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Published ahead of print on March 5, 2003, doi:10.1164/rccm.200207-662OC
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American Journal of Respiratory and Critical Care Medicine Vol 167. pp. 1554-1561, (2003)
© 2003 American Thoracic Society

Original Article

Fibroblast Growth Factor Receptor-1 and Neonatal Compensatory Lung Growth after Exposure to 95% Oxygen

Robert P. Jankov, Xiaoping Luo, Ann Campbell, Rosetta Belcastro, Judy Cabacungan, Leslie Johnstone, Helena Frndova, Stephen J. Lye and A. Keith Tanswell

Canadian Institutes of Health Research (CIHR) Group in Lung Development, Lung Biology Programme, Hospital for Sick Children Research Institute; CIHR Group in Developmental and Fetal Health, Samuel Lunenfeld Research Institute, Mt. Sinai Hospital; and Departments of Obstetrics and Gynaecology, Paediatrics, and Physiology, Faculty of Medicine, University of Toronto, Toronto, Ontario, Canada; and Department of Pediatrics, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, People's Republic of China

Correspondence and requests for reprints should be addressed to Dr. A. Keith Tanswell, Division of Neonatology, The Hospital for Sick Children, 555 University Avenue, Toronto, ON, M5G 1X8 Canada. E-mail: keith.tanswell{at}sickkids.ca


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Neonatal rats exposed to 95% oxygen (O2) for 7 days from birth had inhibited lung growth, DNA synthesis, and secondary septation. These parameters were rapidly restored by a period of recovery in air. Northern and Western blot analysis and immunohistochemistry were used to screen for the fibroblast growth factor receptor-1 (FGF-R1) and its high affinity ligand, basic fibroblast growth factor (bFGF), which could have a role in this recovery process. Expression of bFGF in the lung was significantly reduced at the end of the 7-day exposure to 95% O2 and was increased after 3 days of recovery in air. Expression of FGF-R1 was not affected by exposure to 95% O2 or recovery in air. We hypothesized that the increase in bFGF after removal from 95% O2, acting through the FGF-R1, would be critical for compensatory growth. Intraperitoneal injection of soluble truncated FGF-R1 at the onset of the recovery phase arrested compensatory lung DNA synthesis and secondary septation seen in control animals after 3 days of recovery, confirming a role for FGF-R1 in this model of compensatory neonatal lung growth.

Key Words: pulmonary oxygen toxicity • bronchopulmonary dysplasia • reactive oxygen species • soluble receptors

Bronchopulmonary dysplasia is a chronic neonatal lung injury primarily, though not exclusively, affecting preterm infants in which oxidant stress is believed to play a major role. It has significant mortality and morbidity, and no effective preventive therapy exists. A major pathologic feature of bronchopulmonary dysplasia, as seen in the most severely affected infants, is a failure of secondary septation leading to a marked reduction in alveolar number and surface area (1) and abnormal pulmonary function (2). Secondary septation and alveolarization occurs, in major part, postnatally (3). Despite a large body of information relating to the growth factor regulation of fetal lung growth, there is very little information available relating to the growth factors that control normal postnatal lung growth or abnormal growth in neonatal lung injury (4). Identification of the growth factors that are affected by oxidant injury and the growth factors involved in catch-up growth after injury may facilitate the development of novel therapeutic strategies for bronchopulmonary dysplasia. It is known that mice deficient in platelet-derived growth factor-AA have a failure of postnatal alveolarization related to failure of migration of alveolar smooth muscle progenitors (5). Inhibition of angiogenesis, through inhibition of a vascular endothelial growth factor (VEGF) receptor (KDR/flk)-1, also reduces alveolarization (6). Platelet-derived growth factor-BB may also be involved, in that neonatal rats injected with a truncated soluble platelet-derived growth factor ß-receptor during the period of secondary septation have impaired lung DNA synthesis (7).

Newborn rats exposed to 97% oxygen (O2) or more for 7 days had arrested alveolarization at the level present at birth (8). After a 14-day recovery period in air, the alveolar number increased such that it was not significantly different from newborn rats maintained in air for 21 days (8). The complete recovery of the alveolar number was also observed in neonatal rats exposed to 95% O2 for 7 days followed by recovery in air until 40 days of age (9). Although exposure to 95% O2 or more for prolonged periods differs from normal clinical practice, these models do demonstrate the major feature of modern bronchopulmonary dysplasia, arrested/delayed alveolarization, and can be used for the study of factors that regulate this process. Because the lung is essentially growth arrested during exposure to 95% O2 or more for 7 days, we hypothesized that growth factor expression during recovery in air would be considerably amplified from that present in control animals, where the most rapid phase of alveolarization occurs from 0 to 7 days of age and slows thereafter.

We examined the role of basic fibroblast growth factor (bFGF or FGF-2) and one of its receptors (fibroblast growth factor receptor-1 [FGF-R1] or flg). Both bFGF and FGF-R1 are upregulated in adult rat lung during the proliferation induced by exposure to 85% O2 (10), are highly expressed in the lung during late fetal life (11), and are believed to play a role in both normal and pathologic angiogenesis (12). bFGF is also a potent mitogen for type II pneumocytes isolated during various stages of postnatal life (4). We hypothesized that the expression of both bFGF and FGF-R1 would be upregulated during the accelerated lung growth occurring with recovery in air after a 7-day exposure to 95% O2 and would play a critical role in compensatory catch-up growth.


   METHODS
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 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
In Vivo Interventions
All procedures involving animals were conducted according to criteria established by the Canadian Council for Animal Care. Approval for the study was obtained from the Animal Care Review Committee of the Samuel Lunenfeld Research Institute, Mount Sinai Hospital. Rat pups were maintained in paired chambers (air and 95% O2) for a 7-day exposure period and then allowed to recover in room air for up to 3 weeks. Equal litter sizes (10–12 pups) were maintained between paired chambers. No deaths occurred as a consequence of the exposure to 95% O2. For soluble receptor interventions, seven pups from each litter received either phosphate-buffered saline (PBS) (vehicle control) or truncated soluble receptor (FGF-R1{alpha}(IIIc)/Fc or p75 Neurotrophin-R [NGF-R, nerve growth factor receptor]/Fc chimeric proteins; R&D Systems, Minneapolis, MN) in 100 µl PBS (10 µg/g body weight [BW]). Injections were given once on Day 7 via a 30-gauge needle into the right iliac fossa, and animals were killed after a 3-day recovery in air.

Isolation of Total Lung RNA
The lungs were dissected away from large vessels and airways to be flash frozen in liquid nitrogen immediately after weighing. Total (nuclear and cytoplasmic) RNA was isolated by lysis of tissue in 4 M guanidium thiocyanate, followed by phenol/chloroform extraction according to the method of Chomczynski and Sacchi (13).

RNA Analyses
For Northern blot analyses, 20 µg of total lung RNA was fractionated on 1.0% (wt/vol) agarose gels containing 0.66 M formaldehyde and transferred to a nylon membrane. Probes were labeled with deoxycytidine 5'-[{alpha}-32P] triphosphate by a random priming labeling system. Prehybridization (> 4 hours) and hybridizations (overnight) and washes were performed as described previously (10). All blots were exposed for 24 to 72 hours at -70°C to Kodak XAR-5 film using Dupont Cronex intensifying screens. The films were quantified by an Ultroscan XL laser densitometer (LKB, Bromma, Sweden). To correct for variations in RNA loading of gels and transfer to membranes, all results were normalized to 18S messenger RNA (mRNA), which we determined to be unaffected by O2 exposure.

Isolation and Quantitation of Total Lung DNA
Total DNA was extracted from perfused lung tissue lysates by phenol/chloroform/isoamyl alcohol extraction followed by precipitation in isopropanol as described previously (14). DNA was solubilized in 10 mM ethylenediaminetetraacetic acid, pH 12.3, and neutralized to pH 7.0 with 1 M potassium dihydrogen phosphate. Samples and DNA standards were quantified by fluorescence spectrophotometry as described in detail elsewhere (15).

Western Blot Analyses
Perfused lung tissue lysates were prepared as described previously (16). Samples containing 40 µg protein and standard proteins were fractionated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes. Membranes were blocked with 5% nonfat milk and incubated with 0.2 µg/ml rabbit polyclonal antibodies to bFGF (Chemicon, Temecula, CA) or FGF-R1 flg (Santa Cruz Biotechnology, Santa Cruz, CA) overnight at 4°C followed by secondary antibody for more than 1 hour at room temperature. Blots were imaged and bands quantified by enhanced chemiluminescence detection as described previously (16).

Immunohistochemistry
Perfused lung tissue sections from two animals per group were inflation-fixed as described previously in detail (17). For day-22 fetal lung samples, rats were delivered by hysterotomy on the anticipated day of delivery. Animals were anesthetized with ketamine (80 mg/kg) and xylazine (20 mg/kg) intraperitoneally. The pulmonary circulation was flushed with PBS containing 1 U/ml heparin to clear the lungs of blood and perfusion-fixed with 4% (wt/vol) paraformaldehyde while a constant airway pressure of 10 cm H2O was maintained via a tracheal catheter. The lungs were embedded in paraffin, cut in 5 µm sections, and mounted on {alpha}-aminopropyltriethoxysilane–coated slides. Immunostaining for bFGF and FGF-R1 (0.4 µg/ml), {alpha}-smooth muscle actin using a mouse monoclonal antibody (0.15 µg/ml; Neomarkers, Fremont, CA), and myeloperoxidase using a rabbit polyclonal antibody (1 µg/ml; DAKO, Carpenteria, CA) were performed by an avidin–biotin–peroxidase complex method as described previously (17, 18).

Morphometric Analysis
Morphometric analysis was performed using the mean linear intercept method, as described by Dunnill (19). Lungs were inflation-fixed as described previously. The right lower lobes were embedded in paraffin, cut in 5-µm sections, and stained with hematoxylin and eosin. Intercept numbers (1 count for distal airway intercept and 0.5 for proximal airway or vessel intercept) were evaluated by a light microscope using a x25 objective and x8 ocular lens with a crossed hairline of known length. Total intercepts were counted from 10 random images per section and 3 sections per animal. Mean values from each of three animals per group were expressed in micrometers.

Lung Volume–Pressure Loops
Animals were anesthetized with sodium pentobarbitone (5–10 mg/kg) and subsequently paralyzed with pancuronium bromide (0.3 mg/kg). After tracheotomy, the lungs were degassed and volume–pressure loops in the open chest recorded as described previously (17).

Data Presentation
Unless otherwise stated, all values are for mean ± SEM of four litters. Statistical significance (p < 0.05) was determined by one-way analysis of variance, followed by post hoc analysis using Duncan's multiple range test where significant differences were found between groups (20). Where error bars are not evident in figures, they fall within the plot point.


   RESULTS
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 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
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Exposure to 95% O2 for 1 week after birth, followed by a 3-week exposure to air, had no significant effect (p > 0.05) on BW (Figure 1A) . Lung growth and the lung weight (LW)/BW ratios were significantly reduced (p < 0.05) by a 1-week exposure from birth to 95% O2 (Figures 1B and 1C). As shown in Table 1 , the DNA content of the lungs of pups exposed to 95% O2 for 1 week was significantly different (p < 0.05) from the DNA content of the lungs of pups exposed to air for 1 week but not from those of pups at the time of birth (p > 0.05). This is consistent with a profound global inhibition of DNA synthesis observed previously in the lungs of pups exposed to 95% O2 for 1 week, compared with those in air, as assessed by immunohistochemical staining for proliferating cell nuclear antigen (21). The recovery of DNA mass, after recovery in air after exposure to 95% O2, was rapid with there being no significant difference between air-exposed and 95% O2–exposed pups by Day 10 of life. One potential contributor to this rapid recovery of DNA mass was DNA-derived from a phagocyte influx after an O2-mediated inflammatory response. However, this appeared not to be the case. As shown in Figure 2 , there was a neutrophil influx present at the end of a 1-week exposure to 95% O2 but this had cleared by Day 10 of life and could not account for the increase in DNA mass observed during the 3-day recovery period. The LW/BW ratios (Figure 1C) were consistent with compensatory lung growth at 7 and 14 days after completion of the exposure to 95% O2, with the relative growth rates having normalized by 21 days after completion of the 95% O2 exposure. Exposure of pups to 95% O2 for 1 week had only a modest effect on volume–pressure loops, which were assessed as a functional index of lung injury, with statistically significant differences (p < 0.05) only evident for the inflation loop (Figure 3A) . This was consistent with a modest effect on surfactant function or some degree of pulmonary edema, both of which have been reported for lungs exposed to high concentrations of O2. After the recovery period in air, there were no statistically significant differences (p > 0.05) in volume–pressure loops between groups at 4 weeks of age (Figure 3B). Taken together, these findings are consistent with an essentially complete anatomic and functional recovery of the lungs after a 1-week exposure to 95% O2.



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  		Figure 1. Lung weight (LW) (A), body weight (BW) (B), and LW/BW weight ratios (C) in newborn rats exposed to air for 4 weeks (open bars) or to 95% O2 for 1 week followed by 3 weeks in air (solid bars). Values are means ± SEM for four litters in each group. There was no difference between groups for BW, but exposure to 95% O2 for 1 week caused a significant reduction in LW and LW/BW ratio (*p < 0.05 by one-way analysis of variance [ANOVA]). Recovery in air, after exposure to 95% O2 for 1 week, was associated with a rapid recovery in LW to control values by 2 weeks of age and a significant increase in LW/BW ratios at 2 and 3 weeks of age (*p < 0.05 by one-way ANOVA).

 

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  		TABLE 1. Lung dna content on neonatal rats exposed to air or 95% oxygen from birth*

 


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  		Figure 2. Immunohistochemistry for myeloperoxidase as a marker of neutrophils. (A) The lungs of rat pups exposed to air for 7 days had no neutrophils evident in the interstitium. (B) Exposure of rat pups to 95% O2 for 7 days caused a marked increase in interstitial neutrophil numbers (arrows). (C) No interstitial neutrophils were evident in rat pups exposed to air for 10 days. (D) After a 3-day recovery in air, rat pups previously exposed to 95% O2 for 7 days no longer had increased numbers of neutrophils. Bar = 250 µm.

 


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  		Figure 3. Volume–pressure loops from newborn rats exposed (A) to air (open circles) or to 95% O2 (closed circles) for 1 week or (B) air for 4 weeks (open circles) or to 95% O2 for 1 week followed by 3 weeks in air (closed circles). Values are means ± SEM for 5 to 6 randomly selected animals in each group. Statistically significant differences (*p < 0.05 by one-way ANOVA) were evident in lung volumes at inflation pressures of 5, 15, 20, and 25 cm H2O for animals exposed to 95% O2 for 1 week. After the recovery period, no statistically significant differences were evident.

 
Expression of lung bFGF mRNA was significantly (p < 0.05) inhibited after a 1-week exposure to 95% O2, relative to control animals in air (Figure 4) . An increased expression after 1 week of recovery in air did not achieve statistical significance (p > 0.05). No significant differences between groups (p > 0.05) were evident for the FGF-R1 (flg) mRNA (Figure 4). Because biologically active bFGF can be mobilized from inactive stores within the lung tissue, immunohistochemistry and immunoblot analysis for bFGF and FGF-R1 (flg) were pursued. In accord with the findings for bFGF mRNA, a grossly obvious reduction in lung tissue immunoreactive bFGF was evident after a 1-week exposure to 95% O2 (Figure 5) . A gross increase in immunoreactive bFGF was evident after a 1-week recovery in air, particularly localized to areas of active alveolar septation as was seen in air-exposed animals after 1 week (Figure 5), although the parallel increase in bFGF mRNA had not been statistically significant (Figure 4). In contrast to the lack of change in FGF-R1 (flg) mRNA in response to a 1-week exposure to 95% O2, there was an apparent increase in immunoreactive tissue FGF-R1 (flg) around the airway epithelium at that time point as shown in Figure 6 . Expression of bFGF and FGF-R1 in the total lung after 7 days of air or 95% O2 exposure, as assessed by Western blot analysis (Figure 7) , were consistent with findings for their mRNAs (Figure 4). After 3 days of recovery in air, bFGF expression increased significantly (p < 0.05 compared with animals exposed to 95% O2 for 7 days; Figures 7 and 8) , whereas no differences were seen in FGF-R1 expression (Figures 7 and 8) after a 3-day recovery.



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  		Figure 4. Basic fibroblast growth factor (bFGF) and fibroblast growth factor receptor-1 (FGF-R1) messenger RNA (mRNA) values in the lungs of newborn rats exposed to air for 4 weeks (open circles) or to 95% O2 for 1 week followed by 3 weeks in air (closed circles). Values are means ± SEM for four litters in each group. A statistically significant difference between groups (# p < 0.05 by one-way ANOVA) was only evident for bFGF mRNA at the 1-week time point. Newborn rats exposed to 95% O2 for 1 week followed by 3 weeks in air had a significant reduction in bFGF mRNA expression compared with that present at birth (*p < 0.05 by one-way ANOVA). Inserts from paired samples in air (A) for 1 (bFGF) or 2 (FGF-R1) weeks or 95% O2 (open circles) for 1 week (bFGF) followed by air for 1 week (FGF-R1) are shown to demonstrate the transcript size for the quantitated bands.

 


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  		Figure 5. bFGF immunohistochemistry. Lung tissue from fetal lung of 22 days' gestation was used both as a positive control (F22) and as a negative control, with omission of the primary antibody (C). Bar = 250 µm. Sections were prepared from newborn rat pups after either exposure to air for 7 (A7), 14 (A14), 21 (A21), or 28 (A28) days, or to 95% O2 for 7 (O7) days, followed by air exposure for 7 (O7 + A7), 14 (O7 + A14), or 21 (O7 + A21) days. Expression was largely localized to airway epithelium in fetal lung (F22) and was generalized after 7 days in air-exposed animals (A7). At 2 weeks after birth, expression was localized to the airway epithelium, the vascular wall, and areas of alveolar septation (A14). After 3 and 4 weeks, when septation was advanced, expression was limited to airway and vessels (A21 and A28). A marked overall reduction in bFGF immunoreactivity was evident after 7 days in 95% O2 (O7). There was an apparent increase in bFGF immunoreactivity after 7 days of recovery in air (O7 + A7) similar in degree and localization to air-exposed animals after 7 days.

 


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  		Figure 6. Immunohistochemistry for flg FGF-R1. Lung tissue from fetal lung of 22 days' gestation was used both as a positive control (F22) and as a negative control, with omission of the primary antibody (C). Bar = 250 µm. Sections were prepared from newborn rat pups after either exposure to air for 7 (A7), 14 (A14), 21 (A21), or 28 (A28) days, or to 95% O2 for 7 (O7) days, followed by air exposure for 7 (O7 + A7), 14 (O7 + A14), or 21 (O7 + A21) days. Expression was generalized in fetal lung (F22) and was mainly localized to airway epithelium and a subpopulation of parenchymal cells after birth. An increase in flg FGF-R1 immunoreactivity was evident, largely in airway epithelium, after 7 days in 95% O2 (O7). Expression during recovery in air after 95% O2 exposure was slightly increased in the lung parenchyma after 7 days of recovery (O7 + A7) and was similar to that seen in animals exposed to air from birth after 2 (O7 + A14) and 3 (O7 + A21) weeks recovery.

 


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  		Figure 7. Western blot analyses of bFGF (A) and FGF-R1 (C) in total lung after exposure to air (open circles) or to 95% O2 (closed circles) for 1 week and after 3 days recovery in air. Values are means ± SEM for three litters in each group. A statistically significant decrease in bFGF expression (*p < 0.05 by one-way ANOVA) was found in animals exposed to 95% O2 for 1 week compared with air controls. After 3 days of recovery in air, 95% O2–exposed animals had significantly increased expression of bFGF (#p < 0.05 by one-way ANOVA) compared with the 7-day time point. Animals continuously exposed to air had a significant reduction in bFGF expression over the same time period (#p < 0.05 by one-way ANOVA). No significant differences were found in expression of FGF-R1 between groups or time points. Protein bands were identified at 20 kD for bFGF (B) and 120 kD for FGF-R1 (D).

 


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  		Figure 8. Immunohistochemistry for bFGF and FGF-R1. Sections were prepared from rat pups after 7 and 10 days of exposure to air (A7 and A10) or 95% O2 for 7 days (O7) followed by recovery in air for 3 days (O7 + A3). An apparent increase in bFGF expression was seen in 95% O2–exposed animals after a 3-day recovery in air. No differences were evident in FGF-R1 expression between groups. Bar = 250 µm.

 
These findings with bFGF and the FGF-R1 were consistent with their having a role in early compensatory lung growth after a 1-week exposure to 95% O2. The role of the FGF-R1 (flg) was tested using a truncated soluble receptor approach. The truncated soluble FGF receptor acts as a decoy for the natural receptor ligands, which are not then available to bind to the endogenous receptor. Another soluble receptor not known to bind FGF-R1 ligands, nerve growth factor receptor/Fc, was used as a control. As shown in Figure 9 , animals exposed to 95% O2 that received the truncated soluble FGF-R1 receptor at the beginning of the air recovery phase at Day 7 had an attenuated recovery of lung DNA content at Day 10 when compared with control animals receiving either vehicle alone or a truncated soluble receptor to NGF (p < 0.05). Histologic examination of lung sections from the animals exposed to 95% O2 for 7 days displayed a marked inhibition of secondary septation compared with animals maintained in air for 7 days (Figure 10) . Recovery in air for 3 days allowed a major degree of catch-up in secondary septation, which was prevented by a single intraperitoneal injection of the truncated soluble FGF-R1 receptor on Day 7 (Figure 10). Quantitative assessment of alveolar development, using mean linear intercept, is shown in Figure 11 . Compared with animals exposed to air for the same time period, animals exposed to 95% O2 for 7 days had a profound inhibition of alveolar development as indicated by a greater mean linear intercept distance (p < 0.05). Significant catch-up in alveolar growth was evident after a 3-day recovery in room air, which was inhibited by the truncated soluble FGF-R1 but not the truncated soluble NGF receptor (Figure 11).



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  		Figure 9. Total lung DNA and truncated soluble receptor interventions after a 7-day exposure to 95% O2 and recovery in air for 3 days. Pups received a single intraperitoneal injection (10 µg/g) of either vehicle (closed circle), a truncated soluble recombinant human FGF-R1{alpha}(IIIc)/Fc (open circle) or truncated soluble recombinant human nerve growth factor receptor (NGF-R)/Fc (shaded circle) chimeric proteins at the onset of the recovery phase. Values are means ± SEM for four pups in each group. A statistically significant increase (*p < 0.05 by one-way ANOVA) in lung DNA content occurred between Days 7 and 10 in pups that received vehicle or NGF-R/Fc, but not (p > 0.05 by one-way ANOVA) in pups that received FGF-R1{alpha}(IIIc)/Fc. The increase in lung DNA content of pups that received FGF-R1{alpha}(IIIc)/Fc was significantly different from both other treatment groups (#p < 0.05 by one-way ANOVA). There was no significant difference (p > 0.05 by one-way ANOVA) in the increase in lung DNA content for pups that received vehicle or NGF-R/Fc.

 


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  		Figure 10. Neonatal rat lungs were stained for {alpha}-smooth muscle actin (brown stain) to highlight areas of alveolar septation after pups were exposed to air for 7 (A7) or 10 (A10) days. Specificity of staining was confirmed by omitting the primary antibody (C). Exposure to 95% O2 for 7 days (O7) markedly inhibited secondary septation compared with that evident in pups exposed to air for 7 days (A7). Pups exposed to 95% O2 for 7 days, then allowed to recover in air for 3 days (O7 + A3) while receiving daily intraperitoneal injections of vehicle, had a significant recovery of secondary septation, and an appearance close to that seen in the lungs of pups exposed to air for 10 days (A10). Daily intraperitoneal injections of the FGF-R1{alpha}(IIIc)/Fc chimeric protein attenuated this recovery of structure (O7 + A3 + SR), with the lung having an appearance similar to that present at the beginning of the recovery period (O7). Bar = 1,000 µm.

 


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  		Figure 11. Mean linear intercepts (Lm) from the lungs of rat pups after a 7-day exposure to air (open squares) or 95% O2 (closed circles), followed by recovery in air for 3 days. Pups exposed to 95% O2 received a single intraperitoneal injection of either vehicle (closed circles), 10 µg/g of a truncated soluble recombinant human FGF-R1{alpha}(IIIc)/Fc (open circle) or truncated soluble recombinant human NGF-R/Fc (shaded circle) chimeric proteins at the onset of the recovery phase. Values are mean ± SEM for three pups in each group. A significant increase in Lm was seen in 95% O2–exposed pups after 7 days (*p < 0.05 by one-way ANOVA) compared with air-exposed pups. Significant recovery occurred between Days 7 and 10 in pups that received vehicle or NGF-R/Fc (*p < 0.05 by one-way ANOVA) but not (p > 0.05 by one-way ANOVA) in pups that received FGF-R1{alpha}(IIIc)/Fc. The Lm in pups that received FGF-R1{alpha}(IIIc)/Fc was significantly greater than that from both other treatment groups at the same time point (#p < 0.05 by one-way ANOVA).

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Our findings with respect to changes in LW, DNA content, and histologic appearance after an exposure of newborn rats to 95% O2 for 7 days, followed by recovery in air, are consistent with the findings reported by others (8, 9). Wet LW, total lung DNA, and alveolarization were significantly decreased by exposure to 95% O2 for 7 days, reflecting a global inhibition of lung growth. After 3 days of recovery in air, total lung DNA values in O2-exposed animals had caught up to those from animals exposed to air from birth. Furthermore, the decrease in LW seen in animals exposed to 95% O2 for 1 week had also recovered to be not statistically different from air-exposed animals by 14 days of age. LW/BW ratios were initially decreased after a 7-day exposure to 95% O2, then increased after 7 and 14 days of recovery in air, relative to air controls. This observation is consistent with the small (but not statistically significant) reduction in BW observed in O2-exposed animals during recovery in air, compared with animals exposed to air from birth.

Both bFGF mRNA and protein were reduced in the lung, compared with air-exposed control pups, after a 1-week exposure to 95% O2. After 1 week of recovery in room air, pups that had been exposed to 95% O2 showed a small but not significant increase in bFGF mRNA with an apparent increase in immunoreactive bFGF throughout all lung compartments, and in the whole lung, when compared with pups exposed to air throughout. FGF-R1 mRNA and protein in the total lung were not significantly affected by exposure to 95% O2 but were maintained at a stable level of expression during the early recovery period, compared with animals exposed to air from birth.

On the basis of the observations described previously, we speculated that upregulation of bFGF expression immediately after exposure to 95% O2, through its action on FGF-R1, would be involved in the compensatory lung growth observed immediately after the cessation of exposure to 95% O2. Intraperitoneal injection of a truncated soluble FGF-R1 at the end of the exposure period prevented the compensatory increase in DNA normally seen during the early recovery phase. Treatment with truncated soluble FGF-R1 also prevented the secondary septation occurring in parallel with the increase in DNA synthesis, thus confirming a critical role for the FGF-R1 in the compensatory lung growth occurring after 95% O2–mediated arrest of lung growth. A truncated soluble nerve growth factor receptor, used as a control compound, had no significant effect on compensatory DNA synthesis or alveolarization. Because of its high affinity for the FGF-R1, it is likely that bFGF is acting as a ligand for this receptor during compensatory lung growth, though it may not be the exclusive ligand, as acidic FGF also binds the FGF-R1 with high affinity (22). We have not assessed the expression of acidic FGF in this model as yet and cannot comment on its possible contribution at this time. An additional caveat is that despite there being an increase in immunoreactive bFGF in the early recovery phase after exposure to 95% O2, this does not necessarily mean that this increased bFGF was available for binding to the FGF-R1 because bFGF may have been sequestered in the subcellular matrix in a biologically inactive form, bound to matrix constituents such as heparan sulfate (23, 24). Unfortunately, this also makes targeting interventions against bFGF, such as the use of neutralizing antibodies, problematic due to masking of the ligand from the antibody. An alternative, and widely used, approach for in vivo and in vitro studies has been the use of suramin analogs to bind bFGF (25). However, suramin blocks the effects of several growth factors and does not provide specific inhibition of bFGF (26). The binding of ligands to the FGF-R1 may act directly to increase cell proliferation and stimulate septation and alveolarization, or the effect could be by an indirect pathway involving vascular endothelial growth factor. It is known that increased bFGF expression can result in induction of vascular endothelial growth factor expression (27), the binding of which to the vascular endothelial growth factor receptor-1 is known to be essential for alveolarization (6).

In summary, compensatory neonatal lung growth and septation, after 95% O2–mediated growth arrest, is at least partially mediated through activation of the FGF-R1. The precise ligand or ligands activating the FGF-R1 remain unknown; however, the temporal expression pattern of bFGF implicates this growth factor as a likely candidate.


   FOOTNOTES
 
Dr. Tanswell holds the Hospital for Sick Children Women's Auxiliary Chair in Neonatal Medicine.

Supported by Canadian Institutes of Health Research (Group grants) and Ontario Thoracic Society (an equipment grant), Postdoctoral Research Awards from the Canadian Institutes of Health Research and the Canadian Lung Association as well as a Clinician–Scientist Training Fellowship from the Hospital for Sick Children Research Institute (R.P.J.), and Grant 30125019 from the National Science Fund for Distinguished Young Scholars from the Natural Science Foundation of China (X.L.).

Received in original form July 5, 2002; accepted in final form February 26, 2003


   REFERENCES
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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