCs, including cell morphology, focal adhesions, contraction and migration.

First, we observed a dramatic change in cell morphology after application of blebbistatin. Blebbistatin-treated mouse HSCs became smaller, had less stress fibres and acquired a dendritic morphology. Blebbistatin does not change α-SMA expression, which is a well-accepted marker protein for HSC activation (Ramadori et al., 1990; Rockey et al., 1992), but induces α-SMA filament disassembly, suggesting that myosin II ATPase activity is required for the regulation and stabilization of myosin-α-SMA-containing stress fibres. Those findings are consistent with studies in other cell types; for example, in rat fibroblasts, blebbistatin led to disappearance of actin–myosin bundles (Shutova et al., 2008). In Caco-2 and SK-CO15 intestinal epithelial cells, inhibition of myosin II by blebbistatin resulted in decreased dynamics of cortical F-actin and induced the outgrowth of F-actin-rich surface protrusions (Ivanov et al., 2008). Myosin IIA-deficient SK-CO15 cells failed to assemble F-actin stress fibres (Babbin et al., 2009) and knock-down of myosin IIA expression altered epithelial cell shape and the apical F-actin appeared organized into abnormal aster-like aggregates (Ivanov et al., 2007). These studies also show a decrease in focal adhesions after a blebbistatin treatment or myosin IIA knock-down. We confirmed these observations in mouse HSC cultures in which blebbistatin decreased vinculin-containing focal adhesions in activated HSCs, which are assumed to be essential for HSCs to attach to substrates (Kawai et al., 2003; Melton et al., 2007). Accordingly, blebbistatin application led to reduced numbers of activated HSCs attaching to collagen substrates. This effect was amplified with increasing concentrations of blebbistatin. In contrast, blebbistatin had no such effect in quiescent HSCs, suggesting that the effect of blebbistatin on focal adhesions and mature focal cell-matrix adhesions occurs through disassembly of stress fibres. It has been reported that cells adhere to the ECM via integrin-mediated cell-matrix adhesions that link the ECM to the actin cytoskeleton. These adhesions convert the flow behind into a myosin II-driven mode. Some focal complexes can turn into elongated focal adhesions that are associated with contractile actin–myosin stress fibres (Bershadsky et al., 2006). In this manner, myosin II ATPase inhibition may not suppress formation of focal complexes, but rather block their conversion into mature focal adhesions and further growth of focal adhesion. Furthermore, both classical and supermature focal adhesions occur in myofibroblasts and the formation and stability of supermature focal adhesions depend on α-SMA-containing stress fibres (Hinz et al., 2003; Goffin et al., 2006). These data and our results lead us to conclude that focal adhesions are different between quiescent HSCs (which have no α-SMA stress fibres) and activated HSCs (which have α-SMA stress fibres). Thus, blebbistatin decreases myosin II-α-SMA-containing stress fibres and subsequently reduces vinculin-containing mature focal adhesions. In quiescent and early activated cells without α-SMA (as day 4), cell adhesion may be related to integrin-mediated cell-matrix adhesion or other filaments, such as synemin (Uyama et al., 2006).

Contraction of HSCs contributes to the regulation of sinusoidal blood flow in normal liver and to the degree of portal hypertension in cirrhosis (Rockey and Weisiger, 1996; Rockey, 2001; Marra and Pinzani, 2002; Reynaert et al., 2002; Friedman, 2008). The generation of contractile force by activated HSCs in response to ET-1 has been reported in many studies (Rockey and Weisiger, 1996; Reynaert et al., 2001; Saab et al., 2002; Melton et al., 2006). We found that ET-1-induced HSC contractility was effectively inhibited by blebbistatin both in a silicone wrinkle assay and in a collagen lattice contraction assay. Myosin II-α-SMA-containing stress fibres are major structures essential for HSC contraction (Reynaert et al., 2002; Saab et al., 2002). We showed that the organization of myosin–actin stress fibres was altered in blebbistatin-treated cells, which may explain the reduced contractile capability of HSCs. Two major pathways have been shown to regulate cell contraction: the Rho pathway (Yee et al., 2001; Melton et al., 2006) and Ca2+ signalling (Pinzani et al., 1992b; Bataller et al., 1998). In fact, Y-27632, a selective inhibitor of Rho-associated kinase and ML-7, a myosin light chain kinase inhibitor, can both inhibit myosin light chain phosphorylation, stress fibre formation and ET-1-induced contractile force generation in primary rat HSCs and a HSC cell line (Iwamoto et al., 2000; Tada et al., 2001; Yanase et al., 2003; Melton et al., 2006) and fibroblasts (Katoh et al., 2001). In addition, ET-1-induced HSC contraction is elicited by an increase in intracellular Ca2+ (Pinzani et al., 1992a; Bataller et al., 1998). We did not observe an ET-1-induced [Ca2+]i. increase in blebbistatin-treated cells, suggesting that blebbistatin may also have an effect on HSC contraction by regulating intracellular calcium signalling. The exact mechanism is unknown, but data from different groups show that disruption of the cytoskeleton may impair [Ca2+]i. increase (Tseng et al., 1997; Cotrina et al., 1998). On the other hand, to effectively contract, cells need to attach to the ECM by means of focal adhesions (Lauffenburger and Horwitz, 1996; Melton et al., 2007). We also demonstrated reduced numbers of focal adhesions in activated HSCs upon blebbistatin application.

Migration of stellate cells during hepatic injury is essential for wound healing and the development of liver fibrosis (Friedman, 2000; 2003; Melton and Yee, 2007). In HSCs, treatment with blebbistatin in both the early and later stages of activation did not inhibit – as would be expected – but rather promoted wound-induced migration. This finding is consistent with predictions of a mathematical model for cell migration (DiMilla et al., 1991) and with the finding in human foreskin fibroblasts, mouse embryonic stem cells, human MCF10A breast epithelial cells and primary mouse embryonic fibroblasts, in which blebbistatin treatment or knock-down of myosin IIA also induced substantial increases in migration rates (Even-Ram et al., 2007). The accelerated migration rates in mouse HSCs can be explained by several mechanisms. First, blebbistatin-induced morphological changes may facilitate cell migration (Lauffenburger and Horwitz, 1996). Second, we showed in this study that treatment with blebbistatin results in less stress fibres and mature focal adhesions, which increases cell migration by disrupting strong attachment to the underlying substrate (DiMilla et al., 1991; Even-Ram et al., 2007). Finally, HSCs may use a microtubule-dependent mechanism in cells lacking stress fibres and focal adhesions (Even-Ram et al., 2007; Shutova et al., 2008).

To our knowledge, this is the first study to report on the effects of blebbistatin on HSCs. Our results show that myosin II ATPase activity is necessary for mouse HSCs to display their characteristic contractile myofibroblast phenotype and that myosin II ATPase activity regulates important functions, such as migration and contraction, suggesting that myosin II could be a therapeutic target in the treatment of liver fibrosis and portal hypertension.

Acknowledgments

We remember Professor Albert Geerts who passed away during the finalization of this study. We are grateful to him for all his enthusiasm and support that made the realization of this project possible. This paper is in his honour. We express our warmest thanks to Jan Van Daele for assistance with confocal microscopy and Ca2+-imaging, Inge Mannaerts, Eduardo Guimaraes and Sara Knops for HSC isolations, Christophe Empsen, Danielle Blijweert, Jean-Marc Lazou and Kris Derom for their technical assistance. We are grateful for support from : FWO-V (Fonds voor Wetenschappelijk Onderzoek – Vlaanderen), grants G.0512.04, G.0652.06, G.0229.08, G.0651.06; OZR (Research Council of Vrije Universiteit Brussel) grants OZR1432BOF, OZR1149BOF, GOA48, OZR1600BOF, OZR1226, OZR1219; EASL (European Association for the Study of the Liver) 2007 Sheila Sherlock Entry Level Fellowship; I.A.P. (Belgian Science Policy Bureau) phase VI contract P6/36; IWT/SB/61314; EU 6th Framework program. Roche and Schering-Plough unconditional grant.

Glossary

Abbreviations:

[Ca2+]i
intracellular Ca2+
ECM
extracellular matrix
ET-1
endothelin-1
HSC
hepatic stellate cell
PBS
phosphate buffered saline
α-SMA
α-smooth muscle actin

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