Department of Molecular Biosciences, School of Veterinary Medicine, and Department of Medical Pharmacology and Toxicology, School of Medicine, University of California, Davis, California 95616
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ABSTRACT |
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Pirfenidone (PD) is known for its antifibrotic effects in the bleomycin (BL) hamster model of lung fibrosis. We evaluated whether pretreatment of hamsters with PD could influence the effects of BL-induced overexpression of platelet-derived growth factor (PDGF)-A and PDGF-B genes and proteins in the same model of lung fibrosis. We demonstrate elevated levels of PDGF-A and PDGF-B mRNAs in bronchoalveolar lavage (BAL) cells from lungs of BL-treated compared with saline control hamsters by RT-PCR analysis. However, these levels were not altered in BAL cells obtained from BL-treated hamsters on diets containing 0.5% PD. Western blot analysis of BAL fluid for PDGF isoforms demonstrated that PD treatment inhibited the synthesis of both PDGF-A and PDGF-B isoforms. PD treatment also decreased the mitogenic activity in the BAL fluid from BL-treated hamster lungs. Taken together, these data provide evidence that the protective effects of PD against BL-induced lung fibrosis may be mediated by a reduction in PDGF isoforms produced by lung macrophages.
platelet-derived growth factor; gene regulation
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INTRODUCTION |
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STIMULATION of a set of trophic signals for mesenchymal
cells in the lung interstitium has been identified during
fibroproliferative lung disorders such as chronic idiopathic pulmonary
fibrosis (IPF) and acute lung injury (1, 27, 29, 30). These signals are
known to include platelet-derived growth factors (PDGFs). Active PDGF
protein is composed of a disulfide-linked homodimer or heterodimer of
two peptides that exist in three isoforms (AA, AB, or BB). Both A and B
chains share homology of ~60% at the amino acid level. PDGF
receptors are present on most mesenchymal cells, including those found
in the interstitium of the lung (12). The functional PDGF receptor
exists as a dimer of two subunits ( and
) differing in their
binding specificity for the three isoforms of PDGF. PDGF is a potent
mitogen and chemoattractant for mesenchymal cells and induces gene
expression of cell matrix-related molecules such as fibronectin,
collagen, and glycosaminoglycans (8).
Pulmonary fibroblasts exhibit increased chemotaxis, proliferation, and
extracellular matrix production during the progression of pulmonary
fibrosis caused by bleomycin (BL) (27), asbestos fibers (19), and
silica particles (7). Alveolar macrophages activated by BL instillation
secrete several cytokines including interleukin (IL)-1, IL-1
,
PDGF, transforming growth factor (TGF)-
, TGF-
, basic fibroblast
growth factor (bFGF), and tumor necrosis factor (TNF)-
(2-5, 7,
8, 17, 20, 25, 27, 33); all are known to be involved in increased
fibroproliferative responses.
There is evidence of increased production of PDGF-B, the predominant
form, by macrophages in lung fibrosis in humans. Human alveolar
macrophages appear to have an increased rate of transcription of the
PDGF-B gene (25) and increased PDGF-B mRNA levels (1, 28), and they
exhibit an exaggerated production of PDGF-B protein (24). Further
evidence for the importance of PDGF-B in the pathogenesis of pulmonary
fibrosis is provided by the demonstration of PDGF-B mRNA in alveolar
macrophages by in situ hybridization (30) and in the
bronchoalveolar lavage (BAL) fluid (BALF) obtained from rats treated
with BL to induce pulmonary fibrosis (31). Both PDGF-A and PDGF-B
isoforms have been identified in BALF in the BL-treated rat model of
lung injury. These findings suggest that abnormal expressions of PDGF
play an important role in acute lung injury and chronic IPF. The role
of PDGF in lung fibrosis is further supported by our recent findings
(11) of an overexpression of PDGF-A mRNA in the BL-treated mouse model
and its downregulation by interferon- at the transcriptional level,
which ameliorated the lung fibrosis.
A number of drugs have been used to prevent and/or treat chronic pulmonary fibrosis, although none of these drugs has proven to be efficacious for long-term therapy. Pirfenidone (PD), a newly developed drug (23) currently undergoing clinical trial, has been reported to be effective in both preventing and treating the BL-induced lung fibrosis in hamsters (14, 15). The present study was designed to gain insight into the molecular mechanism by which PD exerts its antifibrotic effect. In this regard, we studied the effect of PD treatment in the BL-treated hamster model of lung fibrosis on PDGF, which is known to be involved in both acute lung injury and IPF.
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MATERIALS AND METHODS |
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Treatment of animals. Male golden
Syrian hamsters weighing 90-110 g were purchased
from Simonsen (Gilroy, CA). Hamsters were housed in
groups of four in facilities provided with filtered air and constant
temperature and humidity. All animal maintenance was in accordance with
National Institutes of Health guidelines for animal welfare. The
hamsters were allowed to acclimate to the environment for 1 wk before
all treatment. A 12:12-h light-dark cycle was maintained, and the
animals had ad libitum access to water and pulverized
rodent laboratory chow 5001 (Purina Mills, St. Louis, MO) with or
without (control) 0.5% (wt/wt) PD. Animals were randomly
divided into four experimental groups:
1) saline-instilled animals fed a
control diet (SA+CD), 2)
saline-instilled animals fed PD in the same diet (SA+PD),
3) BL-instilled animals fed the CD
(BL+CD), and 4) BL-instilled animals
fed PD in the same diet (BL+PD). The animals were fed these diets
starting 3 days before the intratracheal instillation, and the diets
were continued throughout the course of the experiment. Under
pentobarbital sodium anesthesia, hamsters were intratracheally
instilled with saline or BL (5.5 units · 4 ml1 · kg
1)
as described previously by our laboratory (15).
Collection of BALF. Five animals from
each group were killed by injection of 120 mg/kg of
pentobarbital sodium followed by exsanguination at 1, 3, 5, 7, 14, and
21 days after intratracheal instillation. Immediately thereafter, lungs
were lavaged with isotonic saline in
situ according to the method of Giri et
al. (9). BALF was collected and centrifuged at 4°C for 10 min at 1,500 rpm. The supernatant was aspirated for measurement of released PDGF-A and PDGF-B, and the total RNA was extracted from the sedimented cells and stored at 80°C to measure PDGF-A and PDGF-B mRNAs.
RNA extraction and RT-PCR of BALF
cells. BAL cells obtained from lung lavages were washed
in ice-cold saline. A previous study (20) demonstrated that the cell
population in the BALF constituted 95% macrophages. The total cellular
RNA was extracted from these cells by the RNeasy total RNA extraction
protocol (Qiagen, Chatsworth, CA). For the synthesis of
cDNA, 1 µg of RNA measured spectrophotometrically from each sample
was mixed with 1 µl of
oligo(dT)18 primer and heated at
70°C for 2 min. The samples were quenched on ice, and the following
components were added to a final volume of 20 µl: 50 mM
Tris · HCl (pH 8.3), 75 mM KCl, 3 mM
MgCl2, 0.5 µM
3'-deoxynucleotide 5'-triphosphates, 1 U/µl of RNase
inhibitor, and 200 U/µg RNA of Moloney murine leukemia virus RT,
and the reaction mixture was incubated at 42°C for 1 h. The
reaction was terminated by denaturating the enzyme at 94°C for 5 min, and the mixture was diluted with RNase-free water to a volume of
100 µl.
PCR amplification was performed with commercially available PCR primers (Clontech Laboratories, Palo Alto, CA). The primers used for PCR amplification were human, mouse, and rat glyceraldehyde-3-phosphate dehydrogenase (GAPDH): sense, 5'-ACC ACA GTC CAT GCC ATC AC-3', and antisense, 5'-TCC ACC ACC CTG TTG CTG TA-3'; mouse PDGF-A: sense, 5'-GCC CCT GCC CAT TCG GAG GAA GA-3', and antisense, 5'-GGC CAC CTT GAC GCT GCG GTG G-3'; and human PDGF-B: sense, 5'-CTG TCC AGG TGA GAA AGA TCG AGA TTG TGC GG-3', and antisense, 5'-GCC GTC TTG TCA TGC GTG TGC TTG AAT TTC CG-3'. Five-microliter aliquots of the synthesized cDNA were added to 45 µl of PCR mix containing 5 µl of 10× PCR buffer, 1 µl of deoxynucleotides (1 mM each), 1 µl of sense and anti-sense primers (0.15 µM), and 0.25 µl of AmpliTaq DNA polymerase (GeneAmp PCR kit, Perkin-Elmer). The reaction mixture was covered with 50 µl of mineral oil (Perkin-Elmer). Amplification was initiated by denaturation at 94°C for 1 cycle for 5 min followed by 40 cycles at 94°C for 45 s, 55°C for 45 s, and 72°C for 2 min with a GeneAmp PCR 480 DNA thermal cycler (Perkin-Elmer). After the last cycle of amplification, the samples were incubated for 7 min at 72°C. The PCR products were visualized by ultraviolet illumination after electrophoresis through 2.0% agarose (UltraPure, GIBCO BRL) in 1× Tris-acetate-EDTA buffer and stained with ethidium bromide (0.5 µg/ml). The PCR-amplified products of PDGF-A and PDGF-B were extracted by the Qiaquick gel-extraction kit. The isolated cDNA fragments were subcloned and used for Northern blot analysis and DNA sequencing.
PCR products obtained with PDGF-A- and PDGF-B-specific primers were cloned into PCR-Script vector (Stratagene, La Jolla, CA). Sequencing was performed on two independently isolated RT-PCR clones with an Applied Biosystems model 373A sequencer. The Gene Assist Computer program was used to analyze the DNA sequence, and the data were sequentially aligned with the data in GenBank.
Total RNA isolation and hybridization analyses of
lung. Animals were killed at different days after BL or
saline instillation by decapitation, and their lungs were removed,
quickly freeze clamped, dropped in liquid
N2, and then stored at
80°C until they were used for mRNA analysis. The single-step
method of RNA isolation with acid guanidinium
thiocyanate-phenol-chloroform extraction was used to isolate cellular
RNA from hamster whole lung samples (6). Northern blot experiments were
performed to determine the level of PDGF-A and PDGF-B mRNAs in
BL-treated hamster lungs. Briefly, total RNA (10 µg/lane) was
electrophoresed through 1% agarose-2.2 M formaldehyde gels and
transferred to a nylon membrane. The samples were prehybridized at
42°C for 2 h in a solution containing 50% formamide, 5×
saline-sodium phosphate-EDTA, 0.3% SDS, and 200 µg/ml
of sheared salmon sperm DNA. The membrane was hybridized with PCR
products of either PDGF-A or PDGF-B cDNA as a probe (2 × 106
counts · min
1 · ml
hybridization solution
1)
at 42°C for 20 h. Radiolabeled probes were prepared by the
random-primer method (Bio-Rad, Richmond, CA). RNA hybridization and
washings were done as described elsewhere (10). Band intensities were quantified by densitometric scanning with a dual-wavelength flying-spot scanning densitometer (model CS-9301PC, Shimadzu, Columbia, MD).
Analysis of BALF for mitogenic activity. The BALF was concentrated with a Centri/Por centrifuge concentrator (10-kDa cutoff). The growth factor activity of lavage fluid was determined by a lung fibroblast proliferation assay (31). NIH/3T3 cells served as the standard mesenchymal target cell line. Fibroblasts were seeded in 96-well plates at 2 × 104 cells/well in a medium of DMEM containing 0.1% fetal calf serum and incubated for 24 h at 37°C in an atmosphere of 5% CO2-95% air. Baseline cell counts were made on eight wells from each plate. After the addition of the test substance, the cells were cultured for 48 h and then counted.
To count fibroblasts, we decanted the medium from each plate, and 200 µl of a solution of 0.1 M citric acid-0.1% crystal violet (wt/vol) were added to each well for 15 min. A colorimetric assay was used to quantify increases in cell numbers (31). Cells were fixed with 10% Formalin for 30 min and stained with 1% methylene blue in borate buffer for 30 min. Excess stain was removed by repetitive rinses with borate buffer. Cell-associated dye was then eluted in 0.3 ml of ethanol-HCl (1:1). Optical density was read at a wavelength of 658 nm on a Bio Kinetics Reader. Cell number was related to optical absorbance, with a linear standard curve for each cell line.
The proliferative effect of BALF on fibroblasts was blocked by preincubating the fluid samples with either anti-PDGF-A or anti-PDGF-B antibodies for 2 h at 37°C. The neutralizing activity of these antibodies was confirmed by comparing them with a positive control. Antibody (10 µg/ml) was found to completely block half-maximal concentrations (1-2 ng/ml) of PDGF isoforms. In all studies, nonspecific IgG preparations served as negative controls.
Western blot analysis of BALF samples. Initially, the sample of BALF from each animal was concentrated with a Centri/Por centrifuge concentrator (10-kDa cutoff), and the frozen sample was further lyophilized to dryness and reconstituted with 200 µl of water. Each sample (10 µl containing 5 µg of protein) was subjected to SDS-PAGE on 4-20% Tris-glycine minigels (Bio-Rad, Hercules, CA). The gel was electroblotted onto a polyvinylidene difluoride membrane, and the membrane was incubated for 4 h at room temperature with a blocking solution containing Tris-buffered saline (100 mM Tris, 0.9% NaCl, pH 7.5, and 0.1% Tween 20) and 5% nonfat dry milk. The membrane was further incubated with 5 µg/ml of either rabbit anti-PDGF-A or rabbit anti-PDGF-B primary antibodies (Santa Cruz Biotechnology, Santa Cruz, CA) for 18 h. After being washed, the membranes were incubated with a 1:5,000 dilution of horseradish peroxidase sheep anti-human IgG as a secondary antibody in Tris-buffered saline-Tween 20 for 1 h at room temperature. Then, after being washed, the membranes were exposed to the enhanced chemiluminescence developer and X-ray film (Amersham, Arlington Heights, IL). Normal rabbit serum (rabbit IgG) was used in place of the anti-PDGF antibody as a control for nonspecific reactivity.
Statistical analysis. Data are
expressed as means ± SD. Significant differences among SA+CD,
SA+PD, BL+CD, and BL+PD groups at the corresponding times were analyzed
by two-way ANOVA, and a value of P 0.05 was significant.
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RESULTS |
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Expression of PDGF-A and PDGF-B mRNAs in BL-treated
hamster lungs. PDGF-A and PDGF-B cDNAs were amplified
as fragments of 224 and 253 bp, respectively, from the total RNA of
hamster lungs with the use of specific primers (Fig.
1). To validate the PCR-amplified cDNA
sequence of hamster lung PDGF-A and PDGF-B, these fragments were cloned
into a PCR-Script vector and sequenced. A comparative sequence analysis of cloned PDGF-A and PDGF-B with their counterparts from human and mouse revealed that hamster PDGF-A and PDGF-B cDNAs shared substantial homology (>95% at the amino acid level) to PDGF-A
and PDGF-B of human and mouse (Fig. 2). Further
validation was carried out by Northern blot experiments to study the
expression of PDGF-A and PDGF-B mRNAs in hamster lung tissue (Fig.
3). The PDGF-A cDNA fragment, when used as a probe
against the total RNA isolated from hamster lungs, showed hybridization
to 2.9-, 2.3-, and 1.7-kb transcripts similar to mouse PDGF-A, the
2.9-kb band being the major transcript. The cloned PDGF-B cDNA fragment
hybridized to the 3.5-kb transcript (Fig. 3).
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Kinetics of PDGF-A and PDGF-B mRNA expression in
BL-treated hamster lung BAL cells with and without PD
treatment. Experiments were performed to determine the
effect of PD on BL-stimulated expression of PDGF-A and PDGF-B mRNA
during BL-induced pulmonary fibrosis. The effect of PD on PDGF-A and
PDGF-B mRNA accumulation was investigated by RT-PCR on total cellular
RNA isolated from BAL cells after saline or BL instillation.
Steady-state mRNA levels of PDGF-A and PDGF-B from BAL cells after
saline or BL instillation with and without PD treatment are shown in
Fig. 4. The levels of steady-state mRNA expression for
PDGF-A and PDGF-B were different in BAL cells obtained from the BL+CD
group. However, there was no significant difference in
the level of GAPDH mRNA among all four groups analyzed
(Fig. 4A). As shown in Fig.
4B, the instillation of BL caused
dramatic increases in PDGF-A mRNA levels at all time points compared
with those of saline control (SA+CD and SA+PD) groups. The BAL cells
analyzed from the BL+CD group between 1 and 14 days revealed a marked
increase in PDGF-A mRNA expression. However, although PDGF-B mRNA
expression was elevated in the BL+CD group, it was not increased to the
same extent as PDGF-A over the same time period. There were no changes
in the levels of both PDGF-A and PDGF-B mRNAs between the BL+CD and
BL+PD groups, indicating that PD did not exhibit any inhibitory effect
on the BL-induced overexpression of these mRNAs because the intensities
of PCR-amplified products of PDGF-A and PDGF-B remained relatively
unaffected in the BL+PD group (Fig. 4,
B and
C).
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Analysis of PDGF isoforms in BALF.
Western blot analysis was used to determine whether steady-state levels
of mRNA correlated with amounts of immunoreactive proteins.
Immunoblotting was carried out with either anti-PDGF-A or anti-PDGF-B
antibodies. We detected two distinct PDGF isoforms in BALF processed
from BL-treated animals. However, neither PDGF-A nor PDGF-B was
detectable in BALF of saline control groups (SA+CD and SA+PD). On the
other hand, we found significantly higher amounts of PDGF-A and PDGF-B
in BL-treated groups compared with saline control groups. Figure
5 shows the Western blots of
PDGF-A and PDGF-B in BALF at 7 days after saline or BL instillation.
This is the time point at which the increases in the PDGF-A and PDGF-B
mRNAs in BAL cells were maximal in the BL+CD group.
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The Western blots shown are representative of the BALF from one hamster from the SA+CD, SA+PD, BL+CD, and BL+PD groups. BL treatment in the BL+CD group increased the synthesis and secretion of PDGF-A (Fig. 5A). The peptide was a 28-kDa molecule that was detected with anti-human PDGF-A. We also detected another peptide of 29-32 kDa on immunoblots with anti-human PDGF-B from the BALF of animals in the BL+CD group (Fig. 5B). No signal from the 28-kDa or 29- to 32-kDa bands was observed when normal rabbit serum was used in place of the anti-PDGF antibodies, indicating the absence of nonspecific reactivity by these antibodies (Fig. 5C). Densitometry measurements indicated a severalfold increase in PDGF-A and PDGF-B in BALF from the BL+CD group compared with BALF from SA+CD and SA+PD groups. However, treatment with PD completely abolished the BL-induced production of the 28-kDa molecule, i.e. PDGF-A (Fig. 5A), and the 29- to 32-kDa PDGF-B molecule in the BL+PD group (Fig. 5B).
Effect of PD on the mitogenicity of BALF PDGF isoforms
after BL-induced lung injury. Mitogenic activity of
PDGF was demonstrated by testing BALF for its ability to induce
proliferation of confluent cultures of NIH/3T3 fibroblasts. These cells
respond to PDGFs in a concentration-dependent manner over a range of
~1-3 ng/ml (Fig. 6). BALF harvested from
BL-treated animals exhibited a marked increase in fibroblast
proliferation compared with other groups (Fig.
7). The fibroproliferative effect of BALF
obtained from BL-treated animals showed a profound increase in
proliferation from day 1 to
day 7 that declined at 14 and 21 days
after BL instillation. This also parallels the expression of PDGF-A and
PDGF-B mRNAs during the development of BL-induced lung fibrosis (Fig.
4). BALF obtained from animals in the BL+PD group had a significantly
reduced mitogenic activity on NIH/3T3 fibroblast cell proliferation at all times compared with the BL+CD group (Fig. 7).
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To validate the bioassays, we blocked the mitogen biological activity
present in the BALF in vitro with specific antibodies. We evaluated the
degree to which PDGF antibodies block the mitogenic activity of BALF
for fibroblast proliferation. Because we found an elevated level of
mitogenic activity from day 1 to
day 7, we selected these time points
to measure the specificity of each isoform. We tested the volume of
lavage fluid yielding half-maximal stimulation of
NIH/3T3 fibroblast growth to make antibody inhibition studies quantitative. Maximal inhibition with anti-PDGF-A antibodies was 64 ± 7% in the BALF at 5 days, whereas the inhibition with anti-PDGF-B antibodies was 56 ± 11% of the mitogenic activity at 3 days after intratracheal instillation of BL (Table
1).
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Quantification of mitogenic activity in BALF was determined by bioassay
and compared with the activity of known concentrations of PDGF-A and
PDGF-B. Elevated levels of mitogenic activity were observed from
day 1 to day
7 after BL treatment, reaching a peak level at
day 5 equivalent to 3.4 ± 0.45 and
4.3 ± 0.46 ng/ml of PDGF-A and PDGF-B standard, respectively.
However, after BL instillation at 14 and 21 days, the activity dropped
to the level found in BALF from the control group of hamsters (Figs.
8 and 9). The results also indicate
that PD significantly suppressed the mitogenic potency present in BALF
of BL-treated hamsters in BL+PD group (Figs. 8 and 9).
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DISCUSSION |
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PD is an investigational antifibrotic drug that when fed in the diet
(0.5% wt/wt) offers protection against the development of lung
fibrosis in the BL-treated hamster model (14, 15). PD has also been
reported to be effective against cyclophosphamide-induced lung fibrosis
in mice (16). It is known that PD modifies the regulatory actions of
some fibrogenic cytokines, including PDGF, and inhibits the
proliferation of fibroblasts and synthesis of extracellular matrix
proteins in vitro (25). We also found that PD treatment
reduces the BL-induced increase in lung TGF- in vivo
(13). However, the exact mechanism for the antifibrotic effect of PD is not yet clearly understood. The present study was
designed to determine whether the antifibrotic effect of PD involves
the regulation of PDGF transcription and/or translation. Alveolar macrophages activated by BL instillation secrete the following
cytokines that mediate enhanced fibroblast proliferative responses in
the lung: IL-1
, IL-1
, PDGF, TGF-
, TGF-
, bFGF, and TNF-
.
The majority of macrophage-derived cytokine activity is due to PDGF-B
chain homologues (5). Additionally, several other macrophage-derived
cytokines (IL-1
, IL-1
, TGF-
, TNF-
, and bFGF) stimulate
fibroblast proliferation via an autocrine loop by causing the release
of PDGF-A, which then binds to the PDGF-
-receptor subtype on
fibroblasts (2, 3, 26, 32) and initiates signal transduction events.
Because an increased number of activated macrophages are associated with BL-induced inflammatory and fibrotic responses in the lung, we asked whether PD treatment that decreased hydroxyproline content in the BL-treated hamster model of lung fibrosis has the potential to downregulate PDGF isoforms. In view of the fact that macrophages are a principal source of several mediators that may be involved in inflammation and also in the synthesis and accumulation of extracellular matrix of fibrotic lungs (24, 25, 30, 31), we investigated the expression of PDGF-A and PDGF-B genes in BAL cells at various stages during the course of the development of pulmonary fibrosis in hamsters. Because the percentage of macrophages among BAL cells was always >95% in the BALF of BL-instilled hamster lungs except on day 1 after BL treatment in the present study, we did not purify them from the total BAL cell samples (21).
The involvement of PDGF in the pathogenesis of pulmonary fibrosis in
humans has been demonstrated by in situ hybridization analysis. PDGF-B
mRNA expression has been observed in macrophages and epithelial cells
in IPF, whereas no or a weak signal of PDGF-A has been detected (1,
30). Our data also provide convincing evidence for PDGF gene activation
in the hamster model of BL-induced lung fibrosis. In many cases, cells
expressing PDGF were closely associated with the subsequent expression
of procollagen-1 and TGF-
1
mRNA during the fibrotic process (22).
In the present study, elevated levels of PDGF-A and PDGF-B mRNAs were observed in BAL cells obtained from the BL+CD group of hamsters compared with the hamsters of the SA+CD group. In addition, no changes in the relative amounts of both PDGF-A and PDGF-B mRNAs were observed in BAL cells among animals from the BL+CD and BL+PD groups. These findings suggest that the fibroblast proliferation activity in BL-induced lung fibrosis may be partly due to increased release of PDGF-A and PDGF-B from macrophages. The kinetic studies of PDGF-A and PDGF-B mRNA expression in the BAL cells revealed that they were at their peaks at 7 days and declined thereafter. The relative abundance of the "housekeeping" gene (GAPDH) mRNA in BAL cells was similar in both SA- and BL-instilled groups. Overexpression of PDGF mRNAs in both BL+CD and BL+PD groups suggests that these increases could be due to an increase in the stability of PDGF mRNA and/or an increase in PDGF gene transcription.
Fibrosis is the result of proliferation of fibroblasts that synthesize collagen at the inflammatory sites. To test the effects of PD on fibrosis, we evaluated its effects on cell proliferation and collagen synthesis. We found increases in the net mitogenic activity of the BALF on NIH/3T3 fibroblasts at days 1 through 7 after BL instillation. Mitogenic activity in the BALF of BL-treated rats has been demonstrated previously, and the degree of this mitogenic activity for fibroblasts was markedly elevated above the control values (18). Significantly, the BALF from hamsters in BL+PD group had reduced levels of mitogenic activity compared with that in the BL+CD group. This reduction in the mitogenic activity in the BL+PD group may be due to the inhibitory effect of PD on the production of PDGF-A and PDGF-B as well as other growth factors that are known to stimulate fibroblast proliferation.
We detected PDGF proteins in the BALF of hamsters treated with a fibrogenic dose of BL by Western blot analysis and neutralization experiments with anti-PDGF antibodies against PDGF-A and PDGF-B isoforms. However, very low levels of PDGF-A and PDGF-B were detected in the BL+PD group. Therefore, it is likely that PD treatment in the BL+PD group suppresses the translation of PDGF mRNA even after an elevated and sustained level of mRNA expression. The data suggest that if PDGF-A and PDGF-B isoforms are important in BL-induced lung fibrosis, PD is likely to be beneficial in attenuating the lung fibrosis.
The results of the present study demonstrate that PDGF-A and PDGF-B mRNA synthesis and steady-state levels of PDGF-A and PDGF-B mRNAs and PDGF isoforms are elevated in BL-treated hamster lungs. It is obvious from the Western analysis that PD has the potential to suppress the PDGF isoforms that are significantly elevated in BL-treated animals. These findings suggest the involvement of a posttranscriptional or translational mechanism for decreased levels of PDGF-A and PDGF-B proteins in the BL+PD group. On the basis of these results, we conclude that the overexpression of PDGF-A and PDGF-B is closely linked with the development of BL-induced lung fibrosis and that treatment with PD can effectively diminish the BL-induced lung fibrosis by downregulating the expression of PDGF-A as well as of PDGF-B proteins, most probably at the translational level.
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ACKNOWLEDGEMENTS |
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This investigation was supported by National Heart, Lung, and Blood Institute Grant R01-HL-56262-02.
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests: S. N. Giri, Dept. of Molecular Biosciences, School of Veterinary Medicine, Univ. of California, Davis, CA 95616.
Received 20 July 1998; accepted in final form 26 October 1998.
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