Transfer of the Active Form of Transforming Growth Factor-β1 Gene to Newborn Rat Lung Induces Changes Consistent with Bronchopulmonary Dysplasia
2003; Elsevier BV; Volume: 163; Issue: 6 Linguagem: Inglês
10.1016/s0002-9440(10)63612-7
ISSN1525-2191
AutoresJack Gauldie, Tom Galt, Philippe Bonniaud, Clinton S. Robbins, Margaret M. Kelly, David Warburton,
Tópico(s)Medical Imaging and Pathology Studies
ResumoBronchopulmonary dysplasia is a chronic lung disease of premature human infancy that shows pathological features comprising varying sized areas of interstitial fibrosis in association with distorted large alveolar spaces. We have previously shown that transfer of active transforming growth factor (TGF)-β1 (AdTGFβ1223/225) genes by adenovirus vector to embryonic lungs results in inhibition of branching morphogenesis and primitive peripheral lung development, whereas transfer to adult lungs results in progressive interstitial fibrosis. Herein we show that transfer of TGF-β1 to newborn rat pups results in patchy areas of interstitial fibrosis developing throughout a period of 28 days after transfer. These areas of fibrosis appear alongside areas of enlarged alveolar spaces similar to the prealveoli seen at birth, suggesting that postnatal lung development and alveolarization has been inhibited. In rats treated with AdTGFβ1223/225, enlarged alveolar spaces were evident by day 21, and by 28 days, the mean alveolar cord length was nearly twice that in control vector or untreated rats. Hydroxyproline measurements confirmed the presence of fibrosis. These data suggest that overexpression of TGF-β1 during the critical period of postnatal rat lung alveolarization gives rise to pathological, biochemical, and morphological changes consistent with those seen in human bronchopulmonary dysplasia, thus inferring a pathogenic role for TGF-β in this disorder. Bronchopulmonary dysplasia is a chronic lung disease of premature human infancy that shows pathological features comprising varying sized areas of interstitial fibrosis in association with distorted large alveolar spaces. We have previously shown that transfer of active transforming growth factor (TGF)-β1 (AdTGFβ1223/225) genes by adenovirus vector to embryonic lungs results in inhibition of branching morphogenesis and primitive peripheral lung development, whereas transfer to adult lungs results in progressive interstitial fibrosis. Herein we show that transfer of TGF-β1 to newborn rat pups results in patchy areas of interstitial fibrosis developing throughout a period of 28 days after transfer. These areas of fibrosis appear alongside areas of enlarged alveolar spaces similar to the prealveoli seen at birth, suggesting that postnatal lung development and alveolarization has been inhibited. In rats treated with AdTGFβ1223/225, enlarged alveolar spaces were evident by day 21, and by 28 days, the mean alveolar cord length was nearly twice that in control vector or untreated rats. Hydroxyproline measurements confirmed the presence of fibrosis. These data suggest that overexpression of TGF-β1 during the critical period of postnatal rat lung alveolarization gives rise to pathological, biochemical, and morphological changes consistent with those seen in human bronchopulmonary dysplasia, thus inferring a pathogenic role for TGF-β in this disorder. Bronchopulmonary dysplasia (BPD) remains an all too common complication of human premature delivery, despite the advent of artificial surfactants, antenatal and postnatal treatment with glucocorticoids, and the increasing use of gentler modes of mechanical ventilation.1Bancalari E Changes in the pathogenesis and prevention of chronic lung disease of prematurity.Am J Perinatol. 2001; 18: 1-9Crossref PubMed Scopus (130) Google Scholar, 2Jobe AH Ikegami M Prevention of bronchopulmonary dysplasia.Curr Opin Pediatr. 2001; 13: 124-129Crossref PubMed Scopus (101) Google Scholar The term BPD was coined to describe a radiographical entity, comprising patchy interstitial fibrosis along with distortion of peripheral air spaces, which occurs after ventilation of babies with hyaline membrane disease.3Northway WH Rosan RC Porter DY Pulmonary disease following respiratory therapy of hyaline membrane disease. Bronchopulmonary dysplasia.N Engl J Med. 1967; 276: 357-368Crossref PubMed Scopus (2342) Google Scholar This pathology was thought to be a sequela of abnormal repair processes after the resolution of hyaline membrane disease of the newborn. BPD is now more frequently diagnosed in very premature human infants with chronic lung disease who require long-term oxygen therapy.4Jobe AH The new BPD: an arrest of lung development.Pediatr Res. 1999; 46: 641-643Crossref PubMed Scopus (813) Google Scholar BPD is thought to begin as postinjury inflammation, leading to interstitial fibrosis. But, as the lung continues to enlarge, reduced numbers of enlarged distal air spaces also appear.5Erickson AM de la Monte SM Moore GW Hutchins GM The progression of morphologic changes in bronchopulmonary dysplasia.Am J Pathol. 1987; 127: 474-484PubMed Google Scholar The pathogenesis of this disorder is poorly understood despite numerous studies. With the advent of exogenous pulmonary surfactant and more frequent glucocorticoid therapy, the incidence of this form of BPD has fortunately decreased. However, among extremely low birth weight infants, new BPD has emerged as an increasingly common problem.1Bancalari E Changes in the pathogenesis and prevention of chronic lung disease of prematurity.Am J Perinatol. 2001; 18: 1-9Crossref PubMed Scopus (130) Google Scholar, 2Jobe AH Ikegami M Prevention of bronchopulmonary dysplasia.Curr Opin Pediatr. 2001; 13: 124-129Crossref PubMed Scopus (101) Google Scholar, 6Jobe AH Bancalari E Bronchopulmonary dysplasia.Am J Respir Crit Care Med. 2001; 163: 1723-1729Crossref PubMed Scopus (3632) Google Scholar The pathology of the new BPD is characterized primarily by alveolar hypoplasia with some interstitial cellular infiltrates and less marked interstitial fibrosis, and has been considered an arrest of lung development.4Jobe AH The new BPD: an arrest of lung development.Pediatr Res. 1999; 46: 641-643Crossref PubMed Scopus (813) Google Scholar However, recent data showing involvement of growth and repair factors such as transforming growth factor (TGF)-β in adult pulmonary fibrosis,7Selman M King Jr, TE Pardo A Idiopathic pulmonary fibrosis: prevailing and evolving hypotheses about its pathogenesis and implications for therapy.Ann Intern Med. 2001; 134: 136-151Crossref PubMed Scopus (1557) Google Scholar, 8Gauldie J Kolb M Sime PJ A new direction in the pathogenesis of idiopathic pulmonary fibrosis?.Respir Res. 2002; 3: 1-3Crossref PubMed Scopus (97) Google Scholar, 9Sime PJ Xing Z Graham FL Csaky KG Gauldie J Adenovector-mediated gene transfer of active TGF-β1 induces prolonged severe fibrosis in rat lung.J Clin Invest. 1997; 100: 768-776Crossref PubMed Scopus (911) Google Scholar as well as the role of TGF-β as an inhibitor of branching morphogenesis in embryonic lung development,10Zhao J Sime PJ Bringas Jr, P Gauldie J Warburton D Epithelium-specific adenoviral transfer of a dominant-negative mutant TGFβ type II receptor stimulates embryonic lung branching morphogenesis in culture and potentiates EGF and PDGF-AA.Mech Dev. 1998; 72: 89-100Crossref PubMed Scopus (54) Google Scholar, 11Zhao J Sime PJ Bringas P Tefft JD Buckley S Bu D Gauldie J Warburton D Spatial-specific TGF-β1 adenoviral expression determines morphogenetic phenotypes in embryonic mouse lung.Eur J Cell Biol. 1999; 78: 715-725Crossref PubMed Scopus (28) Google Scholar, 12Warburton D Schwarz M Tefft D Flores-Delgado G Anderson KD Cardoso WV The molecular basis of lung morphogenesis.Mech Dev. 2001; 92: 55-81Crossref Scopus (628) Google Scholar suggested to us that factors such as TGF-β may induce similar changes in neonatal lung. We have previously used adenovirus vectors to effect transient gene transfer of active TGF-β1 (AdTGFβ1223/225) to the lung of adult rats9Sime PJ Xing Z Graham FL Csaky KG Gauldie J Adenovector-mediated gene transfer of active TGF-β1 induces prolonged severe fibrosis in rat lung.J Clin Invest. 1997; 100: 768-776Crossref PubMed Scopus (911) Google Scholar resulting in the development of progressive interstitial fibrosis. Herein we have used the same vector to transfer TGF-β1 to the lung of 1-day-old rat pups. We show that this transient overexpression (5 to 10 days) of TGF-β1 causes morphological and biochemical changes consistent with interstitial fibrosis along with large distorted alveoli that appear within a few weeks after birth, pathological features strikingly similar to those seen in human BPD. One-day-old neonatal Sprague-Dawley rats were exposed to isofluorane for 30 to 40 seconds to provide light anesthesia and were administered intranasally with 1 × 108Gauldie J Kolb M Sime PJ A new direction in the pathogenesis of idiopathic pulmonary fibrosis?.Respir Res. 2002; 3: 1-3Crossref PubMed Scopus (97) Google Scholar plaque forming units (PFUs) of either Ad dl70-3 (control vector, no transgene) or AdTGFβ1223/225 (vector expressing the active form of TGF-β1) diluted in 10 μl of sterile phosphate-buffered saline (PBS). Holding the anesthetized rat upright, the adenoviral solution was placed into the nasal passages using a P10 pipette. Using the thumb, slight pressure was placed on the bottom of the jaw to keep the mouth closed and force the animal to inhale the solution through its nose. The rats were held upright for a further 20 to 30 seconds to maximize delivery, and then allowed to recover under supervision before returning them to their mother. Preliminary studies with AdLacZ (vector expressing LacZ) showed this approach gave rise to wide distribution of the vector throughout the lung with uptake in bronchial and bronchiolar epithelial cells, similar to the distribution seen in adult rats with either intranasal or intratracheal instillation (data not shown). The control vector (empty) was chosen to avoid any confusing issues resulting from known immune responses against an expressed foreign transgene, such as LacZ.13Van Ginkel FW Liu C Simecka JW Dong JY Greenway T Frizzell RA Kiyono H McGhee JR Pascual DW Intratracheal gene delivery with adenoviral vector induces elevated systemic IgG and mucosal IgA antibodies to adenovirus and beta-galactosidase.Hum Gene Therapy. 1995; 6: 895-903Crossref PubMed Scopus (114) Google Scholar The TGF-β1 gene is highly homologous and does not engender immune reactions in the host. Because of the small and delicate nature of neonatal rat lungs, bronchoalveolar lavage could not readily be performed in a consistent manner to assess the presence of TGF-β1 transgene. To accomplish this, three to five neonatal rats per group were sacrificed 2 days after adenovector injection. Before excising their lungs, the right ventricle of the heart was perfused with 3 ml of ice-cold PBS to remove all blood from the lungs. The lungs were then excised, weighed, and kept in ice-cold PBS. Lung weights did not vary significantly between individual rats or treated groups throughout the extent of the experiment. Shortly thereafter, lungs were transferred to 2 ml of fresh PBS and homogenized. The homogenate was pelleted by centrifugation at 12,000 rpm for 10 minutes at 4°C and the cytokine-rich supernatant was stored at −70°C until assayed. TGF-β1 levels were determined as described below and expressed as pg of active TGF-β1 per mg wet lung weight. TGF-β1 levels were determined using a human TGF-β1 enzyme-linked immunosorbent assay kit (R&D Systems, Inc., Minneapolis, MN) which is totally cross-reactive with porcine and rodent TGF-β1, but does not cross-react with TGF-β2 or TGF-β3. Total TGF-β1 levels were measured by acid-activating samples as per the manufacturer's protocol. Levels of active TGF-β1 were measured by assaying samples that were not acid-activated. A modified Woessner protocol14Woessner JF The determination of hydroxyproline in tissue and protein samples containing small proportions of this imino acid.Arch Biochem Biophys. 1961; 93: 440-447Crossref PubMed Scopus (3388) Google Scholar was used. Tissue was prepared by immediately snap-freezing the right lobe of excised lungs in liquid nitrogen, then transferring the samples to −70°C for storage until the time of assay. Samples were homogenized in 5 ml of ddH2O. One ml of the resultant homogenate was hydrolyzed in 2 ml of 6 N HCl for 16 hours at 110°C. Samples were adjusted to pH 7.0 and then 400 μl was diluted into 2.0 ml with ddH2O. The colorimetric assay was initiated with the addition of 1 ml of chloramine-T solution to the diluted sample at pH 7.0. One ml of 70% perchloric acid was added, followed by 1 ml of a dimethylbenzaldehyde solution. After 20 minutes at 60°C, the samples were returned to room temperature and the optical density at 557 nm was determined. Using hydroxyproline standards (Sigma Chemical, St. Louis, MO), results were expressed as μg hydroxyproline per mg wet lung weight. In adult mice, on lung excision, the left lobe was inflated and perfused with 10% formalin, and fixed for 24 hours before processing and paraffin embedding. In neonates, <10 days old, tissue was not perfused before fixation because of the small size of vessel for cannulation. Histological sections were stained with hematoxylin and eosin, a general nuclear stain, and elastin van Gieson, which stains collagen and elastin. Sections were also stained for collagen matrix with Sirius Red F3B (CI 35780) (Sigma Aldrich Can. Ltd., Oakville, Ontario, Canada) in saturated picric acid (Picrosirius Red stain). Select sections also underwent immunohistochemical staining for α-smooth muscle actin (α-SMA), as previously described.9Sime PJ Xing Z Graham FL Csaky KG Gauldie J Adenovector-mediated gene transfer of active TGF-β1 induces prolonged severe fibrosis in rat lung.J Clin Invest. 1997; 100: 768-776Crossref PubMed Scopus (911) Google Scholar The size of alveolar space was determined from lung sections stained with Picrosirius Red. Mean chord length (μm) was measured at ×100 magnification and is calculated from automated morphometric analysis using a Leica Q500IW high-grade image analysis system. Chord length increases with alveolar enlargement and is similar to mean linear intercept with the advantage of being independent of the thickness of septa.15Zheng T Zhu Z Wang Z Homer RJ Ma B Riese RJ Chapman HA Shapiro SD Elias JA Inducible targeting of IL-13 to the adult lung causes matrix metalloproteinase- and cathepsin-dependent emphysema.J Clin Invest. 2000; 106: 1081-1093Crossref PubMed Scopus (546) Google Scholar Data were expressed as mean ± SEM or SD. Statistical significance was determined using Student's two-tailed t-test, assuming unequal variances. Differences were considered statistically significant if P was 30-fold) levels of total and active (10-fold) TGF-β1 than those treated with control vector (P < 0.02). There was no significant difference in TGF-β1 levels between control vector-treated and untreated groups. At 7 days after infection (8 days old) normal rat lung showed a hypercellular thick interstitium, made up primarily from noninflammatory cells (Figure 2A) and alveoli that appeared to be smaller in size than those seen at birth. The animals that were treated with the control vector, dL70-3, showed a similar hypercellular interstitium with evidence of some inflammatory cells, neutrophils, and mononuclear cells (Figure 2B). The animals that were treated with AdTGF-β1 also showed a hypercellular interstitium with a number of inflammatory cells, along with areas of apparent consolidation (Figure 2C), seen to be positive for matrix deposition by staining with Picrosirius Red (Figure 2F). Throughout a period of days, the presence of inflammatory cells decreased in the two vector-treated groups, and the alveolar septa decreased in thickness in all three groups and by 29 days (28 days after injection) the lungs of the control vector group demonstrated fundamentally normal lung architecture when compared to untreated lung (Figure 3). Alveoli were divided and subdivided into their saccular network, providing a much larger area for gas exchange than was available at birth when the alveoli had not yet fully compartmentalized. Rat pups injected with the TGF-β1-expressing adenovirus vector developed disperse fibrotic lesions within their lungs (Figure 4), although not to the extent demonstrated in experiments in which the same vector was administered intratracheally to adult rats.9Sime PJ Xing Z Graham FL Csaky KG Gauldie J Adenovector-mediated gene transfer of active TGF-β1 induces prolonged severe fibrosis in rat lung.J Clin Invest. 1997; 100: 768-776Crossref PubMed Scopus (911) Google Scholar There was marked deposition of matrix within the parenchyma localized to defined areas throughout the lung. These were not particularly distributed, for example, in peribronchial areas, but tended to be associated at air interfaces rather than at vascular sites. Analysis of hydroxyproline content at 29 days showed an increase in matrix accumulation within the lungs of the Ad5E1TGFβ1223/225-treated pups compared to the control vector group (Figure 5). Using a one-way analysis of variance, the differences between the three groups showed P = 0.078. Using an independent t-test, day 28 TGF-β1-treated animals differed from day 21 TGF-β1-treated animals (P < 0.05) and day 28 TGF-β1-treated animals differed from control vector-treated animals (P < 0.05). There were no significant differences seen at 21 days, consistent with the progressive nature of fibrosis initiated by TGF-β1 overexpression in adult rats.9Sime PJ Xing Z Graham FL Csaky KG Gauldie J Adenovector-mediated gene transfer of active TGF-β1 induces prolonged severe fibrosis in rat lung.J Clin Invest. 1997; 100: 768-776Crossref PubMed Scopus (911) Google Scholar The areas positive for matrix deposition were also prominent for α-SMA-positive cells, most likely myofibroblasts. In addition, there were prominent α-SMA-positive cells in the alveolar walls indicating an enhanced presence of myofibroblasts throughout the lung parenchyma (Figure 6).Figure 4.Twenty-nine-day-old neonatal rat lung (28 days after treatment). Fibrotic response to AdTGFβ1223/225 vector. A and B were stained with EvG and C was stained with Picrosirius Red. Original magnifications: ×100 (A, C); ×200 (B).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 5.Hydroxyproline content of lungs of rats untreated or after intranasal administration of 1 × 108Gauldie J Kolb M Sime PJ A new direction in the pathogenesis of idiopathic pulmonary fibrosis?.Respir Res. 2002; 3: 1-3Crossref PubMed Scopus (97) Google Scholar PFU AdDL70 or AdTGFβ1223/225. Hydroxyproline content was analyzed 21 days (white bars) and 28 days (black bars) after treatment when the pups were 22 and 29 days old, respectively. TGF-β1 vector-treated group had significantly more hydroxyproline than the control vector group after 28 days, expressed as μg hydroxyproline per mg of wet lung tissue (P = 0.078, one-way analysis of variance; #, P < 0.05, t-test for TGF-β1 versus DL70) and was significantly different from day 21 TGF-β1-treated group (*, P < 0.05, t-test; n = 3 to 5 per group).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 6.Twenty-nine-day-old neonatal rat lung (28 days after treatment). A, C: Treatment with AdTGFβ1223/225. B, D: Untreated normal rat lung. A and B were stained with H&E and C and D were stained for α-SMA content. Original magnifications: ×100 (A, B); ×200 (C, D).View Large Image Figure ViewerDownload Hi-res image Download (PPT) Although excess matrix deposition (fibrosis) is a prominent feature in the Ad5E1TGFβ1223/225-treated lungs, even more striking are the large, apparently undeveloped prealveolar saccules that predominate in the lungs of pups from this group (Figure 6). There were no significant differences in alveolar space sizes at birth between the treatment groups (Table 1). By 21 days, the enlarged spaces were clearly evident with ∼50% increase in mean alveolar chord length from control-treated mice, and at 28 days, the increase was nearly 80% (P > 0.001 for both comparisons against untreated or control vector-treated animals) indicating the presence of markedly enlarged alveoli throughout the lung. The areas of enlarged alveoli are in many instances immediately adjacent to areas showing evidence of marked fibrosis (Figure 7) suggestive of pathology seen in human BPD.5Erickson AM de la Monte SM Moore GW Hutchins GM The progression of morphologic changes in bronchopulmonary dysplasia.Am J Pathol. 1987; 127: 474-484PubMed Google Scholar In isolated cases, inflammatory cells may persist within the alveolar septa, but more often the septa resemble those of a normal lung, without inflammatory infiltrate or they are thickened from an interalveolar fibrotic process.Table 1Mean Alveolar Chord LengthNormal (μm)DL70 treated (μm)TGF-β1 treated (μm)2D36.3 ± 6.041.7 ± 9.039.9 ± 9.521D43.4 ± 5.243.2 ± 6.963.8 ± 10.3*P < 0.0001 versus normal or control vector.28D31.5 ± 9.532.6 ± 8.358.1 ± 9.1*P < 0.0001 versus normal or control vector.Values (μm) are mean ± SD, n = 10.* P < 0.0001 versus normal or control vector. Open table in a new tab Values (μm) are mean ± SD, n = 10. At 29 days of age rat lungs are not quite fully developed. The distal portions of the lung are often not completely alveolarized, demonstrating elastin caps on budding alveolar septa, characteristic of ongoing alveolar septation. We observed many examples of incompletely developed alveolar septa in the TGF-β1-treated lung, identified under high magnification by the presence of elastin caps (Figure 8). The underdeveloped alveoli and localized fibrotic lesions that developed after TGF-β1 exposure are both hallmarks of BPD in human newborns and they were not seen after treating pups with the control vector. Branching morphogenesis occurs during early stages of lung development when the respiratory epithelium invades surrounding mesodermal mesenchyme to form the primitive bronchial tree. Bronchial outgrowths continue to branch and develop throughout gestation, eventually forming the complete bronchial network of the lung, processes that are controlled by finely balanced positive and negative factors.12Warburton D Schwarz M Tefft D Flores-Delgado G Anderson KD Cardoso WV The molecular basis of lung morphogenesis.Mech Dev. 2001; 92: 55-81Crossref Scopus (628) Google Scholar, 16Hilfer S Morphogenesis of the lung: control of embryonic and fetal branching.Annu Rev Physiol. 1996; 58: 93-113Crossref PubMed Scopus (74) Google Scholar In the human, at birth, nonrespiratory airways are well developed, however, the alveolar structure of the lung is not yet mature. Extensive alveolarization greatly increases the surface area available for gas exchange by dividing and subdividing the large, premature peripheral air spaces already formed. During this period of growth, the infant lung is particularly sensitive to perturbations in developmental signals. BPD is a common neonatal lung disease that may be caused by such perturbations and is particularly prevalent in premature, very low birth weight newborns. Infants suffering from BPD exhibit a dramatically reduced lung volume,17Jackson JC Standaert TA Truog WE Murphy JH Palmer S Chi EY Woodrum DE Watchko JF Hodson WA Changes in lung volume and deflation stability in hyaline membrane disease.J Appl Physiol. 1985; 59: 1783-1789PubMed Google Scholar lower levels of alveolarization,18Chambers HN van Velzen D Ventilator-related pathology in the extremely immature lung.Pathology. 1989; 21: 79-83Crossref PubMed Scopus (82) Google Scholar and much larger alveolar spaces19Jobe A Ikegami M Mechanisms initiating lung injury in the preterm.Early Hum Dev. 1998; 53: 81-94Abstract Full Text Full Text PDF PubMed Scopus (364) Google Scholar compared to normal infants.6Jobe AH Bancalari E Bronchopulmonary dysplasia.Am J Respir Crit Care Med. 2001; 163: 1723-1729Crossref PubMed Scopus (3632) Google Scholar Chronic inflammation and abnormal tissue repair can lead to pulmonary fibrosis in mature lungs, and similarly, BPD in infants may be caused by inflammation, illustrated by the positive correlation between the occurrence of BPD and high levels of the inflammatory cytokines tumor necrosis factor-α, interleukin (IL)-1β, IL-6, and IL-8 in amniotic fluid.20Yoon BH Romero R Jun JK Park KH Park JD Ghezzi F Kim BI Amniotic fluid cytokines (interleukin-6, tumor necrosis factor-α, interleukin-1β, and interleukin-8) and the risk for the development of bronchopulmonary dysplasia.Am J Obstet Gynecol. 1997; 177: 825-830Abstract Full Text Full Text PDF PubMed Google Scholar Similar inflammatory mediators are also found in premature neonatal lamb lungs that have been ventilated.21Naik AS Kallapur SG Bachurski CJ Jobe AH Michna J Kramer BW Ikegami M Effects of ventilation with different positive end-expiratory pressures on cytokine expression in the preterm lamb lung.Am J Respir Crit Care Med. 2001; 164: 494-498Crossref PubMed Scopus (153) Google Scholar Tracheal aspirates from human premature infants that have an increased risk of developing BPD contain proinflammatory cytokines such as IL-8, while lacking anti-inflammatory cytokines such as IL-10.22Jones CA Cayabyab RG Kwong KY Stotts C Wong B Hamdan H Minoo P de Lemos RA Undetectable interleukin (IL)-10 and persistent IL-8 expression early in hyaline membrane disease: a possible developmental basis for the predisposition to chronic lung inflammation in preterm newborns.Pediatr Res. 1996; 39: 966-975Crossref PubMed Scopus (233) Google Scholar Increased levels of bioactive TGF-β have also been detected and this can predict the need for prolonged oxygen therapy in extremely premature human infants.23LeCart C Cayabyab R Buckley S Morrison J Kwong KY Warburton D Ramanathan R Jones CA Minoo P Bioactive transforming growth factor-beta in the lungs of extremely low birthweight neonates predicts the need for home oxygen supplementation.Biol Neonate. 2000; 77: 217-223Crossref PubMed Scopus (100) Google Scholar Although high O2 levels may directly contribute to BPD, it has been shown that overinflating the lung during ventilation can also lead to the production of profibrotic cytokines such as tumor necrosis factor-α, basic fibroblast growth factor, and TGF-β1 as well as extracellular matrix components.21Naik AS Kallapur SG Bachurski CJ Jobe AH Michna J Kramer BW Ikegami M Effects of ventilation with different positive end-expiratory pressures on cytokine expression in the preterm lamb lung.Am J Respir Crit Care Med. 2001; 164: 494-498Crossref PubMed Scopus (153) Google Scholar, 24Garland JS Buck RK Allred EN Leviton A Hypocarbia before surfactant therapy appears to increase bronchopulmonary dysplasia risk in infants with respiratory distress syndrome.Arch Pediatr Adolesc Med. 1995; 149: 617-622Crossref PubMed Scopus (159) Google Scholar, 25Berg JT Fu Z Breen EC Tran HC Mathieu-Costello O West JB High lung inflation increases mRNA levels of ECM components and growth factors in lung parenchyma.J Appl Physiol. 1997; 83: 120-128PubMed Google Scholar One might suggest that BPD possibly begins with an inflammatory insult, much like pulmonary fibrosis in adults, followed by the overexpression of TGF-β1 and deposition of excess extracellular matrix components. The early inflammation could be initiated either by hyperventilation and/or overdistension of the lung, by increased levels of O2, as well as by exposure of the lung to inflammatory mediators within the amniotic fluid, which may be present because of invading microbes within the mother.18Chambers HN van Velzen D Ventilator-related pathology in the extremely immature lung.Pathology. 1989; 21: 79-83Crossref PubMed Scopus (82) Google Scholar, 20Yoon BH Romero R Jun JK Park KH Park JD Ghezzi F Kim BI Amniotic fluid cytokines (interleukin-6, tumor necrosis factor-α, interleukin-1β, and interleukin-8) and the risk for the development of bronchopulmonary dysplasia.Am J Obstet Gynecol. 1997; 177: 825-830Abstract Full Text Full Text PDF PubMed Google Scholar, 24Garland JS Buck RK Allred EN Leviton A Hypocarbia before surfactant therapy appears to increase bronchopulmonary dysplasia risk in infants with respiratory distress syndrome.Arch Pediatr Adolesc Med. 1995; 149: 617-622Crossref PubMed Scopus (159) Google Scholar To investigate the theory that overexpression of TGF-β1 plays a key role in the pathogenes
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