Pirfenidone inhibits PDGF isoforms in bleomycin hamster model of lung fibrosis at the translational level

G. Gurujeyalakshmi, M. A. Hollinger, and S. N. Giri

Department of Molecular Biosciences, School of Veterinary Medicine, and Department of Medical Pharmacology and Toxicology, School of Medicine, University of California, Davis, California 95616


    ABSTRACT
Top
Abstract
Introduction
Materials and methods
Results
Discussion
References

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


    INTRODUCTION
Top
Abstract
Introduction
Materials and methods
Results
Discussion
References

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 (alpha  and beta ) 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)-1alpha , IL-1beta , PDGF, transforming growth factor (TGF)-alpha , TGF-beta , basic fibroblast growth factor (bFGF), and tumor necrosis factor (TNF)-alpha (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-gamma 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.


    MATERIALS AND METHODS
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Abstract
Introduction
Materials and methods
Results
Discussion
References

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 ml-1 · 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.


    RESULTS
Top
Abstract
Introduction
Materials and methods
Results
Discussion
References

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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Fig. 1.   Expression of platelet-derived growth factor (PDGF)-A and PDGF-B mRNAs in hamster lungs. RT-PCR of total RNA isolated from bleomycin (BL)-treated lungs was performed with specific primers. Amplified samples were run on 2% agarose gel. Gel was stained with ethidium bromide and photographed. Lane 1, size marker (phi X174 RF DNA/Hae III digest); lanes 2-4, hamster glyceraldehyde-3-phosphate dehydrogenase (GAPDH); lane 5, GAPDH positive control; lanes 6-8, hamster PDGF-A; lane 9, PDGF-A positive control; lanes 10-13, hamster PDGF-B; lane 14, PDGF-B positive control.


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Fig. 2.   Alignment of cDNA and deduced amino acid sequences of lung PDGF-A and PDGF-B isoforms. A: alignment showing identity of hamster PDGF-A to mouse PDGF-A. B: alignment showing identity of hamster PDGF-B to human PDGF-B.


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Fig. 3.   Northern blot analysis of PDGF-A and PDGF-B genes. Total RNA was extracted from BL-treated hamster lungs, fractionated on formaldehyde-agarose gels (10 µg/lane), and transferred to nylon membrane. Membrane was hybridized to radiolabeled cDNA (PCR product obtained with PDGF-A-specific primer) for PDGF-A or to radiolabeled cDNA (PCR product obtained with PDGF-B-specific primer) for PDGF-B chain. Nos. at right, molecular mass.

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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Fig. 4.   RT-PCR analysis of relative increases in PDGF-A and PDGF-B mRNA levels in BAL cells from hamster lungs at 1, 3, 5, 7, and 14 days after intratracheal instillation of saline or BL. Total RNA (1 µg) from BAL cells was reverse transcribed with Moloney murine leukemia virus RT. cDNA (5 µl) was amplified for 40 cycles in presence of GAPDH (A)-, mouse PDGF-A (B)-, or human PDGF-B (C)-specific primers. Products of RT-PCR (5 µl out of 50 µl) were separated by 2% agarose gel electrophoresis. Lane 1, size marker (phi X174 RF DNA/Hae III digest); lanes 2-5, saline instilled with control diet (SA+CD); lanes 6-9, SA + the same diet with pirfenidone (PD); lanes 10-14, BL+CD; lanes 15-19, BL+PD; lane 20, positive control (B) or size marker (A and C).

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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Fig. 5.   Western blot analysis of PDGF isoforms from bronchoalveolar lavage fluid (BALF) of SA- and BL-treated hamster lungs with and without PD treatment. Human recombinant PDGF-A, PDGF-B, and BALF samples were electrophoresed on 4-20% Tris-glycine gels and electroblotted onto polyvinylidene difluoride membrane. Western blotting was performed as described in MATERIALS AND METHODS. Samples were immunoblotted with anti-PDGF-AA (5 µg/ml; A) or anti-PDGF-BB (5 µg/ml; B) or without primary antibody (either PDGF-AA or PDGF-BB; C) as a control that shows minimal staining, ensuring specificity of antibody. Lane 1, molecular-mass marker; lane 2, BALF samples of SA+CD; lane 3, SA+PD; lane 4, BL+CD; lane 5, BL+PD; lane 6, PDGF-A (10 ng); lane 7, PDGF-B (10 ng). Data represent 1 of 3 independent experiments.

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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Fig. 6.   Mitogenic response of target [NIH/3T3 (3T3)] cells to varying concentrations of recombinant PDGF-AA and PDGF-BB. Data are means ± SD of 3 determinations. See MATERIALS AND METHODS for details.


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Fig. 7.   Mitogenic response of NIH/3T3 cells from BALF from SA+CD and BL+CD and BL+PD hamsters. IT, intratracheal. Data are means ± SD; n = 5 hamsters. * Significantly higher than all other groups at corresponding times, P < 0.05. + Significantly lower than BL+CD group at corresponding times, P < 0.05.

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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Table 1.   Neutralization of mitogenic activity in BALF of BL-treated hamster lungs

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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Fig. 8.   Level of mitogenic activity in BALF was determined by bioassay and compared with activity of PDGF-AA. Data are calculated from linear part of concentration-response curves such as those shown in Fig. 6. Data are means ± SD; n = 5 hamsters. * Significantly higher than BL+PD group at corresponding times, P < 0.05.


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Fig. 9.   Level of mitogenic activity in BALF was determined by bioassay and compared with activity of PDGF-BB. Data were calculated from linear part of concentration-response curves such as those shown in Fig. 6. Data are means ± SD; n = 5 hamsters. * Significantly higher than BL+PD group at corresponding times, P < 0.05.


    DISCUSSION
Top
Abstract
Introduction
Materials and methods
Results
Discussion
References

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-beta 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-1alpha , IL-1beta , PDGF, TGF-alpha , TGF-beta , bFGF, and TNF-alpha . The majority of macrophage-derived cytokine activity is due to PDGF-B chain homologues (5). Additionally, several other macrophage-derived cytokines (IL-1alpha , IL-1beta , TGF-beta , TNF-alpha , and bFGF) stimulate fibroblast proliferation via an autocrine loop by causing the release of PDGF-A, which then binds to the PDGF-alpha -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-alpha 1 and TGF-beta 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.


    ACKNOWLEDGEMENTS

This investigation was supported by National Heart, Lung, and Blood Institute Grant R01-HL-56262-02.


    FOOTNOTES

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.


    REFERENCES
Top
Abstract
Introduction
Materials and methods
Results
Discussion
References

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