Inhibition of allergen-induced airway remodeling in Smad 3-deficient mice

Intracellular signaling pathways that converge on Smad 3 are used by both TGF-beta and activin A, key cytokines implicated in the process of fibrogenesis. To determine the role of Smad 3 in allergen-induced airway remodeling, Smad 3-deficient and wild-type (WT) mice were sensitized to OVA and challenged by repetitive administration of OVA for 1 mo. Increased levels of activin A and increased numbers of peribronchial TGF-beta1(+) cells were detected in WT and Smad 3-deficient mice following repetitive OVA challenge. Smad 3-deficient mice challenged with OVA had significantly less peribronchial fibrosis (total lung collagen content and trichrome staining), reduced thickness of the peribronchial smooth muscle layer, and reduced epithelial mucus production compared with WT mice. As TGF-beta and Smad 3 signaling are hypothesized to mediate differentiation of fibroblasts to myofibroblasts in vivo, we determined the number of peribronchial myofibroblasts (Col-1(+) and alpha-smooth muscle actin(+)) as assessed by double-label immunofluorescence microscopy. Although the number of peribronchial myofibroblasts increased significantly in WT mice following OVA challenge, there was a significant reduction in the number of peribronchial myofibroblasts in OVA-challenged Smad 3-deficient mice. There was no difference in levels of eosinophilic airway inflammation or airway responsiveness in Smad 3-deficient compared with WT mice. These results suggest that Smad 3 signaling is required for allergen-induced airway remodeling, as well as allergen-induced accumulation of myofibroblasts in the airway. However, Smad 3 signaling does not contribute significantly to airway responsiveness.     

Coexposure to environmental tobacco smoke increases levels of allergen-induced airway remodeling in mice

Environmental tobacco smoke (ETS) can increase asthma symptoms and the frequency of asthma attacks. However, the contribution of ETS to airway remodeling in asthma is at present unknown. In this study, we have used a mouse model of allergen-induced airway remodeling to determine whether the combination of chronic exposure to ETS and chronic exposure to OVA allergen induces greater levels of airway remodeling than exposure to either chronic ETS or chronic OVA allergen alone. Mice exposed to chronic ETS alone did not develop significant eosinophilic airway inflammation, airway remodeling, or increased airway hyperreactivity to methacholine. In contrast, mice exposed to chronic OVA allergen had significantly increased levels of peribronchial fibrosis, increased thickening of the smooth muscle layer, increased mucus, and increased airway hyperreactivity which was significantly enhanced by coexposure to the combination of chronic ETS and chronic OVA allergen. Mice coexposed to chronic ETS and chronic OVA allergen had significantly increased levels of eotaxin-1 expression in airway epithelium which was associated with increased numbers of peribronchial eosinophils, as well as increased numbers of peribronchial cells expressing TGF-beta1. These studies suggest that chronic coexposure to ETS significantly increases levels of allergen-induced airway remodeling (in particular smooth muscle thickness) and airway responsiveness by up-regulating expression of chemokines such as eotaxin-1 in airway epithelium with resultant recruitment of cells expressing TGF-beta1 to the airway and enhanced airway remodeling.     

Reduced peribronchial fibrosis in allergen-challenged MMP-9-deficient mice

Matrix metalloproteinases (MMPs) are a family of extracellular proteases that are responsible for the degradation of the extracellular matrix during tissue remodeling. We have used a mouse model of allergen-induced airway remodeling to determine whether MMP-9 plays a role in airway remodeling. MMP-9-deficient and wild-type (WT) mice were repetitively challenged intranasally with ovalbumin (OVA) antigen to develop features of airway remodeling including peribronchial fibrosis and increased thickness of the peribronchial smooth muscle layer. OVA-challenged MMP-9-deficient mice had less peribronchial fibrosis and total lung collagen compared with OVA-challenged WT mice. There was no reduction in mucus expression, smooth muscle thickness, or airway responsiveness in OVA-challenged MMP-9-deficient compared with OVA-challenged WT mice. OVA-challenged MMP-9-deficient mice had reduced levels of bronchoalveolar lavage (BAL) regulated on activation, normal T cell expressed, and secreted (RANTES), as well as reduced numbers of BAL and peribronchial eosinophils compared with OVA-challenged WT mice. There were no significant difference in levels of BAL eotaxin, thymus- and activation-regulated chemokine (TARC), or macrophage-derived chemokine (MDC) in OVA-challenged WT compared with MMP-9-deficient mice. Overall, this study demonstrates that MMP-9 may play a role in mediating selected aspects of allergen-induced airway remodeling (i.e., modest reduction in levels of peribronchial fibrosis) but does not play a significant role in mucus expression, smooth muscle thickness, or airway responsiveness.     

Chronic OVA allergen challenged Siglec-F deficient mice have increased mucus,remodeling, and epithelial Siglec-F ligands which are up-regulated by IL-4 and IL-13

Background

In this study we examined the role of Siglec-F, a receptor highly expressed on eosinophils, in contributing to mucus expression, airway remodeling, and Siglec-F ligand expression utilizing Siglec-F deficient mice exposed to chronic allergen challenge.

Methods

Wild type (WT) and Siglec-F deficient mice were sensitized and challenged chronically with OVA for one month. Levels of airway inflammation (eosinophils), Siglec-F ligand expresion and remodeling (mucus, fibrosis, smooth muscle thickness, extracellular matrix protein deposition) were assessed in lung sections by image analysis and immunohistology. Airway hyperreactivity to methacholine was assessed in intubated and ventilated mice.

Results

Siglec-F deficient mice challenged with OVA for one month had significantly increased numbers of BAL and peribronchial eosinophils compared to WT mice which was associated with a significant increase in mucus expression as assessed by the number of periodic acid Schiff positive airway epithelial cells. In addition, OVA challenged Siglec-F deficient mice had significantly increased levels of peribronchial fibrosis (total lung collagen, area of peribronchial trichrome staining), as well as increased numbers of peribronchial TGF-β1+ cells, and increased levels of expression of the extracellular matrix protein fibronectin compared to OVA challenged WT mice. Lung sections immunostained with a Siglec-Fc to detect Siglec-F ligand expression demonstrated higher levels of expression of the Siglec-F ligand in the peribronchial region in OVA challenged Siglec-F deficient mice compared to WT mice. WT and Siglec-F deficient mice challenged intranasally with IL-4 or IL-13 had significantly increased levels of airway epithelial Siglec-F ligand expression, whereas this was not observed in WT or Siglec-F deficient mice challenged with TNF-α. There was a significant increase in the thickness of the peribronchial smooth muscle layer in OVA challenged Siglec-F deficient mice, but this was not associated with significant increased airway hyperreactivity compared to WT mice.

Conclusions

Overall, this study demonstrates an important role for Siglec-F in modulating levels of chronic eosinophilic airway inflammation, peribronchial fibrosis, thickness of the smooth muscle layer, mucus expression, fibronectin, and levels of peribronchial Siglec-F ligands suggesting that Siglec-F may normally function to limit levels of chronic eosinophilic inflammation and remodeling. In addition, IL-4 and IL-13 are important regulators of Siglec-F ligand expression by airway epithelium.     

Modulation of airway remodeling and airway inflammation by peroxisome proliferator-activated receptor gamma in a murine model of toluene diisocyanate-induced asthma

Toluene diisocyanate (TDI) is a leading cause of occupational asthma. Although considerable controversy remains regarding its pathogenesis, TDI-induced asthma is an inflammatory disease of the airways characterized by airway remodeling. Peroxisome proliferator-activated receptor gamma (PPARgamma) has been shown to play a critical role in the control of airway inflammatory responses. However, no data are available on the role of PPARgamma in TDI-induced asthma. We have used a mouse model for TDI-induced asthma to determine the effect of PPARgamma agonist, rosiglitazone, or pioglitazone, and PPARgamma on TDI-induced bronchial inflammation and airway remodeling. This study with the TDI-induced model of asthma revealed the following typical pathophysiological features: increased numbers of inflammatory cells of the airways, airway hyperresponsiveness, increased levels of Th2 cytokines (IL-4, IL-5, and IL-13), adhesion molecules (ICAM-1 and VCAM-1), chemokines (RANTES and eotaxin), TGF-beta1, and NF-kappaB in nuclear protein extracts. In addition, the mice exposed to TDI developed features of airway remodeling, including thickening of the peribronchial smooth muscle layer, subepithelial collagen deposition, and increased airway mucus production. Administration of PPARgamma agonists or adenovirus carrying PPARgamma2 cDNA reduced the pathophysiological symptoms of asthma and decreased the increased levels of Th2 cytokines, adhesion molecules, chemokines, TGF-beta1, and NF-kappaB in nuclear protein extracts after TDI inhalation. In addition, inhibition of NF-kappaB activation decreased the increased levels of Th2 cytokines, adhesion molecules, chemokines, and TGF-beta1 after TDI inhalation. These findings demonstrate a protective role of PPARgamma in the pathogenesis of the TDI-induced asthma phenotype.     

Release of biologically active TGF-beta from airway smooth muscle cells induces autocrine synthesis of collagen

In severe or chronic asthma, there is an increase in airway smooth muscle cell (ASMC) mass as well as an increase in connective tissue proteins in the smooth muscle layer of airways. Transforming growth factor-beta (TGF-beta) exists in three isoforms in mammals and is a potent regulator of connective tissue protein synthesis. Using immunohistochemistry, we had previously demonstrated that ASMCs contain large quantities of TGF-beta1-3. In this study, we demonstrate that bovine ASMC-derived TGF-beta associates with the TGF-beta latency binding protein-1 (LTBP-1) expressed by the same cells. The TGF-beta associated with LTBP-1 localizes TGF-beta extracellularly. Furthermore, plasmin, a serine protease, regulates the secretion of a biologically active form of TGF-beta by ASMCs as well as the release of extracellular TGF-beta. The biologically active TGF-beta released by plasmin induces ASMCs to synthesize collagen I in an autocrine manner. The autocrine induction of collagen expression by ASMCs may contribute to the irreversible fibrosis and remodeling seen in the airways of some asthmatics.     

Opposite effect of corticosteroids and long-acting beta(2)-agonists on serum- and TGF-beta(1)-induced extracellular matrix deposition by primary human lung fibroblasts

Asthma and chronic obstructive pulmonary disease (COPD) are characterized by chronic airway inflammation and major structural lung tissue changes including increased extracellular matrix (ECM) deposition. Inhaled corticosteroids and long-acting beta(2)-agonists (LABA) are the basic treatment for both diseases, but their effect on airway remodeling remains unclear. In this study, we investigated the effect of corticosteroids and LABA, alone or in combination, on total ECM and collagen deposition, gene expression, cell proliferation, and IL-6, IL-8, and TGF-beta(1) levels by primary human lung fibroblasts. In our model, fibroblasts in 0.3% albumin represented a non-inflammatory condition and stimulation with 5% FCS and/or TGF-beta(1) mimicked an inflammatory environment with activation of tissue repair. FCS (5%) increased total ECM, collagen deposition, cell proliferation, and IL-6, IL-8, and TGF-beta(1) levels. In 0.3% albumin, corticosteroids reduced total ECM and collagen deposition, involving the glucocorticoid receptor (GR) and downregulation of collagen, heat shock protein 47 (Hsp47), and Fli1 mRNA expression. In 5% FCS, corticosteroids increased ECM deposition, involving upregulation of COL4A1 and CTGF mRNA expression. LABA reduced total ECM and collagen deposition under all conditions partly via the beta(2)-adrenergic receptor. In combination, the drugs had an additive effect in the presence or absence of TGF-beta(1) further decreasing ECM deposition in 0.3% albumin whereas counteracting each other in 5% FCS. These data suggest that the effect of corticosteroids, but not of LABA, on ECM deposition by fibroblasts is altered by serum. These findings imply that as soon as airway inflammation is resolved, long-term treatment with combined drugs may beneficially reduce pathological tissue remodeling.     

Treatment with Pyranopyran-1, 8-Dione Attenuates Airway Responses in Cockroach Allergen Sensitized Asthma in Mice

Chronic allergic asthma is characterized by Th2-typed inflammation, and contributes to airway remodeling and the deterioration of lung function. Viticis Fructus (VF) has long been used in China and Korea as a traditional herbal remedy for treating various inflammatory diseases. Previously, we have isolated a novel phytochemical, pyranopyran-1, 8-dione (PPY), from VF. This study was conducted to evaluate the ability of PPY to prevent airway inflammation and to attenuate airway responses in a cockroach allergen-induced asthma model in mice. The mice sensitized to and challenged with cockroach allergen were treated with oral administration of PPY. The infiltration of total cells, eosinophils and lymphocytes into the BAL fluid was significantly inhibited in cockroach allergen-induced asthma mice treated with PPY (1, 2, or 10 mg/kg). Th2 cytokines and chemokine, such as IL-4, IL-5, IL-13 and eotaxin in BAL fluid were also reduced to normal levels following treatment with PPY. In addition, the levels of IgE were also markedly suppressed after PPY treatment. Histopathological examination demonstrated that PPY substantially inhibited eosinophil infiltration into the airway, goblet cell hyperplasia and smooth muscle hypertrophy. Taken together, these results demonstrate that PPY possesses a potent efficacy on controlling allergic asthma response such as airway inflammation and remodeling.     

Airway fibrosis in rats induced by vanadium pentoxide

Vanadium pentoxide (V(2)O(5)) is a cause of occupational asthma and bronchitis. We previously reported that intratracheal instillation of rats with V(2)O(5) causes fibrosis of the lung parenchyma (J. C. Bonner, P. M. Lindroos, A. B. Rice, C. R. Moomaw, and D. L. Morgan. Am. J. Physiol. Lung Cell. Mol. Physiol. 274: L72-L80, 1998). In this report, we show that intratracheal instillation of V(2)O(5) induces airway remodeling similar to that observed in individuals with asthma. These changes include airway smooth muscle cell thickening, mucous cell metaplasia, and airway fibrosis. The transient appearance of peribronchiolar myofibroblasts, which were desmin and vimentin positive, coincided with a twofold increase in the thickness of the airway smooth muscle layer at day 6 after instillation and preceded the development of airway fibrosis by day 15. The number of nuclear profiles within the smooth muscle layer also increased twofold after V(2)O(5) instillation, suggesting that hyperplasia accounted for thickening of the smooth muscle layer. The majority of cells incorporating bromodeoxyuridine at day 3 were located in the connective tissue surrounding the airway smooth muscle wall that was positive for vimentin and desmin. These data suggest that myofibroblasts are the principal proliferating cell type that contributes to the progression of airway fibrosis after V(2)O(5) injury.     

Regulation of collagen synthesis by inhibitory Smad7 in cardiac myofibroblasts

Transforming growth factor-beta(1) (TGF-beta(1)) signal and downstream Smads play an important role in tissue fibrosis and matrix remodeling in various etiologies of heart failure. Inhibitory Smad7 (I-Smad7) is an inducible regulatory Smad protein that antagonizes TGF-beta(1) signal mediated via direct abrogation of R-Smad phosphorylation. The effect of ectopic I-Smad7 on net collagen production was investigated using hydroxyproline assay. Adenovirus-mediated I-Smad7 gene (at 100 multiplicity of infection) transfer was associated with significant decrease of collagen synthesis in the presence and absence of TGF-beta(1) in primary rat cardiac myofibroblasts. In I-Smad7-infected cells, we also observed the ablation of TGF-beta(1)-induced R-Smad2 phosphorylation vs. LacZ controls. Overdriven I-Smad7 was associated with significantly increased expression of immunoreactive 65-kDa matrix metalloproteinase-2 (MMP-2) protein in culture medium of myofibroblast compared with LacZ-infected cells. Expression of the 72-kDa MMP-2 variant, e.g., the inactive form, was not altered by exogenous I-Smad7 transfection/overexpression. Furthermore, I-Smad7 overexpression was associated with a significant increase and decrease in expression of p27 and phospho-Rb protein, respectively, as well as reduced [(3)H]thymidine incorporation vs. Ad-LacZ-infected controls. We suggest that negative modulation of R-Smad phosphorylation by ectopic I-Smad7 may contribute to the downregulation of collagen in cardiac myofibroblasts and may suppress the proliferation of these cells. Thus treatments targeting the collagen deposition by overexpression of I-Smad7 may provide a new therapeutic strategy for cardiac fibrosis.     

In Vivo Micro-CT Assessment of Airway Remodeling in a Flexible OVA-Sensitized Murine Model of Asthma

Airway remodeling is a major pathological feature of asthma. Up to now, its quantification still requires invasive methods. In this study, we aimed at determining whether in vivo micro-computed tomography (micro-CT) is able to demonstrate allergen-induced airway remodeling in a flexible mouse model of asthma. Sixty Balb/c mice were challenged intranasally with ovalbumin or saline at 3 different endpoints (Days 35, 75, and 110). All mice underwent plethysmography at baseline and just prior to respiratory-gated micro-CT. Mice were then sacrificed to assess bronchoalveolar lavage and lung histology. From micro-CT images (voxel size = 46×46×46 µm), the numerical values of total lung attenuation, peribronchial attenuation (PBA), and PBA normalized by total lung attenuation were extracted. Each parameter was compared between OVA and control mice and correlation coefficients were calculated between micro-CT and histological data. As compared to control animals, ovalbumin-sensitized mice exhibited inflammation alone (Day 35), remodeling alone (Day 110) or both inflammation and remodeling (Day 75). Normalized PBA was significantly greater in mice exhibiting bronchial remodeling either alone or in combination with inflammation. Normalized PBA correlated with various remodeling markers such as bronchial smooth muscle size or peribronchial fibrosis. These findings suggest that micro-CT may help monitor remodeling non-invasively in asthmatic mice when testing new drugs targeting airway remodeling in pre-clinical studies.     

Mycoplasma pneumoniae infection increases airway collagen deposition in a murine model of allergic airway inflammation

Mycoplasma pneumoniae (Mp) has been linked to chronic asthma. Airway remodeling (e.g., airway collagen deposition or fibrosis) is one of the pathological features of chronic asthma. However, the effects of respiratory Mp infection on airway fibrosis in asthma remain unclear. In the present study, we hypothesized that respiratory Mp infection may increase the airway collagen deposition in a murine model of allergic airway inflammation in part through upregulation of transforming growth factor (TGF)-beta1. Double (2 wk apart) inoculations of Mp or saline (control) were given to mice with or without previous allergen (ovalbumin) challenges. On days 14 and 42 after the last Mp or saline, lung tissue and bronchoalveolar lavage (BAL) fluid were collected for analyses of collagen and TGF-beta1 at protein and mRNA levels. In allergen-na?ve mice, Mp did not alter airway wall collagen. In allergen-challenged mice, Mp infections did not change airway wall collagen deposition on day 14 but increased the airway collagen on day 42; this increase was accompanied by increased TGF-beta1 protein in the airway wall and reduced TGF-beta1 protein release from the lung tissue into BAL fluid. Our results suggest that Mp infections could modulate airway collagen deposition in a murine model of allergic airway inflammation with TGF-beta1 involved in the collagen deposition process.     

GM-CSF increases airway smooth muscle cell connective tissue expression by inducing TGF-beta receptors

Fibrosis around the smooth muscle of asthmatic airway walls leads to irreversible airway obstruction. Bronchial epithelial cells release granulocyte/macrophage colony-stimulating factor (GM-CSF) in asthmatics and are in close proximity to airway smooth muscle cells (ASMC). The findings in this study demonstrate that GM-CSF induces confluent, prolonged, serum-deprived cultures of ASMC to increase expression of collagen I and fibronectin. GM-CSF also induced ASMC to increase the expression of transforming growth factor (TGF)-beta receptors type I, II, and III (TbetaR-I, TbetaR-II, TbetaR-III), but had no detectable effect on the release of TGF-beta1 by the same ASMC. The presence of GM-CSF also induced the association of TGF-beta1 with TbetaR-III, which enhances binding of TGF-beta1 to TbetaR-II. The induction of TbetaRs was parallel to the increased induction of phosphorylated Smad2 (pSmad2) and connective tissue growth factor (CTGF), indicative of TGF-beta-mediated connective tissue synthesis. Dexamethasone decreased GM-CSF-induced TbetaR-I, TbetaR-II, TbetaR-III, pSmad2, CTGF, collagen I, and fibronectin. In conclusion, GM-CSF increases the responsiveness of ASMC to TGF-beta1-mediated connective tissue expression by induction of TbetaRs, which is inhibited by corticosteroids.     

Airway fibrosis and angiogenesis due to eosinophil trafficking in chronic asthma

Asthma is characterized by the presence of increased numbers of inflammatory cells in the airway in particular eosinophils and Th2 lymphocytes. In addition to the presence of inflammatory cells, the airways of patients with asthma exhibit varying levels of structural changes termed airway remodeling. These structural changes include subepithelial fibrosis, smooth muscle hypertrophy/hyperplasia, epithelial cell mucus metaplasia, and increased angiogenesis. This review focuses on the potential role of the eosinophil in promoting features of airway remodeling including fibrosis and neovascularization in chronic asthma. Eosinophils may potentially contribute to airway remodeling through release of eosinophil derived mediators such as TGFbeta which act directly upon target fibroblasts to promote fibrosis. In addition to the potential importance of the eosinophil to remodeling in asthma, eosinophilic esophagitis (EE) is another eosinophil associated disease that is associated with increased levels of esophageal eosinophils, increased levels of TGFbeta expression, and increased levels of fibrosis, suggesting that a similar mechanism of remodeling may contribute to both of these eosinophil associated diseases. However, remodeling in both asthma and EE is likely complex involving both eosinophil dependent and eosinophil independent pathways. Further studies in both humans and animal models will help to increase our knowledge of the contribution of the eosinophil to remodeling in asthma as well as EE.     

IGF-I and TGF-beta1 have distinct effects on phenotype and proliferation of intestinal fibroblasts

Insulin-like growth factor I (IGF-I) and transforming growth factor-beta1 (TGF-beta1) are upregulated in myofibroblasts at sites of fibrosis in experimental enterocolitis and in Crohn's disease (CD). We compared the sites of expression of IGF-I and TGF-beta1 in a rat peptidoglycan-polysaccharide (PG-PS) model of chronic granulomatous enterocolitis and fibrosis. We used the human colonic CCD-18Co fibroblast/myofibroblast cell line to test the hypothesis that TGF-beta1 and IGF-I interact to regulate proliferation, collagen synthesis, and activated phenotype typified by expression of alpha-smooth muscle actin and organization into stress fibers. IGF-I potently stimulated while TGF-beta1 inhibited basal DNA synthesis. TGF-beta1 and IGF-I each had similar but not additive effects to induce type I collagen. TGF-beta1 but not IGF-I potently stimulated expression of alpha-smooth muscle actin and stress fiber formation. IGF-I in combination with TGF-beta1 attenuated stress fiber formation without reducing alpha-smooth muscle actin expression. Stress fibers were not a prerequisite for increased collagen synthesis. TGF-beta1 upregulated IGF-I mRNA, which led us to examine the effects of IGF-I in cells previously activated by TGF-beta1 pretreatment. IGF-I potently stimulated proliferation of TGF-beta1-activated myofibroblasts without reversing activated fibrogenic phenotype. We conclude that TGF-beta1 and IGF-I both stimulate type I collagen synthesis but have differential effects on activated phenotype and proliferation. We propose that during intestinal inflammation, regulation of activated phenotype and proliferation may require sequential actions of TGF-beta1 and IGF-I, but they may act in concert to increase collagen deposition.     

Notch2 negatively regulates myofibroblastic differentiation of myoblasts

Myofibroblasts are one of the key cellular components involved in fibrosis of skeletal muscle as well as in other tissues. Transforming growth factor-beta1 (TGF-beta1) stimulates differentiation of mesenchymal cells into myofibroblasts, but little is known about the regulatory mechanisms of myofibroblastic differentiation. Since Notch2 was shown to be downregulated in TGF-beta1-induced non-muscle fibrogenic tissue, we investigated whether Notch2 also has a distinctive role in myofibroblastic differentiation of myogenic cells induced by TGF-beta1. TGF-beta1 treatment of C2C12 myoblasts led to expression of myofibroblastic marker alpha-smooth muscle actin (alpha-SMA) and collagen I with concomitant downregulation of Notch2 expression. Overexpression of active Notch2 inhibited TGF-beta1-induced expression of alpha-SMA and collagen I. Interestingly, transient knockdown of Notch2 by siRNA in C2C12 myoblasts and primary cultured muscle-derived progenitor cells resulted in differentiation into myofibroblastic cells expressing alpha-SMA and collagen I without TGF-beta1 treatment. Furthermore, we found Notch3 was counter-regulated by Notch2 in C2C12 cells. These findings suggest that Notch2 is inhibiting differentiation of myoblasts into myofibroblasts with downregulation of Notch3 expression.     

Conditional expression of transforming growth factor-alpha in adult mouse lung causes pulmonary fibrosis

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 or 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.     

Transforming growth factor-beta1 stimulates collagen matrix remodeling through increased adhesive and contractive potential by human renal fibroblasts

Renal tubulointerstitial fibrosis is the common final pathway leading to end-stage renal failure. Tubulointerstitial fibrosis is characterized by fibroblast proliferation and excessive matrix accumulation. Transforming growth factor-beta1 (TGF-beta1) has been implicated in the development of renal fibrosis accompanied by alpha-smooth muscle actin (alpha-SMA) expression in renal fibroblasts. To investigate the molecular and cellular mechanisms involved in tubulointerstitial fibrosis, we examined the effect of TGF-beta1 on collagen type I (collagen) gel contraction, an in vitro model of scar collagen remodeling. TGF-beta1 enhanced collagen gel contraction by human renal fibroblasts in a dose- and time-dependent manner. Function-blocking anti-alpha1 or anti-alpha2 integrin subunit antibodies significantly suppressed TGF-beta1-stimulated collagen gel contraction. Scanning electron microscopy showed that TGF-beta1 enhanced the formation of the collagen fibrils by cell attachment to collagen via alpha1beta1 and alpha2beta1 integrins. Flow cytometry and cell adhesion analyses revealed that the stimulation of renal fibroblasts with TGF-beta1 enhanced cell adhesion to collagen via the increased expression of alpha1 and alpha2 integrin subunits within collagen gels. Fibroblast migration to collagen was not up-regulated by TGF-beta1. Furthermore, TGF-beta1 increased the expression of a putative contractile protein, alpha-SMA, by human renal fibroblasts in collagen gels. These results suggest that TGF-beta1 stimulates fibroblast-collagen matrix remodeling by increasing both integrin-mediated cell attachment to collagen and alpha-SMA expression, thereby contributing to pathological tubulointerstitial collagen matrix reorganization in renal fibrosis.