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Am J Physiol Lung Cell Mol Physiol 287: L718-L729, 2004. First published April 16, 2004; doi:10.1152/ajplung.00084.2004
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EDITORIAL FOCUS

Transient induction of TGF-{alpha} disrupts lung morphogenesis, causing pulmonary disease in adulthood

T. D. Le Cras,1 W. D. Hardie,1 G. H. Deutsch,2 K. H. Albertine,3 M. Ikegami,1 J. A. Whitsett,1 and T. R. Korfhagen1

Divisions of 1Pulmonary Biology and 2Pathology, Cincinnati Children's Hospital Medical Center, Department of Pediatrics, University of Cincinnati, Cincinnati, Ohio 45229; and 3Department of Pediatrics, University of Utah, Salt Lake City, Utah 84132

Submitted 10 March 2004 ; accepted in final form 12 April 2004


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Clinical studies have associated increased transforming growth factor (TGF)-{alpha} and EGF receptor with lung remodeling in diseases including bronchopulmonary dysplasia (BPD). BPD is characterized by disrupted alveolar and vascular morphogenesis, inflammation, and remodeling. To determine whether transient increases in TGF-{alpha} are sufficient to disrupt postnatal lung morphogenesis, we utilized neonatal transgenic mice conditionally expressing TGF-{alpha}. Expression of TGF-{alpha} from postnatal days 3 to 5 disrupted postnatal alveologenesis, causing permanent enlargement of distal air spaces in neonatal and adult mice. Lung volume-to-body weight ratios and lung compliance were increased in adult TGF-{alpha} transgenic mice, whereas tissue and airway elastance were reduced. Elastin fibers in the alveolar septae were fragmented and disorganized. Pulmonary vascular morphogenesis was abnormal in TGF-{alpha} mice, with attenuated and occasionally tortuous arterial branching. The ratios of right ventricle weight to left ventricle plus septal weight were increased in TGF-{alpha} mice, indicating pulmonary hypertension. Electron microscopy showed gaps in the capillary endothelium and extravasation of erythrocytes into the alveolar space of TGF-{alpha} mice. Hemorrhage and inflammatory cells were seen in distal air spaces at 1 mo of age. In adult TGF-{alpha} mice, alveolar remodeling, nodules, proteinaceous deposits, and inflammatory cells were seen. Immunostaining for pro-surfactant protein C showed that type II cells were abundant in the nodules, as well as neutrophils and macrophages. Trichrome staining showed that pulmonary fibrosis was minimal, apart from areas of nodular remodeling in adult TGF-{alpha} mice. Transient induction of TGF-{alpha} during early alveologenesis permanently disrupted lung structure and function and caused chronic lung disease.

bronchopulmonary dysplasia; neonatal lung development; epidermal growth factor receptor; pulmonary hypertension; lung remodeling

THE EPIDERMAL GROWTH FACTOR (EGF) receptor (EGFR) and its ligands [transforming growth factor-{alpha} (TGF-{alpha}), EGF, heparin binding EGF, and amphiregulin] contribute to epithelial development, maturation, and repair. Increased TGF-{alpha} and EGFR have been detected in patients with bronchopulmonary dysplasia (BPD) (40, 41), congenital diaphragmatic hernia (16), cystic fibrosis (17), idiopathic pulmonary fibrosis (5), and asthma (3). Mechanisms responsible for the induction of TGF-{alpha} and EGFR signaling in these diseases are unclear, but in experimental studies TGF-{alpha} and EGFR signaling were induced by hyperoxia (45), reactive oxygen species (32), and proinflammatory cytokines, including IL-4, TNF-{alpha}, and IL-13 (9, 29). Secretion of IL-8, which is elevated in patients with chronic obstructive pulmonary disease (24), by bronchial epithelial cells in response to cigarette smoke extract, is dependent on EGFR activation and autocrine production of TGF-{alpha} (34). Collectively, these and other studies suggest that EGFR and its ligands are induced by inflammatory processes and may play a central role in lung remodeling and disruption of lung morphogenesis (8).

In rodents and humans, the alveolar phase of lung development occurs postnatally (11). During this final phase of lung morphogenesis, lung surface area increases markedly due to the generation of alveoli by secondary septation (6, 11). Pulmonary vascular growth also undergoes rapid expansion in close coordination with alveolarization (11, 31). Disruption of postnatal alveolarization and pulmonary vascular growth is a major problem in BPD (13, 22). The etiology of BPD is complex but may include prematurity, ventilation, exposure to hyperoxia, and prenatal infection and inflammation (22, 23, 26, 46). Although the pathogenesis of BPD is still unclear, aberrant growth factor and cytokine signaling are thought to play a role in disruption of lung morphogenesis and lung remodeling (22, 23).

Increased production of TGF-{alpha} and EGFR by epithelial cells, alveolar macrophages, and vascular smooth muscle has been reported in infants with BPD (40, 41). Chronic production of TGF-{alpha} in the lungs of transgenic mice driven by the human surfactant protein-C (SP-C) promoter (SPC-TGF-{alpha} mice) disrupted alveolar and vascular development and caused pulmonary fibrosis and pulmonary hypertension (18, 25, 27). However, whether the alveolar phase of lung development is particularly sensitive to increases in TGF-{alpha} and EGFR signaling is unclear. In previous studies in SPC-TGF-{alpha} transgenic mice, TGF-{alpha} was chronically expressed throughout pre- and postnatal lung development (18, 25, 27). In addition, although the role of TGF-{alpha} and EGFR in the pathogenesis of BPD is still unclear, infants who are exposed to injurious stimuli (such as hyperoxia or infection) for limited periods of time may experience only transient increases in cytokines and growth factors such as TGF-{alpha}. The goal of this study was to determine whether transient induction of TGF-{alpha} during the postnatal phase of lung morphogenesis is sufficient to disrupt alveolar and vascular development and cause chronic lung disease. Utilizing conditional transgenic mice, we transiently induced TGF-{alpha} concentrations during the alveolar phase of postnatal lung morphogenesis (postnatal days 3–5). The acute and long-term effects of this short period of increased TGF-{alpha} on alveolar and vascular structure and function were studied.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Conditional transgenic mice. All animal procedures and protocols were approved by the Animal Care and Use Committee at the Cincinnati Children's Hospital Research Foundation (Cincinnati, OH). All mice were FVB/N strain. A conditional doxycycline (Dox)-regulatable transgenic system was used to induce TGF-{alpha} expression in the lungs of newborn mice during early alveogenesis (19, 33, 43). Generation and characterization of the conditional TGF-{alpha} mice have been previously described (19). To generate conditional transgenic mouse pups for this study, we used two transgenic mouse lines: 1) "activator" mice (CCSP-rtTA) expressing the reverse tetracycline transactivator transcription factor (rtTA) under the control of a 2.3-kb element from the rat Clara cell secretory protein (CCSP) gene promoter (which confers lung specific expression) (33, 42, 43) and 2) "responder" transgenic mice [(tetO)7-TGF-{alpha} line 22.1], in which a human transgene coding for TGF-{alpha} is under the control of tetracycline response elements [(tetO)7] (19). To generate mouse pups, we mated homozygous CCSP-rtTA+/+ mice to heterozygous (tetO)7-TGF-{alpha}+/– mice. The resulting litters contained bitransgenic [CCSP-rtTA+/–/(tetO)7-TGF-{alpha}+/–] and single transgenic (CCSP-rtTA+/–) pups.

Dox induction of TGF-{alpha}. To induce TGF-{alpha} expression in the lungs of bitransgenic pups, we gave nursing dams chow containing Dox (625 mg/kg) from postnatal days 3 to 5 only, during the saccular-to-alveolar transition. Dox is secreted into the milk of lactating dams (33). We ceased Dox treatment after postnatal day 5 by returning the nursing dams to normal mouse chow. Single transgene (CCSP-rtTA+/– only) littermates exposed to Dox were used as controls throughout the study. All mice were genotyped by PCR analysis of tail DNA for CCSP-rtTA (31) and (tetO)7-TGF-{alpha} transgenes (19). Mice were killed with pentobarbital sodium (65 mg/ml) euthanasia solution (Fort Dodge Animal Health, Fort Dodge, IA) after 1 and 2 days of Dox treatment (postnatal days 4 and 5) and 2, 5, and 9 days after Dox treatment for 2 days (Dox treatment postnatal days 3–5, killed at postnatal days 7, 10, and 14). Additional litters were treated with Dox from postnatal days 3 to 5 and then killed at 6 wk of age (adults). Bitransgenic mice from litters of mice not exposed to Dox were also killed at 5 days of age to determine whether there was induction of TGF-{alpha} in the absence of Dox (transgene "leak").

TGF-{alpha} measurements. A TGF-{alpha} ELISA was used to determine both the concentrations of TGF-{alpha} in the lungs of CCSP-rtTA+/–/(tetO)7-TGF-{alpha}+/– and control mice after 1 and 2 days of Dox treatment as well as the time for TGF-{alpha} concentrations to return to control levels after Dox was removed. Litters of mice were treated with Dox from postnatal days 3 to 4 (1 day) and 3 to 5 (2 days) and then killed. Mice were also killed 2 and 9 days after ceasing Dox treatment (Dox treatment postnatal days 3–5, pups killed at 7 and 14 days postnatal age). After performing a thoracotomy, lungs were removed. To measure TGF-{alpha} concentrations, a whole left lung was sonicated in phosphate-buffered saline (1x PBS, pH 7.4) containing protease inhibitors ("Complete" protease inhibitor cocktail; Roche, Indianapolis, IN) and Nonidet P-40 (1%; Sigma). Lung homogenates were centrifuged at low speed (1,000 g) to remove insoluble debris. Supernatants were diluted 1:2 in 1x PBS containing protease inhibitors and assayed with a human TGF-{alpha} ELISA kit following the manufacturer's instructions (Oncogene Research Products, San Diego, CA). Lung homogenate protein levels were determined with a bicinchoninic acid protein assay (Sigma). TGF-{alpha} concentrations in lung homogenates (pg/ml) were normalized to total lung protein levels (mg/ml). TGF-{alpha} concentrations were also measured in lung homogenates from 5-day-old CCSP-rtTA+/–/(tetO)7-TGF-{alpha}+/– mice not exposed to Dox to determine the TGF-{alpha} levels in bitransgenic mice in the absence of Dox.

Lung histology, volume-to-body weight ratios, histochemistry, and immunostaining. Lung histology was performed on CCSP-rtTA+/–/(tetO)7-TGF-{alpha}+/– and control mice treated with Dox (2 days) and at intervals after Dox removal (2, 5, and 9 days), including up to 6 wk of age (adults). Lungs were inflation fixed by tracheal installation of 4% paraformaldehyde in 1x PBS (pH 7.4) at constant pressure (25 cmH2O). After 24 h, lungs were transferred to 70% ethanol. After another 24 h, the lungs were cut into 1-mm sections starting at the point where the bronchus enters the left lung. Three 1-mm sections of lung per animal were embedded in paraffin. Five-micrometer-thick paraffin sections were cut and stained with hematoxylin and eosin. Lung volumes were measured on 6-wk-old adult bitransgenic [CCSP-rtTA+/–/(tetO)7-TGF-{alpha}+/–] and control mice following Dox treatment from postnatal days 3–5. Lungs were inflated with 4% paraformaldehyde in 1x PBS (pH 7.4) at 25 cmH2O, and lung volumes were measured by the volume displacement technique after 24 h (35). Lung volumes (µl) were corrected to body weight (g). Adult lung sections were also stained with Gomori's modified iron stain (Prussian blue) (15, 37) to detect hemosiderin-laden macrophages, Hart's elastin stain to detect elastin (44), and trichrome and pentachrome stains to detect pulmonary fibrosis (19). Trichrome and Gomori's iron stain was also performed on lung sections from adult SPC-TGF-{alpha} mice (25). Immunostaining for pro-SP-C was performed on sections from adult transgenic mice with remodeling as previously described (14).

Pulmonary vascular development. Pulmonary vascular development was assessed by performing barium arteriograms and immunostaining for the endothelial marker platelet endothelial cell adhesion molecule (PECAM) in adult mice in which TGF-{alpha} was induced from postnatal days 3 to 5. Barium arteriograms were performed as previously described (27). Briefly, lungs were gently inflated with air, and blood was flushed from the lungs with heparinized saline through a catheter inserted through the wall of the right ventricle (RV) into the main pulmonary artery. A heated solution of gelatin and barium was infused into the main pulmonary artery catheter at 74 mmHg pressure for at least 5 min. The main pulmonary artery was ligated under pressure, and the lungs were inflation fixed with 4% paraformaldehyde at constant pressure (25 cmH2O). The barium-filled arterial structure in the lungs was imaged by X-ray radiography. PECAM immunostaining was performed as previously described (27), to assess overall vascular structure, as it detects endothelial cells in arteries, veins, and capillaries.

Pulmonary mechanics. Lung mechanics were assessed on adult CCSP-rtTA+/–/(tetO)7-TGF-{alpha}+/– and control mice with a computerized Flexi Vent system (SCIREQ, Montreal, Canada), as previously described (21, 36). Briefly, mice were anesthetized with ketamine and xylazine. The mice were tracheostomized and ventilated with a tidal volume of 8 ml/kg at a rate of 450 breaths/min and positive end-expiratory pressure (PEEP) of 2 cmH2O by a computerized Flexi Vent system (SCIREQ). This machine permits analysis of dynamic lung compliance (36). The ventilation mode on the Flexi Vent was changed to forced oscillatory signal (0.5–19.6 Hz), and respiratory impedance was measured. We obtained estimated tissue resistance or damping and tissue elastance for mice at 2 cmH2O PEEP by fitting a model to each impedance spectrum (36). With this system, the calibration procedure removed the impedance of the equipment and tracheal tube. Hysteresivity describes the mechanical coupling between tissue damping and elastance and is calculated as tissue damping/tissue elastance (36).

Pulmonary hypertension. Right ventricular hypertrophy (RVH) was assessed as an index of pulmonary hypertension in 2- and 6-wk-old CCSP-rtTA+/–/(tetO)7-TGF-{alpha}+/– and control mice, as previously described (27). Briefly, hearts were removed from 2- and 6-wk-old mice and dissected to isolate the free wall of the RV from the left ventricle and septum (LV+S). The ratios of RV weight to LV+S weight (RV/LV+S) were used as an index of RVH, which develops as a result of pulmonary hypertension. The LV+S weight to body weight was calculated to verify that left ventricular hypertrophy was not present.

Electron microscopy of alveolar capillaries. Lungs from 2-wk-old control and CCSP-rtTA+/–/(tetO)7-TGF-{alpha}+/– mice were analyzed by transmission electron microscopy. Briefly, after cannulating the trachea, lungs were inflation fixed with 2.5% glutaraldehyde-1% paraformaldehyde in buffer (pH 7.4, 310 mosmol/kgH2O, 4°C for 24 h) in situ at 25 cmH2O pressure (2). Principles of systematic, uniform, random sampling were used to collect tissue blocks from all lobes (1 mm3, 8–10 per lobe) (7). The tissue blocks were postfixed in 1% osmium tetroxide, dehydrated in a graded acetone series, and infiltrated and embedded in epoxy resin. Thin sections (80 nm thick) were cut with the aid of a diamond knife and counterstained with uranyl acetate and lead citrate. A Philips Tecnai 12 transmission electron microscope was used to observe and photograph the thin sections. Cross sections of capillary profiles were photographed at the same magnification in the upper left corner of each grid square for an entire thin section per tissue block. Thin sections from four tissue blocks per lobe were photographed.

Statistical analysis. Data are presented as means ± SE. Statistical analysis was performed with the Statview software package (Abacus Concepts, Berkeley, CA). Statistical comparisons were made by analysis of variance and Fisher's protected least significant difference test. P < 0.05 was considered significant.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Conditional expression of TGF-{alpha} in the neonatal lung. TGF-{alpha} was measured in lung homogenates of neonatal mice treated with Dox from postnatal days 3 to 4 (1 day Dox, n = 22) and 3 to 5 (2 days Dox, n = 20), as well as 2 and 9 days after ceasing 2 days of Dox treatment (n = 10 and 7, respectively). TGF-{alpha} concentrations in lung homogenates from CCSP-rtTA+/–/(tetO)7-TGF-{alpha}+/– mice were increased relative to controls, after 1 and 2 days of Dox treatment, respectively (P < 0.05, Fig. 1). TGF-{alpha} concentrations decreased 2 days after ceasing Dox treatment (P < 0.05) and were not different from control levels 9 days after ceasing Dox treatment (14 days of age, P > 0.05). TGF-{alpha} levels in lung homogenates of 5-day-old CCSP-rtTA+/–/(tetO)7-TGF-{alpha}+/– mice not treated with Dox (n = 10) were not different from control levels (P > 0.05, Fig. 1), indicating that there was no expression of the TGF-{alpha} transgene in the absence of Dox.



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  		Fig. 1. Conditional expression of transforming growth factor (TGF)-{alpha} in the lungs of neonatal mice. TGF-{alpha} concentrations were measured in lung homogenates from bitransgenic [CCSP-rtTA+/–/(tetO)7-TGF-{alpha}+/–] and control pups after 1 and 2 days of exposure to doxycycline (Dox; 4 and 5 days old, n = 22 and n = 20, respectively), 2 days on Dox then 2 days off (7 days old, n = 10), 2 days on Dox then 9 days off (14 days old, n = 7). Dams were placed on Dox from postnatal day 3 to 5. TGF-{alpha} was also measured in lung homogenates from 5-day-old bitransgenic pups not on Dox (No Dox, n = 5). TGF-{alpha} concentrations were normalized to total lung protein. TGF-{alpha} levels in bitransgenic pups not exposed to Dox were similar to levels in control mice indicating that there was no transgene expression in the absence of Dox (P > 0.05). *P < 0.05 vs. control littermates. {dagger}P < 0.05 vs. CCSP-rtTA+/–/(tetO)7-TGF-{alpha}+/– on Dox for 2 days. CCSP, Clara cell secretory protein; rtTA, reverse tetracycline transactivator transcription factor; d, day.

 
Disrupted alveolarization, increased lung volumes, minimal fibrosis. To determine the effects of transient induction of TGF-{alpha} on postnatal alveolarization, we assessed lung histology on CCSP-rtTA+/–/(tetO)7-TGF-{alpha}+/– and control mice treated with Dox from postnatal days 3 to 5, and 2, 5, and 9 days after ceasing Dox treatment. Normal alveologenesis, characterized by increased secondary septation, was observed from 5 days to 14 days of age in control mice (Fig. 2, top). In contrast, CCSP-rtTA+/–/(tetO)7-TGF-{alpha}+/– mice showed a persistent arrest of septation, resulting in enlarged distal air spaces at 7–14 days of age (Fig. 2, bottom). Distal air spaces were also enlarged in adult CCSP-rtTA+/–/(tetO)7-TGF-{alpha}+/– mice at 6 wk of age, 4 wk after TGF-{alpha} transgene expression returned to control levels (Fig. 3A). Lung volumes and body weights were measured in adult mice after Dox treatment from postnatal days 3 to 5. Lung volumes were increased 1.8-fold in adult CCSP-rtTA+/–/(tetO)7-TGF-{alpha}+/– mice compared with controls (1,235 ± 114 vs. 690 ± 4 µl, respectively; P < 0.05), whereas body weights were not significantly different (20.2 ± 2.7 vs. 23.0 ± 0.6 g, respectively; P > 0.05). Lung volume to body weight ratios were increased 2.1-fold in adult CCSP-rtTA+/–/(tetO)7-TGF-{alpha}+/– mice compared with controls (n = 4 per group, Fig. 3B, P < 0.05). Trichrome staining showed minimal pulmonary fibrosis in adult CCSP-rtTA+/–/(tetO)7-TGF-{alpha}+/– mice, whereas pleural and adventitial fibrosis was present in lungs of SPC-TGF-{alpha} mice (Fig. 4) as previously described (25).



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  		Fig. 2. Distal air space development is disrupted following transient increases in TGF-{alpha}. Histology of distal lungs from bitransgenic [CCSP-rtTA+/–/(tetO)7-TGF-{alpha}+/–] and control pups after 1 and 2 days of exposure to Dox (4 and 5 days old), 2 days on Dox then 2 days off (7 days old), 2 days on Dox then 5 days off (10 days old), and 2 days on Dox then 9 days off (14 days old). Dams were placed on Dox from postnatal day 3 to 5. Lung sections were stained with hematoxylin and eosin (H&E). Differences in distal air space morphogenesis were evident 2 days after Dox treatment for 2 days, and by 14 days of age the normal process of alveolarization was disrupted in the CCSP-rtTA+/–/(tetO)7-TGF-{alpha}+/–. Bar = 100 µm. Photomicrographs are representative of 3–6 CCSP-rtTA+/–/(tetO)7-TGF-{alpha}+/– mice and 3–6 control mice per time point.

 


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  		Fig. 3. Distal air space enlargement and increased lung volumes in adult mice following transient induction of TGF-{alpha} during early alveologenesis. A: histology of distal lung from 6-wk-old adult bitransgenic [CCSP-rtTA+/–/(tetO)7-TGF-{alpha}+/] and control mice following Dox treatment from postnatal days 3 to 5. Lung sections were stained with H&E. Enlarged distal air spaces in the adult CCSP-rtTA+/–/(tetO)7-TGF-{alpha}+/– mice are due to disruption of postnatal alveolarization during the first 2 wk of life. Distal air space septae in bitransgenic mice are thin, with few secondary septae. Bar = 100 µm. Photomicrographs are representative of 9 CCSP-rtTA+/–/(tetO)7-TGF-{alpha}+/– mice and 7 controls. B: lung volumes were measured in 6-wk-old adult bitransgenic CCSP-rtTA+/–/(tetO)7-TGF-{alpha}+/– (n = 4) and control mice (n = 4) following Dox treatment from postnatal days 3 to 5. Lung volumes (µl) were corrected to body weight (g). Lung volume to body weight ratios were increased 2-fold in adult bitransgenic compared with control mice. *P < 0.05 vs. control.

 


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  		Fig. 4. Minimal pulmonary fibrosis in adult mice following transient induction of TGF-{alpha} in the neonatal lung. Trichrome staining was performed on adult mice to detect pulmonary fibrosis. Minimal staining was observed in CCSP-rtTA+/–/(tetO)7-TGF-{alpha}+/– mice following transient induction of TGF-{alpha} during postnatal days 3 to 5. In contrast, pleural and adventitial fibrosis (blue, arrows) was seen in the lung sections from SPC-TGF-{alpha}+/– mice, which were stained at the same time. Micrographs presented are representative of 4 control, 5 CCSP-rtTA+/–/(tetO)7-TGF-{alpha}+/–, and 3 SPC-TGF-{alpha}+/– mice. SPC, surfactant protein C.

 
Abnormal pulmonary mechanics. Pulmonary mechanics studies performed on 6-wk-old adult mice showed that CCSP-rtTA+/–/(tetO)7-TGF-{alpha}+/– mice had increased compliance (1.8-fold) and decreased tissue (50%) and airway (44%) elastance and tissue damping (50%) compared with controls (n = 5 and n = 4, respectively; P < 0.05, Fig. 5). Hysteresivity and airway resistance were similar to controls (P > 0.05, Fig. 5).



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  		Fig. 5. Abnormal pulmonary mechanics in adult conditional TGF-{alpha} transgenic mice following transient induction of TGF-{alpha} from postnatal days 3 to 5. Compliance (A), tissue elastance (B), airway elastance (C), tissue damping (D), hysteresivity (E), and airway resistance (F) were measured in adult CCSP-rtTA+/–/(tetO)7-TGF-{alpha}+/– and control mice (n = 5 and n = 4, respectively). Compliance was increased 2-fold, whereas tissue and airway elastance and tissue damping were reduced 0.5-fold in CCSP-rtTA+/–/(tetO)7-TGF-{alpha}+/– mice. *P < 0.05 vs. control.

 
Abnormal interstitial elastin structure. Hart's elastin staining was performed to assess elastic fiber structure in the lungs of 2- and 6-wk-old mice. In control lungs, elastin fibers were seen running parallel with the alveolar septae and around the alveolar walls (Fig. 6). In contrast, elastin fibers in the alveolar septae and the interstitium were fragmented, tortuous, and irregularly distributed in the alveolar septae of 2- (data not shown) and 6-wk-old CCSP-rtTA+/–/(tetO)7-TGF-{alpha}+/– mice. However, the internal and external elastic lamellae of pulmonary arteries in CCSP-rtTA+/–/(tetO)7-TGF-{alpha}+/– mice were not noticeably abnormal (Fig. 6).



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  		Fig. 6. Abnormal elastin structure in alveolar septae of conditional TGF-{alpha} transgenic mice. Hart's stain detected elastin fibers (black) along alveolar septae (top and bottom, arrows) in 6-wk-old control mice and the internal and external elastic lamellae of vessels (bottom, arrowheads). Elastin fibers in the alveolar septae of 6-wk-old CCSP-rtTA+/–/(tetO)7-TGF-{alpha}+/– mice were fragmented, tortuous, and irregularly distributed in the alveolar septae (top and bottom, arrows). Internal and external elastic lamellae of vessels in CCSP-rtTA+/–/(tetO)7-TGF-{alpha}+/– mice appeared normal (top and bottom, arrowheads). Similar findings were seen in 2-wk-old mice. Bar = 100 µM. Micrographs presented are representative of 7 control and 5 CCSP-rtTA+/–/(tetO)7-TGF-{alpha}+/– mice.

 
Disrupted vascular morphogenesis. To determine the effects of transient induction of TGF-{alpha} on vascular morphogenesis, we performed barium arteriograms and PECAM immunostaining on adult CCSP-rtTA+/–/(tetO)7-TGF-{alpha}+/– and control mice treated with Dox treatment from postnatal days 3 to 5 (n = 7 and n = 6, respectively). Barium arteriograms of left lungs showed that arterial structure was abnormal in CCSP-rtTA+/–/(tetO)7-TGF-{alpha}+/– (Fig. 7A) and arterial branching in particular was attenuated and occasionally tortuous. PECAM immunostaining showed that vascular development was decreased in parallel with the reduction in distal air space morphogenesis (Fig. 7B).



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  		Fig. 7. Abnormal pulmonary vascular structure following transient induction of TGF-{alpha}. A: disrupted arterial morphogenesis: pulmonary arteriograms of left lungs from control mice (top) and CCSP-rtTA+/–/(tetO)7-TGF-{alpha}+/– (bottom) mice. Extensive branching of the pulmonary arteries and an extensive network of distal pulmonary arteries are seen in the control lungs. In contrast, pulmonary arteriograms of CCSP-rtTA+/–/(tetO)7-TGF-{alpha}+/– mice showed abnormal arterial branching and morphogenesis. Figures are representative of arteriograms from 6 adult control and 7 adult CCSP-rtTA+/–/(tetO)7-TGF-{alpha}+/– mice. B: alveolar and vascular simplification: immunostaining for platelet endothelial cell adhesion molecule (PECAM, black), an endothelial marker protein, is shown on lung sections from adult mice. Nuclear fast red was used as a counterstain. Capillary staining for PECAM is present throughout the alveolar septae in both control and CCSP-rtTA+/–/(tetO)7-TGF-{alpha}+/– mice. Vessels (arteries and veins) also stain for PECAM (black) and are marked with arrows. CCSP-rtTA+/–/(tetO)7-TGF-{alpha}+/– mice show a pattern of alveolar and vascular simplification. Bar = 100 µM. Photomicrographs are representative of immunostaining performed on lung sections from 4 CCSP-rtTA+/–/(tetO)7-TGF-{alpha}+/– mice and 4 controls.

 
Pulmonary hypertension and capillary dysfunction. RV weights relative to LV weights (RV/LV+S ratios) were assessed in CCSP-rtTA+/–/(tetO)7-TGF-{alpha}+/– mice and control littermates at 2 and 6 wk of age as an index of pulmonary hypertension (n = 4–8 animals per group). RV/LV+S ratios were increased in CCSP-rtTA+/–/(tetO)7-TGF-{alpha}+/– mice at 2 and 6 wk of age (1.7- and 1.5-fold, respectively; Fig. 8). LV weight-to-body weight ratios were similar in CCSP-rtTA+/–/(tetO)7-TGF-{alpha}+/– and control mice, indicating a lack of left ventricular hypertrophy (P > 0.05). Increased RVH, but not left ventricular hypertrophy, provides evidence of pulmonary hypertension in the 2- and 6-wk-old CCSP-rtTA+/–/(tetO)7-TGF-{alpha}+/–mice. Electron microscopy of lungs from 2-wk-old mice showed normal ultrastructural appearance of alveolar capillaries in control mice, with capillaries lined by a continuous layer of endothelial cells (Fig. 9A). In contrast, evidence of endothelial dysfunction and injury was observed in the alveolar capillaries of CCSP-rtTA+/–/(tetO)7-TGF-{alpha}+/– mice (Fig. 9, B–D). Gaps were seen in the capillary endothelium (Fig. 9, C and D), and extravasation of erythrocytes through the capillary wall into the alveolar space was seen (Fig. 9D). Gomori's iron stain provided further evidence of capillary dysfunction, as hemosiderin-laden macrophages were abundant throughout the distal lung of adult CCSP-rtTA+/–/(tetO)7-TGF-{alpha}+/– mice, particularly adjacent to alveolar septae and capillaries (Fig. 10). The presence of hemosiderin-laden macrophages indicates that vascular leak in the CCSP-rtTA+/–/(tetO)7-TGF-{alpha}+/– mice was a chronic event.



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  		Fig. 8. Pulmonary hypertension following transient induction of TGF-{alpha}. Right ventricular hypertrophy was assessed in 2- and 6-wk bitransgenic [CCSP-rtTA+/–/(tetO)7-TGF-{alpha}+/–] and control mice as the ratio of right ventricle to left ventricle plus septum weights (RV/LV+S). The ratio of RV/LV+S weights was increased in bitransgenic mice at 2 and 6 wk of age, indicating that pulmonary hypertension develops after disruption of postnatal lung development by TGF-{alpha} and persists into adulthood. Data were derived from 4–8 animals in each study group. *Significant difference from control mice, P < 0.05.

 


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  		Fig. 9. Capillary dysfunction following transient induction of TGF-{alpha}. Electron microscopy of conditional TGF-{alpha} mice shows gaps in the capillary endothelium and erythrocyte extravasation. Transmission electron micrographs of alveolar capillaries in 2-wk-old mouse lungs. A: control mouse lung. The capillary profile is normal, in that it is lined by continuous endothelium. B–D: conditional TGF-{alpha} transgenic mice lungs. Capillary profiles are abnormal. An endothelial cell showing signs of injury is marked in B (arrowhead). Portions of the endothelial lining are attenuated (C and D), with multiple, short discontinuities that give a beaded appearance (arrowheads). Extravasation of an erythrocyte through the capillary wall into the alveolar space is shown in D (arrow). All panels are the same magnification. Scale bar = 1 µm.

 


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  		Fig. 10. Hemosiderin-laden macrophages in the distal air spaces of conditional TGF-{alpha} mice. Photomicrographs of Gomori's iron stain of distal lung of adult mice. Hemosiderin-laden macrophages were not detected in the distal lung of control mice (left). Abundant hemosiderin-laden macrophages (blue) were detected along alveolar septae/capillaries and throughout the distal lung of CCSP-rtTA+/–/(tetO)7-TGF-{alpha}+/– mice (center and right). Bar = 100 µm. Micrographs presented are representative of 8 CCSP-rtTA+/– control and 8 CCSP-rtTA+/–/(tetO)7-TGF-{alpha}+/– mice.

 
Hemorrhage, inflammation, and nodular remodeling. At 4 wk of age, all CCSP-rtTA+/–/(tetO)7-TGF-{alpha}+/– mice (n = 4) had areas of hemorrhage, eosinophilic and neutrophilic inflammation, and clusters of macrophages undergoing erythrophagocytosis (Fig. 11, A and B). At 6 wk of age, areas of alveolar remodeling, inflammation, and proteinaceous deposits were seen in 75% of CCSP-rtTA+/–/(tetO)7-TGF-{alpha}+/– mice (6 of n = 8, Fig. 11C). Macrophages containing numerous Charcot-Leyden crystals (Fig. 11D) and mixed eosinophilic and neutrophilic inflammation, and proteinaceous debris were seen in distal air spaces (Fig. 11C). Within some distal air spaces nodular foci, composed of crystal-laden macrophages, inflammatory cells, fibroblasts, and type II cells, were seen (Fig. 11, C, E, and F).



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  		Fig. 11. Inflammation, hemorrhage, and remodeling in a subgroup of conditional TGF-{alpha} mice. Histology of conditional TGF-{alpha} transgenic mice at 4 wk of age showing areas of hemorrhage (A) and abundant neutrophils (arrowheads) and macrophages (arrows) undergoing erythrophagocytosis (B). At 6 wk of age, areas of remodeling, mixed eosinophilic and neutrophilic inflammation, and proteinaceous deposits were seen in a subgroup of CCSP-rtTA+/–/(tetO)7-TGF-{alpha}+/– mice (C). Macrophages containing Charcot-Leyden crystals were seen in these areas in distal air spaces (D, arrows). Nodular remodeling was also observed in these areas (C, E, and F). Immunostaining for pro-SP-C detected abundant type II epithelial cells in the nodules (E, arrows). The inner mass of these nodules also contained neutrophils (E, *) and macrophages containing Charcot-Leyden crystals. Pentachrome staining detected some fibrosis in the nodules (F, arrowheads), consistent with the presence of fibroblasts. Micrographs presented are representative of 4 CCSP-rtTA+/–/(tetO)7-TGF-{alpha}+/– mice at 1 mo of age and 75% (6 of n = 8) of CCSP-rtTA+/–/(tetO)7-TGF-{alpha}+/– mice at 6 wk of age. Bar = 100 µm in all panels.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Transient overexpression of TGF-{alpha} during the early alveolar phase of postnatal lung development disrupted alveolar and vascular morphogenesis. Enlarged distal air spaces and abnormal vascular structure persisted into adulthood, well after TGF-{alpha} levels had returned to baseline. Although pulmonary fibrosis was minimal, lung volumes were increased, and pulmonary mechanics studies showed increased compliance and reduced elastance. Consistent with these functional measurements, elastin fibers in the alveolar septae were fragmented and disorganized. There was evidence of pulmonary hypertension and capillary dysfunction and leak. In addition to enlarged distal air spaces, conditional TGF-{alpha} mice also developed pulmonary hemorrhage, associated with inflammatory cell infiltrates and remodeling.

In previous studies, expression of TGF-{alpha} was driven in transgenic mice throughout pre- and postnatal lung development using the SP-C promoter. This promoter directs transgene expression to type II cells and bronchiolar epithelial cells (14). Overexpression of TGF-{alpha} by the SP-C promoter caused alveolar simplification, disrupted pulmonary vascular development, and caused severe pulmonary fibrosis (18, 25, 27). For this study we used CCSP-rtTA activator mice to drive expression of TGF-{alpha} in the epithelium of conducting airways and distal air spaces (33, 43). The 2.3-kb rat CCSP promoter element used in these activator mice is expressed differently from the native murine CCSP gene (42), in that it drives expression in type II cells, as well as Clara cells (33, 43). This may be due to 1) removal of control elements that normally restrict expression to Clara cells, 2) chromosome insertion effects (33, 43), or 3) a species effect (43). An advantage of the CCSP-rtTA activator line that we used is that it has negligible transgene expression in the absence of Dox (also known as leak) (33). Because the goal of our study was to restrict TGF-{alpha} transgene to a specific developmental time period (the saccular-alveolar transition) and then turn it off, it was essential to use a transgenic system that has no leak. The other advantage of using the CCSP-rtTA system is that this system had already been used to drive conditional TGF-{alpha} expression in adults (19). This enabled a comparison of the effects of conditional TGF-{alpha} expression in adults, which causes primarily pulmonary fibrosis (19), with conditional expression in newborns. Although transient overexpression of TGF-{alpha} in neonates disrupted postnatal lung morphogenesis and severely altered lung structure, it caused minimal pulmonary fibrosis. This alveolar phase of lung morphogenesis is a critical developmental window with sensitivity to TGF-{alpha}-induced injury, such that even relatively brief increases in TGF-{alpha} disrupted alveolar and vascular morphogenesis and caused long-term alterations in lung structure and function, which persisted after TGF-{alpha} levels returned to baseline.

Abnormal elastic fiber architecture was observed in the alveolar septae of 2-wk-old and adult mice following transient induction of TGF-{alpha}. Elastic fibers form a critical component of the extracellular matrix of compliant tissues, allowing tissues such as the lung to recoil after stretch. Elastin is also deposited at the tips of secondary crests and is critical for normal alveolar development and structure (44). Hence, loss of elastic structure probably contributed to abnormal pulmonary mechanics in the adult TGF-{alpha} mice. The precise mechanism causing abnormal elastic fiber structure was not determined but may be due to abnormal synthesis and/or degradation of elastin by elastases and matrix metalloproteinases. Disorganized elastic fiber structure may also have contributed to disrupted alveolar morphogenesis, and loss of elastin structure may have permitted hyperinflation of distal air spaces.

Evidence of pulmonary hypertension was detected in the conditional TGF-{alpha} mice in the present study and in the SPC-TGF-{alpha} mice (27). Because the conditional TGF-{alpha} mice had minimal pulmonary fibrosis, these data show that the development of pulmonary hypertension resulting from increased expression of TGF-{alpha} is not dependent on the presence of pulmonary fibrosis. Furthermore, RVH was not seen in adult mice with pulmonary fibrosis resulting from conditional overexpression of TGF-{alpha} (W. D. Hardie and T. D. Le Cras, unpublished observations). The underlying cause of pulmonary hypertension in the SPC-TGF-{alpha} and the conditional TGF-{alpha} mice is most likely related to the disruption of postnatal lung morphogenesis. Alveolar simplification and disrupted pulmonary vascular development may cause hypoxemia and increase pulmonary vascular resistance. Reduced vascular development may also increase pulmonary vascular resistance and lead to vascular remodeling. In addition to pulmonary hypertension, there was evidence of capillary dysfunction in the conditional TGF-{alpha} mice. Electron microscopy showed that the capillary endothelium was abnormal, and active extravasation of erythrocytes was detected. In addition, hemosiderin-laden macrophages were seen throughout the distal air spaces in the conditional TGF-{alpha} mice, along the alveolar septae/capillaries, providing further evidence of capillary dysfunction and chronic vascular leak. In the Heath-Edwards grading system of pulmonary hypertension, intra-alveolar hemosiderin-filled macrophages are seen in high-grade lesions (20). Hemosiderin-laden macrophages are also reported in scimitar syndrome, which is associated with severe pulmonary hypertension (12).

Hemorrhage and inflammatory cells were detected in distal air spaces of conditional TGF-{alpha} mice at 1 mo of age. By 6 wk of age, extensive remodeling, with nodules, proteinaceous deposits, mixed neutrophilic and eosinophilic inflammation, and macrophages, was seen in these areas. Interestingly, although these macrophages did not stain for hemosiderin (data not shown), they contained Charcot-Leyden crystals. Fibrosis was detected in the nodules in the remodeled areas, consistent with the presence of fibroblasts. Immunostaining for pro-SP-C showed that type II cells were abundant in the nodules, as well as neutrophils and macrophages containing Charcot-Leyden crystals. Neutrophils may contribute to disruption of lung structure by releasing elastase (39). However, in the present study, neutrophil influx was seen after 2 wk of age when elastin structure was already abnormal. In addition, although elastin structure was uniformally abnormal throughout the distal lung of all of the conditional transgenic mice, neutrophils were seen only in the focal areas of hemorrhage and remodeling. Hence, although neutrophils might contribute to elastin degradation in the areas of remodeling, it is unlikely that they caused the overall reduction in secondary septation. The presence of neutrophils in the present study is probably related to vascular leak and areas of subsequent inflammation and remodeling. Macrophages in these areas are most likely playing a role in repair in sites of injury caused by vascular leak and hemorrhage. Macrophages containing Charcot-Leyden-like crystals have also been reported in transgenic mice overexpressing IL-13 (47), and recent studies have shown that the effects of IL-13 are partially mediated through EGFR signaling (29). Nodules associated with increased vascular leak and inflammatory cells were not detected in the SPC-TGF-{alpha} or after conditional induction of TGF-{alpha} in the adult lung (19, 25).

Although the role of macrophages in the pathogenesis of BPD is unclear, they are a notable feature of the lung pathology of this disease. Macrophages have been noted in distal air spaces in association with proteinaceous material (13) and have been reported to increase in the airway secretions of infants with respiratory distress syndrome (RDS) and are the predominant inflammatory cell in the lungs of infants with BPD (38). Currently, macrophages are believed to play a major role in the initiation, regulation, and resolution of inflammation in BPD and in the clearance of neutrophils undergoing apoptosis (38).

Increased TGF-{alpha} and EGFR were detected in the lungs of infants with BPD (40, 41). Some parallels between the lungs of infants with BPD and the transgenic mice in the present study are notable. Increased elastin turnover and abnormal elastic fibers have been reported in the lungs of infants with BPD (10, 30). Pulmonary hypertension with RVH occurs in patients with severe BPD (1). Infants with RDS and BPD have increased numbers of macrophages and neutrophils (28, 38), although the precise role of these inflammatory cells in BPD is unknown. Abnormal lung mechanics in infants with BPD persist into infancy and adolescence (4).

Although the precise role of TGF-{alpha} and EGFR in BPD is not known, mechanisms by which perinatal stress and acute lung injury in premature newborns can permanently alter lung structure are poorly understood. Infants who are exposed to injurious stimuli (such as hyperoxia or infection) for limited periods of time may experience only transient increases in cytokines and growth factors such as TGF-{alpha}. The present study suggests that even transient increases in TGF-{alpha} during the alveolar phase of lung morphogenesis can have severe and long-term consequences on lung structure and function.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by National Heart, Lung, and Blood Institute Grants HL-72894 (T. D. Le Cras), HL-04172 (W. D. Hardie), HL-61646 (M. Ikegami), HL-62875 (K. H. Albertine), and HL-56387 (T. R. Korfhagen and J. A. Whitsett), and an American Lung Association Career Investigator Award CI-31-N (T. D. Le Cras).


   ACKNOWLEDGMENTS
 
The authors thank Brandon Pyles, Patricia Pastura, David Loudy, and Maura Unger in the Div. of Pulmonary Biology, Cincinnati Children's Hospital, for excellent technical assistance and Nancy Chandler at the Health Sciences Center Research Microscopy Facility at the Univ. of Utah for technical assistance with transmission electron microscopy. The authors also thank Dr. Susan Wert (Div. of Pulmonary Biology, Cincinnati Children's) and Dr. Susan Crawford (Pathology Dept., Northwestern Univ.) for help interpreting the lung pathology.


   FOOTNOTES
 

Address for reprint requests and other correspondence: T. Le Cras, Div. of Pulmonary Biology, Cincinnati Children's Hospital Medical Center, 3333 Burnet Ave., Cincinnati, OH 45229-3039 (E-mail: tim.lecras{at}cchmc.org)

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. Section 1734 solely to indicate this fact.


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