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Published ahead of print on June 19, 2003, doi:10.1164/rccm.200210-1254OC
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American Journal of Respiratory and Critical Care Medicine Vol 168. pp. 770-778, (2003)
© 2003 American Thoracic Society

Original Article

Adenoviral Gene Transfer of Connective Tissue Growth Factor in the Lung Induces Transient Fibrosis

Philippe Bonniaud, Peter J. Margetts, Martin Kolb, Thomas Haberberger, Margaret Kelly, Jennifer Robertson and Jack Gauldie

Centre for Gene Therapeutics, Department of Pathology and Molecular Medicine, McMaster University, Hamilton, Ontario, Canada; Service de Pneumologie et Réanimation Respiratoire, CHU du Bocage et Université de Bourgogne, Dijon, France; Medizinische Klinik, Julius-Maximilians-Universitat, Wurzburg, Germany; and FibroGen, Inc., South San Francisco, California

Correspondence and requests for reprints should be addressed to Jack Gauldie, Ph.D., Department of Pathology and Molecular Medicine, Room 2N16, McMaster University, 1200 Main Street West, Hamilton, ON, Canada L8N 3Z5. E-mail: gauldie{at}mcmaster.ca


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Connective tissue growth factor (CTGF) is felt to be one of the key profibrotic factors and is a downstream effector molecule mediating the action of transforming growth factor (TGF)–ß1, a cytokine known to induce severe and progressive fibrosis. However, the in vivo fibrogenic effect of isolated CTGF expression is not well described. We used adenoviral gene transfer to transiently overexpress CTGF in rat lungs after intratracheal administration and compared it with transient overexpression of active TGF-ß1 delivered by a similar adenovirus vector. This high expression of CTGF over 6–10 days induced a moderate but reversible fibrosis. We observed an increase of fibronectin, procollagen 1a2, and endogenous CTGF gene expression at 14 days, which suggested an indirect activation by CTGF. Tissue inhibitor of metalloproteinase–1 was weakly and transiently upregulated after CTGF exposure. These same genes were robustly and persistently stimulated by TGF-ß1 from Day 3 to Day 21. This data suggested that CTGF may act as a TGF-ß1 cofactor rather than a direct fibrogenic factor. We demonstrate that CTGF overexpression can initiate fibrogenic activity but likely requires the presence of additional factors, such as tissue inhibitor of metalloproteinase–1, to maintain a nonfibrolytic environment and to cause progression of fibrosis.

Key Words: extracellular matrix • pulmonary • transforming growth factor–ß • tissue inhibitor of metalloproteinase • fibrotic

Mechanisms that lead to lung fibrosis are poorly understood (1). There is no proven therapy once the process is initiated (2). Different etiologies such as acute lung injury, idiopathic pulmonary fibrosis, and drug-induced lung fibrosis (3) lead to disproportionate increases in extracellular matrix (ECM) deposition throughout the lung parenchyma (4). This increase in ECM is likely due to a subtle imbalance between accumulation and degradation of matrix components.

As potential targets for intervention, two key cytokines have been described: transforming growth factor (TGF)–ß1 and connective tissue growth factor (CTGF) (5).

TGF-ß1 has been widely studied in the context of fibrotic diseases (6). One of the key activities of this growth factor is the induction of matrix proteins and proteoglycans and the inhibition of collagen degradation either by reduction of matrix-degrading proteases such as matrix metalloproteinases or by induction of protease inhibitors such as plasminogen activator inhibitor (PAI)–1 and tissue inhibitors of metalloproteinase (TIMPs) (7). We have demonstrated previously that transient overexpression (6–10 days) of active TGF-ß1 in rat lungs by adenoviral gene transfer induces a marked fibrosis, characterized by extensive deposition of ECM proteins and by accumulation of myofibroblast cells (8). TGF-ß1 gene–induced fibrosis is progressive and persistent for over 60 days.

CTGF is a member of the recently described connective tissue growth factor–cysteine-rich 61–nephroblastoma (CCN) overexpressed gene family (9–11). This 38 kD factor has been shown to be secreted by fibroblasts when stimulated by TGF-ß1 (12). CTGF stimulates in vitro fibroblast growth and matrix deposition by upregulation of collagen and fibronectin gene expression. Its overproduction is proposed to play a major role in fibrogenesis (13) and it is found overexpressed in human fibrotic diseases (14, 15). In a model of murine dermal fibrosis, Mori and colleagues (16) demonstrated that administration of recombinant CTGF is required for the development of persistent fibrosis. More importantly, recombinant CTGF alone was unable to induce this persistent fibrosis and needed the adjunct application of recombinant TGF-ß1. Recently, Abreu and colleagues (17) found in a Xenopus model that CTGF may enhance TGF-ß1 signaling by directly binding to this growth factor, suggesting a cofactor role for CTGF. Aside from these preliminary studies, very little is known about the effect of isolated CTGF overexpression in vivo.

In this study, we used transient overexpression of CTGF by adenoviral gene transfer in rat lungs to evaluate the ability of this growth factor to induce fibrosis. We found that CTGF overexpression induced a moderate but transient fibrosis with kinetics that suggests indirect activation. When the results obtained with CTGF overexpression are compared with those of TGF-ß1 using adenovirus-mediated gene transfer, CTGF induced only minor changes in TIMP-1 expression. We hypothesize that although CTGF may indirectly initiate the fibrogenic response, it is unable to maintain progressive fibrosis. Some of the results of these studies have been published previously as an abstract (18).


   METHODS
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 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Recombinant Adenovirus
Human CTGF (AdCTGF) was obtained from Dr. J. E. Murphy (Bayer Corporation, Berkeley, CA) and has been described previously (19). Mutant TGF-ß1 translated into spontaneously bioactive TGF-ß1 (AdTGF-ß1223/225) and control vectors (AdDL) with no insert in the deleted E1 region are described in detail elsewhere (8, 20).

Animal Treatment
Female Sprague-Dawley rats (Charles River Laboratories, Montreal, QE, Canada) weighing 200–250 g were housed in special pathogen-free conditions. The animals were treated in accordance with the guidelines of the Canadian Council of Animal Care. A total of 1 x 109 plaque-forming units of AdCTGF, AdTGF-ß1223/225, or AdDL were administered intratracheally in a volume of 300 µl phosphate-buffered saline after minor surgery. Rats (three to five per group) were killed on Days 3, 7, 14, 21, and 28. Bronchoalveolar lavage (BAL) and lung tissue processing were performed as described (21).

Evaluation of Fibrosis
After fixation in 10% formalin for 24 hours, sections of the lung were paraffin embedded, sectioned, and stained with hematoxylin and eosin or Masson Trichrome. Immunohistochemistry was performed using {alpha}–smooth muscle actin antibodies (Clone 1A4, Missisauga, ON, Canada) as described (21). Hydroxyproline content was determined as described (22, 23).

CTGF Expression
Human lung epithelial cells (A549) cells were infected with AdCTGF or AdDL (10 or 50 plaque-forming units/cells). Cells were lysed in Trizol (Invitrogen, Carlsbad, CA) at 24 or 48 hours after infection for RNA and protein extraction. Northern blot analysis with a human CTGF probe was used to detect CTGF messenger RNA (mRNA) in treated cells, as well as total lung mRNA, as described previously (23).

Western blot: protein from cell extracts (20 µg) or 25 µl of BAL were separated on a 10% sodium dodecyl sulfate–polyacrylamide gel. The membrane was probed with a 1/2,000 dilution rabbit anti-mouse CTGF IgG antibody (Torrey Pines Biolabs, Houston, TX) followed by a secondary antibody (Santa Cruz Biotechnology, Santa Cruz, CA) and visualized with enhanced chemiluminescence (Amersham Biosciences, Little Chalfont, UK).

Primary rat lung fibroblasts were grown as described (24). Cells were exposed to cell product of A549 cells infected 48 hours with AdCTGF or AdDL or to BAL from AdCTGF or AdDL animals. Fibroblasts were lysed in Trizol for RNA extraction.

Ribonuclease Protection Assay
Frozen lung samples were homogenized in 7 ml of Trizol (Invitrogen) and RNA extracted. RNA (15 µg) was hybridized overnight with a custom rat probe set (Pharmingen, Mississauga, ON, Canada) containing different length probes for rat fibronectin, CTGF, PAI-1, TIMP-1, TGF-ß1, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Hybridized samples were detected with a Storm 820 phosphorimager (Molecular dynamics, Sunnyvale, CA) and band density analyzed using Scion Image software (Scion Corp, Fredrick, MD).

Quantitative Real-time Polymerase Chain Reaction
RNA (1 µg) was DNase treated and then reverse transcribed (Invitrogen). Quantitative polymerase chain reaction (PCR) was performed using an ABI Prism 7700 Sequence Detector. Primers (Mobix, Hamilton, ON, Canada) and probes (Applied Biosystems, Foster City, CA) are shown in Table 1 . Results were normalized to GAPDH-optimized probe and primers (Applied Biosystems).


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  		TABLE 1. Primers and probes used in quantitative real-time pcr reactions

 
Statistical Analysis
Data are shown as mean ± SEM. For group differences, we used Student's t test. A p value less than 0.05 was considered significant.


   RESULTS
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 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Gene Expression and Protein Synthesis of AdCTGF In Vitro and In Vivo
A549 cells infected with AdCTGF showed a positive signal for AdCTGF mRNA by northern gel analysis. There was no difference when cells were infected with 10 or 50 multiplicity of infection, but the signal increased with time after infection (Figure 1A) . CTGF protein (38 kD) was strongly expressed by A549 cells at 24 or 48 hours after infection, as shown by western blot analysis (Figure 1B). Cells infected with the control adenovirus did not express CTGF mRNA or protein. Total lung mRNA from Day 3 and Day 7 AdCTGF-treated animals showed a strong positive signal for AdCTGF compared with AdDL-treated animals. By Day 14 there was no significant signal for AdCTGF compared with AdDL animals (Figure 2A) . BAL from AdCTGF-treated animals showed expression of CTGF protein 3 and 7 days after infection (Figure 2B). CTGF was not detectable in BAL from animals treated with the control virus.




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  		Figure 1. Connective tissue growth factor (CTGF) messenger RNA (mRNA) expression and protein synthesis in vitro: A549 cells were infected with human CTGF (AdCTGF). (A) Total mRNA extracts show a strong positive signal for human CTGF by northern blot analysis. The signal was present at 12 hours and was stronger at 24 hours. There was no difference when the amount of virus was increased by five times. No signal was detectable in the same conditions with the control vector (AdDL). Equal loading was confirmed by ethidium bromide staining for ribosomal RNA. (B) Protein extracts from the same cells showed the presence of CTGF by western blot analysis by 24 and 48 hours, whereas no protein was detectable with the control virus. (C) Primary rat lung fibroblasts have an increased procollagen 1A2 and fibronectin mRNA expression (quantitative real-time–polymerase chain reaction) 48 hours after exposition to cell product of A549 cells infected 48 hours earlier with AdCTGF compared with cell infected with AdDL (10 multiplicity of infection for both). Results are expressed as a mean of two different experiments with five different cell lines. *p < 0.05.

 



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  		Figure 2. Connective tissue growth factor (CTGF) expression in rat lungs: (A) Total lung mRNA was used for northern blot analysis with a human CTGF probe. Marked increase in CTGF expression in human CTGF (AdCTGF)–treated animals compared with control vector (AdDL) is seen at Day 3 and Day 7. (B) An equal amount of bronchoalveolar lavage (BAL) from animals treated with AdCTGF 3 and 7 days earlier was loaded for western blot analysis. CTGF protein was expressed at Day 3, and the signal was stronger by Day 7. No protein was detectable in the BAL from control animals. (C) Primary rat lung fibroblasts have an increased procollagen 1A2 and fibronectin mRNA expression (quantitative real-time–polymerase chain reaction) 24 hours after exposure to BAL fluid from animals treated 7 days earlier with AdCTGF or animals treated in the same manner with AdDL. Results are expressed as a mean of two different experiments with three different cell lines and BAL from four different animals. *p < 0.05.

 
Biological activity of the CTGF transgene has been verified in human lung fibroblasts in culture with a strong elevation of type-1 collagen protein in the media 24 hours after infection (25). In our study, procollagen1A2 and fibronectin mRNA expression were significantly increased (5.7- and 4.5-fold, respectively) in primary rat lung fibroblasts exposed to cell product of A549 cells infected 48 hours before with AdCTGF compared with AdDL (Figure 1C). We have demonstrated that A549 infected with AdCTGF produced a significant level of AdCTGF protein (Figure 1B). Moreover, primary rat fibroblasts exposed to BAL from Day 7 AdCTGF-treated animals, at which time there was no increased expression of rat CTGF mRNA over control animals (Figure 8), showed a significant increase in procollagen1A2 (2.9-fold) and fibronectin (2.3-fold) mRNA compared with fibroblasts exposed to BAL from AdDL-treated animals (Figure 2C).



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  		Figure 8. Rat connective tissue growth factor (CTGF) is autoinduced in human CTGF (AdCTGF)–treated animals at Day 14 and constantly induced in AdTGF-ß1 animals. Quantitative real-time–polymerase chain reaction for rat CTGF mRNA expression: total RNA from four to five animals in each group was used (except Day 21 for control vector [AdDL], three animals). *p < 0.02.

 
Histologic Changes with CTGF Overexpression in the Lung
Microscopic examination of rat lungs infected with the control virus revealed a mild inflammatory reaction at Days 3 and 7 in the perivascular and peribronchial areas without extension into the parenchyma (data not shown). Lung morphology normalized at Day 14 with no evidence of fibrosis at any time point studied (Figures 3A–3C) .



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  		Figure 3. Lung histology: connective tissue growth factor (CTGF) induced a transient fibrosis. Masson Trichrome, representative sections of control vector (AdDL)–treated animals, magnification x50 (A, B, C) show no fibrosis and no change in lung morphology over the course of the experiment. Masson Trichrome–stained section of human CTGF (AdCTGF)–treated animals demonstrates significant collagen accumulation 14 days after adenoviral infection (D: x50; G: x200 magnification) and residual patchy fibrosis at Day 21 (E: x50; H: x200 magnification). Lung morphology has normalized by Day 28 (F: x50 magnification). {alpha}–Smooth muscle actin immunohistochemistry of sections from AdCTGF-treated animals shows accumulation of myofibroblasts (brown) in fibrotic tissue areas at Day 14 decreasing at Day 21 (I, J: x200 magnification).

 
We have already shown that rats receiving AdTGF-ß1223/225 demonstrated markedly abnormal lung histology, with widespread areas of fibrosis beginning as early as Day 3 and markedly from Day 14 to Day 64 (8).

Three to 7 days after infection with AdCTGF, the time of maximal transgene expression from the vector, the rat lungs showed a patchy accumulation of mononuclear cells in peribronchial areas and in adjacent alveolar spaces. There was evidence of enlargement of the bronchial wall with marked edema. At this time, there was no accumulation of collagen with Masson's trichrome stain, suggestive of a lack of fibrosis, and no evidence of fibroblast proliferation with {alpha}–smooth muscle actin immunohistochemistry (data not shown). Fourteen days after AdCTGF infection, at a time when the transgene is no longer expressed, inflammation was no longer evident, but patchy fibrotic areas were identified throughout the parenchyma. Within these obvious areas of collagen accumulation there were also cells strongly positive for {alpha}–smooth muscle actin (Figures 3D, 3G, and 3I). There was no evidence of thickening of the pleural surface as was seen with AdTGF-ß1223/225 (8). By Day 21 after AdCTGF treatment, the larger fibrotic areas had resolved; however, smaller accumulations of collagen and myofibroblasts persisted throughout the parenchyma. There was no evidence of ongoing tissue destruction (Figures 3E, 3H, and 3J). By Day 28, there was no longer evidence of fibrosis (Figure 3F), and at Day 56, the rat lung appeared normal (data not shown).

Hydroxyproline Content in the Lung
The hydroxyproline content of lung homogenates was determined to quantify the collagen content. We have demonstrated previously an increase in the mean hydroxyproline content in the lungs of rats treated with AdTGF-ß1223/225 compared with rats treated with control virus. These changes were significant 14 days after AdTGF-ß1223/225 infection and persisted to Day 64 (8). Animals treated with AdCTGF demonstrated a significant increase in collagen content at Day 14 (+36%, p < 0.02) compared with rats treated with control virus. In agreement with the reversal of histologic changes and contrary to the AdTGF-ß1223/225–treated animal findings, the increased lung collagen content had returned to normal levels by Day 28 (Figures 4A and 4B) .



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  		Figure 4. Lung hydroxyproline concentration (five animals in each human connective tissue growth factor [AdCTGF] group, four in the others) (A) Animals treated with AdCTGF showed a 36% increase in lung hydroxyproline concentration by Day 14 compared with control animals. Hydroxyproline concentration decreased by Day 21 and reached control levels by Day 28. Gray bars indicate AdDL; black bars indicate AdCTGF. *p < 0.05, **p < 0.01. (B) Percentage of increased hydroxyproline level related to control vector (AdDL)–treated animals at the same time points. Transforming growth factor–ß1 data have been published previously (7). Open bars indicate AdTGF-ß1.

 
Gene Expression in Rat Lungs
We isolated mRNA from total lung tissue 4, 7, 14, and 21 days after adenovirus infection and analyzed it using quantitative density analysis and quantitative real-time (RT)–PCR. Quantitative density analysis or quantitative RT-PCR expression indexed to GAPDH and referenced to AdDL-treated animals were used to compare mRNA expression for fibronectin, CTGF, TIMP-1, procollagen1a2, and TGF-ß1.

Fibronectin was significantly expressed in AdCTGF-treated lungs by Day 14 (1.8-fold compared with AdDL animals, p < 0.01). This expression was transient, and the level decreased to that in control animals by Day 21. In AdTGF-ß1223/225–treated animals, we found an upregulation at each time point, sustained at Day 21 (Figure 5A) . Procollagen 1a2 mRNA expression analyzed by quantitative PCR followed a similar pattern with a significant increase at Day 14 (2.9-fold, p < 0.05, Figure 5B) in the AdCTGF group. Meanwhile, quantitative RT-PCR (Figure 6A) revealed an isolated increase in mRNA signal for TIMP-1 at Day 7 (1.7-fold, p < 0.01) in the AdCTGF-treated animal group. These increases declined by Day 14 to baseline. AdTGF-ß1223/225 exposure led to an increased TIMP-1 mRNA by Day 3, which was persistent to Day 21 (Figure 6A). TIMP-1 mRNA expression analyzed by RPA confirmed the quantitative RT-PCR findings (Figure 6A and data not shown). As expected, PAI-1 was upregulated from Day 3 to Day 21 in the AdTGF-ß1223/225–treated animal group, whereas there was only a transient upregulation from Day 7 to Day 14 in the AdCTGF group (Figure 6B).



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  		Figure 5. In human connective tissue growth factor (AdCTGF)–treated animals, fibronectin and procollagen 1a2 are upregulated at Day 14 compared with control animals, whereas there is a sustained upregulation in human transforming growth factor–ß1 (AdTGF-ß1) animals. (A) RNAse protection assay, fibronectin expression: 15 µg of total RNA from animals treated with control vector (AdDL) (n = 3), AdCTGF (n = 4), or AdTGF-ß1 (n = 4) were used per time point. Relative densities compared with glyceraldehyde-3-phosphate dehydrogenase (Days 3, 7, 14, and 21) were calculated as described in METHODS (*p < 0.01). (B) Quantitative real-time–polymerase chain reaction for procollagen 1a2 mRNA expression: total RNA from five (AdCTGF group) to four animals in each group was used (except Day 21 for AdDL, three animals). *p < 0.05.

 


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  		Figure 6. In human connective tissue growth factor (AdCTGF)–treated animals, tissue inhibitor of metalloproteinase–1 (TIMP-1) and plasminogen activator inhibitor–1 (PAI-1) are upregulated at Day 7 compared with control animals, whereas there is a sustained upregulation in AdTGF-ß1 animals. (A) Quantitative real-time–polymerase chain reaction for TIMP-1 mRNA expression: total RNA from five (AdCTGF group) to four animals in each group was used (except Day 21 for control vector [AdDL], three animals). *p < 0.02. (B) RNAse protection assay, PAI-1 expression: 15 µg of total RNA from treated animals with AdDL (n = 3), AdCTGF (n = 4), or AdTGF-ß1 (n = 4) were used per time point. Relative densities compared with glyceraldehyde-3-phosphate dehydrogenase (Days 3, 7, 14, and 21) were calculated as described in METHODS (*p < 0.05, **p < 0.01).

 
TGF-ß1 expression was significantly increased by Day 14 (p < 0.05, Figure 7) in animals with AdCTGF, but this increase did not persist to Day 21. AdTGF-ß1 treatment led to a stronger upregulation of TGF-ß1 expression. This was sustained to Day 21 (Figure 7).



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  		Figure 7. Transforming growth factor–ß1 (TGF-ß1) is induced in human connective tissue growth factor–treated animals at Day 14 and strongly induced in AdTGF-ß1 animals. RNAse protection assay, TGF-ß1 expression: 15 µg of total lung RNA was used with three control vector (AdDL)-, four AdCTGF- or four AdTGF-ß1–treated animals per time point; relative densities compared with glyceraldehyde-3-phosphate dehydrogenase were calculated as described in METHODS *p < 0.05.

 
We used a set of probes and primers specific for rat CTGF for quantitative RT-PCR. This probe did not amplify any signal from the A549 infected with AdCTGF or AdDL used in Figure 1A (data not shown). This probe set was able to specifically detect endogenous rat CTGF. With this specific rat probe (Figure 8) , we did not find any upregulation of rat CTGF mRNA expression in AdCTGF animals at Day 3 and 7 compared with AdDL animals. By Day 14, there was a significant fourfold increase in endogenous rat CTGF mRNA compared with AdDL samples, which is likely due to autoinduction or to transient induction by endogenous TGF-ß. However, this induction was not sustained and disappeared by Day 21. In contrast, rat CTGF mRNA was constantly upregulated in AdTGF-ß1223/225–treated animals (Figure 8).


   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
CTGF appears to be an important growth factor involved in fibrosis. Its expression in lung tissue has been widely described in systemic sclerosis (14), idiopathic pulmonary fibrosis and pulmonary sarcoidosis (15), and bleomycin-induced lung fibrosis (26). Lasky and colleagues (26) have suggested that CTGF mRNA is upregulated in a murine bleomycin-sensitive strain and not in a bleomycin-resistant strain.

Numerous data have shown a strong correlation between TGF-ß1, a key cytokine involved in fibrosis, and CTGF. In vitro CTGF appears to be able to mediate the induction of collagen synthesis by TGF-ß1 (10). CTGF is transcriptionally activated by several factors such as dexamethasone (27) or mechanical stress (28), but TGF-ß1 is the main inducer of CTGF (12, 13). This stimulation seems to be at the transcriptional level, with a TGF-ß responsive element present in the CTGF promoter (29). To our knowledge, the effect of isolated CTGF overexpression in the lung is unknown. In our model of replication-deficient recombinant adenovirus vector, we have repeatedly demonstrated that the transient expression of the transgene molecule in the rodent occurs maximally by 3 days after infection and may last for up to 10 days before the expression of the transgene is no longer detectable. Also, we have shown previously that there was a dramatic and progressive fibrosis induced by overexpression of active TGF-ß1 by adenoviral gene transfer (8).

CTGF Induces Fibrosis
In this study, transient overexpression of CTGF in the lung leads to the induction of fibrosis by Day 14 with a significant increase in hydroxyproline content and myofibroblast accumulation. In vitro, CTGF stimulates ECM components, including fibronectin and collagen I (13, 30), in various fibroblast cultures. In vivo, some data suggest a correlation between fibronectin or collagen expression and CTGF expression in fibrotic tissue (10, 31). To our knowledge, our experiments show for the first time that CTGF overexpression in vivo can result in upregulation of these two ECM components. We also found an upregulation of fibronectin and procollagen1a2 in a similar temporal pattern as the lung collagen content measured by hydroxyproline. However, the increased expression of these ECM components was not seen at times when CTGF was expressed maximally (Day 3 to –Day 10) but was evident at later times (Day 14). Moreover, the ECM stimulation was not as great as what we observed after overexpression of TGF-ß1. This suggests that there is both a CTGF-dependent and a CTGF-independent pathway for the regulation of collagen mRNA levels by TGF-ß, as suggested by Ricupero and colleagues (32) in human embryonic lung fibroblasts. It is possible that this temporary induction of matrix is due to impaired stimulation by AdCTGF in the rat. However, we showed that fibronectin and procollagen1A2 were overexpressed in primary rat lung fibroblasts when stimulated by cell product of AdCTGF-infected A549 cells compared with control AdDL-infected cells, similar to effects seen on human cells (13, 30). We found the same response when we treated the same fibroblast cultures with BAL from Day 7 AdCTGF animals compared with BAL from animals treated with the empty viral vector control (AdDL). We have demonstrated that these animals infected with AdCTGF 7 days before, with a high expression of AdCTGF (Figure 2A) and with a high concentration of CTGF protein in their BAL (Figure 2B), did not show any increase in rat CTGF mRNA expression (Figure 8). Although this stimulation may be caused by a factor other than AdCTGF, the lack of response seen with control vector-treated samples, the stimulation of matrix-related gene expression, and the similar response seen with in vitro cell culture and in vivo BAL fluid would strongly support the identification of the factor as AdCTGF. We also treated rats with increasing doses of AdCTGF (1 to 5 x 109 plaque-forming units) and did not see a dose-dependent difference in hydroxyproline content or procollagen 1a2 mRNA expression at Day 14 (data not shown). This suggests a true qualitative difference in the fibrogenic response between CTGF and TGF-ß1. It is also possible that CTGF, unlike TGF-ß, may be expressed in the epithelium but fails to reach the parenchyma, the presumed target issue. However, in the evaluation of over 20 different cytokines and growth factors by this method, we have not seen such behavior.

CTGF Induces Transient Fibrosis
The fibrosis induced by CTGF was transient, with maximal presence at Day 14 and essentially resolved by Day 28. We believe that other factors are required to develop chronic and progressive fibrosis, as observed with TGF-ß1. In a mouse model of skin fibrosis, isolated recombinant CTGF injection resulted in transient granulated tissue (13, 16), whereas the addition of recombinant TGF-ß1 to CTGF produced a persistent fibrotic tissue (16). We believe that the different fibrogenic response results from the differential gene regulation we observed after exposure to CTGF or TGF-ß1.

Autocrine overexpression of CTGF may maintain fibrosis in certain conditions. CTGF protein is detected in cultured fibroblasts from patients with systemic sclerosis but not in fibroblasts from control patients (30, 33). The available data on autoinduction of CTGF is somewhat contradictory. Riser and colleagues (34) found that recombinant CTGF caused the autoinduction of CTGF mRNA in the absence of elevated TGF-ß1 levels in vitro. However, in a murine model of skin fibrosis, Mori and colleagues (16) found that injection of a small amount of CTGF induced a transient CTGF mRNA expression, although larger doses caused a downregulation of CTGF mRNA. In the current study, we found that rats treated with AdCTGF demonstrated stimulation of native rat CTGF mRNA at Day 14 but not at Day 3 or Day 7 as measured by quantitative RT-PCR with specific rat probe and primers, which does not detect AdCTGF. This autoexpression resolved to normal levels by Day 21. TGF-ß1, on the other hand, can not only cause autoinduction of TGF-ß1 but also appears to lead to prolonged high level expression of rat CTGF mRNA.

Maintenance of enhanced ECM deposition is most likely dependent on downstream collagen metabolism that is regulated in part by the balance between matrix metalloproteinases and TIMPs. Because daily collagen turnover is high, an abrupt change in balance of protease–antiprotease can lead to significant changes in collagen accumulation or degradation (35). High expression of TIMPs, creating a nonfibrolytic environment, has been associated with matrix accumulation in idiopathic pulmonary fibrosis (36) or in murine models of bleomycin (37) and silicosis (38, 39). Recently, we have shown that the different fibrogenic response after transfer of active TGF-ß1 gene to the lung of resistant (Balb/c) and susceptible (C57/Black6) mouse strains was associated with a different level of TIMP-1 expression (24), suggesting that susceptibility to progression of fibrosis is related to events downstream of initiation (40) and proceeds in a microenvironment predisposed to matrix deposition rather than degradation.

To investigate the possibility that CTGF and TGF-ß1 may differentially regulate collagen metabolism, we focused on TIMP-1, which is a major regulator of matrix metalloproteinases and collagenolytic activity. We found a strong and constant TIMP-1 mRNA upregulation from Day 3 to Day 21 in the AdTGF-ß1223/225–treated rats, whereas in the AdCTGF model we found only a transient and lower level of increased TIMP-1 expression, which returned to normal before the onset of observed fibrosis (Day 14). The lack of TIMP-1–enhanced expression in the lung infected by AdCTGF suggests that collagen metabolism may be skewed toward degradation of newly synthesized collagen in the microenvironment of the parenchyma, and this could account for the transient fibrotic response observed. Further to this, Fan and Karnovsky (41) demonstrated that CTGF overexpression in vascular smooth muscle cells induced matrix metalloproteinase–2 and may not induce the necessary nonfibrolytic microenvironment to maintain a fibrotic response.

Finally, in a recent publication, Abreu and colleagues (17) demonstrated in a Xenopus model that CTGF can directly bind to TGF-ß1 and causes synergistic activation of TGF-ß1 signaling. CTGF is present in enhanced amounts in the lung within 24 hours, but we do not see increased expression of TIMP-1 or PAI-1 until 7 days or increased collagen or fibronectin until 14 days, long after peak CTGF expression has ceased. The changes in matrix gene regulation or inhibitor expression correlate best with low level expression of endogenous rat TGF-ß1, at Day 7, likely due to the development of an immune response against the vector. Changes associated with CTGF, but only seen subsequent to the enhanced expression of endogenous TGF-ß1 are consistent, with CTGF acting primarily as a cofactor. CTGF can remain locally within the tissue and provide synergistic enhancement (or inhibition) of other growth factors. The low level of TGF-ß1 resulting from an immune response to adenovectors does not initiate an obvious fibrogenic response (see control vector tissue) but may be enough to cause the rise of an endogenous CTGF signal seen at Day 14. In the presence of additional CTGF, fibrogenesis could occur, but the level of TGF-ß1 would be too low to allow chronic progression. It has been shown that TGF-ß1–mediated induction of matrix gene expression is abrogated by treatment with inhibitory antibody to CTGF (42).

In summary, we have demonstrated that transient overexpression of CTGF in the lung results in a transient reversible fibrosis. Alone, CTGF appears to be able to initiate fibrogenic activity but appears to require other factors to develop chronic and progressive fibrosis, as is observed after exposure to TGF-ß1. Of even greater importance, we believe that the major difference in the fibrogenic response of CTGF and TGF-ß1 is the differential regulation of enzymes and inhibitors involved in collagen metabolism. TGF-ß1 induces a strong expression of TIMP-1, which leads to the suppression of matrix metalloproteinases and a noncollagenolytic microenvironment with preservation of tissue collagen. This preservation may create a growth factor–enriched microenvironment that can perpetuate the fibrogenic process. CTGF does not strongly induce TIMP-1, and collagen induced by CTGF is likely rapidly metabolized by the unopposed collagenolytic enzymes. Insights into these mechanisms may lead to new potential targets for therapy for patients with idiopathic pulmonary fibrosis.


   Acknowledgments
 
The authors thank Jane Ann Schroeder, Carol Lavery, Lisa Yu, Xueya Feng, and Duncan Chong for their invaluable technical help and Mary Jo Smith for outstanding preparation of histology.


   FOOTNOTES
 
Supported by the Bourses Lavoisier du Ministère des Affaires Etrangères, the Ligue Bourguignonne Contre le Cancer, and Bourse de Voyage Boehringer (P.B.), and by the Canadian Institutes of Health Research, St. Joseph's Healthcare, and Hamilton Health Sciences. P.J.M. is a CIHR Clinician Scientist. M. Kolb is a Parker B. Francis Fellow and is also supported by Deutsche Krebshilfe.

Conflict of Interest Statement: P.B. has no declared conflict of interest; P.J.M. has no declared conflict of interest; M.Kolb has no declared conflict of interest; T.H. has no declared conflict of interest; M.Kelly has no declared conflict of interest; J.R. has no declared conflict of interest; J.G. has no declared conflict of interest.

Received in original form October 31, 2002; accepted in final form June 17, 2003


   REFERENCES
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
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