Disrupted pulmonary vascular development and pulmonary hypertension in transgenic mice overexpressing transforming growth factor-{alpha}

Timothy D. Le Cras,1 William D. Hardie,1 Karen Fagan,2 Jeffrey A. Whitsett,1 and Thomas R. Korfhagen1

1Division of Pulmonary Biology, Cincinnati Children's Hospital Medical Center, Cincinnati, Ohio 45229; and 2Department of Medicine, University of Colorado Health Sciences Center, Denver, Colorado 80262

Submitted 14 February 2003 ; accepted in final form 30 July 2003


    ABSTRACT
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 
Pulmonary vascular disease plays a major role in morbidity and mortality in infant and adult lung diseases in which increased levels of transforming growth factor (TGF)-{alpha} and its receptor EGFR have been associated. The aim of this study was to determine whether overexpression of TGF-{alpha} disrupts pulmonary vascular development and causes pulmonary hypertension. Lung-specific expression of TGF-{alpha} in transgenic mice was driven with the human surfactant protein (SP)-C promoter. Pulmonary arteriograms and arterial counts show that pulmonary vascular development was severely disrupted in TGF-{alpha} mice. TGF-{alpha} mice developed severe pulmonary hypertension and vascular remodeling characterized by abnormally extensive muscularization of small pulmonary arteries. Pulmonary vascular development was significantly improved and pulmonary hypertension and vascular remodeling were prevented in bitransgenic mice expressing both TGF-{alpha} and a dominant-negative mutant EGF receptor under the control of the SP-C promoter. Vascular endothelial growth factor (VEGF-A), an important angiogenic factor produced by the distal epithelium, was decreased in the lungs of TGF-{alpha} adults and in the lungs of infant TGF-{alpha} mice before detectable abnormalities in pulmonary vascular development. Hence, overexpression of TGF-{alpha} caused severe pulmonary vascular disease, which was mediated through EGFR signaling in distal epithelial cells. Reductions in VEGF may contribute to the pathogenesis of pulmonary vascular disease in TGF-{alpha} mice.

bronchopulmonary dysplasia; angiogenesis; epidermal growth factor receptor; vascular endothelial growth factor


TRANSFORMING GROWTH FACTOR-{alpha} (TGF-{alpha}) is a member of the epidermal growth factor (EGF) family of polypeptide growth factors that includes TGF-{alpha}, EGF, amphiregulin, and heparin-binding EGF (10). After synthesis as a 160-amino acid precursor, TGF-{alpha} is cleaved by specific elastase-like enzymes to release the mature 50-amino acid peptide (10). TGF-{alpha} shares 42% sequence homology with EGF, and both bind and activate the EGF receptor (EGFR) (9). Clinical studies have associated increased TGF-{alpha} and EGFR with a number of acute and chronic lung diseases (2, 6, 16, 32, 36, 37). TGF-{alpha} was increased in airway and alveolar epithelial cells, alveolar macrophages, and vascular smooth muscle in lungs of infants with bronchopulmonary dysplasia (BPD) (32, 36, 37). Increases in TGF-{alpha} have also been reported in pulmonary epithelial cells of patients with end-stage cystic fibrosis and idiopathic pulmonary fibrosis (2, 16). However, whether TGF-{alpha} signaling through EGFR contributes to the pathogenesis of these infant and adult lung diseases is unclear.

TGF-{alpha} is produced in airway, alveolar epithelial, and vascular smooth muscle cells of the developing lung, with levels highest in the prenatal period and then decreasing postnatally (16, 23, 25, 36-38). TGF-{alpha} has been shown to contribute to the pathogenesis of pulmonary fibrosis in bleomycin-induced lung injury (30, 31) and was increased in neonatal rabbits exposed to hyperoxia (40). Previous studies have shown that chronic expression of TGF-{alpha} under control of the human surfactant protein (SP)-C promoter caused pulmonary fibrosis and severe enlargement of distal air spaces in transgenic mice (17, 19, 24). Although TGF-{alpha} transgenic lungs appeared normal at birth, alveolar simplification occurred during the postnatal phase of lung development and was due to disrupted septation (17, 19). Expression of a dominant-negative mutant EGF receptor (SPC-EGFR-M) in the distal epithelial cells, also driven by the human SP-C promoter, prevented the induction of pulmonary fibrosis by TGF-{alpha} and partially improved postnatal alveolarization (18).

Pulmonary vascular disease is a major cause of morbidity and mortality in both the adult and newborn diseases (1) in which TGF-{alpha} has been implicated, and although previous studies had shown that TGF-{alpha} disrupts postnatal alveogenesis and causes pulmonary fibrosis in transgenic mice, they have not determined whether TGF-{alpha} causes pulmonary vascular disease. Hence, the aim of this study was to determine whether pulmonary vascular development is disrupted in TGF-{alpha} mice and whether these mice develop pulmonary hypertension and vascular remodeling. In addition, SPC-EGFR-M mice were used to block TGF-{alpha} signaling through EGFR to determine whether pulmonary vascular disease is mediated through EGFR signaling in the distal epithelium. Our findings show that TGF-{alpha} mice develop severe pulmonary vascular disease that is primarily mediated through EGFR signaling in the distal epithelium. Vascular endothelial growth factor (VEGF) expression was examined, as it is an important angiogenic factor produced by the distal epithelium and, in previous studies in which VEGF signaling was disrupted, caused severe disruption of pulmonary vascular development and pulmonary hypertension (28).


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 
Animals. All procedures and protocols were approved by the Animal Care and Use Committees at the University of Colorado Health Science Center and the Cincinnati Children's Hospital Research Foundation. All mice were FVB/N strain. TGF-{alpha} transgenic mice (line 28) were previously generated with lung-specific expression of TGF-{alpha} in the distal epithelium driven by the human SP-C promoter (24). Hemizygous TGF-{alpha}(+/-) transgenic mice and wild-type (WT) control mice were studied at 1-2 days and 2-3 mo of age. Lung tissue was also obtained from 2-day-old mice for VEGF measurements. Dominant-negative EGF receptor mutant (EGFR-M) mice were generated as described previously (18) with expression of the EGFR-M also under the control of the human SP-C promoter. Bitransgenic mice, TGF-{alpha} + EGFR-M, were generated by breeding hemizygous TGF-{alpha}(+/-) mice to homozygous EGFR-M(+/+) mice. Genotyping of TGF-{alpha}, bitransgenic TGF-{alpha} + EGFR-M, and EGFR-M mice was performed by PCR and Southern blot analysis of tail DNA as previously described (18, 24). Hematocrits were measured in adult TGF-{alpha} mice and WT controls by standard techniques with heparinized blood (28).

Pulmonary arteriograms, histology, and arterial density counts. Adult mice were killed with a pentobarbital sodium (26%) euthanasia solution (Fort Dodge Animal Health, Fort Dodge, IA). A thoracotomy was rapidly performed, and heparin (10 units) was injected into the right ventricle (RV) to prevent blood from clotting in the lungs. After tracheostomy, the lungs were gently inflated with air from a syringe, and a stainless steel gavage needle was inserted into the trachea. The lungs were inflated with the chest partially open and so that they just filled the thorax. Blood was flushed from the lungs with heparinized saline (1 unit heparin/ml saline) through a catheter inserted through the wall of the RV into the main pulmonary artery catheter. A heated solution of gelatin and barium was infused into the main pulmonary artery catheter at 74-mmHg pressure for at least 5 min as previously described (8). The main pulmonary artery was ligated under pressure with suture, and the lungs were inflation fixed with 4% paraformaldehyde at constant pressure (25 cmH2O) for 48 h. The barium-filled arterial structure in the lungs was imaged by X-ray radiography. The left lungs were subsequently cut into 1-mm sections starting at the point where the bronchus enters the left lung, and at least three 1-mm sections per animal were embedded in paraffin. Five-micrometer-thick paraffin sections were cut and stained with hematoxylin and eosin (H&E). Three to six sections were examined from each animal. H&E sections from every mouse following the barium-gelatin perfusions were carefully examined to verify that there were no arterioles that had not completely filled with barium-gelatin (vessels still containing blood or not filled with barium). If partial filling of the arterial system was found, then the animal was excluded from the study. Barium-filled pulmonary arteries were counted, by an observer blinded to the identity of the slides, in randomly selected high-powered fields of distal lung to determine arterial density. Fields including large airways or large vessels were excluded. All the vessels in a x15 high-powered field were counted. Five high-powered fields were counted per animal from the 3-6 H&E sections.

Measurement of VEGF levels in lung homogenates. VEGF-A protein levels were measured in lung homogenates from adult mice and 2-day-old mice. Briefly, left lungs were sonicated in phosphate-buffered saline containing protease inhibitors (Complete protease inhibitor cocktail; Roche, Indianapolis, IN) and then centrifuged at low speed (1,000 g) to remove insoluble debris. Supernatants were diluted 1:10 and then assayed for VEGF-A (pg/ml) with a mouse VEGF ELISA kit and following the manufacturer's instructions (R&D Systems, Minneapolis, MN). VEGF protein concentrations were corrected to total lung protein to obtain VEGF levels in pg/mg lung protein. The 164-amino acid isoform of VEGF-A (VEGF164) was detected by Western blot analysis of the lung homogenates (25 µg of protein) with a rabbit polyclonal antibody to VEGF-A (SC152 Santa Cruz).

Right and left ventricular systolic pressures and right and left ventricular weight measurements. Right ventricular and left ventricular systolic pressures (RVSP and LVSP, respectively) were measured immediately before death, as previously described (11). Briefly, the mice were anesthetized with ketamine and xylazine and placed in a supine position. The transducer was calibrated before study, and RVSP and LVSP were measured by direct intracardiac puncture with a 26-gauge needle. Measurements were excluded if the heart rate was <300 beats/min due to oversedation. Blood was withdrawn by direct cardiac puncture after RVSP measurements into a heparinized syringe. At death, hearts were removed and dissected to isolate the free wall of the RV from the left ventricle and septum (LV+S). The ratios of RV weight to body weight and RV weight to LV+S weight (RV/LV+S) were used as an index of right ventricular hypertrophy (RVH), which develops as a result of pulmonary hypertension (39). The LV+S weight to body weight was calculated to determine whether left ventricular hypertrophy was present.

Immunohistochemistry for platelet endothelial cell adhesion molecule, smooth muscle {alpha}-actin, and scoring of muscularization of small pulmonary arteries. To determine whether vascular development was abnormal in newborn transgenic mice, we performed immunostaining for the endothelial marker protein platelet endothelial cell adhesion molecule (PECAM) (CD31) on lung sections from 1-day-old TGF-{alpha} mice and WT and compared it with immunostaining of 2-wk-old mice when lung development is essentially complete. PECAM was detected with a mouse anti-CD31 monoclonal antibody (clone MEC13.3; Pharmingen, San Diego, CA). To determine whether abnormal muscularization of small pulmonary arteries contributed to TGF-{alpha}-induced pulmonary vascular disease, we performed immunohistochemical staining for smooth muscle {alpha}-actin with a mouse monoclonal antibody (clone 1A4; Sigma, St. Louis, MO). Immunostaining was performed with a MOM kit (Vector, Burlingame, CA) according to the manufacturer's instructions. The sections were lightly counterstained with either nuclear fast red (for PECAM) or hematoxylin (for smooth muscle {alpha}-actin) before dehydration and mounting. Muscularization of 15- to 50-µm distal pulmonary arteries was assessed by an observer blinded to the identity of the slides. Thirty to forty vessels per animal with an external diameter of >15 µm but <50 µm were scored as nonmuscular (NM, <50% surrounded by smooth muscle cells), partially muscular (PM, >50% surrounded by smooth muscle cells but <100%), or fully muscular (FM, 100% surrounded by smooth muscle cells). Arterioles associated with alveolar ducts (50%) and distal alveolar regions (50%) were randomly selected, measured to determine whether they were 15-50 µm (external diameter), and then scored for muscularization. Of the 30-40 vessels scored, the percentage that was NM vs. PM vs. FM was calculated for each animal.

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 paired least significant difference test. P < 0.05 was considered significant.


    RESULTS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 
Pulmonary arteriograms, histology, and arterial density in TGF-{alpha} mice. Pulmonary arteriograms show that although the large pulmonary arteries were similar, there was an extensive reduction in distal pulmonary arteries in adult TGF-{alpha} transgenic mice (Fig. 1). Histology of barium-infused lungs showed severe enlargement of distal air spaces, a paucity of small pulmonary arteries, and areas of interstitial pulmonary fibrosis and extensive pleural thickening (Fig. 2). Counting of barium-filled pulmonary arteries showed that pulmonary artery density in adult TGF-{alpha} transgenic mice was 70% lower than controls (P < 0.05, Fig. 5C). Immunostaining for the endothelial marker PECAM detected endothelial cells in arteries and capillaries as well as the capillary network in the septae of the distal air spaces (Fig. 3). Immunostaining for PECAM did not detect a difference in vascular structure in the lungs of newborn TGF-{alpha} mice compared with WT mice, whereas in 2-wk-old mice the vascular and alveolar structure was extremely simplified in the TGF-{alpha} mice compared with WT mice.



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Fig. 1. Disruption of pulmonary arterial development in transforming growth factor (TGF)-{alpha} transgenic mice. Pulmonary arteriograms of wild type (WT) mice showed extensive branching of the pulmonary arteries and an extensive network of distal pulmonary arteries. In contrast to WT, arteriograms of TGF-{alpha} transgenic mice showed a reduction in distal pulmonary arteries. Shown are representative arteriograms from 8 adult WT and 8 adult TGF-{alpha} transgenic mice.

 


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Fig. 2. Disruption of distal lung development in TGF-{alpha} transgenic mice. After barium-gelatin infusions into the arterial system, lungs were inflation fixed at constant pressure through the trachea. Lungs were paraffin embedded and sectioned. Shown are representative sections stained with hematoxylin and eosin from a WT mouse and a TGF-{alpha} transgenic mouse. Some examples of pulmonary arteries filled with barium (brown in color) are marked with arrows. Barium-filled pulmonary arteries were reduced in numbers in TGF-{alpha} mice relative to WT. Distal air spaces are severely enlarged, and alveolar septae and the pleura are both thickened in TGF-{alpha} transgenic mice compared with WT mice. Micrographs are representative of sections from 8 adult WT and 8 adult TGF-{alpha} transgenic mice and are at the same magnification (x40). Bar = 100 µm.

 


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Fig. 5. Expression of dominant-negative mutant EGF receptor (EGFR-M) in distal epithelium reduces disruption of pulmonary vascular development by TGF-{alpha}. A: pulmonary arteriograms were performed on WT, TGF-{alpha}, EGFR-M, and bitransgenic TGF-{alpha}+EGFR-M mice. Arteriograms of TGF-{alpha} transgenic mice showed pruning of the arterial system compared with WT. Pulmonary vascular development is substantially improved in bitransgenic mice (TGF-{alpha}+EGFR-M) compared with TGF-{alpha} mice. Shown are 2 examples representative of arteriograms from 4-6 adult animals per study group. B: lung histology of WT, TGF-{alpha}, EGFR-M, and bitransgenic TGF-{alpha}+EGFR-M mice following barium infusion into the pulmonary arteries and lung fixation via tracheal installation of paraformaldehyde at constant pressure. Paraffin sections were stained with hematoxylin and eosin. Barium-filled pulmonary arteries appear brown-green in color (arrows mark some examples). Alveolarization and vascular density are substantially improved in bitransgenic mice (TGF-{alpha}+EGFR-M) compared with TGF-{alpha} mice. Shown are representative micrographs from 4-6 adult animals per study group at the same magnification (x60). Bar = 100 µm. C: pulmonary arterial density in WT, TGF-{alpha} transgenic, bitransgenic TGF-{alpha}+EGFR-M, and EGFR-M mice was determined by counting pulmonary arteries per high-powered field (x150). Arterial density was 70% lower in TGF-{alpha} mice compared with WT controls, but only 18% lower in bitransgenic mice (P < 0.05). Pulmonary artery density in bitransgenic mice was not different from EGFR-M mice (P > 0.05). Data were derived from 4-6 adult animals in each study group. *Significant difference from WT, bitransgenic TGF-{alpha}+EGFR-M, and EGFR-M mice, P < 0.05.

 


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Fig. 3. Platelet endothelial cell adhesion molecule (PECAM) immunostaining of pulmonary vascular structure in 1-day-old newborn and 2-wk-old WT and TGF-{alpha} transgenic mice. Vascular development was assessed by PECAM immunostaining of 1-day-old newborn (1d PN) and 2-wk-old (2wk PN) mice. PECAM staining (brown) detected endothelial cells in arteries, veins, and capillaries. Slides were lightly counterstained with nuclear fast red. Arrows indicate arteries and veins. PECAM staining is also seen marking the capillary network in the septae of the distal air spaces. No remarkable differences in PECAM staining were seen in the 1-day-old newborn TGF-{alpha} mice compared with WT littermates, whereas in 2-wk-old mice the vascular and alveolar structure was extremely simplified in the TGF-{alpha} mice compared with WT mice. Micrographs are representative of sections from 4-5 WT and 4-5 TGF-{alpha} mice and are at the same magnification (x150). Bar = 50 µm.

 

Body weights, hematocrits, pulmonary hypertension, and arterial remodeling in TGF-{alpha} mice. Body weights of adult TGF-{alpha} mice were 24% lower than adult WT controls (P < 0.05, Table 1). Hematocrit was increased by 36% in adult TGF-{alpha} mice compared with WT controls (P < 0.05, Table 1). RVSP was increased 2.1-fold in adult TGF-{alpha} mice (P < 0.05, Table 1). LVSP measurements in TGF-{alpha} mice were not different [P = not significant (NS), Table 1]. The ratio of RV to body weight was 2.3-fold higher, and the ratio of RV to LV+S weight was 1.9-fold higher in TGF-{alpha} transgenic mice (P < 0.05, Table 1). The ratio of LV+S to body weight was not different (Table 1, P = NS). Abnormally extensive muscularization (revealed by immunostaining for smooth muscle {alpha}-actin) of small pulmonary arteries was observed in adult TGF-{alpha} mice (Fig. 4), and morphometric analysis showed a significant increase in the percentage of fully muscularized distal small pulmonary arteries compared with WT (78 ± 6% vs. 3 ± 1%, P < 0.05; Fig. 6B).


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Table 1. Body weight, hematocrit, and pulmonary hypertension in adult TGF-{alpha} mice

 


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Fig. 4. Small pulmonary arteries in TGF-{alpha} mice show increased muscularization. Pulmonary arteries were injected with barium-gelatin mixture, and the lungs were inflation fixed with paraformaldehyde through the trachea. Micrographs show results of immunostaining for smooth muscle {alpha}-actin of distal small pulmonary arteries. Abnormally extensive muscularization of small pulmonary arteries (15-50 µm) in TGF-{alpha} transgenic mice (solid arrows) was detected, compared with WT mice, which showed little or only partial staining (open arrows). Representative micrographs are shown at the same magnification (x100). Bar = 50 µm.

 


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Fig. 6. Expression of EGFR-M in distal epithelium prevents pulmonary hypertension in TGF-{alpha} mice. A: right ventricular hypertrophy was assessed in WT, TGF-{alpha} transgenic, bitransgenic TGF-{alpha}+EGFR-M, and EGFR-M mice as the ratio of right ventricle to left ventricle plus septum weights (RV/LV+S). The ratio of RV/LV+S weights was 2.6-fold higher in TGF-{alpha} transgenic mice compared with WT controls, indicating severe pulmonary hypertension. RV/LV+S weights in bitransgenic and EGFR-M mice were not different from WT. Data were derived from 4-6 adult animals in each study group. *Significant difference from WT, bitransgenic TGF-{alpha}+EGFR-M, and EGFR-M mice, P < 0.05. B: morphometric analysis showed abnormally extensive muscularization of distal pulmonary arteries in adult TGF-{alpha} transgenic mice compared with WT. Abnormal muscularization of small pulmonary arteries was prevented in bitransgenic TGF-{alpha}+EGFR-M mice compared with TGF-{alpha} mice. NM, nonmuscular; PM, partially muscular; FM, fully muscular. Data were derived from 4 adult animals in each study group. *Significant difference from WT, bitransgenic TGF-{alpha}+EGFR-M, and EGFR-M mice, P < 0.05.

 

Arterial density, histology, and RVH in bitransgenic mice. TGF-{alpha} mice were crossed with transgenic mice expressing a dominant-negative EGFR-M to determine whether EGFR signaling in distal epithelial cells contributes to pulmonary vascular disease in the TGF-{alpha} mice. Pulmonary arteriograms (Fig. 5A) and lung histology (Fig. 5B) show that vascular development and alveolarization were improved with expression of EGFR-M and TGF-{alpha} compared with TGF-{alpha} alone. Pulmonary artery density in bitransgenic mice (TGF-{alpha}+EGFR-M) was increased 2.8-fold compared with TGF-{alpha} mice (11.3 ± 0.6 vs. 4.0 ± 0.4, P < 0.05) and was only 18% lower than WT (11.3 ± 0.6 vs. 13.8 ± 1.0, P < 0.05; Fig. 5C). Pulmonary artery density in bitransgenic mice was not different from EGFR-M mice (11.3 ± 0.6 vs. 11.6 ± 0.7; P = NS). The ratios of RV/LV+S weight in bitransgenic and EGFR-M mice were not different from WT (0.29 ± 0.04 and 0.25 ± 0.02 vs. 0.23 ± 0.01, P = NS), indicating that pulmonary hypertension was prevented in bitransgenic TGF-{alpha}+EGFR-M mice compared with TGF-{alpha} mice (Fig. 6A). The ratios of RV/body wt in bitransgenic and EGFR-M mice were not different from WT (0.00070 ± 0.00011 and 0.00073 ± 0.00005 vs. 0.00080 ± 0.00005, P = NS) but were both different compared with TGF-{alpha} mice (0.0018 ± 0.0002, P < 0.05), also indicating that pulmonary hypertension was prevented in bitransgenic TGF-{alpha}+EGFR-M mice. Abnormal muscularization of small pulmonary arteries was also prevented in bitransgenic TGF-{alpha}+EGFR-M mice compared with TGF-{alpha} mice (Fig. 6B).

VEGF protein levels in the lungs of TGF-{alpha} mice. Lung VEGF-A protein levels were decreased by 28% in adult and 45% in 2-day-old TGF-{alpha} mice compared with age-matched WT controls (P < 0.05; Fig. 7, histograms). Western blot analysis also showed that VEGF164 protein levels were reduced in the lungs of 2-day and adult TGF-{alpha} mice (Fig. 7).



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Fig. 7. Lung VEGF protein levels in adult and infant TGF-{alpha} mice are reduced compared with WT controls. A: VEGF levels are reduced in adult TGF-{alpha} mice compared with WT controls. Top: Western blot analysis of lung homogenates for the 164-amino acid isoform of VEGF (VEGF164). Histogram shows data from mouse VEGF ELISA for total lung VEGF-A levels (pg) corrected to total lung protein (mg). Data were derived from 5 animals in each study group. *P < 0.05 vs. WT. B: VEGF levels are reduced in infant TGF-{alpha} mice compared with WT controls. Top: Western blot analysis of lung homogenates for VEGF164. Histogram shows data from mouse VEGF ELISA for total lung VEGF-A levels (pg) corrected to total lung protein (µg). Data were derived from 4-5 animals in each study group. *Significant difference from WT, P < 0.05.

 


    DISCUSSION
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 
A number of clinical studies have suggested that increases in TGF-{alpha} and its receptor EGFR may be playing a role in a number of infant and adult lung diseases (32, 36, 37). Our findings show that expression of TGF-{alpha} in the distal epithelium of transgenic mice disrupted pulmonary vascular development and caused severe pulmonary hypertension. Accompanying the pulmonary hypertension was vascular remodeling with abnormal muscularization of distal pulmonary arteries. Previous findings (17, 19, 24) of disrupted postnatal septation causing alveolar simplification and pulmonary fibrosis were also seen. Immunostaining to detect endothelial cells shows that the vascular structure in newborn TGF-{alpha} mice was not notably abnormal, confirming previous findings that the onset of disrupted lung development induced by TGF-{alpha} appears to occur primarily in the postnatal alveolar phase (17). When TGF-{alpha} mice were crossed with transgenic mice expressing a dominant-negative EGFR-M also under the control of the SP-C promoter, disruption of pulmonary vascular development was reduced and pulmonary hypertension was prevented, demonstrating that the vascular effects of TGF-{alpha} are mediated through EGFR signaling in the distal epithelium.

The reduction in distal pulmonary arteries in the TGF-{alpha} mice may be secondary to reductions in alveolar growth, although a recent study has shown that inhibition of angiogenesis in the developing newborn lung reduces postnatal alveolarization (22). Interactions between the developing distal alveolar epithelium and pulmonary vasculature are likely bidirectional to ensure that there is coordinate development of these functionally dependent structures. It is well known that lung epithelium and mesenchyme produce paracrine factors that influence and regulate the development of each other (35). Although an early study showed that TGF-{alpha} was a potent proangiogenic factor in the hamster cheek pouch assay (34), in the avian embryo TGF-{alpha} and EGF reduced the hemangiopoietic potential and endothelial cell migration from the splanchnopleural mesoderm, a tissue that plays a critical role in lung development (33). Our findings show that disruption of pulmonary vascular development by TGF-{alpha} was inhibited by the targeting of a dominant-negative EGFR to the distal epithelium. This suggests that an indirect mechanism accounts for the disruption of pulmonary vascular development by TGF-{alpha} and that EGFR-dependent autocrine signaling in the epithelium plays a major role in the vascular effects of TGF-{alpha} and that it is not a direct effect of TGF-{alpha} on endothelial cells. Reductions in VEGF may be downstream of TGF-{alpha}-EGFR signaling in the distal epithelium, since we found that VEGF levels were reduced in the lungs of neonatal TGF-{alpha} mice before noticeable signs of disrupted lung development (17). VEGF is critical for normal vascular development in a number of organs, and deletion of even one allele of the VEGF gene is fetal lethal (5, 12), suggesting that even modest reductions in VEGF expression, as in our findings, can have severe consequences. The lung epithelium and type II cells play a critical role in pulmonary vascular development and are major sources of VEGF in the developing and adult lung (13, 15, 20). Although other secondary mediators (such as angiostatic factors) could also be playing a role in this model, inhibition of VEGF signaling has been shown to disrupt pulmonary vascular development and reduce alveolarization and cause severe pulmonary hypertension in infant rats (22, 28).

TGF-{alpha} mice developed severe pulmonary hypertension and vascular remodeling characterized by abnormally extensive muscularization of small pulmonary arteries. The etiology of pulmonary hypertension in the TGF-{alpha} transgenic mice is likely complex. Reduced arterial density and vascular remodeling may contribute to increased pulmonary vascular resistance and then lead to the pulmonary hypertension. Vascular remodeling and muscularization of the small pulmonary arteries may be caused by increased shear stress and/or hypoxemia (suggested by increases in hematocrit), which result from the severe reduction in arterial density. It is also possible that increased perivascular fibrosis, which has been previously reported (24), may also contribute to the pulmonary hypertension and/or hypoxemia. Activation of EGFR has been shown to cause vasoconstriction in the pulmonary circulation of rabbits (21), and so it is also possible that vasoconstriction could have also contributed to the development of pulmonary hypertension in the TGF-{alpha} mice. In other mouse models with pulmonary hypertension (11, 41), it has been suggested that abnormal muscularization of small pulmonary arteries may be due to differentiation of pericytes into smooth muscle cells and/or migration and proliferation of existing smooth muscle cells. Interestingly, expression of the dominant-negative EGFR-M in distal epithelial cells substantially improved arterial density in the bitransgenic mice (TGF-{alpha}+EGFR-M) and was sufficient to prevent both RVH and abnormal muscularization of small pulmonary arteries, despite the fact that arterial density was not completely restored to WT levels.

Although clinical studies have shown high concentrations of TGF-{alpha} in the bronchoalveolar lavage fluid and lungs of infants with BPD (32, 37) and low levels of VEGF (3, 26, 27), whether TGF-{alpha} and VEGF actually contribute to the pathogenesis of BPD is unclear. Although a number of other adverse stimuli, including inflammation and barotrauma, are thought to play a role in the pathogenesis of BPD (4, 7, 23), the chronic production of TGF-{alpha} in the lungs of transgenic mice induces many of the pathological features characteristic of BPD, in the absence of any evidence of inflammation (19). Still, a recent study (29) has shown that inflammatory cytokines (TNF-{alpha}, IL-4 and -13) can induce TGF-{alpha} in bronchial epithelial cells from asthmatics, raising the possibility that TGF-{alpha}-EGFR signaling can be activated and may be downstream of some inflammatory pathways.

In conclusion, in addition to alveolar simplification and pulmonary fibrosis, overexpression of TGF-{alpha} in the distal epithelium of transgenic mice disrupted pulmonary vascular development and resulted in severe pulmonary hypertension and vascular remodeling. EGFR signaling through the distal epithelium played a major role in the pathogenesis of pulmonary vascular disease induced by TGF-{alpha}. VEGF expression was reduced in the lungs of the TGF-{alpha} mice and may play a role.


    DISCLOSURES
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 
This work was supported by National Heart, Lung, and Blood Institute Grants HL-72894 (T. D. Le Cras), HL-04172 (W. D. Hardie), 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 and Patricia Pastura (Pulmonary Biology, Cincinnati Children's Hospital Medical Center) for excellent technical assistance, Rose Martin (Radiology, Cincinnati Children's Hospital Medical Center) for help with radiography, and David Loudy (Morphology Core, Pulmonary Biology, Cincinnati Children's Hospital Medical Center) for help with immunostaining.


    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 (E-mail: tim.lecras{at}chmcc.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.


    REFERENCES
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 

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