Abstract

Acute hyperoxic lung injury remains a major factor in the development of chronic lung disease in neonates. A critical step in the repair of acute lung injury is the proliferation of type II alveolar epithelial cells. Type II cell proliferation is stimulated by keratinocyte growth factor (KGF), an epithelial cell-specific mitogen. We sought to investigate KGF mRNA expression in relation to type II cell proliferation during hyperoxic lung injury. We studied a previously described newborn (NB) rabbit model of acute and chronic hyperoxic injury [C. T. D’Angio, J. N. Finkelstein, M. B. LoMonaco, A. Paxhia, S. A. Wright, R. B. Baggs, R. H. Notter, and R. M. Ryan. Am. J. Physiol. 272 (Lung Cell. Mol. Physiol. 16): L720–L730, 1997]. NB rabbits were placed in 100% O2 for 9 days and then recovered in 60% O2. RT-PCR was used to synthesize and amplify a 267-bp fragment of rabbit KGF cDNA from whole lung RNA. KGF mRNA expression was analyzed by ribonuclease protection assay, and mRNA abundance was quantified by phosphorimaging. Proliferating cell nuclear antigen immunohistochemistry was used on lung sections to identify proliferating cells. The rabbit partial cDNA sequenced was >95% homologous to human cDNA, and all amino acids were conserved. Whole lung KGF mRNA expression was increased 12-fold after 6 days of hyperoxia compared with control lungs, and remained increased throughout the 100% O2 exposure period. Proliferating cell nuclear antigen immunohistochemistry showed an increase in type II cell proliferation after 8–12 days of hyperoxia. NB rabbits exposed to hyperoxic injury exhibit increased whole lung KGF mRNA expression preceding type II cell proliferation. KGF may be an important mitogen in the regulation of alveolar epithelial repair after hyperoxic lung injury.

  • oxygen toxicity
  • alveolar epithelial cell proliferation
  • messenger ribonucleic acid

primary lung injury and subsequent chronic lung disease (CLD) are major causes of mortality and morbidity in both preterm and term neonates. Neonatal CLD presumably results from a combination of acute primary lung injury and hyperoxia with mechanical ventilation at a time when the lung is still developing. The alveolar epithelial lining of the lung is composed of type I and type II pneumocytes. In normal adult lung, a very small number of type II cells continuously proliferate and differentiate into type I cells (36). After hyperoxic lung injury, type I cells are destroyed and type II cells, which are relatively resistant to hyperoxia, proliferate and differentiate into type I cells to restore the disrupted alveolar epithelium (1, 8). Newborns (NBs) of several species are relatively O2 tolerant in comparison to adults (13); one factor that may explain this relative resistance is the ability of NB lung cells to proliferate more rapidly to repair the lung, particularly the alveolar epithelium.

It is presently unknown what regulates type II cell proliferation in the normal developing neonatal lung or in neonatal lung injury repair. Growth factors that are mitogenic for adult rat, rabbit, or human type II cells in vitro include epidermal growth factor, transforming growth factor-α (TGF-α), insulin, and acidic and basic fibroblast growth factor (FGF) (16, 17, 20, 25). Keratinocyte growth factor (KGF; also known as FGF-7), a member of the FGF family, is a potent mitogen specific for epithelial cells (12). KGF mRNA expression is detected in several stromal fibroblast lines derived from embryonic, neonatal, and adult sources (12). It is important in fetal pulmonary growth and differentiation (11, 19, 27). Furthermore, lungs of transgenic mice expressing a dominant negative for the KGF receptor (FGF receptor 2-IIIb) under control of the surfactant protein C promoter have grossly abnormal lung development, with only two primordial epithelial tubes and no branching morphogenesis (24). Transgenic mice overexpressing KGF exhibit lethal papillary cystadenoma, with marked enlargement of the bronchial air spaces (29). KGF induces type II cell proliferation in vitro and in vivo (23, 33). Recently, KGF was found to be protective when given before several forms of lung injury including hyperoxia (22), bleomycin (38), hydrochloric acid (37), and radiation (31, 38).

We have developed and described previously a model of acute and chronic hyperoxic lung injury in neonatal rabbits (9). During the acute period of injury, there is inflammation, abnormal surfactant function, and type II cell hyperplasia. During the chronic phase of injury, there is increased collagen deposition and septal thickening, indicating fibrosis. Because KGF stimulates type II cell proliferation, is induced by proinflammatory cytokines (2, 6), and has a protective effect in lung injury (22, 31, 37, 38), it may be an important regulator of type II cell proliferation in the NB lung after O2 injury. We hypothesized that KGF mRNA would be increased during the alveolar epithelial proliferative phase of lung repair. To test our hypothesis, we measured KGF mRNA expression in the postnatal developing rabbit lung during hyperoxia-induced injury and used staining for proliferating cell nuclear antigen (PCNA) as a marker for cellular proliferation.

MATERIALS AND METHODS

NB rabbit hyperoxia model. We used an NB rabbit hyperoxia model described previously (9), with slight modification. Briefly, New Zealand White term NB rabbit litters were placed in a large Plexiglas chamber and exposed to humidified 100% O2 or room air. The hyperoxia exposure protocol was approved by the University of Rochester (Rochester, NY) Committee on Animal Resources. Previously generated samples from the acute and chronic phases of injury were used. In the acute phase, the day 4 time point and the time at which 50% of the pups died or were killed because of severe respiratory distress (LD50) were assessed. The LD50 was determined to beday 8 or day 11; thus day 10 room air animals were used as controls. The chronic phase of injury was induced by placing the animals in 100% O2 for 8 days and then by allowing them to recover in 60% O2 for up to 36 days of age; samples from day 22 and day 36 time points were studied. To characterize further the acute phase of this model, an experimental group of animals was exposed to 100% O2 for 9 days and then placed in 60% O2 for an additional 5 days. Animals from these litters and room air control age-matched animals were killed at days 2,4, 6,8,10,12, and14 of age. At least two litters (of ≥6 animals each) were exposed to each condition, and no litter contributed more than two animals per condition per time point (except for the LD50). Animals were killed by an intraperitoneal injection of 200 mg/kg of pentobarbital sodium. A total of 41 hyperoxia-exposed and 39 room air-exposed NB rabbits were examined. In addition, three samples from normal fetal rabbit lungs at fetal days 25,28, and31 and one sample atday 0 (at birth) were available for study (full-term gestation is 31 completed days). Adult rabbit exposures were performed as previously described (14).

Sample preparation. Lungs were processed as previously described (10, 25). Briefly, immediately after the animal was killed, a thoracotomy was performed, the right main stem bronchus was clamped, and the right lung was removed and immediately flash-frozen in liquid nitrogen for later RNA extraction. The left lung was instilled in situ with phosphate-buffered 10% Formalin under 25 cmH2O pressure for 30 min. It was then removed for a further 16–24 h of Formalin fixation and preserved in 70% ethanol until embedded in paraffin for later sectioning.

RNA isolation. Total RNA was extracted from NB rabbit lungs by the method of Chomczynski and Sacchi (7) as previously described (10, 25). Briefly, lung tissue was immediately flash-frozen in liquid nitrogen and then minced on ice and homogenized with 4 M guanidinium thiocyanate, 25 mM sodium citrate, pH 7, 0.5% sarcosyl, and 0.1 M 2-mercaptoethanol adjusted to 20 mM sodium acetate, pH 4.0; RNA was extracted with phenol and chloroform and precipitated with isopropanol. RNA was then washed with 75% ethanol and solubilized in diethyl pyrocarbonate-treated water.

Generation of rabbit KGF cDNA clones.Moloney murine leukemia virus reverse transcriptase polymerase (GIBCO BRL, Grand Island, NY) was used to synthesize rabbit lung cDNA, according to the manufacturer’s recommendations, from 2 μg of normal 4-day-old rabbit lung RNA in the presence of oligo(dT)15 (Promega, Madison, WI). PCR amplification with Taq I polymerase (Promega) was used to generate a 267-bp KGF cDNA corresponding to the human KGF nucleotide sequence (nucleotides 654–920) (12). Primer A(5′-TCTGTCGAACACAGTGGTACCTG-3′) and primer B (5′-TGTGTGTCCATTTAGCTGATGC-3′), corresponding to human nucleotide sequences 654–676 and 899–920, respectively, were purchased from Oligos Etc. (Wilsonville, OR). The reaction was allowed to proceed at 94°C for 3 min, then for 39 cycles of 55°C for 1.5 min, 70°C for 1.5 min, and 94°C for 1 min. The reaction was continued for 5 min at 70°C and held at 4°C until further analysis. PCR products were fractionated on a 2% Tris-acetate-EDTA (GIBCO BRL, Gaithersburg, MD) low-melting-point agarose gel (Sigma, St. Louis, MO), and the corresponding rabbit partial KGF (rbKGF) fragment was purified with QIAquick PCR gel purification kit (Qiagen, Santa Clara, CA), subcloned into theSrf I site of pCR-Script SK(+) plasmid, and propagated in Epicurian Coli XL1-Blue MRF′ Kan supercompetent cells (Stratagene, La Jolla, CA). The identity of the cloned rbKGF cDNA was confirmed by DNA sequencing at the Molecular Genetics Core Facility at the University of Rochester Medical Center.

Riboprobe synthesis.Plasmid DNA was isolated with Nucleobond AX plasmid purification according to the manufacturer’s recommendations (Machervy-Nagel, Düren, Germany). The restriction enzymesBamH I and Not I (GIBCO BRL, Grand Island, NY) were used to linearize rbKGF, generating antisense and sense templates, respectively. rbKGF templates were transcribed with32P-labeled UTP (800 Ci/mmol; NEN, Boston, MA) in the presence of either T7 or T3 DNA-dependent RNA polymerases (Ambion, Austin, TX) to synthesize sense and antisense cRNA probes, respectively. Both actin and 28S cRNA probes were synthesized with templates supplied by Ambion. RNA probes were electrophoresed through a 5% polyacrylamide-8 M urea gel. Appropriate full-length32P-labeled cRNAs were localized by autoradiography and eluted from the excised gel into 0.5 M ammonium acetate-1 mM EDTA-0.2% SDS overnight at room temperature.

Ribonuclease protection assay. A total of 80 lungs was examined. Whole lung RNA samples (5 or 10 μg, same within experiments) were hybridized overnight at 45°C in a buffered 50% formamide solution, with 1 × 105 counts/min of gel-purified32P-labeled antisense riboprobes. Hybridization reactions were digested with 1 U of RNase A and 40 U of RNase T1 for 30 min at 37°C. Protected fragments were recovered by ethanol precipitation and analyzed on 5% polyacrylamide-8 M urea gel. All reagents and conditions were supplied by Ambion. Gels were either visualized directly by autoradiography or fixed in 10% acetic acid and 10% methanol in double-distilled water for 20 min and then vacuum-dried at 90°C for 20–30 min before exposure to a phosphorimager screen.

Yeast tRNA was hybridized to rbKGF antisense probe and used as a negative control. The positive control consisted of the protected fragment from the nonradioactive rbKGF sense cRNA hybridized to rbKGF antisense probe. Mouse liver RNA was used as a positive control for the actin antisense probe. For each time point, three or four RNA samples from different animals were tested for each condition (hyperoxia and control). Each ribonuclease protection assay (RPA) was repeated at least three times, and one representative RPA experiment for each time point was analyzed by phosphorimaging.

Phosphorimager. Dried gels exposed to phosphorimager screens (Molecular Dynamics, Sunnyvale, CA) were analyzed, and mRNA abundance was quantified and normalized to 28S rRNA with ImageQuant software (Molecular Dynamics). Measurements were corrected for possible variation in loading by determining the ratio of KGF signal to the internal control 28S rRNA signal (KGF/28S). The signal representing rbKGF mRNA or 28S rRNA protected fragments was integrated over identical areas and background corrected. The relative abundance of rbKGF mRNA to 28S rRNA was calculated as follows: for each protected fragment, the integrated value of background was subtracted from the integrated value of rbKGF and divided by the integrated value of 28S rRNA. The mean for the room air samples at each time was assigned a relative value of 1. Hyperoxia-exposed samples at each time were assigned the KGF/28S value for each sample divided by the average KGF/28S value for the age-matched room air samples. For each time point, results were expressed as means ± SE of the triplicate or quadruplicate sample points.

Densitometry. mRNA abundance was quantified by video densitometry of autoradiograms with MetaMorph software (Universal Imaging, Inner Media, Hollis, NH). A radiogram of one representative RPA experiment for each time point was analyzed. Actin was used as a control for RNA loading. The same measurement procedure was followed by a single person as described inPhosphorimager.

PCNA immunohistochemistry.Lungs were embedded in paraffin, and 4-μm sections were made. After the sections were deparaffinized in Pro-Par (Anatech, Battle Creek, MI) and rehydrated in ethanol, they were treated with 0.3% hydrogen peroxide in methanol for 30 min and then washed in 0.3% Triton X-100 in PBS for 30 min. Sections were stained after blocking in 5% normal horse serum, followed by incubations with a monoclonal anti-rat PCNA antibody, PC10 (Vector Laboratories, Burlingame, CA), a biotinylated horse anti-mouse IgG secondary antibody (Vector Laboratories), and the Vectastain Elite ABC peroxidase kit according to the manufacturer’s protocol (Vector Laboratories). Sections from normal rabbit intestine were used as a positive control. Adult rabbit lung sections were also used as a control: normal adult lung would be expected to show no or minimal PCNA staining, and hyperoxia-injured, room air-recovered adult lungs would be expected to show robust epithelial cell PCNA staining.

Statistical analysis.Statistical analysis was done with the Intercooled Stata program (Stata, College Station, TX). Student’st-test with Bonferroni correction for repeated measures and two-way ANOVA were used to analyze multiple continuous variables. All tests were two sided, andP ≤ 0.05 was considered significant.

RESULTS

Partial rabbit cDNA sequence.A 267-bp rbKGF cDNA was generated from whole lung RNA (222 bp excluding primers A andB). The sequenced fragment was 95% homologous to the human sequence at the base-pair level (nucleotides 654–920) (12). No base-pair differences resulted in a difference at the amino acid level (Fig.1).

Fig. 1.

Partial rabbit (Rab) cDNA nucleotide sequence and encoded 267-bp Rab keratinocyte growth factor (rbKGF) sequence. Nucleotides are numbered from left. Human (Hum) and mouse (Mou) partial KGF cDNA nucleotide sequences are shown for comparison. Underline, primers based on Hum sequence. Identity, same as Hum. The 222 bp (excluding primers) of rbKGF cDNA fragment were 95% homologous to Hum sequence at base-pair level (nucleotides 677–898) and 100% at amino acid (aa) level.

KGF mRNA expression during neonatal hyperoxic lung injury repair. We initially analyzed a total of 31 RNA samples from day 4, LD50, day 22, and day 36. Actin was used as a housekeeper gene, and gels were visualized directly by autoradiography. With the use of densitometry, rbKGF mRNA expression was no different from age-matched room air control rabbits after 96 h of 100% O2 and also was not different atdays 22 or36 during recovery in 60% O2 compared with air-breathing control rabbits. However, rbKGF mRNA expression was increased 16.7-fold at the LD50 (day 8 or11) time of most severe injury compared with day 10air-breathing control rabbits (P < 0.001 by t-test with Bonferroni correction for repeated measures; Fig. 2).

Fig. 2.

KGF mRNA expression during acute injury. Homogenized lung RNA (5 μg) from day 10 room air (RA)-exposed or hyperoxia-exposed newborns (NBs) at time (day 8 or11) at which 50% had died or were killed because of severe respiratory distress (LD50) was hybridized overnight with gel-purified 32P-labeled antisense KGF and actin riboprobes. Samples were then digested with ribonuclease, and protected fragments were analyzed on 5% polyacrylamide-8 M urea gel. A: rbKGF mRNA expression is clearly increased at LD50 time point. Gel is representative of 3 experiments and shows bands for rbKGF mRNA and double bands for actin. n1–n4, Sample no.B: relative rbKGF mRNA expression, quantified by densitometry as described inmaterials and methods, was increased 16.7-fold in LD50 hyperoxia samples compared with air-breathing control Rab (P < 0.001). RA animals were assigned a value of 1.

To further delineate the timing of KGF mRNA changes in this model, we then studied a total of 49 RNA samples from postnataldays 2, 4, 6, 8, 10, 12, and14 and 3 samples from fetal rabbit lungs; 28S rRNA was used as an internal control, and gels were analyzed by phosphorimager. rbKGF mRNA expression was higher in the fetal samples compared with room air control rabbits at all time points (Fig.3 A). rbKGF mRNA expression was increased 12-fold at day 6 of hyperoxia compared with air-breathing control rabbits (P = 0.003); expression was increased sixfold at day 8. After animals were removed from 100% O2and placed in 60% O2, rbKGF mRNA expression dramatically decreased sevenfold at day 12 and returned to baseline at day 14 (Fig. 3 B). Quantitative results are shown in Fig. 4. Two-way ANOVA revealed both a significant effect of hyperoxia (P = 0.004) and a significant change over time of rbKGF mRNA expression (P= 0.05).

Fig. 3.

KGF mRNA expression in normal and hyperoxia-exposed neonatal Rab lung. Homogenized lung RNA (10 μg) from fetal Rab atdays 25 (F25),28 (F28), and31 (F31) and NB rabbits atdays 0, 2, 4, 6, 8 10, 12, and 14 of RA or O2 exposure was hybridized overnight with gel-purified,32P-labeled antisense KGF and 28S rRNA (28S) riboprobes. Samples were then digested with ribonuclease, and protected fragments were analyzed on 5% polyacrylamide-8 M urea gel. Gels are representative of 3 experiments each.A: rbKGF mRNA expression was minimal after birth in RA samples, whereas expression was increased in fetal tissue. B: during acute and chronic injury, rbKGF mRNA expression was increased at days 6 and 8 of injury.

Fig. 4.

Relative changes in KGF mRNA expression in NB Rab at various time points between 2 and 14 days (D) of hyperoxic injury compared with control Rab. A total of 49 RNA samples fromday 2, 4, 6, 8, 10, 12, and14 time points was studied, 28S rRNA was used as housekeeper gene, and gels were analyzed by phosphorimager. For each time point, RA animals were assigned a value of 1. Relative rbKGF mRNA expression was increased 12-fold atday 6of hyperoxia compared with air-breathing control Rab (* P < 0.05). This expression was increased 6-fold at day 8, followed by a 10-fold decrease atday 12 and return to baseline atday 14. There was both a significant effect of hyperoxia (P = 0.004) and a significant change over time (P = 0.05) by two-way ANOVA.

Alveolar epithelial cell proliferation in response to hyperoxia. A total of 82 neonatal lung sections was examined. Adult rabbit sections, neonatal rabbit intestine, and staining in the absence of primary antibody were used as controls. As expected, adult rabbits showed no or rare PCNA staining (Fig.5 A). Adult rabbits exposed to 64 h of 100% O2 and then recovered in room air for 3 days showed the expected robust increase in proliferation, in particular in the alveolar epithelium (Fig. 5,B andC). Neonatal rabbit intestinal sections showed appropriate PCNA staining (data not shown). In neonatal lung sections, PCNA immunostaining was present in nuclei of parenchymal cells in all air-breathing NB rabbits, with staining atdays 4 (Fig.5 E) through14 somewhat greater than atdays 0, 22, and36. Many of the PCNA-positive cells were identified as type II alveolar epithelial cells on the basis of their morphological appearance. During exposure to 100% O2, there was initially a depression in parenchymal PCNA staining [days 2, 4 (Fig. 5 F), and6], but this increased while still in 100% O2 atday 8 (Fig. 5,H andI) and at the LD50 (Fig.5 J). After the switch to 60% O2 at day 9, nuclear PCNA staining increased in intensity, and the number of positive cells appeared increased, particularly in the alveolar epithelium, at days 10 (Fig. 5 L) and12 (data not shown) (1 and 3 days of recovery in 60% O2, respectively) compared with air-breathing control rabbits (Fig.5 K). This was more pronounced in areas of more severe injury and inflammation. The staining decreased atday 14 to levels similar to those in air-breathing animals, although there was still abnormal architecture. During the period when fibrosis was present (days 22 and 36), PCNA staining was generally similar in amount for air-breathing and hyperoxia-exposed neonatal rabbits. At day 22, cells that appeared to be fibroblasts were PCNA positive, and staining remained somewhat increased in focal areas of injury in the hyperoxia-exposed animals (data not shown). Lung sections from neonatal rabbits showed no staining when the full procedure was performed in the absence of the primary antibody (Fig.5 D).

Fig. 5.

Alveolar epithelial cell proliferation assessed by proliferating cell nuclear antigen (PCNA) immunohistochemical staining with anti-PCNA antibodies on lung sections from control and O2-exposed adult and NB Rab.A: normal adult Rab. Magnification, ×20. B andC: adult hyperoxic Rab at 3 days recovery at magnifications of ×20 and ×40, respectively.D: negative control without primary antibody present with no staining. Magnification, ×40.E: normal 4-day-old Rab. Magnification, ×20; F: hyperoxia-exposed 4-day-old Rab. Magnification, ×20.G: normal 8-day-old Rab. Magnification, ×40. H: hyperoxia-exposed 8-day-old Rab. Magnification, ×40.I: hyperoxia-exposed 8-day-old Rab. Magnification, ×20. J: LD50(days 8–11) hyperoxic animal. Magnification, ×40. K: normal 10-day-old Rab. Magnification, ×40.L: hyperoxia-exposed 10-day-old Rab. Magnification, ×20. Adult hyperoxic Rab exposed to 100% O2 for 64 h and then allowed to recover in room air are known to have increased type II cell proliferation during recovery. Note absence of staining in normal lung and increased PCNA-staining positive nuclei denoting cellular proliferation in 3-day recovered lung. Some of these cells are part of a proliferating cuboidal alveolar epithelium, consistent with type II cell proliferation; in NB Rab, there is alveolar cell PCNA staining, denoting proliferation at 4 days. At 4 days of hyperoxia, there is an increase in airway epithelial cell proliferation and a decrease in alveolar cell proliferation. In 8-day-old normal animals, alveolar cell proliferation remains present but is increased in age-matched hyperoxic animals. At LD50, there continues to be airway and alveolar epithelial cell proliferation present, and in some areas, there is evidence for a reparative cuboidal alveolar epithelium, indicating type II cell proliferation. Atday 10 (1 day of recovery in 60% O2), there is increased intensity of nuclear PCNA staining and increased no. of PCNA-positive cells, particularly in alveolar epithelium, compared with air-breathing control Rab.

DISCUSSION

KGF is a potent mitogenic growth factor highly specific for epithelial cells (12). It has been shown to induce type II cell proliferation in vitro (23) and in vivo (33). Also, intratracheal KGF has been found to be protective when given before a variety of toxic agents (31, 37, 38), including hyperoxia (22). Furthermore, KGF has been shown to be essential in wound reepithelialization after skin injury (35) and to enhance corneal epithelial wound healing in vivo (30). Similarly, KGF may play an important role in epithelial lung repair by stimulating type II cell proliferation. In this report, we investigated the endogenous expression of KGF after hyperoxia-induced lung injury in the NB rabbit and its relation to type II cell proliferation. Whole lung KGF mRNA was increased during the acute phase of injury and was followed temporally by type II cell proliferation as demonstrated by PCNA immunohistochemistry.

Hyperoxic lung injury, a major factor in neonatal CLD development, was used to induce fibrosis in an NB rabbit model in our laboratory. Our laboratory has previously reported (9) the physiological, histological, and molecular characteristics of this model. In this report, we show that KGF increased during the acute phase of injury secondary to 100% O2. Temporally, KGF mRNA is first increased several days after the initial influx of inflammatory cells into the lung but before maximal inflammation seen at 8–11 days of 100% O2. After the switch from 100 to 60% O2 atday 9, KGF expression decreased rapidly to become suppressed at day 12before it returned to baseline during the chronic fibrotic phase of injury. An endogenous KGF response after hyperoxic lung injury has not been reported. Several studies to date (2, 6, 18, 32, 34) have examined the regulation of KGF gene expression. KGF mRNA was induced in vitro by interleukin (IL)-1α and IL-1β, platelet-derived growth factor (PDGF)-BB, and TGF-α (2, 6). KGF protein synthesis was increased as well in response to IL-1 and TGF-α. IL-1 and TGF-α are produced by activated macrophages and play an important role during inflammation and wound healing. Activated macrophages are also an important source for PDGF, which might further contribute to the activation of KGF gene expression. In the NB rabbit hyperoxic injury model, our laboratory (9) has shown increased alveolar macrophages in the acute phase of injury. In addition, data from these NB rabbits suggest that IL-1β mRNA and protein increased by 2–4 days of hyperoxia, peaked aroundday 6, and persisted through 10 days of the acute phase of hyperoxic injury (unpublished data). Others have found that IL-1β increased significantly in adult mice after 4 days of exposure to 100% O2 (15; G. Pryhuber, personal communication). Also, data from our laboratory (26) suggested that TGF-α is increased in bronchoalveolar lavage fluid from these animals, starting in the acute phase and continuing in the chronic phase of injury. During hyperoxic injury, increased production of IL-1, TGF-α, and other cytokines produced by alveolar macrophages and other inflammatory cells are potential potent inducers of KGF, which, in turn, could then stimulate type II cell proliferation to repair alveolar damage.

In this study, we used PCNA as a marker for proliferating cells. PCNA is an auxiliary protein for DNA polymerase-δ and is required for DNA replication and repair (3, 28). Although positive PCNA staining could reflect DNA repair, it has been shown to correlate well in hyperoxic lung injury with several other markers of cellular proliferation (5). In the normal growing and developing neonatal rabbit lung, there was ongoing type II cell proliferation at all time points, whereas little proliferation was detected in adult animals. In the hyperoxia-injured neonatal rabbits, we found an initial decrease in PCNA staining of type II cells after 2 and 4 days of 100% O2, similar to McGrath (21) in mice. McGrath showed that exposure of NB mice to 100% O2 resulted in induction of the cyclin-dependent kinase inhibitor p21WAF/C1P1 concomitant to alveolar growth arrest; a similar phenomenon may occur in NB rabbits exposed to hyperoxia. However, we continued the exposure to 100% O2 for a longer period, and there is an actual increase in PCNA staining in the neonates during the more severe acute phase of injury. This is in marked contrast to adult animals that generally do not demonstrate type II cell proliferation while in 100% O2 but do so in recovery. Others (14) have shown that type II cell proliferation in adult rats exposed to 100% O2 and then recovered in room air does not take place until the exposure ends, peaks at 3 days after exposure, and then gradually returns to normal. Recently, KGF was found to induce cell proliferation at the cell cycle level by increasing cyclin-dependent kinase-1 and -4 in the presence of Matrigel (4). Also, KGF was shown to facilitate DNA repair of radiation-induced damage by enhancing DNA polymerase activity (31). It is possible that KGF plays an important role in the hyperoxic-injury repair process by inducing DNA polymerases, in addition to inducing type II cell proliferation. The mechanism of repair and replenishment of the alveolar epithelium by type II cell proliferation and then differentiation into type I cells (36) may also reflect a developmental process, with “injury recapitulating development.” Others (27) have demonstrated that fetal levels of KGF mRNA are higher before birth; this is true for fetal rabbits as well.

It has been proposed that more rapid repair of the alveolar epithelium decreases later fibrosis (36). Our study does not specifically address the role of KGF-mediated type II cell proliferation in the development of subsequent fibrosis. However, it is possible that endogenous upregulation of KGF during the acute phase of hyperoxic lung injury enhances epithelial repair and is important in minimizing later CLD. Similarly, exogenous treatment with KGF may also ameliorate or prevent the development of fibrosis. Further studies are required to investigate those questions.

In summary, we have shown that NB rabbits exposed to 6–11 days of 100% O2 have a significant increase in total lung KGF mRNA expression followed by increased alveolar epithelial cell proliferation. Increased KGF production may contribute to the repairing lung alveolar epithelial cell proliferation prominent several days later in this model of hyperoxia-induced fibrosis.

Acknowledgments

This work was funded by Wyeth Pediatrics Neonatology Research Grants Program (L. Charafeddine); Gilbert Forbes Resident Fellowship Grants Award (to L. Charafeddine); National Heart, Lung, and Blood Institute Grants 5F32-HL-09022 (to C. T. D’Angio) and HL-02630 (to R. M. Ryan); and the March of Dimes (R. M. Ryan).

Footnotes

  • Address for reprint requests: R. M. Ryan, Univ. of Rochester, Children’s Hospital at Strong, 601 Elmwood Ave., Box 651, Rochester, NY 14642.

  • 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.

REFERENCES

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