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Regular Articles |
From the Laboratories of Pulmonary Pathobiology,*
Experimental Pathology,
and
Toxicology,
National Institute of
Environmental Health Sciences, Research Triangle Park, North Carolina
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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.11-13
The deposition of extracellular matrix
proteins such as collagen, mediated largely by TGF-ß family members,
generally follows the proliferative phase of the
disease.5,6
Receptor tyrosine kinases that mediate lung myofibroblast proliferation
include the PDGF receptors (PDGF-Rs)14
and the epidermal
growth factor receptor (EGF-R).15
Ligand binding to the
extracellular domain of the receptor triggers phosphorylation on
tyrosine residues within the intracellular domain. Several ligands bind
to the EGF-R with high affinity, including EGF,16
TGF-,17
and heparin-binding EGF
(HB-EGF).18
The three isoforms of PDGF (PDGF-AA, -AB, and
-BB) bind differentially to two PDGF-Rs termed PDGF-R and PDGF-Rß,
resulting in three possible receptor dimers; , ß, or
ßß.14,19
PDGF-AA binds only PDGF-R, whereas PDGF-AB
and PDGF-BB bind both PDGF-R and PDGF-Rß. Ligand-induced
autophosphorylation of EGF-R and PDGF-Rs allows for the docking of
numerous signaling molecules that initiate the intracellular signaling
cascades leading to cell division and chemotaxis.20
Although several studies have documented the expression of PDGF-R, EGF-R, and their respective ligands during fibroproliferative diseases, the relative contribution of the EGF-R and the PDGF-R system to pulmonary fibrosis in relation to other growth factor receptors remains unknown. Inhibitors of PDGF receptor and EGF receptor tyrosine kinase activity have been reported for low molecular weight compounds of the staurosporine,21 tyrphostin,22,23 and 2-phenylaminopyrimidine24,25 classes of compounds. Specific tyrphostin analogs have been reported to block a variety of pathogenic effects. For example, tyrphostin AG126 blocks lipopolysaccharide-induced septic shock in mice via inhibition of tyrosine phosphorylation of p42 mitogen-activated protein kinase.26 Furthermore, EGF-R- and PDGF-R-specific compounds of the 2-phenylaminopyrimidine class possess antitumor activity in vivo.24,25 In this study we have investigated two tyrphostins, AG1296 and AG1478, that specifically block autophosphorylation of the PDGF-R and EGF-R, respectively. Using a rat model of metal-induced pulmonary fibrosis, we show that both AG1296 and AG1478 inhibit the proliferative response of epithelial and mesenchymal cells within fibrotic lesions, and this results in reduced lung collagen accumulation. This study provides strong evidence that both EGF-R and PDGF-R are important to the progression of pulmonary fibrosis and suggests that targeting autophosphorylation of receptor tyrosine kinases could have potential therapeutic value in treating fibroproliferative lung diseases.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Tyrphostins AG1296 and AG1478 were purchased from Calbiochem (La
Jolla, CA). Vanadium pentoxide was from Aldrich Chemical Co.
(Milwaukee, WI). Recombinant human PDGF-BB, EGF, and fibroblast growth
factor (FGF)-2 were purchased from Upstate Biotechnologies (Lake
Placid, NY). [3H]Thymidine was from Amersham Corp.
(Arlington Heights, IL). Anti-PDGF-R, anti-PDGF-Rß, and EGF-R
antibodies were from Upstate Biotechnologies. Anti-phosphotyrosine
(PY20) antibody and horseradish peroxidase (HRP)-labeled mouse IgG were
from Transduction Laboratories (Lexington, KY). HRP-labeled swine
anti-rabbit and rabbit anti-sheep antibodies were purchased from
DakoPatts (Carpinteria, CA).
Isolation of Rat Lung Myofibroblasts
Early-passage rat lung myofibroblasts were isolated and
characterized as described previously from male Sprague-Dawley
rats.4
Cell isolates at passage 1 or 2 were plated onto
3-amino-propyl-triethoxysilane (APTS)-coated glass chamber slides and
grown to confluence, then fixed briefly in ice-cold acetone. Fixed
cells were then subjected to overnight incubation with a murine
monoclonal antibody to the antigen of interest, followed by a
biotinylated horse anti-mouse antibody (Vector Laboratories,
Burlingame, CA), avidin-biotin immunoperoxidase (Vector), and
3,3'-diaminobenzidine chromogen (Vector). An irrelevant monoclonal
antibody (anti-5-bromo-2'-deoxyuridine, Becton Dickinson, San Jose, CA)
at equivalent IgG concentration served as control for nonspecific
immunoreactivity. Concentrations of primary antibodies were set by
titration on appropriate rat-derived positive control cells. Cells
stained positively for vimentin, desmin, and -smooth muscle actin,
which indicated a myofibroblast phenotype. In addition, examination of
glutaraldehyde-fixed cell pellets by transmission electron microscopy
showed ultrastructural features consistent with a myofibroblast
phenotype (abundant intermediate filaments and rough endoplasmic
reticulum and lack of Weibel-Palade bodies characteristic of
endothelial cells). Cells were grown to confluence in 10% fetal bovine
serum (FBS)/Dulbecco's modified Eagle's medium (DMEM) before being
seeded for the assays described below.
Western Blotting
Myofibroblasts grown to confluency with 10% FBS/DMEM in
75-cm2
tissue culture flasks were rendered quiescent for 24
hours with serum-free defined medium (SFDM) consisting of Ham's F-12
medium supplemented with 0.25% bovine serum albumin and an
insulin/transferrin/selenium mixture (Boehringer Mannheim,
Indianapolis, IN). Cultures were incubated with an increasing
concentration of AG1296 or AG1478 (1 to 100 µmol/L) diluted in SFDM
for 24 hours to block receptor tyrosine kinase activity.
Autophosphorylation was then stimulated by the addition of 50 ng/ml
PDGF-AB, PDGF-BB, EGF, or TGF- for 10 minutes. The cultures were
then washed with ice-cold PBS, and cell lysates were collected by
incubation with 250 µl of lysis buffer consisting of 50 mmol/L
Tris/HCl (pH 7.4), 1% Triton X-100, 150 mmol/L NaCl, l mmol/L EGTA, 1
mmol/L Na3V04, l mmol/L NaF, l mmol/L
phenylmethylsulfonyl fluoride, 0.25% sodium deoxycholate, and 20
µg/ml of each of the following proteinase inhibitors: aprotinin,
leupeptin, and pepstatin. Twenty microliters of each sample was mixed
with 5 µl of sample buffer (0.5 mol/L Tris/HCl, pH 6.8, 10% sodium
dodecyl sulfate (SDS), 0.1% bromphenol blue, 20% glycerol, and 50
mmol/L 2-mercaptoethanol) and separated by SDS-polyacrylamide gel
electrophoresis in a 2% to 15% Tris-glycine gel (Integrated
Separation Systems, Hyde Park, MA). The proteins were transferred to
Hybond nitrocellulose membrane (Amersham), and the membrane was blocked
with 3% milk in PBS for 1 hour before overnight incubation at 4°C
with a 1:500 dilution of rabbit anti-mouse PDGF-R antibody, rabbit
anti-human PDGF-Rß antibody, a sheep anti-human EGF-R antibody
(Upstate Biotechnologies), or a monoclonal phosphotyrosine (PY20)
antibody (Transduction Laboratories). The membranes were washed three
times with PBS/Tween before a 90-minute incubation with a 1:2000
dilution of the appropriate secondary HRP-conjugated antibody:
HRP-swine anti-rabbit for PDGF-R and PDGF-Rß (DakoPatts),
HRP-rabbit anti-sheep for EGF-R (DakoPatts), or HRP-mouse IgG
(Transduction Laboratories). After thoroughly washing in PBS/Tween, the
HRP-labeled proteins were visualized with an ECL kit (Amersham).
[3H]Thymidine Incorporation Assay
Rat lung myofibroblasts were grown to confluence with 10% FBS/DMEM in 24-well tissue culture plates (2-cm2 wells) and then rendered quiescent for 24 hours with SFDM containing 0.5% FBS. The cells were then treated with fresh 0.5% FBS/SFDM containing 100 µmol/L AG1296 or AG1478 in vehicle (dimethylsulfoxide (DMSO)) or vehicle alone for 24 hours. The cultures were rinsed and treated with an increasing concentration of PDGF-AB, EGF, or FGF-2 (0.5 to 100 ng/ml) along with 5 µCi/ml [3H]thymidine (Amersham) for 36 hours. The cells were washed with Ham's F-12 at 25°C, placed on ice, and incubated with 0.5 ml/well 5% trichloroacetic acid for 10 minutes. After washing three times with ice-cold distilled water, solubilization was performed with 0.5 ml/well 0.2 N NaOH containing 0.1% SDS for 30 minutes on an oscillating platform. A 100-µl aliquot of each sample was added to 1 ml of Ecolume (Costa Mesa, CA), and radioactivity was measured on a liquid scintillation counter.
Administration of Tyrphostins in Vivo and Intratracheal Instillation of V2O5
Sprague-Dawley rats (Charles River, Raleigh, NC) weighing ~200 g were injected intraperitoneally (i.p.) with 50 mg/kg AG1296 or AG1478 in vehicle (DMSO) or vehicle alone 1 hour before intratracheal instillation. V2O5 suspensions were vortexed thoroughly, then bath sonicated for 30 minutes at 25°C before instillation. The animals were instilled with saline alone or 1 mg/kg V2O5 in saline as has been described previously.4 Two days after instillation, a second i.p. injection of tyrphostin (50 mg/kg) or vehicle was administered. Groups of rats were sacrificed 3, 6, and 15 days after instillation by overdosing with an i.p. injection of Nembutol. Some groups received a single i.p. injection of 50 mg/kg bromodeoxyuridine (Brdu) 1 hour before sacrifice. The lungs were removed en bloc and inflated with formalin for immunohistochemistry or digested for total lung hydroxyproline as described below.
Bromodeoxyuridine Immunohistochemistry
Lung tissue was fixed overnight in 10% neutral buffered formalin and embedded in paraffin. Immunohistochemistry was performed using the avidin-biotin peroxidase method. Tissue sections (6 µm) were dehydrated through a series of graded alcohol solutions to 1X automation buffer (AB) consisting of 5% NaCl and 2% HCl (Biomeda Corp., Foster City, CA), treated with 2 N HCl at 37°C for 30 minutes and 1 mol/L borate buffer for 1 minute, and then incubated in 0.01% trypsin and 1% calcium chloride for 3 minutes at 37°C. Endogenous peroxidase was blocked in 3% (v/v) H2O2 for 15 minutes. After a 1X AB wash, blocking was performed for 20 minutes, and a monoclonal mouse anti-Brdu antibody (Becton Dickinson, Mountain View, CA) 1:50 was applied for 30 minutes at room temperature. Sections were washed twice with AB, then incubated for 30 minutes with the Elite avidin-biotin complex (Vector) for 30 minutes. Visualization of the antibody complex was done using a diaminobenzidine tablet (10 mg) (Sigma Chemical Co., St. Louis, MO) dissolved in 20 ml of 1X automation buffer containing 12 µl of 30% H2O2 for 6 minutes in the dark. Slides were then rinsed in running tap water, counterstained with Harris hematoxylin (Harelco, Gibbstown, NJ), dehydrated through a series of graded alcohols to xylene, and coverslipped with Permount (Fisher Scientific, Fair Lawn, NJ). Some paraffin-embedded sections were immunostained for desmin as a smooth muscle cell phenotypic marker or vimentin (clone LN6, Accurate Antibodies, Westbury, NY) as a marker of fibroblast phenotype. Mature collagen was detected by Masson's trichrome stain.
Hydroxyproline Assay
The procedure for quantitation of lung hydroxyproline has been described elsewhere.27 For hydrolysis, whole lung tissue was washed in PBS and hydrolyzed for 18 hours in 6 N HCl at 110°C (~40 ml/6 g of tissue). One drop of 1% phenophthalein in ethanol was added to each sample, and the pH was adjusted to 6.0 with NaOH titration. Two milliliters from each sample was centrifuged 5 minutes at 1500 rpm, and the pellet was oxidized with 1 ml of 0.6 mol/L chloramine-T for 30 minutes. Each sample then received 1 ml of 7.5% p-dimethylaminobenzaldehyde and was incubated at 65°C for 15 minutes. The absorbance was measured at 560 nm on a spectrophotometer. Hydroxyproline content was proportional to an increase in absorbance and was evident by a characteristic color change from yellow to red. Lung hyroxyproline was quantitated against a standard curve set up with purified hydroxyproline (Sigma), and values were corrected for total lung wet weight.
Statistical Analysis
The Systat (Evanston, IL) statistical package was used for all analyses. For analysis of [3H]thymidine uptake data, one-way analysis of variance was performed to determine an overall effect of tyrphostin versus control. Two-sample t-tests were performed on Brdu data and hydroxyproline data to determine significant differences among control versus treatment groups.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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AG1296 has been reported to specifically inhibit activation of
PDGF receptors,23
whereas AG1478 has been described as a
selective blocker of EGF-R autophosphorylation.22
To
confirm the specificity of these tyrosine kinase inhibitors in our
system, we stimulated rat lung myofibroblasts with PDGF-R ligands or
EGF-R ligands after pretreating the cells with AG1296 or AG1478 to
block autophosphorylation on tyrosine residues. Phosphotyrosine
immunoblotting showed that autophosphorylation was stimulated in a
time-dependent manner that peaked 5 to 20 minutes after stimulation
with PDGF-R ligands (PDGF-AB and PDGF-BB) or EGF-R ligands (EGF and
TGF-) and declined to undetectable levels by 1 hour after
stimulation (data not shown). Autophosphorylation stimulated by PDGF-AB
or PDGF-BB was completely blocked by 100 µmol/L AG1296, yet
autophosphorylaton stimulated by the addition of either EGF or TGF-
was not affected by AG1296 (Figure 1)
.
Conversely, 100 µmol/L AG1478 completely inhibited EGF- or
TGF--induced autophosphorylation without affecting PDGF-stimulated
autophosphorylation (Figure 2)
. The
levels of PDGF-R, PDGF-Rß, and EGF-R were not affected by the
addition of either AG1296 or AG1478 (Figures 1 and 2)
.
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[3H]Thymidine incorporation assays were undertaken
to establish that the inhibition of autophosphorylation caused by
AG1296 and AG1478 shown in Figures 1 and 2
resulted in inhibition of
mitogenesis in vitro. AG1296 selectively blocked
PDGF-stimulated [3H]thymidine uptake but did not affect
EGF-induced DNA synthesis, whereas AG1478 inhibited EGF-stimulated
mitogenesis without affecting PDGF-induced DNA synthesis (Figure 3)
. [3H]Thymidine uptake
stimulated by FGF-2 was not affected by either AG1296 or AG1478.
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We previously described the progression of
V2O5-induced pulmonary fibrogenesis wherein an
acute inflammatory response was observed by 24 hours after
instillation, a proliferative response of mesenchymal cells staining
positively for vimentin and desmin occurred 3 to 6 days after
instillation, and collagen deposition was evident by day 15 after
instillation as determined by trichrome staining.4
In the
present study, proliferative lesions induced by
V2O5 intratracheal instillation at day 3 after
instillation contained numerous Brdu-positive cells (Figure 4B)
. Day 3 was the peak of Brdu
incorporation, as the number of Brdu-positive cells then declined by
>50% by day 6 after instillation and were reduced by >90% by day 15
after instillation (data not shown). Cells incorporating Brdu after
V2O5 instillation in rats treated with AG1296
were confined mainly to the corners of the alveolar walls, suggesting
proliferating type II epithelial cells (Figure 4D)
. In contrast,
Brdu-positive cells after V2O5 in rats treated
with AG1478 were present in alveolar walls as well as in intra-alveolar
spaces, suggesting the presence of some proliferating mesenchymal cells
(Figure 4F)
. Quantification of Brdu-labeled nuclei showed that
treatment with either AG1296 or AG1478 significantly reduced the number
of Brdu-positive cells at day 3 by ~50% (Figure 5)
.
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Treatment of rats with tyrphostins AG1296 or AG1478 reduced the
severity of V2O5-induced fibrotic lesions in
the lung at day 15 after instillation as assessed by trichrome staining
for collagen (Figure 6)
.
V2O5 caused a fivefold increase in total lung
hydroxyproline content 15 days after instillation (Figure 7)
. In preliminary studies, a single i.p.
injection of AG1296 (50 mg/kg) 1 hour before instillation did not
prevent an increase in hydroxyproline caused by
V2O5-induced lung injury. However, a double
injection of AG1296, 50 mg/kg 1 hour before instillation and again 2
days after instillation, prevented collagen accumulation in
V2O5-instilled animals, resulting in
hydroxyproline measurements that were nearly equivalent to the
saline-instilled group. AG1478 reduced collagen accumulation in
V2O5-instilled animals by ~50%, but
hydroxyproline content was significantly elevated above the saline
control group.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-induced autophosphorylation and
mitogenesis. Both AG1296 and AG1478 prevented an increase in total lung
hydroxyproline (ie, collagen) after lung injury with
V2O5. These data provide strong evidence that
both PDGF-R and EGF-R contribute to the proliferative phase of the
fibrogenic response and that replicating PDGF- and EGF-responsive
mesenchymal cells are the major source of collagen deposited during the
subsequent extracellular matrix deposition phase of fibrogenesis.
Several studies have documented the up-regulation of PDGF isoforms and
EGF receptor ligands during pulmonary fibrogenesis in humans and in
rodent models. PDGF and TGF- are both elevated in alveolar
macrophages or bronchoalveolar lavage fluid from patients with
pulmonary fibrosis.8,9,11,28
In rat models of fibrosis,
PDGF and the PDGF -receptor subtype are increased 2 to 3 days after
lung injury by bleomycin or metals.4,29
EGF and TGF-
are elevated 2 to 4 days in the rat bleomycin model. Asbestos fibers
also stimulate the secretion of PDGF isoforms and TGF- in
vitro and in vivo in rodent
models.10,12,30
Due to the potent mitogenic effect of
these growth factors, these studies suggest a role for PDGF and EGF
receptor ligands in causing a fibrotic response. However, they do not
clearly establish a link between PDGF- or EGF-mediated cell
proliferation and the subsequent deposition of collagen, which defines
the fibrotic lesion. Our data in the present study show that blocking
PDGF- or EGF-receptor-mediated events prevents an increase in total
lung collagen, and this is likely achieved by preventing the
proliferation of mesenchymal cells that have the potential to produce
collagen once stimulated by other growth factors, such as TGF-ß. The
delivery of tyrphostins at day 2 after instillation of
V2O5 was necessary to reduce DNA synthesis and
collagen accumulation in vivo. This time point coincides
with the temporal induction of PDGF and PDGF-R4,29
and
TGF- and the EGF-R13
2 to 4 days after injury.
PDGF-R-specific tyrosine kinase inhibitors have been shown to inhibit intimal hyperplasia after arterial injury in vivo.31-33 Banai and co-workers reported that in vivo delivery of the PDGF-R-specific tyrphostin AG1295 caused a significant reduction (>80%) in the numbers of smooth muscle cells in the intima/media area of porcine femoral arteries after balloon injury.31 Other investigators reported that administration of the PDGF-R-specific 2-phenylaminopyrimidine, CGP53716, inhibited an increase in intimal vascular smooth muscle cell number after balloon injury in rats by inhibiting both the migration and proliferation of smooth muscle cells.33 At least one other blocking strategy that does not involve targeting tyrosine kinases has been reported. Fern and co-workers showed that delivery of a neutralizing antibody to PDGF blocks neointimal smooth muscle accumulation after angioplasty.34 Thus, there is abundance evidence to support a role for PDGF in nonmalignant proliferative diseases such atherosclerosis. Although the data presented in this study are the first to demonstrate reduction of lung fibrosis through inhibition of PDGF-R or EGF-R tyrosine kinase activity, several other investigators have overexpressed PDGF in the lung and observed a fibrotic response. Instillation of exogenous, recombinant PDGF in rats causes obliterative bronchiolitis characterized by the excessive accumulation and proliferation of myofibroblasts in the airways.35 Yoshida and co-workers demonstrated that overexpression of the genes encoding both PDGF-B chain and TGF-ß1 in rats resulted in interstitial lung fibrosis.36 Furthermore, it is noteworthy that PDGF-AA is essential to the development of myofibroblasts in the embryonic lung.37 Collectively, these studies provide strong evidence that the PDGF system is pivotal to mesenchymal cell growth in the lung.
Our study provides additional evidence that PDGF is a key mediator of
the pulmonary fibrotic response and suggests that the EGF-R also plays
an important role in the proliferative phase of lung fibrogenesis.
However, the use of the available tyrosine kinase inhibitors has some
limitations toward dissecting the relative importance of growth factor
isoforms and specific receptor subtypes. For example, as AG1296
inhibits both PDGF-R and PDGF-Rß, this inhibitor does not allow us
to define the relative importance of the two PDGF receptors to the
progression of lung fibrosis. Previous studies from our laboratory have
shown that both PDGF-R and PDGF-Rß are required for maximal
proliferation of myofibroblasts and that the PDGF-R is induced by
fibrogenic agents in vitro and during pulmonary fibrogenesis
in vivo.4,38
In addition, although the AG1478
data indicate that EGF-R activation is important to the progression of
fibrosis, it does not indicate which EGF-R ligand (EGF, TGF-, or
HB-EGF) is more important to the proliferation of mesenchymal cells
during fibrosis, as all of these ligands bind and activate a common
EGF-R. Finally, although the specificity of AG1296 and AG1478 for
PDGF-R- and EGF-R-induced mitogenesis were clearly demonstrated
in vitro, we cannot rule out the possibility that these
tyrosine kinase inhibitors could have inhibitory effects on other
tyrosine kinases in vivo that could contribute to a
reduction in the severity of lung fibrosis. However, the doses of
tyrphostins in this study were within the dose range used by other
investigators investigating tyrosine kinase inhibitors under similar
conditions.25,26,33
Phosphorylation of the EGF-R may also be initiated by oxidant-generating environmental agents in the absence of EGF-R ligands, and in such cases it is important to consider the potential effects of AG1478 on this type of ligand-independent EGF-R activation. For example, asbestos fibers cause stimulation of a mitogen-activated protein (MAP) kinase signaling cascade after phosphorylation of the EGF-R, and asbestos-induced MAP kinase activation is blocked by AG1478.39 Similarly, vanadium compounds cause MAP kinase activation and rapid tyrosine phosphorylation of other key cellular proteins.40 This is likely related to the well known ability of vanadium compounds to inhibit protein tyrosine phosphatases.40 Interestingly, V2O5-induced MAP kinase activation in cultured rat lung myofibroblasts is blocked by pretreatment with AG1478, suggesting a requirement for EGF-R (Y.-Z. Wang and J.C. Bonner, unpublished observation). Therefore, it is conceivable that the beneficial effect of AG1478 could be due in part to blocking the activation of EGF-R by V2O5 in the absence of EGF-R ligands.
In summary, we have shown that tyrosine kinase inhibitors specific for the PDGF-R or EGF-R reduce the proliferative response of lung myofibroblasts in vitro and reduce cell proliferation and collagen deposition during pulmonary fibrogenesis in vivo after injury with V2O5. These findings provide additional evidence that these growth factor receptors play a pivotal role in the proliferation of collagen-producing myofibroblasts in the lung. Additional studies are necessary to define the relative importance of the specific EGF and PDGF ligands to the disease process.
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Acknowledgements |
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Footnotes |
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Accepted for publication March 23, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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in rat myofibroblasts during pulmonary fibrogenesis in vivo. Am J Physiol 1998, 274:L72-L80 levels in bronchoalveolar lavage fluid of patients with acute respiratory distress syndrome. Am J Respir Crit Care Med 1998, 158:424-430 in the bronchiolar-alveolar duct regions of asbestos exposed rats. Am J Pathol 1996, 149:205-217[Abstract]
and epidermal growth factor receptor is increased following bleomycin-induced lung injury in rats. Am J Respir Cell Mol Biol 1994, 1:540-541
in activated human alveolar macrophages. Cell 1988, 53:285-293[Medline]
-receptors for platelet-derived growth factor (PDGF-AA) on rat lung fibroblasts. J Clin Invest 1993, 92:425-430
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W. D. Hardie, T. D. Le Cras, K. Jiang, J. W. Tichelaar, M. Azhar, and T. R. Korfhagen Conditional expression of transforming growth factor-{alpha} in adult mouse lung causes pulmonary fibrosis Am J Physiol Lung Cell Mol Physiol, April 1, 2004; 286(4): L741 - L749. [Abstract] [Full Text] [PDF]
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H. Suzuki, K. Aoshiba, N. Yokohori, and A. Nagai Epidermal Growth Factor Receptor Tyrosine Kinase Inhibition Augments a Murine Model of Pulmonary Fibrosis Cancer Res., August 15, 2003; 63(16): 5054 - 5059. [Abstract] [Full Text] [PDF]
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