Published ahead of print on November 24, 2004, doi:10.1164/rccm.200405-612OC
© 2005 American Thoracic Society doi: 10.1164/rccm.200405-612OC
Progressive Transforming Growth Factor ß1–induced Lung Fibrosis Is Blocked by an Orally Active ALK5 Kinase InhibitorDepartment of Pathology and Molecular Medicine, Centre for Gene Therapeutics, McMaster University, Hamilton, Ontario, Canada; Service de Pneumologie et Réanimation Respiratoire, CHU du Bocage and Université de Bourgogne, Dijon, France; Medizinische Klinik, Julius-Maximilians-Universität, Wurzburg, Germany; Scios, Inc., Fremont, California Correspondence and requests for reprints should be addressed to Jack Gauldie, Ph.D., F.R.S.C., Professor and Chair, Department of Pathology and Molecular Medicine, McMaster University, 1200 Main Street West, Room 2N16, Hamilton, ON, Canada L8N 3Z5. E-mail: gauldie{at}mcmaster.ca
Pulmonary fibrosis is characterized by chronic scar formation and deposition of extracellular matrix, resulting in impaired lung function and respiratory failure. Idiopathic pulmonary fibrosis (IPF) is associated with pronounced morbidity and mortality and responds poorly to known therapeutic interventions; there are no known drugs that effectively block or reverse progressive fibrosis. Transforming growth factor ß (TGF-ß) is known to mediate extracellular matrix gene regulation and appears to be a major player in both the initiation and progression of IPF. TGF-ß mediates its biological effects through members of a family of activin receptor–like kinases (ALK). We have used a gene transfer model of progressive TGF-ß1–induced pulmonary fibrosis in rats to study a newly described orally active small molecular weight drug that is a potent and selective inhibitor of the kinase activity of ALK5, the specific TGF-ß receptor. We show that the drug inhibits the induction of fibrosis when administered at the time of initiation of fibrogenesis and, most important, blocks progressive fibrosis when administered transiently to animals with established fibrosis. These data show promise of the development of an effective therapeutic intervention for IPF and that inhibition of chronic progressive fibrosis may be achieved by blocking TGF-ß receptor activation.
Key Words: fibrogenesis • interstitial lung fibrosis • matrix • TGF-ß receptor
Diseases that involve chronic fibrotic changes to the structure of the lungs, such as toxic interstitial lung diseases (e.g., drug injuries, radiation, environmental damage) or idiopathic pulmonary fibrosis (IPF), are challenging clinical problems. The process of lung fibrosis is not well understood, and there is no proven therapy to prevent or reverse it (1). IPF is one of the most common chronic interstitial lung diseases, with a mortality rate of up to 70% 5 years after diagnosis, and most therapeutic strategies have been based on eliminating or suppressing the inflammatory component without evidence of real efficacy (2). A recent clinical trial of long-term treatment with IFN- Transforming growth factor ß1 (TGF-ß1), among a series of cytokines and chemokines, has been implicated in the initiation and progression of fibrosis (4, 5). TGF-ß promotes myofibroblast proliferation, induces the synthesis of extracellular matrix (ECM) proteins, and inhibits ECM degradation by induction of antiproteinases or reduction of metalloproteases (6). We have previously demonstrated that transient (7–10 days) overexpression of active TGF-ß1 by adenoviral vector gene transfection in rat lungs induces a severe and progressive fibrosis out to 60 days (7). Additional studies have shown that blocking TGF-ß in different animal models, such as with the use of soluble type II receptors for TGF-ß (8), may be effective at reducing fibrosis. The active form of TGF-ß interacts with a series of serine/threonine receptors, which are part of a family of related receptor molecules termed activin receptor–like kinases (ALK) (9). TGF-ß receptor I (ALK5) acts downstream of the TGF-ß type II receptor and interacts with members of Smad, a family of cytoplasmic transducer proteins (10). ALK5 is specific for active TGF-ß and/or activin and phosphorylates Smad2 and Smad3 (11). Smad3 appears to be a crucial element in the TGF-ß signal transduction pathway involved in fibrosis (12–14). This study tests a potent and selective orally active inhibitor of ALK5 kinase activity (SD-208) in an experimental model of pulmonary fibrosis using adenovirus-mediated gene transfer of active TGF-ß1. Using a combination of histologic examinations, biochemical assays, and global gene expression analyses, we demonstrate for the first time that inhibition of ALK5 kinase activity blocks both the early acute fibrogenic response and the progression of established fibrosis. Genes previously implicated in the process of fibrogenesis, such as ECM, growth factors, and proteinase inhibitors, are coordinately regulated in the induction of progressive fibrosis by TGF-ß1 gene overexpression, and regulation of these genes is significantly inhibited by treatment with SD-208. These data indicate an exciting potential role for ALK5 inhibition in the therapy for pulmonary fibrosis. Some of the results of these studies have been previously reported in the form of abstracts (15, 16).
Recombinant Adenovirus AdTGF-ß1223/225 expresses biologically active TGF-ß1, as described previously (7, 17). Control vector (AdDL) has no insert in the E1 region.
Kinase Inhibitor
In Vitro Experiments
Animal Treatment
Early SD-208 treatment.
Delayed SD-208 treatment.
Determination of Cytokine Levels in BAL Fluid
Histology Hydroxyproline content was determined by a colorimetric assay described previously (20, 21). For quantitative lung collagen histomorphology, 25 to 30 random fields (25x) (picrosirius red stain) were image digitized (polarized light) using a Leica DMR microscope with Leica Qwin image-processing software (Leica Imaging Systems, Cambridge, UK).
Quantitative Polymerase Chain Reaction
Microarray Analysis Details of rat microarray and data analysis have been described previously (22). Differential expression values were expressed as the ratio of the median of experimental RNA to the median of the control RNA. The total lung RNA from Day 4–treated animals was extracted using Qiagen's RNeasy kit (Valencia, CA). Arrays were probed in duplicate for a total of six hybridizations: AdDL versus AdTGF-ß1223/225/MC control, AdTGF-ß1223/225/MC control versus AdTGF-ß1223/225/SD-208 (25 mg/kg), and AdTGF-ß1223/225/MC control versus AdTGF-ß1223/225/SD-208 (50 mg/kg).
Statistical Analysis
The animals were treated in accordance with the guidelines of the Canadian Council of Animal Care.
AdTGF-ß1223/225
SD-208 Is Specific for TGF-ß Receptor I and Is a Potent Inhibitor of Fibrosis-related Genes In Vitro Cultures of primary rat lung fibroblasts at Passage 7 were stimulated with 2 ng/ml recombinant human TGF-ß1. SD-208 was added at a concentration of 25 to 1,600 nM. Plasminogen activator inhibitor 1 (PAI-1) protein was measured at 24 hours by ELISA. We demonstrated that SD-208 is a specific kinase inhibitor for TGF-ß receptor I (ALK5), with an IC50 of SD-208 approximately 35 nmol/L against in vitro ALK5 activity, with specificity of more than 100-fold against TGF-ß receptor II kinase and at least 20-fold over a panel of related protein kinases (Table 2). In separate studies, SD-208 blocked both ALK5- and ALK4-mediated signaling with similar IC50 (data not shown). Moreover, in vitro, SD-208 blocks PAI-1 protein expression induced by recombinant TGF-ß1 in a dose-dependent manner in primary rat lung fibroblast cultures (Figure 2).
SD-208 Blocks TGF-ß–induced Lung Fibrosis: Early Treatment To examine the effect of ALK5 kinase inhibition in vivo, AdTGF-ß1223/225 or AdDL as control vector (5 x 108 pfu) were administered intratracheally in Sprague-Dawley rats at Day 0. SD-208 in MC or MC alone as vehicle control was administered orally, twice daily, from Days 1 to 8. The SD-208 dose chosen was on the basis of pharmacodynamic studies after oral dosing, which showed that administration of SD-208 at 60 mg/kg delivered a plasma concentration of approximately 2,500 nM at 4 hours and decreased to undetectable levels by 24 hours. These levels were considered pharmacologic (data not shown). At this level of application, we did not see any apparent side effects over the period of the experiment.
TGF-ß1 levels in BAL fluid.
Animal response. Rats treated with AdDL control vector appeared healthy through the course of the experiment. In contrast, rats treated intratracheally with AdTGF-ß1223/225 (5 x 108 pfu) and MC vehicle were lethargic and breathless, had ruffled fur, and lost considerable body weight—up to 30% between the start of the experiment and Days 14 to 18, as previously described (7). AdTGF-ß1 rats treated with SD-208 (25 or 50 mg/kg) between Days 1 and 8 of the onset of fibrosis lost less weight and appeared to be less breathless and more active during the treatment period.
Cytology in BAL fluid.
Evaluation of lung fibrosis.
To the contrary, as shown previously, rats administered AdTGF-ß1223/225 had markedly abnormal lung histology with widespread areas of fibrosis beginning as early as Day 3 and progressing from Day 7 to beyond Day 60 (termination) (7). Numerous patchy fibrotic areas were seen beginning at Day 4, with prominent collagen accumulation and the presence of fibroblast and myofibroblast foci (Figure 4A). AdTGF-ß1223/225 animals treated with SD-208 from Days 1 to 8 showed a remarkable reduction in myofibroblast accumulation at Day 4. At Day 21, there were large, widespread fibrotic areas in the AdTGF-ß1223/225 /MC vehicle–positive control group. In contrast, in the AdTGF-ß1223/225 /SD-208 group, there was a striking reduction in fibrotic areas, with no evidence of ongoing tissue destruction. Figure 4A shows a comparison of animals treated with 25 mg/kg SD-208. Similar results were seen with treatment at 50 mg/kg. To confirm and validate the histologic findings, tissue fibrosis was quantified by analysis of hydroxyproline content (Figure 4B). AdTGF-ß1223/225/MC vehicle control animals had a significantly higher hydroxyproline content in the lung (87% increase, p < 0.05) at Day 21 compared with control vector–treated rats. In contrast, hydroxyproline concentration in the lungs of AdTGF-ß1223/225 /SD-208 drug–treated rats (25 mg/kg) was not significantly higher at 21 days than in lungs of AdDL/MC-treated control animals (Figure 4B). Similar results were seen with treatment at 50 mg/kg.
Gene expression.
Regulation of representative genes from microarray analysis was validated by quantitative real-time PCR analysis: procollagen 1a2, fibronectin, connective tissue growth factor (CTGF), PAI-1, and tissue inhibitor of metalloproteinase 1 (TIMP-1) gene expression in total lung RNA (Figure 5). Gene transfer of active TGF-ß1 induced a strong upregulation of all these fibrosis-related genes at Day 4. SD-208 treatment significantly abrogated the upregulation of these fibrosis-related genes (Figure 5A). There was a drug dose–response relationship, with lower expression of these genes in the higher SD-208 dose (50 mg/kg) group.
At Day 21, procollagen 1a2, fibronectin, CTGF, PAI-1, and TIMP-1 remained significantly upregulated after AdTGF-ß1223/225 administration compared with AdDL-treated animals. In contrast, after SD-208 administration from Days 1 to 8 (25 mg/kg), fibronectin, CTGF, PAI-1, and TIMP-1 were all expressed in total lung RNA at similar levels to those seen with the AdDL control. Procollagen 1a2 was slightly upregulated in AdTGF-ß1223/225/SD-208 rats compared with AdDL but significantly lower to that of AdTGF-ß1223/225/MC (Figure 5B). Similar results were seen with the 50-mg/kg treatment. Taken together, these results demonstrate that administration of SD-208 throughout the period when the TGF-ß1 transgene is expressed and the fibrogenic process is being initiated results in abrogation of fibrogenesis and prevention of progressive fibrosis.
SD-208 Blocks the Progression of Established Lung Fibrosis: Delayed Treatment
Animal response.
Evaluation of lung fibrosis. By Day 7 after AdTGF-ß1223/225 (2.5 x 108 pfu/animal) and before initiation of drug treatment, we observed characteristic lung fibrosis with marked and widespread collagen accumulation demonstrated by picrosirius red staining (Figure 7).
By Day 21, we found a marked difference in the fibrotic tissue response between AdTGF-ß1223/225/SD-208 delayed drug treatment and AdTGF-ß1223/225/MC vehicle control–treated animals. Lungs from AdTGF-ß1223/225/MC–treated rats showed extensive and progressive ECM accumulation. In contrast, AdTGF-ß1223/225/SD-208 delayed-treatment rats still showed some fibrotic areas present, but these were patchy, widely dispersed, and dramatically decreased when compared with AdTGF-ß1223/225/MC–treated rats (Figure 7A). To confirm these histologic findings, we assessed collagen accumulation by morphometric measurement of picrosirius red staining (Figure 7B). At Day 7 after TGF-ß1 gene administration, at the start of SD-208 treatment, there was a significant increase in collagen concentration in AdTGF-ß1223/225–treated compared with AdDL-treated animals. At Day 21, in AdTGF-ß1223/225/MC, progressive fibrosis was observed, with a significant increase in collagen accumulation compared with Day 7 AdTGF-ß1223/225, the time we started the delayed SD-208 treatment, and significantly increased over control adenovirus–treated animals. By Day 21, the collagen concentration in AdTGF-ß1223/225/SD-208–treated rats was significantly lower than in the AdTGF-ß1223/225/MC group, but was still higher than the AdDL control–treated group. There was no evidence of increased collagen accumulation between Days 7 and 21 in AdTGF-ß1223/225 animals treated with SD-208. To validate the picrosirius red measurement, we performed hydroxyproline content measurements from all Day 21 lungs. We confirmed a highly significant correlation (p < 0.01, R2 = 0.6875) between the two methods (Figure 7C and data not shown).
Gene expression.
TGF-ß1 level in BAL fluid. Analysis of BAL by ELISA at Day 21 after adenovirus infection demonstrated a significantly higher level of total TGF-ß1 in the AdTGF-ß1223/225/MC group compared with the AdTGF-ß1223/225/SD-208 delayed-treatment group. The total TGF-ß1 level in BAL fluid from AdTGF-ß1223/225/SD-208 delayed-treatment animals was similar to that in the AdDL-treated control group (Figure 8B). Although we have not examined this in detail, we suspect that inhibition of TGF-ß signaling interferes with the autocrine induction of TGF-ß. The active TGF-ß1 level was not significantly different between these groups at Day 21 (data not shown).
This study shows that by blocking the activation of TGF-ß receptor I (ALK5), using a novel, orally active, small molecular weight inhibitor of ALK5 kinase activity, we are able to significantly inhibit TGF-ß–induced fibrosis. Furthermore, we provide data demonstrating that the blockade of ALK5 using this orally active agent is able to arrest the progression of fibrosis once the fibrogenic process has been established. Human IPF, as with many fibrotic disorders, is a chronic, progressive, and lethal disease. IPF has an unknown etiology, and there is presently no clearly effective treatment (23). The role of inflammation in the initiation of fibrosis is relatively clear, but the involvement of inflammation in the progression of IPF is not clearly defined (24).
Glucocorticoids have been a standard treatment for IPF for many years; however, despite widespread use, antiinflammatory therapy has not proven very helpful (25). Immunosuppressive or cytotoxic drugs, such as cyclophosphamide, have been studied in clinical trials, which have demonstrated a poor response associated with high toxicity (2). New drugs, such as IFN- TGF-ß has been strongly implicated as a key growth factor in the initiation of fibrosis (28, 29). Overexpression of TGF-ß1 using adenovirus-mediated gene transfer to rat lung has been shown to induce potent and progressive pulmonary fibrosis (7). TGF-ß is also present in the first 7 days after bleomycin administration (30), the most commonly used animal model of lung fibrosis. Several experimental animal studies have demonstrated that it is possible to decrease bleomycin-induced lung fibrosis by inhibiting TGF-ß using TGF-ß soluble type II receptor (8) or TGF-ß neutralizing antibodies (31), or inhibiting TGF-ß pathways using Smad7, a negative regulator of TGF-ß signaling (32). We have already shown that intratracheal adenoviral gene transfer of decorin, a proteoglycan known as an inhibitor of TGF-ß, attenuates bleomycin-induced lung fibrosis in mouse lungs (21). Smad3 null mice, in which one pathway of TGF-ß signaling is interrupted, have shown attenuation of bleomycin-induced fibrosis (33) and are resistant to TGF-ß1–induced lung fibrosis (14). Therefore, therapeutic strategies to inhibit TGF-ß by interfering with the receptor signaling process would appear to be appealing interventions to prevent the initiation of fibrosis. Most previous studies showed attenuation of fibrosis with agents administered at the beginning of the fibrotic process in the experimental animal and are thus not able to clearly differentiate between inhibition of the tissue injury phase, the subsequent inflammatory response, or the prolonged fibrotic response seen in the bleomycin model. No studies have yet been reported on drug intervention in established progressive fibrosis, a situation more akin to the clinical disease. Moreover, the administration of agents, such as Smad7, requires entry into the specific target cell for TGF-ß activity (presumed to be the pulmonary fibroblast) for these to exhibit inhibitory effects on fibrogenesis. This is not readily accomplished with currently available gene transfer approaches or administration of recombinant protein. However, an easily administered, orally active agent, such as SD-208, could readily target the parenchymal cells that respond to TGF-ß, which suggests this approach may have promise as an antifibrotic therapy. SD-208 is a specific and potent inhibitor of TGF-ß1 signaling in vitro. Moreover, preliminary investigations with this drug have shown considerable attenuation of bleomycin-induced lung fibrosis, when administered simultaneously with bleomycin treatment (34). We designed these experiments to analyze the effect of interfering with ALK5 signaling during both the initiation and progression phase of fibrosis. It was important to first ensure that we were able to decrease the initiation of fibrosis in our model with this orally active drug. We have previously demonstrated that, after intratracheal administration of AdTGF-ß1223/225, the expression of the active TGF-ß1 transgene is found in high concentration in the BAL of infected animals as early as at Day 1. TGF-ß1 transgene expression reaches a maximal level between Days 4 and 6 after infection, then rapidly declines, and active TGF-ß1 levels in BAL are not different from those in control animals at Day 14 (7). Nevertheless lung fibrosis remains progressive up to 60 days (termination). Microarray analyses show that numerous fibrosis-related genes are elevated long after the initial TGF-ß1 stimulation has ended (Figure 1). We assumed that treating rats from Days 1 to 8 with SD-208 would cover the animals during the time of most intensive TGF-ß1 transgene expression and therefore the time of initiation of the fibrogenic process. The present study confirms that SD-208 blocked early fibrosis-related gene expression, and, at both doses of drug, dramatically decreased the amount of TGF-ß–induced fibrosis observed at Day 21. By Day 4 after AdTGF-ß1223/225, treatment with SD-208 (50 mg/kg) decreased the neutrophil count in BAL fluid. Early increases in neutrophils and lymphocytes have been associated with higher fibrotic progression in animal models (35) and in humans (36). Microarray analysis demonstrated the global effects of SD-208 to block both induced and downregulated expression of TGF-ß1–responsive genes associated with fibrosis and ECM deposition. Thus, these data establish that SD-208 (at both 25- and 50-mg/kg doses) inhibits acute TGF-ß1–induced lung fibrosis. TGF-ß1 is also strongly associated with later stages of chronic, progressive fibrotic diseases, such as IPF (1) or renal fibrosis (37), and TGF-ß1 autoinduction is believed to play a part in this ongoing process (13). In human IPF, immunohistochemical staining for TGF-ß and detection of TGF-ß mRNA are increased in the lungs, and localized within the sites of ECM accumulation (29). TGF-ß1 has been found to be increased in the development and the progression of radiation-induced fibrosis (38). Progression likely occurs through autoinduction of TGF-ß1 with persistent expression of this fibrogenic molecule, but also through an effect on survival of myofibroblasts and alteration of the matrix environment to a nondegradative state (6). To study the effects of ALK5 inhibition on established fibrosis, we treated rats with a short-term dosage of 50 mg/kg of SD-208 from Days 7 to 11 after AdTGF-ß1223/225 infection. At Day 7, there was already extensive fibrosis with a high level of TGF-ß–responsive gene expression, whereas the transgene expression had decreased to control levels. This later transient treatment with SD-208 in rats with established fibrosis appeared to improve the general clinical condition of these animals, with decreased breathlessness and improved weight gain, and importantly, SD-208 treatment also apparently stopped the progression of fibrosis. On the basis of histology, histomorphometry, and lung hydroxyproline content, the amount of pulmonary fibrosis was identical at 21 days after infection in SD-208–treated animals to that seen at 7 days after infection with AdTGF-ß1223/225 at the time drug treatment was started. Vehicle-treated animals continued to show progressive fibrosis, indicating that SD-208 treatment impaired chronic fibroblast stimulation, and suggests the progression in this model of fibrosis is dependent on ongoing endogenous TGF-ß signaling. Furthermore, despite the short duration of drug administration (Days 7–11), delayed treatment with SD-208 reversed the upregulation of many TGF-ß–responsive genes that were overexpressed at Day 21 in the AdTGF-ß1223/225/MC group, again suggesting the progression seen is caused by ongoing endogenous TGF-ß autoinduction and signaling. Consistent with this suggestion, we observed a higher concentration of total TGF-ß1 in the BAL from AdTGF-ß1223/225/MC– than in drug-treated animals (Figure 8B). Taken together, our data demonstrate that transient treatment of established fibrosis with SD-208 prevented TGF-ß autoinduction and blocked progression of fibrosis. In the pathogenesis of fibrosis, the deposition of ECM is a dynamic process (39). Tissue matrix protein turnover is mediated through a balance of synthesis and degradation through the action of matrix metalloproteinases and their inhibitors, TIMPs (40). Matrix metabolism, in particular the collagen degradation pathways, may define whether a fibrogenic process is progressive or leads to resolution of the deposited matrix (41). High expression of TIMPs, creating a nonfibrolytic or nondegradative environment, has been associated with matrix accumulation in IPF (42). It has been observed that C57BL/6 mice are susceptible and Balb/c mice resistant to induction of lung fibrosis. We have previously shown that this strain difference occurs downstream of TGF-ß or its receptor and that susceptibility to fibrosis is manifest at least at the level of TIMP-1 gene regulation, with more TIMP-1 gene expression in C57BL/6 than in Balb/c mice after TGF-ß1 stimulation (18, 43). This current study demonstrates that, with both early and delayed treatment with SD-208, a complete reversal of TIMP-1 gene upregulation is seen. PAI-1, another important antiproteinase in the process of fibrosis, followed the same pattern as TIMP-1, with upregulation after TGF-ß1 administration and reversal of this regulation by both early and late treatment with SD-208. Inhibition of ALK5 signaling by SD-208 treatment interferes with the creation of an antifibrolytic environment and prevents excessive matrix deposition from occurring. The antifibrotic effect of SD-208 does not appear to have a major effect on apoptosis gene expression because there were only minor changes to p21 gene expression seen early on by microarray analysis but no apparent changes in other apoptosis-related genes (data not shown). The effectiveness of the short-term drug intervention on established fibrosis suggests that similar applications could be envisioned in chronic fibrosis to avoid long-term systemic inhibition of TGF-ß, a cytokine well recognized for regulation of diverse physiologic roles (39). These data indicate that blockade of the kinase activity of the TGF-ß receptor I totally inhibits the matrix deposition and progressive fibrosis induced by gene overexpression with TGF-ß1. Blockade of other kinase-dependent receptors, including that of platelet-derived growth factor or epidermal growth factor, appears to modulate bleomycin-induced fibrosis (44) and, taken together, suggests that drug targeting of growth factor receptors with potent oral-specific kinase inhibitors could provide important and useful therapeutic interventions in human IPF. Moreover, considering that fibrosis tends to be an organ-restricted disorder, short-term local administration of a drug, such as SD-208 (aerosolized delivery for pulmonary fibrosis), may have highly beneficial results. In summary, we have demonstrated that SD-208, an orally active kinase inhibitor of ALK5, a TGF-ß type I receptor, strongly inhibits TGF-ß1–induced initiation of lung fibrosis and also is able to arrest the progression of established pulmonary fibrosis. In certain clinical settings, pulmonary fibrosis is a predictable consequence of therapy, such as radiation or chemotherapy. A drug such as SD-208 could be used to prevent the initiation of fibrosis and may allow higher doses of chemotherapy or radiotherapy to be used. In addition, our research suggests that blocking ALK5 may be useful in preventing the progression of established fibrosis and may therefore have a role in chronic fibrotic diseases such as IPF.
The authors thank Kelly Putzu and Li Yu for their help in animal treatment; Carol Lavery, Duncan Chong, and Xueya Feng for their invaluable technical help; and Mary Jo Smith for outstanding preparation of histology. In addition, the authors thank Andrew Lam, Diana Quon, Gilbert O'Young, and Kimberly Munson for their technical assistance with the RNA processing and microarray analysis. The authors also thank Lisa Garrard, Kip Madden, Jie Hu, and Estelle Tham for their technical and informatics expertise and support.
Supported by the Canadian Institutes of Health Research, St. Joseph's Healthcare, and Hamilton Health Sciences. P.B. is supported by the Bourses Lavoisier du Ministère des Affaires Etrangères, the Ligue Bourguignonne Contre le Cancer, and Bourse de Voyage Boehringer. P.J.M. is a CIHR clinician scientist. M.K. is a Parker B. Francis fellow and is also supported by Deutsche Krebshilfe. Conflict of Interest Statement: P.B. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript; P.J.M. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript; M.K. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript; J.A.S. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript; A.M.K. is an employee of Scios, Inc., and has received Scios, Inc. stock options, and is listed on the following patents: Scios Case 220PRV, 2002; Scios Case 221PRV, 2002; Scios Case 027PR, 2001; Scios Case 194PRV, 1999; Scios Case 193PRV, 1999; D.D. is an employee of Scios, Inc., and has received Scios, Inc. stock options; A.M. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript; S.C. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript; S.D. has submitted patents involving fibrosis; the data covered in the manuscript was not part of those submissions; L.H. is an employee of Scios, Inc., which does not provide direct financial incentives for publication or patents, and Scios is owned by Johnson & Johnson and, as part of the standard compensation package at Scios, L.H. has Johnson & Johnson stock options; A.A.P. has submitted patents involving fibrosis, and the data covered in this manuscript was not part of those submissions; J.G. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. Received in original form May 11, 2004; accepted in final form November 17, 2004
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ABSTRACT
METHODS
1b in IPF showed some promise through effects on mortality, but no impact on the process of fibrogenesis was detected (
–smooth muscle actin antibodies (Dako, Missisauga, ON, Canada), as previously described (
3 ng/ml BAL fluid at Day 4), with no significant difference between SD-208- or MC-treated rats. 
