pneumocyte receptors for bFGF (FGF-R). Increased rat lung. bFGF mRNA, relative to air-exposed control animals, was observed at 4 days of exposure, with no ...
Basic fibroblast growth factor and growth factor receptor gene expression in 85% Oz-exposed rat lung SHILPA BUCH, ROBIN N. N. HAN, JASON LIU, AIDEEN MOORE, JEFFREY D. EDELSON, BRUCE A. FREEMAN, MARTIN POST, AND A. KEITH TANSWELL Medical Research Council Group in Lung Development, Hospital for Sick Children Research Institute and University of Toronto, Toronto M5G 1X8; Department of Medicine, St. Michael’s Hospital and University of Toronto, Toronto, Ontario M5B 1 W8, Canada; and Departments of Anesthesiology, Biochemistry and Pediatrics, University of Alabama at Birmingham, Birmingham, Alabama 35294 Buch, Shilpa, Robin N. N. Han, Jason Liu, Aideen Moore, Jeffrey D. Edelson, Bruce A. Freeman, Martin Post, and A. Keith Tanswell. Basic fibroblast growth factor and growth factor receptor gene expression in 85% 02-exposed rat lung. Am. J. Physiol. 268 (Lung CelZ. Mol. Physiol. 12): L455-L464, 1995.-Lungs exposed to elevated 02 concentrations suffer an initial loss of type I pneumocytes, followed by a reparative type II pneumocyte hyperplasia. We hypothesized that type II pneumocyte hyperplasia after exposure of young adult rats to 85% O2 in vivo would be temporally related to 1) an increased concentration of intrapulmonary basic fibroblast growth factor (bFGF), a potent stimulator of type II pneumocyte DNA synthesis in vitro, and 2) an upregulation of pneumocyte receptors for bFGF (FGF-R). Increased rat lung bFGF mRNA, relative to air-exposed control animals, was observed at 4 days of exposure, with no increase at days 6 and 14 of exposure. Parallel changes were observed with bFGF receptor (fZg) mRNA. Nuclear runoff assays confirmed increased transcription of both bFGF and fig genes in response to 85% 02, whereas increased translation at 6 days of exposure was confirmed by protein immunoanalysis. Immunohistochemistry demonstrated a broad distribution of bFGF throughout the lung, including the alveolar epithelium, which increased after 6 and 14 days of exposure to 85% 02. Our findings are compatible with a role for bFGF in Oz-mediated pneumocyte hyperplasia. fibroblast growth factor receptor; fig; pulmonary ity; type II pneumocyte; pneumocyte hyperplasia
oxygen toxic-
ADVERSE EFFECTS Of pulmonary oxygen toxicity at normobaric pressures were first recognized by J. Lorraine Smith (30) in classic studies with mice. Susceptibility to pulmonary oxygen toxicity has now been recognized in all mammalian species studied, though the degree of susceptibility varies with species (S), age (8), and sex (3). Sustained inhalation of elevated O2 concentrations results in diffuse alveolar damage in which there is an initial necrosis of type I pneumocytes with subsequent or concurrent type II pneumocyte and fibroblast hyperplasia (2). This pattern is observed not only in animal models but also in adult and neonatal humans exposed to prolonged elevated O2 concentrations (2). The adult rat, exposed to 85% O2 for intervals up to 2 wk, develops diffuse alveolar damage that has been well characterized by morphometric techniques (10). In this model, by day 7 of exposure there is a decrease in the number of type I pneumocytes and an increase in the number of type II pneumocytes and fibroblasts, in association with diffuse pulmonary injury (10). We have THE
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previously used this model (13) to describe changes in the pulmonary expression of platelet-derived growth factor (PDGF) and PDGF receptor. PDGF is a known mitogen for mesenchymal cells, and we had hypothesized that it may, at least in part, mediate the fibroblast hyperplasia observed in pulmonary oxygen toxicity. PDGF has not been found to be mitogenic for adult rat type II pneumocytes in vitro (5), suggesting that some other growth factor(s) may mediate the type II pneumocyte hyperplasia that occurs in adult rats exposed to 85% OZ. A likely candidate is one of the fibroblast growth factors (FGF), a family of seven or more structurally related polypeptides that, despite their name, can stimulate proliferation of cells derived from all three embryonic germ layers (1). There are also diverse FGF receptor (FGF-R) forms, resulting from transcription of four different genes that can produce variously spliced transcripts (1). One of the best characterized of the FGFs is basic fibroblast growth factor (bFGF), which we and others (22) have found to increase DNA synthesis in adult type II pneumocytes in vitro. Based on these observations, and the report that bronchoalveolar lavage fluid from patients with acute lung injury is enriched for bFGF (16), we hypothesized a role for bFGF in the pneumocyte hyperplasia observed in pulmonary O2 toxicity. To study the changes that occur in bFGF and FGF-R in the lung after exposure to 85% 02, we have combined studies of gene expression and gene transcription with protein immunoanalysis, immunohistochemistry, and autoradiography. METHODS MateriaZs. Radioisotopes, nylon membranes, and random primed labeling systems were purchased from Amersham Canada (Oakville, Ontario, Canada), and restriction enzymes and dextran sulphate were from Pharmacia (Baie D’Urfe, Quebec, Canada). Bovine serum albumin (BSA) type V, Ficoll 400, polyvinylpyrrolidone, guanidinium thiocyanate, cesium chloride, and salmon sperm DNA were from Sigma (St. Louis, MO). Organic solvents were of high-performance liquid chromatography grade. Bovine bFGF cDNA was a generous gift by Dr. Judith Abraham (California Biotechnology, Mountain View, CA); the mouse FGF-R fig probe was a gift from Dr. Janet Rossant (Mount Sinai Hospital, Toronto, Ontario); and the histone H3 cDNA was a gift from Dr. G. S. Stein (Massachusetts Medical School, Worcester, MA). Gemini vector was from Promega Biotec (Madison, WI). Mouse monoclonal anti-bovine the American
Physiological
Society
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bFGF and rabbit polyclonal anti-human FGF-R were from Upstate Biotechnology (Lake Placid, NY). The rabbit antiserum to FGF-R was raised against the synthetic peptide (DALPSAEDDDEDDSSSEEKEADNTK) corresponding to the deduced cDNA sequence of part of the first immunoglobulin (Ig) domain of the chicken FGF-R1 receptor. Whereas FGF-R1 and FGF-R2 are 73% homologous overall, the first Ig domain has only a 43% homology (11). According to the supplier, this antiserum is specific for fig immunoreactive protein and does not cross-react with FGF-R2 (DALSSGDDEDDNDGSEDFVNDSNQMRAP). However, we must acknowledge that we cannot completely exclude cross-reactivity with the products of other splice variants of the FGF-R gene. Goat anti-mouse peroxidase-conjugated IgG was from Calbiothem (San Diego, CA), and goat anti-rabbit peroxidaseconjugated IgG was from Boehringer Mannheim (Mannheim, Germany). Guinea pig anti-rat surfactant apoprotein A (SP-A) IgG was a generous gift from Dr. J. Whitsett of the University of Cincinnati (Cincinnati, OH). Nitrocellulose membranes were from Schleicher and Schuell (Keene, NH). An avidinbiotin complex staining kit was from Vector Laboratories (Burlingame, CA), and embedding media [ornithine carbamyl transferase (OCT)] compound was from Miles (Elkhart, IN). In vitro studies. Type II pneumocyte isolation, purity, and culture in a defined serum-free medium, as well as the sources of all culture materials, have been described in detail elsewhere (12). Cells were isolated from animals exposed to air or 85% O2 for 72 h. A 72-h exposure period was selected based on preliminary autoradiographic studies to define an early time point with reproducible [3H]thymidine uptake into the nuclei of type II pneumocytes. After 24 h in culture, cells were maintained in defined serum-free medium with or without added transforming growth factor-p1 (TGF-P, 0.1 rig/ml), insulin-like growth factor I (IGF-I, 20 rig/ml), epidermal growth factor (EGF, 50 rig/ml), vitamin D3 (1 nM), or bFGF (20 rig/ml) for 72 h. Uptake of [3H]thymidine into DNA was studied over the last 24 h of the exposure to growth factors
(12). Exposure system. Pathogen-free virgin female SpragueDawley rats of 200-250 g were obtained from Charles River Laboratories (Raleigh, NC). The animals were maintained in a 60 x 48 x 25 cm plastic chamber with a 12:12-h light-dark cycle. Food and water were available ad libitum. Rats (n = 4 for each time point) were exposed to air or 85% O2 for 0, 4, 6, or 14 days for collection of RNA. For some additional studies, the exposure times were 6 or 14 days only. Concentrations of 02 in the exposure chambers were calibrated daily with a Beckman Instruments O2 analyzer (Schiller Park, IL). Gas flow was set to maintain minimal chamber humidity, with a CO2 concentration below 0.5%. For most studies, animals were killed by inhalation of excess chloroform. For immunohistochemical studies, animals were anesthetized with intraperitoneal ketamine (80 mg/kg) and xylazine (20 mg/kg). A tracheal catheter was inserted and sutured in place to facilitate lung inflation. The anterior part of the chest wall was reflected upward. While the heart was beating, a catheter was inserted through the right ventricle into the main pulmonary artery and an incision was made in the left atria1 appendage to allow drainage. The pulmonary circulation was then flushed with phosphate-buffered saline (PBS) containing 1 U/ml heparin during intermittent lung inflations until the lung became white. Isolation of lung RNA. Total lung RNA was isolated as previously described (4). Briefly, the thoracic contents were removed en bloc, and the lungs were dissected away from vessels and large airways to be flash frozen in liquid nitrogen after weighing. Total nuclear and cytoplasmic RNA was
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isolated by lysing the tissue in 4 M guanidinium thiocyanate, followed by centrifugation on a 5.7 M cesium chloride cushion to pellet RNA (6). After extraction with phenol/chloroform (l:l, vol/vol), the RNA was ethanol precipitated and collected by centrifugation. This RNA was lyophilized and dissolved in water. RNA integrity was confirmed after fractionation on a 1.2% (wt/vol) agarose-formaldehyde gel by staining the ribosomal RNA bands with ethidium bromide. Slot-Mot analyses. For slot-blot analyses, aliquots of 0.6-5.0 pg RNA were applied in a final 200 ~1 solution of 6.15 M formaldehyde and 10x SSC (1.5 M NaCl and 0.15 M sodium citrate; pH 7.0). Equal amounts of ribosomal RNA and adult rat lung RNA were loaded onto the slot blot and used as negative and positive controls, respectively. To correct for any minor variations in loading of gels, all results were normalized by taking a ratio of the densitometric values from the autoradiogram and the 28s band of the ethidium bromide-stained gel. The cDNA probes were labeled with deoxycytidine 5’-[a32P] triphosphate by a random-primed labeling system (Amersham, Arlington Heights, IL) with specific activities of 0.52.9 X log counts*mir+* kg DNA-l. Prehybridization (5 h to overnight) and hybridization were performed in 50% (vol/vol) formamide, 750 mM NaCl, 75 mM sodium citrate, 5x Denhardt’s solution [0.4% (wt/vol) each of BSA, Ficoll, and polyvinylpyrrolidone], 10% (wt/vol> dextran sulphate, and 100 pg/ml denatured salmon sperm DNA for 24-48 h at 42°C. The final wash for bFGF and fig probes was 2 x SSC and 0.1% (wt/vol> sodi urn dodecyl sulphate (SDS) at 42°C for 10 min. The blots were exposed for 24-48 h to Kodak XAR-5 film, using DuPont Cronex intensifying screens. The films were quantified by an Ultroscan XL laser densitometer (LKB, Bromma, Sweden). Northern blot analyses. All Northern blot analyses were performed using 15 pg of RNA. The probes used were a gel-purified 1,000 base pair (bp) riboprobe fragment generated from bovine bFGF cDNA, and a random-primed 2-kb EcoR I fragment of mouse FGF-R fig cDNA. The riboprobe was used for studies of bFGF because of limited sensitivity using a cDNA probe for Northern analysis. A DNA template for bovine bFGF cDNA was subcloned into the Gemini vector and sequenced to determine the orientation of the two strands, sense and antisense, with respect to the two phage promoters. The bFGF riboprobe plasmid was linearized with Barn HI, purified, and used as a template for cRNA synthesis incorporating [32P]guanosine 5’-triphosphate (GTP). The full-length antisense transcripts were eluted from 4.5% (wt/vol) polyacrylamide gels, counted, and used as hybridization probes. The Northern blots containing total cellular RNAs from air- and 02-exposed lungs were prehybridized for 15 min in 50% with (vol/vol) for mamide, followed by overnight hybridization the gel-eluted riboprobe (1 x lo6 counts amin-l *ml-l) in the same buffer at 55°C. The blots were washed in 2~ SSC for 5 min at room temperature followed by two washes in PSE buffer [0.25 M sodium phosphate, 2% (wt/vol) SDS, 1 mM EDTA; pH 7.21 at 55°C for 1 h. Subsequent more stringent washes were carried out in PES buffer [0.04 M sodium phosphate, 1% (wt/vol) SDS, 1 mM EDTA; pH 7.21 at 55°C for 30 min. Labeling, prehybridization, and hybridization for the FGF-R probe were as described for the slot-blot analyses. The initial wash of the blots was performed with 5 x SSC with 0.1% (wt/vol) SDS at 42°C for 15 min, followed by 2 x SSC, 0.1% (wt/vol) SDS at 42°C for 10 min. After washes, the blots were exposed for 24-48 h to Kodak XAR-5 film, using DuPont Cronex intensifying screens, and the films were quantified by densitometry.
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1. In vitro 13Hlthymidine uptake into DNA of type Upneumocytes from air- or 85% 02-exposed lungs
Table
Growth
85% 02-exposed
Air-exposed
Factor
Control TGFj3,O.l rig/ml Vitamin Ds, 1 nM IGF-I, 20 rig/ml EGF, 50 ngiml bFGF, 20 ngiml
13.66 r 0.37 16.92 + 1.04 18.30a l.lO* 21.50* 1.10* 25.88*0.78* 91.40 -+ 3.22*
19.76* 2.01* 23.722 l.Ol* 21.66-c 1.21* 25.42t1.88* 31.23t0.44* 80.91 IT 7.78t
Values are means ? SE; n = 4. Results are shown as lo3 counts.min-l.well. TGF-3, transforming growth factor-p, IGF-I, insulin-like growth factor I; EGF, epidermal growth factor; bFGF, basic fibroblast growth factor. *P < 0.05 compared with air-exposed control values. tP i 0.05 compared with 85% Oz-exposed control values.
Nuclear runoff transcription assays. Nuclear runoff transcription reactions were performed with isolated nuclei and [o-32P]UTP, as described by Cochran et al. (9). Briefly, nuclei were incubated with [o.-32P1UTP (800 Ci/mmol) for 30 min at 30°C. Labeled RNA was isolated by lysis in 4 M guanidinium thiocyanate, followed by centrifugation on a 5.7 M cesium chloride cushion (6). Hybridizations were performed with equal numbers of labeled RNA y-counts on replicate filters prepared by slot blotting of 10 kg of linearized plasmid DNA. The filters were hybridized in plastic vials at 65°C for 48 h, then washed. Immunoblot and Westernblot analyses. For bFGF immunoblot analyses, lung tissue was homogenized in lysis buffer [O. 15 M NaCl, 1% (volivol) Triton X-100, 0.01 M tris(hydroxymethylkuninomethane (Tris) buffer; pH 8.01. Homogenization was followed by centrifugation at 10,000 g for 15 min at 4°C. Protein content of the supernatant was determined with the use of a microassay kit (Bio-Rad, Hercules, CA). For FGF-R receptor immunoblot analyses, lung tissue was first homogenized in PBS. Homogenization was followed by centrifugation at 10,000 g for 15 min at 4°C. The supernatant was then centrifuged at 50,000 g for 60 min at 4°C. The membraneenriched pellet was washed once with ice-cold PBS and dissolved in lysis buffer. A sheet of nitrocellulose membrane was soaked in PBS for 30 min and placed in the immunoblot apparatus. Fresh samples, starting with 10 kg protein/well, were applied as a dilution series. Samples were filtered through
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the membrane by gravity flow for 30 min, then vacuum was applied for another 30 min to dry the wells. The membrane was then transferred to 3% (wt/vol) nonfat dry milk in PBS and incubated for 60 min at room temperature. After incubation, the membrane was incubated with mouse anti-bFGF (50 pg/ml) or rabbit anti-FGF-R (1200) for 2 h at room temperature. The membrane was washed three times with PBS and incubated with biotinylated goat antimouse IgG (1:4,000) or biotinylated goat anti-rabbit IgG (12,000) for another 60 min at room temperature. The membrane was then washed again with PBS, followed by an incubation with an avidin-biotin complex (1:3,000) for 1 h. The membrane was washed and developed in 0.0075% (wt/vol) 3,3’-diaminobenzidine tetrahydrochloride in PBS, pH 7.6, containing 0.002% (vol/vol) hydrogen peroxide. The membrane was photographed, and the negative was scanned with a densitometer. The specificity of the antibodies used has been previously validated by immunoprecipitation and Western blot immunoanalysis ( 14). For Western blot analysis, lung tissue was homogenized in chilled extraction buffer (1 M NaCl, 10 mM Tris, 1 mM phenylmethylsulfonyl fluoride, 5 pg/ml pepstatin, and 5 pg/ml leupeptin; pH 7.4). Homogenization was followed by centrifugation at 10,OOOg for 15 min at 4°C. The supernatant was then centrifuged at 100,000 g for 60 min at 4°C. The resultant pellet was discarded, and protein content of supernatant was determined as above. Samples containing equal amounts of proteins were combined with loading buffer 110% (vol/vol) glycerol, 2% (wtivol) SDS, 5% (wt/vol) B-mercaptoethanol, 0.0025% (wtl vol) bromphenol blue, and 0.06 M Tris; pH 8.01, boiled for 5 min, and subjected to 15% SDS-polyacrylamide gel electrophoresis (PAGE) as described by Laemmli (21). Proteins were electrophoretically transferred to nitrocellulose membrane. Nonspecific binding was blocked by incubation with 5% (wt/ vol) BSA in TBS (10 mM Tris, 150 mM NaCl; pH 7.4) at 4°C for 60 min, followed by the addition of monoclonal antibody to bovine bFGF 110 pg/ml in 5% (wt/vol) BSA/TBS]. After an overnight incubation at 4”C, the membrane was washed three times with 0.02% (vol/vol) Tween in TBS, followed by incubation with rabbit anti-mouse IgG [1:1,500 dilution in 5% (wt/vol) BSAITBSI for another 60 min at 4°C. The membrane was washed three times in 0.02% Tween (vol/vol) in TBS, then incubated with 1251-proteinA (4 x lo6 counts.min-l.rnl-I) for 60 min. After several washes with 0.02% (vol/vol) Tween in kb 14.0 7.0 5.1 1.5 1.1 0.8
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Fie. 1. Steadv-state levels of mRNA abundance of bFGF gene in rat lungs exposed to 85% 02 for 0,4,6, or 14 days, as assessed by slot-blot analysis and subsequent densitometry (left). Slot blots were performed with 0.6-2.0 kg total RNA from 4 animals at each time point. Results have been normalized against air-control values and are shown as means ? SE. *Significantly different from control (P < 0.05). To confirm appropriate sizes of transcripts, Northern blot analysis was performed with 15 pg total RNA from each time point (top right). Equal loading was confirmed by ethidium bromide staining (bottom right).
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Fig. 2. Steady-state levels of mRNA abundance of bFGF receptor fig in rat lungs exposed to 85% 02 for 0,4,6, or 14 days, as assessed by slot-blot analysis and subsequent densitometry (left). Slot blots were performed with 0.62.0 fig total RNA from 4 animals at each time point. Results have been normalized against air-control values and are shown as means k SE. Significantly different from control (P < 0.05). To confirm appropriate sizes of transcripts, Northern blot analysis was performed with 15 pg total RNA from each time point (top right). Equal loading was confirmed by ethidium bromide staining (bottom right).
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TBS, the membrane was air dried and exposed to Kodak-XAR film. Immunohistochemistry. Lungs from adult female rats that had been exposed to air or 85% 02 for 6 and 14 days were perfusion fixed with a paraformaldehyde-based fixative (13) after blood had been flushed from the pulmonary circulation. Tissues were embedded in OCT compound, and 5-bm sections were cut on a cryotome. Sections were mounted on o-aminopropyltriethoxysilane-coated slides. Immunohistochemical studies were conducted using an avidin-biotin-peroxidase complex method (14). Primary and secondary antibody dilutions for bFGF and FGF-R immunolocalization studies were as reported for previous studies on fetal rat lungs (14). Antibody specificity was verified by demonstrating an absence of immunostaining both when preimmune or nonimmune sera and when a blocking solution [5% (vol/vol) normal goat serum and 1% (wt/vol) BSA] without antibody were substituted for the primary antibody. Immunoabsorption studies were also conducted by incubating the primary antibodies with excess (double the amount) bFGF peptide and synthetic chicken FGF-R peptide for 4 h at 4°C before immunostainingprocedures. Negative immunoreactivity was observed with these studies, indicating that the antibodies were highly specific. After completion of immunohistochemical procedures, slides were lightly counterstained with Carazzi hematoxylin.
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Autoradiography. In some experiments, animals were exposed to air or 85% 0s for 6 days. The choice of a 6-day time point was based on the assumption that any increase in protein synthesis would postdate maximal mRNA levels by 24-48 h. The animals received 1 l&i/g [3H]thymidine intraperitoneally 2 h before lung fixation. The lungs were cleared of blood as previously described. For combined autoradiography and immunolocalization, lungs were fixed with a paraformaldehyde-based fixative, and 5-pm sections were cut on a cryotome. Sections were mounted on o-aminopropyltriethoxysilanecoated slides. After completion of immunohistochemical procedures, slides were lightly counterstained with Carazzi hematoxylin. Slides were coated with Kodak NBT-3 emulsion for autoradiography and developed after 2 wk for examination by light microscopy to identify dense areas of silver granule concentration over cell nuclei undergoing active DNA synthesis. Positive labeling was defined using the criteria of Chwalinski et al. (7). Datapresentation. Values are presented as the means * SE of arbitrary densitometric units for scans from four animals at each time point. No differences were observed between animals before exposure or animals exposed to air for various periods as controls. For the purposes of presentation, only preexposure values are shown. Statistical significance (P < 0.05) was determined by an analysis of variance followed by
IDAY
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Fig. 3. Densitometric scans (left) and original (right) nuclear runoff transcription analyses of bFGF, fig, and histone H3 genes in lungs from animals exposed to 85% 02 for 0, 4, 6, or 14 days. Controls consisted of plasmid pBR322.
-
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Fip.. 4. Alveolar euithelial cells with SP-A exposure to 85% 6,. Original magnification,
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assessment of differences using the Dunnett’s or Duncan’s multiple-range test (31).
IN 85%
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RESULTS
Changes in lung weight, body weight, and total extracted RNA during the exposure period in this model have been reported previously (13). A significant increase in lung weight, lung weightibody weight ratio, and total lung RNA occurred in animals exposed to 85% O2 for 4, 6, or 14 days when compared with air-exposed control animals. In an initial series of studies, type II pneumocytes were isolated from air- or 85% 02-exposed lungs to determine whether cells exposed to 85% O2 in vivo had an increased response to a variety of growth factors when studied in vitro. Although no enhanced response to growth factors by cells from 85% 02-exposed lungs was observed, the relative response to the different growth factors studied was of interest (Table 1). Significant increases in DNA synthesis by air-exposed type II pneumocytes were observed with IGF-I, EGF, vitamin D3, and bFGF, but not TGF+. By far the most potent stimulator of type II pneumocyte DNA synthesis was bFGF. Significantly increased DNA synthesis was seen in cells from 85% 02-exposed lungs. No significant further increase was seen on the addition of IGF-I, EGF, or vitamin D3, but the marked response to bFGF was still evident. As bFGF is such a potent stimulator of pneumocyte DNA synthesis in vitro, we explored the possibility that increased bFGF synthesis might be involved in the pulmonary cellular hyperplasias seen in vivo after exposure to 85% Oz.
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stain)
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The abundance of the bFGF message, as assessed by slot blot and Northern blot, is shown in Fig. 1. There was a significant increase in message by day 4 of exposure to 85% 02, with a subsequent fall to control values by 14 days of exposure. Northern analysis confirmed the presence of the well-described major transcript at 7.0 kb (1) and additional transcripts at 14, 5.1, 1.5, 1.1, and 0.8 kb. Additional multiple transcripts for
0
6
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DAYS OF EXPOSURE Fig. 5. Western blot analysis of bFGF protein m lung extracts of rats exposed to 85% 02 for 0,6, or 14 days. Each lane was loaded wkh 50 Kg total protein.
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pg PROTEIN 10 5 Fig. 6. Immunoblot analysis of FGF-R protein in membrane-enriched lung extracts of rats exposed to 85% 0s for 0,6, or 14 days. Densitometric scans (1.25 kgprotein) are shown on left and dilution series on right.
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bFGF representing alternate splice sites, but utilizing the same start site, have been demonstrated in other systems (1). The message for FGF fZg receptor, as assessed by slot blots and Northern blots, was significantly increased by day 4 of exposure, with a fall to below control values by 14 days of exposure (Fig. 2). Northern analysis confirmed the presence of a 4.4 kb transcript for the FGF fZg receptor. To determine whether the increased bFGF and FGF fig mRNAs observed after 4 days of 85% O2 exposure reflected increased mRNA transcription or reduced mRNA breakdown, nuclear runoff transcription assays were conducted. Consistent with the slot-blot analyses, there was increased transcription of both bFGF and fig on days 4 and 6 of exposure to 85% 02, with a subsequent decrease to control values by 14 days of exposure (Fig. 3). This contrasted with the findings for transcription of the histone H3 gene, which remained elevated throughout the exposure period. Consistent with the sustained transcription of the histone H3 gene was the finding of multiple binucleated cells, immunoreactive for SP-A, in the alveolar epithelium (Fig. 4). To determine whether the increased expression of bFGF and fZg genes was translated into increased protein synthesis, immunoblot analyses of the lung content of bFGF and FGF-R were performed. Based on the mRNA studies, we isolated cellular proteins and enriched membrane fractions from the lungs of animals exposed to either air or
85% O2 for 6 and 14 days. As assessed by immunoblot, the lung content of bFGF increased by 37% after 6 days of exposure to 85% O2 but had declined to 59% of control values by 14 days of exposure (data not shown). These findings were not entirely consistent with the observed changes in mRNA (Fig. 1). To exclude the possibility that the results of this immunoblot analysis were due either to proteolytic degredation or to an inefficient extraction procedure, extraction was repeated using a more rigorous extraction procedure and antiproteinases for Western analysis. This analysis was consistent with mRNA findings, revealing a marked increase in the lung content of bFGF after 6 days of exposure to 85% 02, with a decline in bFGF content by 14 days of exposure, though not down to control values (Fig. 5). Similarly, fZg in membraneenriched fractions was increased by 35% after 6 days of exposure to 85% O2 but had declined to 50% of control values by 14 days of exposure (Fig. 6). Immunohistochemical findings were, for the most part, consistent with the observations of gene expression and protein immunoanalysis. In contrast to our findings with PDGF (13) and TGF-P (24), immunohistochemistry revealed the presence of bFGF widely dispersed in lung tissue from control animals. In general, except for areas surrounding airways and vessels, staining was faint (Fig. 7B) in air-exposed animals. Although subtle, more intense
Fig. 7. Immunolocalization of bFGF and bFGF-R in lungs of rats exposed to air or 02 for 6 or 14 days. A: negative control without primary antibody. Original magnification x 220. B: immunoreactivity of bFGF in lungs from animals exposed to air for 6 days. Moderate staining is evident around airways (top right) and vessels (left), with light staining over rest of parenchyma. Original magnification x 220. C: immunoreactivity of bFGF in lungs from animals exposed to 85% 02 for 6 days. More intense staining is evident in alveolar regions, particularly over epithelium. [3H]thymidine autoradiography (black granules over cell nuclei) is not well correlated with bFGF immunoreactivity. Original magnification x 280. D: immunoreactivity of bFGF in lungs from animals exposed to 85% 02 for 14 days. Intense and generalized staining is evident. Original magnification x220. E: lack of immunoreactivity of FGF-R in lungs from animals exposed to air for 6 days. Original magnification x220. F: immunoreactivity of FGF-R in lungs from animals exposed to 85% 02 for 6 days. Staining is evident in alveolar regions, particularly over epithelium. [3H]thymidine autoradiography is occasionally correlated with FGF-R immunoreactivity. Original magnification x 280. G: immunoreactivity of FGF-R in lungs from animals exposed to 85% 02 for 6 days. Staining is less evident in this area of lung. [3H]thymidine autoradiography is not well correlated with FGF-R immunoreactivity. Original magnification x280. H: immunoreactivity of FGF-R in lungs from animals exposed to 85% 0s for 14 days. Only occasional cell shows immunoreactivity. Original magnification x280.
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staining over individual cells was evident after 6 days of exposure to 85% O2 (Fig. 7C). In particular, some alveolar epithelial cells were stained while others were not. This change in the staining pattern after 6 days of exposure to 85% O2 coincides with the period in which there is considerable remodeling of the alveolar epithelium as damaged type I pneumocytes are replaced by proliferating type II pneumocytes. By 14 days (Fig. 70) of exposure to 85% 02, staining for bFGF had again become generalized. Though immunohistochemistry is not quantitative, the degree of staining after 14 days of exposure to 85% OZ was more than we would have expected from the immunoanalysis. A very different pattern from that seen with bFGF was evident for FGF-R, with no detectable immunostaining in control samples (Fig. 7E). FGF-R staining evident after 6 days of exposure to 85% O2 (Fig. 7, F and G) was, except for an occasional cell, no longer evident after 14 days of exposure (Fig. 7H). The techniques we have used do not allow us to identify the cell types stained for bFGF or FGF-R with certainty, but many of the cells stained for both the growth factor and the receptor were situated in the alveolar epithelium (Fig. 7, C and F). Autoradiography with [3H]thymidine showed no clear relationship between expression of the FGF-R and active DNA synthesis, as illustrated in Fig. 7, F and G. DISCUSSION
A number of polypeptide growth factors, including PDGF (4), bFGF (16), EGF (17), IGF (19), and TGF-PlV3 (15, 26), are b e1ieved to play a role in the rapid cellular hyperplasias that occur during fetal lung development. However, much less information is available about the role of these factors in the pneumocyte, fibroblast, and endothelial cell hyperplasias that occur after lung injury or in the compensatory growth which follows partial pneumonectomy. A role for EGF has been suggested in chronic human neonatal lung injury and for TGF-P both in animal models of lung injury (27) and in human pulmonary fibrosis (18). We (13) and others have previously reported expression of the normally latent PDGF-B chain and PDGF-B-receptor genes in the lungs of adult rats exposed to 85% OZ. PDGF-BB, a known competencetype growth factor for cells of mesenchymal origin, was localized to the interstitium, suggesting that it may mediate the early interstitial fibroblast proliferation observed in this model (13). Late changes of fibroblast hyperplasia and increased type I collagen synthesis in this model are temporally related to a late increase in TGF-p expression (24). The presence and distribution of bFGF in the lungs of air-exposed control animals confirm the recent findings of Sannes et al. (29), though we did not hyaluronidasedigest our tissue preparations. Since free bFGF appears in the alveolar space as a consequence of lung injury (16) and is mitogenic for adult rat type II pneumocytes in vitro, we hypothesized that bFGF may be a competencetype growth factor involved in the type II pneumocyte hyperplasia also seen with lung injury. Our findings are compatible with such a role for bFGF in the early pneumocyte hyperplasia that results from exposure of
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the adult rat to 85% OZ. There was an upregulation of both bFGF mRNA and of its fig receptor mRNA after 4 days of exposure to 85% 02, after which there was a decline in the message levels by 14 days of exposure. Nuclear runoff analyses demonstrated that this increase in the mRNAs of bFGF and its receptor resulted from increased gene transcription, whereas immunoanalysis and immunohistochemistry demonstrate a resultant increase in protein synthesis. The distribution of bFGF, as assessed by immunohistochemistry, becomes less evenly distributed around the alveolar epithelium after 6 days of exposure to 85% OZ. One explanation of this change is proteolytic degradation of matrix-associated bFGF during the process of remodeling as damaged type I pneumocytes are replaced by proliferating type II pneumocytes. While fig mRNA levels fall on day 6 of exposure to 85% OZ, compared with day 4 of exposure, transcription of the fig gene is increased on day 6. This may be attributed to an increase in total lung RNA (13) and/or to a decrease in fig mRNA half-life. The cellular sites of increased bFGF and FGF-R synthesis cannot be determined with certainty by the techniques used in this study. Although FGFs can be produced by macrophages and such a source cannot be excluded, this production has been reported to be arrested by elevated O2 concentrations (20). Endothelial cells can deposit bFGF into extracellular matrix, which can then be made available for autocrine (l), and presumably paracrine, stimulation of growth. Some cells demonstrating immunoreactivity for FGF-R were localized to the epithelial surface (Fig. 6F). We have previously reported that FGF-R is present in fetal lung epithelium (14), and it is entirely possible that the type II pneumocyte in lung injury reexpresses some aspects of the fetal phenotype. Identification of the cells synthesizing bFGF will require immunohistochemistry with cell-specific markers, and in situ hybridization to detect bFGF mRNA. The altered distribution of bFGF evident in lung tissue after exposure to 85% O2 may to some extent reflect deposition after lethal cell injury by exocytosis (23). Enhan ce d production of bFGF-binding proteoglycans occurs in response to TGF-P (25), which we have found to increase after 14 days of exposure to 85% O2 (24). The discrepancy between direct measures of bFGF content and apparent content by immunohistochemistry after 14 days of exposure to 85% O2 is difficult to reconcile. Immunohistochemistry is not quantitative, and antigenic sites on bFGF may be less accessible to antibody at different times during exposure. Alternatively, it is possible that neither of the extraction procedures used for immunoanalysis completely dissociated bFGF from insoluble matrix components. Double-labeling immunohistochemistry with antibodies to cell-specific markers and to FGF-R will be required to definitively identify those cells expressing the receptor. However, bFGF uptake by cells can occur through receptor-dependent and receptor-independent pathways (28). This suggests that mitogenic effects can still occur, even in the absence of demonstrable receptors. Such a mechanism may account for the observed lack of correla-
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tion between cells immunoreactive for FGF-R and cells undergoing DNA synthesis, as assessed by [3H]thymidine autoradiography. Alternatively, downregulation of FGF-R may precede DNA synthesis by cells responding to bFGF through the FGF-R. Of interest was the transience of increased mRNA for both bFGF and FGF-R. This is understandable for the bFGF mRNA, since it can be deposited intact in matrix for subsequent release. The transience of the increased mRNA for FGF-R suggests that bFGF-mediated cellular hyperplasia may only occur early in the injury process or that uptake may not be exclusively receptor-mediated. We had observed a similar phenomenon with PDGF and the PDGF-B-receptor (13). These findings are consistent with the morphometric analysis of cellular proliferative changes occurring in this model by Crapo et al. (10). The maximal number of type II pneumocytes and interstitial cells was observed at 7 days of exposure with a 30% reduction in the number of type II pneumocytes and with a 46% reduction in the number of interstitial cells between 7 and 14 days of exposure to 85% OZ. Our analysis of cell types labeled with [3H]thymidine had shown (13), as expected, a significant reduction in the number of labeled interstitial cells between 6 and 14 days of exposure to 85% OZ. Surprisingly, we had seen a sustained increase in the number of type II pneumocytes labeled by [3H]thymidine (13). We also noted a sustained increase in histone H3 mRNA (13). Histone H3 gene expression is tightly coupled to DNA synthesis and peaks at the S phase of the cell cycle, then disappears toward the end of the G2 phase (32). In this study, we confirmed that the previously reported increased histone H3 mRNA (13) reflected increased gene transcription. Active DNA synthesis in the distal lung epithelium, as suggested by both the autoradiography data and the expression of histone H3, is contrary to what one might expect in a model in which morphometric analysis has shown a significant reduction in pneumocyte numbers between 7 and 14 days of exposure to 85% O2 (10). Even though active DNA synthesis in type II pneumocytes is only present in a relatively small percentage of the total pool of type II pneumocytes at 14 days of exposure (13), the purpose of such an event in the face of a declining pneumocyte pool is unclear. One explanation, suggested by the observation of Crapo et al. (10) that the type II pneumocyte volume increases 61% after a 14-day exposure to 85% 02, is that division of some pneumocytes is arrested or delayed at or after the S phase of the cell cycle. Our finding of binucleated cells that are SP-A immunoreactive is consistent with such a phenomenon. Increased DNA synthesis without completion of cell division is a well-recognized response of growth factor-stimulated type II pneumocytes in vitro (5,22).
In summary, exposure to 85% O2 results in increased bFGF and FGF-R gene expression and protein synthesis. Given the known properties of bFGF, it is likely that this growth factor plays a role in the pneumocyte hyperplasia observed in pulmonary oxygen toxicity.
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The authors wish to thank T. Cosgrove for his expert technical assistance. This work was supported by a Group Grant (to S. Buch, M. Post, and A. K. Tanswell) and Grant MT-10545 (to J. D. Edelson) from the Medical Research Council of Canada; National Heart, Lung, and Blood Institute Grant ROl-HL-45476 (to B. A. Freeman and A. K. Tanswell); a Grant from Physicians’ Services Incorporated Foundation (to A. Moore and M. Post); and an equipment grant from the Ontario Thoracic Society (A. K. Tanswell). Address for reprint requests: S. Buch, Neonatology Research, Hospital for Sick Children, 555 University Ave., Toronto, Ontario M5G 1X8, Canada. Received
19 October
1993; accepted
in final
form
27 October
1994.
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