One of the pathways activated during liver fibrosis is the Rho kinase pathway, which regulates activation, migration, and contraction of hepatic stellate cells (HSC). Inhibition of this kinase by the Rho kinase inhibitor Y27632 [(+)-(R)-trans- 4-(1-aminoethyl)-N-(4-pyridyl)cyclohexanecarboxamide dihydrochloride] has been shown to reduce fibrosis in animal models. However, kinase expression is ubiquitous, so any inhibitor may affect many cell types. We hypothesize that cell-specific delivery of a kinase inhibitor will be beneficial. Therefore, we conjugated Y27632 to the carrier mannose-6-phosphate (M6P) human serum albumin (HSA), which is taken up specifically in activated HSC through the M6P/insulin-like growth factor II receptor. This conjugate decreased protein expression of phosphorylated myosin light chain 2 (pMLC2) and vinculin, downstream of Rho kinase, in activated primary HSC and decreased the migration and contraction of HSC. In an ex vivo model, free Y27632 decreased contractility of rat aortas, whereas the Y27-conjugate did not, showing that the Y27-conjugate does not affect nontarget tissue. In chronic CCl4-induced liver fibrosis, both free drug and conjugate reduced HSC activation; however, only the Y27-conjugate significantly reduced collagen deposition. Treatment with the Y27-conjugate, but not with free drug, reduced pMLC2 expression in livers 24 h after injection, demonstrating prolonged inhibition of the Rho kinase pathway. The Rho kinase inhibitor Y27632 can be specifically targeted to HSC using M6PHSA, decreasing its effects in nontarget tissues. The targeted drug effectively reduced fibrotic parameters in vivo via the inhibition of the Rho kinase pathway.
Liver fibrosis is the final stage of various forms of chronic liver disease, irrespective of the original cause of liver injury (Schuppan and Afdhal, 2008). During these pathological conditions, hepatic stellate cells (HSC) are activated and form a major source of myofibroblasts. These cells are responsible for the production of profibrotic cytokines, such as TGF-β and PDGF, and extracellular matrix proteins, such as collagen and fibronectin. Increased production and decreased breakdown of extracellular matrix lead to scar formation (Friedman, 2008a,b), the hallmark of fibrosis. The disease can be induced by viral infections (hepatitis B and C), obesity (nonalcoholic steatohepatitis), and toxic compounds such as alcohol, cholestasis, and genetic disorders (Schuppan and Afdhal, 2008). Although millions of people are at risk worldwide, no pharmacotherapy is available to attenuate fibrogenesis (Thompson and Patel, 2010).
Activation of HSC during fibrosis is induced by several growth factor-mediated pathways; foremost among them are TGF-β and PDGF (Kawada et al., 1999; Melton and Yee, 2007; Friedman, 2008b). One of the points of convergence of these profibrotic pathways is Rho-GTPase, which is activated by several of the cytokines produced during fibrogenesis. Rho-GTPase regulates cell migration, contraction, and transdifferentiation of HSC to myofibroblasts through its control of cytoskeletal proteins (Yee, 1998; Charest and Firtel, 2007). Rho-associated coiled-coil-forming protein kinase (Rho kinase) is the most extensively studied downstream mediator of Rho-GTPase (Wettchureck and Offermanns, 2002; Riento and Ridley, 2004; Kitamura et al., 2007). Rho kinase regulates its downstream mediators, such as myosin light chain (MLC), through phosphorylation. The phosphorylation of MLC can be either a direct or an indirect effect (Pellegrin and Mellor, 2007). Phosphorylated MLC (pMLC2) binds to actin and stabilizes the stress fibers necessary for migration and contraction. Inhibition of Rho kinase reduces MLC2 phosphorylation and indirectly reduces vinculin recruitment to focal adhesions (Dumbauld et al., 2010), thus regulating contraction and migration of fibroblasts. Rho kinase inhibition also leads to decreased activity of the promoter for myofibroblast marker α-smooth muscle actin (α-SMA) (Mack et al., 2001). Rho kinase inhibitors have been shown to reduce fibrotic parameters in activated HSC (Iwamoto et al., 2000; Murata et al., 2001; Mizunuma et al., 2003; Tangkijvanich et al., 2003; Fukushima et al., 2005) and rat models of liver fibrosis (Murata et al., 2001, 2003; Tada et al., 2001).
Kinase-regulated pathways are present in every cell type of the body, so their inhibition in nondiseased organs may lead to serious side effects (Force et al., 2007). To elicit a strong and more specific effect, two different strategies are possible: inhibition of a kinase downstream in a particular pathway, to prevent inhibition of vital processes in nontarget cells, or inhibition of an upstream kinase only in the target cell, using a cell-specific carrier. The first approach might inhibit only a redundant downstream mediator and not affect the disease process. The second strategy does not have this disadvantage, but cell-specific carriers are not always available. However, we have developed a drug carrier, mannose-6-phosphate (M6P) human serum albumin (HSA), which is selective for the key cell type in fibrogenesis, the hepatic stellate cell. To explore the strategy of cell-specific inhibition, we coupled a Rho kinase inhibitor to this carrier. M6PHSA binds specifically to the M6P/insulin-like growth factor II (M6P/IGFII) receptor. The receptor-ligand complex thus formed is taken up in the cell through endocytosis (Beljaars et al., 1999). The M6P/IGFII receptor is a multifunctional receptor that traffics between the Golgi and the endosomal-lysosomal network and shuttles to the plasma membrane (Ghosh et al., 2003). It has also been associated with the activation of latent TGF-β to its active form (Dennis and Rifkin, 1991; de Bleser et al., 1995). Because the receptor is up-regulated on the plasma membrane of activated HSC during liver fibrosis (de Bleser et al., 1995; Greupink et al., 2006), drugs coupled to M6PHSA will be taken up by activated HSC.
We used the Universal Linkage System (ULS) conjugation technology to couple the Rho kinase inhibitor Y27632 [(+)-(R)-trans-4-(1-aminoethyl)-N-(4-pyridyl)cyclohexanecarboxamide dihydrochloride] to M6PHSA. This offers a rapid coupling method, applicable to different kinase inhibitors (Gonzalo et al., 2007) and a slow intracellular release of the active drug from the conjugate over several days (Prakash et al., 2006).
We compared the conjugate and the free Rho kinase inhibitor in several in vitro systems. In vivo, we examined the intrahepatic distribution of the conjugate and its therapeutic effects in a CCl4-induced chronic liver injury mouse model. This model is characterized by HSC activation and extensive collagen deposition, resembling advanced fibrosis. We propose that cell-specific delivery will lead to a more effective inhibition of fibrosis by the HSC-targeted inhibitor.
Materials and Methods
Y27632 was purchased from Tocris Bioscience (Bristol, UK), PDGF-BB was from PeproTech (Rocky Hill, NJ), TGF-β was from Roche Diagnostics (Mannheim, Germany), latent TGF-β was from R&D Systems (Minneapolis, MN), and Transwell plates were from Corning Life Sciences (Lowell, MA). Primary antibodies were as follows: mouse anti-α-SMA, anti-desmin and anti-vinculin (Sigma-Aldrich, St. Louis, MO), rabbit anti-human serum albumin (Cappel, Zoetermeer, Netherlands), goat anti-collagen I (Southern Biotechnology Associates, Birmingham, AL), and mouse anti-pMLC2 (Ser19) (Cell Signaling Technology, Danvers, MA). Species-specific horseradish peroxidase, tetramethylrhodamine B isothiocyanate, or fluorescein isothiocyanate-coupled secondary antibodies were purchased from Dako Denmark A/S (Glostrup, Denmark).
Synthesis of Y27632-ULS-M6PHSA.
The ULS, developed by Kreatech Diagnostics, Amsterdam, The Netherlands, is a platinum-based linkage technology that facilitates the coupling of molecules directly to each other through the formation of a coordinative bond. The ULS technology has been proven to have important applications in the area of genomics, proteomics, diagnostics, and therapeutics. The linker was conjugated to Y27632 as reported previously (Prakash et al., 2008). M6P28HSA was synthesized and characterized as described previously (Beljaars et al., 2001). ULS-Y27632 (2.1 μmol) was subsequently reacted with M6PHSA (0.14 μmol) in tricine buffer at 37°C, and the resulting conjugate was extensively dialyzed against PBS at 4°C and purified.
Characterization of Y27632-ULS-M6PHSA.
The amount of Y27632 coupled to M6PHSA was determined by HPLC analysis as reported previously (Prakash et al., 2008) after incubation with 0.5 M potassium thiocyanate at 80°C to chemically displace the drug from the carrier. The M6PHSA protein concentration was determined by Lowry protein assay (Bio-Rad Laboratories, Hercules, CA).
In Vitro Assays.
HSC were isolated from the livers of male Wistar rats (>500 g; Harlan, Zeist, The Netherlands) according to previously published methods (Geerts et al., 1998). After isolation, HSC were cultured on plastic for 10 days until activation and then used for experiments. For some experiments HSC were used that were cryopreserved on day 7 and thawed later for use in experiments.
NIH-3T3 fibroblasts were cultured in Dulbecco's modified Eagle's medium (Lonza Verviers SPRL, Verviers, Belgium) containing 10% fetal calf serum and penicillin/streptomycin.
Rat HSC Contraction Assay.
Hydrated collagen gels were prepared by mixing rat tail collagen I (BD Biosciences, San Jose, CA) with 1 N NaOH, 10× PBS, and 0.2 M HEPES to a final collagen concentration of 1.2 mg/ml. This was incubated for 1 h at 37°C in a humidified atmosphere to allow gelation. Isolated HSC were allowed to adhere to the gel for 3 h, and then incubated with 10 μM Y27632, Y27-conjugate, or M6PHSA in medium with 10% fetal calf serum to induce contraction. After 24 h, the gels were photographed, and the total gel area was calculated and compared with controls.
Rat HSC Migration Assay.
To examine the migration of HSC 6 × 104 cells per well were added to the upper compartment of a Transwell chamber, together with 10 μM Y27632-conjugate, free Y27632 (equimolar), or carrier. PDGF-BB (20 ng/ml) was added to the lower chamber to stimulate migration. The cells were then incubated for 24 h, fixed, and stained with Mayer's hematoxylin. Cells on both sides of the membrane were counted in five fields per membrane at 40× magnification. Migration was calculated as the percentage of cells on the lower side of the membrane relative to the total number of cells in each field.
All animal experiments were approved by the Animal Ethics Committee of the University of Groningen, The Netherlands. All animals were purchased from Harlan and kept at 12-h light/12-h dark cycles with ad libitum chow and water.
Contraction of Aorta Rings.
For measurement of contractility of isolated rat aortic rings, freshly excised aortas from Wistar rats (200–300 g) were used. Aortas were excised, cleaned of adherent and connective tissue, and cut in 3- to 4-mm-wide rings. Rings were mounted for isometric recording in organ bath chambers filled with pregassed (95% O2/5% CO2) Krebs-Henseleit buffer maintained at pH 7.4 and 37°C with an outer water jacket. Passive tension was adjusted to 0.5 g. After 60-min equilibration, rings were contracted using increasing concentrations of phenylephrine (10−8 to 10−5 M) followed by washing. Finally, after re-equilibration in Krebs-Henseleit buffer, rings were contracted with 10−7 M phenylephrine. After 15-min equilibration, increasing concentrations (10−8 to 10−5 M) of Y27632, Y27-conjugate, or M6PHSA were added, and the relaxation of the precontracted aortas was monitored using a Grass FT03 transducer (Grass Instruments, West Warwick, RI).
Chronic CCl4-Induced Liver Fibrosis Model.
For studies in the chronic model, male BALB/c mice (20–22 g) were injected twice a week intraperitoneally with increasing doses of CCl4 diluted in olive oil (week 1, 0.5 ml/kg; week 2, 0.8 ml/kg; weeks 3–8, 1 ml/kg). Control mice received olive oil. Drug administration was started in week 7 after the start of the CCl4 injections. The mice were divided into five groups: 1) control + vehicle (PBS), 2) CCl4 + vehicle (PBS), 3) CCl4 + Y27632-M6PHSA (equivalent to 1.5 mg/kg/day Y27632 and 45 mg/kg/day M6PHSA), 4) CCl4 + Y27632 (1.5 mg/kg/day), and 5) CCl4 + M6PHSA (45 mg/kg/day). All treatment groups received a total of six injections of Y27632, Y27-conjugate, or carrier on alternate days for 2 weeks. Blood cell counts were performed and serum markers were determined according to standard clinical procedures by the University Medical Centre, Groningen, The Netherlands.
Immunohistochemistry was performed on 4-μm cryo and paraffin sections. Stainings were visualized using 3,3′-diamino-benzidine tetrahydrochloride or 3-amino-9-ethylcarbazole. Immunofluorescent staining in sections was visualized using a M.O.M. kit (Vector Laboratories, Burlingame, CA) according to the manufacturer's instructions. Cells were counterstained with phalloidin-tetramethylrhodamine B isothiocyanate (Sigma-Aldrich) in immunofluorescent staining, and nuclei were counterstained with 4,6-diamidino-2-phenylindole or Mayer's hematoxylin. Immunohistochemical stainings were quantitated using the Cell D computer program (Olympus, Hamburg, Germany).
Results are expressed as the mean ± S.D., unless otherwise specified. Statistical analyses were performed using Student's t test or one-way analysis of variance with post hoc Bonferroni test. p < 0.05 was considered as the minimum level of significance.
Synthesis and Characteristics of Y27-Conjugate.
Y27632 (Fig. 1A) was coupled to M6PHSA using the ULS linker. The average molar coupling ratio of Y27632 to M6PHSA, calculated from HPLC analysis after chemical release of the drug (Fig. 1B), was seven molecules of drug per molecule of protein. HPLC analysis also showed that in the final conjugate no free drug was present (Fig. 1B). After two freeze-thaw cycles only 2% of drug was released, demonstrating the stability of the conjugate.
Y27-Conjugate Inhibits Fibrotic Parameters in Activated HSC.
Rho kinase pathway activation is known to regulate the migration and contraction of HSC. Therefore, we examined the effect of our conjugate on the migration of primary isolated HSC in vitro using a two-chamber (Transwell) system. We found that treatment with free Y27632 or conjugate reduced PDGF-BB-induced cell migration by approximately 50% (Fig. 2A). The expression of focal adhesion marker vinculin, which is involved in cell migration processes, was also inhibited (Fig. 2B). In parallel experiments, both free Y27632 and Y27-conjugate significantly inhibited the contraction of primary HSC seeded on collagen gels by approximately 60 and 20%, respectively (Fig. 2C). The significant effects of the conjugate on both functionalities of HSC demonstrate that the conjugate is pharmacologically active within the target cell.
To determine whether the effects seen with the conjugate were associated with the inhibition of Rho kinase, we examined the expression of the downstream mediator pMLC2. We found that pMLC2 was inhibited by both free Y27632 and the Y27-conjugate (Fig. 2D). The effects on HSC activation were thus paralleled by a decrease in Rho kinase activity in HSC.
Effects of the Carrier on Fibroblast Activation.
Because the M6P/IGFII receptor, the target receptor for our drug carrier, was reported to be involved in the binding and subsequent activation of latent TGF-β (Dennis and Rifkin, 1991; de Bleser et al., 1995), we examined whether inhibition of the M6P/IGFII receptor by M6PHSA leads to an antifibrotic effect through attenuation of the activity of latent TGF-β. We found that 0.2 mg/ml M6PHSA attenuated collagen production induced after incubation of fibroblasts with latent TGF-β (Fig. 3).
In Vitro Contraction of Rat Aorta.
To test whether our construct has pharmacological effects in tissue that is responsive to Rho kinase inhibition, yet lacks the M6P/IGFII receptor, we examined the effects of free and carrier-conjugated drug on vascular contraction. Y27632 is known to decrease vascular contraction (Hennenberg et al., 2006). We used freshly prepared rat aortas and measured contraction using standard isometric recording. The M6P/IGFII receptor was not expressed in this vascular tissue (data not shown). We incubated precontracted rat aorta rings with increasing concentrations of free drug or conjugate and found that free drug strongly affected the vascular tonus, i.e., induced a decrease of phenylephrine-induced vascular contraction, whereas Y27-conjugate did not have any effect at all (Fig. 4).
Intrahepatic Distribution of Y27-Conjugate.
To examine the intrahepatic distribution of the Y27-conjugate, we studied the distribution of HSA within livers of CCl4 mice. Mice received a dose of Y27-conjugate just before sacrifice (10 min), thus excluding any pharmacological effect of the construct on the fibrotic process. HSA staining was found to colocalize with the staining for desmin (Fig. 5), a marker for HSC, reflecting uptake in our target cell. In addition, staining for the Y27-conjugate was seen within the sinusoids, which is in accordance with previous results on the pharmacokinetics of M6PHSA (Beljaars et al., 1999), showing that the construct is still present in the blood 10 min after injection. PBS-treated mice did not show any positive staining for HSA.
Y27-Conjugate Reduces Chronic CCl4-Induced Liver Fibrosis in Mice.
Having demonstrated the pharmacological activities of our construct in vitro and its distribution pattern in vivo, we set out to examine the effects of Y27-conjugate in a chronic model of established liver fibrosis. In this model, fibrosis was induced by 8 weeks of administration of CCl4. Treatments were started after 6 weeks of CCl4 treatment, that is, when extensive fibrosis was already present. Animals received intravenous injections three times a week for 2 weeks. No toxicity of treatment with Y27-conjugate was found by examination of the blood cell counts, although treatment with the carrier alone resulted in a slight reduction in platelet count (Supplemental Table 1). None of the treatments led to kidney toxicity, as measured by plasma creatinine levels (Supplemental Table 2). Treatments with Y27-conjugate and free drug both led to a decrease in alanine aminotransferase and aspartate aminotransferase, which are serum markers for liver injury (Supplemental Figure 1).
The antifibrotic effects of the conjugate were assessed by analysis of two important fibrotic parameters, α-SMA and collagen I, by performing immunohistochemical stainings (Fig. 6, A and B). Treatment with the conjugate inhibited the activation of HSC, as determined by quantitation of α-SMA staining, to 61% (p < 0.01) of the PBS-treated group (Fig. 6C). Free Y27632 also significantly inhibited α-SMA expression to 68% (p < 0.05). However, only the conjugate reduced the intrahepatic deposition of collagen to 68% (p < 0.05) compared with the PBS-treated group, whereas the free drug had no significant effect on this parameter (Fig. 6D).
Furthermore, to examine whether the antifibrotic effects of the conjugate were related to the inhibition of the Rho kinase pathway, we examined the expression of pMLC2 in liver by immunofluorescent staining. We found an up-regulation of pMLC2 in the fibrotic septa of CCl4-treated livers compared with control livers. The pMLC2 staining colocalized with the staining for the HSC marker desmin (Fig. 7). This up-regulation was blocked by Y27-conjugate. However, the free drug had no detectable effect on pMLC2 staining in any of the fibrotic livers (Fig. 7).
In this study we have shown that cell-specific inhibition of Rho kinase in activated HSC leads to a reduction of liver fibrosis in mice. Cell specificity is conferred by coupling of the Rho kinase inhibitor to a drug carrier in a reversible way. We found that the carrier-conjugate colocalizes with desmin-positive cells in the fibrotic liver, and this is associated with a reduction in HSC activation. The conjugate reduced the most important parameter determining the extent of fibrosis, namely the deposition of collagen. In addition, the use of conjugate could reduce the effects of Rho kinase inhibitor on other tissues outside the liver, i.e., aorta. The strategy described here leads to a stronger reduction of fibrotic parameters than the use of an untargeted drug, as has been shown in previous articles examining cell-specific inhibition of fibrosis-related pathways in HSC (Gonzalo et al., 2006, 2007; Moreno et al., 2010). Here, we used a conjugate of Rho kinase inhibitor Y27632 coupled to the HSC-specific carrier M6PHSA, which has been found to accumulate in HSC during liver fibrosis (Beljaars et al., 1999).
The effect studies in primary HSC showed that the efficacy of the conjugate in vitro is comparable with that of an equivalent dose of the free drug, demonstrating that the drug is released from the conjugate in its active form. Both the conjugate and free drug inhibited pMLC2, which is a downstream target of Rho kinase. In addition to this, we examined two functional parameters that represent key activities of activated HSC. We showed that the inhibition of Rho kinase inhibitor-induced pMLC2 staining is paralleled by an inhibition of HSC migration and HSC contraction. Previous reports stated that the effect of inhibition of Rho kinase on the migrational ability of a cell depends on the concentration of the inhibitor and the cell type (Ridley, 2001). Rho kinase inhibition leads to reduced contraction of the cell, which inhibits migration, but also to reduced adhesion, facilitating migration (Ridley, 2001). The final effect on migration depends on which of these opposite effects prevails in the cell type studied. In accordance with earlier articles studying the effect of Rho kinase inhibition on HSC migration (Tangkijvanich et al., 2003; Melton and Yee, 2007), in our studies the Rho kinase inhibitor strongly inhibited migration in primary HSC, as did the conjugate.
The target receptor for our carrier is the M6P/IGFII receptor, which is highly up-regulated on activated HSC (de Bleser et al., 1995; Greupink et al., 2006) and causes rapid endocytosis of the drug-carrier complex (Beljaars et al., 1999). The M6P/IGFII receptor may also play a role in fibrogenesis: it binds latent TGF-β, thereby facilitating proteolytic cleavage and subsequent conversion of latent TGF-β into its active form (Dennis and Rifkin, 1991; de Bleser et al., 1995). This notion is supported by our in vitro studies, which show that blocking of the receptor by our carrier attenuated collagen production induced in fibroblasts after incubation with latent TGF-β. This effect may further add to the pharmacological effects of the drug-carrier complex in vivo, although no effect of the carrier alone was noted on protein expression levels of the parameters examined here, i.e., α-SMA or collagen I.
Kinase inhibitors represent highly relevant new options for the treatment of liver fibrosis (Parsons et al., 2007). Different pathways have been investigated in recent years, and several have been shown to be an interesting target for treatment (Son et al., 2009; Kluwe et al., 2010). In the last decade, a key role of the Rho kinase pathway in liver disease has been established, because it affects the activation, contraction, and migration of HSC. The inhibition of Rho kinase has been shown to be a feasible way to reduce liver fibrosis in two different rat models (Murata et al., 2001, 2003; Tada et al., 2001), although not yet in mice. By targeting the drug specifically to HSC, one of the main cell types responsible for the production of extracellular matrix and profibrotic cytokines can be selectively inhibited while using lower doses of drugs. Indeed, we now have used doses of 1.5 mg/kg in this study and established significant pharmacological effects.
One of the most important side effects to be expected of Y27632 is a decrease in vascular smooth muscle contractility (Riento and Ridley, 2004; Hennenberg et al., 2006). This could lead to a further deterioration of the mean arterial pressure during cirrhosis. This effect of Y27632 has been described in cirrhotic rats (Hennenberg et al., 2006) and was replicated by us in an ex vivo aorta model. However, in our studies, only free drug induced a relaxation in aortas, whereas Y27-conjugate did not have any effect on aorta contraction. The conjugate therefore seems to have no effect in this nontarget tissue, which lacks the M6P/IGFII receptor. In the mouse CCl4, model we did not detect any effects on pMLC2 expression in aortas, either from CCl4 or the treatments. However, because mouse aorta also lacks the M6P/IGFII receptor, we do not expect any effects of the conjugate in mouse aorta either.
In the chronic liver injury model, which is a model of established fibrosis, protein levels for α-SMA, an activation marker for HSC, were inhibited by both the free drug and the conjugate, whereas only the conjugate induced a decrease in collagen deposition. Previously, we found that the ULS linker between the drug and the drug carrier caused a slow release of the drug (Prakash et al., 2006). Apparently prolonged inhibition of Rho kinase activity by continuous release of the drug is necessary to reduce collagen production by HSC during fibrosis. In this context it is also striking that only the Y27-conjugate could reduce pMLC2 expression in liver, whereas in Y27632-treated mice there was no inhibition of pMLC2 staining in the liver. This indicates that drug levels in the livers of conjugate-treated mice were still sufficiently high to inhibit phosphorylation of MLC2 at the moment of sample collection, that is, 24 h after the last injection, whereas the direct effects of the free drug were not detectable anymore at this time point. The difference between the effect of free Y27632 on α-SMA and pMLC2 probably can be explained by the fact that phosphorylation of proteins is rapidly reversible, whereas any effects on expression of proteins, in this case α-SMA, tend to last longer.
In conclusion, we present here evidence that coupling of the Rho kinase inhibitor Y27632 to the HSC-specific carrier M6PHSA results in a conjugate that displays antifibrotic properties in vitro and in vivo. Targeted Rho kinase inhibitor has a stronger antifibrotic effect than the free inhibitor in vivo in a mouse model of advanced liver fibrosis. This study shows the benefits of a cell-specific approach, and it may open new opportunities for the treatment of liver fibrosis.
Participated in research design: van Beuge, Prakash, Lacombe, Beljaars, and Poelstra.
Conducted experiments: van Beuge, Lacombe, Gosens, Post, and Reker-Smit.
Contributed new reagents or analytic tools: Prakash, Lacombe, and Gosens.
Performed data analysis: van Beuge, Lacombe, Reker-Smit, and Poelstra.
Wrote or contributed to the writing of the manuscript: van Beuge, Prakash, Lacombe, Beljaars, and Poelstra.
This work was supported by the European Framework Program FP6 [Grant LSHB-CT-2007-036644].
Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
- hepatic stellate cells
- transforming growth factor β
- platelet-derived growth factor
- human serum albumin
- insulin-like growth factor II
- Universal Linkage System
- α-smooth muscle actin
- myosin light chain
- phosphorylated MLC
- (+)-(R)-trans-4-(1-aminoethyl)-N-(4-pyridyl)cyclohexanecarboxamide dihydrochloride
- phosphate-buffered saline: HPLC, high-performance liquid chromatography.
- Received January 10, 2011.
- Accepted March 3, 2011.
- Copyright © 2011 by The American Society for Pharmacology and Experimental Therapeutics