Published ahead of print on July 15, 2004, doi:10.1165/rcmb.2004-0158OC
© 2004 American Thoracic Society DOI: 10.1165/rcmb.2004-0158OC Connective Tissue Growth Factor Is Crucial to Inducing a Profibrotic Environment in "Fibrosis-Resistant" Balb/c Mouse LungsDepartments of Medicine, Pathology and Molecular Medicine, Center for Gene Therapeutics, McMaster University, Hamilton, Ontario, Canada; Service de Pneumologie et Réanimation Respiratoire, CHU du Bocage et Université de Bourgogne, Dijon, France; Medizinische Klinik, Julius-Maximilians-Universität Würzburg, Würzburg, Germany Address correspondence to: Dr. Martin Kolb, M.D., P.D., Departments of Medicine, Pathology and Molecular Medicine, McMaster University, Firestone Institute for Respiratory Health, 50 Charlton Ave. East, Room H 325, Hamilton, ON, Canada L8N 4A6. E-mail: kolbm{at}mcmaster.ca
The individual susceptibility to pulmonary fibrosis (PF) remains a mystery, suggesting a role for genetic predisposition. The pathogenesis of PF involves a multitude of factors mediating crosstalk between various tissue components. Some factors, such as transforming growth factor ß, are recognized as key elements in the process, whereas the role of others, such as connective tissue growth factor (CTGF), is unclear. We investigated if Balb/c mice, known to be fibrosis resistant partly due to lack of CTGF induction upon stimulation with bleomycin, can be transformed into fibrosis-sensitive individuals by generation of a CTGF-rich environment using transient overexpression of CTGF by adenoviral gene transfer (AdCTGF). We show that AdCTGF is not sufficient to cause fibrosis, and that bleomycin challenge results in inflammation, but not fibrosis, in Balb/c mouse lungs. This inflammation is accompanied by lower levels of CTGF and tissue inhibitor of metalloproteinase–1 gene expression compared with fibrosis-prone C57BL/6 mice. However, concomitant administration of AdCTGF and bleomycin leads to a persistent upregulation of tissue inhibitor of metalloproteinase–1 gene and a significant fibrotic response in Balb/c similar to that in C57BL/6 mice. We propose that CTGF is an important mediator in the pathogenesis of PF in that it provides a local microenvironment in the lung that causes individual susceptibility. CTGF should be considered as a novel drug target and as a potential marker for identifying individuals at risk.
Abbreviations: connective tissue growth factor ß adenoviral gene transfer, AdCTGF-ß • control vector (no transgene), AdDL • transforming growth factor-ß adenoviral gene transfer, AdTGF-ß • bronchoalveolar lavage, BAL • connective tissue growth factor, CTGF • extracellular matrix, ECM • phosphate-buffered saline, PBS • pulmonary fibrosis, PF • transforming growth factor–ß, TGF-ß • tissue inhibitor of metalloproteinase, TIMP
Fibrosis is characterized by exaggerated extracellular matrix (ECM) accumulation due to excessive tissue repair and impaired matrix turnover (1). Although some agents capable of inducing this process are well described (e.g., drug-induced lung disease, irradiation damage), the causative agent frequently remains unknown, and the disorder seen in the lung is named idiopathic pulmonary fibrosis (PF) (2). The best-characterized drug-induced PF is caused by the antibiotic bleomycin, which is commonly integrated in chemotherapy protocols of leukemia and testicular cancer (3). In animal models, bleomycin is a helpful tool for studying the pathogenesis of lung fibrosis and for investigating the efficacy of new antifibrotic drugs (4). In humans, the individual susceptibility to PF remains a mystery, suggesting a role for genetic predisposition (5). In animal models, the strain variability in lung fibrosis susceptibility is well known and easy to assess. Some strains, such as C57BL/6 mice, develop severe fibrosis after exposure to various stimuli, such as bleomycin, irradiation, or silica. In contrast, in Balb/c mice and some other murine strains, the fibrotic response to identical stimuli is either absent or markedly reduced (6). The biological mechanism for these differences is incompletely understood, but may play an important role in the pathogenesis of PF. Understanding this mechanism could lead to the development of novel treatments for this disease (7).
Fibrosis-prone mice have higher levels of chemokines (regulated upon activation, normal T cell expressed and secreted, interferon-inducible protein 10, macrophage inhibitory protein–1 Connective tissue growth factor (CTGF) is a cysteine-rich protein that stimulates fibroblast growth and in vitro collagen and fibronectin upregulation (12). The most potent CTGF inducer is TGF-ß, and it has been proposed that CTGF overproduction plays a major role in pathways that lead to fibrosis (13, 14); however, little is known about CTGF function in vivo. CTGF overexpression has been described in human fibrotic diseases (15, 16), and Lasky and colleagues have reported that "fibrosis-resistant" Balb/c mice do not upregulate CTGF expression in the bleomycin model compared with "fibrosis-prone" C57BL/6 mice (17). Recently, we have shown that CTGF overexpression in rat lungs is followed by a temporal induction of procollagen gene expression and transient matrix accumulation, but is not sufficient to induce progressive fibrosis (18). The purpose of the current study was to assess the bleomycin-induced response in "fibrosis-resistant" Balb/c mice in the concomitant presence of high levels of CTGF using adenoviral gene transfer (AdCTGF). We show here that neither bleomycin nor CTGF alone were able to induce a fibrotic response in Balb/c mice when applied individually. However, the combination of bleomycin and CTGF resulted in a similar fibrotic response as bleomycin alone in susceptible C57BL/6 mice, associated with a strong increase in TIMP-1 gene expression. We propose that the restoration of CTGF in this otherwise resistant mouse strain has created a profibrotic environment, and that CTGF may play a role as an important cofactor in the pathways of fibrogenesis downstream of TGF-ß1.
Recombinant Adenovirus The construction of adenoviral vectors is described in detail elsewhere (19). AdCTGF was obtained from Dr. Murphy (Department of Molecular Technologies, Bayer Corporation, Berkeley, CA) and has been previously described (20). In short, the full-length human CTGF cDNA was cloned into a p73 shuttle vector with a human cytomegalovirus (CMV) promoter and cotransfected with a virus-rescuing vector. The resulting replication-deficient virus (AdCTGF) was amplified and purified by cesium chloride (CsCl) gradient centrifugation and over a Sephadex PD-10 chromatography column, and finally plaque-titred on 293 cells. The control vectors, AdDL, with no insert in the deleted E1 region, or adenoviral vector expressing the ß-galactosidase gene (AdLacZ), coding for ß-galactosidase, were produced in the same way (19, 21).
Animal Treatment
Histology
Determination of TGF-ß1 in BAL Fluid
Quantitative Polymerase Chain Reaction
Hydroxyproline Assay Frozen lungs were homogenized in 5 ml of deionized water. One milliliter of the homogenate was hydrolyzed in 2 ml 6 N HCL for 16 h at 110°C. Hydroxyproline content was determined by a colorimetric assay described previously (23). The results were calculated as µg hydroxyproline per mg wet lung weight using hydroxyproline standards (Sigma, Oakville, ON, Canada).
Statistical Analysis
CTGF Gene Expression in Mouse Lungs To ensure effective transfection, we determined CTGF mRNA in Balb/c lungs. Balb/c lungs treated with AdCTGF/PBS compared with AdDL/PBS showed a significant, four-fold increase in CTGF mRNA expression in total lung RNA 7 d after vector administration. CTGF overexpression was transient, as we did not see any difference in CTGF mRNA expression between AdCTGF/PBS- and AdDL/PBS-treated Balb/c mice at Day 14 (Figure 1A, left panel). In lungs from Balb/c mice, there was no increase in CTGF mRNA expression by Day 7 after AdDL/bleomycin administration and only a relatively mild increase by Day 14 compared with AdDL/PBS-treated animals. In contrast, C57BL/6 mice showed a significant, almost 3-fold increase in CTGF mRNA expression after bleomycin at Day 7 and 14 compared with AdDL/PBS-treated mice (Figure 1A, right panel). Balb/c mice treated with AdCTGF plus bleomycin showed a more that 4-fold increase of CTGF mRNA by Day 7 that persisted through Day 14 with a 2-fold increase above control lungs (Figure 1A, left panel). To ensure that transgene expression was maintained in the context of acute lung injury after bleomycin injection, we infected lungs from Balb/c mice with AdLacZ and treated them after 3 d with bleomycin. Four days after bleomycin treatment, the transgene ß-galactosidase protein was still expressed in airway epithelium, as confirmed by histochemistry (Figure 1B).
Differential Cytology and TGF-ß1 in BAL Fluid All animals treated with AdDL/bleomycin and AdCTGF/bleomycin showed a significant increase in total cell numbers in BAL fluid (BALF) after injection of bleomycin compared with AdDL/PBS-treated animals (Table 2). Four days after bleomycin (corresponding to Day 7 of the experiment), we observed no difference in total cell numbers between AdDL/bleomycin- and AdCTGF/bleomycin-treated Balb/c mice, but we did observe an increase in total cells when we compared both groups to AdDL/bleomycin-treated C57BL/6 mice. AdDL/bleomycin-treated Balb/c mice had higher lymphocyte counts than AdCTGF/bleomycin-treated Balb/c mice by Day 7, possibly related to the antiviral immune response against the specific adenoviral vectors (e.g., due to replication-competent virus in the AdDL preparation). All these differences between BAL cell counts between the strains were transient and no longer present by Day 14.
TGF-ß1 in BALF of all mice treated with bleomycin was elevated by Days 7 and 14 compared with PBS-treated control animals (levels < 20 pg/ml), independent of the underlying mouse strain. Animals exposed to AdCTGF had increased TGF-ß1 in BALF compared with AdDL (Table 3). No differences in TGF-ß1 were observed between Balb/c mice treated with bleomycin plus AdDL or AdCTGF, and TGF-ß1 in BALF was not different between Balb/c and C57BL/6 mice treated with AdDL/bleomycin.
Evaluation of Histology and Fibrosis Both Balb/c and C57BL/6 mice showed marked inflammatory changes in the lungs at Days 7 and 14 of the experiment, irrespective of the concomitant application of adenovectors. Individual mice exposed to AdCTGF/PBS or AdDL/PBS demonstrated minor and patchy peribronchial inflammation by Day 7 as previously observed, which resolved completely by Day 14.
All bleomycin-treated Balb/c mice (AdDL/bleomycin and AdCTGF/bleomycin) had persistent and severe inflammation within the interstitium and alveolar space, with numerous neutrophils and foamy macrophages by Day 21 (Figure 2, left panels). Masson's trichrome and Picrosirius red staining revealed little collagen within those inflammatory areas in AdDL/bleomycin-treated Balb/c mice, whereas there was significant collagen accumulation in AdCTGF/bleomycin-treated Balb/c lungs (Figure 2, compare panels G/H with J/K). Collagen accumulation in AdCTGF/bleomycin Balb/c mice was similar to that in C57BL/6 mice after AdDL/bleomycin (Figures 2M and 2N). Accordingly, there was substantial myofibroblast accumulation, as suggested by positive
To confirm the histologic findings, we determined lung collagen by hydroxyproline analysis (Figure 3). No significant difference in hydroxyproline concentration between Balb/c mice treated with AdCTGF/PBS, AdDL/bleomycin, or AdCTGF/bleomycin was seen by Day 7. By Days 14 and 21, there was a significant increase of  30% in AdCTGF/bleomycin-treated Balb/c mice compared with AdDL/PBS and AdDL/bleomycin-treated Balb/c mice (Figure 3, left panel). C57BL/6 mice showed a very similar (41%) increase in hydroxyproline content by Day 21 when treated with AdDL/bleomycin when compared to AdDL/PBS-treated C57BL/6 mice (Figure 3, right panel). The lung hydroxyproline content of AdCTGF/PBS-treated Balb/c mice was not different from that of AdDL/PBS-treated Balb/c mice at Days 14 and 21.
CTGF Induces a Profibrotic Environment Procollagen 3a1 mRNA expression was increased after bleomycin administration in AdDL/bleomycin Balb/c mouse lungs compared with AdDL/PBS Balb/c mouse lungs, and a similar increase was observed in AdCTGF/PBS-treated Balb/c (Figure 4, left panel), and in AdDL/PBS-treated C57BL/6 mouse lungs (Figure 4, right panel). Balb/c mice treated with AdCTGF/bleomycin had a significantly higher increase than AdDL/bleomycin animals at Days 7 and 14 (2.6- and 3.3-fold, respectively; P < 0.05). There was no difference in procollagen gene expression between Balb/c and C57BL/6 mice 7 and 14 d after AdDL/bleomycin exposure.
TIMP-1 gene expression was strongly upregulated in lungs from AdCTGF/PBS-treated Balb/c mice by Day 7 compared with AdDL/PBS-treated Balb/c mice (15-fold mRNA increase; P < 0.01). As seen previously, this effect was transient, and decreased to baseline by Day 14 after virus administration (Figure 5, left panel, left bars). TIMP-1 mRNA expression was also increased in Balb/c mice treated with AdDL/bleomycin by Day 7, but returned to near-baseline values by Day 14 (Figure 5, left panel, center bars). In contrast, AdCTGF/bleomycin administration to Balb/c mice resulted in doubling of TIMP-1 gene expression compared with AdDL/bleomycin (19-fold increase compared with AdDL/PBS), that persisted until Day 14 (5.2-fold increase in AdCTGF/bleomycin versus AdDL/PBS, P < 0.04, and 2-fold versus AdDL/bleomycin, P < 0.05, Figure 5, left panel, right bars). The effect of AdCTGF/bleomycin on TIMP-1 gene expression in Balb/c mice was similar to the effect of AdDL/bleomycin in C57BL/6 mice, both in extent and duration (Figure 5, right panel).
In this study, we investigated whether the local environment in the lungs of a fibrosis-resistant mouse strain can be altered to promote a more profibrotic phenotype by enhancing the presence of CTGF using adenoviral gene transfer. We demonstrate here that Balb/c mice, known to be resistant to fibrogenic stimuli, such as intratracheal administration of bleomycin in the lung, develop severe progressive PF in response to bleomycin in the presence of enhanced CTGF. CTGF is a 38 kD member of the CCN (Connective tissue growth factor, Cystein-rich protein, and Nephroblastoma overexpressed gene) family of proteins, and enhances fibroblast proliferation and ECM production in vitro (12). TGF-ß, one of the key components in induction and progression of fibrosis (24), is the most potent stimulator of CTGF, and it is suggested that the profibrotic effects of TGF-ß are partially mediated through CTGF (25). Several studies have shown that CTGF alone is not sufficient to cause ongoing fibrotic changes (17, 18). However, it is possible that CTGF is required for the development of persistent fibrosis. Mori and colleagues have demonstrated in a skin fibrosis model that administration of recombinant CTGF is required for the development of persistent fibrosis, and—more importantly—that recombinant CTGF alone is unable to induce this fibrosis and needs the simultaneous application of recombinant TGF-ß1 (26). We have recently used AdCTGF in the rat lung and found that, whereas CTGF causes a rapid proliferation of myofibroblasts and expression of procollagen and fibronectin genes, these changes were only transient and resolved almost completely within 4 wk. In contrast, overexpression of TGF-ß with the same vector expression technique (TGF-ß adenoviral gene transfer [AdTGF-ß]), results in pronounced, severe, and progressive fibrosis. We have previously described a marked effect on gene expression of the proteinase inhibitor TIMP-1 in rat lungs after both AdTGF-ß and AdCTGF treatment, with persistent upregulation in AdTGF-ß–treated animals compared with a temporary effect in AdCTGF-treated animals (18). In animal models, strain variability for the development of lung fibrosis is well known and suggests some degree of genetic predisposition. C57BL/6 mice are mostly used for lung fibrosis research because they develop severe fibrotic changes after various stimuli, such as bleomycin, asbestos, irradiation, or silica. In contrast, the response to identical stimuli is either absent or markedly reduced in Balb/c mice and some other murine strains (9, 10, 27). Bleomycin treatment is known to increase TGF-ß mRNA (28), and this increase is more pronounced in C57BL/6 than in Balb/c mice (10). Similar findings were reported in an irradiation model, where C57BL/6 mice showed an upregulation of TGF-ß mRNA after 8 wk in contrast to C3H/He mice, a fibrosis-resistant strain (29). We have recently addressed the question of whether differences in TGF-ß induction are a major reason for strain variability (6). We have overexpressed TGF-ß equally in both C57BL/6 and Balb/c mice and have found, surprisingly, that Balb/c mice do not develop PF even in the presence of abundant, active TGF-ß1 in the lungs. This nonresponse was not caused by a lack of TGF-ß1 responsiveness of pulmonary mesenchymal cells in the resistant mice, but was related to a significantly lower level of TIMP-1 gene expression in Balb/c mice compared with fibrosis-prone animals, suggesting impaired matrix degradation as a central component in the pathogenesis of fibrosis. The results presented here are the first to demonstrate that overexpression of a single factor, CTGF, is sufficient to generate a profibrotic lung environment in an otherwise resistant individual. Overexpression of CTGF or bleomycin alone caused a significant upregulation of procollagen genes in Balb/c mice similar to that in C57BL/6 mice. However, in Balb/c mice, this upregulation is transient and only bleomycin-treated C57BL/6 mice developed fibrosis, as determined by histology and hydroxyproline analysis, supporting the hypothesis that collagen accumulation is a result of the balance between synthesis and degradation. Consistent with our previous experiments in mouse strains exposed to TGF-ß (6), and with CTGF overexpression in rat lung (18), we again observed major differences in TIMP-1 gene expression between Balb/c and C57BL/6 mice. TIMP-1 upregulation in the fibrosis-prone strain was significantly higher and of greater duration than in the resistant strain. Balb/c mice treated with bleomycin alone also failed to respond with increased CTGF mRNA expression, whereas C57BL/6 mice did respond with such an increase, confirming previous reports by Lasky and coworkers (17). Interestingly, the combination of CTGF overexpression plus bleomycin in Balb/c mice resulted in the development of PF that was not distinguished from control vector plus bleomycin-treated C57BL/6 mice. With the combined treatment, the levels of increased TIMP-1 mRNA in Balb/c mice were as high and prolonged as in bleomycin-treated C57BL/6 mice. These findings have two important implications: they suggest (i) that CTGF is a crucial mediator in bleomycin induced lung fibrosis in animals; and (ii) that strain differences in the susceptibility to fibrogenic stimuli are related to the ability to upregulate CTGF and TIMP-1 in the lung. It is possible that it is the presence of CTGF and TGF-ß at the same time that causes increased ECM protein synthesis and reduced matrix degradation through upregulation of TIMP. In accordance with our previous findings (22), we observed an increase in total TGF-ß1 protein level in all mice treated with bleomycin, independent of the underlying mouse strain. Surprisingly, animals exposed to AdCTGF alone also revealed elevated levels of TGF-ß1 in BALF, a finding that has not been described previously and requires further exploration. The administration of AdCTGF before bleomycin exposure did not change total TGF-ß1 in the BALF of Balb/c mice compared with control vector AdDL. However, BALF concentration of growth factors does not necessarily reflect the levels present in the interstitium, where fibroproliferative responses occur. In this study, we have expressed CTGF by adenovirus along with increased expression of TGF-ß1 induced by bleomycin. Under these conditions, the balance of matrix deposition and degradation creates a TIMP-1–rich microenvironment that favors decreased degradation, resulting in matrix accumulation and fibrosis. This is in accordance with the skin fibrosis model cited above (26), in which it was demonstrated that CTGF is necessary but needs association with another factor (presumably TGF-ß) to create a profibrotic environment. Recently, Abreu and colleagues (30) found in a Xenopus model that CTGF may enhance TGF-ß1 signaling by direct binding to this growth factor, supporting a cofactor role for CTGF. The exact mechanisms of the interaction between TGF-ß and CTGF have yet to be investigated. However, the context of CTGF and its role in PF is probably complex, and factors other than TGFs have to be considered. It was recently reported that a relationship between thrombin, protease-activated receptor-1 (PAR-1) receptors, and CTGF induction exists (31). The clinical relevance of our findings is related to two different aspects: first, although CTGF may not be sufficient to induce PF alone, it is possibly crucial in the progression of the disease, and thus is a major target for novel therapeutic intervention in PF; second, aberrant expression of CTGF may be one factor that predisposes an individual to develop PF in response to otherwise harmless or reversible stimuli. Several studies have demonstrated that CTGF mRNA is elevated in BAL cells from patients with usual interstitial pneumonia and chronic sarcoidosis (15), and in human lung tissue from patients with idiopathic pulmonary fibrosis (32). To date, it is unclear whether determination of growth factor levels in BAL or human tissue is useful for the identification of patients at risk for disease progression, but this remains a challenging task to pursue in future studies. In summary, we demonstrate that transient overexpression of CTGF in otherwise fibrosis-resistant Balb/c mice can create a profibrotic microenvironment in the lung, leading to the development of progressive fibrosis in response to bleomycin. This profibrotic milieu is related to a sustained expression of the antiproteinase TIMP-1. This suggests that CTGF is a crucial mediator in the pathogenesis of PF, and—although being unable to induce progressive and severe fibrosis by itself—remains a key target in the development of therapeutic treatments for fibrotic disease.
The authors thank Jane Ann Schroeder, Carol Lavery, Duncan Chong, and Xueya Feng for their invaluable technical help, and Mary Jo Smith for outstanding preparation of histology. This work is supported by Canadian Institutes of Health Research, St. Joseph's Healthcare, and Hamilton Health Sciences. P.B. is supported by the Bourses Lavoisier and the Ligue Bourguignonne Contre le Cancer; P.J.M. is a Canadian Institutes of Health Research Clinician Scientist; and M.K. is a Parker B. Francis Fellow. Received in original form May 10, 2004 Received in final form June 28, 2004
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Abstract
Introduction
) (
–smooth muscle actin antibodies (DAKO Corp., Carpinteria, CA). 
C57BL/6 versus Balb/c mice after AdDL/bleomycin treatment; 
AdCTGF/bleomycin- versus AdDL/bleomycin-treated Balb/c mice; for all comparisons, significance set at P < 0.05.
