Cynthia Davidson

Rapamycin Prevents Transforming Growth Factor-α–Induced Pulmonary Fibrosis

Transforming growth factor (TGF)-alpha is a ligand for the epidermal growth factor receptor (EGFR). EGFR activation is associated with fibroproliferative processes in human lung disease and animal models of pulmonary fibrosis. Overexpression of TGF-alpha in transgenic mice causes progressive and severe pulmonary fibrosis; however, the intracellular signaling pathways downstream of EGFR mediating this response are unknown. Using a doxycycline-regulatable transgenic mouse model of lung-specific TGF-alpha expression, we observed increased PCNA protein and phosphorylation of Akt and p70S6K in whole lung homogenates in association with induction of TGF-alpha. Induction in the lung of TGF-alpha caused progressive pulmonary fibrosis over a 7-week period. Daily administration of rapamycin prevented accumulation of total lung collagen, weight loss, and changes in pulmonary mechanics. Treatment of mice with rapamycin 4 weeks after the induction of TGF-alpha prevented additional weight loss, increases in total collagen, and changes in pulmonary mechanics. Rapamycin prevented further increases in established pulmonary fibrosis induced by EGFR activation. This study demonstrates that mammalian target of rapamycin (mTOR) is a major effector of EGFR-induced pulmonary fibrosis, providing support for further studies to determine the role of mTOR in the pathogenesis and treatment of pulmonary fibrosis.

EGF receptor tyrosine kinase inhibitors diminish transforming growth factor-α-induced pulmonary fibrosis

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.

MEK-ERK pathway modulation ameliorates pulmonary fibrosis associated with epidermal growth factor receptor activation.

Pulmonary fibrosis remains a significant public health burden with no proven therapies. The mitogen-activated protein kinase (MAPK)/MAPK kinase (MEK)/extracellular signal-regulated kinase (ERK) signaling cascade is a major pathway controlling cellular processes associated with fibrogenesis, including growth, proliferation, and survival. Activation of the MAPK/ERK pathway is detected in the lungs of human fibrosis samples; however, the effect of modulating the pathway in vivo is unknown. Overexpression of transforming growth factor (TGF)-α in the lung epithelium of transgenic mice causes a progressive pulmonary fibrosis associated with increased MEK/ERK activation localized primarily in mesenchymal cells. To determine the role of the MEK pathway in the induction of TGF-α-induced lung fibrosis, TGF-α was overexpressed for 4 weeks while mice were simultaneously treated with the specific MEK inhibitor, ARRY-142886 (ARRY). Treatment with ARRY prevented increases in lung cell proliferation and total lung collagen, attenuated production of extracellular matrix genes, and protected mice from changes in lung function. ARRY administered as a rescue treatment after fibrosis was already established inhibited fibrosis progression, as assessed by lung histology, changes in body weights, extracellular matrix gene expression, and lung mechanics. These findings demonstrate that MEK inhibition prevents progression of established fibrosis in the TGF-α model, and provides proof of concept of targeting the MEK pathway in fibrotic lung disease.

Inhibition of the αvβ6 integrin leads to limited alteration of TGF-α-induced pulmonary fibrosis

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.

Bone Marrow–Derived Stromal Cells Are Invasive and Hyperproliferative and Alter Transforming Growth Factor-α–Induced Pulmonary Fibrosis

Pulmonary fibrosis is caused by excessive proliferation and accumulation of stromal cells. Fibrocytes are bone marrow (BM)-derived cells that contribute to pathologic stromal cell accumulation in human lung disease. However, the cellular source for these stromal cells and the degree of fibrocyte contribution to pulmonary fibrosis remain unclear. To determine the etiology of stromal cell excess during pulmonary fibrosis, we measured fibrocytes during the progression of fibrosis in the transforming growth factor (TGF)-α transgenic mouse model. Lung epithelial-specific overexpression of TGF-α led to progressive pulmonary fibrosis associated with increased accumulation of fibrocytes in the fibrotic lesions. Although reconstitution of BM cells into TGF-α mice demonstrated accumulation of these cells in fibrotic lesions, the majority of the cells did not express α-smooth muscle actin, suggesting that fibrocytes did not transform into myofibroblasts. To explore the mechanisms of fibrocytes in pulmonary fibrogenesis, adoptive cell-transfer experiments were performed. Purified fibrocytes were transferred intravenously into TGF-α transgenic mice, and fibrosis endpoints were compared with controls. Analysis of lung histology and hydroxyproline levels demonstrated that fibrocyte transfers augment TGF-α-induced lung fibrosis. A major subset of TGF-α-induced fibrocytes expressed CD44 and displayed excessive invasiveness, which is attenuated in the presence of anti-CD44 antibodies. Coculture experiments of resident fibroblasts with fibrocytes demonstrated that fibrocytes stimulate proliferation of resident fibroblasts. In summary, fibrocytes are increased in the progressive, fibrotic lesions of TGF-α-transgenic mice and activate resident fibroblasts to cause severe lung disease.

Rapamycin Regulates Bleomycin-Induced Lung Damage in SP-C-Deficient Mice

Injury to the distal respiratory epithelium has been implicated as an underlying cause of idiopathic lung diseases. Mutations that result in SP-C deficiencies are linked to a small subset of spontaneous and familial cases of interstitial lung disease (ILD) and interstitial pulmonary fibrosis (IPF). Gene-targeted mice that lack SP-C (Sftpc −/−) develop an irregular ILD-like disease with age and are a model of the human SP-C related disease. In the current study, we investigated whether rapamycin could ameliorate bleomycin-induced fibrosis in the lungs of Sftpc −/− mice. Sftpc +/+ and −/− mice were exposed to bleomycin with either preventative administration of rapamycin or therapeutic administration beginning eight days after the bleomycin injury. Rapamycin-treatment increased weight loss and decreased survival of bleomycin-treated Sftpc +/+ and Sftpc −/− mice. Rapamycin did not reduce the fibrotic disease in the prophylactic or rescue experiments of either genotype of mice. Further, rapamycin treatment augmented airway resistance and reduced lung compliance of bleomycin-treated Sftpc −/− mice. Rapamycin treatment was associated with an increased expression of profibrotic Th2 cytokines and reduced expression of INF-γ. These findings indicate that novel therapeutics will be required to treat individuals with SP-C deficient ILD/IPF.

Resistin-like molecule alpha1 (Fizz1) recruits lung dendritic cells without causing pulmonary fibrosis

BackgroundResistin-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.MethodsFizz1 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.ResultsWhen 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.ConclusionsThe 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.

Dual targeting of MEK and PI3K pathways attenuates established and progressive pulmonary fibrosis.

Pulmonary fibrosis is often triggered by an epithelial injury resulting in the formation of fibrotic lesions in the lung, which progress to impair gas exchange and ultimately cause death. Recent clinical trials using drugs that target either inflammation or a specific molecule have failed, suggesting that multiple pathways and cellular processes need to be attenuated for effective reversal of established and progressive fibrosis. Although activation of MAPK and PI3K pathways have been detected in human fibrotic lung samples, the therapeutic benefits of in vivo modulation of the MAPK and PI3K pathways in combination are unknown. Overexpression of TGFα in the lung epithelium of transgenic mice results in the formation of fibrotic lesions similar to those found in human pulmonary fibrosis, and previous work from our group shows that inhibitors of either the MAPK or PI3K pathway can alter the progression of fibrosis. In this study, we sought to determine whether simultaneous inhibition of the MAPK and PI3K signaling pathways is a more effective therapeutic strategy for established and progressive pulmonary fibrosis. Our results showed that inhibiting both pathways had additive effects compared to inhibiting either pathway alone in reducing fibrotic burden, including reducing lung weight, pleural thickness, and total collagen in the lungs of TGFα mice. This study demonstrates that inhibiting MEK and PI3K in combination abolishes proliferative changes associated with fibrosis and myfibroblast accumulation and thus may serve as a therapeutic option in the treatment of human fibrotic lung disease where these pathways play a role.

p70 ribosomal S6 kinase regulates subpleural fibrosis following transforming growth factor-α expression in the lung

The p70 ribosomal S6 kinase (S6K) is a downstream substrate that is phosphorylated and activated by the mammalian target of rapamycin complex and regulates multiple cellular processes associated with fibrogenesis. Recent studies demonstrate that aberrant mTORC1-S6K signaling contributes to various pathological conditions, but a direct role in pulmonary fibroproliferation has not been established. Increased phosphorylation of the S6K pathway is detected immediately following transforming growth factor-α (TGF-α) expression in a transgenic model of progressive lung fibrosis. To test the hypothesis that the S6K directly regulates pulmonary fibroproliferative disease we determined the cellular sites of S6K phosphorylation during the induction of fibrosis in the TGF-α model and tested the efficacy of specific pharmacological inhibition of the S6K pathway to prevent and reverse fibrotic disease. Following TGF-α expression increased phosphorylation of the S6K was detected in the airway and alveolar epithelium and the mesenchyme of advanced subpleural fibrotic regions. Specific inhibition of the S6K with the small molecule inhibitor LY-2584702 decreased TGF-α and platelet-derived growth factor-β-induced proliferation of lung fibroblasts in vitro. Administration of S6K inhibitors to TGF-α mice prevented the development of extensive subpleural fibrosis and alterations in lung mechanics, and attenuated the increase in total lung hydroxyproline. S6K inhibition after fibrosis was established attenuated the progression of subpleural fibrosis. Together these studies demonstrate targeting the S6K pathway selectively modifies the progression of pulmonary fibrosis in the subpleural compartment of the lung.

Subacute TGFβ expression drives inflammation, goblet cell hyperplasia, and pulmonary function abnormalities in mice with effects dependent on CFTR function

Cystic fibrosis (CF) produces variable lung disease phenotypes that are, in part, independent of the CF transmembrane conductance regulator ( CFTR) genotype. Transforming growth factor-β (TGFβ) is the best described genetic modifier of the CF phenotype, but its mechanism of action is unknown. We hypothesized that TGFβ is sufficient to drive pathognomonic features of CF lung disease in vivo and that CFTR deficiency enhances susceptibility to pathological TGFβ effects. A CF mouse model and littermate controls were exposed intratracheally to an adenoviral vector containing the TGFβ1 cDNA (Ad-TGFβ), empty vector, or PBS only. Studies were performed 1 wk after treatment, including lung mechanics, collection of bronchoalveolar lavage fluid, and analysis of lung histology, RNA, and protein. CF and non-CF mice showed similar weight loss, inflammation, goblet cell hyperplasia, and Smad pathway activation after Ad-TGFβ treatment. Ad-TGFβ produced greater abnormalities in lung mechanics in CF versus control mice, which was uniquely associated with induction of phosphoinositide 3-kinase and mitogen-activated protein kinase signaling. CFTR transcripts were reduced, and epithelial sodium channel transcripts were increased in CF and non-CF mice, whereas the goblet cell transcription factors, forkhead ortholog A3 and SAM-pointed domain-containing ETS-like factor, were increased in non-CF but not CF mice following Ad-TGFβ treatment. Pulmonary TGFβ1 expression was sufficient to produce pulmonary remodeling and abnormalities in lung mechanics that were associated with both shared and unique cell signaling pathway activation in CF and non-CF mice. These results highlight the multifunctional impact of TGFβ on pulmonary pathology in vivo and identify cellular-response differences that may impact CF lung pathology.