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Published ahead of print on January 27, 2005, doi:10.1165/rcmb.2004-0127OC
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American Journal of Respiratory Cell and Molecular Biology. Vol. 32, pp. 290-300, 2005
© 2005 American Thoracic Society
DOI: 10.1165/rcmb.2004-0127OC

Simvastatin Inhibits Growth Factor Expression and Modulates Profibrogenic Markers in Lung Fibroblasts

Keira L. Watts, Edith M. Sampson, Gregory S. Schultz and Monica A. Spiteri

Lung Research, Institute of Science and Technology in Medicine, University Hospital of North Staffordshire/Keele University, Stoke on Trent, Staffordshire, United Kingdom; and Department of Obstetrics and Gynecology, Institute of Wound Repair, Health Science Center, University of Florida, Gainesville, Florida

Correspondence and requests for reprints should be addressed to Keira Watts, Ph.D., Lung Research, Institute of Science and Technology in Medicine, School of Postgraduate Medicine, Thornburrow Drive, Hartshill, Stoke on Trent ST4 7NQ, UK. E-mail: keira_watts{at}yahoo.co.uk


   Abstract
Top
 Abstract
Introduction
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
Simvastatin is best known for its antilipidemic action and use in cardiovascular disease due to its inhibition of 3-hydroxy-3-methylglutaryl CoenzymeA (HMG CoA) reductase, a key enzyme in the cholesterol synthesis pathway. Inhibition of biological precursors in this pathway also enables pleiotrophic immunomodulatory and anti-inflammatory capabilities, including modulation of growth factor expression. Connective tissue growth factor (CTGF) and persistent myofibroblast formation are major determinants of the aggressive fibrotic disease, idiopathic pulmonary fibrosis (IPF). In this study we used human lung fibroblasts derived from healthy and IPF lungs to examine Simvastatin effects on CTGF gene and protein expression, analyzed by RT-PCR and ELISA, respectively. Simvastatin significantly inhibited (P < 0.05) CTGF gene and protein expression, overriding the induction by transforming growth factor-ß1, a known potent inducer of CTGF. Such Simvastatin suppressor action on growth factor interaction was reflected functionally on recognized phenotypes of fibrosis. {alpha}-smooth muscle actin expression was downregulated and collagen gel contraction reduced by 4.94- and 7.58-fold in IMR90 and HIPF lung fibroblasts, respectively, when preconditioned with 10 µM Simvastatin compared with transforming growth factor-ß1 treatment alone after 24 h. Our data suggest that Simvastatin can modify critical determinants of the profibrogenic machinery responsible for the aggressive clinical profile of IPF, and potentially prevents adverse lung parenchymal remodeling associated with persistent myofibroblast formation.

Key Words: {alpha}-SMA • CTGF • myofibroblasts • Simvastatin • TGF-ß1


   Introduction
Top
 Abstract
 Introduction
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
Statins are widely used in first line management of cardiovascular diseases due to their known efficacy in improving lipid profiles and direct vascular actions (1, 2). However, statins also possess pleiotrophic immunomodulatory and anti-inflammatory capabilities, which are comparatively poorly recognized. Statins have been shown to repress major histocompatibility complex class II (MHC-II)–mediated T cell activation (3), to modulate host inflammatory cell recruitment (4), and to downregulate activation of the early inflammatory response gene nuclear factor-{kappa}B (5). These additional effects of statins occur independently of their lipid-lowering abilities, as a direct inhibition of their target enzyme 3-hydroxy-3-methylglutaryl Coenzyme A (HMG-CoA) reductase at the subcellular level. This is a key enzyme in the cholesterol synthesis pathway. Simvastatin inhibits the synthesis of isoprenoid intermediates of cholesterol biosynthesis farnesylpyrophosphate (FPP) and geranylgeranyl-pyrophosphate (GGPP), which are used in the post-translation modification and prenylation of the key Ras and Rho family of proteins (Figure 1).



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  		Figure 1. Simvastatin acts early in the cholesterol synthesis pathway, inhibiting the rate-limiting enzyme HMG-CoA reductase. Downregulation of other biological precursors in this pathway, including geranylgeranylpyrophosphate (GGPP), causes disruption of signal transduction, protein synthesis, and energy metabolism. The intermediate GGPP is essential for the post-translational modification of the signaling molecule RhoA.

 
Of particular interest in the study of fibrogenesis are the recently reported observations that statins can induce regression of cardiac hypertrophy and fibrosis in transgenic rabbits with human hypertrophic cardiomyopathy (6). In addition, statin use may have substantial benefits after pulmonary transplantation, improving survival and reducing the incidence of obliterative bronchiolitis (7). Statins have also been shown to attenuate the expression of key profibrogenic mediators such as transforming growth factor (TGF)-ß1 in glomeruli of diabetic rats (8) and rat mesangial cells (9). TGF-ß1 is a pluripotent growth factor, and is produced locally at wound sites and within fibrotic lesions (10). Many of its actions are mediated through connective tissue growth factor (CTGF), an autocrine growth factor that is overexpressed in established fibrosis of the lung (11), kidney (12), and liver (13), and which acts downstream of TGF-ß1 to facilitate cell proliferation, collagen deposition, angiogenesis, and fibroblast differentiation into the profibrogenic myofibroblast phenotype. All these features are major determinants in the pathogenesis of idiopathic pulmonary fibrosis (IPF), an aggressive interstitial lung disease commonly affecting adults from middle age onwards and in which prognosis is invariably poor; median survival is 3–5 yr from diagnosis (14), with no currently available efficacious treatment.

Pooling the above information, we hypothesized that Simvastatin, an established pleiotrophic HMG-CoA reductase inhibitor, could selectively modulate crucial CTGF–TGF-ß1 interactions in human lung fibroblast models. In addition, Simvastatin can inhibit key functional markers of pulmonary fibrosis and block the transition of fibroblasts to the pro-fibrotic myofibroblast phenotype. We tested this hypothesis by examining the effect of a range of concentrations of Simvastatin on CTGF gene and protein expression in established culture models for normal lung fibroblasts and those derived from patients with IPF, in the presence/absence of TGF-ß1 exposure. We demonstrate that Simvastatin is able to override TGF-ß1 induction of CTGF in lung fibroblasts and downregulate CTGF at both gene and protein level. To determine whether the actions of Simvastatin on CTGF–TGF-ß1 interrelationship are reflected functionally on recognized phenotypes of fibrosis, we examined drug effects on collagen gel contraction and {alpha}-smooth muscle actin (SMA) gene and protein expression. We show that Simvastatin can effectively inhibit both collagen gel contraction and {alpha}-SMA expression. We also show that these effects on myofibroblast markers are likely to result from the inhibitory actions of Simvastatin on the RhoA signaling pathway, as demonstrated by the use of C3 exotoxin, a specific Rho inhibitor. These myofibroblast-mediated profibrogenic phenotypes are responsible for much of the tissue remodeling and clinical morbidity associated with IPF (15). These regressive effects of Simvastatin on key phenotypes of pulmonary fibrogenesis have not been reported before; they merit further detailed investigation. If confirmed in vivo, these findings could open novel avenues for anti-IPF strategies targeted toward halting or reversing the adverse lung parenchymal remodeling associated with growth factor overexpression and consequent persistent myofibroblast formation.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 References
 
Human Lung Fibroblast Cell Culture
Normal human lung fibroblasts (IMR90; ATCC, Manassas, VA) and three separate human lung fibroblast cell lines isolated from patients with IPF (LL29 and LL97a were from ATCC; HIPF were a generous gift from R. J. McAnulty, UCL London, London, UK) were cultured in Dulbecco's modified Eagle's medium (DMEM; Labtech, Sussex, UK). Medium was supplemented with penicillin/streptomycin (100 U/ml; Gibco BRL, Paisley, Scotland) and L-glutamine (2 mM, Gibco BRL, Paisley, Scotland) with 10% fetal calf serum (FCS; Labtech). For experiments, medium was replaced with serum-free DMEM (SF-DMEM) for 48 h to induce quiescence before treatment.

Simvastatin Treatment of Lung Fibroblasts
Simvastatin (Merck, Sharp and Dohme Ltd, Hertfordshire, UK) was dissolved and filter-sterilized before use (16). Quiescent lung fibroblasts were incubated overnight (18 h) with Simvastatin in SF-DMEM at concentrations of 0.1, 1, and 10 µM. The Simvastatin concentrations used (0.1–10 µM) are within physiologically relevant levels of the drug, as used in clinical practice (in which doses vary from 10–80 mg/d).

C3 Exotoxin Treatment of Lung Fibroblasts
Quiescent lung fibroblasts were incubated overnight (16 h) with Clostridium botulinum C3 exotoxin (Upstate Cell Signaling Solutions, Waltham, MA) in SF-DMEM. C3 exotoxin was used at concentrations of 0.5, 1, and 5 µg/ml; these doses have been previously shown to inhibit Rho signaling pathways in similar fibroblast lines.

TGF-ß1 Treatment
After Simvastatin preconditioning, cells were stimulated with human recombinant TGF-ß1 (R&D Systems, Oxford, UK) at a dose of 5 ng/ml for up to 24 h. This chosen concentration was determined from ongoing studies within our laboratories, in which 5 ng/ml showed significant induction of CTGF. Fibroblasts exposed to Simvastatin and/or TGF-ß1 were harvested for gene and protein analysis.

Real-Time PCR
Stored cDNA samples isolated from normal and IPF isolated lung fibroblasts were used to assess CTGF and {alpha}-SMA gene expression. Two microliters of undiluted cDNA was used per reaction; the primer and probe sets were "predesigned assay on demand" probes (Applied Biosystems, Foster City, CA); these predesigned primers are tested and standardized for reproducible expression analysis. Primer and cDNA were added to the TaqMan universal PCR master mix (Applied Biosystems) containing all the reagents for PCR. Absolute quantification of the PCR products was performed with an ABI prism 7,000 (Applied Biosystems) using the relative standard curve method. cDNA that positively expresses the gene of interest is used to create a dilution series with arbitrary units. To ensure reproducibility, quantitative data were taken at a point in which each sample was in the exponential phase of amplification. The mean quantity of target gene expression was determined from the generated standard curve; then all samples were normalized against an internal standard ß-actin in all quantitative PCR reactions. All data are presented as the fold-change over control in {alpha}-SMA gene expression.

CTGF Protein Detection by Immunofluorescence
Treated lung fibroblasts were washed and fixed in situ in ice-cold methanol for 5 min. A primary rabbit monoclonal anti-human CTGF antibody (Fibrogen, San Francisco, CA) was used to detect CTGF protein in fixed lung fibroblasts using a dilution of 1:1,000 in PBS for 30 min at room temperature. After washing thoroughly with PBS, a secondary antibody, fluorescein isothiocyanate (FITC)-conjugated goat anti-rabbit IgG (Sigma Aldrich, Dorset, UK), was used at a 1:1,000 dilution for 30 min at room temperature. The samples were then washed and mounted onto slides using Vectashield mounting medium containing 4',6-Diamidino-2-phenylindole (DAPI), a specific nucleic acid stain (Vector Laboratories, Peterborough, UK). Each field of view was analyzed first for the total number of cells present as determined by DAPI staining of the nuclei. From the exact same field of view, the FITC-labeled CTGF protein was analyzed and the number of cells staining positively for fluorescence counted according to the published procedure (17), and five random fields of view were selected and imaged for each treatment. Slides were viewed under epifluorescence with filter set 450–490 nm for FITC and 340–380 nm for DAPI. Images were obtained using a Leica DC200 digital camera and software (Leica Microsystems, Heerbrugg, Switzerland). Camera exposure setting remained constant throughout. The results were expressed as the percentage of cells positively expressing the protein within the fields of view; the mean ± SEM was calculated.

CTGF Sandwich Enzyme-Linked Immunosorbent Assay
CTGF protein was measured in the conditioned medium of cultured cells and cell lysates by capture sandwich enzyme-linked immunosorbent assay (ELISA) with biotinylated and nonbiotinylated affinity-purified goat polyclonal antibodies to human CTGF as described previously (18). Cell lysates were prepared by solubilizing cell cultures in TBS containing 0.1% Triton X-100 and a protease inhibitor cocktail added (Sigma Aldrich, Dorset, UK). The solubilized samples were centrifuged at 15,000 x g for 15 min and the supernatant solution assayed in the ELISA. The ELISA was performed as described previously (19); briefly, this involved a flat-bottomed ELISA plate being coated with 50 µl of goat anti-human CTGF antibody at a concentration of 10 µg/ml in PBS and 0.02% sodium azide for 1 h at 37°C. Wells were washed four times and incubated with 300 µl of blocking buffer (PBS, 0.02% sodium azide and 1% bovine serum albumin) for 1 h at room temperature. The wells were rewashed four times and 50 µl of recombinant human CTGF protein (0.1–100 ng/ml) or sample was added and incubated at room temperature for 1 h. After washing, 50 µl of alkaline phosphatase–conjugated streptavidin (1.5 µg/ml; Zymed, South San Francisco, CA) was added and incubated at room temperature for 1 h. The wells were rewashed and incubated in alkaline phosphatase substrate solution (Sigma). Absorbance was measured at 405 nm with a microplate reader (Molecular Devices, Sunnyvale, CA); CTGF protein levels were normalized for total protein content using a bicinchoninic acid (BCA) protein assay (Bio-Rad, Hercules, CA) and were expressed as ng/mg protein from three replicate samples for each condition.

{alpha}-SMA Protein Expression by Immunofluorescence
Lung fibroblasts treated with Simvastatin (0.1–10 µM) and/or TGF-ß1 (5 ng/ml) were washed in PBS and fixed in situ using ice-cold methanol (5 min). After further washing in PBS the samples were incubated with FITC-conjugated monoclonal anti–{alpha}-SMA antibody (clone 1A4; Sigma Aldrich), diluted 1:250 in PBS for 30 min at room temperature. Samples were analyzed as described above.

Collagen Gel Contraction Assays
Collagen lattices were prepared using type I collagen from rat tail tendon (Stratech Scientific, Luton, UK) adjusted to final concentration 2.5 mg/ml with 10x DMEM and 1N NaOH. Lung fibroblasts were suspended in SF-DMEM and mixed with the neutralized collagen solution to give a final cell density of 1 x 106 cells/ml and final collagen concentration of 1.25 mg/ml. Aliquots (0.3 ml/well) of this solution were seeded into each well of a 24-well plate. Collagen lattices were polymerized for 20 min in humidified 5% CO2 atmosphere at 37°C, then incubated with DMEM containing 10% FCS for 4 h, followed by overnight incubation in SF-DMEM. Simvastatin was added overnight to precondition lung fibroblasts before TGF-ß1 stimulation. To initiate collagen contraction, polymerized gels were gently released from the underlying culture dish and fibroblasts immediately stimulated with TGF-ß1 (5 ng/ml) in serum-free media. The degree of collagen gel contraction was determined after 0.5, 1, 2, 4, 8, and 24 h. To determine the degree of collagen gel contraction, the area of each gel after release was measured in mm by scanning the gel with an imaging densitometer (Bio-Rad); data were expressed as a percentage of the uncontracted gel size.

Statistical Analysis
Data are shown as a mean ± SEM. An unpaired Student's t test was employed for comparing two groups of data. Multiple comparisons were made using ANOVA followed by Tukey's pairwise comparison. All P values < 0.05 were considered significant.


   RESULTS
Top
 Abstract
 Introduction
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 References
 
Simvastatin Overrides and Downregulates the TGF-ß1 Induction of CTGF
CTGF gene expression. The effect of Simvastatin (0.1–10 µM) on basal and TGF-ß1–induced expression of the CTGF gene in normal and IPF-diseased lung fibroblasts was analyzed by real-time RT-PCR; the results are presented in Figure 2. After serum deprivation for 48 h, the cells became quiescent; these cells were then exposed to Simvastatin overnight. At basal levels Simvastatin alone induces a 3.3-fold and 10-fold decrease in CTGF gene expression in normal lung fibroblasts at 1 µM and 10 µM, respectively (P < 0.05). Responses were comparable in IPF-derived fibroblasts in which Simvastatin induced a 1.25- and 2.0-fold decrease at the same concentrations. TGF-ß1 is a known potent CTGF molecule inducer. Accordingly, compared with untreated controls, there is a marked increase in CTGF gene expression after TGF-ß1 treatment (P < 0.05) in both normal and IPF-derived lung fibroblasts. However, fibroblast preconditioning with Simvastatin overnight, before TGF-ß1 treatment, induces a significant dose-dependent downregulation of the CTGF gene in all cell lines (P < 0.05), suppressing levels to below basal conditions at higher concentrations. These data imply that Simvastatin overrides the inductive effects of TGF-ß1 on CTGF gene expression. Although results in Figure 2 are those from HIPF fibroblast cultures, the same pattern of Simvastatin downregulation was seen in the other two IPF-derived (LL29 and LL97a) fibroblast lines.



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  		Figure 2. CTGF gene expression in normal lung fibroblasts; IMR90 and human lung fibroblasts derived from patients with IPF; HIPF was determined by quantitative real-time RT-PCR. Serum-deprived cells were grown in SF-DMEM for 48 h to ensure quiescence; cells were then preconditioned with Simvastatin (0.1–10 µM) overnight (16 h). TGF-ß1 treatment at 5 ng/ml was performed after Simvastatin preconditioning for 4 h. Experiments were performed in triplicate and data are expressed as a fold change relative to the control CTGF transcript: ß-actin transcript ± SEM. Significant differences (P < 0.05) compared with serum-deprived cells are represented by *, and compared with TGF-ß1 treatment denoted by {dagger}.

 
CTGF protein expression. CTGF protein expression was determined by two independent methods: immunofluorescent cytology and ELISA. CTGF protein was FITC-labeled for immunofluorescent analysis, with additional nucleic acid staining with DAPI. The percentage number of cells staining positive for CTGF protein are presented in Figure 3A; representative images of lung fibroblasts stained for CTGF protein are presented in Figure 3B (IMR90) and Figure 3C (HIPF). A significant increase in percentage number of cells expressing CTGF after TGF-ß1 exposure compared with untreated controls in both normal and IPF-derived fibroblasts (P < 0.05) was observed. In contrast, preconditioning the same cells with Simvastatin resulted in a decrease in the number of positively staining cells in a concentration-dependent manner (Figure 3A). This supports the above findings at the gene level; the response to Simvastatin preconditioning occurs irrespective of TGF-ß1 stimulation. Comparable results were also observed in the other two IPF-derived cell lines (LL29 and LL97a).







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  		Figure 3. (A) Quiescent serum-deprived lung fibroblasts were preconditioned for 16 h with Simvastatin (0.1–10 µM) and then stimulated with TGF-ß1 (5 ng/ml) for 24 h. CTGF protein was detected using immunofluorescent cytology; the data are presented as the % number of cells staining positive for CTGF protein within the population ± SEM. Experiments were performed in triplicate. Significant differences (P < 0.05) compared with untreated control cells are represented by *, differences compared with TGF-ß1–treated cells are denoted by {dagger}. (B) Representative images of normal lung fibroblasts; IMR90 stained for CTGF protein. (a) Untreated controls. (b) –Cells treated with 5 ng/ml TGF-ß1 for 24 h. (c) Cells preconditioned with 0.1 µM Simvastatin for 16 h before 5 ng/ml TGF-ß1 treatment. (d) Cells preconditioned with 10 µM Simvastatin for 16 h before 5 ng/ml TGF-ß1 treatment. (C) Representative images of IPF-derived lung fibroblasts; HIPF stained for CTGF protein. (a) Untreated controls. (b) –Cells treated with 5 ng/ml TGF-ß1 for 24 h. (c) Cells preconditioned with 0.1 µM Simvastatin for 16 h before 5 ng/ml TGF-ß1 treatment. (d) Cells preconditioned with 10 µM Simvastatin for 16 h before 5 ng/ml TGF-ß1 treatment. (D) Expression of CTGF protein in the conditioned media from human lung fibroblasts (IMR90 and HIPF) was detected by ELISA. Cells were grown until confluent and starved of serum for 48 h. Cells were then incubated in Simvastatin (0.1–10 µM) overnight followed by 24 h TGF-ß1 treatment as indicated on the graph. Each treatment group was assayed in triplicate. Results are presented as ng CTGF per mg of total protein, and are expressed as the mean ± SEM. Significant differences (P < 0.05) are expressed as *compared with control, {dagger} compared with TGF-ß1 in HIPF and {ddagger} compared with TGF-ß1 in IMR90. (E) Expression of CTGF protein in cell lysates of human lung fibroblasts (IMR90 and HIPF) was detected by ELISA. Cells were grown until confluent and starved of serum for 48 h. Cells were then incubated in Simvastatin (0.1–10 µM) overnight followed by 24 h TGF-ß1 treatment as indicated on the graph. Each treatment group was assayed in triplicate. Results are presented as ng CTGF per mg of total protein, and are expressed as the mean ± SEM. Significant differences (P < 0.05) are expressed as *compared with control, {dagger} compared with TGF-ß1 in both HIPF and IMR90.

 
These findings were also supported using CTGF ELISA; conditioned media from lung fibroblast cultures (Figure 3D) were analyzed for secreted CTGF protein. In addition, cell lysates (Figure 3E) were analyzed for CTGF protein remaining within the cells. Results show that CTGF release from lung fibroblasts is induced by TGF-ß1 stimulation and that this can be significantly reduced by Simvastatin (P < 0.05 at 1 µM and 10 µM in IMR90 cells, and at 0.1 µM and 10 µM in HIPF cells). A reduction is also seen in CTGF protein relative to the control after Simvastatin treatment, indicating a reduction in basal CTGF expression. Similarly, the same trends are observed in cell lysates. We show that CTGF protein levels are abrogated in both cell lines upon Simvastatin preconditioning irrespective of TGF-ß1 treatment (P < 0.05 at all concentrations 0.1–10 µM). The CTGF protein detected is markedly higher in cell lysates compared with conditioned media; as expected, overall CTGF production is greatest in the IPF-derived cell line. These observations suggests that CTGF production differs between the cell lines; in HIPF cells, only a fraction of the CTGF produced is secreted as a large proportion of CTGF is retained within the cells.

Simvastatin Inhibits Collagen Gel Contraction
During the course of fibrogenesis, in situ fibroblast populations differentiate to adopt a different morphology that is more aggressive and contractile in nature, known as the myofibroblast (20). Collagen gel contraction assays enable functional differentiation to be assessed as contraction directly relates to the myofibroblast phenotype (21). In these studies normal and IPF-derived lung fibroblasts were seeded into a collagen matrix. The fibroblasts were preconditioned with Simvastatin at 0.1 µM and 10 µM before the matrices were released and stimulated with the profibrotic mediator TGF-ß1 (5 ng/ml); results are shown in Figures 4A and 4B. Generally, lower levels of contraction are observed in normal lung fibroblasts (IMR90) regardless of treatment, compared with equivalent IPF-derived fibroblasts (HIPF). Contraction occurs at low levels in the control, unstimulated fibroblasts of both cell types; TGF-ß1 exposure induced maximal contraction of the gel matrix, which is significant at later time points (P < 0.05). In contrast, gels preconditioned with Simvastatin before TGF-ß1 treatment consistently inhibited contraction, with pronounced suppression observed at 10 µM Simvastatin, where collagen gel contraction was reduced to near-basal levels. At 24 h, collagen gel contraction in IMR90 (Figure 4A) and HIPF (Figure 4B) was reduced by 4.94- and 7.58-fold, respectively, in the presence of 10 µM Simvastatin, irrespective of the addition of TGF-ß1 (5 ng/ml). In addition, although contraction increased with time as cells were exposed to longer periods of TGF-ß1, the overriding suppression induced by the presence of Simvastatin remains evident throughout the experimental period. Reproducible results were obtained from another two separate IPF-derived cell lines (LL29 and LL97a).




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  		Figure 4. Normal lung fibroblasts, IMR90 (A), and IPF-derived lung fibroblasts, HIPF (B), were grown in a collagen matrix before overnight (16 h) incubation with 0.1 or 10 µM Simvastatin (Sim) followed by 5 ng/ml TGF-ß1 treatment. The controls were untreated, and the contraction of the collagen matrix was measured over a 24-h time course. Experiments were performed in triplicate and the data are representative of the % decrease in collagen gel area ± SEM. Significant differences (P < 0.05) are denoted by * with respect to TGF-ß1–induced contraction over controls and 10 µM Simvastatin treatment at the same time points.

 
Simvastatin Downregulates {alpha}-SMA Gene and Protein Expression; Marker of the Myofibroblast Phenotype
Fibroblasts expressing higher levels of {alpha}-SMA have an enhanced ability to contract collagen gels and reflect the myofibroblast phenotype. {alpha}-SMA gene expression in normal and IPF-derived lung fibroblast cell lines was analyzed by real-time PCR; the results are presented in Figure 5A. Gene expression of this myofibroblast marker was significantly upregulated upon addition of TGF-ß1 (5 ng/ml); this induction was, however, abrogated in a concentration-dependent manner after preincubation with Simvastatin (0.1–10 µM). Simvastatin significantly (P < 0.05) suppressed levels of {alpha}-SMA gene expression in all fibroblast cell lines at all concentrations used. Interestingly, we observed significant differences in {alpha}-SMA production between normal and IPF-derived lung fibroblasts (P < 0.01); this would be expected as IPF-derived fibroblasts are recognized to be associated with the aggressive contractile, myofibroblast phenotype characterized by {alpha}-SMA expression. Similar {alpha}-SMA expression profiles were observed in a further two IPF-derived fibroblast cell lines (LL29 and LL97a).





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  		Figure 5. (A) {alpha}-SMA gene expression in human lung fibroblasts (HIPF and IMR90) was determined by real-time PCR. The serum deprived cells were grown in SF-DMEM for 48 h to ensure quiescence; cells were then preconditioned with Simvastatin (0.1–10 µM) overnight (16 h), before TGF-ß1 (5 ng/ml) treatment. Experiments were performed in triplicate and data are expressed as the fold change in {alpha}-SMA:ß-actin transcript expression relative to the control ± SEM. *Significant increase above the control (P < 0.05); {dagger} significant decrease compared with TGF-ß1 treatment (P < 0.05). (B and C) Quiescent serum-deprived lung fibroblasts (normal IMR90, B; IPF-derived HIPF, C) were stained for presence of {alpha}-SMA protein, and cells were preconditioned for 16 h with Simvastatin (0.1–10 µM) and then stimulated with 5 ng/ml TGF-ß1 for up to 48 h. {alpha}-SMA protein was detected using immunofluorescent cytology; the data are presented as the percentage of cells positively expressing the protein within the population ± SEM. Experiments were performed in triplicate, significant differences (P < 0.05) are denoted by * with respect to TGF-ß1 treatment above control and Simvastatin treatments at these time points.

 
To further support these findings, immunofluorescent staining of {alpha}-SMA protein was performed in response to TGF-ß1 (5 ng/ml) and Simvastatin (0.1–10 µM) over 48 h. The percentage number of cells positively expressing this protein were calculated; the results for normal lung fibroblasts (IMR90) are presented in Figure 5B, IPF-derived lung fibroblasts (HIPF) in Figure 5C. In control unstimulated fibroblasts, {alpha}-SMA decreased over the time course, whereas with addition of TGF-ß1 the {alpha}-SMA expression was markedly induced over the same time (P < 0.05). Cells preconditioned with either 0.1 or 10 µM Simvastatin before exposure to TGF-ß1 showed a reduction in {alpha}-SMA–expressing cells. As with the contraction studies above, we observed that the percentage number of {alpha}-SMA positively stained cells at the early time points (before 4 h) was lower than equivalent controls. However, as cells were exposed to longer periods of TGF-ß1, the relevant proportions of positive stained cells increases, but never reach those of cultures treated with TGF-ß1 alone. Thus, the overriding suppression induced by the presence of Simvastatin remains evident throughout the experimental period.

Effect of C3 Exotoxin, a Potent Inhibitor of Rho, on {alpha}-SMA Gene and Protein Expression
Clostridium botulinum C3 exotoxin is an enzyme that specifically ADP ribosylates and inactivates Rho. It is known that the Rho pathway is disrupted after Simvastatin treatment, and we have recently shown that Rho signaling mechanisms can modulate CTGF–TGF-ß1 interaction, which has important implications in IPF (22). To confirm this, in this present study separate experiments using C3 exotoxin were conducted to explore the effects of inhibiting Rho on {alpha}-SMA gene (Figure 6A) and protein (Figure 6B) in lung fibroblasts, assessing whether this would replicate the responses previously observed with Simvastatin preconditioning. {alpha}-SMA gene expression was determined by real-time RT-PCR; compared with control levels, 5 µg/ml of C3 exotoxin induced a 5.6- and 1.75-fold inhibition of {alpha}-SMA expression in IMR90 and HIPF, respectively (P < 0.05). TGF-ß1 induces induction of the {alpha}-SMA gene, while C3 exotoxin at the highest concentration initiated a 2.3- and 1.9-fold decrease in {alpha}-SMA expression in IMR90 and HIPF lung fibroblasts (P < 0.05), irrespective of subsequent TGF-ß1 stimulation. Thus, this specific Rho inhibitor was able to induce inhibition of this myofibroblast marker in a similar pattern as previously seen by the use of Simvastatin. This is also translated to the protein level; as seen previously TGF-ß1 causes significant (P < 0.05) induction of the myofibroblast phenotype as determined by expression of {alpha}-SMA protein in both cell types but most noticeably in IPF derived fibroblasts. Treatment of cells with C3 exotoxin induces a reduction in {alpha}-SMA–expressing cells; this occurs even in the presence of TGF-ß1 and is significant (P < 0.05) at all concentrations of inhibitor in HIPF and at the highest concentration (5 µg/ml) in IMR90. We observed reduction in the percentage of cells expressing the protein correlated with a reduction in fluorescent emissions from the cells (Figures 6C and 6D), with maximal inhibition observed after administration of 5 µg/ml of C3 exotoxin. This reduction in {alpha}-SMA at both the gene and protein level is in line with the inhibition also seen by the treatment of the same fibroblasts with Simvastatin.






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  		Figure 6. (A) {alpha}-SMA gene expression in normal lung fibroblasts (IMR90) and human lung fibroblasts derived from patients with IPF (HIPF) was determined by quantitative real-time RT-PCR. Serum-deprived cells were grown in SF-DMEM for 48 h to ensure quiescence; cells were then preconditioned with C3 exotoxin (0.5–5 µg/ml) overnight (16 h). TGF-ß1 treatment at 5 ng/ml was performed after Simvastatin preconditioning for 4 h. Experiments were performed in triplicate and data are expressed as a mean expression of the {alpha}-SMA transcript:ß-actin transcript ± SEM. Significant differences (P < 0.05) are represented by *. (B) Quiescent serum-deprived lung fibroblasts (normal IMR90 and IPF-derived HIPF) were stained for the presence of {alpha}-SMA protein. Cells were preconditioned for 16 h with C3 exotoxin (0.5–5 µg/ml) and then stimulated with TGF-ß1 (5 ng/ml) for 24 h. The mean number of cells expressing the {alpha}-SMA protein as determined by immunofluorescent cytology ± SEM is presented. Significant difference in expression is denoted by *(P < 0.05) compared with control and {dagger} compared with TGF-ß1. Data are representative of images obtained from three independent experiments. (C) Representative immunofluorescent cytology images of IMR90 lung fibroblasts stained for {alpha}-SMA protein. (a) Untreated control cells. (b) Cells treated with 5 ng/ml TGF-ß1 for 24 h. (c) Cells treated with 5 µM C3 exotoxin for 16 h. (d) Cells treated with 5 µM C3 exotoxin for 16 h before TGF-ß1 (5 ng/ml) treatment for 24 h. (D) Representative immunofluorescent cytology images of HIPF lung fibroblasts stained for {alpha}-SMA protein. (a) Untreated control. (b) Cells treated with 5 ng/ml TGF-ß1 for 24 h. (c) Cells treated with 5 µM C3 exotoxin for 16 h. (d) Cells treated with 5 µM C3 exotoxin for 16 h before TGF-ß1 (5 ng/ml) treatment for 24 h.

 

   DISCUSSION
Top
 Abstract
 Introduction
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
References
 
Dysregulation of CTGF expression and responses to TGF-ß1 are central factors in influencing development of fibrosis, and consequently the irreversible remodeling associated with fibrogenic diseases such as IPF. Conventional immunosuppressive therapy for IPF has been shown to be ineffective; thus future therapeutic strategies need to veer away from anti-inflammatory drugs and concentrate on targeting key mediators (23) to reverse/halt profibrogenic events. The ability to modulate intimate growth factor interactions such as the relationship between TGF-ß1 and CTGF, and to selectively disrupt consequent functional/structural profibrogenic determinants, may provide a new therapeutic avenue for fibrotic lung diseases. Recent literature has highlighted the effect of Simvastatin (a widely used lipid-lowering agent) in blocking CTGF actions (24) as well as its antifibrotic potential in renal disease (25). In the present study, we demonstrate that Simvastatin can downregulate both CTGF gene and protein expression, irrespective of TGF-ß1 stimulation, in a dose-dependent manner in both normal and IPF lung fibroblasts. Simultaneously, Simvastatin blocks functional markers of fibrogenesis, specifically the differentiation of the myofibroblast as demonstrated by an abrogation of collagen gel contraction and reduction in {alpha}-SMA expression. These effects are likely to result from inhibition of Rho signaling as determined by treatment with C3 exotoxin; a known potent inhibitor of Rho. This article provides indirect evidence that Rho inhibition is responsible for the inhibition of myofibroblast markers caused by Simvastatin. We have confirmed an influential role of Rho inhibition by Simvastatin in abrogating the profibrotic mediator CTGF in human lung fibroblasts using similar techniques (22).

Despite the recognized pivotal role played by myofibroblasts in the tissue remodeling processes underlying fibrotic lung diseases (20), the effect of statins on human lung fibroblast function has hitherto not been explored. Thus, our culture models were designed to examine the effects of Simvastatin on human fibroblasts derived from both healthy and IPF-diseased lungs, and in particular analyze drug influence on established growth factors. Myofibroblasts differentiate from precursor quiescent fibroblasts during tissue healing, and persist when tissue repair becomes aberrant such as observed in IPF. The myofibroblast trait possesses the requisite aggressive, contractile properties to promote deranged deposition of ECM proteins in situ (26) and induce tissue structural remodeling. Thus, hallmarks of the myofibroblast phenotype include the expression of contractile proteins such as {alpha}-SMA (27) and their ability to contract collagen gels. The collagen lattice contraction studies used in our experiments involved the growth of normal and IPF-derived lung fibroblasts in a three-dimensional network of collagen ECM protein; this is considered to be a suitable in vitro model of wound healing and scar formation. Fibroblasts seeded into the collagen matrix are able to exert contractile functions upon stimulation with mediators such as TGF-ß1, and there is a correlation between the level of {alpha}-SMA expression and fibroblast contraction (21). TGF-ß1 was shown to significantly enhance the contraction of collagen lattices using HIPF (2.10-fold increase) and IMR90 (1.6-fold increase) fibroblasts over unstimulated controls at 24 h. In contrast, equivalent gel matrices preconditioned with Simvastatin overnight (before TGF-ß1 exposure) presented an inhibited contractile ability. The ability of Simvastatin to override the potent inductive effects of the growth factor TGF-ß1 and modify contractile properties was simultaneously associated with a significant downregulation in {alpha}-SMA gene expression analyzed by real-time PCR. Fibroblasts selectively preconditioned with Simvastatin (0.1–10 µM) compared with those exposed to TGF-ß1 alone showed significant (P < 0.05) downregulation of {alpha}-SMA. The same trends were seen when {alpha}-SMA protein expression was analyzed using immunofluorescent cytology. Simvastatin-induced inhibition of markers of fibrosis was associated with a direct downregulation of CTGF at the gene and protein level in both normal and IPF-derived fibroblasts. Interestingly, Simvastatin also modulated basal levels of CTGF; this may suggest that Simvastatin can directly affect CTGF gene transcription, irrespective of any induction from TGF-ß1.

The weight of our data suggests that therapeutic concentrations of Simvastatin beneficially modulate the major determinants of morbidity associated with IPF, an action/s occurring independently of the cholesterol-lowering properties of this drug class. These observations are supported by findings in other cell types. Simvastatin is able to reduce the proliferation of cultured human atrial myofibroblasts (28) and vascular smooth muscle cells (29), and to block CTGF actions in human mesangial cells (24). Simvastatin has also been found to prevent the differentiation of hepatic stellate cells into myofibroblasts, modulating the myofibroblast phenotype (30) with implications for liver fibrosis and liver cancer. Others have shown that Lovastatin, another member of the 3-HMG-CoA reductase inhibitors, can induce fibroblast apoptosis (31). In separate studies, we have also shown that Simvastatin in high concentrations (50–100 µM) promotes lung fibroblast apoptosis, while reducing collagen production (32). Within the context of fibrogenesis, such proapoptotic actions would be expected to reverse myofibroblast persistence, enhance their clearance, and allow normal tissue repair processes to progress, potentially limiting lung damage.

Interestingly, this beneficial protective effect of Simvastatin against the proliferative and invasive myofibroblast phenotype is reflected in both animal and human studies.

Simvastatin treatment of transgenic rabbits with hypertropic cardiomyopathy induced regression of the cardiac hypertrophy and fibrosis, with improvement in cardiac function (6). Simvastatin has also been recently associated with the improved function and survival of lung allografts, consequent on reduced incidence of obliterative bronchiolitis in recipients taking statins (7). Bronchiolitis obliterans syndrome (BOS) is an obstructive distal airways disease which invariably afflicts lung transplant patients; it is a fibroproliferative process involving the accumulation of mesenchymal cells and their connective tissue products within the bronchiolar lumina, resulting in progressive airflow obstruction and graft failure (33). Based on our study results and other quoted observations, it is plausible to suggest that Simvastatin's ability to downregulate CTGF expression profiles and override TGF-ß1, thus modulating markers of the myofibroblast phenotype, may have prevented the development of obliterative bronchiolitis in all the 15 recipients receiving statins during their first postoperative year. Interestingly, similar findings were observed in the development of allograft arteriosclerosis; transplant graft arterial disease (GAD) is a major limitation in graft survival, leading to ischemia and graft failure. HMG CoA reductase inhibitors such as Simvastatin reduce GAD incidence in human cardiac allografts, and diminish host inflammatory cell recruitment (34).

We postulate that the mechanism/s by which Simvastatin is able to effect its antifibrogenic potential occur partly through direct inhibition of the Rho-signaling pathway. We have recently shown that Simvastatin disrupts intracellular signal transduction via inhibition of isoprenoid synthesis, namely the geranylgeranylation of Rho, resulting in the inhibition of CTGF (22). Recently, it has been shown that the small G protein Rho is also involved in lung myofibroblast differentiation, and constitutively active RhoA induces collagen gel contraction and {alpha}-SMA organization (35). This is supported by our data in which C3 exotoxin (a specific inhibitor of Rho) was used and was found to inhibit {alpha}-SMA expression at both the gene and protein level. The pattern of inhibition reflects that achieved with the use of Simvastatin, therefore suggesting that inhibition of Rho signaling by Simvastatin is implicated in the beneficial effects of reducing myofibroblastic markers in lung fibroblasts. Simvastatin has also been reported to prevent adverse cardiac remodeling associated with the cardiac myofibroblast phenotype via a mechanism involving inhibition of Rho geranylation (28). Patel and coworkers have implicated other critical subcellular effects of Simvastatin, involving reduction in levels of activated stress-responsive signaling kinases, which are activated in fibrosed tissue (6).

These secondary actions of statins, independent of their hitherto established antilipidemic properties, now need to be dissected and therapeutically exploited in an attempt to prevent/reverse the adverse lung parenchymal remodeling associated with fibrotic lung diseases. This will greatly reduce the significant morbidity and mortality associated with IPF.


   Acknowledgments
 
The authors thank Dr. Robin McAnulty (UCL, London, UK) for provision of the HIPF cell line used in this study.


   Footnotes
 
This work was generously supported by the British Lung Foundation (grant number P02/5); K.L.W. is a BLF research fellow.

Conflict of Interest Statement: K.L.W. has no declared conflicts of interest; E.M.S. has no declared conflicts of interest; G.S.S. has no declared conflicts of interest; and M.A.S. has no declared conflicts of interest.

Received in original form April 22, 2004

Received in final form January 14, 2005


   References
Top
 Abstract
 Introduction
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 

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