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A number of growth factors and signaling pathways regulate matrix deposition and fibroblast proliferation in the lung. The epidermal growth factor receptor (EGFR) family of receptors and the transforming growth factor-β (TGF-β) family are active in diverse biological processes and are central mediators in the initiation and maintenance of fibrosis in many diseases. Transforming growth factor-α (TGF-α) is a ligand for the EGFR, and doxycycline (Dox)-inducible transgenic mice conditionally expressing TGF-α specifically in the lung epithelium develop progressive fibrosis accompanied with cachexia, changes in lung mechanics, and marked pleural thickening. Although recent studies demonstrate that EGFR activation modulates the fibroproliferative effects involved in the pathogenesis of TGF-β induced pulmonary fibrosis, in converse, the direct role of EGFR induction of the TGF-β pathway in the lung is unknown. The αvβ6 integrin is an important in vivo activator of TGF-β activation in the lung. Immunohistochemical analysis of αvβ6 protein expression and bronchoalveolar analysis of TGF-β pathway signaling indicates activation of the αvβ6/TGF-β pathway only at later time points after lung fibrosis was already established in the TGF-α model. To determine the contribution of the αvβ6/TGF-β pathway on the progression of established fibrotic disease, TGF-α transgenic mice were administered Dox for 4 wk, which leads to extensive fibrosis; these mice were then treated with a function-blocking anti-αvβ6 antibody with continued administration of Dox for an additional 4 wk. Compared with TGF-α transgenic mice treated with control antibody, αvβ6 inhibition significantly attenuated pleural thickening and altered the decline in lung mechanics. To test the effects of genetic loss of the β6 integrin, TGF-α transgenic mice were mated with β6-null mice and the degree of fibrosis was compared in adult mice following 8 wk of Dox administration. Genetic ablation of the β6 integrin attenuated histological and physiological changes in the lungs of TGF-α transgenic mice although a significant degree of fibrosis still developed. In summary, inhibition of the β6 integrin led to a modest, albeit significant, effect on pleural thickening and lung function decline observed with TGF-α-induced pulmonary fibrosis. These data support activation of the αvβ6/TGF-β pathway as a secondary effect contributing to TGF-α-induced pleural fibrosis and suggest a complex contribution of multiple mediators to the maintenance of progressive fibrosis in the lung.

Idiopathic pulmonary fibrosis (IPF) is marked by the activation of fibroblasts, leading to excessive production and deposition of extracellular matrix (ECM) within the lung parenchyma. Despite the pivotal role of ECM overexpression in IPF, potential negative regulators of ECM production in fibroblasts have yet to be identified. Semaphorin class 3B (SEMA3B), a secreted protein highly expressed in lung tissues, has established roles in axonal guidance and tumor suppression. However, the role of SEMA3B in ECM production by fibroblasts in the pathogenesis of IPF remains unexplored. Here, we show the downregulation of SEMA3B and its cognate binding receptor, neuropilin 1 (NRP1), in IPF lungs compared with healthy controls. Notably, the reduced expression of SEMA3B and NRP1 is associated with a decline in lung function in IPF. The downregulation of SEMA3B and NRP1 transcripts was validated in the lung tissues of patients with IPF, and two alternative mouse models of pulmonary fibrosis. In addition, we show that transforming growth factor-β (TGFβ) functions as a negative regulator of SEMA3B and NRP1 expression in lung fibroblasts. Furthermore, we demonstrate the antifibrotic effects of SEMA3B against TGFβ-induced ECM production in IPF lung fibroblasts. Overall, our findings uncovered a novel role of SEMA3B in the pathogenesis of pulmonary fibrosis and provided novel insights into modulating the SEMA3B-NRP1 axis to attenuate pulmonary fibrosis. NEW & NOTEWORTHY: The excessive production and secretion of collagens and other extracellular matrix proteins by fibroblasts lead to the scarring of the lung in severe fibrotic lung diseases. This study unveils an antifibrotic role for semaphorin class 3B (SEMA3B) in the pathogenesis of idiopathic pulmonary fibrosis. SEMA3B functions as an inhibitor of transforming growth factor-β-driven fibroblast activation and reduced levels of SEMA3B and its receptor, neuropilin 1, are associated with decreased lung function in idiopathic pulmonary fibrosis. [ABSTRACT FROM AUTHOR]

Transforming growth factor-[alpha] (TGF-[alpha]) is a ligand for the EGF receptor (EGFR). EGFR activation is associated with fibroproliferative processes in human lung disease and animal models of pulmonary fibrosis. We determined the effects of EGFR tyrosine kinase inhibitors gefitinib (Iressa) and erlotinib (Tarceva) on the development and progression of TGF-[alpha]-induced pulmonary fibrosis. Using a doxycycline-regulatable transgenic mouse model of lung-specific TGF-[alpha] expression, we determined effects of treatment with gefitinib and erlotinib on changes in lung histology, total lung collagen, pulmonary mechanics, pulmonary hypertension, and expression of genes associated with synthesis of ECM and vascular remodeling. Induction in the lung of TGF-[alpha] caused progressive pulmonary fibrosis over an 8-wk period. Daily administration of gefitinib or erlotinib prevented development of fibrosis, reduced accumulation of total lung collagen, prevented weight loss, and prevented changes in pulmonary mechanics. Treatment of mice with gefitinib 4 wk after the induction of TGF-[alpha] prevented further increases in and partially reversed total collagen levels and changes in pulmonary mechanics and pulmonary hypertension. Increases in expression of genes associated with synthesis of ECM as well as decreases of genes associated with vascular remodeling were also prevented or partially reversed. Administration of gefitinib or erlotinib did not cause interstitial fibrosis or increases in lavage cell counts. Administration of small molecule EGFR tyrosine kinase inhibitors prevented further increases in and partially reversed pulmonary fibrosis induced directly by EGFR activation without inducing inflammatory cell influx or additional lung injury. gefitinib; erlotinib; pulmonary hypertension

Transforming growth factor-[alpha] (TGF-[alpha]) and its receptor, the epithelial growth factor receptor (EGFR), have been associated with lung remodeling in premature infants with bronchopulmonary dysplasia (BPD). The goal of this study was to target TGF-[alpha] overexpression to the saccular phase of lung morphogenesis and determine early alterations in gene expression. Conditional lung-specific TGF-[alpha] bitransgenic mice and single-transgene control mice were generated. TGF-[alpha] overexpression was induced by doxycycline (Dox) treatment from embryonic day 16.5 (E16.5) to E18.5. After birth, all bitransgenic pups died by postnatal day 7 (P7). Lung histology at E18.5 and P1 showed abnormal lung morphogenesis in bitransgenic mice, characterized by mesenchymal thickening, vascular remodeling, and poor apposition of capillaries to distal air spaces. Surfactant levels (saturated phosphatidylcholine) were not reduced in bitransgenic mice. Microarray analysis was performed after 1 or 2 days of Dox treatment during the saccular (E17.5, E18.5) and alveolar phases (P4, P5) to identify genes induced by EGFR signaling that were shared or unique to each phase. We found 196 genes to be altered (> 1.5-fold change; P < 0.01 for at least 2 time points), with only 32% similarly altered in both saccular and alveolar phases. Western blot analysis and immunostaining showed that five genes selected from the microarrays (egr-1, SP-B, SP-D, S100A4, and pleiotrophin) were also increased at the protein level. Pathological changes in TGF-[alpha]-overexpressing mice bore similarities to premature infants born in the saccular phase who develop BPD, including remodeling of the distal lung septae and arteries. bronchopulmonary dysplasia; epidermal growth factor receptor; lung remodeling; lung development; transforming growth factor-[alpha]

To determine potential relationships between transforming growth factor (TGF)-[alpha] and surfactant homeostasis, the metabolism, function, and composition of surfactant phospholipid and proteins were assessed in transgenic mice in which TGF-[alpha] was expressed in respiratory epithelial cells. Secretion of saturated phosphatidylcholine was decreased 40-60% by expression of TGF-[alpha]. Although SP-A, SP-B, and SP-C mRNA levels were unchanged by expression of TGF-[alpha] SP-A and SP-B content in bronchoalveolar lavage fluid was decreased. The minimum surface tension of surfactant isolated from the transgenic mice was significantly increased. Incubation of cultured normal mice type II cells with TGF-[alpha] in vitro did not change secretion of surfactant phosphatidylcholine and SP-B, indicating that TGF-[alpha] does not directly influence surfactant secretion. Expression of a dominant negative (mutant) EGF receptor in the respiratory epithelium blocked the TGF-[alpha]-induced changes in lung morphology and surfactant secretion, indicating that EGF receptor signaling in distal epithelial cells was required for TGF-[alpha] effects on surfactant homeostasis. Because many epithelial cells were embedded in fibrotic lesions caused by TGF-[alpha], changes in surfactant homeostasis may at least in part be influenced by tissue remodeling that results in decreased surfactant secretion. The number of nonembedded type II cells was decreased 30% when TGF-[alpha] was expressed during development and was increased threefold by TGF-[alpha] expression in adulthood, suggesting possible alteration of type II cells on surfactant metabolism in the adult lung. Abnormalities in surfactant function and decreased surfactant level in the airways may contribute to the pathophysiology induced by TGF-[alpha] in both the developing and adult lung. bronchopulmonary dysplasia; acute respiratory distress syndrome; pulmonary fibrosis; surface activity of surfactant

Hardie, William D., Timothy D. Le Cras, Kenny Jiang, Jay W. Tichelaar, Mohamad Azhar, and Thomas R. Korfhagen. Conditional expression of transforming growth factor-[alpha] in adult mouse lung causes pulmonary fibrosis. Am J Physiol Lung Cell Mol Physiol 286: L741-L749, 2004. First published December 5, 2003; 10.1152/ajplung.00208.2003.--To determine whether overexpression of transforming growth factor (TGF)-[alpha] in the adult lung causes remodeling independently of developmental influences, we generated conditional transgenic mice expressing TGF-[alpha] in the epithelium under control of the doxycycline (Dox)-regulatable Clara cell secretory protein promoter. Two transgenic lines were generated, and following 4 days of Dox-induction TGF-[alpha] levels in whole lung homogenate were increased 13- to 18-fold above nontransgenic levels. After TGF-[alpha] induction, transgenic mice developed progressive pulmonary fibrosis and body weight loss, with mice losing 15% of their weight after 6 wk of TGF-[alpha] induction. Fibrosis was detected within 4 days of TGF-[alpha] induction and developed initially in the perivascular, peribronchial, and pleural regions but later extended into the interstitium. Fibrotic regions were composed of increased collagen and cellular proliferation and were adjacent to airway and alveolar epithelial sites of TGF-[alpha] expression. Fibrosis progressed in the absence of inflammatory cell infiltrates as determined by histology, without changes in bronchiolar alveolar lavage total of differential cell counts and without changes in proinflammatory cytokines TNF-[alpha] or IL-6. Active TGF-[beta] in whole lung homogenate was not altered 1 and 4 days after TGF-[alpha] induction, and immunostaining was not increased in the peribronchial/perivascular areas at all time points. Chronic epithelial expression of TGF-[alpha] in adult mice caused progressive pulmonary fibrosis associated with increased collagen and extracellular matrix deposition and increased cellular proliferation. Induction of pulmonary fibrosis by TGF-[alpha] was independent of inflammation or early activation of TGF-[beta]. collagen; proliferation; epidermal growth factor receptor; transforming growth factor-[beta]

Le Cras, Timothy D., William D. Hardie, Karen Fagan, Jeffrey A. Whitsett, and Thomas R. Korfhagen. Disrupted pulmonary vascular development and pulmonary hypertension in transgenic mice overexpressing transforming growth factor-[alpha]. Am J Physiol Lung Cell Mol Physio1285: L1046-L1054, 2003. First published August 1, 2003; 10.1152/ajplung.00045.2003.--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

Byline: William D. Hardie (1), Pablo A. Bejarano (2), Mary Ann Miller (2), James R. Yankaskas (3), Jon H. Ritter (4), Jeffrey A. Whitsett (1), Thomas R. Korfhagen (1) Keywords: Key words: cystic fibrosis, epidermal growth factor receptor, transforming growth factor [alpha] Abstract: Transforming growth factor [alpha] (TGF-[alpha]) is expressed in respiratory epithelial cells and alveolar macrophages during development and following lung injury. In the present study, the presence and sites of synthesis of TGF-[alpha] and its receptor, the epidermal growth factor receptor (EGF-R), were assessed in lung tissue from patients with severe lung disease caused by cystic fibrosis (CF). Lung sections from 24 individuals with CF, obtained at the time of lung transplantation, were compared to lung sections from five lung donors without CF. Cellular sites of TGF-[alpha], EGF-R, and cellular sites of proliferation were assessed by immunohistochemistry. All CF lung sections contained multiple cell types with detectable TGF-[alpha]. Compared to control sections, intensity of TGF-[alpha] immunostaining in macrophages, airway epithelial cells, and peribronchial submucosal cells was increased. EGF-R was detected in respiratory epithelial and peribronchial stromal cells but not in alveolar macrophages. The intensity of EGF-R staining in CF lung tissue did not differ from that of controls. An increased number of cells expressing Ki-67 nuclear antigen was detected in peribronchial submucosal cells but not bronchiolar epithelial cells in the CF lungs. The increased expression of TGF-[alpha] in CF lung tissue supports the concept that TGF-[alpha] plays a role in paracrine/autocrine regulation of lung remodeling associated with injury and repair in the lungs of individuals with cystic fibrosis. Author Affiliation: (1) Division of Pulmonary Biology, Children's Hospital Medical Center, 3333 Burnet Avenue, Cincinnati, OH 45229-3039, USA , US (2) Department of Pathology, University of Cincinnati School of Medicine, 231 Bethesda Avenue, Cincinnati, OH 45267, USA , US (3) Division of Pulmonary and Critical Care Medicine, The University of North Carolina at Chapel Hill School of Medicine, CB 7248, 7007 Thurston-Bowles Building, Chapel Hill, NC 27599, USA , US (4) Department of Pathology, Washington University School of Medicine, One Barnes Hospital Plaza, St. Louis, MO 63110, USA , US

The etiology of complicated parapneumonic effusions (CPE) in children seems to have come full circle in the United States during the past 60 years. In the preantibiotic era, Streptococcus pneumoniae [...]

Abstract Background Resistin-like molecule alpha or found in inflammatory zone protein (Fizz1) is increased in pulmonary epithelial cells and also in limited amounts by other lung cells during various lung injuries and fibrosis. However, the direct role of Fizz1 produced in the pulmonary epithelium has not been determined. Methods Fizz1 Transgenic mice (CCSP/Fizz1) were generated that overexpress Fizz1 in the lung epithelium under the control of a doxycycline (Dox) inducible lung epithelial cell specific promoter Scgb1a1 (Clara cell secretory protein, CCSP). Histology and FACS analysis of lung cells were used to identify the direct effects of Fizz1 in the transgenic mice (Dox treated) when compared with control (CCSP/-) mice. Intratracheal bleomycin sulfate or silica in saline and saline alone were used to study the role of Fizz1 during bleomycin- and silica-induced pulmonary fibrosis in CCSP/Fizz1 and CCSP/- mice. Weight change, pulmonary inflammation, and fibrosis were assessed 10 days post bleomycin or 28 days post silica challenge. Results When CCSP/Fizz1 mice were fed Dox food, elevated Fizz1 protein was detected in lung homogenates by western blot. Lungs of mice in which Fizz1 was induced in the epithelium contained increased lung cells staining for CD11c and F4/80 by FACS analysis consistent with increased dendritic cells however, no changes were observed in the percentage of interstitial macrophages compared to CCSP/- controls. No significant changes were found in the lung histology of CCSP/Fizz1 mice after up to 8 weeks of overexpression compared to CCSP/- controls. Overexpression of Fizz1 prior to challenge or following challenge with bleomycin or silica did not significantly alter airway inflammation or fibrosis compared to control mice. Conclusions The current study demonstrates that epithelial cell derived Fizz1 is sufficient to increase the bone-marrow derived dendritic cells in the lungs, but it is not sufficient to cause lung fibrosis or alter chemical or particle-induced fibrosis.