transforming-growth-factor-beta and Pulmonary-Fibrosis

Very few studies and limited information are available regarding the mechanism of fibrosis in tuberculosis (TB). This study aimed to identify, describe and synthesise potential biomarkers of the development of tissue fibrosis induced by TB through a systematic method and meta-analysis.. A literature search was performed using keywords according to the topic from electronic databases (ScienceDirect and PubMed) and other methods (websites, organisations and citations). Studies that matched predetermined eligibility criteria were included. The quality assessment tool used was the Quality Assessment of Diagnostic Accuracy Score 2, and the data obtained were processed using Review Manager V.5.3.. Of the 305 studies, 7 met the eligibility criteria with a total sample of 365. The results of the meta-analysis showed that the post-TB group of patients with pulmonary parenchymal fibrosis had a higher transforming growth factor (TGF)-β level (6.09) than the control group (1.82), with a 4.27 (95% CI: 0.92 to 7.61) mean difference. Moreover, patients with residual pleural thickening post-TB had a higher mean of TGF-β (0.61) than the control group (0.56), with a 0.05 (95% CI: 0.04 to 0.06) mean difference. Besides TGF-β, our qualitative synthesis also found that matrix metalloproteinase-1 might have a role in forming and developing pulmonary tissue fibrosis, thus, could be used as a predictor marker in the formation of fibrotic lesions in patients with TB. In addition, several other biomarkers were assessed in the included studies, such as tumour necrosis factor-α, interleukin (IL)-4, IL-8, IL-10, plasminogen activator inhibitor-1 and platelet-derived growth factor. However, this study is not intended to examine these biomarkers.. There were differences in the results of TGF-β levels in patients with fibrotic lesions compared with controls. TGF-β might be a biomarker of fibrotic tissue formation or increased pulmonary tissue fibrosis in post-TB patients. However, further studies are needed on a larger scale.

Topics: Biomarkers; Fibrosis; Humans; Matrix Metalloproteinases; Pulmonary Fibrosis; Transforming Growth Factor beta; Transforming Growth Factors; Tuberculosis

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), like other viruses, can induce proliferation of myofibroblasts and even lead to fibrosis in the lung. Epithelial-mesenchymal transition (EMT) is thought to play an essential role in the pathogenesis of Coronavirus disease 19 (COVID-19). EMT is originally a critical process that regulates the development of different tissues in the embryo, but in inflammatory situations, EMT tries to be activated again to control inflammation or even heal inflammatory damage. However, in pathological situations, such as chronic viral infections (e.g., COVID-19) or pulmonary fibrosis initiation, this benign healing transforms into sinister nature, pushing the lung into the fibrotic process. Notably, the cytokines released by inflammatory cells and the chronic inflammatory microenvironment shared by fibrotic cells promote each other as critical factors in the induction of pathological EMT. In the induction of SARS-CoV-2 virus, cytokines are an essential mediator of EMT transformation, and a summary of whether COVID-19 patients, during the infection phase, have many persistent inflammatory mediators (cytokines) that are a causative factor of EMT has not yet appeared. The following common signaling drivers, including Transforming growth factor beta (TGF-β), cytokines, Notch signaling pathway, Wnt and hypoxia signaling pathways, drive the regulation of EMT. In this review, we will focus on 3 key EMT signaling pathways: TGF-β, Leucine zipper transcription factor like 1 (LZTFL1) and the common interleukin family expressed in the lung. TGF-β-induced SNAIL and LZTFL1 were identified as regulatory EMT in COVID-19. For cytokines, the interleukin family is a common inducer of EMT and plays an essential role in the formation of the microenvironment of fibrosis. We sought to demonstrate that cytokines act as "communicators" and build the "microenvironment" of fibrosis together with EMT as a "bridge" to induce EMT in fibrosis. The mechanisms utilized by these two pathways could serve as templates for other mesenchymal transformations and provide new potential therapeutic targets.

Topics: COVID-19; Cytokines; Epithelial-Mesenchymal Transition; Fibrosis; Humans; Interleukins; Pulmonary Fibrosis; SARS-CoV-2; Transforming Growth Factor beta; Transforming Growth Factor beta1

Air pollution is a big ecumenical problem associated with public health around the world. The rapid development of nanotechnology worldwide resulted in a significant increase in human exposure with unknown particles, and ultimately leading to an increase in acute and chronic diseases. The effect of nanoparticles on pulmonary fibrosis has been reported in vivo and in vitro studies; however, the results are inconsistent. The present systematic review and meta-analysis of animal preclinical studies was conducted to assess the effect of nanoparticles on pulmonary fibrosis. A systematic search of online databases and gray literature as well as reference lists of retrieved studies was performed up to February 2019 to identify preclinical animal studies. Studies were assessed for methodological quality using the SYstematic Review Center for Laboratory animal Experimentation bias risk tool (SYRCLE's ROB tool). Pooled standardized mean difference (SMD) estimate with corresponding 95% CI was calculated using inverse-variance weights method while random effects meta-analysis was used, taking into account conceptual heterogeneity. To assess the robustness of pooled estimates as well as heterogeneity across studies, sensitivity analysis and Cochran

Topics: Air Pollution; Animals; Nanoparticles; Pulmonary Fibrosis; Research Design; Transforming Growth Factor beta

Peroxisome proliferator-activated receptor gamma (PPARγ) is a type II nuclear receptor, initially recognized in adipose tissue for its role in fatty acid storage and glucose metabolism. It promotes lipid uptake and adipogenesis by increasing insulin sensitivity and adiponectin release. Later, PPARγ was implicated in cardiac development and in critical conditions such as pulmonary arterial hypertension (PAH) and kidney failure. Recently, a cluster of different papers linked PPARγ signaling with another superfamily, the transforming growth factor beta (TGFβ), and its receptors, all of which play a major role in PAH and kidney failure. TGFβ is a multifunctional cytokine that drives inflammation, fibrosis, and cell differentiation while PPARγ activation reverses these adverse events in many models. Such opposite biological effects emphasize the delicate balance and complex crosstalk between PPARγ and TGFβ. Based on solid experimental and clinical evidence, the present review summarizes connections and their implications for PAH and kidney failure, highlighting the similarities and differences between lung and kidney mechanisms as well as discussing the therapeutic potential of PPARγ agonist pioglitazone.

Topics: Animals; Humans; Kidney; Lung; Pioglitazone; PPAR gamma; Pulmonary Arterial Hypertension; Pulmonary Fibrosis; Renal Insufficiency; Signal Transduction; Transforming Growth Factor beta

Radiation-induced lung injury (RILI) is a form of radiation damage to normal lung tissue caused by radiotherapy (RT) for thoracic cancers, which is most commonly comprised of radiation pneumonitis (RP) and radiation pulmonary fibrosis (RPF). Moreover, with the widespread utilization of immunotherapies such as immune checkpoint inhibitors as first- and second-line treatments for various cancers, the incidence of immunotherapy-related lung injury (IRLI), a severe immune-related adverse event (irAE), has rapidly increased. To date, we know relatively little about the underlying mechanisms and signaling pathways of these complications. A better understanding of the signaling pathways may facilitate the prevention of lung injury and exploration of potential therapeutic targets. Therefore, this review provides an overview of the signaling pathways of RILI and IRLI and focuses on their crosstalk in diverse signaling pathways as well as on possible mechanisms of adverse events resulting from combined radiotherapy and immunotherapy. Furthermore, this review proposes potential therapeutic targets and avenues of further research based on signaling pathways. Many new studies on pyroptosis have renewed appreciation for the value and importance of pyroptosis in lung injury. Therefore, the authors posit that pyroptosis may be the common downstream pathway of RILI and IRLI; discussion is also conducted regarding further perspectives on pyroptosis as a crucial signaling pathway in lung injury treatment.

Topics: HMGB1 Protein; Humans; Immune Checkpoint Inhibitors; Lung Injury; NF-E2-Related Factor 2; Pulmonary Fibrosis; Pyroptosis; Radiation Pneumonitis; Signal Transduction; Transforming Growth Factor beta

Pulmonary fibrosis (PF) is the most common chronic, progressive interstitial lung disease, mainly occurring in the elderly, with a median survival of 2-4 years after diagnosis. Its high mortality rate attributes to the delay in diagnosis due to its generic symptoms, and more importantly, to the lack of effective treatments. MicroRNAs (miRNAs) are a class of small non-coding RNAs that are involved in many essential cellular processes, including extracellular matrix remodeling, alveolar epithelial cell apoptosis, epithelial-mesenchymal transition, etc. We summarized the dysregulated miRNAs in TGF-β signaling pathway-mediated PF in recent years with dual effects, such as anti-fibrotic let-7 family and pro-fibrotic miR-21 members. Therefore, this review will set out the latest application of miRNAs to provide a new direction for PF treatment.

Topics: Humans; MicroRNAs; Pulmonary Fibrosis; Signal Transduction; Transforming Growth Factor beta

Coronavirus disease 2019 (COVID-19) has rapidly spread around the world causing global public health emergency. In the last twenty years, we have witnessed several viral epidemics such as severe acute respiratory syndrome coronavirus (SARS-CoV), Influenza A virus subtype H1N1 and most recently Middle East respiratory syndrome coronavirus (MERS-CoV). There were tremendous efforts endeavoured globally by scientists to combat these viral diseases and now for SARS-CoV-2. Several drugs such as chloroquine, arbidol, remdesivir, favipiravir and dexamethasone are adopted for use against COVID-19 and currently clinical studies are underway to test their safety and efficacy for treating COVID-19 patients. As per World Health Organization reports, so far more than 16 million people are affected by COVID-19 with a recovery of close to 10 million and deaths at 600,000 globally. SARS-CoV-2 infection is reported to cause extensive pulmonary damages in affected people. Given the large number of recoveries, it is important to follow-up the recovered patients for apparent lung function abnormalities. In this review, we discuss our understanding about the development of long-term pulmonary abnormalities such as lung fibrosis observed in patients recovered from coronavirus infections (SARS-CoV and MERS-CoV) and probable epigenetic therapeutic strategy to prevent the development of similar pulmonary abnormalities in SARS-CoV-2 recovered patients. In this regard, we address the use of U.S. Food and Drug Administration (FDA) approved histone deacetylase (HDAC) inhibitors therapy to manage pulmonary fibrosis and their underlying molecular mechanisms in managing the pathologic processes in COVID-19 recovered patients.

Topics: Adult; Aged; Coronavirus Infections; COVID-19; Drug Repositioning; Extracellular Matrix; Histone Deacetylase Inhibitors; Humans; Middle Aged; Pulmonary Fibrosis; Risk Factors; Signal Transduction; Survivors; Transforming Growth Factor beta

Idiopathic pulmonary fibrosis (IPF) is a progressive and fatal interstitial lung disease. During the past decade, novel pathogenic mechanisms of IPF have been elucidated that have shifted the concept of IPF from an inflammatory-driven to an epithelial-driven disease. Dysregulated repair responses induced by recurrent epithelial cell damage and excessive extracellular matrix accumulation result in pulmonary fibrosis. Although there is currently no curative therapy for IPF, two medications, pirfenidone and nintedanib, have been introduced based on understanding the pathogenesis of the disease. In this review, we discuss advances in understanding IPF pathogenesis, highlighting epithelial-mesenchymal transition (EMT), the ubiquitin-proteasome system, and endothelial cells. TGF-β is a central regulator involved in EMT and pulmonary fibrosis. HECT-, RING finger-, and U-box-type E3 ubiquitin ligases regulate TGF-β-Smad pathway-mediated EMT via the ubiquitin-proteasome pathway. p27 degradation mediated by the SCF-type E3 ligase, Skp2, contributes to the progression of pulmonary fibrosis by promotion of either mesenchymal fibroblast proliferation, EMT, or both. In addition to fibroblasts as key effector cells in myofibroblast differentiation and extracellular matrix deposition, endothelial cells also play a role in the processes of IPF. Endothelial cells can transform into myofibroblasts; therefore, endothelial-mesenchymal transition can be another source of myofibroblasts.

Topics: Epithelial-Mesenchymal Transition; Humans; Proliferating Cell Nuclear Antigen; Proteasome Endopeptidase Complex; Pulmonary Fibrosis; S-Phase Kinase-Associated Proteins; Signal Transduction; Transforming Growth Factor beta; Ubiquitin

Periostin is known to be a useful biomarker for various diseases. In this article, we focus on allergic diseases and pulmonary fibrosis, for which we and others are now developing detection systems for periostin as a biomarker. Biomarker-based precision medicine in the management of type 2 inflammation and fibrotic diseases since heterogeneity is of utmost importance. Periostin expression is induced by type 2 cytokines (interleukin-4/-13) or transforming growth factor-β, and plays a vital role in the pathogenesis of allergic inflammation or interstitial lung disease, respectively, andits serum levels are correlated disease severity, prognosis and responsiveness to the treatment. We first summarise the importance of type 2 biomarker and then describe the pathological role of periostin in the development and progression of type 2 allergic inflammation and pulmonary fibrosis. In addition, then, we summarise the recent development of assay methods for periostin detection, and analyse the diseases in which periostin concentration is elevated in serum and local biological fluids and its usefulness as a biomarker. Furthermore, we describe recent findings of periostin as a biomarker in the use of biologics or anti-fibrotic therapy. Finally, we describe the factors that influence the change in periostin concentration under the healthy conditions.

Topics: Biomarkers; Cell Adhesion Molecules; Chronic Disease; Cytokines; Eosinophilia; Fibrosis; Humans; Idiopathic Pulmonary Fibrosis; Immunoglobulin E; Inflammation; Interleukin-13; Lung; Precision Medicine; Prognosis; Pulmonary Fibrosis; Rhinitis; Sinusitis; Transforming Growth Factor beta

Idiopathic pulmonary fibrosis (IPF) is a progressive and terminal lung disease with no known cure. IPF is a disease of aging, with median age of diagnosis over 65 years. Median survival is between 3 and 5 years after diagnosis. IPF is characterized primarily by excessive deposition of extracellular matrix (ECM) proteins by activated lung fibroblasts and myofibroblasts, resulting in reduced gas exchange and impaired pulmonary function. Growing evidence supports the concept of a pro-fibrotic environment orchestrated by underlying factors such as genetic predisposition, chronic injury and aging, oxidative stress, and impaired regenerative responses may account for disease development and persistence. Currently, two FDA approved drugs have limited efficacy in the treatment of IPF. Many of the genes and gene networks associated with lung development are induced or activated in IPF. In this review, we analyze current knowledge in the field, gained from both basic and clinical research, to provide new insights into the disease process, and potential approaches to treatment of pulmonary fibrosis.

Topics: Animals; Biomarkers; Cellular Microenvironment; Disease Susceptibility; Homeodomain Proteins; Humans; Myofibroblasts; Pulmonary Fibrosis; Signal Transduction; Stromal Cells; Trans-Activators; Transforming Growth Factor beta

Transforming growth factor (TGF)-β is an evolutionarily conserved pleiotropic factor that regulates a myriad of biological processes including development, tissue regeneration, immune responses, and tumorigenesis. TGF-β is necessary for lung organogenesis and homeostasis as evidenced by genetically engineered mouse models. TGF-β is crucial for epithelial-mesenchymal interactions during lung branching morphogenesis and alveolarization. Expression and activation of the three TGF-β ligand isoforms in the lungs are temporally and spatially regulated by multiple mechanisms. The lungs are structurally exposed to extrinsic stimuli and pathogens, and are susceptible to inflammation, allergic reactions, and carcinogenesis. Upregulation of TGF-β ligands is observed in major pulmonary diseases, including pulmonary fibrosis, emphysema, bronchial asthma, and lung cancer. TGF-β regulates multiple cellular processes such as growth suppression of epithelial cells, alveolar epithelial cell differentiation, fibroblast activation, and extracellular matrix organization. These effects are closely associated with tissue remodeling in pulmonary fibrosis and emphysema. TGF-β is also central to T cell homeostasis and is deeply involved in asthmatic airway inflammation. TGF-β is the most potent inducer of epithelial-mesenchymal transition in non-small cell lung cancer cells and is pivotal to the development of tumor-promoting microenvironment in the lung cancer tissue. This review summarizes and integrates the current knowledge of TGF-β signaling relevant to lung health and disease.

Topics: Animals; Asthma; Humans; Lung; Lung Diseases; Lung Neoplasms; Pulmonary Emphysema; Pulmonary Fibrosis; Signal Transduction; Transforming Growth Factor beta

Pulmonary fibrosis is the most common form of interstitial lung disease. The transforming growth factor-β (TGF-β) signaling pathway is extensively involved in the development of pulmonary fibrosis by inducing cell differentiation, migration, invasion, or hyperplastic changes. Accumulating evidence indicates that microRNAs (miRNAs) are dysregulated during the initiation of pulmonary fibrosis. miRNAs are small noncoding RNAs functioning as negative regulators of gene expression at the post-transcriptional level. A number of miRNAs have been reported to regulate the TGF-β signaling pathway and consequently affect the process of pulmonary fibrosis. A better understanding of the pro-fibrotic role of the TGF-β signaling pathway and relevant miRNA regulation will shed light on biomedical research of pulmonary fibrosis. This review summarizes the current knowledge of miRNAs regulating the TGF-β signaling pathway with relevance to pulmonary fibrosis.

Topics: Animals; Humans; MicroRNAs; Pulmonary Fibrosis; Signal Transduction; Transforming Growth Factor beta

Transforming growth factor-β (TGFB) regulates cell proliferation, differentiation, apoptosis, and matrix homeostasis and is intimately involved in fibrosis. TGFB expression is increased in fibrotic lung diseases, such as idiopathic pulmonary fibrosis, and in chronic inflammatory conditions, such as chronic obstructive pulmonary disease and asthma. In addition to exhibiting profibrotic activities, the protein exhibits profound immune-suppressive actions involving both innate and adaptive responses, but often this aspect of TGFB biology is overlooked. Recent investigations have demonstrated that TGFB causes wide-ranging immune suppression, including blunting of pivotal early innate IFN responses. These activities permit severe virus infections, often followed by secondary bacterial infections, which may last longer, with augmented inflammation, scarring, fibrosis, and loss of lung function. Strategies to oppose TGFB actions or to enhance IFN responses may help ameliorate the detrimental consequences of infection in patients with diseases characterized by TGFB overexpression, inflammation, and fibrosis.

Topics: Animals; Humans; Immunity, Innate; Lung; Models, Biological; Pulmonary Fibrosis; Signal Transduction; Transforming Growth Factor beta

Idiopathic pulmonary fibrosis (IPF) is a devastating disease characterized by alveolar epithelial cell injury, accumulation of fibroblasts/myofibroblasts and deposition of extracellular matrix proteins. Levels of sphingosine-1-phosphate (S1P), a naturally occurring bioactive lipid, are elevated in bronchoalveolar fluids and lung tissues from IPF patients and animal models of pulmonary fibrosis. However, the in vivo contribution of S1P, regulated by its synthesis catalyzed by Sphingosine kinases (SphKs) 1 & 2 and catabolism by S1P phosphatases and S1P lyase (S1PL), in the pathogenesis of pulmonary fibrosis is not well defined. Microarray analysis of blood mononuclear cells from patients with IPF and SphK1-, SphK2- or S1PL-knockout mice and SphK inhibitor were used to assess the role of S1P in fibrogenesis. The expression of SphK1 negatively correlated with lung function and survival of patients with IPF. Further, the expressions of SphK1 and S1PL were increased in lung tissues from patients with IPF and bleomycin-challenged mice. Genetic knockdown of SphK1, but not SphK2, ameliorated bleomycin-induced pulmonary fibrosis in mice while deletion of S1PL (SGPL1(+/-)) in mice potentiated fibrosis post-bleomycin challenge. TGF-β increased the expression of SphK1 and S1PL in human lung fibroblasts and knockdown of SphK1 or treatment with SphK inhibitor attenuated S1P generation and TGF-β mediated signal transduction. Over-expression of S1PL attenuated bleomycin-induced TGF-β secretion and S1P mediated differentiation of human lung fibroblasts through regulation of autophagy. Administration of SphK1 inhibitor 8 days post-bleomycin challenge reduced bleomycin-induced mortality and pulmonary fibrosis. Our results suggest that SphK1 and S1PL play critical roles in the pathology of lung fibrosis and may be novel therapeutic targets.

Topics: Aldehyde-Lyases; Animals; Antibiotics, Antineoplastic; Bleomycin; Disease Models, Animal; Humans; Mice; Mice, Knockout; Phosphotransferases (Alcohol Group Acceptor); Pulmonary Alveoli; Pulmonary Fibrosis; Signal Transduction; Sphingolipids; Transforming Growth Factor beta

Transforming growth factor-β (TGF-β) plays a central role in driving tissue fibrosis. TGF-β is secreted in a latent form, held latent by noncovalent association of the active cytokine with a peptide derived from cleavage of the N-terminal domain of the same gene product, and needs to be activated extracellularly to exert any of its diverse biological effects. We have shown that two of the three mammalian isoforms of TGF-β, TGF-β1 and TGF-β3, depend on interactions with cell surface integrins for activation. We found that the integrin αvβ6 is highly induced on injured alveolar epithelial cells, potently induces TGF-β activation, and is critical for the development of pulmonary fibrosis and acute lung injury. However, although TGF-β drives fibrosis in virtually every anatomic site, αvβ6-mediated TGF-β activation is much more restricted. For example, αvβ6 is not induced on injured hepatocytes and plays little or no role in cirrhosis induced by repetitive hepatocyte injury. Fibroblasts are highly contractile cells that express multiple integrins closely related to αvβ6, which share the promiscuous αv subunit, so we reasoned that perhaps one or more of these αv integrins on fibroblasts might substitute for αvβ6 and activate the TGF-β required to drive liver fibrosis. Indeed, deletion of the αv subunit from activated fibroblasts protected mice from carbon tetrachloride-induced liver fibrosis. Importantly, these same mice were protected from bleomycin-induced pulmonary fibrosis and renal fibrosis caused by unilateral ureteral obstruction, despite the presence of epithelial αvβ6 in these mice. These results suggest that the generation and maintenance of sufficient quantities of active TGF-β to cause tissue fibrosis in multiple organs probably depends on at least two sources-TGF-β activation by injured epithelial cells that drives fibroblast expansion and activation and an amplification step that involves TGF-β activation by an αv integrin on activated fibroblasts. These results suggest that intervening at either of these steps could be useful for the treatment of fibrotic diseases.

Topics: Animals; Antigens, Neoplasm; Epithelial Cells; Fibroblasts; Humans; Integrins; Liver Cirrhosis; Mice; Pulmonary Fibrosis; Transforming Growth Factor beta

Within the lungs, fibrosis can affect both the parenchyma and the airways. Fibrosis is a hallmark pathological change in the parenchyma in patients with idiopathic pulmonary fibrosis (IPF), whilst in asthma or chronic obstructive pulmonary disease (COPD) fibrosis is a component of the remodelling of the airways. In the past decade, significant advances have been made in understanding the disease behaviour and pathogenesis of parenchymal and airway fibrosis and as a result a variety of novel therapeutic targets for slowing or preventing progression of these fibrotic changes have been identified. This review highlights a number of these targets and discusses the potential for treating parenchymal or airway fibrosis through these mediators/pathways in the future.

Topics: Airway Remodeling; Animals; Humans; Lung; Pulmonary Fibrosis; Signal Transduction; Transforming Growth Factor beta

There are no effective treatments for fibrosis, an out-of-control wound-healing process in which excessive deposition of extracellular matrix (ECM) such as collagen, resulting in significant morbidity and mortality. Endostatin is a natural proteolytic fragment of collagen XVIII, which is known to possess potent antiangiogenic activity. Many clinical trials of endostatin have been conducted for anti-cancer therapy. In addition to antiangiogenic effects, recent studies have revealed that endostatin may suppress aberrant tissue remodeling and scarring. Neutralization of endogenous endostatin in rat myocardial infarction (MI) model worsened the outcomes of MI, indicating that endostatin may have protective role against left ventricular remodeling and heart failure after MI. Recently, we also reported inhibitory effects of peptides derived from endostatin on fibrosis. A peptide derived from the C-terminus of endostatin suppressed ECM production in fibroblasts in the presence of transforming growth factor-β (TGF-β), prevented TGF-β-induced dermal fibrosis ex vivo in human skin, and ameliorated skin and pulmonary fibrosis induced by bleomycin in vivo. The antifibrotic capacity was accompanied by reduced cell apoptosis and lower levels of lysyl oxidase and early growth response gene-1. Endostation may have the therapeutic potential for inhibiting fibrosis.

Topics: Angiogenesis Inhibitors; Animals; Apoptosis; Bleomycin; Cicatrix; Early Growth Response Protein 1; Endostatins; Extracellular Matrix; Fibroblasts; Fibrosis; Heart Failure; Humans; Myocardial Infarction; Protein-Lysine 6-Oxidase; Pulmonary Fibrosis; Rats; Scleroderma, Systemic; Skin; Transforming Growth Factor beta; Ventricular Remodeling

Topics: Animals; Bone Density Conservation Agents; Calcitriol; Calcium Channel Agonists; Humans; Pulmonary Fibrosis; Signal Transduction; Transforming Growth Factor beta; Wnt Proteins

It is well accepted that TGF-β signaling has critical functional roles in lung development, injury, and repair. We showed previously that null mutation of Smad3, a critical node in the TGF-β pathway, protects mice against fibrosis induced by bleomycin. However, more recently we noticed that abnormal alveolarization also occurs in Smad3-deficient mice and that this is followed by progressive emphysema-like alveolar wall destruction mediated by MMP9. We now know that Smad3 cooperates with c-Jun to synergistically regulate a protein deacetylase SIRT1, by binding to an AP-1 site in the SIRT1 promoter. Consistently, Smad3 knockout lung at postnatal day 28 had reduced SIRT1 expression, which in turn resulted in increased histone acetylation at the binding sites of the transcription factors AP-1, NF-κB, and Pea3 on the MMP9 promoter, as well as increased acetylation of NF-κB. Thus, upon TGF-β activation, phosphorylated Smad3 can be translocated into the nucleus with Smad4, whereat Smad3 in turn collaborates with c-Jun to activate SIRT1 transcription. SIRT1 can deacetylate NF-κB at lysine 30, as well as histones adjacent to the transcription factor AP-1, NF-κB, and Pea3 binding sites of the MMP9 promoter, thereby suppressing MMP9 transcription, hence fixing MMP9 in the OFF mode. Conversely, when Smad3 is missing, this regulatory pathway is inactivated so that MMP9 is epigenetically turned ON. We postulate that these developmental epigenetic mechanisms by which Smad3 regulates MMP9 transcription cell autonomously may be important in modulating both emphysema and pulmonary fibrosis and that this could explain why both pathologies can appear within the same lung specimen.

Topics: Animals; Epigenomics; Humans; Matrix Metalloproteinase 9; Mice; Pulmonary Alveoli; Pulmonary Emphysema; Pulmonary Fibrosis; Rats; Signal Transduction; Smad3 Protein; Transforming Growth Factor beta

Fibrosis is one of the key pathological features of airway remodeling in asthma. In the normal airway the amount of collagen and other extracellular matrix components is kept in equilibrium by regulation of synthesis and degradation. In asthma this homeostasis is disrupted due to genetic and environmental factors. In the airways of patients with the disease there is increased extracellular matrix deposition, particularly in the reticular basement membrane region, lamina propria and submucosa. Fibrosis is important as it can occur early in the pathogenesis of asthma, be associated with severity and resistant to therapy. In this review we will discuss current knowledge of relaxin and other key regulators of fibrosis in the airway including TGFβ, Smad2/3 and matrix metalloproteinases. As fibrosis is not directly targeted or effectively treated by current asthma drugs including corticosteroids, characterization of airway fibrosis and how it is regulated will be essential for the development of novel therapies for asthma.

Topics: Airway Remodeling; Asthma; Collagen; Extracellular Matrix; Humans; Matrix Metalloproteinase Inhibitors; Matrix Metalloproteinases; Pulmonary Fibrosis; Relaxin; Respiratory System; Smad2 Protein; Smad3 Protein; Tissue Inhibitor of Metalloproteinases; Transforming Growth Factor beta

Pathogenic mechanisms involved in fibrosis of various organs share many common features. Myofibroblasts are thought to play a major role in fibrosis through excessive deposition of extracellular matrix during wound healing processes. Myofibroblasts are observed in fibrotic lesions, and whereas these derive from the hepatic stellate cells in liver, in lung they appear to originate from fibroblasts. The source of these fibroblasts has been the object of numerous studies over the recent years and points towards multiple sources. First of all, resident fibroblasts are thought to differentiate into the more contractile myofibroblasts, secreting many extracellular matrix proteins. Secondly, the epithelial to mesenchymal transition (EMT) of epithelial cells may also account for increased numbers of fibroblasts, though in vivo evidence in patient tissue is still scarce. Thirdly, the enigmatic fibrocytes, stemming from the bone marrow, may also account for increasing numbers of fibroblasts in fibrotic lesions. These pathogenic processes are further augmented by the generation of so-called alternatively activated macrophages, which have direct and indirect effects on myofibroblast accumulation and collagen deposition. TGFβ, which is produced predominantly by macrophages, plays a central role in all these processes by inducing EMT, driving differentiation of fibrocytes, and differentiation towards myofibroblasts. This review describes the potential origins and roles of these fibrotic cells in the lung and discusses models to study these cells in vitro. These models offer innovative approaches in target and drug discovery, aiming to uncover novel therapeutic targets that regulate the profibrotic phenotype of these cells.

Topics: Animals; Cell Differentiation; Collagen; Drug Design; Extracellular Matrix; Fibroblasts; Humans; Idiopathic Pulmonary Fibrosis; Macrophages; Models, Biological; Myofibroblasts; Pulmonary Fibrosis; Transforming Growth Factor beta; Wound Healing

Tissue injury incites a repair response with a key mesenchymal component that provides the essential connective tissue for subsequent regeneration or pathological fibrosis. The fibroblast is the major mesenchymal cell type to be implicated in this connective tissue response, and it is in its activated or differentiated form that it participates in the repair process. The myofibroblast represents such an activated mesenchymal cell and is a key source of extracellular matrix and inflammatory/fibrogenic cytokines as well as participating in wound contraction. Although successful healing results in gradual disappearance of myofibroblasts, their persistence is associated with chronic and progressive fibrosis. Thus, elucidation of the mechanism involved in the genesis of the myofibroblast should provide insight into both pathogenesis of chronic fibrotic diseases and therapeutic strategies for their management and control. Although the fibroblast is a well-documented progenitor cell for the myofibroblast, recent studies have suggested additional precursor cells that have the potential to give rise to the myofibroblast. Many of the studies focused on mechanisms and factors that regulate induction of α-smooth muscle actin expression, a key and commonly used marker of the myofibroblast. These reveal complex and multifactorial mechanisms involving transcriptional and epigenetic regulation and implicating diverse cell-signaling pathways, including those activated by the potent fibrogenic cytokine transforming growth factor β. Despite these extensive studies, many aspects remain poorly understood, with the suggestion that additional novel mechanisms remain to be discovered. Future studies with the help of newly developed technical advancements should expedite discovery in this direction.

Topics: Actins; Cell Differentiation; Epigenesis, Genetic; Epithelial-Mesenchymal Transition; Fibroblasts; Gene Expression Regulation; Humans; Lung Injury; Mesenchymal Stem Cells; Myofibroblasts; Pulmonary Fibrosis; Transforming Growth Factor beta; Wound Healing

Organ fibrosis has been viewed as one of the major medical problems, which can lead to progressive dysfunction of the liver, lung, kidney, skin, heart, and eventually death of patients. Fibrosis is initiated by a variety of pathological, physiological, biochemical, and physical factors. Regardless of their different etiologies, they all share a common pathogenetic process: excessive activation of the key profibrotic cytokine, transforming growth factor-beta (TGF-beta). Peroxisome proliferator-activated receptor gamma (PPARgamma), a ligand-activated transcription factor of the nuclear receptor superfamily, has received particular attention in recent years, because the activation of PPARgamma by both natural and synthetic agonists could effectively inhibit TGF-beta-induced profibrotic effects in many organs.. The English-language medical databases, PubMed, Elsevier and SpringerLink were searched for articles on PPARgamma, TGF-beta, and fibrosis, and related topics.. TGF-beta is recognized as a key profibrotic cytokine. Excessive activation of TGF-beta increases synthesis of extracellular matrix proteins and decreases their degradation, associated with a gradual destruction of normal tissue architecture and function, whereas PPARgamma agonists inhibit TGF-beta signal transduction and are effective antifibrogenic agents in many organs including the liver, lung, kidney, skin and heart.. The main antifibrotic activity of PPARgamma agonists is to suppress the TGF-beta signaling pathway by so-called PPARgamma-dependent effect. In addition, PPARgamma agonists, especially 15d-PGJ2, also exert potentially antifibrotic activity independent of PPARgamma activation. TGF-beta1/Smads signaling not only plays many essential roles in multiple developmental processes, but also forms cross-talk networks with other signal pathways, and their inhibition by PPARgamma agonists certainly affects the cytokine networks and causes non-suspected side-effects. Anti-TGF-beta therapies with PPARgamma agonists may have to be carefully tailored to be tissue- and target gene-specific to minimize side-effects, indicating a great challenge to the medical research at present.

Topics: Epithelial-Mesenchymal Transition; Fibrosis; Humans; Keloid; Kidney; Liver Cirrhosis; Myocardium; PPAR gamma; Pulmonary Fibrosis; Signal Transduction; Transforming Growth Factor beta

TGF-β is one of the main mediators involved in tissue remodeling in the asthmatic lung. This profibrotic cytokine is produced by a number of cells, including macrophages, epithelial cells, fibroblasts, and eosinophils. High expression of TGF-β in patients with asthma was reported by many investigators. However, controversy remains whether the concentration of TGF-β correlates with disease severity. TGF-β is believed to play an important role in most of the cellular biological processes leading to airway remodeling. It was shown to be involved in epithelial changes, subepithelial fibrosis, airway smooth muscle remodeling, and microvascular changes. Here, sources of TGF-β, as well as its role in the development of airway remodeling, will be reviewed. Therapeutic strategies that modulate TGF-β will also be discussed.

Topics: Airway Remodeling; Animals; Asthma; Eosinophils; Goblet Cells; Humans; Inflammation Mediators; Lung; Microcirculation; Mucus; Muscle, Smooth; Pulmonary Circulation; Pulmonary Fibrosis; Respiratory Mucosa; Signal Transduction; Transforming Growth Factor beta

Prostaglandin (PG)E(2) is a bioactive eicosanoid that regulates many biologically important processes in part due to its ability to signal through four distinct G-protein-coupled receptors with differential signaling activity and unique expression patterns in different cell types. Although PGE(2) has been linked to malignancy in many organs, it is believed to play a beneficial role in the setting of fibrotic lung disease. This is in part due to the ability of PGE(2) to limit many of the pathobiologic features of lung fibroblasts and myofibroblasts, including the ability of PGE(2) to limit fibroblast proliferation, migration, collagen secretion, and, as originally reported in the Journal by us in 2003, the ability to limit transforming growth factor (TGF)-β-induced myofibroblast differentiation. In the setting of lung fibrosis, PGE(2) production and signaling is often diminished. In the last 8 years, significant advances have been made to better understand the dysregulation of PGE(2) production and signaling in the setting of lung fibrosis. We also have a clearer picture of how PGE(2) inhibits myofibroblast differentiation and the receptor signaling pathways that can influence fibroblast proliferation. This review highlights these recent advances and offers new insights into the potential ways that PGE(2) and its downstream signals can be regulated for therapeutic benefit in a disease that has no validated treatment options.

Topics: Animals; Cell Adhesion; Cell Differentiation; Cell Movement; Cell Proliferation; Dinoprostone; Epigenesis, Genetic; Fibroblasts; Gene Expression Regulation; Humans; Models, Biological; Protein Structure, Tertiary; Pulmonary Fibrosis; Signal Transduction; Transforming Growth Factor beta

Pirfenidone is an orally active small molecule that has recently been evaluated in large clinical trials for the treatment of idiopathic pulmonary fibrosis, a fatal disease in which the uncontrolled deposition of extracellular matrix leads to progressive loss of lung function. This review describes the activity of pirfenidone in several well-characterised animal models of fibrosis in the lung, liver, heart and kidney. In these studies, treatment-related reductions in fibrosis are associated with modulation of cytokines and growth factors, with the most commonly reported effect being reduction of transforming growth factor-β. The consistent antifibrotic activity of pirfenidone in a broad array of animal models provides a strong preclinical rationale for the clinical characterisation of pirfenidone in pulmonary fibrosis and, potentially, other conditions with a significant fibrotic component.

Topics: Administration, Oral; Animals; Cardiomyopathies; Disease Models, Animal; Extracellular Matrix Proteins; Humans; Kidney Diseases; Liver Cirrhosis; Pulmonary Fibrosis; Pyridones; Signal Transduction; Transforming Growth Factor beta

Pulmonary fibrosis is a highly heterogeneous and lethal pathological process with limited therapeutic options. Although research on the pathogenesis of pulmonary fibrosis has frequently focused on the mechanisms that regulate the proliferation, activation, and differentiation of collagen-secreting myofibroblasts, recent studies have identified new pathogenic mechanisms that are critically involved in the initiation and progression of fibrosis in a variety of settings. A more detailed and integrated understanding of the cellular and molecular mechanisms of pulmonary fibrosis could help pave the way for effective therapeutics for this devastating and complex disease.

Topics: Animals; Cell Differentiation; Cell Proliferation; Chemokines; Humans; Inflammation Mediators; Interleukin-13; Leukocytes; Macrophages; Models, Biological; Myofibroblasts; Pulmonary Fibrosis; Th2 Cells; Transforming Growth Factor beta; Wound Healing

Fibrogenic lung reactions occur as a common phenotype shared among disorders of heterogeneous etiologies. Even with a common etiology, the extent and pattern of fibrosis vary greatly among individuals, even within families, suggesting complex gene-environment interactions. The search for mechanisms shared among all fibrotic lung diseases would represent a major advance in the identification of therapeutic targets that could have a broad impact on lung health. Although it is difficult to grasp all of the complexities of the varied cell types and cytokine networks involved in lung fibrogenic responses, and to predict the biologic responses to the overexpression or deficiency of individual cytokines, a large body of evidence converges on a single common theme: the central importance of the transforming growth factor beta (TGF-beta) pathway. Therapies that act upstream or downstream of TGF-beta activation have the therapeutic potential to treat all fibrogenic responses in the lung.

Topics: Asthma; Emphysema; Fibroblasts; Humans; Lung Diseases; Pulmonary Disease, Chronic Obstructive; Pulmonary Fibrosis; Transforming Growth Factor beta

Transforming growth factor (TGF)-β is a pleiotropic cytokine with beneficial and detrimental effects posthematopoietic stem-cell transplantation. TGF-β is increased in specific sites postengraftment and can suppress immune responses and maintain peripheral tolerance. Thus, TGF-β may promote allograft acceptance. However, TGF-β is also the central pathogenic cytokine in fibrotic disease and likely promotes pneumonitis. Although TGF-β can enhance leukocyte recruitment and IgA production, it inhibits both innate and adaptive immune cell function and antiviral host defense posthematopoietic stem-cell transplantation. This review will focus on the current understanding of TGF-β biology and the numerous ways it can impact outcomes posttransplant.

Topics: Animals; Cell Survival; Graft Rejection; Graft Survival; Graft vs Host Disease; Hematopoietic Stem Cell Transplantation; Hematopoietic Stem Cells; Humans; Pulmonary Fibrosis; T-Lymphocytes, Regulatory; Time Factors; Transforming Growth Factor beta; Transplantation Tolerance; Transplantation, Homologous; Treatment Outcome

Pulmonary fibrosis complicates a number of disease processes and leads to substantial morbidity and mortality. Idiopathic pulmonary fibrosis (IPF) is perhaps the most pernicious and enigmatic form of the greater problem of lung fibrogenesis with a median survival of three years from diagnosis in affected patients. In this review, we will focus on the pathology of IPF as a model of pulmonary fibrotic processes, review possible cellular mechanisms, review current treatment approaches and review two transgenic mouse models of lung fibrosis to provide insight into processes that cause lung fibrosis. We will also summarize the potential utility of signaling pathway inhibitors as a future treatment in pulmonary fibrosis. Finally, we will present data demonstrating a minimal contribution of epithelial-mesenchymal transition in the development of fibrotic lesions in the transforming growth factor-alpha transgenic model of lung fibrosis.

Topics: Animals; Disease Models, Animal; Epithelial Cells; ErbB Receptors; Humans; Mesoderm; Mice; Pulmonary Fibrosis; Pulmonary Surfactant-Associated Protein C; Signal Transduction; Transforming Growth Factor beta

IPF (idiopathic pulmonary fibrosis) is a chronic progressive disease of unknown aetiology without effective treatment. IPF is characterized by excessive collagen deposition within the lung. Recent evidence suggests that the lung epithelium plays a key role in driving the fibrotic response. The current paradigm suggests that, after epithelial injury, there is impaired epithelial proliferation and enhanced epithelial apoptosis. This in turn promotes lung fibrosis through impaired basement membrane repair and increased epithelial-mesenchymal transition. Furthermore, fibroblasts are recruited to the wounded area and adopt a myofibroblast phenotype, with the up-regulation of matrix-synthesizing genes and down-regulation of matrix-degradation genes. There is compelling evidence that the cytokine TGFbeta (transforming growth factor beta) plays a central role in this process. In normal lung, TGFbeta is maintained in an inactive state that is tightly regulated temporally and spatially. One of the major TGFbeta-activation pathways involves integrins, and the role of the (alpha)vbeta6 integrin has been particularly well described in the pathogenesis of IPF. Owing to the pleiotropic nature of TGFbeta, strategies that inhibit activation of TGFbeta in a cell- or disease-specific manner are attractive for the treatment of chronic fibrotic lung conditions. Therefore the molecular pathways that lead to integrin-mediated TGFbeta activation must be precisely defined to identify and fully exploit novel therapeutic targets that might ultimately improve the prognosis for patients with IPF.

Topics: Animals; Gene Expression Regulation; Humans; Integrins; Models, Biological; Pulmonary Fibrosis; Transforming Growth Factor beta

The complex structure of the lung is developed sequentially, initially by epithelial tube branching and later by septation of terminal air sacs with accompanying coordinated growth of a variety of lung epithelial and mesenchymal cells. Groups of transcriptional factors, peptide growth factors and their intracellular signaling regulators, as well as extracellular matrix proteins are programmed to be expressed at appropriate levels in the right place at the right time to control normal lung formation. Studies of lung development and lung repair/fibrosis to date have discovered that many of the same factors that control normal development are also key players in lung injury repair and fibrosis. Transforming growth factor-beta (TGF-beta) family peptide signaling is a prime example. Lack of TGF-beta signaling results in abnormal lung branching morphogenesis and alveolarization during development, whereas excessive amounts of TGF-beta signaling cause severe hypoplasia in the immature lung and fibrosis in mature lung. This leads us to propose the 'Goldilocks' hypothesis of regulatory signaling in lung development and injury repair that everything must be done just right!

Topics: Extracellular Matrix; Humans; Lung; Lung Injury; Pulmonary Fibrosis; Signal Transduction; Transforming Growth Factor beta

Transforming growth factor-betas (TGF-beta) regulate a wide variety of cellular functions in normal development and are involved in both tissue homeostasis and disease pathogenesis. The regulation of the TGF-beta family of growth factors is unique because they are targeted to the extracellular matrix in a biologically inactive form. The release from pericellular matrices and the activation of TGF-beta are important mechanisms in several pathophysiologic conditions. Reactive oxygen species (ROS) can activate TGF-beta either directly or indirectly via the activation of proteases. In addition, TGF-beta itself induces ROS production as part of its signal-transduction pathway. The lung is a unique organ, because its structures act as boundaries between gaseous and aqueous phases, allowing the utilization of inhaled oxygen. However, this renders pulmonary tissues vulnerable to the toxic effects of inhaled air. The oxidant pathways are especially relevant in the lung, where TGF-beta is known to have a role in tissue repair and connective tissue turnover. In pulmonary fibrosis, TGF-beta activation is considered as a hallmark of disease progression. More recently, the oxidative effects of cigarette smoking have been found to activate TGF-beta in chronic obstructive pulmonary disease (COPD), a disease consisting of emphysema, airway fibrosis, and focal lung fibrosis.

Topics: Antioxidants; Extracellular Matrix; Humans; Lung; Models, Biological; Pulmonary Fibrosis; Reactive Oxygen Species; Signal Transduction; Transforming Growth Factor beta

Transient adenovirus-mediated gene transfer of active TGF-beta1 (transforming growth factor-beta1) induces severe and progressive fibrosis in rodent lung without apparent inflammation. Alternatively, transfer of IL-1beta (interleukin 1beta) induces marked tissue injury and inflammation, which develops into progressive fibrosis, associated with an increase in TGF-beta1 concentrations in lung fluid and tissue. Both vector treatments induce a fibrotic response involving myofibroblasts and progressive matrix deposition starting at the peri-bronchial site of expression and extending over days to involve the entire lung and pleural surface. Administration of the TGF-beta1 vector to the pleural space induces progressive pleural fibrosis, which minimally extends into the lung parenchyma. The mechanisms involved in progressive fibrosis need to account for the limitation of fibrosis to specific organs (lung fibrosis and not liver fibrosis or vice versa) and the lack of effect of anti-inflammatory treatments in regulating progressive fibrosis. TGF-beta1 is a key cytokine in the process of fibrogenesis, using intracellular signalling pathways involving the ALK5 receptor and signalling molecules Smad2 and Smad3. Transient gene transfer of either TGF-beta1 or IL-1beta to Smad3-null mouse lung provides little evidence of progressive fibrosis and no fibrogenesis-associated genes are induced. These results suggest that mechanisms of progressive fibrosis involve factors presented within the context of the matrix that define the microenvironment for progressive matrix deposition.

Topics: Animals; Humans; Pulmonary Fibrosis; Smad3 Protein; Transforming Growth Factor beta

Idiopathic pulmonary fibrosis (IPF) is one of a group of interstitial lung diseases that are characterized by excessive matrix deposition and destruction of the normal lung architecture. Long-term survival of IPF patients is poor, with a 5-year survival rate of only 20%. Despite a lack of evidence-based benefit, IPF has historically been treated with corticosteroids and/or cytotoxic agents such as prednisone. Given the poor efficacy of these drugs, novel therapeutic strategies are required for the management of IPF. This demands a better understanding of the molecular mechanisms underlying the pathogenesis and progression of this disease. The primary effector cell in fibrosis is the myofibroblast; these cells are highly synthetic for collagen, have a contractile phenotype, and are characterized by the presence of alpha-smooth muscle actin stress fibers. They may be derived by activation/proliferation of resident lung fibroblasts, epithelial-mesenchymal differentiation, or recruitment of circulating fibroblastic stem cells (fibrocytes). From a therapeutic viewpoint, interfering with the pathways that lead to myofibroblast expansion should be of considerable benefit in the treatment of IPF. This review will highlight some of the key molecules involved in this process and the clinical trials that have ensued.

Topics: Animals; Cell Differentiation; Chemokine CXCL12; Connective Tissue Growth Factor; Disease Progression; Extracellular Matrix; Fibroblasts; Humans; Immediate-Early Proteins; Intercellular Signaling Peptides and Proteins; Interleukin-13; Platelet-Derived Growth Factor; Pulmonary Fibrosis; Receptor, PAR-1; Receptors, CXCR4; Transforming Growth Factor beta; Wnt Proteins

Multiple investigators have undertaken genetic studies in idiopathic pulmonary fibrosis populations in attempts to define genetic links to disease in hopes that this would improve understanding of disease pathogenesis and target pathways for therapy. Multiple genes have been evaluated using a candidate gene approach with limited success, with results suggesting a disease modifier effect rather than a disease causing effect. Using this approach, associations have been observed between idiopathic pulmonary fibrosis and specific polymorphisms in genes encoding interleukin-1 receptor antagonist, tumor necrosis factor-alpha, and complement receptor 1. Recently investigators have used familial pulmonary fibrosis cohorts to evaluate for genetic mutations associated with idiopathic pulmonary fibrosis. Using one pulmonary fibrosis kindred, a mutation in the gene encoding surfactant protein C was identified as the cause of pulmonary fibrosis in this family. Subsequently, another individual with idiopathic pulmonary fibrosis was identified with a different mutation in surfactant protein C. Though rarely found in patients with idiopathic pulmonary fibrosis, these surfactant protein C mutations highlight the importance of the alveolar epithelium in disease pathogenesis. A recent collaboration between investigators at three major centers has resulted in the largest collection of families with pulmonary fibrosis to date, with hopes that this effort will identify genetic mutations associated with idiopathic pulmonary fibrosis. If genetic links to idiopathic pulmonary fibrosis are defined in this study, then the pathways involved with these genes and gene products can be targeted by investigators to help identify potential treatment options for this disease.

Topics: Cytokines; Genetic Predisposition to Disease; Humans; Mutation; Peptidyl-Dipeptidase A; Polymorphism, Genetic; Pulmonary Fibrosis; Pulmonary Surfactant-Associated Proteins; Receptors, Complement; Transforming Growth Factor beta

An understanding of the mechanisms underlying pulmonary fibrosis remains elusive. Once believed to result primarily from chronic inflammation, it is now clear that inflammation and chronic fibrosis, especially in diseases such as idiopathic pulmonary fibrosis/usual interstitial pneumonia, are often dissociated, and that inflammation is neither necessary nor sufficient to induce fibrosis. The origin of the primary effector cell of fibrosis in the lung, the myofibroblast, is not clearly established. Three potential sources have been hypothesized. Although conversion of resident fibroblasts and differentiation of circulating bone marrow-derived progenitors likely play a role, the possible contribution of alveolar epithelial cells (AECs), through a process termed "epithelial-mesenchymal transition" (EMT), has only recently received consideration. A process by which epithelial cells lose cell-cell attachment, polarity and epithelial-specific markers, undergo cytoskeletal remodeling, and gain a mesenchymal phenotype, EMT plays a prominent role in fibrogenesis in adult tissues such as the kidney. This review summarizes the evidence supporting a central role for EMT in the pathogenesis of lung fibrosis, the potential for EMT in AECs in vitro and in vivo and role of transforming growth factor-beta1 in this process, and the implications of epithelium-driven fibrosis on future research and treatment. Potential pathways involved in EMT are also discussed. It is hoped that a major shift in current paradigms regarding the genesis of pulmonary fibrosis and dissection of the relevant pathways may allow development of targeted interventions that could potentially reverse the process and ameliorate the debilitating effects of abnormal repair and progressive fibrosis.

Topics: Animals; Apoptosis; Biomarkers; Cell Line; Epithelial Cells; Fibroblasts; Membrane Proteins; Mesoderm; Myocytes, Smooth Muscle; Pulmonary Alveoli; Pulmonary Fibrosis; Rats; Transforming Growth Factor beta; Transforming Growth Factor beta1

The currently accepted approach to treatment of idiopathic pulmonary fibrosis (IPF) is based on the assumption that it is a chronic inflammatory disease, and most available antiinflammatory drugs target numerous biological processes involving multiple genes, but are not often beneficial. More novel therapeutic strategies take recent findings about the underlying molecular mechanisms of fibrogenesis into account, and ongoing and as yet unpublished clinical trials in IPF aim to block single gene targets believed to play major roles in disease progression. Characterization of the mechanisms involved in the pathogenesis of IPF has largely come from the use of animal disease models in rodents. Most data suggest, from among the different factors, a prominent role for the transforming growth factor (TGF)-beta1 and platelet-derived growth factor pathways. Inflammation is a critical element of the initiation of fibrosis and data indicate that the Smad pathway is a necessary link to fibrosis through TGF-beta and Smad3 signaling, which introduces matrix regulation as a new target for therapeutic intervention. Regardless, gene targeted therapy has numerous pitfalls that have to be addressed before we see a real therapeutic advance.

Topics: Clinical Trials as Topic; Connective Tissue Growth Factor; Etanercept; Extracellular Matrix; Humans; Immediate-Early Proteins; Immunoglobulin G; Intercellular Signaling Peptides and Proteins; Platelet-Derived Growth Factor; Pulmonary Fibrosis; Receptors, Tumor Necrosis Factor; Signal Transduction; Smad3 Protein; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha

Idiopathic pulmonary fibrosis (IPF) is an under-recognised, rare, progressive disease of the lungs with unknown aetiology and high mortality. The currently advocated pathogenic mechanism is represented by progressive multifocal fibrosis. It is diagnosed based on clinical, radiographic, physiological and histopathological criteria. Existing therapeutic guidelines recommend anti-inflammatory and immunosuppressive combinations, despite proven limited efficacy. There is no therapy approved specifically for IPF, but several antifibrotic agents are currently under development for this indication. Pirfenidone is an antifibrotic agent potentially effective for IPF therapy, and preclinical and available clinical data support its use in IPF. Future clinical studies are expected to provide more consistent information on survival benefit, lung function and health-related quality of life.

Topics: Administration, Oral; Amiodarone; Animals; Anti-Inflammatory Agents, Non-Steroidal; Bleomycin; Cell Line; Clinical Trials, Phase II as Topic; Connective Tissue Growth Factor; Cricetinae; Cyclophosphamide; Drug Evaluation, Preclinical; Drugs, Investigational; Fibroblasts; Hermanski-Pudlak Syndrome; Humans; Immediate-Early Proteins; Intercellular Signaling Peptides and Proteins; Lung Transplantation; Mice; Orphan Drug Production; Photosensitivity Disorders; Pulmonary Fibrosis; Pyridones; Randomized Controlled Trials as Topic; Scleroderma, Systemic; Transforming Growth Factor beta; Treatment Outcome; Tumor Necrosis Factor-alpha

The requirement for precise geometric organization of endothelial cells and epithelial cells makes the gas-exchange region of the lung especially vulnerable to the adverse consequences of toxic products released from inflammatory cells. However, as a filter for large volumes of atmospheric gas, the lung is continually exposed to microorganisms and other toxic insults that require robust inflammatory defense. Enhanced production of extracellular matrix proteins is one important mechanism for restricting tissue damage, but excessive matrix production also has serious adverse effects on gas exchange. The amazing ability of the lung to recover from a barrage of environmental insults depends on precisely regulating both inflammation and extracellular matrix production in space and time. Below I review some of the evidence implicating members of the transforming growth factor beta family as critical mediators of this delicate dance and describe examples of how disruption of this balance by alterations in the magnitude of spatially restricted transforming growth factor beta activation can contribute to pathologic consequences of alveolar and airway injury and inflammation.

Topics: Animals; Bronchitis; Humans; Lung Diseases, Interstitial; Pulmonary Fibrosis; Transforming Growth Factor beta

Inflammation and tissue remodeling with pathologic fibrosis are common consequences of Th2 responses in the lung and other organs. Interleukin (IL)-13 and transforming growth factor-beta1 (TGF-beta(1)) are frequently coexpressed in these responses and are believed to play important roles in the pathogenesis of Th2-induced pathologies. To shed light on the mechanisms of these responses, overexpression transgenic approaches were used to selectively target each of these cytokines to the murine lung. IL-13 proved to be a potent stimulator of eosinophilic inflammation, mucus metaplasia, tissue fibrosis, and alveolar remodeling. CC chemokines, specific chemokine receptors (CCR2, CCR1), adenosine metabolism, vascular endothelial growth factor, and IL-11 contributed to the genesis of these responses. IL-13 also induced tissue fibrosis, at least in part, via its ability to induce and activate TGF-beta(1). In the TGF-beta(1) transgenic mouse, epithelial apoptosis preceded the onset of tissue fibrosis and alveolar remodeling. In addition, chemical (Z-VAD-fmk) and genetic (null mutations of early growth response gene 1) interventions blocked apoptosis and ameliorated TGF-beta(1)-induced fibrosis and alveolar restructuring. These studies define an IL-13-TGF-beta(1) pathway of tissue remodeling that regulates inflammation, mucus metaplasia, apoptosis, vascular responses, and fibrosis in the lung. They also highlight the intimate relationship between apoptosis and fibrosis induced by TGF-beta(1). By defining the complexities of this pathway, these studies highlight sites at which therapies can be directed to control these important responses.

Topics: Animals; Apoptosis; DNA; Gene Expression; Humans; Pulmonary Alveoli; Pulmonary Fibrosis; Transforming Growth Factor beta; Transforming Growth Factor beta1

The incidence of finding evidence of both emphysema and pulmonary fibrosis in the same patient has received increased attention. Several investigators have found on biopsy the presence of emphysema of the upper zones and diffuse parenchymal disease with fibrosis of the lower zones of the lung, especially associated with current or previous heavy smokers. Believed previously to be two different disease mechanisms, there are now data to implicate some common pathways of cell and molecular activation leading to the different morphologic and physiologic outcomes. According to a current view, emphysema may originate from a protease/antiprotease imbalance, whereas a role for antiproteases has been proposed in the modulation of fibrosis. Overexpression of transforming growth factor beta (TGF-beta) in experimental rodent models leads to progressive pulmonary fibrosis, accompanied with marked up-regulation of protease inhibitors, such as tissue inhibitor of metalloproteinases (TIMP) and plasminogen activator inhibitor-1 (PAI-1) genes, along with excessive matrix accumulation. It may be that a "matrix degrading" pulmonary microenvironment, one in which metalloproteinase activities prevail, favors the development of emphysema, whereas a "matrix nondegrading" microenvironment, with enhanced presence of TIMPs, would lead to matrix accumulation and fibrosis. Surprisingly, although Smad3 null mice, deficient in TGF-beta signal transmission, are resistant to bleomycin- and TGF-beta-mediated fibrosis, they develop spontaneous age-related airspace enlargement, consistent with emphysema, with a lack of ability to repair tissue damage appropriately. A common element is tissue damage and repair, with TGF-beta and the Smad signaling pathway playing prominent molecular roles. Both changes can be followed in experimental models with noninvasive imaging and physiologic measurements.

Topics: Animals; Humans; Inflammation; Lung; Mice; Models, Animal; Pulmonary Emphysema; Pulmonary Fibrosis; Rats; Signal Transduction; Smad3 Protein; Smoking; Transforming Growth Factor beta

Idiopathic ulmonary fibrosis (histopathology of usual interstitial pneumonia) is a progressive lung disease of unknown etiology. No treatment has been shown to improve the prognosis of the patients with this disease. Recent evidence, including the observations that the patients with idiopathic pulmonary fibrosis have higher levels of oxidant stress than control patients, and a recent multicenter European study examining the effect of the antioxidant N-acetylcysteine on the progression of idiopathic pulmonary fibrosis suggest that the cellular redox state may play a significant role in the progression of this disease. These complex mechanisms include activation of growth factors as well as regulation of matrix metalloproteinases and protease inhibitors. Potential future approaches for the therapy of interstitial pulmonary fibrosis may involve synthetic agents able to modulate cellular redox state. Investigation into therapeutic approaches to inhibit oxidant-mediated reactions in the initiation and progression of pulmonary fibrosis may provide hope for the future treatment of this disease.

Topics: Animals; Antioxidants; Disease Models, Animal; Free Radicals; Humans; Lung; Oxidation-Reduction; Oxidative Stress; Pulmonary Fibrosis; Reactive Oxygen Species; Transforming Growth Factor beta

Topics: Angiotensin II; Animals; Apoptosis; Caspases; Cell Cycle Proteins; Cell Proliferation; Cyclin-Dependent Kinase Inhibitor p21; Endothelial Cells; Epithelial Cells; Fas Ligand Protein; Fibroblasts; Fibrosis; Humans; Lung; Membrane Glycoproteins; Pulmonary Fibrosis; Reactive Oxygen Species; Regeneration; Transforming Growth Factor beta; Tumor Suppressor Protein p53

Topics: Animals; Biomarkers; Bone Regeneration; Humans; Immunoenzyme Techniques; Kidney Diseases; Osteogenesis; Pulmonary Fibrosis; Transforming Growth Factor alpha; Transforming Growth Factor beta; Wound Healing

A set of lung diseases share the tendency for the development of progressive fibrosis ultimately leading to respiratory failure. This review examines the common pathogenetic features of these disorders in light of recent observations in both humans and animal models of disease, which reveal important pathways of lung matrix remodeling.

Topics: Animals; Collagen; Disease Models, Animal; Disease Progression; Epithelium; Fibrosis; Humans; Inflammation; Lung; Lung Diseases; Mice; Models, Anatomic; Models, Biological; Mutation; Pulmonary Fibrosis; Signal Transduction; Transforming Growth Factor beta; Transforming Growth Factor beta1

Transforming growth factor (TGF) beta plays an important role in normal pulmonary morphogenesis and function and in the pathogenesis of lung disease. The effect of TGFbeta is regulated via a selective pathway of TGFbeta synthesis and signaling that involves activation of latent TGFbeta, specific TGFbeta receptors, and intracellular signaling via Smad molecules. All three isoforms of TGFbeta are expressed at high levels during normal lung development, being particularly important for branching morphogenesis and epithelial cell differentiation with maturation of surfactant synthesis. Small amounts of TGFbeta are still present in the adult lung, and TGFbeta is involved in normal tissue repair following lung injury. However, in a variety of forms of pulmonary pathology, the expression of TGFbeta is increased. These include chronic lung disease of prematurity as well as several forms of acute and chronic adult lung disease. While TGFbeta1 appears to be the predominant isoform involved, elevated levels of all three isoforms have been demonstrated. The increase in TGFbeta precedes abnormalities in lung function and detectable lung pathology, but correlates with the severity of the disease. TGFbeta plays a key role in mediating fibrotic tissue remodeling by increasing the production and decreasing the degradation of connective tissue via several mechanisms.

Topics: Animals; Female; Humans; Lung; Lung Diseases; Male; Morphogenesis; Pulmonary Fibrosis; Risk Factors; Sensitivity and Specificity; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha

Topics: Angiotensin II; Animals; Cell Division; Collagen; Endothelin-1; Extracellular Matrix; Fibroblast Growth Factor 2; Fibroblasts; Glomerulosclerosis, Focal Segmental; Humans; Hypertension; Liver Cirrhosis; Myoblasts, Cardiac; Myocardial Infarction; Pulmonary Fibrosis; Receptors, Endothelin; Transforming Growth Factor beta; Ventricular Remodeling

Topics: Humans; Polymorphism, Genetic; Pulmonary Fibrosis; Transforming Growth Factor beta

A diagnosis of idiopathic pulmonary fibrosis (IPF) carries a poor prognosis, with our currently available therapies offering little clinical benefit. Unfortunately, recent major advances in our understanding of the clinical and biologic features of this disease have not been matched by similar advances in treatment. This is likely because of the complex cascade of biologic and pathobiologic events that occurs in IPF. The necessary, and desperately needed, next generation of therapies, focused on specific molecular targets thought to play pivotal roles in the development and progression of fibrosis, are under active investigation.

Topics: Antioxidants; Clinical Trials as Topic; Cytokines; Eicosanoids; Humans; Immunosuppressive Agents; Interferons; Pulmonary Fibrosis; Risk Factors; Transforming Growth Factor beta

Idiopathic Pulmonary Fibrosis (IPF) is a chronic interstitial lung disease which results in end-stage fibrosis. The pathogenesis is believed to be related to a dysregulation in cross-talk between inflammatory and structural cells, mediated by various cytokines, chemokines and growth factors, which are responsible for the maintenance of tissue homeostasis and which coordinate the response to injury. The large number of mediators involved and the complexity of their interaction makes it difficult to identify the factors responsible for initiation of fibrogenesis and progression to chronicity. Whether a mediator's presence in fibrotic lung is as a result of tissue injury or if it playsan active role in disease onset and progression has been partly answered by the use of transient and/or permanent transgenic and gene knock-out approaches to over-express single factors at a time. Chemokines such as interleukin-8 (IL-8), RANTES, IP-10, MIG or lymphotactin, do not appear to induce fibrosis when over-expressed in rodent lung. Amongst many tested, four cytokines and growth factors have been found to be pro-fibrotic; IL-1beta, which demonstrates marked inflammation, tissue damage and chronic fibrosis, TNF-alpha, which induces inflammation and mild fibrosis, and GM-CSF, which induces moderate inflammation and fibrosis. A common finding with these cytokines are increased lung TGF-beta levels, proportionate to the degree of fibrosis generated, while TGF-beta itself causes minor inflammation but marked progressive chronic fibrosis. A growth factor 'downstream' from the pro-fibrotic effects of TGF-beta, CTGF, is a likely critical mediator. However, over-expression of CTGF produces only mild and reversible fibrosis.

Topics: Animals; Chemokines; Connective Tissue Growth Factor; Cytokines; Granulocyte-Macrophage Colony-Stimulating Factor; Humans; Immediate-Early Proteins; Intercellular Signaling Peptides and Proteins; Lung; Pulmonary Fibrosis; Transforming Growth Factor beta

To review recent advances in the use of transforming growth factor (TGF)-beta in acute lung injury and to apply this knowledge to understanding the pathophysiology of this syndrome.. Published research and review articles in the English language related to the role of TGF-beta in acute lung injury.. The cytokine TGF-beta plays a critical role in the resolution of tissue injury in multiple organs, including the lung. Following injury, TGF-beta has been most thoroughly evaluated during the late phases of tissue repair, where it plays a critical role in the development of pulmonary fibrosis. In contrast, recent animal studies showed that expression levels of several TGF-beta-inducible genes were dramatically increased as early as 2 days after the induction of injury. The integrin alpha(v)beta(6) activates latent TGF-beta in the lungs. Mice lacking this integrin were completely protected from pulmonary edema in a model of bleomycin-induced acute lung injury. Pharmacologic inhibition of TGF-beta also protected wild-type mice from pulmonary edema induced by bleomycin or Escherichia coli endotoxin. Similar findings also have been reported in patients in a clinical study evaluating TGF-beta in the bronchoalveolar lavage fluid during the course of acute respiratory distress syndrome (ARDS). Indeed, the bronchoalveolar lavage concentrations were dramatically increased as early as 1 day after the initiation of ARDS criteria and were correlated with decreases in the Pao(2)/Fio(2) ratio, suggesting an important role for TGF-b1 in the development of ARDS in humans.. These studies suggest that TGF-beta not only participates in the late phase of acute lung injury, but also might be active early in acute lung injury and potentially could contribute to the development of pulmonary edema. Integrin-mediated local activation of TGF-beta is critical to the development of pulmonary edema in ARDS, and blocking TGF-beta or its activation could be an effective treatment for this disorder.

Topics: Animals; Humans; Mice; Pulmonary Fibrosis; Respiratory Distress Syndrome; Signal Transduction; Transforming Growth Factor beta

Topics: Animals; Fibroblasts; Humans; Pulmonary Alveoli; Pulmonary Fibrosis; Respiratory Mucosa; Transforming Growth Factor beta

Topics: Animals; Apoptosis; Humans; Inflammation; Lung Diseases, Interstitial; Models, Biological; Phagocytosis; Pulmonary Fibrosis; Transforming Growth Factor beta

Molecular investigation into the physiopathology of interstitial lung diseases has gained special interest through the trials carried out in the last decade. These trials seem to point at the role played by certain molecules, such as cytokines (transforming growth factor, platelet derived growth factor) and integrins, in the processes that lead to pulmonary fibrosis during the course of interstitial lung disease. They also demonstrate the important role that angiotensin II plays in increasing the secretion of transforming growth factor by several cells. The above-mentioned studies allow new therapeutic approaches to be considered which will possibly improve the serious prognosis of such diseases once they have reached the last stage of their course: pulmonary fibrosis.

Topics: Angiotensin II; Humans; Integrins; Lung Diseases, Interstitial; Pulmonary Fibrosis; Transforming Growth Factor beta

Pulmonary fibrosis occurs in up to 70% of scleroderma patients and progresses to cause severe restrictive lung disease in about 15% of patients. The mechanisms that cause pulmonary fibrosis in scleroderma remain incompletely understood. Increased amounts of mRNA or protein for multiple profibrotic cytokines and chemokines have been identified in lung tissue or broncholveolar lavage samples from scleroderma patients, when compared to healthy controls. These cytokines include transforming growth factor (TGF)-beta, connective tissue growth factor (CTGF), platelet-derived growth factor (PDGF), oncostatin M (OSM), monocyte chemotactic factor-1 and pulmonary and activation-regulated chemokine (PARC). Potential cellular sources of these profibrotic cytokines and chemokines in scleroderma lung disease include alternatively activated macrophages, activated CD8+ T cells, eosinophils, mast cells, epithelial cells and fibroblasts themselves. This review summarizes the literature on involvement of cytokines and chemokines in the development of pulmonary fibrosis in scleroderma.

Topics: Adult; CD8-Positive T-Lymphocytes; Chemokine CCL2; Chemokines; Connective Tissue Growth Factor; Cytokines; DNA Topoisomerase IV; Eosinophils; Epithelial Cells; Female; Fibroblasts; Fibrosis; Humans; Immediate-Early Proteins; Intercellular Signaling Peptides and Proteins; Interleukin-1; Lung; Male; Mast Cells; Oncostatin M; Peptides; Platelet-Derived Growth Factor; Pulmonary Fibrosis; RNA, Messenger; Scleroderma, Diffuse; Transforming Growth Factor beta

The pathogenesis of pulmonary fibrosis remains incompletely understood. Studies of associated inflammation have led to the discovery of a number of cytokines and chemokines that are found to be important either directly or indirectly for the fibrotic process. However, the importance of inflammation in pulmonary fibrosis is unclear, and at the time of diagnosis the inflammatory component is variable and usually not responsive to anti-inflammatory therapeutic agents. Patients usually exhibit evidence of active fibrosis with increased numbers of activated fibroblasts, many of which have the phenotypic characteristics of myofibroblasts. At these sites, increased amounts of extracellular matrix deposition are evident with effacement of the normal alveolar architecture. Animal model studies show the myofibroblast to be the primary source of type I collagen gene expression in active fibrotic sites. In vitro studies show differentiation of these cells from fibroblasts under the influence of certain cytokines but indicate their susceptibility to nitric oxide-mediated apoptosis. In addition to promoting myofibroblast differentiation, transforming growth factor-beta1 provides protection against apoptosis. Thus, this well-known fibrogenic cytokine is important both for the emergence of the myofibroblast and its survival against apoptotic stimuli. This is consistent with the critical importance of this cytokine in diverse models of fibrosis in various tissues. In view of these properties, the persistence or prolonged survival of the myofibroblast may be key to understanding why certain forms of lung injury may result in progressive disease, terminating in end-stage disease.

Topics: Apoptosis; Cell Differentiation; Fibroblasts; Humans; Inflammation; Pulmonary Alveoli; Pulmonary Fibrosis; Transforming Growth Factor beta; Transforming Growth Factor beta1

Idiopathic pulmonary fibrosis (IPF) is a lung disease that is characterized by epithelial cell damage and areas of denuded basement membrane resulting in inflammation, fibroblast proliferation, excessive extracellular matrix (ECM) deposition, and remodeling of alveolar gas exchange units. The progressive loss of lung gas exchange units in patients with IPF leads to respiratory failure and eventually to death. While the etiology of this disease is unknown, for many years studies suggested that chronic inflammation was the underlying factor that caused fibroproliferation and structural alterations of the lung. Recent data show that fibroproliferation and fibrosis can occur independently of inflammation, suggesting that IPF is a disease caused by a mesenchymal, rather than an immune disorder. Mesenchymal growth factors, including transforming growth factor (TGF)-beta, insulin-like growth factor (IGF)-I, platelet-derived growth factor, connective tissue growth factor, fibroblast growth factors, and keratinocyte growth factors, as well as proinflammatory cytokines such as tumor necrosis factor-alpha and interleukin-1beta, have been shown to be exaggerated in several fibrotic lung disorders including IPF, ARDS, sarcoidosis, and bronchopulmonary dysplasia, as well as pulmonary manifestations of systemic diseases such as rheumatoid arthritis or progressive systemic sclerosis (scleroderma). We argue that inflammation is required to initiate growth factor production and repair of the damaged alveolar epithelial lining in fibrotic lung diseases and that exaggerated TGF-beta production may be responsible for the fibrotic response seen in diseases such as IPF. We recognize the potential role of several growth factors in the fibroproliferative process in the lung, and in this brief report we focus on the possible roles of the growth factors IGF-I and TGF-beta in cell migration, proliferation, and ECM synthesis in patients with IPF.

Topics: Humans; Inflammation; Insulin-Like Growth Factor I; Pulmonary Fibrosis; Respiratory Mucosa; Transforming Growth Factor beta

Topics: Acute-Phase Reaction; Genes, p53; Genes, ras; Humans; Lung Neoplasms; Mutation; Precancerous Conditions; Pulmonary Fibrosis; Receptors, Transforming Growth Factor beta; Risk Factors; Smoking; Transforming Growth Factor beta

Tissue fibrosis can lead to significant organ dysfunction and resulting patient morbidity and mortality. Unfortunately, the therapeutic repertoire is currently limited, nonspecific, and largely ineffective. While the pathogenesis is incompletely understood, evidence is accumulating that immune and cytokine mediated mechanisms are critical. In this review, data will be provided to support the role of Type 2 cytokines in the pathogenesis of fibrosis. The importance of the role of the pro-fibrogenic cytokine TGF-beta and CD40-CD40 ligand mediated fibroblast activation will also be evaluated. Finally, novel therapeutic options based on inhibiting these pathways will be described.

Topics: Animals; CD40 Antigens; CD40 Ligand; Cytokines; Fibrosis; Humans; Interferon-gamma; Pulmonary Fibrosis; Transforming Growth Factor beta

Topics: Animals; Cell Division; Chemokines; Cytokines; Disease Progression; Extracellular Matrix; Humans; Inflammation; Interferons; Lung; Matrix Metalloproteinase 7; Matrix Metalloproteinases; Pulmonary Fibrosis; Transforming Growth Factor beta; Transforming Growth Factor beta1

Better understanding of the hemodynamic-independent actions of the renin-angiotensin system (RAS) may lead to improved therapies for heart, kidney, and liver fibrosis. The conventional view of the RAS is that its role is solely hemodynamic. Pharmacologic blockade of the RAS is beneficial in treating hypertension, as well as primary renal and cardiac diseases. Recent findings from clinical trials and several laboratories that used different experimental approaches have revealed a whole new dimension to the RAS that is beyond the realm of hemodynamics. The RAS is best viewed as part of a system of interconnected molecules biologically designed to be activated after tissue injury to promote tissue repair and, when in excess, tissue fibrosis. This new understanding of the RAS has important clinical implications. It predicts and explains why blockade of the RAS with angiotensin-converting enzyme inhibitors (ACEI), the newer receptor antagonists, or both together, will significantly slow the progression of fibrotic disease. However, it further suggests that higher doses and/or a combination of angiotensin II blockade with another agent or agents might truly halt progressive fibrosis.

Topics: Angiotensin II; Angiotensin-Converting Enzyme Inhibitors; Fibrosis; Humans; Kidney Diseases; Liver Cirrhosis; Pulmonary Fibrosis; Renin-Angiotensin System; Transforming Growth Factor beta

Topics: Collagen; Cytokines; Dermatology; Esophagus; Humans; Pulmonary Fibrosis; Scleroderma, Systemic; Severity of Illness Index; Skin; Transforming Growth Factor beta

The pathology of progressive pulmonary fibrosis combines injury, chronic inflammation and exaggerated, but futile organ repair. Models of experimental organ fibrosis such as bleomycin- or irradiation-induced lung fibrosis indicate that the continuous overexpression of major growth factors such as transforming growth factor beta 1 plays a major role in the tissue reorganization process and the modulation of the accompanying immune response. Moreover, this process is combined with a reorganization of the extracellular matrix that is likely to allow for the secondary loss of transcription of the interferon gamma gene. As a result, the cytokine pattern of the evolving chronic cellular immune response shifts to the so-called T helper 2 type. Recent investigations have demonstrated that this poorly balanced immune response is a characteristic feature of human progressive lung fibrosis such as idiopathic pulmonary fibrosis. Based on the strong antifibrotic properties of interferon gamma, we combined low-dose glucocorticoids with interferon gamma-1b for the treatment of idiopathic pulmonary fibrosis, a relentlessly progressive form of human pulmonary fibrosis. This pilot investigation demonstrated that interferon gamma is able to improve pulmonary function in patients with idiopathic pulmonary fibrosis while at the same time counterbalancing mechanisms of exaggerated wound repair, such as the overinduction of transforming growth factor beta 1.

Topics: Animals; Drug Therapy, Combination; Glucocorticoids; Humans; Interferon-gamma; Pulmonary Fibrosis; Recombinant Proteins; Th2 Cells; Transforming Growth Factor beta; Transforming Growth Factor beta1; Treatment Outcome

Topics: Collagen; Fibroblasts; Humans; Lung; Platelet-Derived Growth Factor; Pulmonary Fibrosis; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha

Topics: Animals; Controlled Clinical Trials as Topic; Dose-Response Relationship, Drug; Drug Administration Schedule; Female; Humans; Interferon-gamma; Interferons; Male; Prognosis; Pulmonary Fibrosis; Scleroderma, Systemic; Sensitivity and Specificity; Severity of Illness Index; Transforming Growth Factor beta; Treatment Outcome

Immune response and restructuring of tissue during organ fibrosis mutually influence each other. It has become evident that the immunomodulatory properties of lining cells of the lung, such as bronchial or alveolar epithelial cells or pulmonary endothelial cells exert a major influence on the acute and chronic activation of the immune system. On the other hand, recent data obtained under in vivo conditions, suggest that the process of mesenchymal organ remodelling during inflammation not only causes organ fibrosis, but may actually perpetuate the process of chronic pulmonary inflammation due to its immunosuppressive effects. In this short review, two examples for this reciprocal influence are discussed.

Topics: Adjuvants, Immunologic; Animals; Humans; Immune Tolerance; Immunity, Cellular; Pneumonia, Pneumococcal; Pulmonary Fibrosis; Transforming Growth Factor beta

Considerable progress has been made in defining the phenotype of contractile cells of the lung during development, in the adult and during the remodeling process. The high degree of phenotypic heterogeneity of subgroups of these cells, such as SMCs, is appreciated. Recent studies also have explored the relationship between phenotype and cell function, though this remains an important area for research in the coming years. Similarly, though our understanding of the regulation of cell phenotype is expanding rapidly, much remains to be done, particularly at the level of gene regulation. New transgenic models, coupled with gene-promotor analyses in transgenic animals and in cultured cells should allow rapid progress. Studies of the regulation of specific contractile and cytoskeletal proteins at the gene level by specific cytokines and extracellular-matrix elements will be particularly important.

Topics: Actins; Animals; Fibroblasts; Gene Expression Regulation; Humans; Lung; Muscle, Smooth; Phenotype; Pulmonary Fibrosis; Transforming Growth Factor beta

Topics: Actins; Animals; Fibroblasts; Gene Transfer Techniques; Granulocyte-Macrophage Colony-Stimulating Factor; Humans; Lung; Muscle, Smooth; Mutagenesis; Pulmonary Fibrosis; Transforming Growth Factor beta

Pulmonary fibrosis can complicate diverse pulmonary and systemic pathologies. In many cases the underlying cause remains unidentified. Mortality from the disease is increasing steadily in the UK and USA. The clinical features are well-described, but patients frequently present at an advanced stage, and current treatments have not improved the poor prognosis. There is a compelling need to identify the fibrotic process earlier and to develop new therapeutic agents. Increased collagen deposition is central to the pathology and interest over the last decade has focused on the role of cytokines in this process. These polypeptide mediators are believed to be released from both circulating inflammatory and resident lung cells in response to endothelial and epithelial injury. Key cytokines currently implicated in the fibrotic process are transforming growth factor-beta, tumour necrosis factor-alpha and endothelin-1. This article outlines the evidence implicating these mediators in the pathogenesis of pulmonary fibrosis and also considers the possible role of cytokines with antifibrotic effects, such as interferon-gamma. The "balance" of positively and negatively regulating cytokines is discussed, and the potential for interaction with other factors including viruses, hormones and altered antioxidant status is also considered. Finally, potential novel therapeutic approaches are discussed, together with suggestions for future studies and clinical trials. As the outcomes of different avenues of research over the last ten years are brought together, it is clear that there is now a hitherto unrivalled opportunity to begin to tackle the treatment of this devastating disease.

Topics: Animals; Cytokines; Endothelin-1; Humans; Pulmonary Fibrosis; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha

Fibrosis is a pathological process characterized by the replacement of normal tissue by mesenchymal cells and the extracellular matrix produced by these cells. The sequence of events leading to fibrosis of an organ involves the subsequent processes of injury with inflammation and disruption of the normal tissue architecture, followed by tissue repair with accumulation of mesenchymal cells in the area of derangement. The same sequence of events occurs in wound healing with normal granulation tissue and scar formation, but, while normal scar formation is very localized and transient, in contrast, in fibrosis, the repair process is exaggerated and usually widespread and can be chronic. Inflammatory cells (mainly mononuclear phagocytes), platelets, endothelial cells, and type II pneumocytes play a direct and indirect role in tissue injury and repair. The evaluation of three human fibrotic lung diseases, two diffuse [idiopathic pulmonary fibrosis (IPF), and the adult respiratory distress syndrome (ARDS)], and one focal (tumor stroma in lung cancer), has shown that several cytokines participate to the local injury and inflammatory reaction [interleukin-1 (IL-1), interleukin-8 (IL-8), monocyte chemotactic protein-1 (MCP-1), tumor necrosis factor-alpha (TNF-alpha)], while other cytokines are involved in tissue repair and fibrosis [platelet-derived growth factor (PDGF), insulin-like growth factor-1 (IGF-1), transforming growth factor-beta (TGF-beta), and basic-fibroblast growth factor (b-FGF)]. A better understanding of the cytokines and cytokine networks involved in lung fibrosis leads to the possibility of new therapeutic approaches.

Topics: Cytokines; Humans; Interleukin-1; Interleukin-8; Molecular Weight; Platelet-Derived Growth Factor; Pulmonary Fibrosis; Respiratory Distress Syndrome; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha; Wound Healing

To elucidate the relevance of transforming growth factor (TGF)-beta 1 and platelet-derived growth factor (PDGF)-B to the pathogenesis of pulmonary fibrosis, we introduced each of these expression vectors via trachea into Wistar rat to overexpress them locally in the lungs by hemagglutinating virus of Japan (HVJ)-liposome method. The TGF-beta 1 gene induced significant proliferatin of fibroblasts and deposition of collagen fibrils with mild cellular infiltration. The PDGF-B gene induced mild fibrotic changes with some cellular infiltration. These findings suggest that both factors may be very closely relevant to the pathogenesis of lung fibrosis.

Topics: Animals; Gene Transfer Techniques; Humans; Platelet-Derived Growth Factor; Pulmonary Fibrosis; Rats; Rats, Wistar; Transforming Growth Factor beta

Chronic lung disease of prematurity (CLD) is a common respiratory disorder of preterm infants. At autopsy, fibroblast proliferation, and components of the extracellular matrix, including collagen and fibronectin, are markedly increased in the lungs of infants who die from CLD. Examination of broncho-alveolar fluid suggests that the persistence of neutrophils is associated with the development of CLD. In our studies, the pro-inflammatory cytokines, interleukin-1 beta (IL-1 beta) and interleukin-6, (IL-6) and mediators which reflect neutrophil recruitment and activation, including soluble intercellular adhesion molecule, interleukin-8 (IL-8) and neutrophil elastase, were increased in lavage fluid obtained from infants who developed CLD when compared to infants who did not. Furthermore, semiquantitative reverse transcriptase-polymerase chain reaction of mRNA extracted from lavage cells suggested that luminal cells may be the source of IL-6 detected in lavage fluid but non-luminal cells may be the sources of IL-1 beta and IL-8. Fibrosis is thought to be mediated by the pro-fibrotic cytokines including transforming growth factor-beta1 (TGF-beta 1). Both active and total TGF-beta 1 were increased in lavage fluid from infants who developed CLD. Furthermore, both type I procollagen and TGF-beta were increased qualitatively in lung tissue obtained at autopsy from infants who died from respiratory failure. The increase in inflammatory mediators was maximal at 10 days of age. By contrast, the increase in TGF-beta 1 was maximal at 4 days of age. This suggests that the interaction between inflammation and fibrosis in CLD is complex, and that prenatal factors may be important in the pathogenesis of CLD.

Topics: Cytokines; Humans; Infant, Newborn; Infant, Premature; Infant, Premature, Diseases; Inflammation; Lung; Lung Diseases; Pulmonary Fibrosis; Transforming Growth Factor beta

Interstitial pneumonia is characterized by alveolitis that results in interstitial fibrosis. To study the role of humoral factors in the pathogenesis of interstitial fibrosis, we introduced expression vectors into Wistar rats via the trachea, to cause local overexpression of these humoral factors in the lung. Genes for human interleukin (IL)-6 and for the IL-6 receptor caused lymphocytic alveolitis without marked proliferation of fibroblasts. In contrast, overexpression of the genes for human transforming growth factor (TGF)-beta 1 and for human platelet-derived growth factor (PDGF)-B caused only mild cellular infiltration in the alveoli. However, both caused marked proliferation of fibroblasts and deposition of collagen fibrils. Introducing an expression vector that coded for a mutant form of the PDGF beta receptor that lacks its cytoplasmic domain markedly alleviated the pathohistologic changes caused by bleomycin in murine lungs. These findings show that TGF- and PDGF-B may be closely related to fibrosis in the lung, and that artificial regulation of them may be effective for treatment of lung fibrosis.

Topics: Animals; Humans; Interleukin-6; Mice; Platelet-Derived Growth Factor; Pulmonary Fibrosis; Rats; Rats, Wistar; Receptors, Cytokine; Recombination, Genetic; Transforming Growth Factor beta

Fibrotic process affecting the lung and other tissues is characterized by stimulation of fibroblast proliferation and connective tissue deposition. Conventional therapy consisting of glucocorticoids or cytotoxic agents is usually ineffective in blocking progression of disease. Potential new therapies have emerged from the use of animal models of pulmonary fibrosis and recent advances in the cellular and molecular biology of inflammatory reactions. Such therapies involve the use of substances directed against the action of certain growth factors, cytokines, or oxidants that are elaborated during the fibrotic reaction. In this article, we review possible therapeutic applications of these advances.

Topics: Angiotensin-Converting Enzyme Inhibitors; Animals; Antioxidants; Collagen; Disease Models, Animal; Granulocyte-Macrophage Colony-Stimulating Factor; Humans; Interleukin-1; Pulmonary Fibrosis; Reactive Oxygen Species; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha

Topics: Animals; Cytokines; Fibrosis; Humans; Kidney; Liver Cirrhosis; Pulmonary Fibrosis; Transforming Growth Factor beta; Wound Healing

Chronically inflamed and fibrotic tissue of the respiratory tract can be shown to actively express the genes and products of a number of powerful growth and differentiating factors. The initial activation of lung inflammatory cells, including alveolar macrophages, is presumed to result in the release of early acting cytokines such as IL-1 and TNF. Subsequent activation and possible phenotype alteration of the structural cells results in release of other growth factors and accumulation of blood derived inflammatory cells. These cells, once they have entered the tissue and become further activated, may begin to release their own autocrine factors and "feed back" some of the similar signals to the tissue cells in a paracrine manner, further inducing differentiation and phenotype change. These internal tissue cell and cytokine cascades could account for the chronic nature of the inflammation. Therapeutic intervention must therefore take into account the inflammatory component as well as the nature of the cytokines and structural cells involved in the propagation of the disease.

Topics: Cytokines; Granulocyte-Macrophage Colony-Stimulating Factor; Humans; Platelet-Derived Growth Factor; Pulmonary Fibrosis; Transforming Growth Factor beta

Pulmonary fibrosis is an irreversible accumulation of connective tissue in the interstitium of the lung. The pathogenesis of pulmonary fibrosis is not well understood. Research on animal models and studies of human lung disease suggest the initiating events may be a combination of pulmonary injury and the recruitment of inflammatory cells, mainly macrophages. A number of well characterized cytokines, including TGF-beta, have been either found in the injured lung or produced by inflammatory cells removed from the lung. In an animal model of pulmonary fibrosis, TGF-beta production is increased prior to collagen synthesis and is mainly produced by alveolar macrophages. In advanced idiopathic pulmonary fibrosis, a human fibrotic lung disease, extensive TGF-beta deposition can be detected by immunohistochemical staining, primarily in epithelial cells in areas of lung regeneration and remodelling. This suggests that the pathogenesis of the progressive fibrosis characteristic of this lung disease may be an aberrant repair process.

Topics: Bleomycin; Blood Platelets; Chemotaxis; Extracellular Matrix Proteins; Fibroblasts; Fibrosis; Humans; Lung; Macrophages; Models, Biological; Pulmonary Fibrosis; Transforming Growth Factor beta; Wound Healing

Topics: Animals; Extracellular Matrix; Granulation Tissue; Humans; Inflammation; Pulmonary Fibrosis; Transforming Growth Factor beta; Wound Healing

Topics: Animals; Antineoplastic Agents; Bleomycin; Cyclophosphamide; DNA Damage; Fibronectins; Lung; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Procollagen; Pulmonary Fibrosis; RNA, Messenger; Transforming Growth Factor beta

Pulmonary fibrosis (PF) is considered as an end stage for many lung diseases. Mesenchymal stem cells (MSC) as regenerative therapy have become a remarkably valuable therapeutic strategy in different diseases. Hydrogen sulfide has been recently introduced into the medical field for its antifibrotic properties in addition to enhancement of MSC stemness and function. The aim of the present study was to investigate the ability of BM-MSC in combination with NaHS to attenuate Bleomycin induced pulmonary fibrosis was studied in rats. A special emphasis was given to miR-21 and GAS5 as important players in the development of PF.. PF was induced in 32 Wistar male rats by single endotracheal injection of bleomycin, those were randomly divided into four groups (8 rats each): (untreated PF group) - (PF + MSC) treated group- (PF + NaHS treated group) - PF + combined (NAHS + MSC) treated group.. Induction of PF was associated with increased miR-21 and decreased lncRNA-GAS5 expression. Treatment with either NaHS or BM-MSC leads to an inhibitory effect on pulmonary fibrosis as evidenced by improvement of histopathological studies, pulmonary function tests, reduction of inflammatory and fibrotic markers like Hydroxyproline, TNF α, TGF-β and caspase -3 together with downregulation miR-21 and increase lncRNA-GAS5 expression.. The current work revealed the inhibitory effect of combined NaHS and BM-MSC on pulmonary fibrosis with concomitant modulation of miR-21 and lncRNA-GAS5 expression.

Topics: Animals; Bleomycin; Bone Marrow; Caspases; Hydrogen Sulfide; Hydroxyproline; Male; Mesenchymal Stem Cells; MicroRNAs; Pulmonary Fibrosis; Rats; Rats, Wistar; RNA, Long Noncoding; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha

Pathological fibrosis is driven by a feedback loop in which the fibrotic extracellular matrix is both a cause and consequence of fibroblast activation. However, the molecular mechanisms underlying this process remain poorly understood. Here we identify yes-associated protein (YAP) (homolog of drosophila Yki) and transcriptional coactivator with PDZ-binding motif (TAZ) (also known as Wwtr1), transcriptional effectors of the Hippo pathway, as key matrix stiffness-regulated coordinators of fibroblast activation and matrix synthesis. YAP and TAZ are prominently expressed in fibrotic but not healthy lung tissue, with particularly pronounced nuclear expression of TAZ in spindle-shaped fibroblastic cells. In culture, both YAP and TAZ accumulate in the nuclei of fibroblasts grown on pathologically stiff matrices but not physiologically compliant matrices. Knockdown of YAP and TAZ together in vitro attenuates key fibroblast functions, including matrix synthesis, contraction, and proliferation, and does so exclusively on pathologically stiff matrices. Profibrotic effects of YAP and TAZ operate, in part, through their transcriptional target plasminogen activator inhibitor-1, which is regulated by matrix stiffness independent of transforming growth factor-β signaling. Immortalized fibroblasts conditionally expressing active YAP or TAZ mutant proteins overcome soft matrix limitations on growth and promote fibrosis when adoptively transferred to the murine lung, demonstrating the ability of fibroblast YAP/TAZ activation to drive a profibrotic response in vivo. Together, these results identify YAP and TAZ as mechanoactivated coordinators of the matrix-driven feedback loop that amplifies and sustains fibrosis.

Topics: Adaptor Proteins, Signal Transducing; Animals; Cell Cycle Proteins; Female; Fibroblasts; Gene Knockdown Techniques; Humans; Intracellular Signaling Peptides and Proteins; Lung; Male; Mechanotransduction, Cellular; Mice; Mice, Inbred NOD; Mutation; NIH 3T3 Cells; Phosphoproteins; Plasminogen Activator Inhibitor 1; Pulmonary Fibrosis; Serpin E2; Trans-Activators; Transcription Factors; Transcriptional Coactivator with PDZ-Binding Motif Proteins; Transforming Growth Factor beta; YAP-Signaling Proteins

Idiopathic pulmonary fibrosis (IPF) is a deadly disease, largely unresponsive to treatment with corticosteroids and immunosuppressives. The aim of this randomized, prospective, open-label study was to characterize the molecular effects of IFN-gamma-1b and colchicine, on biomarkers expression associated with fibrosis (TGF-beta, CTGF) and immunomodulatory/antimicrobial activity (IFN-gamma), in the lungs of patients with IPF. Fourteen (14) patients with an established diagnosis of IPF received either 200 microg of IFN-gamma-1b subcutaneously three times per week, or 1mg of oral colchicine per day, for 24 months. Using RT-PCR assay, we evaluated the transcription levels of transforming growth factor beta1 (TGF-beta1), connective-tissue growth factor (CTGF), and interferon-gamma (IFN-gamma) genes in lung tissue before and after treatment with IFN-gamma-1b or colchicine. Marked mRNA expression of TGF-beta1 and CTGF, but complete lack of interferon-gamma was detected in fibrotic lung tissue at entry. After treatment, both groups exhibited increased expression of IFN-gamma gene at 6 months that was sustained at 24 months. The expression of CTGF and TGF-beta1 remained almost stable before and after treatment, in the IFN-gamma-1b group, while TGF-beta1 was statistically decreased after therapy, in the colchicine group (p=0.0002). Significant difference in DLCO (% pred), was found between the two treatment groups in favor of IFN-gamma-1b group (p=0.04). In addition, the IFN-gamma-1b group showed stability in arterial PO2 while the colchicine group significantly deteriorated (p=0.02). In conclusion, we report the effect of antifibrotic agents (IFN-gamma-1b and colchicine) in TGF-beta, CTGF, and endogenous IFN-gamma gene expression, in human fibrosis. However, extended studies are needed to verify the pathophysiological consequences of these findings.

Topics: Aged; Aged, 80 and over; Antineoplastic Agents; Colchicine; Connective Tissue Growth Factor; Female; Humans; Immediate-Early Proteins; Intercellular Signaling Peptides and Proteins; Interferon-gamma; Male; Middle Aged; Prednisolone; Prospective Studies; Pulmonary Fibrosis; Recombinant Proteins; Respiratory Function Tests; Transforming Growth Factor beta; Tubulin Modulators

Patients with idiopathic pulmonary fibrosis have progressive scarring of the lung and usually die within four to five years after symptoms develop. Treatment with oral glucocorticoids is often ineffective. We conducted an open, randomized trial of treatment with a combination of interferon gamma-1b, which has antifibrotic properties, and an oral glucocorticoid. We studied 18 patients with idiopathic pulmonary fibrosis who had not had responses to glucocorticoids or other immunosuppressive agents. Nine patients were treated for 12 months with oral prednisolone alone (7.5 mg daily, which could be increased to 25 to 50 mg daily), and nine with a combination of 200 microg of interferon gamma-1b (given three times per week subcutaneously) and 7.5 mg of prednisolone (given once a day).. All the patients completed the study. Lung function deteriorated in all nine patients in the group given prednisolone alone: total lung capacity decreased from a mean (+/-SD) of 66+/-8 percent of the predicted value at base line to 62+/-6 percent at 12 months. In contrast, in the group receiving interferon gamma-1b plus prednisolone, total lung capacity increased (from 70+/-6 percent of the predicted value at base line to 79+/-12 percent at 12 months, P<0.001 for the difference between the groups). In the group that received interferon gamma-1b plus prednisolone, the partial pressure of arterial oxygen at rest increased from 65+/-9 mm Hg at base line to 76+/-8 mm Hg at 12 months, whereas in the group that received prednisolone alone it decreased from 65+/-6 to 62+/-4 mm Hg (P<0.001 for the difference in the change from baseline values between the two groups); on maximal exertion, the value increased from 55+/-6 to 65+/-8 mm Hg in the group that received combined treatment and decreased from 55+/-6 mm Hg to 52+/-5 mm Hg in the group given prednisolone alone (P<0.001). The side effects of interferon gamma-1b, such as fever, chills, and muscle pain, subsided within the first 9 to 12 weeks.. In a preliminary study, 12 months of treatment with interferon gamma-1b plus prednisolone was associated with substantial improvements in the condition of patients with idiopathic pulmonary fibrosis who had had no response to glucocorticoids.

Topics: Carrier Proteins; Connective Tissue Growth Factor; Drug Therapy, Combination; Female; Glucocorticoids; Growth Substances; Humans; Immediate-Early Proteins; Intercellular Signaling Peptides and Proteins; Interferon-gamma; Male; Middle Aged; Prednisolone; Pulmonary Fibrosis; Pulmonary Gas Exchange; Recombinant Proteins; Total Lung Capacity; Transcription, Genetic; Transforming Growth Factor beta

Bronchiolitis obliterans syndrome (BOS) develops in one-third of lung transplant recipients. A fibroproliferative process involving mesenchymal cells is observed histopathologically. In order further to evaluate the pathomechanisms of BOS, the gene expression of platelet-derived growth factor (PDGF)-B and transforming growth factor (TGF)-beta 1 in bronchoalveolar lavage (BAL) cells of six lung transplant recipients and appropriate controls was studied. Equal amounts of total RNA were submitted to semiquantitative reverse transcription/polymerase chain reaction (RT-PCR), amplifying actin, PDGF-B and TGF-beta 1 using established protocols and primer sets. The signal/actin ratio was calculated based on laser densitometry measurements. TGF-beta 1 transcripts were detected in all samples, and a slight increase in BOS patients was observed. PDGF-B mRNA was increased in BAL samples from BOS patients compared to unaffected recipients and controls. Plotting the FEV1 in percent of vital capacity and the PDGF expression in BOS patients revealed an increased PDGF signal preceding lung function deterioration. The data were consistent with the hypothesis based mainly on in vitro findings that PDGF and TGF-beta contribute to the development of BOS.

Topics: Adult; Bronchiolitis Obliterans; Bronchoalveolar Lavage Fluid; Bronchoscopy; Cytomegalovirus Infections; Female; Gene Expression Regulation; Graft vs Host Reaction; Humans; Leukocyte Count; Lung Transplantation; Male; Middle Aged; Platelet-Derived Growth Factor; Polymerase Chain Reaction; Pulmonary Fibrosis; Respiratory Distress Syndrome; Respiratory Function Tests; RNA, Messenger; Transcription, Genetic; Transforming Growth Factor beta

Transforming growth factor-beta (TGF-beta) is a cytokine that promotes extracellular matrix accumulation and inhibits matrix degradation. Although the natural course of sarcoidosis is usually favourable, granuloma healing in the lung may result in pulmonary fibrosis and respiratory impairment in some patients. In this study TGF-beta1 was evaluated in bronchoalveolar lavage (BAL) fluid and culture supernatants of alveolar macrophages (AM) from 73 patients with biopsy-proven sarcoidosis. Disease activity was defined when patients recently developed or increased symptoms (cough, dyspnoea, systemic symptoms) and/or demonstrated increasing opacities on chest radiography. Pulmonary function tests were performed in all patients including forced expiratory volume in one second (FEV1), forced vital capacity (FVC), total lung capacity (TLC) and the diffusing capacity of the lung for carbon monoxide (DL,CO). Fourteen patients with idiopathic pulmonary fibrosis (IPF) and 14 healthy subjects were investigated as a control group. Immunohistochemistry was used to evaluate the cell distribution of TGF-beta1 on lung specimens. TGF-beta1 levels in BAL and in AM supernatants were not different between sarcoidosis and healthy subjects, whereas they were markedly increased in IPF. However, the TGF-beta1 level was significantly increased in BAL fluid but not in AM supernatants from sarcoidosis with altered lung function, compared with patients with normal lung function. The TGF-beta1 level in BAL was increased in active sarcoidosis but this increased level was mainly related to the higher level observed in patients with altered lung function. TGF-beta1 levels in BAL correlated significantly with the lymphocyte percentage. TGF-beta1 staining assessed by immunohistochemistry was intense in epithelioid histiocytes comprising non-necrotizing granuloma and in bronchiolar epithelial cells, in hyperplastic type II pneumocytes and occasionally in AM. This study supports the hypothesis that overproduction of transforming growth factor-beta1 is associated with functional impairment in patients with pulmonary sarcoidosis.

Topics: Adult; Analysis of Variance; Biopsy, Needle; Bronchoalveolar Lavage Fluid; Cells, Cultured; Female; Humans; Immunohistochemistry; Logistic Models; Macrophages, Alveolar; Male; Middle Aged; Prospective Studies; Pulmonary Fibrosis; Reference Values; Respiratory Function Tests; Sarcoidosis, Pulmonary; Transforming Growth Factor beta

Tamoxifen is an anti-estrogen with proven efficacy and low toxicity in the treatment of breast cancer. However, tamoxifen has been shown to exert a number of nonhormonal as well as hormonal effects. One nonhormonal effect of tamoxifen is the induction of transforming growth factor-beta (TGF-beta) secretion. TGF-beta has been implicated in the pathogenesis of radiation-induced fibrosis.. We investigated the development of lung fibrosis in breast cancer patients who were treated after mastectomy with radiotherapy, with or without simultaneous adjuvant treatment with tamoxifen.. Data from 196 women were included in the analysis. Eighty-four women were postmenopausal patients who participated in a randomized trial testing tamoxifen as an adjuvant to postmastectomy radiotherapy. The radiotherapy technique employed an 8-MV photon field covering the axillary and the infraclavicular and supraclavicular regions of the affected side of the chest; the chest wall was treated with an abutted electron field. Optical density changes in pretreatment and post-treatment chest x-ray films were used to monitor the development of lung fibrosis; lung reactions were assessed in the photon-irradiated field only. Logistic regression analysis was used to explore relationships between radiation dose and the development of lung fibrosis in patients either receiving or not receiving tamoxifen. All P values are from two-sided tests.. Among the 84 women who participated in the randomized trial of radiotherapy plus tamoxifen (n = 38) versus radiotherapy alone (n = 46), there was a significant association between tamoxifen treatment and the incidence of marked lung fibrosis (relative risk = 2.0; 95% confidence interval [CI] = 1.2-3.5; P = .01). When logistic regression analysis was used to evaluate data from all 196 patients, a highly significant relationship was found between the incidence of lung fibrosis and total radiation dose (P = .0005). In the full analysis, an increased risk of marked lung fibrosis was found again for patients who received tamoxifen simultaneously with radiotherapy (with patients receiving radiotherapy alone as the referent, odds ratio = 2.9; 95% CI = 1.3-6.3; P = .007). Patient age and menopausal status did not significantly influence the results.. Tamoxifen treatment during postmastectomy radiotherapy enhances the risk of radiation-induced lung fibrosis.. In view of pre-existing data, we hypothesize that tamoxifen mediates the enhancement of radiation-induced lung fibrosis through the induction of TGF-beta secretion. If this hypothesis is correct, new strategies might be devised for preventing or reducing radiation-induced fibrosis. Because we studied a relatively small portion of the irradiated lung, we cannot recommend changes in current therapeutic measures; however, we strongly encourage additional studies of lung fibrosis in patients receiving tamoxifen and radiotherapy.

Topics: Aged; Antineoplastic Agents, Hormonal; Breast Neoplasms; Chemotherapy, Adjuvant; Female; Humans; Mastectomy; Middle Aged; Pulmonary Fibrosis; Radiotherapy Dosage; Radiotherapy, Adjuvant; Tamoxifen; Transforming Growth Factor beta

Pulmonary injury and fibrosis can be caused by various factors because of their inflammatory nature, both can lead to serious clinical consequences. Inula japonica Thunb. is used in traditional Chinese medicine for the treatment of lung diseases. However, the effect and mechanism of action of the essential oil of I. japonica (EOI) on pulmonary injury and fibrosis are not well understood.. To investigate the therapeutic effects of EOI on mice with bleomycin (BLM)-induced acute pulmonary injury and chronic fibrosis formation, as well as its potential mechanism.. A short-term mouse model of pulmonary injury was established by intratracheal injection of BLM to investigate the anti-inflammatory effect of EOI, and a long-term model of pulmonary fibrosis was used to explore the anti-fibrosis effect of EOI. High-dose EOI (200 mg/kg) was administered intragastrically, and low-dose (50 mg/kg) was administered by intratracheal injection. Gas chromatography-mass spectrometry (GC-MS) was used to identify the ingredients in EOI, and high-performance liquid chromatography (HPLC) was performed for the preparation of EOI compounds. Western blot and real-time qPCR were used to verify the effects of EOI and its active composition on inflammation, oxidative stress and fibrosis signaling pathway.. Treatment with EOI significantly reduced the inflammation and oxidative stress by reducing the levels of inflammatory and oxidative cytokines such as tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and malondialdehyde in BLM-treated mice with acute pulmonary injury. EOI treatment could also suppress the formation of fibrous tissue in mice with BLM-induced pulmonary fibrosis through inhibiting TGF-β/Smad and PI3K/Akt pathways. Chromatographic analysis and preparation suggested that fatty acid and phenol derivatives are present in EOI. Based on cellular inflammation and fibrosis models, the phenolic compounds in EOI can represent the anti-inflammatory and anti-fibrotic effects of EOI by regulating pro-inflammatory and pro-fibrotic cytokines such as NO, TNF-α, IL-6, TGF-β1, and α-SMA.. EOI ameliorated BLM-induced pulmonary injury and fibrosis in mice by inhibiting the inflammatory response and regulating the redox equilibrium, as well as by mediating TGFβ/Smad and PI3K/Akt, which suggested that EOI has potential to treat pulmonary diseases.

Topics: Animals; Anti-Inflammatory Agents; Bleomycin; Cytokines; Fibrosis; Inflammation; Interleukin-6; Inula; Lung; Lung Injury; Mice; Oils, Volatile; Phenols; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; Pulmonary Fibrosis; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha

Pulmonary fibrosis is characterized by damage to the epithelial cells and alveolar-capillary basement membrane. The increased expression levels of transforming growth factor β (TGF-β) and TGF-β-receptor-1 induced differentiation of lung fibroblasts to myofibroblasts, an alarming sign and considered the hallmark event development of pulmonary fibrosis. In the current study, the stability of phytochemicals of

Topics: Curcuma; Molecular Docking Simulation; Pulmonary Fibrosis; Tinospora; Transforming Growth Factor beta

Lung fibrosis is the leading cause of death in SSc, with no cure currently available. Antifibrotic Endostatin (ES) production does not reach therapeutic levels in SSc patients, suggesting a deficit in its release from Collagen XVIII by the main cleavage enzyme, Cathepsin L (CTSL). Thus, elucidating a potential deficit in CTSL expression and activity unravels an underlying molecular cause for SSc-driven lung fibrosis.. Fibrosis was induced experimentally using TGF-β in vitro, in primary human lung fibroblasts (pLFs), and ex vivo, in human lung tissues. ES and CTSL expression was quantified using ELISA, RT-qPCR, immunoblotting or immunofluorescence. Recombinant NC1-FLAG peptide was used to assess CTSL cleavage activity. CTSL expression was also compared between SSc vs normal (NL)-derived pLFs and lung tissues.. ES levels were significantly reduced in media conditioned by TGF-β-induced pLFs. TGF-β-stimulated pLFs significantly reduced expression and secretion of CTSL into the extracellular matrix (ECM). CTSL was also sequestered in its inactive form into extracellular vesicles, further reducing its availability in the ECM. Media conditioned by TGF-β-induced pLFs showed reduced cleavage of NC1-Flag and reduced release of the antifibrotic ES fragment. SSc-derived pLFs and lung tissues expressed significantly lower levels of CTSL compared with NL.. Our findings identify CTSL as a protein protective against lung fibrosis via its activation of antifibrotic ES, and whose expression in SSc pLFs and lung tissues is suppressed. Identifying strategies to boost CTSL endogenous levels in SSc patients could serve as a viable therapeutic strategy.

Topics: Cathepsin L; Cells, Cultured; Fibroblasts; Fibrosis; Humans; Pulmonary Fibrosis; Scleroderma, Systemic; Skin; Transforming Growth Factor beta

Pulmonary fibrosis involves various types of immune cells and soluble mediators, including TGF-β and IL-35, a recently identified heterodimeric cytokine that belongs to the IL-12 cytokine family. However, the effect of regulatory IL-35 may play an important role in fibrotic diseases. The aim of this paper is to explore the immunoregulatory role of IL-35 in the development of fibrosis in interstitial lung disease (ILD). To gain a better understanding of this issue, the concentrations of IL-35 and different profibrotic cytokines in fibrotic (F-ILD) and non-fibrotic (NF-ILD) patients by ELISA were compared to that of intracellular IL-35 and IL-17 on CD4+ T cells stimulated in the presence of BAL or with different ratios of recombinant IL-35 (rIL-35) and TGF-β (rTGF-β), which were evaluated by flow cytometry. We observed that BAL concentration of IL-35 was lower in F patients (p < 0.001) and was negatively correlated with concentrations of TGF-β (p < 0.001) and IL-17 (p < 0.001). In supplemented cell cultures, BAL from NF but not F patients enhanced the percentage of IL-35 + CD4+ T (p < 0.001) cells and decreased the percentage of IL-17 + CD4+ T cells (p < 0.001). The percentage of IL-35 + CD4+ T cells correlated positively with BAL concentration of IL-35 (p = 0.02), but correlated negatively with BAL concentrations of IL-17 (p = 0.007) and TGF-β (p = 0.01). After adjusting the concentrations of recombinant cytokines to establish a TGF-β: IL-35 ratio of 1:4, an enhanced percentage of IL-35 + CD4+ T cells (p < 0.001) but a decreased percentage of IL-17 + CD4+ T cells (p < 0.001) was observed. After adding recombinant IL-35 to the BAL from F patients until a 1:4 ratio of TGF-β: IL-35 was reached, a significantly increased percentage of IL-35 + CD4+ T cells (p < 0.001) and a decreased percentage of IL-17 + CD4+ T cells (p = 0.003) was found. These results suggest that IL-35 may induce an anti-fibrotic response, regulating the effect of TGF-β and the inflammatory response on CD4+ T cells. In addition, the TGF-β: IL-35 ratio in BAL has been shown to be a potential biomarker to predict the outcome of F patients with ILD.

Topics: Bronchoalveolar Lavage Fluid; Cytokines; Humans; Interleukin-17; Lung Diseases, Interstitial; Pulmonary Fibrosis; Transforming Growth Factor beta

Idiopathic pulmonary fibrosis is a terminal lung ailment that shares several pathological and genetic mechanisms with severe COVID-19. Thymol (THY) is a dietary compound found in thyme species that showed therapeutic effects against various diseases. However, the effect of THY against bleomycin (BLM)-induced lung fibrosis was not previously investigated. The current study investigated the ability of THY to modulate oxidative stress, inflammation, miR-29a/TGF-β expression, and PI3K/phospho-Akt signaling in lung fibrosis. Mice were divided into Normal, THY (100 mg/kg, p.o.), BLM (15 mg/kg, i.p.), BLM + THY (50 mg/kg, p.o.), and BLM + THY (100 mg/kg, p.o.) groups and treated for four weeks. The obtained results showed that BLM + THY (50 mg/kg) and BLM + THY (100 mg/kg) reduced fibrotic markers; α-SMA and fibronectin, inflammatory mediators; TNF-α, IL-1β, IL-6, and NF-kB and oxidative stress biomarkers; MDA, GSH, and SOD, relative to BLM group. Lung histopathological examination by H&E and Masson's trichrome stains confirmed the obtained results. Remarkably, expression levels of TGF-β, PI3K, and phospho-Akt were decreased while miR-29a expression was elevated. In conclusion, THY effectively prevented BLM-induced pulmonary fibrosis by exerting significant anti-oxidant and anti-inflammatory effects. Our novel findings that THY upregulated lung miR-29a expression while decreased TGF-β and PI3K/Akt signaling are worthy of further investigation as a possible molecular mechanism for THY's anti-fibrotic actions.

Topics: Animals; Bleomycin; COVID-19; Fibrosis; Inflammation; Lung; Mice; MicroRNAs; Oxidative Stress; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; Pulmonary Fibrosis; Thymol; Transforming Growth Factor beta

Yangqing Chenfei formula (YCF) has been demonstrated its clinical efficiency on silicosis patients. However, the effect of YCF against silicotic fibrosis and its mechanism remain unclear.. This study is aimed to investigate active compounds and molecular mechanism of YCF in treating silicosis.. YCF was orally administrated to silicosis rats induced by crystalline silica. The effective fraction of YCF and the compounds was isolated and identified by using macroporous resin and HPLC-MS, respectively. The targets and potential molecular mechanism of YCF against silicotic fibrosis were investigated through pharmacological network and RNA-sequencing analysis and in vitro-experimental validation.. YCF could remarkably improve the lung function and pathological changes of silicotic rats, reduce the aggregation of fibrocytes and deposition of ECM, such as collagen I, III, FN, and α-SMA, and suppress the TGF-β/Smad3 signaling. Furthermore, YCF6, the effective fraction derived from YCF, could significantly inhibit fibroblast activation induced by TGF-β. Then, 135 compounds were identified from YCF6 by using HPLC-MS, and Network pharmacology analysis predicted total 941 targets for these compounds. Moreover, 409 differentially expressed genes of fibroblast activation induced by TGF-β were identified. Then, integrated analysis of the 941 targets with 409 differentially expressed genes showed that YCF6 contains multiple compounds, such as tangeretin, L-Malic acid, 2-Monolinolein etc., which inhibits fibroblast activation probably by targeting different proteins, such as PIK3CA, AKT1, JAK2, STAT3, GSK3β, leading to regulate the signal network, such as PI3K/AKT signaling pathway, JAK/STAT signaling pathway, and Wnt signaling pathway. Finally, in vitro experiment indicated that tangeretin, the active compound contained in YCF6, could significantly inhibit TGF-β induced fibroblast activation. Moreover, YCF6 and tangeretin could markedly inhibit the activation of PI3K/AKT, JAK/STAT, and Wnt pathway.. YCF contained multiple compounds and targeted various proteins that regulated the fibroblast activation, which might be the molecular mechanisms of it in treating silicosis.

Topics: Animals; Fibroblasts; Fibrosis; Janus Kinases; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; Pulmonary Fibrosis; Rats; Silicon Dioxide; Silicosis; STAT Transcription Factors; Transforming Growth Factor beta; Wnt Signaling Pathway

To evaluate the effects of the Qingwen Gupi decoction (QGT) in a rat model of bleomycin-induced pulmonary fibrosis (PF), and explore the underlying mechanisms by integrating UPLC-Q-TOF/MS metabolomics and 16S rDNA sequencing of gut microbiota.. The animals were randomly divided into the control, PF model, pirfenidone-treated, and low-, medium-, and high-dose QGT groups. The lung tissues were examined and the expression of TGF-β, SMAD-3, and SMAD-7 mRNAs in the lung tissues were analyzed. Metabolomic profiles were analyzed by UPLC-QTOF/MS, and the intestinal flora were examined by prokaryotic 16 rDNA sequencing. Pathological examination and biochemical indices revealed that QGT treatment improved the symptoms of PF by varying degrees. Furthermore, QGT significantly downregulated TGF-β1 and Smad-3 mRNAs and increased the expression levels of Smad-7. QGT-L in particular increased the levels of 18 key metabolic biomarkers that were associated with nine gut microbial species and may exert antifibrosis effects through arachidonic acid metabolism, glycerophospholipid metabolism, and phenylalanine metabolism.. QGT alleviated PF in a rat model through its anti-inflammatory, antioxidant, and anti-fibrotic effects, and by reversing bleomycin-induced gut dysbiosis.This study lays the foundation for further research on the pathological mechanisms of PF and the development of new drug candidates.

Topics: Animals; Bleomycin; Gastrointestinal Microbiome; Lung; Metabolomics; Pulmonary Fibrosis; Rats; Transforming Growth Factor beta

Pulmonary fibrosis (PF) is a progressive and fatal interstitial lung disease with limited therapeutic options at present, and epithelial-mesenchymal transition (EMT) is recognized as a major cause of lung fibrosis. Our previous work has confirmed that total extract of Anemarrhena asphodeloides Bunge [Asparagaceae] exerted the effect of anti-PF. As a main constituent of Anemarrhena asphodeloides Bunge [Asparagaceae], the effect of timosaponin BII (TS BII) on drug-induced EMT process in PF animals and alveolar epithelial cells remains unknown. In this study, we evaluated the effect of TS BII on bleomycin (BLM)-induced PF. The results showed that TS BII could restore the structure of lung architecture and MMP-9/TIMP-1 balance in fibrotic rat lung and inhibit collagen deposition. Moreover, we found that TS BII could reverse the abnormal expression of TGF-β1 and EMT-related marker proteins including E-cadherin, vimentin, and α-SMA. Besides, aberrant TGF-β1 expression and phosphorylation of Smad2 and Smad3 in BLM-induced animal model and TGF-β1-induced cell model were downregulated by TS BII treatment, indicating that EMT in fibrosis was suppressed by inhibition of TGF-β/Smad pathway both in vivo and in vitro. In summary, our study suggested that TS BII could be a promising candidate for PF treatment.

Topics: Animals; Bleomycin; Epithelial-Mesenchymal Transition; Fibrosis; Lung; Pulmonary Fibrosis; Rats; Transforming Growth Factor beta; Transforming Growth Factor beta1

Pulmonary fibrosis (PF) is a type of fatal respiratory diseases with limited therapeutic options and poor prognosis. The chemokine CCL17 plays crucial roles in the pathogenesis of immune diseases. Bronchoalveolar lavage fluid (BALF) CCL17 levels are significantly higher in patients with idiopathic PF (IPF) than in healthy volunteers. However, the source and function of CCL17 in PF remain unclear. Here, we demonstrated that the levels of CCL17 were increased in the lungs of IPF patients and mice with bleomycin (BLM)-induced PF. In particular, CCL17 were upregulated in alveolar macrophages (AMs) and antibody blockade of CCL17 protected mice against BLM-induced fibrosis and significantly reduced fibroblast activation. Mechanistic studies revealed that CCL17 interacted with its receptor CCR4 on fibroblasts, thereby activating the TGF-β/Smad signaling pathway to promote fibroblast activation and tissue fibrosis. Moreover, the knockdown of CCR4 by CCR4-siRNA or blockade by CCR4 antagonist C-021 was able to ameliorate PF pathology in mice. In summary, the CCL17-CCR4 axis is involved in the progression of PF, and targeting of CCL17 or CCR4 inhibits fibroblast activation and tissue fibrosis and may benefit patients with fibroproliferative lung diseases.

Topics: Animals; Bleomycin; Chemokine CCL17; Fibroblasts; Lung; Mice; Mice, Inbred C57BL; Pulmonary Fibrosis; Smad Proteins; Transforming Growth Factor beta

Pulmonary fibrosis (PF) is a progressive pulmonary disease with no effective treatment and high mortality. Resveratrol has shown promising benefits in the treatment of PF. However, the probable efficacy and underlying mechanism of resveratrol in PF treatment remain unclear. This study investigates the intervention effects and potential mechanisms underpinning the treatment of PF with resveratrol. The histopathological analysis of lung tissues in PF rats showed that resveratrol improved collagen deposition and reduced inflammation. Resveratrol decreased the levels of collagen, glutathione, superoxide dismutase, myeloperoxidase, and hydroxyproline, lowered total anti-oxidant capacity, and suppressed the migration of TGF-[Formula: see text]1 and LPS-induced 3T6 fibroblasts. With resveratrol intervention, the protein and RNA expressions of TGF-[Formula: see text]1, a-SMA, Smad3/4, p-Smad3/4, CTGF, and p-ERK1/2 were markedly downregulated. Similarly, the protein and RNA expression levels of Col-1 and Col-3 were significantly downregulated. However, Smad7 and ERK1/2 were evidently upregulated. The protein and mRNA expression levels of TGF-[Formula: see text], Smad, and p-ERK correlated positively with the lung index, while the protein and mRNA expression levels of ERK correlated negatively with the lung index. These results reveal that resveratrol may have therapeutic effects on PF by reducing collagen deposition, oxidation, and inflammation. The mechanism is associated with the regulation of the TGF-[Formula: see text]/Smad/ERK signaling pathway.

Topics: Animals; Inflammation; Pulmonary Fibrosis; Rats; Resveratrol; RNA; RNA, Messenger; Signal Transduction; Transforming Growth Factor beta

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection continues to pose threats to public health. The clinical manifestations of lung pathology in COVID-19 patients include sustained inflammation and pulmonary fibrosis. The macrocyclic diterpenoid ovatodiolide (OVA) has been reported to have anti-inflammatory, anti-cancer, anti-allergic, and analgesic activities. Here, we investigated the pharmacological mechanism of OVA in suppressing SARS-CoV-2 infection and pulmonary fibrosis in vitro and in vivo. Our results revealed that OVA was an effective SARS-CoV-2 3CLpro inhibitor and showed remarkable inhibitory activity against SARS-CoV-2 infection. On the other hand, OVA ameliorated pulmonary fibrosis in bleomycin (BLM)-induced mice, reducing inflammatory cell infiltration and collagen deposition in the lung. OVA decreased the levels of pulmonary hydroxyproline and myeloperoxidase, as well as lung and serum TNF-ɑ, IL-1β, IL-6, and TGF-β in BLM-induced pulmonary fibrotic mice. Meanwhile, OVA reduced the migration and fibroblast-to-myofibroblast conversion of TGF-β1-induced fibrotic human lung fibroblasts. Consistently, OVA downregulated TGF-β/TβRs signaling. In computational analysis, OVA resembles the chemical structures of the kinase inhibitors TβRI and TβRII and was shown to interact with the key pharmacophores and putative ATP-binding domains of TβRI and TβRII, showing the potential of OVA as an inhibitor of TβRI and TβRII kinase. In conclusion, the dual function of OVA highlights its potential for not only fighting SARS-CoV-2 infection but also managing injury-induced pulmonary fibrosis.

Topics: Animals; Bleomycin; COVID-19; Diterpenes; Fibroblasts; Humans; Lung; Mice; Pulmonary Fibrosis; SARS-CoV-2; Signal Transduction; Transforming Growth Factor beta; Transforming Growth Factor beta1

Topics: Electron Transport; Humans; Pulmonary Fibrosis; Ribosomes; Transforming Growth Factor beta

Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive disorder that severely impairs lung function, by increasing lung stiffness. Sesamol, a phenolic Phyto-molecule isolated from sesame seeds, possess a rich source of protein and is known to have extensive nutritional and health effects. Here we investigated the effect of sesamol on TGF-β/periostin-induced fibroblast differentiation in in vitro and bleomycin-induced pulmonary fibrosis in an in vivo model. Our results demonstrated that activation of (DHLF, LL29, NHLF and A549) cells with TGF-β, elevates the epithelial to mesenchymal transition, extracellular matrix, and collagen deposition and periostin signaling marker's expression, further treatment with sesamol attenuated these markers significantly. In addition, sesamol treatment improved the TGF-β-induced contraction and migration of cells. Mechanistic studies showed that activation of IPF cells with periostin increased the TGF-β signaling and treatment with sesamol significantly abrogated the periostin-induced TGF-β activation and its downstream fibrotic marker's expression. In in vivo, sesamol treatment attenuated the lung inflammation, infiltration of cells, wall thickening and the formation of fibrous bands significantly in BLM-induced fibrosis rats. Molecular studies revealed that sesamol treatment reduced the bleomycin-induced fibrotic, inflammatory, apoptotic marker's expression by modulating the TGF-β/periostin crosstalk signaling in a dose-dependent manner. Further, treatment with sesamol dramatically improved lung function and decreased mortality. Our study first time reports the sesamol's inhibitory effects on periostin signalling. Collectively, our study demonstrated that periostin and TGF-β seem to work in a positive-feedback loop, inducing the other, therefore, targeting TGF-β/periostin signaling may provide a better therapeutic approach against IPF and other fibrotic disorders.

Topics: Animals; Bleomycin; Epithelial-Mesenchymal Transition; Fibroblasts; Lung; Pulmonary Fibrosis; Rats; Transforming Growth Factor beta

The pathogenesis of pulmonary fibrosis involves complex interplay between cell types and signaling pathways. Recurrent alveolar epithelial injury can occur during pulmonary inflammation, causing dysregulation of epithelial repair. Dysregulated repair interacts with mesenchymal, inflammatory, and endothelial cells to trigger fibroblast-to-myofibroblast activation. CD26/dipeptidyl peptidase-4 (DPP4) is a type II membrane protein mediating pleiotropic effect. However, the mechanistic role of CD26/DPP4 in pulmonary fibrosis remains unclear. In this study, we aimed to characterize Dpp4 deficiency in a mouse bleomycin (BLM)-induced pulmonary fibrosis model and in cell culture systems of human lung fibroblasts (HLFs). Dpp4 knockout (Dpp4 KO) mouse lungs exhibited lower Ashcroft scale indices, collagen content, and numbers of fibroblasts and myofibroblasts compared with those in C57BL/6 wild-type (WT) mice. Upregulation of Tgfb1 and Tgfb2 mRNA levels in the lungs after BLM treatment was lower in Dpp4 KO mice compared with those in WT mice. Although TGF-β-driven endothelial-to-mesenchymal transition (EndMT) has been implicated as one of the mechanisms of pulmonary fibrosis, a number of partial EndMT cells in lungs did not differ between Dpp4 KO mice and WT mice. The proliferation capacity and mRNA levels of COL1A1, a collagen deposition-related gene, in cultured HLFs were suppressed in DPP4 small interfering RNA-treated cells. This study indicates that the genetic deficiency of DPP4 has protective effects against BLM-induced pulmonary fibrosis, partly through the reduction in TGF-β expression and inhibition of fibroblast activation in the lung. Our study suggests that CD26/DPP4 inhibition is a potential therapeutic strategy for pulmonary fibrosis.

Topics: Animals; Bleomycin; Collagen; Dipeptidyl Peptidase 4; Endothelial Cells; Fibroblasts; Humans; Lung; Mice; Mice, Inbred C57BL; Mice, Knockout; Pulmonary Fibrosis; RNA, Messenger; Transforming Growth Factor beta

Pulmonary fibrosis is a destructive, progressive disease that dramatically reduces life quality of patients, ultimately leading to death. Therapeutic regimens for pulmonary fibrosis have shown limited benefits, hence justifying the efforts to evaluate the outcome of alternative treatments.. Using a mouse model of bleomycin (BLM)-induced lung fibrosis, in the current work we asked whether treatment with pro-resolution molecules, such as pro-resolving lipid mediators (SPMs) could ameliorate pulmonary fibrosis. To this end, we injected aspirin-triggered resolvin D1 (7S,8R,17R-trihydroxy-4Z,9E,11E,13Z,15E19Z-docosahexaenoic acid; ATRvD1; i.v.) 7 and 10 days after BLM (intratracheal) challenge and samples were two weeks later.. Assessment of outcome in the lung tissues revealed that ATRvD1 partially restored lung architecture, reduced leukocyte infiltration, and inhibited formation of interstitial edema. In addition, lung tissues from BLM-induced mice treated with ATRvD1 displayed reduced levels of TNF-α, MCP-1, IL-1-β, and TGF-β. Of further interest, ATRvD1 decreased lung tissue expression of MMP-9, without affecting TIMP-1. Highlighting the beneficial effects of ATRvD1, we found reduced deposition of collagen and fibronectin in the lung tissues. Congruent with the anti-fibrotic effects that ATRvD1 exerted in lung tissues, α-SMA expression was decreased, suggesting that myofibroblast differentiation was inhibited by ATRvD1. Turning to culture systems, we next showed that ATRvD1 impaired TGF-β-induced fibroblast differentiation into myofibroblast. After showing that ATRvD1 hampered extracellular vesicles (EVs) release in the supernatants from TGF-β-stimulated cultures of mouse macrophages, we verified that ATRvD1 also inhibited the release of EVs in the bronco-alveolar lavage (BAL) fluid of BLM-induced mice. Motivated by studies showing that BLM-induced lung fibrosis is linked to angiogenesis, we asked whether ATRvD1 could blunt BLM-induced angiogenesis in the hamster cheek pouch model (HCP). Indeed, our intravital microscopy studies confirmed that ATRvD1 abrogates BLM-induced angiogenesis. Collectively, our findings suggest that treatment of pulmonary fibrosis patients with ATRvD1 deserves to be explored as a therapeutic option in the clinical setting.

Topics: Aspirin; Bleomycin; Docosahexaenoic Acids; Humans; Lung; Pulmonary Fibrosis; Transforming Growth Factor beta

Fibrosing pneumonia (FP) is classified into usual interstitial pneumonia (UIP) and nonspecific interstitial pneumonia (NSIP), each having its own etiology and prognosis. Both types of FP are progressive and chronic conditions with distinct etiologies. Cytokines and inflammatory mediators play critical roles in the pathogenesis of FP. Among them, the role of transforming growth factor beta-1 (TGF-β1) and modulators triggering fibrosis are not well understood. In this study, the expression of triggering receptor expressed on myeloid cells-1 (TREM-1) as a stimulator for the production of TGF-β1 and also CD4+CD25+Foxp3+ regulatory cells were investigted in FP patients. Sixteen UIP, 14 NSIP and 4 pulmonary fibrosis following Mycobacterium tuberculosis (TB) infection patients, were compared with 12 healthy controls. The frequency of blood CD14+TGF-β1+ and CD14+TREM1+-gated monocytes and CD4+CD25+Foxp3+ regulatory T cells (Treg), as well as the plasma levels of TGF-β1 and IL‑10 were measured. Fibrosis patients compared to healthy controls had a greater frequency of CD14+TGF-β1+ [15.9 (0.2-88.2) vs. 0.6 (0.2-11.0)] and CD14+TREM1+ [21.1 (2.3-91.2) vs. 10.3 (3.1-28.6)]-gated monocytes, and CD4+CD25+Foxp3+ [1.2 (0.3-3.6) vs. 0.2 (0.1-0.4)]-gated lymphocytes. Plasma TGF-β1 were also significantly increased in patients with fibrosis compared to healthy controls [9316.2 (±5554.4) vs. 3787.5 (±2255.6)]. These results confirm the importance of TGF-β1 and TREM1 in pulmonary fibrosis. It seems that this reciprocal cycle in healthy people is modulated by the production of IL‑10 by Treg cells, thus limiting fibrosis, as observed in patients following TB infection. Further investigations are recommended to evaluate possible immunomodulatory mechanisms defects in pulmonary fibrosis.

Topics: Forkhead Transcription Factors; Humans; Interleukin-10; Pulmonary Fibrosis; T-Lymphocytes, Regulatory; Transforming Growth Factor beta; Transforming Growth Factor beta1; Triggering Receptor Expressed on Myeloid Cells-1

A subset of severe COVID19 patients develop pulmonary fibrosis, but the pathophysiology of this complication is still unclear. We previously described the possibility to isolate lung mesenchymal cells (LMC) by culturing broncho-alveolar lavage (BAL) cells from patients with pulmonary fibrosis or chronic lung allograft dysfunction. Aim of this study was to investigate the possibility to isolate and characterize LMC from BAL of patients that, two months after discharge for severe COVID19, show CT signs of post-COVID19 fibrosis (Post-COVID) and in some cases has been considered transplant indication. Results were compared with those from BAL of patients with collagen tissue disease-associated interstitial fibrosis (CTD-ILD). BAL fluid levels of TGFβ, VEGF, TIMP2, RANTES, IL6, IL8, and PAI1 were assessed. LMC were cultured and expanded, phenotyped by flow cytometry, and tested for osteogenic and adipogenic differentiation. Finally, we tested immunomodulatory and proliferative capabilities, collagen I production + /- TGF-beta stimulation. BAL cytokine and growth factor levels were comparable in the two groups. Efficiency of isolation from BAL was 100% in post-COVID compared to 63% in CTD-ILD. LMC from post-COVID were positive for CD105, CD73, CD90, and negative for CD45, CD34, CD19 and HLA-DR as in CTD-ILD samples. Post-COVID LMC displayed higher collagen production with respect to CTD-ILD LMC. Immunomodulatory capacity towards lymphocytes was very low, while Post-COVID LMC significantly upregulated pro-inflammatory cytokine production by healthy PBMCs. Our preliminary data suggest that LMC from post-COVID19 fibrosis patients share several features with CTD-ILD ones but might have a higher response to fibrogenic signals and pro-inflammatory profile.

Topics: COVID-19; Cytokines; Fibrosis; Humans; Lung; Lung Diseases, Interstitial; Pulmonary Fibrosis; Transforming Growth Factor beta

Sunitinib (SUN) is an FDA approved first line drug for management of metastatic renal cancers and advanced cancerous states of gastrointestinal tract, however, side effects including fibrosis has been reported. Secukinumab (Secu) is an immunoglobulin G1 monoclonal antibody that exhibits anti-inflammatory activity by inhibiting several cellular signaling molecules. This study aimed to examine pulmonary protective potential of Secu in SUN-induced pulmonary fibrosis mediated through inhibition of inflammation via targeting IL-17A associated signaling pathway and using pirfenidone (PFD), an antifibrotic drug approved in 2014 for treatment of pulmonary fibrosis with IL-17A as one of its targets, as a reference drug. Wistar rats (160-200 g) were divided randomly into 4 groups (n = 6); Group 1 served as normal control; Group 2 served as disease control where it was exposed to SUN (25 mg/kg; 3 times weekly orally for 28 days); Group 3 was administered SUN and Secu (3 mg/kg subcutaneous at 0,14 and 28 days) and Group 4 was administered SUN and PFD (100 mg/kg/day orally for 28 days). Pro-inflammatory cytokines IL-1β, IL-6 and TNF-α were measured in addition to components of IL-17A signaling pathway (TGF-β, collagen, hydroxyproline). Results revealed that IL-17A-associated signaling pathway was activated in fibrotic lung tissue induced by SUN. Relative to normal control, SUN administration significantly elevated lung organ coefficient, IL-1β, IL-6, TNF-α, IL-17A, TGF-β, hydroxyproline and collagen expression. Secu or PFD treatment restored the altered levels to nearly normal values. Our study indicates that IL-17A participates in the development and progression of pulmonary fibrosis in a TGF-β dependent manner. Hence, components of IL-17A signaling pathway represent potential therapeutic targets for protection and treatment of fibro-proliferative lung disease.

Topics: Animals; Collagen; Fibrosis; Hydroxyproline; Interleukin-17; Interleukin-6; Pulmonary Fibrosis; Rats; Rats, Wistar; Sunitinib; Transforming Growth Factor beta; Transforming Growth Factor beta1; Tumor Necrosis Factor-alpha

The periphery of malignant tumors and the leading edge of fibrotic tissue have analogous metabolic pathways. Both use glycolysis as the primary source of energy to produce biomass with consequential acidification of the microenvironment. A low PH has been shown to increase the ability of cancer cells to invade the surrounding tissue in both in vitro and in vivo studies. The pH-dependent activation of TGF-B leading to myofibroblast activation is an important step in the initiation and progression of fibrosis. Markers of accelerated cell proliferation have also been reported in the periphery of malignant tumors and the leading edge of fibrosis. Understanding the shared molecular and metabolic characteristics of these conditions may explain the increased prevalence of cancer among patients with fibrosis.

Topics: Cell Differentiation; Fibroblasts; Fibrosis; Humans; Myofibroblasts; Neoplasms; Pulmonary Fibrosis; Transforming Growth Factor beta; Transforming Growth Factor beta1; Tumor Microenvironment

Topics: COVID-19; Epithelium; Fibrosis; Humans; Pulmonary Fibrosis; Transforming Growth Factor beta; Transforming Growth Factor beta1

Endothelial-to-mesenchymal transition (EndMT), the process by which endothelial cells lose their characteristics and acquire mesenchymal phenotypes, participates in the pathogenic mechanism of idiopathic pulmonary fibrosis. Recently, exosomes derived from human umbilical cord mesenchymal stem cells (hucMSC-Exos) has been introduced as a promising treatment in organ fibrosis. This study aimed to explore the effects as well as the molecular mechanism for hucMSC-Exo in pulmonary fibrosis. The intravenous administration of hucMSC-Exos alleviated bleomycin-induced pulmonary fibrosis in vivo. Moreover, hucMSC-Exos elevated miR-218 expression and restored endothelial properties weakened by TGF-β in endothelial cells. Knockdown of miR-218 partially abrogated the inhibition effect of hucMSC-Exos on EndMT. Our mechanistic study further demonstrated that MeCP2 was the direct target of miR-218. Overexpressing MeCP2 aggravated EndMT and caused increased CpG islands methylation at BMP2 promoter, which lead to BMP2 post-transcriptional gene silence. Transfection of miR-218 mimic increased BMP2 expression as well, which was downregulated by overexpression of MeCP2. Taken together, these findings indicate exosomal miR-218 derived from hucMSCs may possess anti-fibrotic properties and inhibit EndMT through MeCP2/BMP2 pathway, providing a new avenue of preventive application in pulmonary fibrosis.

Topics: Bone Morphogenetic Protein 2; Endothelial Cells; Fibrosis; Humans; Mesenchymal Stem Cells; MicroRNAs; Pulmonary Fibrosis; Transforming Growth Factor beta

Pulmonary fibrosis is a progressive and fatal fibrotic lung disease and associated with a high mortality rate. In the study, the prevention and treatment effects of fibroblast growth factor-21 (FGF-21) in bleomycin (BLM)-induced pulmonary fibrosis were investigated in vivo and vitro. In the prevention of pulmonary fibrosis studies, the results showed that interdict of FGF-21 could reduce the related gene and protein expression levels of pulmonary fibrosis. In addition, FGF-21 significantly reduced both the aggregation of inflammatory cells and deposition of collagen in the lung by histopathology. In therapy of pulmonary fibrosis studies, the results indicated that treatment with FGF-21 resulted in an amelioration of the pulmonary fibrosis in mice with reductions of the pathological score, collagen deposition and transforming growth factor (TGF)-β and α-smooth muscle actin (α-SMA) expressions in the lung tissues at fibrotic stage, and late administration was also able to reduce the degree of pulmonary fibrosis and even better than these in the prevention group. Furthermore, BLM-induced THP-1 macrophage model was verified using FGF-21; the result showed that FGF-21 decreased the related gene expression level of pulmonary fibrosis. FGF-21 may have preventive and therapeutic effects on BLM-induced pulmonary fibrosis via inhibiting myofibroblast differentiation and inflammatory. Thus, FGF-21 represents a potential drug for the prevention and treatment of pulmonary fibrosis.

Topics: Animals; Bleomycin; Collagen; Fibroblast Growth Factors; Fibroblasts; Fibrosis; Lung; Mice; Mice, Inbred C57BL; Pulmonary Fibrosis; Transforming Growth Factor beta; Transforming Growth Factor beta1

Silica-induced pulmonary fibrosis (silicosis) is a diffuse interstitial fibrotic disease characterized by the massive deposition of extracellular matrix in lung tissue. Fibroblast to myofibroblast differentiation is crucial for the disease progression. Inhibiting myofibroblast differentiation may be an effective way for pulmonary fibrosis treatment.. The experiments were conducted in TGF-β treated human lung fibroblasts to induce myofibroblast differentiation in vitro and silica treated mice to induce pulmonary fibrosis in vivo.. By quantitative mass spectrometry, we revealed that proteins involved in mitochondrial folate metabolism were specifically upregulated during myofibroblast differentiation following TGF-β stimulation. The expression level of proteins in mitochondrial folate pathway, MTHFD2 and SLC25A32, negatively regulated myofibroblast differentiation. Moreover, plasma folate concentration was significantly reduced in patients and mice with silicosis. Folate supplementation elevated the expression of MTHFD2 and SLC25A32, alleviated oxidative stress and effectively suppressed myofibroblast differentiation and silica-induced pulmonary fibrosis in mice.. Our study suggests that mitochondrial folate pathway regulates myofibroblast differentiation and could serve as a potential target for ameliorating silica-induced pulmonary fibrosis.

Topics: Animals; Cell Differentiation; Fibroblasts; Humans; Lung; Mice; Mice, Inbred C57BL; Myofibroblasts; Pulmonary Fibrosis; Silicon Dioxide; Silicosis; Transforming Growth Factor beta

Pulmonary fibrosis (PF) is a fibrotic interstitial pneumonia with poor prognosis and limited treatment methods. Inhibition of integrin α

Topics: Antibodies, Blocking; Antigens, Neoplasm; Humans; Hydrogen Peroxide; Integrins; Pulmonary Fibrosis; Reactive Oxygen Species; Transforming Growth Factor beta

Systemic sclerosis (SSc) has the highest mortality rate among the rheumatic diseases, with lung fibrosis leading as the cause of death. A characteristic of severe SSc-related lung fibrosis is its progressive nature. Although most research has focused on the pathology of the fibrosis, the mechanism mediating the fibrotic spread remains unclear. We hypothesized that extracellular vesicle (EV) communication drives the propagation of SSc lung fibrosis.. EVs were isolated from normal (NL) or SSc-derived human lungs and primary lung fibroblasts (pLFs). EVs were also isolated from human fibrotic lungs and pLFs induced experimentally with transforming growth factor-β (TGFβ). Fibrotic potency of EVs was assessed using functional assays in vitro and in vivo. Transmission electron microscopy, nanoparticle tracking analysis, real-time quantitative polymerase chain reaction (RT-qPCR), immunoblotting, and immunofluorescence were used to analyze EVs, their cargo, extracellular matrix (ECM) fractions, and conditioned media.. SSc lungs and pLFs released significantly more EVs than NL lungs, and their EVs showed increased fibrotic content and activity. TGFβ-stimulated NL lung cores and pLFs increased packaging of fibrotic proteins, including fibronectin, collagens, and TGFβ, into released EVs. The EVs induced a fibrotic phenotype in recipient pLFs and in vivo in mouse lungs. Furthermore, EVs interacted with and contributed to the ECM. Finally, suppressing EV release in vivo reduced severity of murine lung fibrosis.. Our findings highlight EV communication as a novel mechanism for propagation of SSc lung fibrosis. Identifying therapies that reduce EV release, activity, and/or fibrotic cargo in SSc patient lungs may be a viable therapeutic strategy to improve fibrosis.

Topics: Animals; Extracellular Vesicles; Fibroblasts; Fibrosis; Humans; Lung; Mice; Pulmonary Fibrosis; Scleroderma, Systemic; Signal Transduction; Transforming Growth Factor beta

Idiopathic pulmonary fibrosis (IPF) is a type of interstitial pneumonia characterized by chronic and progressive fibrosis with an unknown etiology. Previous pharmacological studies have shown that Sanghuangporus sanghuang possesses various beneficial properties including immunomodulatory, hepatoprotective, antitumor, antidiabetic, anti-inflammatory, and neuroprotective effects. This study used a bleomycin (BLM)-induced IPF mouse model to illustrate the possible benefits of SS in ameliorating IPF. BLM was administered on day 1 to establish a pulmonary fibrosis mouse model, and SS was administered through oral gavage for 21 d. Hematoxylin and eosin (H&E) and Masson's trichrome staining results showed that SS significantly reduced tissue damage and decreased fibrosis expression. We observed that SS treatment resulted in a substantial lowering in the level of pro-inflammatory cytokines like TGF-β, TNF-α, IL-1β, and IL-6 as well as MPO. In addition, we observed a notable increase in glutathione (GSH) levels. Western blot analysis of SS showed that it reduces inflammatory factors (TWEAK, iNOS, and COX-2), MAPK (JNK, p-ERK, and p-38), fibrosis-related molecules (TGF-β, SMAD3, fibronectin, collagen, α-SMA, MMP2, and MMP9), apoptosis (p53, p21, and Bax), and autophagy (Beclin-1, LC3A/B-I/II, and p62), and notably increases caspase 3, Bcl-2, and antioxidant (Catalase, GPx3, and SOD-1) levels. SS alleviates IPF by regulating the TLR4/NF-κB/MAPK, Keap1/Nrf2/HO-1, CaMKK/AMPK/Sirt1, and TGF-β/SMAD3 pathways. These results suggest that SS has a pharmacological activity that protects the lungs and has the potential to improve pulmonary fibrosis.

Topics: AMP-Activated Protein Kinases; Animals; Apoptosis; Autophagy; Bleomycin; Calcium-Calmodulin-Dependent Protein Kinase Kinase; Disease Models, Animal; Fibrosis; Kelch-Like ECH-Associated Protein 1; Lung; Mice; NF-E2-Related Factor 2; NF-kappa B; Pulmonary Fibrosis; Signal Transduction; Sirtuin 1; Toll-Like Receptor 4; Transforming Growth Factor beta

Fibrosis is a response to ongoing cellular injury, disruption, and tissue remodeling, the pathogenesis of which is unknown, and is characterized by extracellular matrix deposition. The antifibrotic effect of Geranylgeranylacetone (GGA), as an inducer of Heat shock protein 70 (HSP70), in liver, kidney and pulmonary fibrosis has been supported by multiple preclinical evidence. However, despite advances in our understanding, the precise roles of HSP70 in fibrosis require further investigation. The purpose of this study was to investigate whether GGA could participate in the progression of pulmonary fibrosis in mice through apoptosis, oxidative stress and inflammation.. B-cell lymphoma-2(Bcl-2) and Bcl2-Associated X (Bax) are two proteins related to apoptosis. Anti-apoptotic factor Bcl-2 and pro-apoptotic factor Bax are often involved in the apoptotic process in the form of dimer. Immunofluorescence and Western blot results showed that bleomycin (BLM) and transforming growth factor-β (TGF-β) inhibited Bcl-2 expression and promoted Bax expression in vitro and in vivo, respectively. In contrast, GGA treatment reverses this change. Reactive oxygen species (ROS), Malondialdehyde (MDA) and superoxide dismutase (SOD) are markers of oxidative stress, which often reflect oxidative injury of cells. The detection of ROS, MDA and SOD expression showed that TGF-β and BLM treatment could significantly promote oxidative stress, while GGA treatment could alleviate oxidative stress damage. In addition, BLM significantly elevated Tumor necrosis factor-α(TNF-α), Interleukin1β (IL-1β) and Interleukin 6 (IL-6), while scutellarin reversed the above alterations except for that of GGA.. Taken together, GGA suppressed apoptotic, oxidative stress and inflammation in BLM-induced pulmonary fibrosis.

Topics: Animals; Apoptosis; bcl-2-Associated X Protein; Bleomycin; Fibrosis; Inflammation; Lung; Mice; Oxidative Stress; Pneumonia; Pulmonary Fibrosis; Reactive Oxygen Species; Superoxide Dismutase; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha

Pulmonary fibrosis is a chronic and progressive disease with limited therapeutic options. Nitric oxide (NO) is suggested to reduce the progression of pulmonary fibrosis via NO-sensitive guanylyl cyclase (NO-GC). The exact effects of NO-GC during pulmonary fibrosis are still elusive. Here, we used a NO-GC knockout mouse (GCKO) and examined fibrosis and inflammation after bleomycin treatment. Compared to wildtype (WT), GCKO mice showed an increased fibrotic reaction, as myofibroblast occurrence (

Topics: Animals; Anti-Inflammatory Agents; Bleomycin; Bronchoalveolar Lavage Fluid; Lung; Mice; Mice, Inbred C57BL; Mice, Knockout; Pulmonary Fibrosis; Soluble Guanylyl Cyclase; Transforming Growth Factor beta

Topics: Alkaloids; Animals; Fibroblasts; Macrophages; MAP Kinase Signaling System; Mice; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; Pulmonary Fibrosis; Transforming Growth Factor beta

The incidence of pulmonary fibrosis is on the rise, and existing treatments have limited efficacy in improving patient survival. The purpose of this study was to reveal the potential of Krüppel-like factor (KLF)15 activation in alleviating pulmonary fibrosis. Transforming growth factor beta (TGF-β) was utilized to induce lung fibroblasts to establish an in vitro model of pulmonary fibrosis. The impacts of TGF-β and KLF15 level on cell proliferation, migration, extracellular matrix (ECM) accumulation, and endoplasmic reticulum stress (ERS) were assessed. Additionally, tunicamycin, an ERS agonist, was used to investigate the role of ERS in KLF15 regulation. The results showed that KLF15 was dropped in response to TGF-β treatment. However, KLF15 overexpression reduced cell proliferation, migration, ECM accumulation, and ERS, alleviating the effects of TGF-β stimulation. Subsequent treatment with tunicamycin diminished the effects of KLF15 overexpression, demonstrating that ERS mediated the modulation of KLF15. KLF15 acts against ERS and suppresses excessive proliferation and ECM accumulation in lung fibroblast. These findings suggest that activating KLF15 is a promising strategy for alleviating pulmonary fibrosis.

Topics: Cell Proliferation; Cells, Cultured; Endoplasmic Reticulum Stress; Extracellular Matrix; Fibroblasts; Fibrosis; Humans; Kruppel-Like Transcription Factors; Lung; Pulmonary Fibrosis; Transforming Growth Factor beta; Tunicamycin

Diesel exhaust (DE) exposure contributes to the progression of chronic respiratory diseases and is associated with dysregulation of microRNA expression. The present study aims to investigate the involvement of miRNAs and target genes in DE-induced lung fibrosis.. C57BL/6 mice were divided into three groups. Group 1 mice were exposed to filtered air (Control). Group 2 mice were exposed to DE for 30 min per day, 5 days per week, for 8 weeks (DE). Group 3 mice received DE exposure along with resveratrol on alternate days for the last 2 weeks (DE + RES). Mice were sacrificed to isolate RNA from lung tissue for miRNA microarray profiling. Bronchoalveolar lavage fluid and lung tissues were collected for cell count and biochemical analysis.. DE exposure resulted in differential expression of 28 miRNAs with fold change >2 (p < 0.05). The upregulated miR-212-3p was selected for further analysis. Consensus analysis revealed enrichment of SIRT1 in the FoxO pathway, along with a co-annotation of reduced body weight (p < 0.05). A549 cells transfected with a miR-212-3p inhibitor showed a dose-dependent increase in SIRT1 expression, indicating SIRT1 as a direct target. Treatment with resveratrol restored SIRT1 and miR-212-3p expression and led to a reduction in inflammatory cytokines (p < 0.05). The modulation of SIRT1 correlated negatively with macrophage infiltration, confirming its role in regulating cellular infiltration and lung inflammation. Fibronectin, alpha-SMA, and collagen levels were significantly decreased in DE + RES compared to DE group suggesting modulation of cellular functions and resolution of lung fibrosis. Furthermore, a significant decrease in FoxO3a and TGF-β gene expressions was observed upon resveratrol administration thereby downregulating pro-fibrotic pathway.. The present study demonstrates resveratrol treatment stabilizes SIRT1 gene expression by attenuating miR-212-3p in DE-exposed mice, leading to downregulation of TGF-β and FoxO3a expressions. The study highlights the therapeutic role of resveratrol in the treatment of DE-induced pulmonary fibrosis.

Topics: Animals; Cytokines; Mice; Mice, Inbred C57BL; MicroRNAs; Pulmonary Fibrosis; Resveratrol; Sirtuin 1; Transforming Growth Factor beta; Vehicle Emissions

Fibrotic extracellular matrix (ECM) remodeling characterized different types of pulmonary fibrosis, and its regulation could be a potential shared treatment strategy for pulmonary fibrosis.. We aimed to investigate the effect of triptolide on pulmonary fibrosis through the inhibition of several important aspects of fibrotic ECM remodeling.. Bleomycin-induced pulmonary fibrosis mice and TGF-β. Triptolide significantly alleviated bleomycin-induced pulmonary fibrosis in mice. It inhibited the expression of fibrotic genes α-SMA, collagen I, fibronectin, and vimentin and blocked the TGF-β-SMAD signaling pathway both in vivo and in vitro. In addition, triptolide regulated the expression and activity of MMPs during fibrosis. Interestingly, it suppressed the expression of lysyl oxidase, which was responsible for matrix cross-linking and elevated ECM stiffness. Furthermore, triptolide blocked the biomechanical stress transduction pathway integrin-β1-FAK-YAP signaling and attenuated the pro-fibrotic feedback of fibrotic ECM on fibroblasts via integrin inhibition.. These findings show that triptolide prevents the key linkages of fibrotic ECM remodeling, including deposition, degradation, cross-linking, and pro-fibrotic feedback and, therefore, has potential therapeutic value for pulmonary fibrosis.

Topics: Animals; Bleomycin; Extracellular Matrix; Integrins; Matrix Metalloproteinases; Mice; Protein-Lysine 6-Oxidase; Pulmonary Fibrosis; Transforming Growth Factor beta

Pulmonary fibrosis (PF) is one of the common manifestations of end-stage lung disease. Chronic lung failure after lung transplantation is mainly caused by bronchiolitis obliterans syndrome (BOS) and is mainly characterized by lung tissue fibrosis. Pulmonary epithelial-mesenchymal transformation (EMT) is crucial for pulmonary fibrosis. Telocytes (TCs), a new type of mesenchymal cells, play a protective role in various acute injuries. For exploring the anti-pulmonary fibrosis effect of TCs in the BOS model in vitro and the related mechanism, rat tracheal epithelial (RTE) cells were treated with transforming growth factor-β (TGF-β) to simulate lung tissue fibrosis in vitro. The RTE cells were then co-cultured with TCs primarily extracted from rat lung tissue. Western blot, Seahorse XF Analysers and enzyme-linked immunosorbent assay were used to detect the level of EMT and aerobic respiration of RTE cells. Furthermore, anti-hepatocyte growth factor (anti-HGF) antibody was exogenously added to the cultured cells to explore further mechanisms. Moreover, hexokinase 2 (HK2) in RTE cells was knocked down to assess whether it influences the blocking effect of the anti-HGF antibody. TGF-β could induce lung tissue fibrosis in RTE cells in vitro. Nevertheless, TCs co-culture decreased the level of EMT, glucose metabolic indicators (lactate and ATP) and oxygen levels. Furthermore, TCs released hepatocyte growth factor (HGF). Therefore, the exogenous addition of anti-HGF antibody in the co-culture system blocked the anti-lung tissue fibrosis effect. However, HK2 knockdown attenuated the blocking effect of the anti-HGF antibody. In conclusion, TCs can protect against lung tissue fibrosis by releasing HGF, a process dependent on HK2.

Topics: Animals; Fibrosis; Hepatocyte Growth Factor; Hexokinase; Lung; Pulmonary Fibrosis; Rats; Telocytes; Transforming Growth Factor beta; Transforming Growth Factor beta1

This study investigates the interactions between histaminergic system and glucocorticoid-induced leucin zipper (GILZ) in the inflammatory process and glucocorticoid modulation in lung fibrosis.. Wild-type (WT) and GILZ Knock-Out (KO) mice were treated with bleomycin (0.05 IU) or saline, delivered by intra-tracheal injection. After surgery, mice received a continuous infusion of JNJ7777120 (JNJ, 2 mg/kg b.wt.) or vehicle for 21 days. Lung function was studied by measuring airway resistance to air insufflation through the analysis of pressure at airway opening (PAO). Lung samples were collected to evaluate the expression of histamine H. Airway fibrosis and remodeling were assessed by measuring TGF-β production and α-SMA deposition. JNJ reduces PAO in WT but not in GILZ KO mice (from 22 ± 1 mm to 15 ± 0.5 and from 24 ± 1.5 to 19 ± 0.5 respectively), MPO activity (from 204 ± 3.13 pmol/mg to 73.88 ± 2.63 in WT and from 221 ± 4.46 pmol/mg to 107 ± 5.54 in GILZ KO), the inflammatory response, TGF-β production, and α-SMA deposition in comparison to WT and GILZ KO vehicle groups.. In conclusion, the role of H

Topics: Animals; Anti-Inflammatory Agents; Glucocorticoids; Histamine; Mice; Pulmonary Fibrosis; Receptors, Histamine; Transcription Factors; Transforming Growth Factor beta

Topics: Animals; Bacillus amyloliquefaciens; Bleomycin; Lung; Mice; Pulmonary Fibrosis; Spores, Bacterial; Superoxide Dismutase; Transforming Growth Factor beta; Transforming Growth Factor beta1

Topics: Animals; Bleomycin; Lung; Paraquat; Peptides; Pulmonary Fibrosis; Rats; Signal Transduction; Transforming Growth Factor beta; Transforming Growth Factor beta1

Lung fibrosis is a major concern in severe COVID-19 patients undergoing mechanical ventilation (MV). Lung fibrosis frequency in post-COVID syndrome is highly variable and even if the risk is proportionally small, many patients could be affected. However, there is still no data on lung extracellular matrix (ECM) composition in severe COVID-19 and whether it is different from other aetiologies of ARDS.. We have quantified different ECM elements and TGF-β expression in lung tissue of 28 fatal COVID-19 cases and compared to 27 patients that died of other causes of ARDS, divided according to MV duration (up to six days or seven days or more). In COVID-19 cases, ECM elements were correlated with lung transcriptomics and cytokines profile.. We observed that COVID-19 cases presented significant increased deposition of collagen, fibronectin, versican, and TGF-β, and decreased decorin density when compared to non-COVID-19 cases of similar MV duration. TGF-β was precociously increased in COVID-19 patients with MV duration up to six days. Lung collagen was higher in women with COVID-19, with a transition of upregulated genes related to fibrillogenesis to collagen production and ECM disassembly along the MV course.. Fatal COVID-19 is associated with an early TGF-β expression lung environment after the MV onset, followed by a disordered ECM assembly. This uncontrolled process resulted in a prominent collagen deposition when compared to other causes of ARDS. Our data provides pathological substrates to better understand the high prevalence of pulmonary abnormalities in patients surviving COVID-19.

Topics: Collagen; COVID-19; Extracellular Matrix; Female; Humans; Lung; Pulmonary Fibrosis; Respiratory Distress Syndrome; Transforming Growth Factor beta

The purpose of this study was to investigate the parenchymal changes in idiopathic pulmonary fibrosis (IPF) caused by massive fibroblastic infiltration and proliferation in lung tissue. Galectin-1 (Gal-1) has been reported to be involved in angiogenesis and fibrosis via modification of TGF-b receptor signaling pathways. However, it remains unknown whether Galectin-1 plays a critical role in IPF. In the current study, we aimed to identify Gal-1 as a crucial fibrotic protein in IPF process. Murine lung fibroblast was pre-treated using Gal-1 inhibitor OTX-008 or overexpression of Gal-1 and then activated using transforming growth factor-beta (TGF-β). Adult male C57BL/6J mice were conducted intratracheal injection of bleomycin (BLM) for lung fibrosis. Mice were conducted OTX-008 administration. Gal-1 expression, fibroblast activation and proliferation, extracellular matrix (ECM), lung fibrosis, lung histology and pulmonary function were investigated respectively. We demonstrated that Gal-1, as a positive pro-fibrotic marker, could promote lung fibroblast activation and proliferation. Inhibition of Gal-1 reduced fibroblast activation and proliferation through negative regulation of TGF-β/Erk1/2 and AKT pathway. In vivo, Gal-1 inhibition ameliorates lung fibroblast accumulation and protects lung histology and function. Gal-1 is verified to be a pro-fibrotic gene in IPF pathogenesis, which promotes fibroblast activation and proliferation via TGF-β/Erk1/2 and AKT pathway. Moreover, inhibition of Gal-1 in lung fibrosis model attenuates lung fibroblast bioactivity and reduces ECM, leading to improved pulmonary histology and function. Hence, knockdown of Gal-1 in IPF may be a promising target therapy.

Topics: Animals; Cell Proliferation; Fibroblasts; Galectin 1; Lung; Male; Mice; Mice, Inbred C57BL; Proto-Oncogene Proteins c-akt; Pulmonary Fibrosis; Transforming Growth Factor beta

Epidemiological studies suggest increased risk of lung cancer associated with diesel exhaust (DE) exposure. However, DE-induced lung fibrosis may lead to cancer and needs investigation.. To study the mechanism involved in the initiation of DE- induced lung fibrosis.. C57BL/6 mice were exposed to DE for 30 min/day for 5 days/weeks for 8 weeks. Pulmonary function test was performed to measure lung function. Mice were euthanized to collect BALF, blood, and lung tissue. BALF was used for cell count and cytokine analysis. Lung tissue slides were stained to examine structural integrity. RNA from lung tissue was used for RT-PCR. Immunoblots were performed to study fibrosis and EMT pathway.. Mice exposed to DE increase lung resistance and tissue elastance with decrease in inspiratory capacity (p < 0.05) suggesting lung function impairment. BALF showed significantly increased macrophages, neutrophils and monocytes (p < 0.01). Additionally, there was an increase in inflammation and alveolar wall thickening in lungs (p < 0.01) correlates with cellular infiltration. Macrophages had black soot deposition in lung tissue of DE exposed mice. Lung section staining revealed increase in mucus producing goblet cells for clearance of soot in lung. DE exposed lung showed increased collagen deposition and hydroxyproline residue (p < 0.01). Repetitive exposure of DE in mice lead to tissue remodeling in lung, demonstrated by fibrotic foci and smooth muscles. A significant increase in α-SMA and fibronectin (p < 0.05) in lung indicate progression of pulmonary fibrosis. TGF-β/Smad3 signaling was activated with increase in P-smad3 expression in DE exposed mice. Decreased expression of E-cadherin and increased vimentin (p < 0.05) in lungs of DE exposed mice indicate epithelial to mesenchymal transition.. DE exposure to mice induced lung injury and pulmonary fibrosis thereby remodeling tissue. The study demonstrates TGF-β/SMAD3 pathway involvement with an activation of EMT in DE exposed mice.

Topics: Animals; Epithelial-Mesenchymal Transition; Lung; Mice; Mice, Inbred C57BL; Pulmonary Fibrosis; Signal Transduction; Transforming Growth Factor beta; Vehicle Emissions

Topics: Animals; Antibiotics, Antineoplastic; Bleomycin; Case-Control Studies; Disease Models, Animal; Extracellular Matrix Proteins; Gene Expression Profiling; Gene Regulatory Networks; Humans; Lung; Mice; Mice, Inbred C57BL; Myofibroblasts; Protein Interaction Maps; Pulmonary Fibrosis; rab GTP-Binding Proteins; Receptor, Transforming Growth Factor-beta Type I; Signal Transduction; Transforming Growth Factor beta

The epithelial-mesenchymal transition (EMT) is an important pathological process in the occurrence of pulmonary fibrosis. Changes in histone methylation modifications of key genes play an important role in this process. As a histone methyltransferase, the regulatory mechanism and role of SET domain bifurcated 1 (SETDB1) in pulmonary fibrosis remain unclear. We found that SETDB1 inhibited EMT and that cells attenuated the expression of SETDB1 to relieve this inhibition during transforming growth factor-β (TGF-β)-induced EMT. Silencing SETDB1 expression significantly enhanced the mesenchymal phenotype induced by TGF-β and the expression and deposition of fibronectin and significantly reduced the expression of E-cadherin. The decrease in E-cadherin expression and the induction of EMT led to increased lipid reactive oxygen species (ROS) and ferrous ions, which induced ferroptosis. Chromatin immunoprecipitation (ChIP) results showed that SETDB1 regulates the expression of Snai1 by catalyzing the histone H3 lysine 9 trimethylation (H3K9me3) of Snai1, the main transcription factor that initiates the process of EMT, and thus, indirectly regulates E-cadherin. Surprisingly, when examining the effect of overexpressed SETDB1 on EMT, we found that overexpressed SETDB1 alleviated EMT and also caused ferroptosis. We suggest that the overexpression of SETDB1 partially reverses the mesenchymal phenotype to an epithelial state, while those cells that fail to reverse are depleted by ferroptosis. In conclusion, the histone methylase SETDB1 regulates Snai1 epigenetically, driving EMT gene reprogramming and ferroptosis in response to TGF-β. However, there are unexplored links between the epigenetic reprogramming and transcriptional processes that regulate EMT in a TGF-β-dependent manner.

Topics: A549 Cells; Animals; Bleomycin; Epithelial-Mesenchymal Transition; Ferroptosis; Gene Knockdown Techniques; Histone-Lysine N-Methyltransferase; Humans; Promoter Regions, Genetic; Pulmonary Fibrosis; Rats, Sprague-Dawley; Snail Family Transcription Factors; Transforming Growth Factor beta

We investigated BMPR2 expression in pulmonary fibrosis and TGF-β/BMP signaling in lung fibroblasts. Then we evaluated the impact of BMPR2 upregulation using adenoviral transduction on TGF-β-induced Smad2/3 phosphorylation and fibronectin production in lung fibroblasts.. BMPR2 was distributed in airway epithelium and alveolar walls in rat lungs. BMPR2 expression was decreased in fibrotic lesions in the lungs of rats with bleomycin-induced pulmonary fibrosis and in human lung fibroblasts (HLFs) stimulated with TGF-β. Although Smad2/3 phosphorylation and fibronectin production were not suppressed solely by BMPs, phosphorylated Smad2/3 was decreased in BMPR2-transduced cells even without BMP stimulation. Fibronectin was decreased only when BMPR2-transduced HLFs were stimulated with BMP7 (but not BMP4). Similar results were also observed in IPF patient HLFs and rat lung fibroblasts.. BMPR2 expression was reduced in fibrotic lungs and lung fibroblasts stimulated with TGF-β. BMPR2 transduction to lung fibroblasts reduced Smad2/3 phosphorylation, and reduced fibronectin production when treated with BMP7. Upregulation of BMPR2 may be a possible strategy for treating pulmonary fibrosis.

Topics: Animals; Bone Morphogenetic Protein Receptors, Type II; Fibroblasts; Fibronectins; Humans; Lung; Pulmonary Fibrosis; Rats; Transforming Growth Factor beta; Transforming Growth Factor beta1

Schisandra chinensis decoction derived from the book of Waitai Miyao (Tao Wang, Tang dynasty) is often used in the treatment of idiopathic pulmonary fibrosis (IPF), which is included in the Grand Ceremony of Chinese formulae (Huairen Peng, 1994). Schisandrae Chinensis Fructus (Sch) is one of the most important herbs in this formula. According to the "Shennong's Herbal Classicherbal" of the Han Dynasty, Sch has sour taste, warm nature, which has the effect of tonifying qi and curing cough. In addition, according to the "Compendium of Materia Medica" of the Ming Dynasty, Sch is used to treat cough and asthma, which has the effect of moistening the lung and tonifying the kidney. However, the active ingredients of Sch absorption into the plasma and its pharmacological mechanism of treatment for IPF still remained unclear.. Our research aimed at identifying the absorbed active ingredients and metabolized of Sch in rat plasma and the mechanism of anti-IPF based on serum pharmacochemistry.. First, the rats were divided into control group and Sch group. Sch sample was orally administrated to the rats for seven days. The blood samples were drawn into an Eppendorf tube after the last dosing. The ultrahigh performance liquid chromatography coupled with quadrupole-time of flight mass spectrometry (UPLC-Q-TOF/MS) was applied to identify the absorption components and metabolites of Sch in rat plasma. Second, the network pharmacology combined with molecular docking analysis was further investigated to illuminate its potential mechanism of treatment for IPF by the biological targets regulating related pathways. Finally, the mechanism of action was verified by experimental in vitro and in vivo.. A total of 78 compounds, consist of 13 prototype lignans and 65 metabolites (including isomers) were identified. Network pharmacology study and molecular docking analysis indicated that schisandrol A (L1) play an anti-fibrosis role by regulating the TGF-β signaling pathway. Experimental in vitro and in vivo verified that the schisandrol A could inhibiting pulmonary fibrosis through TGF-β signaling pathway. The effect and mechanism of schisandrol A inhibiting pulmonary fibrosis were reported for the first time.. In this study, the absorption active ingredients of Sch in rat plasma were combined with the network pharmacology investigation and experimental in vitro and in vivo to elucidate its biological mechanism of treatment for IPF. The results provided a theoretical support for understanding the bioactive compounds and the pharmacological mechanism of Sch.

Topics: Animals; Chromatography, High Pressure Liquid; Cyclooctanes; Female; Fruit; Lignans; Male; Mass Spectrometry; Mice; Mice, Inbred C57BL; Molecular Docking Simulation; Network Pharmacology; Pulmonary Fibrosis; Rats; Rats, Sprague-Dawley; Schisandra; Signal Transduction; Transforming Growth Factor beta

Extracellular matrix (ECM) stiffening with downstream activation of mechanosensitive pathways is strongly implicated in fibrosis. We previously reported that altered collagen nanoarchitecture is a key determinant of pathogenetic ECM structure-function in human fibrosis (Jones et al., 2018). Here, through human tissue, bioinformatic and ex vivo studies we provide evidence that hypoxia-inducible factor (HIF) pathway activation is a critical pathway for this process regardless of the oxygen status (pseudohypoxia). Whilst TGFβ increased the rate of fibrillar collagen synthesis, HIF pathway activation was required to dysregulate post-translational modification of fibrillar collagen, promoting pyridinoline cross-linking, altering collagen nanostructure, and increasing tissue stiffness. In vitro, knockdown of Factor Inhibiting HIF (FIH), which modulates HIF activity, or oxidative stress caused pseudohypoxic HIF activation in the normal fibroblasts. By contrast, endogenous FIH activity was reduced in fibroblasts from patients with lung fibrosis in association with significantly increased normoxic HIF pathway activation. In human lung fibrosis tissue, HIF-mediated signalling was increased at sites of active fibrogenesis whilst subpopulations of human lung fibrosis mesenchymal cells had increases in both HIF and oxidative stress scores. Our data demonstrate that oxidative stress can drive pseudohypoxic HIF pathway activation which is a critical regulator of pathogenetic collagen structure-function in fibrosis.

Topics: Biomarkers; Cells, Cultured; Collagen; Fibroblasts; Gene Expression Regulation; Humans; Hypoxia-Inducible Factor 1; Mixed Function Oxygenases; Oxidative Stress; Pulmonary Fibrosis; Repressor Proteins; Transforming Growth Factor beta

Pulmonary fibrosis (PF) is a chronic, progressive, and, ultimately, terminal interstitial disease caused by a variety of factors, ranging from genetics, bacterial, and viral infections, to drugs and other influences. Varying degrees of PF and its rapid progress have been widely reported in post-COVID-19 patients and there is consequently an urgent need to develop an appropriate, cost-effective approach for the prevention and management of PF.. The potential "therapeutic" effect of the tocotrienol-rich fraction (TRF) and carotene against bleomycin (BLM)-induced lung fibrosis was investigated in rats via the modulation of TGF-β/Smad, PI3K/Akt/mTOR, and NF-κB signaling pathways.. Lung fibrosis was induced in Sprague-Dawley rats by a single intratracheal BLM (5 mg/kg) injection. These rats were subsequently treated with TRF (50, 100, and 200 mg/kg body wt/day), carotene (10 mg/kg body wt/day), or a combination of TRF (200 mg/kg body wt/day) and carotene (10 mg/kg body wt/day) for 28 days by gavage administration. A group of normal rats was provided with saline as a substitute for BLM as the control. Lung function and biochemical, histopathological, and molecular alterations were studied in the lung tissues.. Both the TRF and carotene treatments were found to significantly restore the BLM-induced alterations in anti-inflammatory and antioxidant functions. The treatments appeared to show pneumoprotective effects through the upregulation of antioxidant status, downregulation of MMP-7 and inflammatory cytokine expressions, and reduction in collagen accumulation (hydroxyproline). We demonstrated that TRF and carotene ameliorate BLM-induced lung injuries through the inhibition of apoptosis, the induction of TGF-β1/Smad, PI3K/Akt/mTOR, and NF-κB signaling pathways. Furthermore, the increased expression levels were shown to be significantly and dose-dependently downregulated by TRF (50, 100, and 200 mg/kg body wt/day) treatment in high probability. The histopathological findings further confirmed that the TRF and carotene treatments had significantly attenuated the BLM-induced lung injury in rats.. The results of this study clearly indicate the ability of TRF and carotene to restore the antioxidant system and to inhibit proinflammatory cytokines. These findings, thus, revealed the potential of TRF and carotene as preventive candidates for the treatment of PF in the future.

Topics: Animals; Bleomycin; Carotenoids; COVID-19; Humans; NF-kappa B; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; Pulmonary Fibrosis; Rats; Rats, Sprague-Dawley; SARS-CoV-2; Signal Transduction; Tocotrienols; TOR Serine-Threonine Kinases; Transforming Growth Factor beta

Statins have anti-inflammatory and antifibrotic effects in addition to cholesterol-lowering effect. We aimed to investigate the effect of atorvastatin (ATR) in fibrotic mouse lung and human lung fibroblasts (MRC5s). Pulmonary fibrosis was induced by a single dose of bleomycin by intratracheal instillation in adult mice. ATR was administered (20 mg/kg ip) to mice with healthy and pulmonary fibrosis for 10 days from Day 7 of the experiment. Mice were dissected on the 21st day. The levels of alpha-smooth muscle actin (α-SMA), pSMAD2/3, LOXL2, and p-Src were determined by Western blot analysis in the lungs. Furthermore, a group of MRC5 was differentiated into myofibroblasts by transforming growth factor-beta (TGF-β). Another group of MRC5s was treated with 10 µM ATR at 24 h after TGF-β stimulation. Cells were collected at 0, 24, 48, and 72 h. The effects of ATR on myofibroblast differentiation, apoptosis, and TGF-β and Wnt/β-catenin signaling activations were examined by Western blot analysis and flow cytometry in MRC5s. ATR attenuated pulmonary fibrosis by regulating myofibroblast differentiation and interstitial accumulation of collagen, by acting on LOXL2, p-Src, and pSMAD2/3 in mice lungs. Additionally, it blocked myofibroblast differentiation via reduced TGF-β and Wnt/β-catenin signaling and decreased α-SMA in MRC5s stimulated with TGF-β. Moreover, ATR caused myofibroblast apoptosis via caspase-3 activation. ATR treatment attenuates pulmonary fibrosis in mice treated with bleomycin. It also inhibits fibroblast/myofibroblast activation, by both reducing myofibroblasts differentiation and inducing myofibroblast apoptosis.

Topics: Animals; Apoptosis; Atorvastatin; beta Catenin; Bleomycin; Cell Differentiation; Fibroblasts; Humans; Lung; Mice; Mice, Inbred C57BL; Myofibroblasts; Pulmonary Fibrosis; Transforming Growth Factor beta

Idiopathic pulmonary fibrosis (IPF) is an interstitial lung disease characterised by irreversible fibrosis and destruction of the alveolar structure. Receptor for advanced glycation end products (RAGE) has been identified as one of the key molecules involved in IPF pathogenesis. A RAGE-antagonist peptide (RAP) was developed based on the RAGE-binding domain of high mobility group box-1 (HMGB-1). Anti-IPF effects of RAP were evaluated in a bleomycin-induced mouse model of IPF. Bleomycin was administered intratracheally, and then RAP was administrated twice by intratracheal instillation, 1 and 3 d after bleomycin challenge. Seven days after the bleomycin challenge, the mice were sacrificed and the lungs were harvested. The results showed that pulmonary hydroxyproline was reduced in mice administered RAP compared with the control group. Tumour growth factor-β (TGF-β), α-smooth muscle actin (α-SMA) and collagen were also reduced by RAP administration in a dose-dependent manner. Longer-term effects of RAP were investigated in mice challenged with bleomycin. RAP was administered intratracheally every 7 d for 28 d, after which lung samples were harvested and analysed. The results showed that hydroxyproline, TGF-β, α-SMA and collagen were reduced by repeated RAP administration. Taken together, the results suggest that RAP is useful for treatment of IPF.

Topics: Animals; Bleomycin; Collagen; Disease Models, Animal; Hydroxyproline; Lung; Mice; Mice, Inbred C57BL; Pulmonary Fibrosis; Receptor for Advanced Glycation End Products; Transforming Growth Factor beta

Fibrosis is an energy-intensive process requiring the activation of fibroblasts to myofibroblasts, resulting in the increased synthesis of extracellular matrix proteins. Little is known about the transcriptional control of energy metabolism in cardiac fibroblast activation, but glutaminolysis has been implicated in liver and lung fibrosis. Here we explored how pro-fibrotic TGFβ and its effector scleraxis, which drive cardiac fibroblast activation, regulate genes involved in glutaminolysis, particularly the rate-limiting enzyme glutaminase (GLS1). The GLS1 inhibitor CB-839 attenuated TGFβ-induced fibroblast activation. Cardiac fibroblast activation to myofibroblasts by scleraxis overexpression increased glutaminolysis gene expression, including GLS1, while cardiac fibroblasts from scleraxis-null mice showed reduced expression. TGFβ induced GLS1 expression and increased intracellular glutamine and glutamate levels, indicative of increased glutaminolysis, but in scleraxis knockout cells, these measures were attenuated, and the response to TGFβ was lost. The knockdown of scleraxis in activated cardiac fibroblasts reduced GLS1 expression by 75%. Scleraxis transactivated the human GLS1 promoter in luciferase reporter assays, and this effect was dependent on a key scleraxis-binding E-box motif. These results implicate scleraxis-mediated GLS1 expression as a key regulator of glutaminolysis in cardiac fibroblast activation, and blocking scleraxis in this process may provide a means of starving fibroblasts of the energy required for fibrosis.

Topics: Animals; Basic Helix-Loop-Helix Transcription Factors; Fibroblasts; Glutaminase; Mice; Myofibroblasts; Pulmonary Fibrosis; Transforming Growth Factor beta

The acute respiratory distress syndrome (ARDS) is a major healthcare problem, accounting for significant mortality and long-term disability. Approximately 25% of patients with ARDS will develop an overexuberant fibrotic response, termed fibroproliferative ARDS (FP-ARDS) that portends a poor prognosis and increased mortality. The cellular pathological processes that drive FP-ARDS remain incompletely understood. We have previously shown that the transmembrane receptor-type tyrosine phosphatase protein tyrosine phosphatase-α (PTPα) promotes pulmonary fibrosis in preclinical murine models through regulation of transforming growth factor-β (TGF-β) signaling. In this study, we examine the role of PTPα in the pathogenesis of FP-ARDS in a preclinical murine model of acid (HCl)-induced acute lung injury. We demonstrate that although mice genetically deficient in PTPα (

Topics: Acute Lung Injury; Animals; Lung; Mice; Phosphoric Monoester Hydrolases; Pulmonary Fibrosis; Receptor-Like Protein Tyrosine Phosphatases, Class 4; Respiratory Distress Syndrome; Transforming Growth Factor beta

Pulmonary fibrosis is a devastating lung disease with few therapeutic options. CHIT1 (chitinase 1), an 18 glycosyl hydrolase family member, contributes to the pathogenesis of pulmonary fibrosis through the regulation of TGF-β (transforming growth factor-β) signaling and effector function. Therefore, CHIT1 is a potential therapeutic target for pulmonary fibrosis. This study aimed to identify and characterize a druggable CHIT1 inhibitor with strong antifibrotic activity and minimal toxicity for therapeutic application to pulmonary fibrosis. Extensive screening of small molecule libraries identified the aminoglycoside antibiotic kasugamycin (KSM) as a potent CHIT1 inhibitor. Elevated concentrations of CHIT1 were detected in the lungs of patients with pulmonary fibrosis. In

Topics: Aminoglycosides; Animals; Antifibrotic Agents; Bleomycin; Chitinases; Fibroblasts; Humans; Lung; Mice; Mice, Inbred C57BL; Pulmonary Fibrosis; Transforming Growth Factor beta

Fibroblasts play a central role in the lung fibrotic process. Our recent study identified a novel subpopulation of lung fibroblasts expressing meflin (mesenchymal stromal cell- and fibroblast-expressing Linx paralogue), antifibrotic properties of which were confirmed by murine lung fibrosis model. Meflin-expressing fibroblasts were resistant to fibrogenesis induced by TGF-β (transforming growth factor-β), but its underlying mechanisms remain unknown. In this study, evaluation of a silica-nanoparticle-induced lung fibrosis model confirmed the antifibrotic effect of meflin via the regulation of TGF-β signaling. We conducted comparative gene expression profiling in lung fibroblasts, which identified growth differentiation factor 10 (

Topics: Animals; Fibroblasts; Growth Differentiation Factor 10; Lung; Mice; Mice, Knockout; Pulmonary Fibrosis; Silicon Dioxide; Transforming Growth Factor beta; Transforming Growth Factors

Severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2), responsible for COVID-19, has caused a global pandemic. Observational studies revealed a condition, herein called as Long-COVID syndrome (PC), that affects both moderately and severely infected patients, reducing quality-of-life. The mechanism/s underlying the onset of fibrotic-like changes in PC are still not well defined. The goal of this study was to understand the involvement of the Absent in melanoma-2 (AIM2) inflammasome in PC-associated lung fibrosis-like changes revealed by chest CT scans. Peripheral blood mononuclear cells (PBMCs) obtained from PC patients who did not develop signs of lung fibrosis were not responsive to AIM2 activation by Poly dA:dT. In sharp contrast, PBMCs from PC patients with signs of lung fibrosis were highly responsive to AIM2 activation, which induced the release of IL-1α, IFN-α and TGF-β. The recognition of Poly dA:dT was not due to the activation of cyclic GMP-AMP (cGAMP) synthase, a stimulator of interferon response (cGAS-STING) pathways, implying a role for AIM2 in PC conditions. The release of IFN-α was caspase-1- and caspase-4-dependent when AIM2 was triggered. Instead, the release of pro-inflammatory IL-1α and pro-fibrogenic TGF-β were inflammasome independent because the inhibition of caspase-1 and caspase-4 did not alter the levels of the two cytokines. Moreover, the responsiveness of AIM2 correlated with higher expression of the receptor in circulating CD14+ cells in PBMCs from patients with signs of lung fibrosis.

Topics: Carrier Proteins; Caspase 1; COVID-19; DNA-Binding Proteins; Humans; Inflammasomes; Interferon-alpha; Leukocytes, Mononuclear; Post-Acute COVID-19 Syndrome; Pulmonary Fibrosis; SARS-CoV-2; Transforming Growth Factor beta

Despite the recent therapeutic developments for the treatment of pulmonary fibrosis, its prognosis is still not well controlled, and a novel therapeutic agent is needed. Recently, the critical role of Toll-like receptors (TLRs) in the pathophysiology of pulmonary fibrosis has been reported; however, the effects of multiple TLR signaling inhibition are still unknown. Here, we examined how the inhibition of multiple TLRs affects pulmonary fibrosis using a novel synthetic receptor activator of nuclear factor κB ligand (RANKL) partial peptide, MHP1-AcN, which could suppress TLR2, 3, 4, 7, and 9 signaling through CD14 and RANK. When MHP1-AcN was administered in the bleomycin-induced lung fibrosis model, reduced collagen deposition was observed, with suppressed fibrosis-related gene expression including Col1a1, Col1a2, Acta2, Tgfb1 and Tgfbr2. MHP1-AcN also decreased proinflammatory M1 and profibrotic M2 macrophage marker expression. Furthermore, MHP1-AcN treatment inhibited transforming growth factor (TGF-β)-induced Smad2/3 phosphorylation and myofibroblast differentiation in human fetal lung fibroblast (MRC-5) cells. This effect was associated with decreased TGF-β receptor levels and the upregulated Bmp7 and Smad7 expression. These findings suggest that MHP1-AcN protects mice against bleomycin-induced pulmonary fibrosis. MHP1-AcN might provide a novel therapeutic strategy for the pulmonary fibrosis.

Topics: Animals; Bleomycin; Fibroblasts; Humans; Lung; Mice; Mice, Inbred C57BL; Peptides; Pulmonary Fibrosis; RANK Ligand; Transforming Growth Factor beta

Pulmonary fibrosis, a severe condition that can progress to respiratory failure and death, is characterized by aberrant activation/proliferation of fibroblasts and excessive extracellular matrix (ECM) deposition and has limited therapeutic options. Identifying novel mediators of pulmonary fibrosis is currently needed to facilitate the development of more effective therapeutic strategies targeting pulmonary fibrosis. The present study was designed to investigate whether transforming growth factor-β (TGF-β) induced protein (TGFBI), an extracellular matrix protein, regulates pulmonary fibrosis in vitro and in vivo and the possible mechanism of actions. It was found that protein expressions of TGFBI were significantly upregulated and G-protein signaling modulator 2 (GPSM2) expression downregulated in fibrotic lung tissues from bleomycin (BLM)-induced rats and TGF-β1-stimulated human lung IMR-90 fibroblasts. Either silencing TGFBI with specific siRNA or treatment with the TGF-β signaling inhibitor SB431542 significantly inhibited TGF-β1-induced fibrotic effects and dysregulation of GPSM2 and Snail expressions in IMR-90 fibroblasts. Moreover, GPSM2 overexpression also inhibited TGF-β1-induced fibrotic effects and Snail upregulation in IMR-90 fibroblasts. Silencing Snail with specific siRNA attenuated TGF-β1-induced fibrotic effects. Therefore, our findings suggest that the extracellular matrix protein TGFBI mediates pulmonary fibrosis through regulation of the GPSM2/Snail axis, which identifies TGFBI as a novel mediator of pulmonary fibrosis and may be a potential therapeutic target for the treatment of pulmonary fibrosis.

Topics: Animals; Extracellular Matrix Proteins; GTP-Binding Proteins; Humans; Lung; Pulmonary Fibrosis; Rats; RNA, Small Interfering; Transforming Growth Factor beta; Transforming Growth Factor beta1

The online version contains supplementary material available at 10.1007/s00531-022-02219-9.

Topics: Accidents, Occupational; Adult; Animals; Anxiety; beta Catenin; Chromatography, High Pressure Liquid; Chronic Disease; Cities; Depression; Drugs, Chinese Herbal; Flavonoids; Heat Stroke; Hesperidin; Humans; Hyperplasia; Hypoxia-Inducible Factor 1, alpha Subunit; Interleukin-10; Interleukin-6; Kruppel-Like Factor 4; Macrophages, Alveolar; Medical Staff; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Occupational Health; Occupational Injuries; Occupational Stress; Occupations; PPAR gamma; Pulmonary Fibrosis; RNA, Messenger; Sanitation; Silicon Dioxide; Sinusitis; Stress, Psychological; Surveys and Questionnaires; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha; X-Box Binding Protein 1; Young Adult

Bleomycin is a well-recognized antineoplastic drug. However, pulmonary fibrosis (PF) is considered to be the principal drawback that greatly limits its use. Here, we sought to investigate ability of the neurokinin receptor 1 blocker, aprepitant, to prevent PF caused by bleomycin. Male adult Wistar rat groups were given a single intratracheal injection of bleomycin, either alone or in combination with aprepitant therapy for 3 or 14 days. Collagen deposition and a rise in transforming growth factor beta (TGF-β) immunoreactivity in lung tissue serve as evidence of bleomycin-induced PF. The serum levels of lactate dehydrogenase, alkaline phosphatase, and total antioxidant improved after aprepitant therapy.Additionally, it reduced the protein expressions of interferon alpha, tumor necrosis factor alpha, and lung lipid peroxidation. Moreover, aprepitant treatment led to an increase in the antioxidant indices glutathione, glutathione peroxidase, and catalase. Aprepitant is postulated to protect against bleomycin-induced PF by decreasing TGF-β, phosphorylating Smad3, and increasing interleukin 37, an anti-fibrotic cytokine, and G Protein-coupled Receptor Kinase 2. Aprepitant for 14 days considerably exceeded aprepitant for 3 days in terms of improving lung damage and having an anti-fibrotic impact. In conclusion, aprepitant treatment for 14 days may be used as an adjuvant to bleomycin therapy to prevent PF, mostly through inhibiting the TGF-/p-Smad3 fibrotic pathway.

Topics: Alkaline Phosphatase; Animals; Antioxidants; Aprepitant; Bleomycin; Catalase; Collagen; Cytokines; Glutathione; Glutathione Peroxidase; Interferon-alpha; Interleukins; Lactate Dehydrogenases; Lung; Male; Pulmonary Fibrosis; Rats; Rats, Wistar; Receptors, G-Protein-Coupled; Transforming Growth Factor beta; Transforming Growth Factor beta1; Tumor Necrosis Factor-alpha

Pulmonary fibrosis (PF) is characterized by oxidative injury and excessive collagen synthesis in lung fibroblasts, causing impaired pulmonary function and chronic lung injury. Piceatannol, a dietary polyphenol, possesses vital pharmacological effects in metabolic disorders, cancers, cardiovascular disease and infectious disease; however, its role in PF is still not completely elucidated. Mice (8 to 10 weeks old) were administered bleomycin (BLM) intratracheally (2 U/kg) to establish an in vivo PF model. Murine primary lung fibroblasts were isolated and stimulated with TGF-β (10 ng/mL) for 48 h to induce its activation. Meanwhile, mice or primary lung fibroblasts were treated with different doses of piceatannol to observe its protective roles. Pulmonary function and arterial blood gas were detected to assess pulmonary physiological status. Collagen deposition and the mRNA levels of profibrotic genes were determined by H&E staining and RT-PCR. Meanwhile, the protein and mRNA markers, as well as end-product of oxidative stress were detected in vivo and in vitro. The results showed that pulmonary function was significantly impaired in BLM-induced mice, accompanied by elevated oxidative stress and excessive collagen synthesis. Piceatannol significantly improved pulmonary function and decreased oxidative injury as well as collagen synthesis in mice with PF. Mechanically, piceatannol treatment significantly inhibited the activation of JAK2/STAT3 signaling pathway in BLM-induced mice and TGF-β-induced lung fibroblasts. Additional findings also demonstrated that coumermycin A1 (C-A1), an agonist of JAK2, could abolish the effects of piceatannol on TGF-β-induced lung fibroblasts and reactivated the phosphorylation STAT3. Taken together, our study demonstrated that piceatannol could protect against oxidative injury and collagen synthesis during PF in a JAK2/STAT3 signaling pathway-dependent manner.

Topics: Animals; Bleomycin; Collagen; Fibroblasts; Janus Kinase 2; Lung; Mice; Mice, Inbred C57BL; Oxidative Stress; Pulmonary Fibrosis; RNA, Messenger; Signal Transduction; Stilbenes; Transforming Growth Factor beta

Fibrosing interstitial lung disease (ILD) develops due to the impaired reparative processes following lung tissue damage. Cellular senescence has been reported to contribute to the progression of fibrosis. However, the mechanisms by which these senescent cells initiate and/or drive the progression of lung tissue fibrosis are not yet fully understood. We demonstrated that p21

Topics: Alveolar Epithelial Cells; Animals; Bleomycin; Cyclin-Dependent Kinase Inhibitor p16; Cyclin-Dependent Kinase Inhibitor p21; Fibrosis; Mice; Pulmonary Fibrosis; Transforming Growth Factor beta

Topics: Alkaloids; Delphinium; Diterpenes; Fibrosis; Lipopolysaccharides; Molecular Docking Simulation; Pulmonary Fibrosis; Receptor, Transforming Growth Factor-beta Type I; Signal Transduction; Smad Proteins; Transforming Growth Factor beta; Transforming Growth Factor beta1

Pulmonary fibrosis (PF) is a chronic, irreversible, and debilitating lung disease that typically leads to respiratory failure, and is a major cause of morbidity and mortality. Few drugs are effective for the treatment of patients with PF or for reducing the rate of disease progression.. Transforming growth factor-β1 (TGF-β1) is a profibrotic cytokine that signals through Smad and non-Smad pathways. Verbascoside (VB) and isoverbascoside (isoVB) exhibit anti-oxidative and anti-inflammatory activities, however, their anti-fibrotic effects remain unclear. This study evaluated the effects of VB and isoVB on TGF-β1-stimulated murine lung fibroblasts (MLg 2908) and also human lung fibroblasts (confirmed by immunostaining).. Neither VB nor isoVB had a cytotoxic effect on MLg 2908 fibroblasts. Both compounds (10 μM) reduced intracellular reactive oxygen species and markedly attenuated collagen I expression in TGF-β1 (5 ng/ml)-induced MLg 2908 cells compared to TGF-β1 alone. Both compounds suppressed the TGF-β1-induced phosphorylation of Smad2/3 and ERK/p38 mitogen-activated protein kinases (MAPKs). VB and isoVB, but not pirfenidone and nintedanib, inhibited TGF-β1-induced pSmad2/3, ERK/p38 MAPK, and collagen I expression. VB and isoVB also decreased collagen I deposition in TGF-β1-induced MLg 2908 cells. Only isoVB significantly suppressed collagen I deposition in TGF-β1-induced human pulmonary cells. Our results indicated that VB and isoVB may exert antifibrotic effects by inhibiting TGF-β1-induced collagen I expression via inhibition of oxidative stress and downregulation of the Smad/non-Smad pathway.. The present findings suggest that VB or isoVB may be used as a supplement to alleviate PF.

Topics: Animals; Collagen; Fibroblasts; Glucosides; Humans; Lung; Mice; p38 Mitogen-Activated Protein Kinases; Phenols; Polyphenols; Pulmonary Fibrosis; Reactive Oxygen Species; Signal Transduction; Smad Proteins; Transforming Growth Factor beta; Transforming Growth Factor beta1

The abnormal modification of chondroitin sulfate is one of the leading causes of disease, including cancer progression. During chondroitin sulfate biosynthesis, the CHST11 enzyme plays a vital role in its modification, but its role in cancer is not fully understood. Therefore, understanding the relationship between CHST11 and pulmonary-related diseases through clinically relevant information may be useful for diagnosis or treatment.. A variety of pulmonary fibrosis clinical gene expression omnibus (GEO) datasets were used to assess the association between CHST11-related manifestations and fibrosis. Multiple lung cancer-related databases, including The Cancer Genome Atlas, GEO datasets, UCSC Xena, GEPIA2, Cbioportal and ingenuity pathway analysis were used to evaluate the clinical correlation between CHST11 and lung cancer and potential molecular mechanisms. For drug repurposing prediction, the molecules that correlated with CHST11 were subjected to the LINCS L1000 algorithm. A variety of in vitro assays were performed to evaluate the in-silico models, including RNA and protein expression, proliferation, migration and invasion.. Clinical analyses indicate that the levels of CHST11 are significantly elevated in cases of pulmonary-related diseases, including fibrosis and lung cancer. According to multiple lung cancer cohorts, CHST11 is the only member of the carbohydrate sulfotransferase family associated with overall survival for lung adenocarcinomas, and it is highly related to smoking-induced lung cancer patients. Based on the results of in vitro experiments, CHST11 expression contributes to tumor malignancy and promotes multiple fibrotic activators. Correlation-based ingenuity pathway analysis indicated that CHST11-related molecules contributed to pulmonary fibrosis or lung adenocarcinomas via similar upstream stimulators. Based on known molecular regulatory relationships, CHST11 has been associated with the regulation of TGF-β and INFγ as important molecules contributing to fibrosis and cancer progression. Interestingly, WordCloud analysis revealed that CHST11-related molecules are involved in regulation primarily by integrin signaling, and these relationships were consistently reflected in the analysis of cell lines and the clinical correlation. A CHST11 signature-based drug repurposing analysis demonstrated that the CHST11/integrin axis could be targeted by AG-1478 (Tyrphostin AG 1478), brefeldin A, geldanamycin and importazole.. This study provides the first demonstration that CHST11 may be used as a biomarker for pulmonary fibrosis or lung cancer, and the levels of CHST11 were increased by TGF-β and INFγ. The molecular simulation analyses demonstrate that the CHST11/integrin axis is a potential therapeutic target for treating lung cancer.

Topics: Adenocarcinoma of Lung; Chondroitin Sulfates; Humans; Integrins; Lung Neoplasms; Pulmonary Fibrosis; Sulfotransferases; Transforming Growth Factor beta

Lymphocyte-specific protein tyrosine kinase (Lck) is a member of the Src family of tyrosine kinases. The significance of Lck inhibition in lung fibrosis has not yet been fully elucidated, even though lung fibrosis is commonly preceded by inflammation caused by infiltration of T-cells expressing Lck. In this study, we examined the effect of Lck inhibition in an experimental mouse model of lung fibrosis. We also evaluated the effect of Lck inhibition on the expression of TGF-β1, an inhibitory cytokine regulating the immune function, in regulatory T-cells (Tregs).. Lung fibrosis was induced in mice by intratracheal administration of bleomycin. A-770041, a Lck-specific inhibitor, was administrated daily by gavage. Tregs were isolated from the lung using a CD4+CD25+ Regulatory T-cell Isolation Kit. The expression of Tgfb on Tregs was examined by flow cytometry and quantitative polymerase chain reaction. The concentration of TGF-β in bronchoalveolar lavage fluid (BALF) and cell culture supernatant from Tregs was quantified by an enzyme-linked immunosorbent assay.. A-770041 inhibited the phosphorylation of Lck in murine lymphocytes to the same degree as nintedanib. A-770041 attenuated lung fibrosis in bleomycin-treated mice and reduced the concentration of TGF-β in BALF. A flow-cytometry analysis showed that A-770041 reduced the number of Tregs producing TGF-β1 in the lung. In isolated Tregs, Lck inhibition by A-770041 decreased the Tgfb mRNA level as well as the concentration of TGF-β in the supernatant.. These results suggest that Lck inhibition attenuated lung fibrosis by suppressing TGF-β production in Tregs and support the role of Tregs in the pathogenesis of lung fibrosis.

Topics: Animals; Bleomycin; Lymphocyte Specific Protein Tyrosine Kinase p56(lck); Mice; Mice, Inbred C57BL; Pulmonary Fibrosis; T-Lymphocytes, Regulatory; Transforming Growth Factor beta; Transforming Growth Factor beta1

Adenosine receptors (ARs) are involved in the suppression and development of inflammatory and fibrotic conditions. Specifically, AR activation promotes differentiation of lung fibroblasts into myofibroblasts, typical of a fibrotic event. Pulmonary fibrosis is a severe disease characterized by inflammation and fibrosis of unknown etiology and lacking an effective treatment. The present investigation explored the action of MRS5980, a new, highly potent and selective A

Topics: Adenosine; Animals; Bleomycin; Fibroblasts; Fibrosis; Inflammation; Lung; Mice; Mice, Inbred C57BL; Pulmonary Fibrosis; Receptors, Purinergic P1; Transforming Growth Factor beta

To investigate the effect of digoxin on bleomycin-induced pulmonary fibrosis in mice, and investigate its possible mechanism through in vitro and in vivo experiments.. (1) In vivo experiment: 60 C57/BL6J mice were randomly divided into control group, pulmonary fibrosis model group (model group), pirfenidone (300 mg/kg) group, digoxin 1.0 mg/kg and 0.2 mg/kg groups, with 12 mice in each group. The pulmonary fibrosis model of mice was reproduced by single intratracheal infusion of bleomycin (5 mg/kg). The control group was given the same amount of sterile normal saline. From the next day after modeling, each group was received corresponding drugs by intragastric administration once a day for 28 days. Control group and model group were given the same amount of normal saline. The mice were sacrificed and the lung tissue was collected to detect the lung coefficient. After hematoxylin-eosin (HE) and Masson staining, the lung tissue morphology and collagen changes were observed under light microscope. Immunohistochemistry was used to detect the positive expressions of α-smooth muscle actin (α-SMA) and extracellular matrix (ECM) collagen (COL-I and COL-III) in lung tissue. The protein expressions of ECM fibronectin (FN), transforming growth factor-β (TGF-β) and phosphorylation of Smad3 (p-Smad3) in lung tissue were detected by Western blotting. (2) In vitro experiment: human embryonic lung fibroblast-1 (HFL-1) cells were cultured and divided into blank control group, fibroblast activation model group (model group), pirfenidone (2.5 mmol/L) group and digoxin 100 nmol/L and 50 nmol/L groups when cell density reached 70%-90%. After 3-hour treatment with corresponding drugs, except blank control group, the other groups were treated with TGF-β for 48 hours to establish fibroblast activation model. The expressions of α-SMA, FN and p-Smad3 proteins and the phosphorylations of phosphatidylinositol-3-kinase (PI3K)/Akt pathway proteins PI3K and Akt (p-PI3K, p-Akt) were detected by Western blotting.. (1) In vivo, compared with the control group, the alveolar structure of mice in the model group was significantly damaged, a large number of inflammatory cells infiltrated, collagen deposition in the lung interstitium was increased, the deposition of ECM in the lung tissue was also increased, and the expressions of α-SMA, FN, TGF-β and p-Smad3 protein were increased, indicating that the model of bleomycin-induced pulmonary fibrosis in mice was successfully prepared. Compared with the model group, digoxin significantly inhibited airway inflammation and collagen fiber deposition, reduced ECM deposition, and decreased the protein expressions of α-SMA, FN, TGF-β and p-Smad3, while the effect was better than that of the pirfenidone group, and the digoxin 1.0 mg/kg group had a better effect except FN [α-SMA (A value): 5.37±1.10 vs. 9.51±1.66, TGF-β protein (TGF-β/GAPDH): 0.09±0.04 vs. 0.33±0.23, p-Smad3 protein (p-Smad3/GAPDH): 0.05±0.01 vs. 0.20±0.07, all P < 0.01]. (2) In vitro, compared with the blank control group, the expressions of FN, α-SMA, p-Smad3 and PI3K/Akt signaling proteins in the model group were increased, indicating that the fibroblast activation model induced by TGF-β was successfully reproduced. Compared with the model group, digoxin significantly inhibited fibroblast activation, and decreased the expressions of FN, α-SMA, p-Smad3, and PI3K/Akt pathway proteins, moreover, the effect was better than that of the pirfenidone group, and decreased FN, SMA and p-Akt protein expressions were more obvious in digoxin 100 nmol/L group [FN protein (FN/GAPDH): 0.21±0.15 vs. 0.88±0.22, α-SMA protein (α-SMA/GAPDH): 0.20±0.01 vs. 0.50±0.08, p-Akt protein (p-Akt/GAPDH): 0.30±0.01 vs. 0.65±0.10, all P < 0.01].. Digoxin could suppress the pulmonary fibrosis in mice induced by bleomycin, which might be associated with the regulation of fibroblast activation via suppressing PI3K/Akt signaling pathway in a dose-dependent manner.

Topics: Animals; Bleomycin; Collagen; Digoxin; Fibroblasts; Humans; Mice; Phosphatidylinositol 3-Kinases; Phosphatidylinositols; Proto-Oncogene Proteins c-akt; Pulmonary Fibrosis; Saline Solution; Signal Transduction; Smad3 Protein; Transforming Growth Factor beta; Transforming Growth Factor beta1

In this paper, three optimal control problems are proposed to prevent forming lung fibrosis while control is transforming growth factor-β (TGF-β) in the myofibroblast diffusion process. Two diffusion equations for fibroblast and myofibroblast are mathematically formulated as the system's dynamic, while different optimal control model problems are proposed to find the optimal TGF-β. During solving the first optimal control problem with the regulator objection function, it is understood that the control function gets unexpected negative values. Thus, in the second optimal control problem, for the control function, the non-negative constraint is imposed. This problem is solved successfully using the extended canonical Hamiltonian equations with no flux boundary conditions. Pontryagin's minimum principle is used to solve the related optimal control problems successfully. In the third optimal control problem, the fibroblast equation is added to a dynamic system consisting of the partial differential equation. The two-dimensional diffusion equations for fibroblast and myofibroblast are transferred to a system of ordinary differential equations using the central finite differences explicit method. Three theorems and two propositions are proved using extended Pontryagin's minimum principle and the extended Hamiltonian equations. Numerical results are given. We believe that this optimal strategy can help practitioners apply some medication to reduce the TGF-β in preventing the formation of pulmonary fibrosis.

Topics: Fibroblasts; Fibrosis; Humans; Myofibroblasts; Pulmonary Fibrosis; Transforming Growth Factor beta

To investigate the effects of chronic intermittent hypoxia (CIH) on the expression of transforming growth factor-β (TGF-β), P-samd3, serum laminin (LN) and hyaluronidase (HA) in mouse lung tissues and the protective effects of Bu Zhong Yi Qi decoction on lung interstitial deposition damage in CIH mice.. Fifty SPF-grade C57BL mice were randomly divided into five groups (. HE staining showed alveolar collapse, septal thickening and epithelial cell necrosis in CIH mice, Masson showed massive collagen fiber proliferation and deposition in lung interstitium, while the above changes in lung tissues were significantly improved in the CIH + Bu Zhong Yi Qi decoction groups compared with the CIH group. TGF-β1, P-smad3 and Collagen I, Collagen Ⅲ, and α-SMA expression levels were increased compared with the blank control group (. Bu Zhong Yi Qi decoction can inhibit alveolar structural changes and excessive collagen deposition in the interstitium of CIH mice, and then improve lung function in CIH mice. The mechanism may be related to the down-regulation of protein expression related to TGF-β/smads signaling pathway by Bu Zhong Yi Qi decoction.

Topics: Animals; Drugs, Chinese Herbal; Fibrosis; Mice; Mice, Inbred C57BL; Pulmonary Fibrosis; Transforming Growth Factor beta; Transforming Growth Factor beta1

Idiopathic Pulmonary Fibrosis (IPF) is a grave disease characterized by abnormal wound healing associated with chronic, progressive, irreversible fatal lung disease, leading to persistent injuries to the alveolar epithelium. A consequent disturbance of fibroblast proliferation and apoptosis results in subsequent release of pro-inflammatory and pro-fibrotic mediators coupled with accumulation of extracellular matrix within the interstitium. Inexorable distortion of lung alveolar architecture leads to respiratory failure with a median survival rate of 3-5 years. Currently available drugs can only slowdown the progression of fibrosis and novel drugs are warranted to treat this disease. In this study, we demonstrate the fibro-protective effect of diosgenin in experimental lung fibrosis through regulation of Epithelial Mesenchymal Transition (EMT). A single dose of 3 U/kg body weight (b.wt) Bleomycin (BLM) was administered intratracheally in Wistar male albino rats and fibrotic animals were treated with diosgenin (100 mg/kg b.wt) orally for 28 days. BLM administered rat show histological alteration with increased mast cell and collagen accumulation. BLM induced abnormalities were significantly reduced upon treatment with diosgenin. Western blot analysis revealed an increased level of pro-inflammatory and pro-fibrotic molecules such as IL-1β and TGF-β in BLM induced rats. Rats supplemented with diosgenin showed a decreased expression of inflammatory and pro-fibrotic mediators. Markers of EMT molecules were evaluated by immunoblot. The results of immunoblot demonstrate that diosgenin regulated the expression of TGF-β mediated EMT. Hence, from the overall study, administration of diosgenin prevents pulmonary fibrosis by restraint inflammation and EMT.

Topics: Animals; Bleomycin; Diosgenin; Epithelial-Mesenchymal Transition; Lung; Male; Pulmonary Fibrosis; Rats; Rats, Wistar; Transforming Growth Factor beta; Transforming Growth Factor beta1

The U.S. Food and Drug Administration-approved proteasomal inhibitor bortezomib (BTZ) has attracted interest for its potential antifibrotic actions. However, neither its

Topics: Adult; Apoptosis; Bleomycin; Bortezomib; Cell Dedifferentiation; Cell Line; Cell Proliferation; Dual Specificity Phosphatase 1; fas Receptor; Fibroblast Growth Factor 2; Fibroblasts; Humans; Myofibroblasts; NF-kappa B; Prostaglandins; Proteasome Inhibitors; Pulmonary Fibrosis; Signal Transduction; Transcription, Genetic; Transforming Growth Factor beta

The TβRII∆k-fib transgenic (TG) mouse model of scleroderma replicates key fibrotic and vasculopathic complications of systemic sclerosis through fibroblast-directed upregulation of TGFβ signalling. We have examined peroxisome proliferator-activated receptor (PPAR) pathway perturbation in this model and explored the impact of the pan-PPAR agonist lanifibranor on the cardiorespiratory phenotype.. PPAR pathway gene and protein expression differences from TG and WT sex-matched littermate mice were determined at baseline and following administration of one of two doses of lanifibranor (30 mg/kg or 100 mg/kg) or vehicle administered by daily oral gavage up to 4 weeks. The prevention of bleomycin-induced lung fibrosis and SU5416-induced pulmonary hypertension by lanifibranor was explored.. Gene expression data were consistent with the downregulation of the PPAR pathway in the TβRII∆k-fib mouse model. TG mice treated with high-dose lanifibranor demonstrated significant protection from lung fibrosis after bleomycin and from right ventricular hypertrophy following induction of pulmonary hypertension by SU5416, despite no significant change in right ventricular systolic pressure.. In the TβRII∆k-fib mouse strain, treatment with 100 mg/kg lanifibranor reduces the development of lung fibrosis and right ventricular hypertrophy induced by bleomycin or SU5416, respectively. Reduced PPAR activity may contribute to the exaggerated fibroproliferative response to tissue injury in this transgenic model of scleroderma and its pulmonary complications.

Topics: Animals; Benzothiazoles; Mice; Mice, Transgenic; PPAR gamma; Pulmonary Fibrosis; Scleroderma, Systemic; Signal Transduction; Sulfonamides; Transforming Growth Factor beta

Idiopathic pulmonary fibrosis (IPF) induces significant morbidity and mortality, for which there are limited therapeutic options available. Here, we found that tetraethylthiuram disulphide (disulfiram, DSF), a derivative of thiuram, used in the treatment of alcohol abuse, has an inhibitory effect on bleomycin (BLM)-induced pulmonary fibrosis via the attenuation of the fibroblast-to-myofibroblast transition, migration, and proliferation of fibroblasts. Furthermore, DSF inhibited the activation of primary pulmonary fibroblasts and fibroblast cell line under transforming growth factor-β 1 (TGF-β1) challenge. Mechanistically, the anti-fibrotic effect of DSF on fibroblasts depends on the inhibition of TGF-β signalling. We further determined that DSF interrupts the interaction between SMAD3 and TGF-β receptor Ι (TBR Ι), and identified that DSF directly binds with SMAD3, in which Trp326, Thr330, and Cys332 of SMAD3 are critical binding sites for DSF. Collectively, our results reveal a powerful anti-fibrotic function of DSF in pulmonary fibrosis through the inhibition of TGF-β/SMAD signalling in pulmonary fibroblasts, indicating that DSF is a promising therapeutic candidate for IPF.

Topics: Actins; Alcohol Deterrents; Animals; Bleomycin; Collagen Type I, alpha 1 Chain; Disulfiram; Fibronectins; HEK293 Cells; Humans; Lung; Male; Mice; Mice, Inbred C57BL; NIH 3T3 Cells; Pulmonary Fibrosis; Signal Transduction; Transforming Growth Factor beta

We previously reported that female mice exhibit protection against chemically induced pulmonary fibrosis and suggested a potential role of estrogen. Phytoestrogens act, at least in part, via stimulation of estrogen receptors; furthermore, compared to residents of Western countries, residents of East Asian countries consume higher amounts of phytoestrogens and exhibit lower rates of pulmonary fibrosis. Therefore, we tested the hypothesis that dietary phytoestrogens ameliorate the severity of experimentally induced pulmonary fibrosis. Male mice placed on either regular soybean diet or phytoestrogen-free diet were instilled with 0.1 N HCl to provoke pulmonary fibrosis. Thirty days later, lung mechanics were measured as indices of lung function and bronchoalveolar lavage fluid (BALF) and lung tissue were analyzed for biomarkers of fibrosis. Mice on phytoestrogen-free diet demonstrated increased mortality and stronger signs of chronic lung injury and pulmonary fibrosis, as reflected in the expression of collagen, extracellular matrix deposition, histology, and lung mechanics, compared to mice on regular diet. We conclude that dietary phytoestrogens play an important role in the pathogenesis of pulmonary fibrosis and suggest that phytoestrogens (e.g., genistein) may be useful as part of a therapeutic regimen against hydrochloric acid-induced lung fibrosis and chronic lung dysfunction.

Topics: Animals; Chronic Disease; Diet; Extracellular Matrix Proteins; Hydrochloric Acid; Inflammation; Leukocyte Count; Lung; Lung Injury; Male; Mice, Inbred C57BL; Models, Biological; Phytoestrogens; Pulmonary Fibrosis; RNA, Messenger; Signal Transduction; Transforming Growth Factor beta

Fibrosis is the formation of scar tissue due to injury or long-term inflammation and is a leading cause of morbidity and mortality. Activation of the pro-fibrotic cytokine transforming growth factor-β (TGFβ) via the alpha-V beta-6 (αvβ6) integrin has been identified as playing a key role in the development of fibrosis. Therefore, a drug discovery programme to identify an orally bioavailable small molecule αvβ6 arginyl-glycinyl-aspartic acid (RGD)-mimetic was initiated. As part of a medicinal chemistry programme GSK3335103 was identified and profiled in a range of pre-clinical in vitro and in vivo systems. GSK3335103 was shown to bind to the αvβ6 with high affinity and demonstrated fast binding kinetics. In primary human lung epithelial cells, GSK3335103-induced concentration- and time-dependent internalisation of αvβ6 with a rapid return of integrin to the cell surface observed after washout. Following sustained engagement of the αvβ6 integrin in vitro, lysosomal degradation was induced by GSK3335103. GSK3335103 was shown to engage with the αvβ6 integrin and inhibit the activation of TGFβ in both ex vivo IPF tissue and in a murine model of bleomycin-induced lung fibrosis, as measured by αvβ6 engagement, TGFβ signalling and collagen deposition, with a prolonged duration of action observed in vivo. In summary, GSK3335103 is a potent αvβ6 inhibitor that attenuates TGFβ signalling in vitro and in vivo with a well-defined pharmacokinetic/pharmacodynamic relationship. This translates to a significant reduction of collagen deposition in vivo and therefore GSK3335103 represents a potential novel oral therapy for fibrotic disorders.

Topics: Administration, Oral; Animals; Antifibrotic Agents; Antigens, Neoplasm; Biological Availability; Bleomycin; Cells, Cultured; Disease Models, Animal; Epithelial Cells; Humans; Integrins; Lung; Lysosomes; Male; Mice; Oligopeptides; Primary Cell Culture; Proteolysis; Pulmonary Fibrosis; Transforming Growth Factor beta

Initially, endothelin (ET)-2 was described as an endothelium-derived vasoconstrictor. However, accumulating evidence suggests the involvement of ET-2 in non-cardiovascular physiology and disease pathophysiology. The deficiency of ET-2 in mice can be lethal, and such mice exhibit a distinct developmental abnormality in the lungs. Nonetheless, the definite role of ET-2 in the lungs remains unclear. The ET-2 isoform, ET-1, promotes pulmonary fibrosis in mice. Although endothelin receptor antagonists (ERAs) show improvements in bleomycin-induced pulmonary fibrosis in mouse models, clinical trials examining ERAs for pulmonary fibrosis treatment have been unsuccessful, even showing harmful effects in patients. We hypothesized that ET-2, which activates the same receptor as ET-1, plays a distinct role in pulmonary fibrosis. In this study, we showed that ET-2 is expressed in the lung epithelium, and ET-2 deletion in epithelial cells of mice results in the exacerbation of bleomycin-induced pulmonary fibrosis. ET-2 knockdown in lung epithelial cell lines resulted in increased apoptosis mediated via oxidative stress induction. In contrast to the effects of ET-1, which induced fibroblast activation, ET-2 hampered fibroblast activation in primary mouse lung fibroblast cells by inhibiting the TGF-β-SMAD2/3 pathway. Our results demonstrated the divergent roles of ET-1 and ET-2 in pulmonary fibrosis pathophysiology and suggested that ET-2, expressed in epithelial cells, exerts protective effects against the development of pulmonary fibrosis in mice.

Topics: Animals; Bleomycin; Endothelin-2; Epithelial Cells; Epithelium; Lung; Mice; Pulmonary Fibrosis; Transforming Growth Factor beta

Acute respiratory distress syndrome (ARDS) followed by repair with lung remodeling is observed in COVID-19. These findings can lead to pulmonary terminal fibrosis, a form of irreversible sequelae. There is evidence that TGF-β is intimately involved in the fibrogenic process. When activated, TGF-β promotes the differentiation of fibroblasts into myofibroblasts and regulates the remodeling of the extracellular matrix (ECM). In this sense, the present study evaluated the histopathological features and immunohistochemical biomarkers (ACE-2, AKT-1, Caveolin-1, CD44v6, IL-4, MMP-9, α-SMA, Sphingosine-1, and TGF-β1 tissue expression) involved in the TGF-β1 signaling pathways and pulmonary fibrosis. The study consisted of 24 paraffin lung samples from patients who died of COVID-19 (COVID-19 group), compared to 10 lung samples from patients who died of H1N1pdm09 (H1N1 group) and 11 lung samples from patients who died of different causes, with no lung injury (CONTROL group). In addition to the presence of alveolar septal fibrosis, diffuse alveolar damage (DAD) was found to be significantly increased in the COVID-19 group, associated with a higher density of Collagen I (mature) and III (immature). There was also a significant increase observed in the immunoexpression of tissue biomarkers ACE-2, AKT-1, CD44v6, IL-4, MMP-9, α-SMA, Sphingosine-1, and TGF-β1 in the COVID-19 group. A significantly lower expression of Caveolin-1 was also found in this group. The results suggest the participation of TGF-β pathways in the development process of pulmonary fibrosis. Thus, it would be plausible to consider therapy with TGF-β inhibitors in those patients recovered from COVID-19 to mitigate a possible development of pulmonary fibrosis and its consequences for post-COVID-19 life quality.

Topics: Actins; Adrenal Cortex Hormones; Adult; Aged; Aged, 80 and over; Angiotensin-Converting Enzyme 2; Caveolin 1; Collagen Type I; Collagen Type III; COVID-19; COVID-19 Drug Treatment; Female; Humans; Hyaluronan Receptors; Immunohistochemistry; Influenza A Virus, H1N1 Subtype; Influenza, Human; Interleukin-4; Male; Matrix Metalloproteinase 9; Middle Aged; Proto-Oncogene Proteins c-akt; Pulmonary Fibrosis; Retrospective Studies; Signal Transduction; Transforming Growth Factor beta; Transforming Growth Factor beta1

Few reports have documented the ability of phosphodiesterase-5 inhibitors (PDE-5-Is) to ameliorate idiopathic pulmonary fibrosis (IPF) mainly by their anti-inflammatory/antioxidant capacities, without unveiling the possible molecular mechanisms involved. Because of the recent role of miR-200 family and Sonic Hedgehog (SHH) trajectory in IPF, we have studied their impact on the anti-fibrotic potential of tadalafil against bleomycin-induced pulmonary fibrosis. Animals were allocated into normal-control, bleomycin-fibrotic control, and bleomycin post-treated with tadalafil or dexamethasone, as the reference drug. On the molecular level, tadalafil has reverted the bleomycin effect on all the assessed parameters. Tadalafil upregulated the gene expression of miR-200a, but decreased the smoothened (SMO) and the transcription factors glioma-associated oncogene homolog (Gli-1, Gli-2), members of SHH pathway. Additionally, tadalafil ebbed transforming growth factor (TGF)-β, its canonical (SMAD-3/alpha smooth muscle actin [α-SMA] and Snail), and non-canonical (p-Akt/p-Forkhead box O3 (FOXO3) a) pathways. Besides, a strong negative correlation between miR-200a and the analyzed pathways was proved. The effect of tadalafil was further confirmed by the improved lung structure and the reduced Ashcroft score/collagen deposition. The results were comparable to that of dexamethasone. In conclusion, our study has highlighted the involvement of miR-200a in the anti-fibrotic effect of tadalafil with the inhibition of SHH hub and the pro-fibrotic pathways (TGF-β/ SMAD-3/α-SMA, Snail and p-AKT/p-FOXO3a). Potential anti-fibrotic effect of tadalafil. Modulation of miR200a/SHH/canonical and non-canonical TGF-β trajectories. → : stimulatory effect; ┴: inhibitory effect.

Topics: Animals; Bleomycin; Dexamethasone; Disease Models, Animal; Hedgehog Proteins; Male; MicroRNAs; Phosphodiesterase 5 Inhibitors; Pulmonary Fibrosis; Rats; Rats, Wistar; Tadalafil; Transforming Growth Factor beta

Alstonia scholaris (L.) R. Br. (Apocynaceae) is a Dai folk medicine for the treatment of lung diseases in China.. The present study investigated the anti-pulmonary fibrosis effects of total alkaloids (TA) and the potential active ingredients and its possible mechanism.. After intratracheal instillation of bleomycin (BLM, 5 mg/kg), mice were divided into ten groups, and orally treated with the corresponding samples once daily for 28 days. The effect of indole alkaloids was determined through analysis of cytokines, as well as histopathological examinations and gene expressions.. Severe lung fibrosis was observed in the BLM-treated mice on day 28. However, the administration of TA significantly ameliorated the pathological changes in the lungs, decreased the content of Krebs von den Lungen-6, lactate dehydrogenase, transforming growth factor-β (TGF-β), hydroxyproline, type I collagen, and malonaldehyde, and enhanced the activity of superoxide dismutase in the serum and lung tissues. In addition, the enhanced TGF-β and matrix metalloproteinase-1 (MMP-1) expressions in BLM-induced mice were obviously weakened by indole alkaloids, as well as the ratio of matrix metalloproteinase-1 to tissue inhibitor of metalloproteinase-1 was decreased. Moreover, picrinine and scholaricine yielded markedly better values in the aforementioned indices than those in other samples, indicating that they may be the active ingredients of alkaloids.. TA exerted protective effects against BLM-induced pulmonary fibrosis by reducing collagen deposition through TGF-β/MMP-1 pathway.

Topics: Alstonia; Animals; Bleomycin; Collagen; Cytokines; Disease Models, Animal; Gene Expression Regulation; Indole Alkaloids; Inflammation Mediators; Lung; Male; Matrix Metalloproteinase 13; Mice, Inbred ICR; Plant Extracts; Pulmonary Fibrosis; Signal Transduction; Tissue Inhibitor of Metalloproteinase-1; Transforming Growth Factor beta

Mushroom Inonotus sanghuang has been characterized as a traditional medicine in China and has pharmacological activities to treat inflammation, gastroenteric dysfunction, and cancer. Recently, we reported the impact of Inonotus sanghuang extract (ISE) from ethyl acetate fraction on bleomycin (BLM)-induced acute lung injury in mice. Here, we aimed to investigate ISE's impact on pulmonary fibrosis using in vivo and in vitro models and the underlying mechanisms. To evaluate pulmonary fibrosis, female C57BL/6 mice fed ISE (0% or 0.6% in diet) for 4 weeks were instilled intratracheally with BLM and then continued the same diet before the end of the experiment. A549 cells were used to evaluate the epithelial-mesenchymal transition (EMT). Feeding ISE improved BLM-treated mice's survival via decreasing lung infiltrating cells and fibrosis, followed by reducing hydroxyproline content, collagen deposition, and mesenchymal markers (α-SMA and vimentin) while increasing epithelial marker E-cadherin. ISE also suppressed the TGF-β expression, Smad2/3 phosphorylation, and EMT-related transcription factor Snail upon BLM instillation. Iin vitro study demonstrated that ISE inhibited TGF-β-induced EMT-like phenotype and cell behaviors, the expression of α-SMA and vimentin, and prevented E-cadherin reduction of A549 cells. Consistent with in vivo study, ISE abrogated p-Smad2/3, and Snail expression. Finally, the influence of ISE on EMT was not due to ISE toxicity. Our findings indicated that ISE effectively attenuated BLM-induced lung fibrosis. These ISE properties were thought to be involved in interfering TGF-β, Smad2/3 phosphorylation, and EMT process, suggesting that the material has the potential health benefits to improve lung fibrosis.

Topics: A549 Cells; Animals; Basidiomycota; Bleomycin; Bronchoalveolar Lavage Fluid; Cell Movement; Collagen Type I; Disease Models, Animal; Epithelial-Mesenchymal Transition; Female; Humans; Lung; Mice, Inbred C57BL; Phosphorylation; Pulmonary Fibrosis; Respiratory System Agents; Signal Transduction; Smad2 Protein; Smad3 Protein; Snail Family Transcription Factors; Transforming Growth Factor beta

Abnormal activation of transforming growth factor (TGF)-β is a common cause of fibroblast activation and fibrosis. In bleomycin (BLM)-induced lung fibrosis, the marked expression of phospho-Src homology-2 domain-containing phosphatase (SHP) 2, phospho-signal transducer and activator of transcription (STAT) 3, and suppressor of cytokine signaling (SOCS) 3 was highly associated with pulmonary parenchymal lesions and collagen deposition. Human pulmonary fibroblasts differentiated into myofibroblasts exhibited activation of SHP2, SOCS3, protein inhibitor of activated STAT1, STAT3, interleukin (IL)-6, and IL-10. The significant retardation of interferon (IFN)-γ signaling in myofibroblasts was revealed by the decreased expression of phospho-STAT1, IFN-γ-associated genes, and IFN-γ-inducible protein (IP) 10. Microarray analysis showed an induction of fibrotic genes in TGF-β1-differentiated myofibroblasts, whereas IFN-γ-regulated anti-fibrotic genes were suppressed. Interestingly, BIBF 1120 treatment effectively inhibited both STAT3 and SHP2 phosphorylation in TGF-β1-differentiated myofibroblasts and BLM fibrotic lung tissues, which was accompanied by suppression of fibroblast-myofibroblast transition. Moreover, the combined treatment of BIBF 1120 plus IFN-γ or SHP2 inhibitor PHPS1 plus IFN-γ markedly reduced TGF-β1-induced α-smooth muscle actin and further ameliorated BLM lung fibrosis. Accordingly, myofibroblasts were hyporesponsiveness to IFN-γ, while blockade of SHP2 contributed to the anti-fibrotic efficacy of IFN-γ.

Topics: Animals; Antibiotics, Antineoplastic; Bleomycin; Fibroblasts; Humans; Interferon-gamma; Lung; Male; Mice; Mice, Inbred C57BL; Myofibroblasts; Pulmonary Fibrosis; Rats; Rats, Sprague-Dawley; Transforming Growth Factor beta

Natural products are one of the best sources for the discovery of novel drugs and compounds for multiple diseases. Pulmonary fibrosis (PF) is a chronic, progressive, irreversible, and fatal fibrotic disorder of lungs with unknown etiology and finite therapeutic choices. The use of naturally occurring phytomedicines has emerged to counteract many fibrotic disorders involving oxidative stress and inflammation. In the present study, we evaluated the protective effects of ferulic acid (FA), in an animal model of silica-induced PF. Pulmonary function of mice was evaluated by performing radiological analysis, bronchoalveolar lavage fluid (BALF), inflammatory cytokines, histology and protein expression studies. Our findings revealed that mice challenged with silica displayed characteristic features of pulmonary injury and fibrosis. However, treatment with FA significantly restored the accumulation of inflammatory cells in BALF. FA led to a partial reversal of silica-induced fibrotic changes in the pulmonary tissue. Subsequently, FA halts the progression of PF in a dose-dependent manner by ameliorating the expression of fibrotic proteins including collagen-I, TGF-β, p-smad2/3 and prevented epithelial-mesenchymal transition (EMT). Collectively, the present study suggests that the inhibition of oxidative stress, inflammatory and TGF-β/smad signalling might be involved in the observed anti-fibrotic benefits of FA against silica-induced PF in mice.

Topics: Animals; Bronchoalveolar Lavage Fluid; Coumaric Acids; Gene Expression Regulation; Inflammation; Male; Mice; Oxidative Stress; Pulmonary Fibrosis; Random Allocation; Silicon Dioxide; Smad Proteins; Transforming Growth Factor beta

Systemc sclerosis (SSc) is an autoimmune disorder characterized by fibrosis of the skin and internal organs. Recently, it has been shown that leucine-rich α-2 glycoprotein (LRG) functions as a modulator of transforming growth factor-β (TGF-β) signaling in fibrosis. We aimed to characterize the effect of LRG in SSc model and SSc patients.. Histological analysis was performed on LRG knockout (KO) and wild type (WT) mouse in the skin and the lung after bleomycin administration. Serum LRG levels were measured during the injection period. Gene expression analysis of the skin and lung tissue from LRG KO and WT mice was performed. In addition, serum LRG levels were determined in SSc patients and healthy controls.. LRG KO mice display an inhibition of fibrosis in the skin in association with a decrease of dermal thickness, collagen deposition, and phospho-Smad3 expression after bleomycin. Serum LRG concentration significantly increased in WT mice after bleomycin. There was also a suppression of inflammation and fibrosis in the LRG KO mouse lung indicated by a reduction of lung weight, collagen content, and phospho-Smad3 expression after bleomycin. Gene expressions of TGF-β and Smad2/3 were significantly reduced in LRG KO mice. Serum LRG levels in SSc patients were significantly higher than those in controls.. LRG promotes fibrotic processes in SSc model through TGF-β-Smad3 signaling, and LRG can be a biomarker for SSc in humans and also a potential therapeutic target for SSc.

Topics: Animals; Bleomycin; Disease Models, Animal; Fibroblasts; Fibrosis; Glycoproteins; Humans; Mice; Pulmonary Fibrosis; Scleroderma, Systemic; Skin; Transforming Growth Factor beta

Connective tissue growth factor (CTGF, CCN2) is a matricellular protein which plays key roles in normal mammalian development and in tissue homeostasis and repair. In pathological conditions, dysregulated CCN2 has been associated with cancer, cardiovascular disease, and tissue fibrosis. In this study, genetic manipulation of the CCN2 gene was employed to investigate the role of CCN2 expression in vitro and in experimentally-induced models of pulmonary fibrosis and pulmonary arterial hypertension (PAH). Knocking down CCN2 using siRNA reduced expression of pro-fibrotic markers (fibronectin p < 0.01, collagen type I p < 0.05, α-SMA p < 0.0001, TIMP-1 p < 0.05 and IL-6 p < 0.05) in TGF-β-treated lung fibroblasts derived from systemic sclerosis patients. In vivo studies were performed in mice using a conditional gene deletion strategy targeting CCN2 in a fibroblast-specific and time-dependent manner in two models of lung disease. CCN2 deletion significantly reduced pulmonary interstitial scarring and fibrosis following bleomycin-instillation, as assessed by fibrotic scores (wildtype bleomycin 3.733 ± 0.2667 vs CCN2 knockout (KO) bleomycin 4.917 ± 0.3436, p < 0.05) and micro-CT. In the well-established chronic hypoxia/Sugen model of pulmonary hypertension, CCN2 gene deletion resulted in a significant decrease in pulmonary vessel remodelling, less right ventricular hypertrophy and a reduction in the haemodynamic measurements characteristic of PAH (RVSP and RV/LV + S were significantly reduced (p < 0.05) in CCN2 KO compared to WT mice in hypoxic/SU5416 conditions). These results support a prominent role for CCN2 in pulmonary fibrosis and in vessel remodelling associated with PAH. Therefore, therapeutics aimed at blocking CCN2 function are likely to benefit several forms of severe lung disease.

Topics: Animals; Antibiotics, Antineoplastic; Bleomycin; Cells, Cultured; Collagen Type I; Connective Tissue Growth Factor; Disease Models, Animal; Gene Deletion; Humans; Mice; Mice, Knockout; Pulmonary Arterial Hypertension; Pulmonary Fibrosis; Signal Transduction; Transforming Growth Factor beta

Pulmonary fibrosis (PF), the end point of interstitial lung diseases, is characterized by myofibroblast over differentiation and excessive extracellular matrix accumulation, leading to progressive organ dysfunction and usually a terminal outcome. Studies have shown that umbilical cord-derived mesenchymal stromal cells (uMSCs) could alleviate PF; however, the underlying mechanism remains to be elucidated.. The therapeutic effects of uMSC-derived extracellular vesicles (uMSC-EVs) on PF were evaluated using bleomycin (BLM)-induced mouse models. Then, the role and mechanism of uMSC-EVs in inhibiting myofibroblast differentiation were investigated in vivo and in vitro.. Treatment with uMSC-EVs alleviated the PF and enhanced the proliferation of alveolar epithelial cells in BLM-induced mice, thus improved the life quality, including the survival rate, body weight, fibrosis degree, and myofibroblast over differentiation of lung tissue. Moreover, these effects of uMSC-EVs on PF are likely achieved by inhibiting the transforming growth factor-β (TGF-β) signaling pathway, evidenced by decreased expression levels of TGF-β2 and TGF-βR2. Using mimics of uMSC-EV-specific miRNAs, we found that miR-21 and miR-23, which are highly enriched in uMSC-EVs, played a critical role in inhibiting TGF-β2 and TGF-βR2, respectively.. The effects of uMSCs on PF alleviation are likely achieved via EVs, which reveals a new role of uMSC-EV-derived miRNAs, opening a novel strategy for PF treatment in the clinical setting.

Topics: Animals; Bleomycin; Extracellular Vesicles; Mesenchymal Stem Cells; Mice; Pulmonary Fibrosis; Signal Transduction; Transforming Growth Factor beta; Transforming Growth Factors; Umbilical Cord

Radiation-induced lung injury (RILI) is a progressive condition with an early phase (radiation pneumonitis) and a late phase (lung fibrosis). RILI may occur after partial-body ionizing radiation exposures or internal radioisotope exposure, with wide individual variability in timing and extent of lung injury. This study aimed to provide new insights into the pathogenesis and progression of RILI in the nonhuman primate (NHP) rhesus macaque model.. We used an integrative approach to understand RILI and its evolution at clinical and molecular levels in 17 NHPs exposed to 10 Gy of whole-thorax irradiation in comparison with 3 sham-irradiated control NHPs. Clinically, we monitored respiratory rates, computed tomography (CT) scans, plasma cytokine levels, and bronchoalveolar lavage (BAL) over 8 months and lung samples collected at necropsy for molecular and histopathologic analyses using RNA sequencing and immunohistochemistry.. Elevated respiratory rates, greater CT density, and more severe pneumonitis with increased macrophage content were associated with early mortality. Radiation-induced lung fibrosis included polarization of macrophages toward the M2-like phenotype, TGF-β signaling, expression of CDKN1A/p21 in epithelial cells, and expression of α-SMA in lung stroma. RNA sequencing analysis of lung tissue revealed SERPINA3, ATP12A, GJB2, CLDN10, TOX3, and LPA as top dysregulated transcripts in irradiated animals. In addition to transcriptomic data, we observed increased protein expression of SERPINA3, TGF-β1, CCL2, and CCL11 in BAL and plasma samples.. Our combined clinical, imaging, histologic, and transcriptomic analysis provides new insights into the early and late phases of RILI and highlights possible biomarkers and potential therapeutic targets of RILI. Activation of TGF-β and macrophage polarization appear to be key mechanisms involved in RILI.

Topics: Animals; Cell Cycle Checkpoints; Cytokines; Gene Expression Profiling; Lung; Lung Injury; Macaca mulatta; Macrophages; Pulmonary Fibrosis; Radiation Injuries, Experimental; Radiation Pneumonitis; Tomography, X-Ray Computed; Transforming Growth Factor beta

Topics: COVID-19; Dietary Supplements; Down-Regulation; Drug Discovery; Fibroblasts; Gene Expression Profiling; Humans; Pulmonary Fibrosis; Research Design; SARS-CoV-2; Signal Transduction; Transforming Growth Factor beta

Pulmonary fibrosis is an irreversible, potentially fatal disease. Adrenomedullin (AM) is a multifunctional peptide whose activity is regulated by receptor activity-modifying protein 2 (RAMP2). In the present study, we used the bleomycin (BLM)-induced mouse pulmonary fibrosis model to investigate the pathophysiological significance of the AM-RAMP2 system in the lung. In heterozygous AM knockout mice (AM+/-), hydroxyproline content and Ashcroft scores reflecting the fibrosis severity were significantly higher than in wild-type mice (WT). During the acute phase after BLM administration, FACS analysis showed significant increases in eosinophil, monocyte, and neutrophil infiltration into the lungs of AM+/-. During the chronic phase, fibrosis-related molecules were upregulated in AM+/-. Notably, nearly identical changes were observed in RAMP2+/-. AM administration reduced fibrosis severity. In the lungs of BLM-administered AM+/-, the activation level of Smad3, a receptor-activated Smad, was higher than in WT. In addition, Smad7, an antagonistic Smad, was downregulated and microRNA-21, which targets Smad7, was upregulated compared to WT. Isolated AM+/- lung fibroblasts showed less proliferation and migration capacity than WT fibroblasts. Stimulation with TGF-β increased the numbers of α-SMA-positive myofibroblasts, which were more prominent among AM+/- cells. TGF-β-stimulated AM+/- myofibroblasts were larger and exhibited greater contractility and extracellular matrix production than WT cells. These cells were α-SMA (+), F-actin (+), and Ki-67(-) and appeared to be nonproliferating myofibroblasts (non-p-MyoFbs), which contribute to the severity of fibrosis. Our findings suggest that in addition to suppressing inflammation, the AM-RAMP2 system ameliorates pulmonary fibrosis by suppressing TGF-β-Smad3 signaling, microRNA-21 activity and differentiation into non-p-MyoFbs.

Topics: Adrenomedullin; Animals; Bleomycin; Cell Differentiation; Disease Models, Animal; Drug Evaluation, Preclinical; Infusions, Intravenous; Male; Mice, Inbred C57BL; Mice, Knockout; MicroRNAs; Myofibroblasts; Pulmonary Fibrosis; Receptor Activity-Modifying Protein 2; Smad7 Protein; Transforming Growth Factor beta

Dysregulation of non-coding RNAs (ncRNAs) has been proved to play pivotal roles in epithelial-mesenchymal transition (EMT) and fibrosis. We have previously demonstrated the crucial function of long non-coding RNA (lncRNA) ATB in silica-induced pulmonary fibrosis-related EMT progression. However, the underlying molecular mechanism has not been fully elucidated. Here, we verified miR-29b-2-5p and miR-34c-3p as two vital downstream targets of lncRNA-ATB. As opposed to lncRNA-ATB, a significant reduction of both miR-29b-2-5p and miR-34c-3p was observed in lung epithelial cells treated with TGF-β1 and a murine silicosis model. Overexpression miR-29b-2-5p or miR-34c-3p inhibited EMT process and abrogated the pro-fibrotic effects of lncRNA-ATB in vitro. Further, the ectopic expression of miR-29b-2-5p and miR-34c-3p with chemotherapy attenuated silica-induced pulmonary fibrosis in vivo. Mechanistically, TGF-β1-induced lncRNA-ATB accelerated EMT as a sponge of miR-29b-2-5p and miR-34c-3p and shared miRNA response elements with MEKK2 and NOTCH2, thus relieving these two molecules from miRNA-mediated translational repression. Interestingly, the co-transfection of miR-29b-2-5p and miR-34c-3p showed a synergistic suppression effect on EMT in vitro. Furthermore, the co-expression of these two miRNAs by using adeno-associated virus (AAV) better alleviated silica-induced fibrogenesis than single miRNA. Approaches aiming at lncRNA-ATB and its downstream effectors may represent new effective therapeutic strategies in pulmonary fibrosis.

Topics: A549 Cells; Animals; Cell Line; Epithelial-Mesenchymal Transition; Humans; Male; MAP Kinase Kinase Kinase 2; Mice; Mice, Inbred C57BL; MicroRNAs; Pulmonary Fibrosis; Receptor, Notch2; Response Elements; RNA, Long Noncoding; Transforming Growth Factor beta

It is well known that supplemental oxygen used to treat preterm infants in respiratory distress is associated with permanently disrupting lung development and the host response to influenza A virus (IAV). However, many infants who go home with normally functioning lungs are also at risk for hyperreactivity after a respiratory viral infection. We recently reported a new, low-dose hyperoxia mouse model (40% for 8 days; 40×8) that causes a transient change in lung function that resolves, rendering 40×8 adult animals functionally indistinguishable from room air controls. Here we report that when infected with IAV, 40×8 mice display an early transient activation of TGFβ signaling and later airway hyperreactivity associated with peribronchial inflammation (profibrotic macrophages) and fibrosis compared with infected room air controls, suggesting neonatal oxygen induced hidden molecular changes that prime the lung for hyperreactive airways disease. Although searching for potential activators of TGFβ signaling, we discovered that thrombospondin-1 (TSP-1) is elevated in naïve 40×8 mice compared with controls and localized to lung megakaryocytes and platelets before and during IAV infection. Elevated TSP-1 was also identified in human autopsy samples of former preterm infants with bronchopulmonary dysplasia. These findings reveal how low doses of oxygen that do not durably change lung function may prime it for hyperreactive airways disease by changing expression of genes, such as TSP-1, thus helping to explain why former preterm infants who have normal lung function are susceptible to airway obstruction and increased morbidity after viral infection.

Topics: Animals; Bronchial Hyperreactivity; Bronchopulmonary Dysplasia; Cell Line; Disease Models, Animal; Dogs; Female; Humans; Hyperoxia; Influenza A virus; Influenza, Human; Madin Darby Canine Kidney Cells; Male; Mice; Mice, Inbred C57BL; Orthomyxoviridae Infections; Pulmonary Fibrosis; Thrombospondin 1; Transforming Growth Factor beta

Idiopathic pulmonary fibrosis (IPF) is a severe disorder leading to progressive and irreversible loss of pulmonary function. In this study we investigated the anti-fibrotic effect of vitamin D using a mouse model of IPF. Lung fibrosis was induced with bleomycin in vitamin D-sufficient and vitamin D-deficient C57BL/6 mice. We found that treatment with active vitamin D analog paricalcitol prevented mouse body weight loss and alleviated lung fibrosis, whereas vitamin D deficiency severely aggravated lung injury. At the molecular level, paricalcitol treatment suppressed the induction of fibrotic inducer TGF-β and extracellular matrix proteins α-SMA, collagen type I and fibronectin in the lung, whereas vitamin D deficiency exacerbated the induction of these proteins. Interestingly, bleomycin treatment activated the local renin-angiotensin system (RAS) in the lung, manifested by the induction of renin, angiotensinogen, angiotensin II and angiotensin receptor type 1 (AT1R). Paricalcitol treatment suppressed the induction of these RAS components, whereas vitamin D deficiency enhanced the activation of the lung RAS. We also showed that treatment of bleomycin-induced vitamin D-deficient mice with AT1R antagonist losartan relieved weight loss, substantially ameliorated lung fibrosis and markedly blocked TGF-β induction in the lung. Moreover, we demonstrated that in lung fibroblast cultures, TGF-β and angiotensin II synergistically induced TGF-β, AT1R, α-SMA, collagen type I and fibronectin, whereas 1,25-dihydroxyvitamin D markedly suppressed the induction of these fibrotic markers. Collectively, these observations strongly suggest that vitamin D mitigates lung fibrosis by blocking the activation of the lung RAS in this mouse model of IPF.

Topics: Angiotensin II; Angiotensin II Type 1 Receptor Blockers; Angiotensinogen; Animals; Bleomycin; Disease Models, Animal; Ergocalciferols; Losartan; Lung; Mice; Mice, Inbred C57BL; Mice, Knockout; Pulmonary Fibrosis; Receptor, Angiotensin, Type 1; Renin; Renin-Angiotensin System; Transforming Growth Factor beta; Vitamin D

Idiopathic pulmonary fibrosis (IPF) is the prototypic progressive fibrotic lung disease with a median survival of 2 to 4 years. Injury to and/or dysfunction of the alveolar epithelium is strongly implicated in IPF disease initiation, but the factors that determine whether fibrosis progresses rather than normal tissue repair occurs remain poorly understood. We previously demonstrated that zinc finger E-box-binding homeobox 1-mediated epithelial-mesenchymal transition in human alveolar epithelial type II (ATII) cells augments transforming growth factor-β-induced profibrogenic responses in underlying lung fibroblasts via paracrine signaling. Here, we investigated bidirectional epithelial-mesenchymal crosstalk and its potential to drive fibrosis progression. RNA-Seq of lung fibroblasts exposed to conditioned media from ATII cells undergoing RAS-induced epithelial-mesenchymal transition identified many differentially expressed genes including those involved in cell migration and extracellular matrix regulation. We confirmed that paracrine signaling between RAS-activated ATII cells and fibroblasts augmented fibroblast recruitment and demonstrated that this involved a zinc finger E-box-binding homeobox 1-tissue plasminogen activator axis. In a reciprocal fashion, paracrine signaling from transforming growth factor-β-activated lung fibroblasts or IPF fibroblasts induced RAS activation in ATII cells, at least partially through the secreted protein acidic and rich in cysteine, which may signal via the epithelial growth factor receptor via epithelial growth factor-like repeats. Together, these data identify that aberrant bidirectional epithelial-mesenchymal crosstalk in IPF drives a chronic feedback loop that maintains a wound-healing phenotype and provides self-sustaining profibrotic signals.

Topics: Cell Movement; Epithelial Cells; Epithelial-Mesenchymal Transition; Extracellular Matrix; Female; Fibroblasts; Fibrosis; Humans; Idiopathic Pulmonary Fibrosis; Lung; Male; Primary Cell Culture; Pulmonary Fibrosis; Tissue Plasminogen Activator; Transforming Growth Factor beta; Zinc Finger E-box-Binding Homeobox 1

Topics: Animals; Bleomycin; Cell Line; Epithelial-Mesenchymal Transition; Flavonoids; Humans; Male; Mice, Inbred BALB C; Pulmonary Fibrosis; Signal Transduction; Transforming Growth Factor beta

This study aimed to examine whether lung tissue extracellular matrix (ECM) hydrogels have protective effects on radiation-induced lung injury (RILI). The cytocompatibility and histocompatibility were tested for the obtained ECM-derived hydrogel. Sprague-Dawley rats were randomly divided into three groups (n = 18): control group (control); rats receiving irradiation and intratracheal injection of normal saline (IR + NS); and rats receiving irradiation and intratracheal injection of lung ECM-derived hydrogel (IR + ECM). The wet/dry weight ratio was used to evaluate the congestion and edema of the lungs. Histopathological analysis of lung tissues was performed using hemotoxylin and eosin staining and Masson's trichrome staining. Immunohistochemical staining and western blot analyses were carried out to determine the expression of epithelial-mesenchymal transition (EMT)-related proteins in lung tissues (E-cadherin, α-smooth muscle actin [α-SMA], and vimentin). In addition, tumor necrosis factor-α (TNF-α), transforming growth factor-β1 (TGF-β1) and interleukin-6 (IL-6), hydroxyproline, malondialdehyde (MDA), and superoxide dismutase (SOD) levels were also evaluated. The ECM-derived hydrogels had good cytocompatibility and histocompatibility. ECM-derived hydrogel treatment improved lung histopathology injury and pulmonary edema. Higher expression of E-cadherin and lower expression of vimentin and α-SMA were found in the IR + ECM group compared with those in the IR + NS group. Hydroxyproline levels were reduced by ECM-derived hydrogel treatment compared with those in the IR + NS group. Obvious increases of TNF-α, IL-6, and TGF-β1 were identified following irradiation. Marked reductions in MDA content and increases in SOD were induced by ECM-derived hydrogel treatment in rats after radiation. ECM-derived hydrogels were shown to protect against RILI, potentially by reducing EMT, inflammation, and oxidative damage.

Topics: Animals; Epithelial-Mesenchymal Transition; Extracellular Matrix; Gene Expression Regulation; Humans; Hydrogels; Interleukin-6; Lung; Lung Injury; Protective Agents; Pulmonary Fibrosis; Radio Waves; Rats; Rats, Sprague-Dawley; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha

With poor prognosis and aberrant lung remodeling, pulmonary fibrosis exhibits worldwide prevalence accompanied by an increase in burden in terms of hospitalization and death. Apart from genetic and non-genetic factors, fibrosis occurs as a side effect of bleomycin antineoplastic activity. Elucidating the cellular and molecular mechanism could help in the development of effective anti-fibrotic treatment strategies. In the present study, we investigated the underlying mechanism behind bleomycin-induced fibrosis using human alveolar epithelial cells (A549 cells). On the basis of the experimental observation, it was demonstrated that with transforming growth factor-β (TGF-β) as a central mediator of fibrosis progression, a cross-talk between epithelial-mesenchymal transition (EMT) and senescence upon bleomycin treatment occurs. This results in the advancement of this serious fibrotic condition. Fibrosis was initiated through integrin activation and imbalance in the redox state (NOX expression) of the cell. It progressed along the TGF-β-mediated non-canonical pathway (via ERK phosphorylation) followed by the upregulation of α-smooth muscle actin and collagen synthesis. Additionally, in this process, the loss of the epithelial marker E-cadherin was observed. Furthermore, the expressions of senescence markers, such as p21 and p53, were upregulated upon bleomycin treatment, thereby intensifying the fibrotic condition. Accordingly, the molecular pathway mediating the bleomycin-induced fibrosis was explored in the current study.

Topics: A549 Cells; Antibiotics, Antineoplastic; Apoptosis; Bleomycin; Cell Movement; Cell Proliferation; Cellular Senescence; Epithelial-Mesenchymal Transition; Humans; Pulmonary Fibrosis; Transforming Growth Factor beta

Pulmonary fibrosis (PF) is chronic and irreversible damage to the lung characterized by fibroblast activation and matrix deposition. Although recently approved novel anti-fibrotic agents can improve the lung function and survival of patients with PF, the overall outcomes remain poor. In this study, a novel imidazopurine compound, 3-(2-chloro-6-fluorobenzyl)-1,6,7-trimethyl-1H-imidazo[2,1-f]purine-2,4(3H,8H)-dione (IM-1918), markedly inhibited transforming growth factor (TGF)-β-stimulated reporter activity and reduced the expression of representative fibrotic markers, such as connective tissue growth factor, fibronectin, collagen and α-smooth muscle actin, on human lung fibroblasts. However, IM-1918 neither decreased Smad-2 and Smad-3 nor affected p38MAPK and JNK. Instead, IM-1918 reduced Akt and extracellular signal-regulated kinase 1/2 phosphorylation increased by TGF-β. Additionally, IM-1918 inhibited the phosphorylation of fibroblast growth factor receptors 1 and 3. In a bleomycin-induced murine lung fibrosis model, IM-1918 profoundly reduced fibrotic areas and decreased collagen and α-smooth muscle actin accumulation. These results suggest that IM-1918 can be applied to treat lung fibrosis.

Topics: Animals; Antibiotics, Antineoplastic; Bleomycin; Enzyme Inhibitors; Fibronectins; Imidazoles; Male; Mice; Mice, Inbred C57BL; Pulmonary Fibrosis; Receptor, Fibroblast Growth Factor, Type 1; Receptor, Fibroblast Growth Factor, Type 3; Transforming Growth Factor beta

Galectin-9 is a β-galactoside-binding protein with two carbohydrate recognition domains. Recent studies have revealed that galectin-9 regulates cellular biological reactions and plays a pivotal role in fibrosis. The aim of this study was to determine the role of galectin-9 in the pathogenesis of bleomycin-induced systemic sclerosis (SSc).. Human galectin-9 levels in the serum of patients with SSc and mouse sera galectin-9 levels were measured by a Bio-Plex immunoassay and enzyme-linked immunosorbent assay. Lung fibrosis was induced using bleomycin in galectin-9 wild-type and knockout mice. The effects of galectin-9 on the fibrosis markers and signaling molecules in the mouse lung tissues and primary lung fibroblast cells were assessed with western blotting and quantitative polymerase chain reaction.. Galectin-9 levels in the serum were significantly higher (9-fold) in patients compared to those of healthy individuals. Galectin-9 deficiency in mice prominently ameliorated epithelial proliferation, collagen I accumulation, and α-smooth muscle actin expression. In addition, the galectin-9 knockout mice showed reduced protein expression levels of fibrosis markers such as Smad2/3, connective tissue growth factor, and endothelin-1. Differences between the wild-type and knockout groups were also observed in the AKT, mitogen-activated protein kinase, and c-Jun N-terminal kinase signaling pathways. Galectin-9 deficiency decreased the signal activation induced by transforming growth factor-beta in mouse primary fibroblasts, which plays a critical role in fibroblast activation and aberrant catabolism of the extracellular matrix.. Our findings suggest that lack of galectin-9 protects against bleomycin-induced SSc. Moreover, galectin-9 might be involved in regulating the progression of fibrosis in multiple pathways.

Topics: Animals; Biomarkers; Bleomycin; Fibroblasts; Galectins; Lung; Mice; Mice, Knockout; Pulmonary Fibrosis; Scleroderma, Systemic; Signal Transduction; Smad Proteins; Transforming Growth Factor beta

To study the protective effects of ganoderic acid A (GAA) on bleomycin (BLM)-induced pulmonary fibrosis.. ICR mice were intratracheally instilled with BLM to induce pulmonary fibrosis on day 0. Then the mice were orally given GAA (25, 50 mg/kg) or dexamethasone (2 mg/kg). After treatment for 21 days, the mice were sacrificed. Wet dry weight (W/D) ratio of lung was used to detect pulmonary edema. Myeloperoxidase (MPO), interleukin-1β (IL-1β), IL-6, tumor necrosis factor-α (TNF-α), malondialdehyde (MDA), and superoxide dismutase (SOD) were detected by enzyme-linked immunosorbent assay. Hematoxylin and eosin staining was used to evaluate the pathological changes. The levels of transforming growth factor β (TGF-β), phosphorylated-smad3 (p-smad3), p-IκB, and p-nuclear factor-kappa B (NF-κB) in lung tissue were detected by western blot.. GAA treatment significantly improved MPO activity, W/D ratio, and lung histopathology. The protective effect of GAA may be related to downregulation of TNF-α, IL-1β, IL-6, MDA and upregulation of SOD. In addition, GAA significantly decreased the levels of TGF-β, p-smad3, p-IκB, and p-NF-κB, compared with those in BLM group.. GAA has protective effect on BLM-induced lung injury, and TGF-β/Smad-3/NF-κB signaling pathway may play an important role in the pathogenesis of BLM-induced lung injury.

Topics: Animals; Bleomycin; Cytokines; Heptanoic Acids; Lanosterol; Lung; Male; Malondialdehyde; Mice; Mice, Inbred ICR; NF-kappa B; Peroxidase; Phytotherapy; Plant Extracts; Protective Agents; Pulmonary Edema; Pulmonary Fibrosis; Smad3 Protein; Superoxide Dismutase; Transforming Growth Factor beta

Occupational exposure to silica dust particles was the major cause of pulmonary fibrosis, and many miRNAs have been demonstrated to regulate target mRNAs in silicosis. In the present study, we found that a decreasing level of miR-411-3p in silicosis rats and lung fibroblasts induced by TGF-β1. Enlargement of miR-411-3p could inhibit the cell proliferation and migration in lung fibroblasts with TGF-β1 treatment and attenuate lung fibrosis in silicotic mice. In addition, a mechanistic study showed that miR-411-3p exert its inhibitory effect on Smad ubiquitination regulatory factor 2 (Smurf2) expression and decrease ubiquitination degradation of Smad7 regulated by smurf2, result in blocking of TGF-β/Smad signaling. We proposed that increased expression of miR-411-3p abrogates silicosis by blocking activation of TGF-β/Smad signaling through decreasing ubiquitination degradation effect of smurf2 on Smad7.

Topics: Animals; Gene Expression Regulation; Male; MicroRNAs; Pulmonary Fibrosis; Rats; Rats, Wistar; Silicon Dioxide; Silicosis; Transforming Growth Factor beta; Ubiquitin-Protein Ligases

Pulmonary fibrosis (PF) is an epithelial/fibroblastic crosstalk disorder of the lungs with highly complex etiopathogenesis. Limited treatment possibilities are responsible for poor prognosis and mean survival rate of 3 to 5 years of PF patients after definite diagnosis. Once thought to be an irreversible disorder, recent evidences have brought into existence the concept of organ fibrosis reversibility due to plastic nature of fibrotic tissues. These findings have kindled interest among the scientific community and given a new direction for research in the arena of fibrosis for developing new anti-fibrotic therapies. The current study is designed to evaluate the anti-fibrotic effects of Honokiol (HNK), a neolignan active constituent from Magnolia officinalis. This study has been conducted in TGF-β1 induced in vitro model and 21 day in vivo murine model of Bleomycin induced PF. The findings of our study suggest that HNK was able to inhibit fundamental pathways of epithelial to mesenchymal transition (EMT) and TGF-β/Smad signaling both in vitro and in vivo. Additionally, HNK also attenuated collagen deposition and inflammation associated with fibrosis. We also hypothesized that HNK interfered with IL-6/CD44/STAT3 axis. As hypothesized, HNK significantly mitigated IL-6/CD44/STAT3 axis both in vitro and in vivo as evident from outcomes of various protein expression studies like western blotting, immunohistochemistry and ELISA. Taken together, it can be concluded that HNK reversed pulmonary fibrotic changes in both in vitro and in vivo experimental models of PF and exerted anti-fibrotic effects majorly by attenuating EMT, TGF-β/Smad signaling and partly by inhibiting IL-6/CD44/STAT3 signaling axis.

Topics: Animals; Biphenyl Compounds; Bleomycin; Bronchoalveolar Lavage Fluid; Cell Line; Cell Movement; Collagen; Cytokines; Epithelial-Mesenchymal Transition; Humans; Hyaluronan Receptors; Interleukin-6; Lignans; Lung; Mice; Pulmonary Fibrosis; Signal Transduction; Smad Proteins; STAT3 Transcription Factor; Transforming Growth Factor beta

Uncontrolled activation of transforming growth factor (TGF)-β results in a wide range of pathologic conditions. Therapeutic interventions to regulate TGF-β signaling during fibrosis have been developed but the effectiveness is still limited. Here, we show that developmental endothelial locus-1 (Del-1) ameliorates fibrosis in mice by inhibiting α

Topics: Animals; Calcium-Binding Proteins; Cell Adhesion Molecules; Mice; Mice, Inbred C57BL; Pulmonary Fibrosis; Signal Transduction; Transforming Growth Factor beta

In interstitial fibrosis, alveolar epithelial type II (AE2) cells fail to repair damaged epithelium. However, whether this dysfunction is related to fibroblast growth factor (FGF) signal pathway and how it affects the fibrotic process remains unclear. In our study, the medium of the human foetal lung fibroblast cell line MRC-5 (Med) can induce epithelial-to-mesenchymal transition (EMT) in AE2 cells, we also found that TGF-β in Med can induce FGF-2 and CTGF expression in AE2 cells. TGF-β or CTGF exposure trigger a FGFR2 subtype b to c transition which can be supressed by siRNA-CTGF. All together, since FGFR2IIIc have the highest affinity with FGF-2 in all of the FGFRs, we indicate the activation of FGF2 signal pathway was induced by TGF-β, which is the key component of Med Here, we also find the inhibitory effect of msFGFR2c (S252W mutant of soluble FGFR2IIIc extracellular domain) on EMT of mouse primary AE2 cells in pulmonary fibrotic process. In a bleomycin-induced mouse pulmonary fibrosis model, msFGFR2c alleviate pulmonary fibrosis and suppress the decrease in pro-SPC levels. Thus, msFGFR2c can inhibit EMT-induced fibrosis of AE2 cells via FGF-2 signal and AE2 cells is suggested to play an important role in the lung fibrotic process.

Topics: Alveolar Epithelial Cells; Animals; Cell Line; Connective Tissue Growth Factor; Culture Media, Conditioned; Disease Models, Animal; Epithelial-Mesenchymal Transition; Fibroblasts; Humans; Mice; Pulmonary Fibrosis; Receptor, Fibroblast Growth Factor, Type 2; Signal Transduction; Transforming Growth Factor beta

Intraflagellar transport protein 88 (Ift88) is required for ciliogenesis and shear stress-induced dissolution of cilia in embryonic endothelial cells coincides with endothelial-to-mesenchymal transition (EndMT) in the developing heart. EndMT is also suggested to underlie heart and lung fibrosis, however, the mechanism linking endothelial Ift88, its effect on EndMT and organ fibrosis remains mainly unexplored. We silenced Ift88 in endothelial cells (ECs) in vitro and generated endothelial cell-specific Ift88-knockout mice (Ift88

Topics: Animals; Biopsy; Bleomycin; Cell Movement; Cell Proliferation; Disease Susceptibility; Epithelial-Mesenchymal Transition; Gene Knockout Techniques; Hedgehog Proteins; Humans; Mice; Pulmonary Fibrosis; Pulmonary Heart Disease; Respiratory Mucosa; Signal Transduction; Transforming Growth Factor beta; Tumor Suppressor Proteins; Wnt Signaling Pathway

Pulmonary fibrosis is a serious respiratory disease, with limited therapeutic options. Since TGF-β is a critical factor in the fibrotic process, downregulation of this cytokine has been considered a potential approach for disease treatment. Herein, we designed a new lung-targeted delivery technology based on the complexation of polymeric antisense oligonucleotides (pASO) and dimeric human β-defensin 23 (DhBD23). Antisense oligonucleotides targeting TGF-β mRNA were polymerized by rolling circle amplification and complexed with DhBD23. After complexation with DhBD23, pASO showed improved serum stability and enhanced uptake by fibroblasts in vitro and lung-specific accumulation upon intravenous injection in vivo. The pASO/DhBD23 complex delivered into the lung downregulated target mRNA, and subsequently alleviated lung fibrosis in mice, as demonstrated by western blotting, quantitative reverse-transcriptase PCR (qRT-PCR), immunohistochemistry, and immunofluorescence imaging. Moreover, as the complex was prepared only with highly biocompatible materials such as DNA and human-derived peptides, no systemic toxicity was observed in major organs. Therefore, the pASO/DhBD23 complex is a promising gene therapy platform with lung-targeting ability to treat various pulmonary diseases, including pulmonary fibrosis, with low side effects.

Topics: Animals; Bleomycin; Fibroblasts; Lung; Mice; Oligonucleotides, Antisense; Pulmonary Fibrosis; Transforming Growth Factor beta

In the current work we show that the profibrotic actions of TGF-β are mediated, at least in part, through a metabolic maladaptation in glutamine metabolism and how the inhibition of glutaminase 1 (GLS1) reverses pulmonary fibrosis. GLS1 was found to be highly expressed in fibrotic vs normal lung fibroblasts and the expression of profibrotic targets, cell migration, and soft agar colony formation stimulated by TGF-β required GLS1 activity. Moreover, knockdown of SMAD2 or SMAD3 as well as inhibition of PI3K, mTORC2, and PDGFR abrogated the induction of GLS1 by TGF-β. We further demonstrated that the NAD-dependent protein deacetylase, SIRT7, and the FOXO4 transcription factor acted as endogenous brakes for GLS1 expression, which are inhibited by TGF-β. Lastly, administration of the GLS1 inhibitor CB-839 attenuated bleomycin-induced pulmonary fibrosis. Our study points to an exciting and unexplored connection between epigenetic and transcriptional processes that regulate glutamine metabolism and fibrotic development in a TGF-β-dependent manner.

Topics: Animals; Antibiotics, Antineoplastic; Bleomycin; Cell Movement; Cells, Cultured; Female; Fibroblasts; Gene Expression Regulation; Glutaminase; Mice; Mice, Inbred C57BL; Pulmonary Fibrosis; Signal Transduction; Sirtuins; Smad Proteins; Transforming Growth Factor beta

Idiopathic pulmonary fibrosis is a progressive-fibrosing lung disease with high mortality and limited therapy, which characterized by myofibroblasts proliferation and extracellular matrix deposition. Myricetin, a natural flavonoid, has been shown to possess a variety of biological characteristics including anti-inflammatory and anti-tumor. In this study we explored the potential effect and mechanisms of myricetin on pulmonary fibrosis in vivo and vitro. The in vivo studies showed that myricetin effectively alleviated bleomycin (BLM)-induced pulmonary fibrosis. KEGG analysis of RNA-seq data indicated that myricetin could regulate the transforming growth factor (TGF)-β signaling pathway. In vitro studies indicated that myricetin could dose-dependently suppress TGF-β1/Smad signaling and attenuate TGF-β1-induced fibroblast activation and epithelial-mesenchymal transition (EMT). Molecular docking indicated that heat shock protein (HSP) 90β may be a potential target of myricetin, and MST assay demonstrated that the dissociation constant (Kd) of myricetin and HSP90β was 331.59 nM. We demonstrated that myricetin interfered with the binding of HSP90β and TGF-β receptor II and impeded fibroblast activation and EMT. In conclusion, myricetin impedes TGF-β1-induced lung fibroblast activation and EMT via targeting HSP90β and attenuates BLM-induced pulmonary fibrosis in mice.

Topics: A549 Cells; Animals; Binding Sites; Bleomycin; Cadherins; Cell Proliferation; Epithelial-Mesenchymal Transition; Extracellular Matrix; Fibronectins; Flavonoids; Gene Expression Regulation; HSP90 Heat-Shock Proteins; Humans; Lung; Male; Mice; Mice, Inbred C57BL; Molecular Docking Simulation; Protective Agents; Protein Binding; Pulmonary Fibrosis; Signal Transduction; Smad Proteins; Transforming Growth Factor beta; Vimentin

Topics: Alveolar Epithelial Cells; Angiotensin I; Angiotensin II; Angiotensin-Converting Enzyme 2; Betacoronavirus; Coronavirus Infections; COVID-19; Host Microbial Interactions; Humans; Lung; Pandemics; Peptide Fragments; Peptidyl-Dipeptidase A; Pneumonia, Viral; Pulmonary Fibrosis; Receptors, Coronavirus; Receptors, Virus; Renin-Angiotensin System; SARS-CoV-2; Transforming Growth Factor beta

TGF-β is a master regulator of fibrosis, driving the differentiation of fibroblasts into apoptosis-resistant myofibroblasts and sustaining the production of extracellular matrix (ECM) components. Here, we identified the nuclear long noncoding RNA (lncRNA) H19X as a master regulator of TGF-β-driven tissue fibrosis. H19X was consistently upregulated in a wide variety of human fibrotic tissues and diseases and was strongly induced by TGF-β, particularly in fibroblasts and fibroblast-related cells. Functional experiments following H19X silencing revealed that H19X was an obligatory factor for TGF-β-induced ECM synthesis as well as differentiation and survival of ECM-producing myofibroblasts. We showed that H19X regulates DDIT4L gene expression, specifically interacting with a region upstream of the DDIT4L gene and changing the chromatin accessibility of a DDIT4L enhancer. These events resulted in transcriptional repression of DDIT4L and, in turn, in increased collagen expression and fibrosis. Our results shed light on key effectors of TGF-β-induced ECM remodeling and fibrosis.

Topics: Adaptor Proteins, Signal Transducing; Animals; Cell Line; Extracellular Matrix; Humans; Mice; Myofibroblasts; Pulmonary Fibrosis; RNA, Long Noncoding; Transforming Growth Factor beta

Vitamin D (VitD) has an anti-fibrotic effect on fibrotic lungs. It reduces epithelial-mesenchymal transition (EMT) on tumors. We aimed to investigate target proteins of VitD for the regression of EMT-mediated myofibroblast differentiation. A group of A549 cells were treated with 5 % cigarette smoke extract (CSE) and 5 %CSE + TGF-β (5 ng/ml) to induce EMT. The others were treated with 50 nM VitD 30 min before %5CSE and TGF-β treatments. All cells were collected at 24, 48 and 72 h following 5 %CSE and TGF-β administrations. The expression of p120ctn and NEDD9 proteins acted on E-cadherin turnover in addition to activations of TGF-β and Wnt pathways were examined in these cells and fibrotic human lungs. CSE and TGF-β induced EMT by reducing E-cadherin, p-VDR, SMAD7 and DKK1, increasing α-SMA, p120ctn, Kaiso, NEDD9 and stimulating TGF-β and Wnt/β-catenin signalings in A549 cells. VitD administration reversed these alterations and regressed EMT. Co-immunoprecipitation analysis revealed p-VDR interaction with β-catenin and Kaiso in fibrotic and non-fibrotic human lungs. VitD pre-treatments reduced TGF-β and Wnt/β-catenin signalings by increasing p-VDR, protected from E-cadherin degradation and led to the regression of EMT in A549 cells treated with CSE and TGF-β. Finally, VitD supplementation combined with anti-fibrotic therapeutics can be suggested for treatment of pulmonary fibrosis, which may be developed by smoking, in cases of VitD deficiency.

Topics: A549 Cells; Antigens, CD; Cadherins; Cell Differentiation; Epithelial-Mesenchymal Transition; Humans; Lung; Myofibroblasts; Pulmonary Fibrosis; Receptors, Calcitriol; Smoke; Tobacco Products; Transforming Growth Factor beta; Vitamin D; Vitamins; Wnt Signaling Pathway

Macrophages in lung, including resident alveolar macrophages (AMs) and interstitial macrophages (IMs), and monocyte-derived macrophages, play important roles in pulmonary fibrosis (PF), but mechanisms underlying their differential regulation remain unclear. Recombination signal-binding protein Jκ (RBP-J)-mediated Notch signaling regulates macrophage development and phenotype. Here, using bleomycin-induced fibrosis model combined with myeloid-specific RBP-J disruption (RBP-J

Topics: Animals; Bodily Secretions; Histocompatibility Antigens Class II; Immunoglobulin J Recombination Signal Sequence-Binding Protein; Inflammation; Macrophages, Alveolar; Matrix Metalloproteinases; Mice; Mice, Inbred C57BL; Monocytes; Myofibroblasts; Pulmonary Fibrosis; Receptors, Notch; Signal Transduction; Transforming Growth Factor beta

Fine particulate matter (PM2.5) is the primary air pollutant that is able to induce airway injury. Compelling evidence has shown the involvement of IL-17A in lung injury, while its contribution to PM2.5-induced lung injury remains largely unknown. Here, we probed into the possible role of IL-17A in mouse models of PM2.5-induced lung injury. Mice were instilled with PM2.5 to construct a lung injury model. Flow cytometry was carried out to isolate γδT and Th17 cells. ELISA was adopted to detect the expression of inflammatory factors in the supernatant of lavage fluid. Primary bronchial epithelial cells (mBECs) were extracted, and the expression of TGF signalling pathway-, autophagy- and PI3K/Akt/mTOR signalling pathway-related proteins in mBECs was detected by immunofluorescence assay and Western blot analysis. The mitochondrial function was also evaluated. PM2.5 aggravated the inflammatory response through enhancing the secretion of IL-17A by γδT/Th17 cells. Meanwhile, PM2.5 activated the TGF signalling pathway and induced EMT progression in bronchial epithelial cells, thereby contributing to pulmonary fibrosis. Besides, PM2.5 suppressed autophagy of bronchial epithelial cells by up-regulating IL-17A, which in turn activated the PI3K/Akt/mTOR signalling pathway. Furthermore, IL-17A impaired the energy metabolism of airway epithelial cells in the PM2.5-induced models. This study suggested that PM2.5 could inhibit autophagy of bronchial epithelial cells and promote pulmonary inflammation and fibrosis by inducing the secretion of IL-17A in γδT and Th17 cells and regulating the PI3K/Akt/mTOR signalling pathway.

Topics: Animals; Cells, Cultured; Disease Models, Animal; Disease Susceptibility; Epithelial Cells; Humans; Interleukin-17; Male; Mice; Particulate Matter; Phosphatidylinositol 3-Kinases; Pneumonia; Proto-Oncogene Proteins c-akt; Pulmonary Fibrosis; Receptors, Antigen, T-Cell, gamma-delta; Signal Transduction; T Cell Transcription Factor 1; Th17 Cells; TOR Serine-Threonine Kinases; Transforming Growth Factor beta

Acute lung injury (ALI), a common condition in critically ill patients, has limited treatments and high mortality. Aging is a risk factor for ALI. Sirtuins (SIRTs), central regulators of the aging process, decrease during normal aging and in aging-related diseases. We recently showed decreased SIRT7 expression in lung tissues and fibroblasts from patients with pulmonary fibrosis compared to controls. To gain insight into aging-related mechanisms in ALI, we investigated the effects of SIRT7 depletion on lipopolysaccharide (LPS)-induced inflammatory responses and endothelial barrier permeability in human primary pulmonary endothelial cells. Silencing SIRT7 in pulmonary artery or microvascular endothelial cells attenuated LPS-induced increases in ICAM1, VCAM1, IL8, and IL6 and induced endomesenchymal transition (EndoMT) with decreases in VE-Cadherin and PECAM1 and increases in collagen, alpha-smooth muscle actin, TGFβ receptor 1, and the transcription factor Snail. Loss of endothelial adhesion molecules was accompanied by increased F-actin stress fibers and increased endothelial barrier permeability. Together, these results show that an aging phenotype induced by SIRT7 deficiency promotes EndoMT with impaired inflammatory responses and dysfunction of the lung vascular barrier.

Topics: Adult; Animals; Bleomycin; Capillary Permeability; Cell Membrane Permeability; Cells, Cultured; Endothelial Cells; Epithelium; Gene Expression Regulation; Gene Silencing; Humans; Inflammation; Inflammation Mediators; Lipopolysaccharides; Lung; Mice, Inbred C57BL; NF-kappa B; Pulmonary Fibrosis; RNA, Messenger; Signal Transduction; Sirtuins; Transforming Growth Factor beta

Idiopathic pulmonary fibrosis is a debilitating lung disease. CD26/DPP4 plays promotive roles in pulmonary damage and fibrosis. This study aimed to explore the roles of vildagliptin in bleomycin-induced pulmonary fibrosis, and to address its ameliorative effect on the extracellular matrix (ECM).. Idiopathic pulmonary fibrosis mice models were induced by intratracheal injection of bleomycin. DPP4 activity was evaluated, and the fibrosis was investigated by Hematoxylin-eosin, Masson's trichrome staining and hydroxyproline assay. Expression of extracellular matrix proteins including α-SMA, collagen IV, collagen I, FN and TGF-β were analyzed by immunochemistry and western blot. Percentages of the numbers of monocytes, leukocytes, basophils and lymphocytes were classified, and inflammatory factors in plasma as well as lung tissues were examined by enzyme-linked immunosorbent assay and western blot. The influences of vildagliptin on TGF-β1-induced cell proliferation, differentiation and inflammatory factors in MRC-5 cells were detected.. Vildagliptin effectively attenuated inflammation and fibrosis in bleomycin-induced pulmonary tissue via inhibiting the activity of CD26/DPP4. extracellular matrix proteins were suppressed by vildagliptin. Thus, lung tissue fibrosis was efficiently alleviated by vildagliptin.. As an inhibitor of CD26/DPP4, Vildagliptin could be a promising therapeutic candidate for idiopathic pulmonary fibrosis.

Topics: Actins; Animals; Bleomycin; Cell Line; Cell Survival; Collagen; Dipeptidyl Peptidase 4; Dipeptidyl-Peptidase IV Inhibitors; Extracellular Matrix; Fibronectins; Humans; Inflammation; Lung; Male; Mice, Inbred C57BL; Pulmonary Fibrosis; Transforming Growth Factor beta; Vildagliptin

Idiopathic Pulmonary Fibrosis (IPF) is a progressive inflammatory disorder driven by a fibrotic cascade of events such as epithelial to mesenchymal transition, extracellular matrix production and collagen formation in the lungs in a sequential manner. IPF incidences were raising rapidly across the world. FDA approved pirfenidone and nintedanib (tyrosine kinase inhibitors) are being used as a first-line treatment drugs for IPF, however, neither the quality of life nor survival rates have been improved because of patient noncompliance due to multiple side effects. Thus, the development of novel therapeutic approaches targeting TGF-β mediated cascade of fibrotic events is urgently needed to improve the survival of the patients suffering from devastating disease.. The aim of this study was to investigate and validate the anti-fibrotic properties of Biochanin-A (isoflavone) against TGF-β mediated fibrosis in in vitro, ex vivo, in vivo models and to determine the molecular mechanisms that mediate these anti-fibrotic effects.. The therapeutic activity of BCA was determined in in vitro/ex vivo models. Cells were pre-treated with BCA and incubated in presence or absence of recombinant-TGF-β to stimulate the fibrotic cascade of events. Pulmonary fibrosis was developed by intratracheal administration of bleomycin in rats. BCA treatment was given for 14 days from post bleomycin instillation and then various investigations (collagen content, fibrosis gene/protein expression and histopathological changes) were performed to assess the anti-fibrotic activity of BCA.. In vitro/ex vivo (Primary normal, IPF cell line and primary IPF cells/ Precision cut mouse lung slices) experiments revealed that, BCA treatment significantly (p < 0.001) reduced the expression of TGF-β modulated fibrotic genes/protein expressions (including their functions) which are involved in the cascade of fibrotic events. BCA treatment significantly (p < 0.01) reduced the bleomycin-induced inflammatory cell-infiltration, inflammatory markers expression, collagen deposition and expression of fibrotic markers in lung tissues equivalent or better than pirfenidone treatment. In addition, BCA treatment significantly (p < 0.001) attenuated the TGF-β1/BLM-mediated increase of TGF-β/Smad2/3 phosphorylation and resulted in the reduction of pathological abnormalities in lung tissues determined by histopathology observations.. Collectively, BCA treatment demonstrated the remarkable therapeutic effects on TGF-β/BLM mediated pulmonary fibrosis using IPF cells and rodent models. This current study may offer a novel treatment approach to halt and may be even rescue the devastating lung scarring of IPF.

Topics: Animals; Bleomycin; Cell Differentiation; Collagen; Epithelial-Mesenchymal Transition; Extracellular Matrix; Female; Genistein; Humans; Mice, Inbred C57BL; Myofibroblasts; Pulmonary Fibrosis; Rats, Wistar; Reproducibility of Results; Smad Proteins; Transforming Growth Factor beta

Radiation therapy is an important modality in the treatment of lung cancer, but it can lead to radiation pneumonitis, and eventually radiation fibrosis. To date, only few available drugs can effectively manage radiation-induced pulmonary fibrosis. Lipoxins are endogenous molecules exhibit anti-inflammatory and pro-resolving effects. These molecules play a vital role in reducing excessive tissue injury and chronic inflammation; however, their effects on radiation-induced lung injury (RILI) are unknown. In this study, we investigated the effects of lipoxin A

Topics: Animals; Anti-Inflammatory Agents; Cytokines; Fibrosis; Humans; Lipoxins; Lung; Male; Mice; Mice, Inbred C57BL; Pulmonary Fibrosis; Radiation; Radiotherapy; Receptor Cross-Talk; Receptors, Formyl Peptide; Receptors, Lipoxin; Signal Transduction; Smad Proteins; Transforming Growth Factor beta

Idiopathic pulmonary fibrosis (IPF) is an intractable disease with poor prognosis, and therapeutic options are limited. While the pathogenic mechanism is unknown, cytokines, such as transforming growth factor (TGF)-β, and immune cells, such as monocytes and macrophages, that produce them, seem to be involved in fibrosis. Some phosphodiesterase 4 (PDE4) inhibitors reportedly have anti-fibrotic potential by acting on these disease-related factors. Therefore, we evaluated the effect of a novel PDE4 inhibitor, AA6216, on nonclinical IPF-related models and samples from IPF patients. First, we examined the inhibitory effect of AA6216 on the production of TGF-β1 from a human monocytic cell line, THP-1. Second, we analyzed the impact of AA6216 on TNF-α production by human alveolar macrophages collected from patients with IPF. Finally, we investigated the anti-fibrotic potency of AA6216 on bleomycin-induced lung fibrosis in mice. We found that AA6216 significantly inhibited TGF-β1 production by THP-1 cells. It also significantly suppressed TNF-α production by alveolar macrophages from patients with IPF. In the mouse model of bleomycin-induced pulmonary fibrosis, therapeutic administration of AA6216 significantly reduced fibrosis scores, collagen-stained areas, and TGF-β1 in bronchoalveolar lavage fluid. AA6216 may represent a new agent for the treatment of IPF with a distinct mechanism of action from that of conventional anti-fibrotic agents.

Topics: Animals; Bleomycin; Bronchoalveolar Lavage Fluid; Cell Line; Female; Humans; Macrophages; Macrophages, Alveolar; Male; Mice; Mice, Inbred C57BL; Mice, Inbred ICR; Monocytes; Phosphodiesterase 4 Inhibitors; Pulmonary Fibrosis; Transforming Growth Factor beta

Pulmonary fibrosis is the end stage of many interstitial lung diseases, characterized by the deposition of excess extracellular matrix (ECM), destruction of normal alveolar structure, and resulting in the obstruction of gas exchange and respiratory failure. The idiopathic pulmonary fibrosis (IPF) is the most common form of pulmonary fibrosis with little effective therapies. 5-Methoxytryptophan (5-MTP) is a newly found tryptophan metabolite. Previous studies suggested that 5-MTP has the effects of anti-inflammatory, anti-tumorigenesis, vascular protection and anti-fibrosis in renal disease. Whether 5-MTP has therapeutic effect on pulmonary fibrosis is not clear. In our study, we used TGF-β1 to stimulate human lung fibroblasts (HLFs) and bleomycin (BLM) induced pulmonary fibrosis model to investigate the effect of 5-MTP on pulmonary fibrosis. Our study demonstrated that 5-MTP could improve the lung function and attenuate the destruction of alveolar structure in BLM-induced pulmonary fibrosis mice. Furthermore, 5-MTP significantly decreased accumulation of myofibroblasts and the deposition of ECM by inhibiting the differentiation of fibroblasts to myofibroblasts and suppressing the protein expression of the ECM both in vivo and in vitro. Our results also revealed 5-MTP could inhibit the proliferation and migration of the fibroblasts in vitro, which played an important role in the progressive pulmonary fibrosis. To further investigate the mechanism of the anti-fibrosis of 5-MTP, several canonical and noncanonical signaling pathways were examined. Our results revealed that 5-MTP could inhibit the pulmonary fibrosis through downregulating the phosphorylation of TGF-β/SMAD3, PI3K/AKT signaling pathways. Together, our study indicated that 5-MTP promises to be therapeutic agent of pulmonary fibrosis.

Topics: Animals; Antibiotics, Antineoplastic; Bleomycin; Cell Differentiation; Extracellular Matrix; Gene Expression Regulation; Humans; Lung; Male; Mice; Mice, Inbred C57BL; Myofibroblasts; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; Pulmonary Fibrosis; Smad3 Protein; Transforming Growth Factor beta; Tryptophan

Pulmonary fibrosis is a progressive disease characterized by lung remodeling due to excessive deposition of extracellular matrix. In this study, the bleomycin experimental model of pulmonary fibrosis was employed to investigate the anti-fibrotic and immunomodulatory activity of the inhibition of MALT1 protease activity. Mice received a single intra-tracheal administration of bleomycin (1 mg/kg) in the presence or absence of MI-2, a selective MALT1 inhibitor, (a dose of 30 mg/kg administered intra-peritoneally 1 h after bleomycin and daily until the end of the experiment). Seven days after bleomycin instillation mice were sacrificed and bronchoalveolar lavage fluid analysis, measurement of collagen content in the lung, histology, molecular analysis and immunohistochemistry were performed. To evaluate mortality and body weight gain a subset of mice was administered daily with MI-2 for 21 days. Mice that received MI-2 showed decreased weight loss and mortality, inflammatory cells infiltration, cytokines overexpression and tissue injury. Moreover, biochemical and immunohistochemical analysis displayed that MI-2 was able to modulate the excessive production of reactive oxygen species and the inflammatory mediator upregulation induced by bleomycin instillation. Additionally, MI-2 demonstrated anti-fibrotic activity by reducing transforming growth factor-β (TGF-β), α-smooth muscle actin (α-SMA) and receptor associated factor 6 (TRAF6) expression. The underlying mechanisms for the protective effect of MI-2 bleomycin induced pulmonary fibrosis may be attributed to its inhibition on NF-κB pathway. This is the first report showing the therapeutic role of MALT1 inhibition in a bleomycin model of pulmonary fibrosis, thus supporting further preclinical and clinical studies.

Topics: Animals; Bleomycin; Bronchoalveolar Lavage Fluid; Collagen; Cytokines; Enzyme Inhibitors; Lung; Lung Injury; Male; Mice; Mucosa-Associated Lymphoid Tissue Lymphoma Translocation 1 Protein; NF-kappa B; Pulmonary Fibrosis; Signal Transduction; TNF Receptor-Associated Factor 6; Transforming Growth Factor beta

Heart failure with reduced ejection fraction (HFrEF) causes lung remodelling with myofibroblasts proliferation and fibrosis leading to a restrictive lung syndrome with pulmonary hypertension (PH) and right ventricular (RV) dysfunction. PBI-4050 is a first-in-class anti-fibrotic, anti-inflammatory, and anti-proliferative compound. The present study evaluated the therapeutic impact of PBI-4050 on PH in an HFrEF model.. HFrEF was induced after myocardial infarction (MI) in rats. Two weeks later, sham-operated and MI groups received PBI-4050 (200 mg/kg/day by gavage) or saline for 3 weeks. Animals were analysed according to infarct size as large (≥30% left ventricle) or medium MI (<30%). Large MI caused PH and RV hypertrophy (RVH) with a restrictive lung syndrome. PBI-4050 did not adversely affect left ventricular (LV) function but markedly reduced PH and RVH and improved RV dysfunction. PBI-4050 reduced lung remodelling and improved respiratory compliance with decreased lung fibrosis, alveolar wall cellular proliferation and α-smooth muscle actin expression. The increased expression of endothelin-1 (ET-1), transforming growth factor beta (TGF-β), interleukin-6 (IL-6) and of tissue inhibitor of metalloprotease-1 in the lungs from HFrEF were reduced with PBI-4050 therapy. Activation of isolated human lung fibroblasts (HLFs) to a myofibroblastic pro-fibrogenic phenotype was markedly reduced by PBI-4050. The fatty acid receptor GPR84 was increased in HFrEF lungs and in activated HLFs, and reduced by PBI-4050. GPR84 agonists activated fibrogenesis in HLFs and finally, PBI-4050 reduced ERK1/2 phosphorylation.. PBI-4050 reduces PH and RVH in HFrEF by decreasing lung fibrosis and remodelling. This novel agent decreases the associated restrictive lung syndrome and recovers RV function. A contributing mechanism involves reducing the activation of lung fibroblasts by IL-6, TGF-β, and ET-1 by antagonism of GPR84 and reduced ERK1/2 phosphorylation. PBI-4050 is a novel promising therapy for targeting lung remodelling in group II PH.

Topics: Acetates; Animals; Cells, Cultured; Disease Models, Animal; Endothelin-1; Extracellular Signal-Regulated MAP Kinases; Fibroblasts; Fibrosis; Heart Failure; Heart Ventricles; Humans; Hypertension, Pulmonary; Hypertrophy, Right Ventricular; Interleukin-6; Lung; Male; Phosphorylation; Pulmonary Fibrosis; Rats, Wistar; Receptors, G-Protein-Coupled; Signal Transduction; Transforming Growth Factor beta; Ventricular Dysfunction, Right; Ventricular Function, Right; Ventricular Remodeling

The immunogenic collagen V (Col V) and the proinflammatory cytokine interleukin (IL)-17 have been implicated in the pathogenesis of multiple autoimmune diseases. Col V is also up-regulated during adipogenesis and can stimulate adipocyte differentiation in vitro. Conditioned medium (CM) generated from adipose-derived mesenchymal stem cells (MSCs) reduces bleomycin (BLM)-induced lung injury in rats, suggesting a crucial role in situ of immunomodulatory factors secreted by MSCs in these beneficial effects. In the present work, we investigated this hypothesis, analyzing levels of plasma inflammatory mediators and inflammatory and fibrotic mediators in the lung tissue of BLM-injured rats after treatment with MSCs and CM. Pulmonary fibrosis was intratracheally induced by BLM. After 10 days, BLM animals were further randomized into subgroups receiving saline, MSCs, or CM intravenously. On days 14 and 21, the animals were euthanized, and the lungs were examined through protein expression of nitric oxide synthase (NOS), IL-17, transforming growth factor-β (TGF-β), vascular endothelial growth factor, endothelin-1, and the immunogenic Col V through histological quantitative evaluation and plasma levels of fibrinogen, Von Willebrand factor, and platelet-derived growth factor (PDGF). Rats that had been injected with MSCs and CM showed a significant increase in weight and significant improvements at 14 and 21 days after intravenous injection at both time points of analysis of plasma fibrinogen, PDGF, and Von Willebrand factor and NOS-2 expression, supporting an early anti-inflammatory action, thus reducing TGF-β and collagen I fibers. In contrast, intravenous injection of CM was able to significantly increase the deposition of Col V fibers and IL-17 on both day 14 and day 21 as compared with the amount observed in rats from the BLM group and MSC groups. In conclusion, this study reinforces previous observations on the therapeutic properties of MSCs and CM and is the first report to demonstrate the association of its actions with immunomodulatory biomarkers on lung tissue. We concluded that adipose-derived stem cells and adipose-derived stem cells-CM modulate an in situ imbalance between collagen I- and Col V-mediated IL-17 immune response, emerging as a promising therapeutic option for recovering from BLM pulmonary fibrosis.

Topics: Adipose Tissue; Animals; Biomarkers; Bleomycin; Collagen Type I; Collagen Type V; Culture Media, Conditioned; Immune System; Interleukin-17; Lung; Pulmonary Fibrosis; Randomized Controlled Trials as Topic; Rats; Rats, Wistar; Stem Cells; Transforming Growth Factor beta

Topics: A549 Cells; Animals; Antibiotics, Antineoplastic; Bleomycin; Dose-Response Relationship, Drug; Humans; MAP Kinase Signaling System; Mice; NIH 3T3 Cells; Oxidative Stress; Peptide Fragments; Pulmonary Fibrosis; Transforming Growth Factor beta; Treatment Outcome

Collagen Tissue Disease-associated Interstitial Lung Fibrosis (CTD-ILDs) and Bronchiolitis Obliterans Syndrome (BOS) represent severe lung fibrogenic disorders, characterized by fibro-proliferation with uncontrolled extracellular matrix deposition. Hyaluronic acid (HA) plays a key role in fibrosis with its specific receptor, CD44, overexpressed by CTD-ILD and BOS cells. The aim is to use HA-liposomes to develop an inhalatory treatment for these diseases. Liposomes with HA of two molecular weights were prepared and characterized. Targeting efficiency was assessed toward CTD-ILD and BOS cells by flow cytometry and confocal microscopy and immune modulation by RT-PCR and ELISA techniques. HA-liposomes were internalized by CTD-ILD and BOS cells expressing CD44, and this effect increased with higher HA MW. In THP-1 cells, HA-liposomes decreased pro-inflammatory cytokines IL-1β, IL-12, and anti-fibrotic VEGF transcripts but increased TGF-β mRNA. However, upon analyzing TGF-β release from healthy donors-derived monocytes, we found liposomes did not alter the release of active pro-fibrotic cytokine. All liposomes induced mild activation of neutrophils regardless of the presence of HA. HA liposomes could be also applied for lung fibrotic diseases, being endowed with low pro-inflammatory activity, and results confirmed that higher MW HA are associated to an increased targeting efficiency for CD44 expressing LFs-derived from BOS and CTD-ILD patients.

Topics: A549 Cells; Adult; Bronchiolitis Obliterans; Drug Delivery Systems; Gene Expression Regulation; Healthy Volunteers; Humans; Hyaluronan Receptors; Hyaluronic Acid; Liposomes; Microscopy, Confocal; Monocytes; Pulmonary Fibrosis; Transforming Growth Factor beta; Vascular Endothelial Growth Factor A

Tissue fibrosis is a chronic disease driven by persistent fibroblast activation that has recently been linked to epigenetic modifications. Here, we screened a small library of epigenetic small-molecule modulators to identify compounds capable of inhibiting or reversing TGFβ-mediated fibroblast activation. We identified pracinostat, an HDAC inhibitor, as a potent attenuator of lung fibroblast activation and confirmed its efficacy in patient-derived fibroblasts isolated from fibrotic lung tissue. Mechanistically, we found that HDAC-dependent transcriptional repression was an early and essential event in TGFβ-mediated fibroblast activation. Treatment of lung fibroblasts with pracinostat broadly attenuated TGFβ-mediated epigenetic repression and promoted fibroblast quiescence. We confirmed a specific role for HDAC-dependent histone deacetylation in the promoter region of the anti-fibrotic gene

Topics: Cell Line; Down-Regulation; Fibroblasts; Histone Deacetylases; Humans; Lung; Peroxisome Proliferator-Activated Receptor Gamma Coactivator 1-alpha; Promoter Regions, Genetic; Pulmonary Fibrosis; Transforming Growth Factor beta

Idiopathic pulmonary fibrosis (IPF) is a chronic interstitial lung disease with a high mortality and poor prognosis. Transforming growth factor (TGF)-β plays crucial roles in the pathogenesis of IPF. To investigate the role of sodium arsenite (SA) on fibroblast differentiation and pulmonary fibrosis, we checked the effects of SA on TGF-β-induced normal human lung fibroblasts (NHLFs) differentiation, and the anti-fibrotic effect of SA on bleomycin (BLM)-induced pulmonary fibrosis in mouse. SA treatment significantly inhibits α-smooth muscle actin and fibronectin (FN) expression in TGF-β treated NHLFs; and SA also inhibits TGF-β stimulated expression of NADPH oxidase 4 and accumulation of intracellular reactive oxygen species. TGF-β-induced the phosphorylation of ERK and Smad3 were also blocked by SA. The administration of SA (IP) suppressed BLM-induced lung fibrosis characterized as the inhibition of collagen deposition, TGF-β accumulation in bronchoalveolar lavage fluid, and the expression of FN and collagen 1a2 in lung tissue. This study revealed that SA inhibits TGF-β-induced lung fibroblast differentiation and BLM-induced pulmonary fibrosis in mice, suggesting that SA could be a potential therapeutic approach to IPF.

Topics: Animals; Arsenites; Bleomycin; Cell Differentiation; Fibroblasts; Humans; Lung; Male; MAP Kinase Signaling System; Mice, Inbred C57BL; Pulmonary Fibrosis; Smad3 Protein; Sodium Compounds; Transforming Growth Factor beta

Syndecan-4 is a transmembrane heparan sulfate proteoglycan expressed in a variety of cells, and its heparan sulfate glycosaminoglycan side chains bind to several proteins exhibiting various biological roles. The authors have previously demonstrated syndecan-4's critical roles in pulmonary inflammation. In the current study, however, its role in pulmonary fibrosis was evaluated. Wild-type and syndecan-4-deficient mice were injected with bleomycin, and several parameters of inflammation and fibrosis were analyzed. The mRNA expression of collagen and α-smooth muscle action (α-SMA) in lung tissues, as well as the histopathological lung fibrosis score and collagen content in lung tissues, were significantly higher in the syndecan-4-deficient mice. However, the total cell count and cell differentiation in bronchoalveolar lavage fluid were equivalent between the wild-type and syndecan-4-deficient mice. Although there was no difference in the TGF-β expression in lung tissues between the wild-type and syndecan-4-deficient mice, significantly more activation of Smad3 in lung tissues was observed in the syndecan-4-deficient mice compared to the wild-type mice. Furthermore, in the in vitro experiments using lung fibroblasts, the co-incubation of syndecan-4 significantly inhibited TGF-β-induced Smad3 activation, collagen and α-SMA upregulation. Moreover, syndecan-4 knock-down by siRNA increased TGF-β-induced Smad3 activation and upregulated collagen and α-SMA expression. These findings showed that syndecan-4 inhibits the development of pulmonary fibrosis, at least in part, through attenuating TGF-β signaling.

Topics: Actins; Animals; Collagen; Disease Models, Animal; Fibroblasts; Lung; Mice; Mice, Knockout; Pulmonary Fibrosis; Signal Transduction; Syndecan-4; Transforming Growth Factor beta

Pulmonary fibrosis (PF), a progressive and life-threatening pulmonary disease, is the main pathological basis of interstitial lung disease (ILD) which includes the idiopathic pulmonary fibrosis (IPF). No effective therapeutic strategy for pulmonary fibrosis has been established. TGF-

Topics: Animals; Cell Line; Cell Proliferation; Cyclin-Dependent Kinase Inhibitor p27; Gene Knockdown Techniques; Humans; Male; Mechanistic Target of Rapamycin Complex 2; Mice, Inbred C57BL; Pulmonary Fibrosis; Transforming Growth Factor beta

Myofibroblasts are key contributors to pathological fibrotic conditions of several major organs. The transdifferentiation of fibroblasts into myofibroblasts requires both a mechanical signal and transforming growth factor-β (TGF-β) signaling. The cation channel transient receptor potential vanilloid 4 (TRPV4) is a critical mediator of myofibroblast transdifferentiation and in vivo fibrosis through its mechanosensitivity to extracellular matrix stiffness. Here, we showed that TRPV4 promoted the transdifferentiation of human and mouse lung fibroblasts through its interaction with phosphoinositide 3-kinase γ (PI3Kγ), forming nanomolar-affinity, intracellular TRPV4-PI3Kγ complexes. TGF-β induced the recruitment of TRPV4-PI3Kγ complexes to the plasma membrane and increased the activities of both TRPV4 and PI3Kγ. Using gain- and loss-of-function approaches, we showed that both TRPV4 and PI3Kγ were required for myofibroblast transdifferentiation as assessed by the increased production of α-smooth muscle actin and its incorporation into stress fibers, cytoskeletal changes, collagen-1 production, and contractile force. Expression of various mutant forms of the PI3Kγ catalytic subunit (p110γ) in cells lacking PI3Kγ revealed that only the noncatalytic, amino-terminal domain of p110γ was necessary and sufficient for TGF-β-induced TRPV4 plasma membrane recruitment and myofibroblast transdifferentiation. These data suggest that TGF-β stimulates a noncanonical scaffolding action of PI3Kγ, which recruits TRPV4-PI3Kγ complexes to the plasma membrane, thereby increasing myofibroblast transdifferentiation. Given that both TRPV4 and PI3Kγ have pleiotropic actions, targeting the interaction between them could provide a specific therapeutic approach for inhibiting myofibroblast transdifferentiation.

Topics: Animals; Cell Line; Cell Membrane; Cell Transdifferentiation; Class Ib Phosphatidylinositol 3-Kinase; Humans; Lung; Mice; Myofibroblasts; Pulmonary Fibrosis; Transforming Growth Factor beta; TRPV Cation Channels

Enhancing the therapeutic efficacy of drugs for inflammatory diseases is of high demand. One possible approach is targeting drugs to the extracellular matrix of the inflamed area. Here, we target collagens in the matrix, which are inaccessible in most tissues yet are exposed to the bloodstream in the inflamed area because of vascular hyperpermeability. We conferred collagen affinity to anti-tumor necrosis factor-α (α-TNF) antibody by conjugating a collagen-binding peptide (CBP) derived from the sequence of decorin. CBP-α-TNF accumulated in the inflamed paw of the arthritis model, and arthritis development was significantly suppressed by treatment with CBP-α-TNF compared with the unmodified antibody. Similarly, CBP-anti-transforming growth factor-β (α-TGF-β) accumulated in the inflamed lung of pulmonary fibrosis model and significantly suppressed pulmonary fibrosis compared with the unmodified antibody. Together, collagen affinity enables the anticytokine antibodies to target arthritis and pulmonary fibrosis accompanied by inflammation, demonstrating a clinically translational approach to treat inflammatory diseases.

Topics: Animals; Anti-Inflammatory Agents; Antibodies; Collagen; Humans; Inflammation; Lung; Mice, Inbred BALB C; Mice, Inbred C57BL; Molecular Targeted Therapy; Peptide Fragments; Pulmonary Fibrosis; Sialoglycoproteins; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha

Type 2 insulin-like growth factor (IGF-II) levels are increased in fibrosing lung diseases such as idiopathic pulmonary fibrosis (IPF) and scleroderma/systemic sclerosis-associated pulmonary fibrosis (SSc). Our goal was to investigate the contribution of IGF receptors to IGF-II-mediated fibrosis in these diseases and identify other potential mechanisms key to the fibrotic process. Cognate receptor gene and protein expression were analyzed with qRT-PCR and immunoblot in primary fibroblasts derived from lung tissues of normal donors (NL) and patients with IPF or SSc. Compared to NL, steady-state receptor gene expression was decreased in SSc but not in IPF. IGF-II stimulation differentially decreased receptor mRNA and protein levels in NL, IPF, and SSc fibroblasts. Neutralizing antibody, siRNA, and receptor inhibition targeting endogenous IGF-II and its primary receptors, type 1 IGF receptor (IGF1R), IGF2R, and insulin receptor (IR) resulted in loss of the IGF-II response. IGF-II tipped the TIMP:MMP balance, promoting a fibrotic environment both intracellularly and extracellularly. Differentiation of fibroblasts into myofibroblasts by IGF-II was blocked with a TGFβ1 receptor inhibitor. IGF-II also increased TGFβ2 and TGFβ3 expression, with subsequent activation of canonical SMAD2/3 signaling. Therefore, IGF-II promoted fibrosis through IGF1R, IR, and IGF1R/IR, differentiated fibroblasts into myofibroblasts, decreased protease production and extracellular matrix degradation, and stimulated expression of two TGFβ isoforms, suggesting that IGF-II exerts pro-fibrotic effects via multiple mechanisms.

Topics: Cells, Cultured; Fibroblasts; Humans; Insulin-Like Growth Factor II; Matrix Metalloproteinases; Pulmonary Fibrosis; Receptor, IGF Type 1; Receptor, Insulin; Signal Transduction; Tissue Inhibitor of Metalloproteinase-1; Transforming Growth Factor beta

Our earlier report indicated that active vitamin D3 inhibited epithelial-mesenchymal transition (EMT) in bleomycin (BLM)-induced pulmonary fibrosis. The objective of this study was to further investigate whether vitamin D deficiency exacerbates BLM-induced pulmonary fibrosis.. The relative weight of lungs was elevated in BLM-treated mice. Col1α1 and Col1α2, two collagen protein genes, were upregulated, and collagen deposition, as determined by Sirius red staining, was observed in the lungs of BLM-treated mice. E-cadherin, an epithelial marker, was downregulated. By contrast, vimentin and α-SMA, two EMT markers, were upregulated in the lungs of BLM-treated mice. Pulmonary TGF-β/Smad3 signaling was activated in BLM-induced lung fibrosis. Further analysis showed that feeding VDD diet, leading to vitamin D deficiency, aggravated elevation of BLM-induced relative lung weight. Moreover, feeding VDD diet aggravated BLM-induced TGF-β/Smad3 activation and subsequent EMT in the lungs. In addition, feeding VDD diet exacerbated BLM-induced pulmonary fibrosis. Additional experiment showed that Cyp27b1 gene knockout, leading to active vitamin D3 deficiency, exacerbated BLM-induced pulmonary fibrosis. Moreover, Cyp27b1 gene knockout aggravated pulmonary TGF-β/Smad2/3 activation and subsequent EMT in BLM-induced lung fibrosis.. Vitamin D deficiency exacerbates BLM-induced pulmonary fibrosis partially through aggravating TGF-β/Smad2/3-mediated EMT in the lungs.

Topics: Animals; Biopsy, Needle; Bleomycin; Blotting, Western; Disease Models, Animal; Immunohistochemistry; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Pulmonary Fibrosis; Random Allocation; Real-Time Polymerase Chain Reaction; Reference Values; RNA, Messenger; Sensitivity and Specificity; Signal Transduction; Smad3 Protein; Transforming Growth Factor beta; Up-Regulation; Vitamin B Deficiency

Progressive organ fibrosis accounts for one-third of all deaths worldwide, yet preclinical models that mimic the complex, progressive nature of the disease are lacking, and hence, there are no curative therapies. Progressive fibrosis across organs shares common cellular and molecular pathways involving chronic injury, inflammation, and aberrant repair resulting in deposition of extracellular matrix, organ remodeling, and ultimately organ failure. We describe the generation and characterization of an in vitro progressive fibrosis model that uses cell types derived from induced pluripotent stem cells. Our model produces endogenous activated transforming growth factor β (TGF-β) and contains activated fibroblastic aggregates that progressively increase in size and stiffness with activation of known fibrotic molecular and cellular changes. We used this model as a phenotypic drug discovery platform for modulators of fibrosis. We validated this platform by identifying a compound that promotes resolution of fibrosis in in vivo and ex vivo models of ocular and lung fibrosis.

Topics: Animals; Cell Line; Cells, Cultured; Drug Discovery; Female; Humans; Induced Pluripotent Stem Cells; Male; Mice; Mice, Inbred C57BL; Pulmonary Fibrosis; Small Molecule Libraries; Transforming Growth Factor beta

We aimed to investigate in the present work, using metabonomics approaches, the scalability of lung fibrosis-biomarkers, in bleomycin (BLM) model of pulmonary fibrosis in rats. Sixty male Wistar rats, weighing 250 ± 10 g, were randomly divided into three groups: a negative control group receiving normal saline treatment (G

Topics: Aerosols; Animals; Antioxidants; Biomarkers; Bleomycin; Bronchoalveolar Lavage Fluid; Disease Models, Animal; Fibroblasts; Inhalation Exposure; Lipid Peroxidation; Lung; Male; Metabolomics; Oxidative Stress; Proton Magnetic Resonance Spectroscopy; Pulmonary Fibrosis; Rats, Wistar; Time Factors; Transforming Growth Factor beta

The differentiation of interstitial lung fibroblasts into contractile myofibroblasts that proliferate and secrete excessive extracellular matrix is critical for the pathogenesis of pulmonary fibrosis. Certain lipid signaling molecules, such as prostaglandins (PGs), can inhibit myofibroblast differentiation. However, the sources and delivery mechanisms of endogenous PGs are undefined. Activated primary human lung fibroblasts (HLFs) produce PGs such as PGE

Topics: Antifibrinolytic Agents; Cell Differentiation; Cells, Cultured; Dinoprostone; Exosomes; Extracellular Matrix; Extracellular Vesicles; Female; Fibroblasts; Humans; Interleukin-1beta; Lung; Male; Myofibroblasts; Prostaglandins; Pulmonary Fibrosis; Signal Transduction; Transforming Growth Factor beta

Topics: Cicatrix; Humans; Myofibroblasts; Pulmonary Fibrosis; Transforming Growth Factor beta; Transforming Growth Factor beta1; Transforming Growth Factors

Idiopathic pulmonary fibrosis (IPF) is a chronic and deadly disease with a poor prognosis and few treatment options. Pathological remodeling of the extracellular matrix (ECM) by myofibroblasts is a key factor that drives disease pathogenesis, although the underlying mechanisms remain unknown. Alternative polyadenylation (APA) has recently been shown to play a major role in cellular responses to stress by driving the expression of fibrotic factors and ECMs through altering microRNA sensitivity, but a connection to IPF has not been established. Here, we demonstrate that CFIm25, a global regulator of APA, is down-regulated in the lungs of patients with IPF and mice with pulmonary fibrosis, with its expression selectively reduced in alpha-smooth muscle actin (α-SMA) positive fibroblasts. Following the knockdown of CFIm25 in normal human lung fibroblasts, we identified 808 genes with shortened 3'UTRs, including those involved in the transforming growth factor-β signaling pathway, the Wnt signaling pathway, and cancer pathways. The expression of key pro-fibrotic factors can be suppressed by CFIm25 overexpression in IPF fibroblasts. Finally, we demonstrate that deletion of CFIm25 in fibroblasts or myofibroblast precursors using either the Col1a1 or the Foxd1 promoter enhances pulmonary fibrosis after bleomycin exposure in mice. Taken together, our results identified CFIm25 down-regulation as a novel mechanism to elevate pro-fibrotic gene expression in pulmonary fibrosis.

Topics: 3' Untranslated Regions; Actins; Adult; Aged; Animals; Bleomycin; Cleavage And Polyadenylation Specificity Factor; Disease Progression; Down-Regulation; Extracellular Matrix; Female; Fibroblasts; Humans; Lung; Male; Mice; Mice, Inbred C57BL; MicroRNAs; Middle Aged; Muscle, Smooth; Myofibroblasts; Polyadenylation; Pulmonary Fibrosis; RNA, Small Interfering; Signal Transduction; Transforming Growth Factor beta

Idiopathic pulmonary fibrosis (IPF) is a progressive and fatal lung disorder driven by unrelenting extracellular matrix deposition. Fibroblasts are recognized as the central mediators of extracellular matrix production in IPF; however, the characteristics of the underlying fibroblast cell populations in IPF remain poorly understood. Here, we use an unbiased single-cell RNA sequencing analysis of a bleomycin-induced pulmonary fibrosis model to characterize molecular responses to fibrotic injury. Lung cells were isolated on Day 11 to capture emerging fibrosis and gene expression was analyzed by three complementary techniques, which, together, generated a 49-gene signature that defined an activated subpopulation of fibroblasts. However, none of the identified genes were specific to the activated cells or to the disease setting, implying that the activated fibroblasts are not uniquely defined, but exhibit a similar, yet amplified, gene expression pattern to control cells. Our findings have important implications for fibrosis research, including:

Topics: Actins; Animals; Biomarkers; Bleomycin; Disease Models, Animal; Fibroblasts; Gene Expression Profiling; Gene Expression Regulation; Lung; Male; Mice, Inbred C57BL; Muscle, Smooth; Pulmonary Fibrosis; Sequence Analysis, DNA; Signal Transduction; Single-Cell Analysis; Time Factors; Transforming Growth Factor beta

It has been established that lanatoside C, a FDA-approved cardiac glycoside, reduces proliferation of cancer cell lines. The proliferation of fibroblasts is critical to the pathogenesis of pulmonary fibrosis (PF), a progressive and fatal fibrotic lung disease lacking effective treatment. In this study we have investigated the impact of lanatoside C on a bleomycin (BLM)-induced mouse model of PF and through the evaluation of fibroblast proliferation and activation in vitro. We evaluated explanted lung tissue by histological staining, western blot analysis, qRT-PCR and survival analysis, demonstrating that lanatoside C was able to protect mice against BLM-induced pulmonary fibrosis. The proliferation of cultured pulmonary fibroblasts isolated from BLM-induced PF mice was suppressed by lanatoside C, as hypothesized, through the induction of cell apoptosis and cell cycle arrest at the G2/M phase. The Akt signalling pathway was involved in this process. Interestingly, the production of α-SMA, fibronectin, and collagen I and III in response to TGF-β1 in healthy mouse fibroblasts was suppressed following lanatoside C administration by inhibition of TGF-β1/Smad signalling. In addition, TGF-β1-induced migration in lung fibroblasts was also impeded after lanatoside C treatment. Together, our data revealed that lanatoside C alleviated BLM-induced pulmonary fibrosis in mice via attenuation of growth and differentiation of fibroblasts, suggesting that it has potential as a candidate therapy for PF patients.

Topics: Animals; Apoptosis; Bleomycin; Cell Cycle Checkpoints; Cell Differentiation; Cell Proliferation; Cyclin D1; Cyclin E; Cytoprotection; Down-Regulation; Fibroblasts; Forkhead Box Protein O1; Lanatosides; Male; Mice; Mice, Inbred C57BL; Phosphorylation; Proto-Oncogene Proteins c-akt; Proto-Oncogene Proteins c-bcl-2; Pulmonary Fibrosis; Signal Transduction; Smad Proteins; Transforming Growth Factor beta

Fibrosis is a universal finding in chronic allograft dysfunction, and it is characterized by an accumulation of extracellular matrix. The precise source of the myofibroblasts responsible for matrix deposition is not understood, and pharmacological strategies for prevention or treatment of fibrosis remain limited. One source of myofibroblasts in fibrosis is an endothelial-to-mesenchymal transition (EndMT), a process first described in heart development and involving endothelial cells undergoing a phenotypic change to become more like mesenchymal cells. Recently, lineage tracing of endothelial cells in mouse models allowed studies of EndMT in vivo and reported 27% to 35% of myofibroblasts involved in cardiac fibrosis and 16% of isolated fibroblasts in bleomycin-induced pulmonary fibrosis to be of endothelial origin. Over the past decade, mature microRNAs (miRNAs) have increasingly been described as key regulators of biological processes through repression or degradation of targeted mRNA. The stability and abundance of miRNAs in body fluids make them attractive as potential biomarkers, and progress is being made in developing miRNA targeted therapeutics. In this review, we will discuss the evidence of miRNA regulation of EndMT from in vitro and in vivo studies and the potential relevance of this to heart, lung, and kidney allograft dysfunction.

Topics: Animals; Chronic Disease; Endothelial Cells; Fibrosis; Humans; Kidney; Mesoderm; MicroRNAs; Myocardium; Myofibroblasts; Pulmonary Fibrosis; Transforming Growth Factor beta; Transplantation, Homologous

TGF-β1 is a critical mediator of tissue fibrosis in health and disease whose effects are augmented by chitinase 1 (CHIT1). However, the mechanisms that CHIT1 uses to regulate TGF-β1-mediated fibrotic responses have not been defined. Here, we demonstrate that CHIT1 enhances TGF-β1-stimulated fibrotic cellular and tissue responses and TGF-β1 signaling. Importantly, we also demonstrate that these effects are mediated by the ability of CHIT1 to inhibit TGF-β1 induction of its feedback inhibitor, SMAD7. CHIT1 also interacted with TGF-β receptor associated protein 1 (TGFBRAP1) and forkhead box O3 (FOXO3) with TGFBRAP1 playing a critical role in CHIT1 enhancement of TGF-β1 signaling and effector responses and FOXO3 playing a critical role in TGF-β1 induction of SMAD7. These pathways were disease relevant because the levels of CHIT1 were increased and inversely correlated with SMAD7 in tissues from patients with idiopathic pulmonary fibrosis or scleroderma-associated interstitial lung disease. These studies demonstrate that CHIT1 regulates TGF-β1/SMAD7 axis via TGFBRAP1 and FOXO3 and highlight the importance of these pathways in the pathogenesis of pulmonary fibrosis.

Topics: Fibroblasts; Forkhead Box Protein O3; Gene Expression Regulation; Genes, Reporter; Hexosaminidases; Humans; Immunohistochemistry; Intracellular Signaling Peptides and Proteins; Promoter Regions, Genetic; Pulmonary Fibrosis; RNA, Small Interfering; Signal Transduction; Smad7 Protein; Transforming Growth Factor beta

P120-catenin (P120) was known to function in adhesion between cells and signal transduction in many types of cells. In this study, we investigated the expression and role of P120 in pulmonary fibrosis and transforming growth factor beta (TGF-β) mediated lung fibroblast-to-myofibroblast differentiation (fibroblast differentiation). Our data indicated that P120 expression increased in lung fibrotic foci and primary lung fibroblasts isolated from bleomycin- (BLM) challenged mice, compared to controls. In vitro, TGF-β induced P120 expression in human lung fibroblasts, and siRNA-mediated SMAD3 depletion inhibited TGF-β stimulated P120 expression. Blocking nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB) pathway through chemical inhibitor or knockdown of NF-kB p65 subunit also suppressed TGF-β induced P120 expression in human lung fibroblast. Knockdown of P120 expression inhibited TGF-β induced human lung fibroblast differentiation, as well as suppressed the activation of SMAD and ERK signaling pathways. Administration of lentivirus coding mouse P120 sh-RNA into mouse lung tissue dramatically attenuated the expression of P120 in lung tissue and lung fibroblast, suppressed BLM induced increase of TGF-β, alpha smooth muscle actin (α-SMA) and fibronectin (FN) expression, and decreased the deposition of collagen and pulmonary fibrosis. Collectively, these results suggested that P120 involved in lung fibroblast differentiation and pulmonary fibrosis, and inhibition of P120 expression decreased pulmonary fibrosis in BLM challenged mice. Thus, attenuation of P120 expression might be a potential therapeutic strategy for human lung fibrosis.

Topics: Animals; Bleomycin; Catenins; Cell Differentiation; Cell Line; Cells, Cultured; Delta Catenin; Fibroblasts; Fibronectins; Humans; Lung; Male; Mice; Mice, Inbred BALB C; NF-kappa B; Pulmonary Fibrosis; Signal Transduction; Smad3 Protein; Transforming Growth Factor beta; Transforming Growth Factor beta1

Early pulmonary fibrosis is the leading cause of poor prognosis in patients with acute respiratory distress syndrome (ARDS). However, whether the renin-angiotensin system (RAS) can serve as a therapeutic target is unknown. In this study, an animal model of early pulmonary fibrosis was established via the LPS three-hit regimen. Afterwards, the animals were treated with intraperitoneal injections of Ang-(1-7), AVE0991, or A779 once per day for 20 days. The plasma and BALF AngII levels of the animals were increased, while there were no significant changes in Ang-(1-7) levels in lung tissue after LPS treatment. Furthermore, the AT1R protein levels were significantly increased and the Mas levels were significantly decreased on days 14 and 21. Administration of Ang-(1-7) downregulated LPS-induced AT1R mRNA expression, which was upregulated by A779. The expression of Mas mRNA responded in the opposite direction relative to AT1R. Moreover, LPS caused decreased levels of Mas and E-cadherin and increased AT1R, Vimentin, and Src phosphorylation levels. Ang-(1-7) or AVE0991 blocked these effects but was counteracted by A779 treatment. Our findings suggested that AngII and AT1R levels exhibit opposite dynamic trends during LPS-induced early pulmonary fibrosis, as do Ang-(1-7) and Mas. Ang-(1-7) exerts protective effects against early pulmonary fibrosis, mainly by regulating the balance between AngII and AT1R and between Ang-(1-7) and Mas and by inhibiting Src kinase activation.

Topics: Angiotensin I; Angiotensin II; Animals; Bronchoalveolar Lavage Fluid; Cadherins; Drug Evaluation, Preclinical; Imidazoles; Lipopolysaccharides; Lung; Peptide Fragments; Proto-Oncogene Mas; Proto-Oncogene Proteins; Pulmonary Fibrosis; Random Allocation; Rats, Sprague-Dawley; Receptor, Angiotensin, Type 1; Receptors, G-Protein-Coupled; Transforming Growth Factor beta; Vasodilator Agents; Vimentin

Topics: Animals; c-Mer Tyrosine Kinase; Collagen Type I; Disease Models, Animal; Intercellular Signaling Peptides and Proteins; Lung; Male; Mice, Inbred C57BL; Mice, Knockout; Pneumonia; Pulmonary Fibrosis; Signal Transduction; Silicosis; Suppressor of Cytokine Signaling 1 Protein; Time Factors; Transforming Growth Factor beta

The plant Arenaria kansuensis is used in traditional medicine to treat lung inflammation for a long time. However, the anti-pulmonary fibrosis effect and its corresponding bioactive constituents of this plant have not been studied extensively.. The purpose of this study was to investigate the anti-pulmonary fibrosis effect and its corresponding bioactive constituents of A. kansuensis and its possible mechanism.. In vivo experiment, the anti-pulmonary fibrosis effects of the fraction (Part1) enriched from ethyl acetate extracts of the whole plant A. kansuensis were evaluated through bleomycin (BLM)-induced pulmonary fibrosis mice (five groups, n = 10) daily at doses of 50, 100 and 150 mg/kg for 15 days. In vitro experiment, the anti-inflammation and reversed epithelial-mesenchymal transition (EMT) effect of 12 β-carboline alkaloids isolated from Part1 were evaluated through lipopolysaccharide (LPS)-induced RAW264.7 inflammatory cell model and TGF-β1 induced A549 cell model.. In this study, a fraction named Part1 extracted from Arenaria kansuensis presented strong anti-pulmonary fibrosis effect at the dose of 150 mg/kg. Vivo experiments showed that the survival rate and body weight of mice significantly increased after Part1 treatment. Part1 could significantly inhibit the initial of inflammation, deposition of collagen and expression of TGF-β1 and α-SMA, moreover, the expression of E-cadherin was significantly elevated after administration of Part1. All the cure effects of Part1 were in dose dependent manner. A total of 12 β-carboline alkaloids were identified in Part1 and they all showed suppressive effect on inflammatory cytokines including MCP-1, TNF-α, IL-6 and IL-1β through inhibition of NF-kb/p65 phosphorylation, and that epithelial-mesenchymal transition (EMT) process was reversed by different compounds in different levels. The expression of indicators of EMT including α-SMA, vimentin and E-cadherin was significantly improved after given different β-carboline alkaloids.. This study showed that antifibrogenic effect of β-carboline alkaloids was due to inhibiting the initial of inflammation through NF-kb/p65 pathway and reversing the process of EMT.

Topics: Alkaloids; Animals; Anti-Inflammatory Agents; Arenaria Plant; Bleomycin; Carbolines; Cell Line; Cytokines; Epithelial-Mesenchymal Transition; Female; Humans; Lung; Male; Mice; NF-kappa B; Phosphorylation; Plant Extracts; Pulmonary Fibrosis; Transforming Growth Factor beta

Pulmonary fibrosis is a result of an abnormal wound healing in lung tissue triggered by an excessive accumulation of extracellular matrix proteins, loss of tissue elasticity, and debit of ventilatory function. NKT cells are a major source of Th1 and Th2 cytokines and may be crucial in the polarization of M1/M2 macrophages in pulmonary fibrogenesis. Although there appears to be constant scientific progress in that field, pulmonary fibrosis still exhibits no current cure. From these facts, we hypothesized that NKT cells could influence the development of pulmonary fibrosis via modulation of macrophage activation. Wild type (WT) and NKT type I cell-deficient mice (Jα18

Topics: Animals; Bleomycin; Collagen; Cytokines; Disease Models, Animal; Galactosylceramides; Inflammation; Lung; Macrophages; Mice; Mice, Inbred C57BL; Mice, Knockout; Natural Killer T-Cells; Phenotype; Pulmonary Fibrosis; Th1 Cells; Th2 Cells; Transforming Growth Factor beta; Vimentin

Radiation-induced pulmonary fibrosis is a severe complication of patients treated with thoracic irradiation. We have previously shown that syndecan-2 reduces fibrosis by exerting alveolar epithelial cytoprotective effects. Here, we investigate whether syndecan-2 attenuates radiation-induced pulmonary fibrosis by inhibiting fibroblast activation. C57BL/6 wild-type mice and transgenic mice that overexpress human syndecan-2 in alveolar macrophages were exposed to 14 Gy whole-thoracic radiation. At 24 weeks after irradiation, lungs were collected for histological, protein, and mRNA evaluation of pulmonary fibrosis, profibrotic gene expression, and α-smooth muscle actin (α-SMA) expression. Mouse lung fibroblasts were activated with transforming growth factor (TGF)-β1 in the presence or absence of syndecan-2. Cell proliferation, migration, and gel contraction were assessed at different time points. Irradiation resulted in significantly increased mortality and pulmonary fibrosis in wild-type mice that was associated with elevated lung expression of TGF-β1 downstream target genes and cell death compared with irradiated syndecan-2 transgenic mice. In mouse lung fibroblasts, syndecan-2 inhibited α-SMA expression, cell contraction, proliferation, and migration induced by TGF-β1. Syndecan-2 attenuated phosphoinositide 3-kinase/serine/threonine kinase/Rho-associated coiled-coil kinase signaling and serum response factor binding to the α-SMA promoter. Syndecan-2 attenuates pulmonary fibrosis in mice exposed to radiation and inhibits TGF-β1-induced fibroblast-myofibroblast differentiation, migration, and proliferation by down-regulating phosphoinositide 3-kinase/serine/threonine kinase/Rho-associated coiled-coil kinase signaling and blocking serum response factor binding to the α-SMA promoter via CD148. These findings suggest that syndecan-2 has potential as an antifibrotic therapy in radiation-induced lung fibrosis.

Topics: Animals; Cell Movement; Cell Proliferation; Female; Mice; Mice, Inbred C57BL; Mice, Transgenic; Myofibroblasts; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; Pulmonary Fibrosis; Radiation Injuries; Receptor-Like Protein Tyrosine Phosphatases, Class 3; rho-Associated Kinases; Syndecan-2; Thorax; Transforming Growth Factor beta

Heparin-binding epidermal growth factor (EGF)-like growth factor (HB-EGF) is a member of the EGF family growth factors, which affects multiple aspects of the wound healing process such as epithelialization, wound contraction and angiogenesis. In our study, we measured the serum HB-EGF levels of 51 systemic sclerosis (SSc) patients, which showed a significant increase compared with those of 20 normal subjects. Further analysis revealed a positive correlation between the HB-EGF level and pulmonary ground-glass score but no correlation between the former and pulmonary fibrosis score. Other findings include: a significant increase of serum sialylated carbohydrate antigen KL-6 levels and significant shortness of disease duration in the diffuse cutaneous SSc patients with elevated HB-EGF levels; and significantly higher HB-EGF levels in the presence of Raynaud's phenomenon, in that of telangiectasia, and in the absence of contracture of phalanges in all SSc patients. We then evaluated HB-EGF mRNA levels of fibroblasts harvested from skin samples of the SSc patients and those of foreskin-derived fibroblasts treated with transforming growth factor-β, both of which were significantly higher than each control. In conclusion, we speculate that HB-EGF plays a pro-inflammatory role in the active skin and lung lesions of SSc.

Topics: Adult; Aged; Biopsy; Cells, Cultured; Female; Fibroblasts; Fibrosis; Heparin-binding EGF-like Growth Factor; Humans; Lung; Male; Middle Aged; Mucin-1; Pulmonary Fibrosis; Respiratory Function Tests; RNA, Messenger; Scleroderma, Systemic; Skin; Transforming Growth Factor beta

Pulmonary fibrosis triggered during the early stage of acute respiratory distress syndrome (ARDS) contributes to poor prognosis in patients. However, whether microRNAs (miRNAs) can serve as therapeutic targets for early pulmonary fibrosis during ARDS is still largely unknown. In this study, we evaluated the effects and mechanisms of miR-200s and its targets ZEB1/2 in lung tissue. An early pulmonary fibrosis mouse model caused by ARDS was established via a lipopolysaccharide (LPS) three-hit regimen. Lentiviral packaged miR-200b/c cDNA or ZEB1/2 shRNA was intratracheally administered into the lungs of C57BL/6 mice 1 day before an LPS injection was administered. In vitro, following a 30-min pretreatment with miR-200b/c or SB203580/SIS3, RLE-6TN cells were stimulated by LPS or LPS + transforming growth factor-β (TGF-β) for 24 h. miR-200b/c and E-cadherin protein expression declined, whereas ZEB1/2 mRNA and protein and vimentin and α-smooth muscle actin (α-SMA) protein levels gradually increased during the development of pulmonary fibrosis. Furthermore, both the overexpression of miR-200b/c and the silencing of ZEB1/2 significantly alleviated pulmonary inflammation and fibrosis, reduced vimentin and α-SMA expression, and increased E-cadherin protein levels. In RLE-6TN cells, LPS combined with TGF-β exerts synergistic effects of increasing vimentin and α-SMA protein levels, increasing p38 and smad3 phosphorylation and reducing E-cadherin protein levels, which were reversed by pretreatment with miR-200b/c or SB203580/SIS3. Our findings demonstrate that miR-200b/c was downregulated, whereas ZEB1/2 was upregulated in the development of LPS-induced early pulmonary fibrosis. miR-200b/c exerts a protective effect by targeting ZEB1/2, which may be associated with the inhibition of p38 MAPK and TGF-β /smad3 signaling pathways.

Topics: Actins; Animals; Bronchoalveolar Lavage Fluid; Cadherins; Cell Line; Disease Models, Animal; Epithelial-Mesenchymal Transition; Lipopolysaccharides; Lung; MAP Kinase Signaling System; Mice, Inbred C57BL; MicroRNAs; Pulmonary Fibrosis; Random Allocation; Rats; Respiratory Distress Syndrome; RNA, Small Interfering; Smad3 Protein; Transforming Growth Factor beta; Vimentin; Zinc Finger E-box Binding Homeobox 2; Zinc Finger E-box-Binding Homeobox 1

Radiation-induced lung injury has restricted radiotherapy for thoracic cancer. The purpose of this study was to investigate the radioprotective effects of bromodomain and extra terminal (BET) inhibitor JQ1 in a murine model of pulmonary damage. Chest computed tomography (CT) was performed in a rat model after 20 Gy radiation of the right thorax. And histological evaluation and protein expressions of irradiated tissue were analyzed to confirm the potential anti-fibrosis effect of JQ1 and its underlying mechanisms. Moreover, colony formation assays were used to explore the effects of JQ1 on esophageal cancer Eca109 and breast cancer MCF7. JQ1 attenuated radiologic and histologic presentations of radiation-induced fibrosis, inflammatory reaction and pulmonary structural changes and the increase of Hounsfield units (HU) density and hydroxyproline content after radiation. Additionally, JQ1 suppressed BRD4, c-MYC, Collagen I, TGF-β, p-NF-κB p65, p-Smad2 and p-Smad3 expressions after irradiation, repressed proliferation and transdifferentiation of lung fibroblasts, and impaired clonogenic survival of thoracic cancer cells. Collectively, our study demonstrated for the first time that BET Bromodomain inhibitor JQ1 protected normal lung tissue after radiation, and exerted a radiosensitizing effect in thoracic cancer cells.

Topics: Animals; Azepines; Cell Line, Tumor; Cell Proliferation; Collagen Type I; Female; Fibroblasts; Gamma Rays; Gene Expression Regulation; Humans; Hydroxyproline; Lung; MCF-7 Cells; Molecular Targeted Therapy; NF-kappa B; Nuclear Proteins; Proteins; Proto-Oncogene Proteins c-myc; Pulmonary Fibrosis; Rats; Rats, Sprague-Dawley; Smad2 Protein; Smad3 Protein; Transcription Factors; Transforming Growth Factor beta; Triazoles

In pulmonary fibrosis (PF), fibroblasts and myofibroblasts proliferate and deposit excessive extracellular matrix in the interstitium, impairing normal lung function. Because most forms of PF have a poor prognosis and limited treatment options, PF represents an urgent unmet need for novel, effective therapeutics. Although the role of immune cells in lung fibrosis is unclear, recent studies suggest that T lymphocyte (T cell) activation may be impaired in PF patients. Furthermore, we have previously shown that activated T cells can produce prostaglandins with anti-scarring potential. Here, we test the hypothesis that activated T cells directly inhibit myofibroblast differentiation using a coculture system. Coculture with activated primary blood-derived T cells, from both healthy human donors and PF patients, inhibited transforming growth factor β-induced myofibroblast differentiation in primary human lung fibroblasts isolated from either normal or PF lung tissue. Coculture supernatants contained anti-fibrotic prostaglandins D

Topics: Cell Differentiation; Cells, Cultured; Coculture Techniques; Dinoprostone; Fibroblasts; Humans; Myofibroblasts; Prostaglandin D2; Pulmonary Fibrosis; T-Lymphocytes; Transforming Growth Factor beta

The respiratory disease pulmonary fibrosis (PF), which is characterized by scar formation throughout the lung, imposes a serious health burden. No effective drug without side effects has been proven to prevent this fatal lung disease. In this context, this study was undertaken to elucidate the protective effect of celastrol, a quinine methide pentacyclic triterpenoid from a Chinese medicinal plant 'thunder god vine' against bleomycin (BLM)-induced PF. We also attempted to study how the cytokine transforming growth factor-β (TGF-β) stimulates fibrosis through the induction of epithelial-mesenchymal transition (EMT) and the role of celastrol in regulating EMT. TGF-β (5 ng/ml) was administered to human alveolar epithelial adenocarcinoma A549 cells to induce fibrotic response in cells. Induction of EMT was analysed in cells through morphological analysis and expression of epithelial and mesenchymal markers by Western blotting. Bleomycin at a concentration of 3 U/Kg b.w was used to induce fibrosis in adult male rat lungs. Celastrol (5 mg/kg b.w) was given to rats twice a week after BLM administration for a period of 28 days. Western blot and immunofluorescence analyses were performed with lung tissue sample to find out the potential of celastrol in regulating EMT during the progression of fibrosis. TGF-β induces EMT in A549 cells as demonstrated by changes in epithelial cell morphology and expression of epithelial and mesenchymal marker proteins. The expressions of epithelial marker proteins E-cadherin and claudin were found to be reduced in the BLM-induced group of rats. Expression of mesenchymal markers, such as N-cadherin, snail, slug, vimentin and β-catenin, was enhanced in BLM-induced rat lungs. Celastrol reverts these cellular changes in rat lungs, and it was found that celastrol regulates EMT through the inhibition of heat shock protein 90 (HSP 90). Together, the results indicate that EMT is a crucial phenomenon for the progression of fibrosis, and celastrol provides protection against PF through the regulation of EMT.

Topics: A549 Cells; Animals; Bleomycin; Cadherins; Disease Models, Animal; Drugs, Chinese Herbal; Epithelial Cells; Epithelial-Mesenchymal Transition; Humans; Lung; Male; Pentacyclic Triterpenes; Pulmonary Fibrosis; Rats; Rats, Wistar; Signal Transduction; Smad Proteins; Transforming Growth Factor beta; Tripterygium; Triterpenes

Evidence is provided that the fibroproliferative actions of TGF-β are dependent on a metabolic adaptation that sustains pathologic growth. Specifically, profibrotic TGF-β signaling is shown to require fatty acid synthase (FASN), an essential anabolic enzyme responsible for the de novo synthesis of fatty acids. With the use of pharmacologic and genetic approaches, we show that TGF-β-stimulated FASN expression is independent of Smad2/3 and is mediated via mammalian target of rapamycin complex 1. In the absence of FASN activity or protein, TGF-β-driven fibrogenic processes are reduced with no apparent toxicity. Furthermore, as increased FASN expression was also observed to correlate with the degree of lung fibrosis in bleomycin-treated mice, inhibition of FASN was examined in a murine-treatment model of pulmonary fibrosis. Remarkably, inhibition of FASN not only decreased expression of profibrotic targets, but lung function was also stabilized/improved, as assessed by peripheral blood oxygenation.-Jung, M.-Y., Kang, J.-H., Hernandez, D. M., Yin, X., Andrianifahanana, M., Wang, Y., Gonzalez-Guerrico, A., Limper, A. H., Lupu, R., Leof, E. B. Fatty acid synthase is required for profibrotic TGF-β signaling.

Topics: Animals; Bleomycin; Cell Line; Fatty Acid Synthase, Type I; Mice; Mice, Inbred C57BL; Pulmonary Fibrosis; Signal Transduction; Smad Proteins; Transforming Growth Factor beta

Idiopathic pulmonary fibrosis (IPF) is a progressive and fatal disease with no effective treatment. The epithelial-mesenchymal transition (EMT) is a critical stage during the development of fibrosis. To assess the effect of sulforaphane (SFN) on the EMT and fibrosis using an in vitro transforming growth factor (TGF)-β1-induced model and an in vivo bleomycin (BLM)-induced model.. In vitro studies, cell viability, and cytotoxicity were measured using a Cell Counting Kit-8. The functional TGF-β1-induced EMT and fibrosis were assessed using western blotting and a quantitative real-time polymerase chain reaction. The lungs were analyzed histopathologically in vivo using hematoxylin and eosin and Masson's trichrome staining. The BLM-induced fibrosis was characterized by western blotting and immunohistochemical analyses for fibronectin, TGF-β1, E-cadherin (E-cad), and α-smooth muscle actin (SMA) in lung tissues.. SFN reversed mesenchymal-like changes induced by TGF-β1 and restored cells to their epithelial-like morphology. The results confirmed that the expression of the epithelial marker, E-cadherin, increased after SFN treatment, while expression of the mesenchymal markers, N-cadherin, vimentin, and α-SMA decreased in A549 cells after SFN treatment. In addition, SFN inhibited TGF-β1-induced mRNA expression of the EMT-related transcription factors, Slug, Snail, and Twist. The SFN treatment attenuated TGF-β1-induced expression of fibrosis-related proteins, such as fibronection, collagen I, collagen IV, and α-SMA in MRC-5 cells. Furthermore, SFN reduced the TGF-β1-induced phosphorylation of SMAD2/3 protein in A549 cells and MRC-5 cells. BLM induced fibrosis in mouse lungs that was also attenuated by SFN treatment, and SFN treatment decreased BLM-induced fibronectin expression, TGF-β1 expression, and the levels of collagen I in the lungs of mice.. SFN showed a significant anti-fibrotic effect in TGF-β-treated cell lines and BLM-induced fibrosis in mice. These findings showed that SFN has anti-fibrotic activity that may be considered in the treatment of IPF.

Topics: Animals; Bleomycin; Cell Line; Collagen Type I; Collagen Type IV; Epithelial-Mesenchymal Transition; Humans; Isothiocyanates; Lung; Male; Mice, Inbred C57BL; Pulmonary Fibrosis; Smad2 Protein; Smad3 Protein; Sulfoxides; Transforming Growth Factor beta

The nature of pulmonary fibrosis involves inadequate repair of the epithelial cell barrier accompanied by impaired regulation of the fibroblast. Moreover, pulmonary fibrosis currently lacks an effective therapeutic drug. This study targets the protection of the epithelial cell and fibroblast to identify a novel, potentially therapeutic drug (i.e., astilbin).. In this study, the cytotoxicity of astilbin was firstly detected using CCK-8. A real-time proliferation/migration analysis system was used to test the inhibitory proliferation and migration of astilbin in vitro. The expression of mesenchymal markers and the loss of epithelial cell markers were analyzed to evaluate the antifibrotic activity of astilbin on TGF-β1-treated AEC-II and L929 cells and bleomycin-treated mice. Then, in fibrosis-associated signaling pathways, the regulation of astilbin was tested using RNA sequencing and Cignal Finder 45-Pathway system. Rescue and other experiments were used to confirm this pathway regulation further.. The data showed that astilbin inhibited proliferation and migration of cell samples. Its treatment resulted in the reduction of pathological score and collagen deposition, with a decrease in α-SMA and Snail and an increase in E-cadherin and SP-C in vivo and in vitro. The fibrosis-associated aberrant genes are some of the most notable components of the Hedgehog signaling pathway.. Astilbin ameliorates pulmonary fibrosis via blockade of Hedgehog signaling pathway and has potential therapeutic value for lung fibrosis treatment.

Topics: Alveolar Epithelial Cells; Animals; Bleomycin; Cadherins; Cell Line; Collagen Type I; Collagen Type III; Fibroblasts; Flavonols; Hedgehog Proteins; Humans; Lung; Mice; Pulmonary Fibrosis; Random Allocation; RNA, Long Noncoding; RNA, Messenger; Signal Transduction; Transforming Growth Factor beta; Zinc Finger Protein GLI1

When reclaimed water is used as municipal miscellaneous water, acute exposure of the generated aerosol with high levels of endotoxins can cause severe inflammation in the lungs. However, the potential risks of long-term inhalation of reclaimed water remains unclear. To identify the adverse effects of sub-chronic reclaimed water inhalation and explain the underlying mechanisms, a mouse model of 12-week sub-chronic exposure was established, and wastewater before a membrane bioreactor (MBR, positive control) and the MBR effluent (reclaimed water, which met the quality standard of urban use and was currently used for landscape irrigation) were tested in this study. The exposure dose was set to approach the real working scenarios. Lung lavage and histology were analyzed. Obvious epithelial cell apoptosis in the bronchi was observed, along with the accumulation of myofibroblasts and the collagen deposition both in main bronchi and terminal bronchioles. All these symptoms were persistent after 4 weeks of recovery. Inflammation and induced bronchus-associated lymphoid tissues (iBALT) were also observed but diminished after recovery indicating inflammation may not be the direct cause of the symptom. Furthermore, two fibrogenic cytokines (TNF-α and TGF-β) were constantly high in the lung during the study. They might be the biomarkers of lung damage after the inhalation of reclaimed water. Adaptive immune responses were also detected as elevated levels of IgG and IgA, but not for IgE. Inhalation of reclaimed water causes sustained fibrotic lesions in the lungs, which suggests potential health risks during urban application where aerosols generated.

Topics: Administration, Inhalation; Aerosols; Animals; Endotoxins; Immunoglobulins; Lung; Male; Mice, Inbred ICR; Pulmonary Fibrosis; Recycling; Toxicity Tests, Subchronic; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha; Wastewater; Water Pollutants

Aloperine is a quinolizidine alkaloid extracted from Sophora alopecuroides. It has been proven to alleviate oxidative stress and effectively promote tumor cell apoptosis in mice. Herein, we investigated whether aloperine could also mediate its protective effects on bleomycin (BLM)-induced pulmonary fibrosis. Pathological staining, western blot, RT-PCR and flow cytometry were used to evaluate the impact of aloperine on the development of pulmonary fibrosis. The effect of aloperine on fibroblast proliferation, differentiation and related signaling pathways were next investigated to demonstrate the underlying mechanisms. In the present report, we showed that aloperine provided protection for mice against BLM-induced pulmonary fibrosis as manifested by the attenuated lung injury and reduced fibrosis along with alleviated fibroblast proliferation and differentiation. Additionally, we provided in vitro evidence revealing that aloperine inhibited cellular proliferation in PDGF-BB-stimulated mouse lung fibroblasts by repressed PI3K/AKT/mTOR signaling and fibroblast to myofibroblast differentiation by repressed TGF-β/Smad signaling. Overall, our data showed that aloperine could protect the mice against BLM-induced pulmonary fibrosis by attenuated fibroblast proliferation and differentiation, which indicated that aloperine may be therapeutically beneficial for IPF patients.

Topics: Animals; Bleomycin; Cell Differentiation; Cell Proliferation; Fibroblasts; Mice; Phosphatidylinositol 3-Kinases; Piperidines; Pulmonary Fibrosis; Quinolizidines; Signal Transduction; Transforming Growth Factor beta

Topics: Animals; Cell Lineage; Cells, Cultured; Coculture Techniques; Collagen Type I; Disease Models, Animal; Female; Fibroblasts; Immunity, Innate; Immunity, Mucosal; Interleukin-1 Receptor-Like 1 Protein; Interleukin-33; Lung; Lymphoid Progenitor Cells; Mice, Inbred C57BL; Mice, Knockout; Paracrine Communication; Phenotype; Pulmonary Fibrosis; Signal Transduction; Transforming Growth Factor beta

Anomalous histoarchitecture with increased levels of type-V collagen (Col V) in lungs of human idiopathic pulmonary fibrosis (IPF) and bleomycin (BLM) airway-centered interstitial fibrosis suggest that this collagen can be a possible trigger involved in the pathogenesis of these diseases. Butylated hydroxytoluene (BHT) injury model revealed a distal involvement of lung parenchyma with significant endothelial injury and fibrotic response, contrasting with the BLM airway-centered insult. We undertook this study to analyze whether BHT alters distal airway/alveolar epithelial cells (AECs) and extracellular matrix (ECM) signaling involved in the initiation and progression of pulmonary fibrosis in a different pathway concerning overexpression of Col V. Female mice C57BL/6 (n=6) were instilled intraperitoneally with 400 mg/kg of BHT dissolved in 1 mL of corn oil and euthanized at day 14 or 21 after BHT administration. Morphometry, immunohistochemistry and transmission electron microscopy were performed to characterize microscopic and submicroscopic changes of AECs and endothelial cells through transforming growth factor beta (TGF-β) basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF) expression. Immunofluorescence and immunogold electron microscopy were performed to characterize Col V. Quantitative polymerase chain reaction (qPCR) was used to confirm differential levels of RNA messenger. BHT lungs showed marked fibrotic areas and hyperplastic AECs. The alveolar damage caused destruction of elastic fibers and a critical increase of Col V in ECM of distal lung parenchyma. Fibrogenesis-promoting markers TGF-β, bFGF and VEGF were also overexpressed in situ, coinciding with up-regulation in remodeling enzymes, growth factors, cytokines, transduction and transcription genes. BHT alters distal lung parenchyma signaling involved in pulmonary fibrosis highlighted similarities to human IPF in a pathway involving Col V arising as a promissory model to identify effective therapeutic targets.

Topics: Animals; Butylated Hydroxytoluene; Collagen Type IV; Disease Models, Animal; Epithelial Cells; Extracellular Matrix; Female; Fibroblast Growth Factor 2; Lung; Mice, Inbred C57BL; Pulmonary Fibrosis; Signal Transduction; Transforming Growth Factor beta; Up-Regulation; Vascular Endothelial Growth Factor A

To evaluate the therapeutic effects of decorin (DCN)-modified mesenchymal stem cells (MSCs) on radiation-induced lung injuries (RILIs) and to clarify the underlying mechanisms.. Umbilical cord-derived mesenchymal stem cells (MSCs) were modified with Ad(E1-).DCN to generate DCN-expressing MSCs (DCN-modified MSCs [MSCs.DCN]). In an experimental mouse model of RILI, MSCs.DCN and MSCs.Null [MSCs modified with Ad(E1-).Null] were intravenously engrafted at 6 hours or 28 days after irradiation. The therapeutic effects on lung inflammation and fibrosis were evaluated by histopathologic analysis at 28 days and 3 months after irradiation. Inflammatory cytokines and chemokines were analyzed in both sera and lung tissues, and subtypes of T lymphocytes including regulatory T cells (Tregs) were analyzed in the peripheral blood and spleen.. Both MSC treatments could alleviate histopathologic injuries by reducing lymphocyte infiltration, decreasing apoptosis, increasing proliferation of epithelial cells, and inhibiting fibrosis in the later phase. However, treatment with MSCs.DCN resulted in much more impressive therapeutic effects. Moreover, we discovered that MSC treatment reduced the expression of chemokines and inflammatory cytokines and increased the expression of anti-inflammatory cytokines in both the peripheral blood and local pulmonary tissues. An important finding was that MSCs.DCN were much more effective in inducing interferon-γ expression, inhibiting collagen type III α1 expression in pulmonary tissues, and decreasing the proportion of Tregs. Furthermore, our data suggested that treatment during the acute phase (6 hours) after irradiation evoked much stronger responses both in attenuating inflammation and in inhibiting fibrosis than in the later phase (28 days).. MSCs.DCN could attenuate acute inflammation after irradiation and significantly inhibit later fibrosis. Likewise, DCN enhanced the functions of MSCs by targeting profibrotic factors and Tregs.

Topics: Adenoviridae; Animals; Apoptosis; Cell Proliferation; Chemokines; Collagen; Collagen Type I; Collagen Type III; Cytokines; Decorin; Disease Progression; Down-Regulation; Gamma Rays; Genetic Vectors; Humans; Immunohistochemistry; In Situ Nick-End Labeling; Interferon-gamma; Lung; Male; Mesenchymal Stem Cell Transplantation; Mesenchymal Stem Cells; Mice; Mice, Inbred C57BL; Pneumonia; Pulmonary Fibrosis; Radiation Injuries, Experimental; T-Lymphocytes, Regulatory; Time Factors; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha; Umbilical Cord; Vascular Endothelial Growth Factor A

To investigate the regulatory mechanism of MEN1 gene in radiation-induced lung fibrosis in mice and provide a new theoretical basis for the clinical treatment of radiation pulmonary fibrosis.. First, 80 C57BL/6 mice aged 8 weeks and weighing 18-22 g were selected, half of them were male and the other half were female. The mice were divided into control group and irradiation group (40 mice in each group) according to the method of the random number table. A radiation-induced lung fibrosis mouse model was established in which a single X-ray irradiation of 20 Gy was applied to the right lung in the irradiation group; H&E and Masson staining were used to verify whether the model was successful at 4, 8, 16 and 24 weeks after irradiation. The expression of MEN1, smooth muscle actin (α-SMA), Collagen-1 and transforming growth factor (TGF-β) in lung tissue were detected by Western blot and qPCR. Secondly, in the mouse embryonic fibroblast cell line (MEF) and mouse lung epithelial cell line (MLE-12), we constructed cell models of MEN1 knockout and interference separately with the irradiation of 10 Gy X-rays. The expression of α-SMA, Collagen-1, and TGF-β/Smads signaling pathway molecules was detected by qPCR. Finally, using the immunoprecipitation (IP) method, we can detect the interaction between Smad2 and the protein menin encoded by the MEN1 gene.. The results of the radiation pulmonary fibrosis model in mice showed that compared with the control group, the alveolar septum widens, the alveolar integrity decreases, the lung tissue slightly thickens, and a small amount of collagen deposits appear after 4-8 weeks in the model group. At twenty-fourth weeks, a large number of cells in the interstitial space of the lung tissue and a localized focal fibrosis area were observed. Further study found that radiation induced fibrogenic inflammatory cytokines TGF-β up-regulation, down-regulation of MEN1 gene expression, and then enhanced the expression of α-SMA and promotes the transformation of fibroblasts to myofibroblasts; At the same time, the expression of Collagen-1 was enhanced, which suggested that the extracellular matrix was overconcentrated and eventually promoted the formation of pulmonary fibrosis. In vitro, we found that knockout and interference of MEN1 gene can significantly enhance radiation-induced fibrosis, and up-regulate the expression of downstream molecules Smad2 and Smad3 of TGF-β signaling pathway, and down-regulate the expression of Smad7. Furthermore, it played an important role in regulating the process of radionuclide fibrosis.. MEN1 plays a key role in the formation of pulmonary fibrosis by regulating the secretion of TGF-β and the activation of TGF-β/Smads signaling pathway.

Topics: Actins; Animals; Cell Line; Collagen Type I; Disease Models, Animal; Female; Gene Expression Regulation; Gene Knockout Techniques; Male; Mice; Mice, Inbred C57BL; Proto-Oncogene Proteins; Pulmonary Fibrosis; Signal Transduction; Smad Proteins; Transforming Growth Factor beta; X-Rays

Pulmonary fibrosis is a multifaceted disease with high mortality and morbidity, and it is commonly nonresponsive to conventional therapy.. We explore the possible discourse of sinapic acid (SA) against the prevention of bleomycin (BLM)-instigated lung fibrosis in rats through modulation of Nrf2/HO-1 and NF-κB signaling pathways.. Lung fibrosis was persuaded in Sprague-Dawley rats by a single intratracheal BLM (6.5 U/kg) injection. Then, these rats were treated with SA (10 and 20 mg/kg, p.o.) for 28 days. The normal control rats provided saline as a substitute of BLM. The lung function and biochemical, histopathological, and molecular alterations were studied in serum, bronchoalveolar lavage fluid (BALF), and the lungs tissues.. SA treatment significantly restored BLM-induced alterations in body weight index and serum biomarkers [lactate dehydrogenase (LDH) and alkaline phosphatase (ALP)]. SA (10 and 20 mg/kg) treatment appeared to show a pneumoprotective effect through upregulation of antioxidant status, downregulation of inflammatory cytokines and MMP-7 expression, and reduction of collagen accumulation (hydroxyproline). Nrf2, HO-1, and TGF-β expression was downregulated in BLM-induced fibrosis model, while the reduced expression levels were significantly and dose-dependently upregulated by SA (10 and 20 mg/kg) treatment. We demonstrated that SA ameliorates BLM-induced lung injuries through inhibition of apoptosis and induction of Nrf2/HO-1-mediated antioxidant enzymes via NF-κB inhibition. The histopathological findings also revealed that SA treatment (10 and 20 mg/kg) significantly ameliorated BLM-induced lung injury.. The present results showed the ability of SA to restore the antioxidant system and to inhibit oxidative stress, proinflammatory cytokines, extracellular matrix, and TGF-β. This is first report demonstrating that SA amoleriates BLM induced lung injuries through inhibition of apoptosis and induction of Nrf2 and HO-1 mediated antioxidant enzyme via NF-κB inhibition. The histopathological finding reveals that SA treatment (10 and 20 mg/kg) significantly ameliorates BLM induced lung injuries.

Topics: Animals; Antioxidants; Apoptosis; Biomarkers; Bleomycin; Bronchoalveolar Lavage Fluid; Coumaric Acids; Disease Models, Animal; Heme Oxygenase-1; Hydroxyproline; Inflammation; Lung; Matrix Metalloproteinase 7; NF-E2-Related Factor 2; NF-kappa B; Organ Size; Oxidative Stress; Pulmonary Fibrosis; Rats, Sprague-Dawley; Transforming Growth Factor beta; Weight Gain

Nicorandil, an antianginal and potassium channel opener agent, has different useful impacts on cardiovascular and respiratory systems. Its effect against silicosis has not been discussed yet, therefore, this is an attempt to decide whether nicorandil can reduce silica-induced lung injury in rats. Silica model was induced by intranasal instillation of silica dust once. Rats were given nicorandil for 56 days after exposure to silica. Results showed that nicorandil significantly alleviated silica-induced inflammation as it decreased the elevated levels of total and differential cell counts, pulmonary edema (revealed by decreased lung/body weight ratio and W/D weight ratio), LDH and total protein levels in BALF. Notably, nicorandil decreased collagen deposition as evidenced by reduction in levels of hydroxyproline and collagen in lung tissues as well as obvious alleviation in silica-induced fibrosis in histopathological findings. Nicorandil effectively reduced the increased expression of NF-κB and iNOS and decreased MPO levels in lung tissues. Moreover, nicorandil abolished oxidative and nitrosative stress via reducing levels of pulmonary MDA and NOx concomitant with elevating levels of pulmonary GSH and SOD. Meanwhile, nicorandil decreased the levels of TNF-α and TGF-β, up regulated Nrf-2 and HO-1 levels in BALF suggesting antioxidant, anti-inflammatory and antifibrotic properties. In summary, nicorandil can confer protection against silica-induced lung inflammation and fibrosis. This impact might be due to its ability to down regulate the production of inflammatory and fibrotic cytokines in addition to restoring oxidant/antioxidant balance.

Topics: Animals; Collagen; Disease Models, Animal; Male; Nicorandil; Nitric Oxide Synthase Type II; Pneumonia; Pulmonary Fibrosis; Rats; Rats, Sprague-Dawley; Silicosis; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha

A previous study demonstrated that Lysyl oxidase‑like 2 (LOXL2) serves an essential role in matrix remodeling and fibrogenesis, thus indicating its involvement in fibrosis‑associated diseases. Our previous studies revealed a novel association between LOXL2 expression and pulmonary fibrosis in mice. However, the exact role and mechanisms of LOXL2 in interstitial lung disease remain poorly understood. The present study aimed to detect LOXL2 expression in mice with bleomycin (BLM)‑induced pulmonary fibrosis, and explore the effects of silencing LOXL2 on the proliferation, activation and fibrosis process of mouse lung fibroblasts (MLFs). In addition, the present study investigated the association between LOXL2 and the transforming growth factor-β (TGF‑β)/Smad signaling pathway to identify the mechanism underlying the role of LOXL2 in fibrosis progression. An animal model of pulmonary fibrosis was established by administering an intratracheal injection of 5 mg/kg BLM to C57BL/6 mice. ELISA and immunohistochemical examination were used to detect the LOXL2 level in the serum, lung homogenate and pulmonary tissues in mice. Pulmonary tissues of mice were extracted to culture primary MLFs, and a LOXL2 small interfering RNA adenovirus vector was established to silence LOXL2 in MLFs. Cell proliferation was detected using the cell counting kit‑8 assay. Reverse transcription‑quantitative polymerase chain reaction and western blotting were used to measure the expression of LOXL2, TGF‑β1, Smad2/3, phosphorylated (p)Smad2/3, Smad4, and Smad7 and Snail in cells. Interleukin‑6 (IL‑6) and type 1 collagen α1 (COL1A1) in the supernatant of cells were analyzed by ELISA. It was demonstrated that LOXL2 expression was significantly increased in serum, lung homogenate and pulmonary tissues of mice with BLM‑induced pulmonary fibrosis compared with control mice. Furthermore, silencing LOXL2 significantly decreased MLF proliferation, and the levels of IL‑6 and COL1A1 in the supernatant of cells. Furthermore, silencing LOXL2 inhibited the expression of pSmad2/3, Smad4 and Snail, while it promoted Smad7 expression. The present data provides a comprehensive analysis of the LOXL2 in pulmonary fibrosis and indicates prominent roles for LOXL2 in fibrogenesis via regulation of the TGF‑β/Smad signaling pathway.

Topics: Adenoviridae; Amino Acid Oxidoreductases; Animals; Bleomycin; Cell Proliferation; Copper; Disease Models, Animal; Disease Progression; Fibroblasts; Gene Silencing; Genetic Vectors; Lung; Male; Mice, Inbred C57BL; Monoamine Oxidase; Pulmonary Fibrosis; RNA, Small Interfering; Signal Transduction; Smad Proteins; Transforming Growth Factor beta

Radiation-induced lung fibrosis (RILF) is a complication of radiotherapy in thoracic cancer patients. Thalidomide (THD) has a therapeutic effect on fibrotic and inflammatory disorders. The purpose of the current study was to investigate the therapeutic effect of THD on RILF in mice and better understand the underlying regulatory mechanisms of the therapeutic effect. We found that THD mitigated the fibrosis caused by irradiation in mice. The action of THD on RILF was related to the elevation of low levels reactive oxygen species (ROS), which inhibited the transforming growth factor‑β (TGF‑β)/Smad3 signaling pathway through activation of nuclear factor (erythroid-derived 2)-like 2 (Nrf2). Analysis of the therapeutic effect of THD using Nrf2-/- mouse model confirmed the role of Nrf2 in vivo. In addition, no radioprotective effect of THD on thoracic cancer cell lines was observed. In conclusion, these data showed that THD attenuated RILF in mice, which was mediated by Nrf2-dependent down-regulation of the TGF-β/Smad3 pathway, suggesting THD as a potential novel agent for RILF prevention.

Topics: A549 Cells; Animals; Cell Line, Tumor; Epithelial Cells; Female; Gene Expression Regulation; Humans; Lung; Mice; Mice, Inbred C57BL; Mice, Knockout; NF-E2-Related Factor 2; Pulmonary Fibrosis; Radiation Injuries, Experimental; Radiation-Protective Agents; Reactive Oxygen Species; Signal Transduction; Smad3 Protein; Thalidomide; THP-1 Cells; Transforming Growth Factor beta; X-Rays

BACKGROUND Idiopathic pulmonary fibrosis (IPF) is a progressive disease with unknow. etiology and a high mortality rate. Oridonin is a diterpenoid isolated from the Rabdosia rubesecens with diverse biological functions. However, whether oridonin possess potential protective activity on IPF is still unclear. MATERIAL AND METHODS The aim of the present study was to explore the therapeutic effects of oridonin on IPF. First, TGF-β1-induced MRC-5 cells were employed for the evaluation of inhibitory activity in vitro. Then, a bleomycin (BLM)-induced mice pulmonary fibrosis model was used to verify the activity of oridonin in vivo. Several pathological changes, including alveolar space collapse, emphysema, and infiltration of inflammatory cells, were observed in the BLM‑treated mice. RESULTS Oridonin could significantly inhibit the mRNA and protein expression levels of α-SMA and COL1A1 in TGF-β1-induced MRC-5 cells. Oridonin could attenuate pathological changes, including alveolar space collapse, emphysema, and infiltration of inflammatory cells induced by BLM. In addition, oridonin could significantly inhibit BLM-induced upregulation of α-SMA and COL1A1 and the phosphorylation of Smad2/3 in lung tissues of mice. CONCLUSIONS Oridonin could be used as a potential therapeutic agent in treatment for patients with IPF. The mechanisms of anti-fibrosis effect of oridonin might be inhibition of the TGF-β/Smad pathway.

Topics: Animals; Bleomycin; Cell Differentiation; Cell Line, Tumor; Diterpenes, Kaurane; Humans; Idiopathic Pulmonary Fibrosis; Lung; Mice; Myofibroblasts; Phosphorylation; Pulmonary Fibrosis; Signal Transduction; Smad Proteins; Transforming Growth Factor beta; Transforming Growth Factor beta1

Oxidative stress is one of the important factors involved in the pathogenesis of idiopathic pulmonary fibrosis (IPF). The equilibrium of Nuclear factor-erythroid-related factor 2 (Nrf2)/[BTB (broad-complex, tramtrack and bric-a-brac) and CNC (cap'n'collar protein) homology 1, Bach1] determines the expression level of antioxidant factors, further regulating the function of oxidation/antioxidation capacity. Pirfenidone (PFD) is one of two currently for IPF therapy approved drugs. PFD regulates intracellular antioxidants, inhibits secretion of inflammatory cytokines and collagen synthesis. However the mechanisms of its antioxidant effects remain elusive.. Effects of PFD treatment were studied in mouse lung fibroblasts (MLF) following induction by transforming-growth factor beta 1 (TGF-β1) and in mice following bleomycin-induced lung fibrosis. The mRNA and protein levels of oxidative stress-related factors Nrf2/Bach1 and their downstream antioxidant factors heme oxygenase-1 (Ho-1) and glutathione peroxidase 1 (Gpx1) were determined by RT-PCR and Western blot. Fibrosis-related cytokines interleukin-6 (IL-6) and myofibroblast markers type 1 collagen α1 (COL1A1) levels in supernate of MLF, serum, and bronchoalveolar lavage fluid (BALF) as well as malondialdehyde (MDA) in serum and BALF were detected by ELISA, reactive oxygen species (ROS) generation was measured by 2',7'- dichlorofluorescin diacetate (DCFH-DA) assay and lung pathological/morphological alterations in mice were observed by HE and Masson to assess the antioxidant mechanism and therapeutic effects on pulmonary fibrosis induced by bleomycin.. PFD inhibited Bach1 mRNA and protein expressions in mouse lung fibroblasts induced by TGF-β1 and lung tissues with pulmonary fibrosis induced by bleomycin. Furthermore, it improved Nrf2, Ho-1 and Gpx1 mRNA and protein expressions. After PFD treatment, COL1A1and IL-6 levels in supernate of MLF, serum, and BALF as well as ROS in lung tissues and MDA in serum and BALF from a mouse with pulmonary fibrosis were significantly decreased, and the infiltration of lung inflammatory cells and fibrosis degree were alleviated.. Theraputic effects of PFD for IPF were involved in Nrf2/Bach1 equilibrium which regulated the capacity of oxidative stress. The study provided new insights into the antioxidant mechanism of PFD.

Topics: Animals; Antioxidants; Basic-Leucine Zipper Transcription Factors; Bleomycin; Bronchoalveolar Lavage Fluid; Collagen Type I; Fibroblasts; Glutathione Peroxidase; Glutathione Peroxidase GPX1; Heme Oxygenase-1; Interleukin-6; Lung; Male; Malondialdehyde; Membrane Proteins; Mice; Mice, Inbred C57BL; Myofibroblasts; NF-E2-Related Factor 2; Oxidative Stress; Pulmonary Fibrosis; Pyridones; RNA, Messenger; Transforming Growth Factor beta

Janus kinase 2 (JAK2) has recently been described as a novel downstream mediator of the pro-fibrotic effects of transforming growth factor-β. Although JAK2 inhibitors are in clinical use for myelodysplastic syndromes, patients often rapidly develop resistance. Tumour cells can escape the therapeutic effects of selective JAK2 inhibitors by mutation-independent transactivation of JAK2 by JAK1. Here, we used selective JAK2 inhibition as a model to test the hypothesis that chronic treatment may provoke resistance by facilitating non-physiological signalling pathways in fibroblasts.. The antifibrotic effects of long-term treatment with selective JAK2 inhibitors and reactivation of JAK2 signalling by JAK1-dependent transphosphorylation was analysed in cultured fibroblasts and experimental dermal and pulmonary fibrosis. Combined JAK1/JAK2 inhibition and co-treatment with an HSP90 inhibitor were evaluated as strategies to overcome resistance.. The antifibrotic effects of selective JAK2 inhibitors on fibroblasts decreased with prolonged treatment as JAK2 signalling was reactivated by JAK1-dependent transphosphorylation of JAK2. This reactivation could be prevented by HSP90 inhibition, which destabilised JAK2 protein, or with combined JAK1/JAK2 inhibitors. Treatment with combined JAK1/JAK2 inhibitors or with JAK2 inhibitors in combination with HSP90 inhibitors was more effective than monotherapy with JAK2 inhibitors in bleomycin-induced pulmonary fibrosis and in adTBR-induced dermal fibrosis.. Fibroblasts can develop resistance to chronic treatment with JAK2 inhibitors by induction of non-physiological JAK1-dependent transactivation of JAK2 and that inhibition of this compensatory signalling pathway, for example, by co-inhibition of JAK1 or HSP90 is important to maintain the antifibrotic effects of JAK2 inhibition with long-term treatment.

Topics: Adult; Animals; Antibiotics, Antineoplastic; Benzoquinones; Bleomycin; Blotting, Western; Disease Models, Animal; Fibroblasts; Fibrosis; HSP90 Heat-Shock Proteins; Humans; Immunohistochemistry; Janus Kinase 1; Janus Kinase 2; Lactams, Macrocyclic; Lung; Male; Mice; Middle Aged; Nitriles; Phosphorylation; Protein Kinase Inhibitors; Pulmonary Fibrosis; Pyrazoles; Pyrimidines; Real-Time Polymerase Chain Reaction; Scleroderma, Systemic; Sulfonamides; Transforming Growth Factor beta

IPF is characterized by fibroblast accumulation, collagen deposition, and ECM remodeling, with myofibroblasts believed to be the effector cell type. Myofibroblasts develop due to EMT of lung alveolar epithelial cells, which can be induced by TGF-β. M2 macrophages, a macrophage subpopulation, secrete large amounts of TGF-β. To clarify the relationship between IPF, EMT, TGF-β, and M2 macrophages, a bleomycin-induced pulmonary fibrosis mouse model was used. Seventeen days after mice were treated with bleomycin, the successful establishment of a pulmonary fibrosis model was confirmed by HE stain and Masson's trichrome stain. We found evidence in support of EMT, such as elevated protein levels of α-SMA in lung tissue and decreased levels of E-cadherin and CK-18. Additionally, increased TGF-β levels and TGF-β/Smad2 signaling activation was observed. Macrophages were recruited to pulmonary alveoli. Alveolar macrophages were phenotyped and identified as M2 macrophages, with up-regulated CD206 on the cell surfaces. For in vitro studies, we treated RAW 264.7 cells with IL-4 for 24 h, and the cells were then utilized as M2 macrophages. TGF-β levels increased significantly in the culture supernatant. Forty-eight hours after lung epithelial cells (MLE-12) were co-cultured with the M2 macrophages, the expression of α-SMA increased, and E-cadherin and CK-18 decreased. When a TGF-β receptor inhibitor, LY2109761 was used, the EMT induced by M2 macrophages was blocked. In conclusion, we demonstrated that M2 macrophages induce EMT through the TGF-β/Smad2 signaling pathway.

Topics: Animals; Bleomycin; Disease Models, Animal; Epithelial Cells; Epithelial-Mesenchymal Transition; Fibroblasts; Idiopathic Pulmonary Fibrosis; Keratin-18; Macrophages; Male; Mice; Mice, Inbred C57BL; Myofibroblasts; Protein Serine-Threonine Kinases; Pulmonary Alveoli; Pulmonary Fibrosis; RAW 264.7 Cells; Receptor, Transforming Growth Factor-beta Type II; Receptors, Transforming Growth Factor beta; Signal Transduction; Smad2 Protein; Transforming Growth Factor beta

Treg abnormalities have been implicated in the pathogenesis of systemic sclerosis (SSc). Treg subpopulations and their cytokines, IL-10 and TGF-β in the peripheral blood of early stage SSc patients were investigated. We hypothesized that epigenetically regulated methylation of the FOXP3 promoter and enhancer regions are altered in Tregs of SSc patients, which might be involved in the T cell imbalance. CD4+CD25+Foxp3+CD127- Treg cells were significantly elevated in patients with diffuse cutaneous SSc and in patients with anti-Scl-70/RNA-Pol-III autoantibody positivity and with lung fibrosis. Increased CD62L+ Treg cells were present in all SSc subgroups. The production of immunosuppressive cytokines by both CD127- and CD62L+ Tregs was diminished. We observed reduced methylation of Treg specific FOXP3 enhancer regions, and elevated FOXP3 gene expression in active SSc cases with negative correlation in the frequency of CD62L+IL-10+ Tregs. Our data indicate an inappropriate distribution and cytokine production of Treg cells in early form SSc.

Topics: Adult; Aged; Antibodies, Antinuclear; DNA Methylation; DNA Topoisomerases, Type I; Epigenesis, Genetic; Female; Forkhead Transcription Factors; Gene Expression; Gene Expression Regulation; Humans; Interleukin-10; Middle Aged; Nuclear Proteins; Promoter Regions, Genetic; Pulmonary Fibrosis; RNA Polymerase III; Scleroderma, Diffuse; Scleroderma, Limited; Scleroderma, Systemic; T-Lymphocyte Subsets; T-Lymphocytes, Regulatory; Transforming Growth Factor beta

TGF-β is considered a master switch in the pathogenesis of organ fibrosis. The primary mediators of this activity are the SMAD proteins, particularly SMAD3. In the current study, we have developed a cell-penetrating peptide (CPP) conjugate of the HIV TAT protein that is fused to an aminoterminal sequence of sorting nexin 9 (SNX9), which was previously shown to bind phosphorylated SMAD3 (pSMAD3). We determined that specifically preventing the nuclear import of pSMAD3 using the TAT-SNX9 peptide inhibited profibrotic TGF-β activity in murine cells and human lung fibroblasts as well as in vivo with no demonstrable toxicity. TGF-β signaling mediated by pSMAD2, bone morphogenetic protein 4 (BMP4), EGF, or PDGF was unaffected by the TAT-SNX9 peptide. Furthermore, while the TAT-SNX9 peptide prevented TGF-β's profibrotic activity in vitro as well as in 2 murine treatment models of pulmonary fibrosis, a 3-amino acid point mutant that was unable to bind pSMAD3 proved ineffective. These findings indicate that specifically targeting pSMAD3 can ameliorate both the direct and indirect fibroproliferative actions of TGF-β.

Topics: Animals; Cell Line; Cell-Penetrating Peptides; Disease Models, Animal; Female; Mice; Pulmonary Fibrosis; Signal Transduction; Smad3 Protein; Transforming Growth Factor beta

Pulmonary fibrosis is a kind of devastating interstitial lung disease due to the limited therapeutic strategies. Traditional Chinese medicine (TCM) practices have put forth Shenks as a promising treatment approach. Here, we performed in vivo study and in vitro study to delineate the anti-fibrotic mechanisms behind Shenks treatment for pulmonary fibrosis. We found that regardless of the prophylactic or therapeutic treatment, Shenks was able to attenuate BLM-induced-fibrosis in mice, down regulate extracellular matrix genes expression, and reduce collagen production. The aberrantly high Smad3 phosphorylation levels and SBE activity in TGF-β-induced fibroblasts were dramatically decreased as a result of Shenks treatment. At the same time, Shenks was able to increase the expression of antioxidant-related genes, including Gclc and Ec-sod, while reduce the transcription levels of oxidative-related genes, such as Rac1 and Nox4 demonstrated by both in vivo and in vitro studies. Further investigations found that Shenks could decrease the oxidative productions of protein (3-nitrotyrosine) and lipid (malondialdehyde) and increase GSH content both in bleomycin treated mouse lungs and TGF-β stimulated fibroblasts, as well as inhibit the production of ROS stimulated by TGF-β to fight against oxidative stress. Overall, Shenks inhibited fibrosis by blocking TGF-β pathway and modulating the oxidant/antioxidant balance.

Topics: Animals; Antibiotics, Antineoplastic; Biomarkers; Bleomycin; Cells, Cultured; Collagen; Disease Models, Animal; Female; Fibroblasts; Gene Expression Regulation; Medicine, Chinese Traditional; Mice; Oxidation-Reduction; Oxidative Stress; Phosphorylation; Promoter Regions, Genetic; Pulmonary Fibrosis; Reactive Oxygen Species; Signal Transduction; Smad3 Protein; Transforming Growth Factor beta

Pulmonary fibrosis is a progressive lung disease that its pathogenic mechanism currently is incompletely understood. Toll-like receptor (TLR) signaling has recently been identified as a regulator of inflammation and pulmonary fibrosis. In addition, mesenchymal stem cells (MSCs) of different origins offer a great promise in treatment of idiopathic pulmonary fibrosis (IPF). However mechanisms of pathogenic roles of TLR signaling and therapeutic effects of MSCs in the IPF remain elusive. In present study, the involvement of TLR signaling and the therapeutic role of MSCs were interrogated in MyD88-deficient mice using human placental MSCs of fetal origins (hfPMSCs). The results showed an alleviated pulmonary inflammation and fibrosis in myeloid differentiation primary response gene 88 (MyD88)-deficient mice treated with bleomycin (BLM), accompanied with a reduced TGF-β signaling and production of pro-fibrotic cytokines, including TNF-α, IL-1β. An exposure of HLF1 lung fibroblasts, A549 epithelial cells and RAW264.7 macrophages to BLM led an increased expression of key components of MyD88 and TGF-β signaling cascades. Of interest, enforced expression and inhibition of MyD88 protein resulted in an enhanced and a reduced TGF-β signaling in above cells in the presence of BLM, respectively. However, the addition of TGF-β1 showed a marginally inhibitory effect on MyD88 signaling in these cells in the absence of BLM. Importantly, the administration of hfPMSCs could significantly attenuate BLM-induced pulmonary fibrosis in mice, along with a reduced hydroxyproline (HYP) deposition, MyD88 and TGF-β signaling activation, and production of pro-fibrotic cytokines. These results may suggest an importance of MyD88/TGF-β signaling axis in the tissue homeostasis and functional integrity of lung in response to injury, which may offer a novel target for treatment of pulmonary fibrosis.

Topics: A549 Cells; Animals; Bleomycin; Cell Line; Female; Humans; Hydroxyproline; Inflammation; Interleukin-1beta; Mesenchymal Stem Cell Transplantation; Mesenchymal Stem Cells; Mice; Mice, Inbred C57BL; Mice, Knockout; Myeloid Differentiation Factor 88; Placenta; Pregnancy; Pulmonary Fibrosis; Signal Transduction; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha

Pulmonary fibrosis is a progressive and often fatal lung disease characterized by fibroblast proliferation and excessive deposition of extracellular matrix. Both TGF-β and Wnt signaling have been implicated in the regulation of organ fibrosis. However little is known about whether TGF-β-induced gene expression changes in Wnt signaling pathway could predict disease progression. In the study, we investigated the interaction between TGF-β and Wnt signaling in mediating pulmonary fibrosis by big data analysis, in vitro and in vivo experimental studies and clinical data analysis. We found that TGF-β1 treatment induces a marked upregulation of Frizzled-7 (FZD7) in human lung fibroblasts. FZD7 expression is also increased in animal models of TGF-β1-induced pulmonary fibrosis. TGF-β1 upregulated FZD7 expression in a Smad3-dependent manner. Functionally, knockdown of FZD7 inhibits TGF-β1-induced expression of α-smooth muscle actin (α-SMA), collagen I (Col I), fibronectin and connective tissue growth factor (CTGF). FZD7 inhibition further attenuates TGF-β1-induced pulmonary fibrosis in vivo. Finally our data demonstrated that FZD7 transmits non-canonical Wnt signaling by interacting Wnt5A in the regulation of ECM expression.. These results suggest that FZD7-targeted therapeutic strategies may be applicable for treating an array of fibrotic diseases.

Topics: Animals; Extracellular Matrix; Fibroblasts; Frizzled Receptors; Gene Expression Profiling; Gene Silencing; Humans; Lung; Mice, Inbred BALB C; Pulmonary Fibrosis; Receptors, G-Protein-Coupled; Smad3 Protein; Transforming Growth Factor beta; Wnt Signaling Pathway

Renal interstitial fibrosis (RIF) is the main process that leads to renal failure. It is necessary to investigate the mechanism of RIF and identify appropriate methods of regulating it. Furthermore, unilateral ureteral obstruction is a frequently used model for the study of RIF. The morphological damage associated with kidney and lung dysfunction was detected using histopathological experiments. Subsequently, high expression of reactive oxygen species (ROS) modulator 1 (ROMO1) and ROS was measured in blood serum. In addition, epithelial‑mesenchymal transition marker, transforming growth factor β (TGF‑β) and mothers against decapentaplegic homolog 2/3 expression was evaluated using the reverse transcription‑quantitative polymerase chain reaction and western blotting. All serious symptoms were relieved to a certain extent following oxidation inhibitor intervention using three common antioxidants. HK‑2 cells were treated with H2O2 to cause oxidative stress, and ROMO1 and fibrosis marker expression increased; however, activation was suppressed byROMO1 knockout. The present study provides evidence that the expression of ROMO1 induces ROS production and activates the TGF‑β signaling pathway. It may be concluded that ROMO1 helps to provide a molecular basis for improved clinical intervention and prognosis of patients.

Topics: Animals; Biopsy; Disease Models, Animal; Epithelial-Mesenchymal Transition; Extracellular Matrix; Fibrosis; Gene Expression; Hydrogen Peroxide; Immunohistochemistry; Kidney Diseases; Male; Mice, Knockout; Mitochondrial Proteins; Oxidative Stress; Pulmonary Fibrosis; Rats; Reactive Oxygen Species; Signal Transduction; Transforming Growth Factor beta; Ureteral Obstruction

To evaluate the antifibrotic effects of the pan-peroxisome proliferator-activated receptor (PPAR) agonist IVA337 in preclinical mouse models of pulmonary fibrosis and related pulmonary hypertension (PH).. IVA337 has been evaluated in the mouse model of bleomycin-induced pulmonary fibrosis and in Fra-2 transgenic mice, this latter being characterised by non-specific interstitial pneumonia and severe vascular remodelling of pulmonary arteries leading to PH. Mice received two doses of IVA337 (30 mg/kg or 100 mg/kg) or vehicle administered by daily oral gavage up to 4 weeks.. IVA337 demonstrated at a dose of 100 mg/kg a marked protection from the development of lung fibrosis in both mouse models compared with mice receiving 30 mg/kg of IVA337 or vehicle. Histological score was markedly reduced by 61% in the bleomycin model and by 50% in Fra-2 transgenic mice, and total lung hydroxyproline concentrations decreased by 28% and 48%, respectively, as compared with vehicle-treated mice. IVA337 at 100 mg/kg also significantly decreased levels of fibrogenic markers in lesional lungs of both mouse models. In addition, IVA337 substantially alleviated PH in Fra-2 transgenic mice by improving haemodynamic measurements and vascular remodelling. In primary human lung fibroblasts, IVA337 inhibited in a dose-dependent manner fibroblast to myofibroblasts transition induced by TGF-β and fibroblast proliferation mediated by PDGF.. We demonstrate that treatment with 100 mg/kg IVA337 prevents lung fibrosis in two complementary animal models and substantially attenuates PH in the Fra-2 mouse model. These findings confirm that the pan-PPAR agonist IVA337 is an appealing therapeutic candidate for these cardiopulmonary involvements.

Topics: Animals; Benzothiazoles; Bleomycin; Cell Proliferation; Disease Models, Animal; Fibroblasts; Fos-Related Antigen-2; Hypertension, Pulmonary; Mice; Mice, Transgenic; Myofibroblasts; Pulmonary Artery; Pulmonary Fibrosis; Severity of Illness Index; Sulfonamides; Transforming Growth Factor beta; Treatment Outcome; Vascular Remodeling

Idiopathic pulmonary fibrosis (IPF) is a fatal disease with a median survival of 3-4 years after diagnosis. It is the most frequent form of a group of interstitial pneumonias of unknown etiology. Current available therapies prevent deterioration of lung function but no therapy has shown to improve survival. Periostin is a matricellular protein of the fasciclin 1 family. There is increased deposition of periostin in lung fibrotic tissues. Here we evaluated whether small interfering RNA or antisense oligonucleotide against periostin inhibits lung fibrosis by direct administration into the lung by intranasal route. Pulmonary fibrosis was induced with bleomycin and RNA therapeutics was administered during both acute and chronic phases of the disease. The levels of periostin and transforming growth factor-β1 in airway fluid and lung tissue, the deposition of collagen in lung tissue and the lung fibrosis score were significantly reduced in mice treated with siRNA and antisense against periostin compared to control mice. These findings suggest that direct administration of siRNA or antisense oligonucleotides against periostin into the lungs is a promising alternative therapeutic approach for the management of pulmonary fibrosis.

Topics: Administration, Intranasal; Animals; Bleomycin; Cell Adhesion Molecules; Collagen; Female; Fibroblasts; Fibrosis; Idiopathic Pulmonary Fibrosis; Lung; Mice; Mice, Inbred C57BL; Oligonucleotides; Oligonucleotides, Antisense; Pulmonary Fibrosis; RNA, Small Interfering; Transforming Growth Factor beta

Lung fibrosis is a progressive and often fatal lung disease characterized by fibroblast proliferation and excessive deposition of extracellular matrix in the lungs. The chemokine receptor CXCR7 has been shown to control cell adhesion, migration and proliferation by regulating the epithelial-to-mesenchymal transition (EMT), but the role of CXCR7 in regulating the endothelial-to-mesenchymal transition (EndMT) and lung fibrosis remains largely unclear. In this study, we investigated the interrelation of CXCR7 and TGF-β, a crucial player in lung fibrogenesis. We report herein that CXCR7 expression is significantly increased in animal models of TGF-β1-induced pulmonary fibrosis and in TGF-β1-treated endothelial cells. TGF-β1 up-regulates CXCR7 expression in a Smad2/3-dependent manner in endothelial cells. The overexpression of CXCR7 effectively attenuates TGF-β1-induced EndMT in lung endothelial cells, whereas CXCR7 knockdown in endothelial cells further promotes TGF-β1-induced EndMT. Mechanically, CXCR7 attenuates EndMT by inhibiting the Jag1-Notch pathway. CXCR7 overexpression in mice also results in a significant enhancement in endothelial markers and a decrease in mesenchymal markers, indicating a decreased susceptibility to TGF-β1-induced lung fibrosis and deposition of extracellular matrix and collagen. These data suggest that CXCR7 upregulation induced by TGF-β is a feedback mechanism to regulate TGF-β-induced EndMT and pulmonary fibrosis.

Topics: Animals; Cell Adhesion; Cell Movement; Cell Proliferation; Epithelial-Mesenchymal Transition; Mice; Mice, Inbred BALB C; Pulmonary Fibrosis; Receptors, CXCR; Signal Transduction; Transforming Growth Factor beta; Transforming Growth Factor beta1

Pulmonary fibrosis is a progressive fibrotic lung disease of persisting lung injury and ineffective wound repair, with poor prognosis. Epithelial-mesenchymal transition (EMT) of alveolar epithelia cells is an early event in the development of pulmonary fibrosis, and transforming growth factor β (TGF-β) is an acknowledged inducer of EMT. Epidemiological studies demonstrated that serum levels of 25-hydroxy-vitamin D were associated with the presence of fibrosis diseases. We investigated whether vitamin D attenuated TGF-β-induced pro-fibrotic effects through inhibiting EMT in human alveolar epithelia A549 cells. A549 cells were cultured with TGF-β alone or in combination with 1α,25-dihydroxyvitamin D3 (1α,25(OH)₂D₃). TGF-β increased the expression of the mesenchymal markers (N-cadherin and Vimentin), and decreased the expression of epithelial markers (E-cadherin). 1α,25(OH)₂D₃ attenuated these TGF-β-induced alterations. Furthermore, the EMT-related transcription factors (Snail and β-catenin) and the extracellular matrix genes (Collagen I and fibronectin) were inhibited by 1α,25(OH)₂D₃, while the expression of vitamin D receptor (VDR) was elevated. In addition, 1α,25(OH)₂D₃ alleviated the cell migration and the invasion abilities in TGF-β-stimulated A549 cells, determined by the scratch wound healing and transwell assays. Our findings suggested that 1α,25(OH)₂D₃ inhibited the pro-fibrotic phenotype of lung epithelial cells under TGF-β stimulation and provided new clues in the clinical management of pulmonary fibrosis.

Topics: A549 Cells; Antigens, CD; beta Catenin; Cadherins; Calcitriol; Cell Movement; Cell Shape; Collagen Type I; Epithelial Cells; Epithelial-Mesenchymal Transition; Fibronectins; Humans; Lung; Phenotype; Pulmonary Fibrosis; Receptors, Calcitriol; Signal Transduction; Snail Family Transcription Factors; Time Factors; Transforming Growth Factor beta; Vimentin

TGF-

Topics: Animals; Fibroblasts; Glycoproteins; Mice; Mice, Inbred C57BL; Mice, Knockout; Pulmonary Fibrosis; Signal Transduction; Transforming Growth Factor beta

To evaluate effects of halofuginone (H) on radiation-induced lung injury (RILI), 60 rats were divided into six groups: Group (G) 1 control, G2 radiotherapy (RT) only, G3 and G4 2. 5 and 5 μg H and G5 and G6 RT + 2.5 and 5 μg H groups, respectively. A single dose of 12 Gy RT was given to both lungs. H was applied intraperitoneally with daily doses, until animals were killed at 6 and 16 weeks after RT. At 6th and 16th weeks of RT, five rats from each group were killed. Lung tissues were dissected for light and electron microscopy. Chronic inflammation, fibrosis and transforming growth factor-beta (TGF)-β scores of all study groups were significantly different at 6th and 16th week ( p < 0.001). Chronic inflammation, fibrosis and TGF-β scores of G2 were higher than G5 and G6 at 6th and 16th weeks of RT. At 16th week, fibrosis and TGF-β scores of G5 were higher than G6 ( p = 0.040 and 0.028, respectively). Electron microscopical findings also supported these results. Therefore, H may ameliorate RILI. The effect of the H was more prominent at higher dose and after long-term follow-up. These findings should be clarified with further studies.

Topics: Animals; Female; Lung; Lung Injury; Piperidines; Pulmonary Fibrosis; Quinazolinones; Radiation Injuries, Experimental; Radiation-Protective Agents; Radiation, Ionizing; Rats, Wistar; Transforming Growth Factor beta

Idiopathic pulmonary fibrosis is a severe disease characterized by excessive myofibroblast proliferation, extracellular matrix and fibrils deposition, remodelling of lung parenchyma and pulmonary insufficiency. Drugs able to reduce disease progression are available, but therapeutic results are unsatisfactory; new and safe treatments are urgently needed. Poly(ADP-ribose) polymerases-1 (PARP-1) is an abundant nuclear enzyme involved in key biological processes: DNA repair, gene expression control, and cell survival or death. In liver and heart, PARP-1 activity facilitates oxidative damage, collagen deposition and fibrosis development. In this study, we investigated the effects of HYDAMTIQ, a potent PARP-1 inhibitor, in a murine model of lung fibrosis. We evaluated the role of PARP on transforming growth factor-β (TGF-β) expression and TGF-β/SMAD signalling pathway in lungs. Mice were intratracheally injected with bleomycin and then treated with either vehicle or different doses of HYDAMTIQ for 21 days. Airway resistance to inflation and lung static compliance, markers of lung stiffness, were assayed. Histochemical and biochemical parameters to evaluate TGF-β/SMAD signalling pathway with alpha-smooth muscle actin (αSMA) deposition and the levels of a number of inflammatory markers (tumour necrosis factor-α, interleukin-1β, iNOS and COX-2) were performed. Bleomycin administration increased lung stiffness. It also increased lung PARP activity, TGF-β levels, pSMAD3 expression, αSMA deposition and content of inflammatory markers. HYDAMTIQ attenuated all the above-mentioned physiological, biochemical and histopathological markers. Our findings support the proposal that PARP inhibitors could have a therapeutic potential in reducing the progression of signs and symptoms of the disease by decreasing TGF-β expression and the TGF-β/SMAD transduction pathway.

Topics: Actins; Animals; Biomarkers; Bleomycin; Fibroblasts; Hydroxyproline; Inflammation Mediators; Isoquinolines; Lung; Male; Mice, Inbred C57BL; Oxidative Stress; Poly(ADP-ribose) Polymerase Inhibitors; Poly(ADP-ribose) Polymerases; Pulmonary Fibrosis; Signal Transduction; Smad Proteins; Thiophenes; Transforming Growth Factor beta

Hedgehog signalling plays a critical role during the pathogenesis of fibrosis in systemic sclerosis (SSc). Besides canonical hedgehog signalling with smoothened (SMO)-dependent activation of GLI transcription factors, GLI can be activated independently of classical hedgehog ligands and receptors (so-called non-canonical pathways). Here, we aimed to evaluate the role of non-canonical hedgehog signalling in SSc and to test the efficacy of direct GLI inhibitors that target simultaneously canonical and non-canonical hedgehog pathways.. The GLI inhibitor GANT-61 was used to inhibit canonical as well as non-canonical hedgehog signalling, while the SMO inhibitor vismodegib was used to selectively target canonical hedgehog signalling. Furthermore, GLI2 was selectively depleted in fibroblasts using the Cre-LoxP system. The effects of pharmacological or genetic of GLI2 on transforming growth factor-β (TGF-β) signalling were analysed in cultured fibroblasts, in bleomycin-induced pulmonary fibrosis and in mice with overexpression of a constitutively active TGF-β receptor I.. TGF-β upregulated GLI2 in a Smad3-dependent manner and induced nuclear accumulation and DNA binding of GLI2. Fibroblast-specific knockout of GLI2 protected mice from TBR. Our data demonstrate that hedgehog pathways and TGF-β signalling both converge to GLI2 and that GLI2 integrates those signalling to promote tissue fibrosis. These findings may have translational implications as non-selective inhibitors of GLI2 are in clinical use and selective molecules are currently in development.

Topics: Adult; Aged; Anilides; Animals; Cells, Cultured; Collagen Type I; Connective Tissue Growth Factor; Female; Fibroblasts; Fibrosis; Gene Knockout Techniques; Hedgehog Proteins; Humans; Kruppel-Like Transcription Factors; Male; Mice; Mice, Knockout; Mice, Transgenic; Middle Aged; Plasminogen Activator Inhibitor 1; Protein Serine-Threonine Kinases; Pteridines; Pulmonary Fibrosis; Pyridines; Pyrimidines; Receptor, Transforming Growth Factor-beta Type I; Receptors, Transforming Growth Factor beta; Recombinant Proteins; RNA, Messenger; Scleroderma, Systemic; Signal Transduction; Skin; Smad3 Protein; Smoothened Receptor; Transforming Growth Factor beta; Young Adult; Zinc Finger Protein Gli2

The efficacy and feasibility of targeting transforming growth factor-β (TGFβ) in pulmonary fibrosis and lung vascular remodeling in systemic sclerosis (SSc) have not been well elucidated. In this study we analyzed how blocking TGFβ signaling affects pulmonary abnormalities in Fos-related antigen 2 (Fra-2) transgenic (Tg) mice, a murine model that manifests three important lung pathological features of SSc: fibrosis, inflammation, and vascular remodeling. To interrupt TGFβ signaling in the Fra-2 Tg mice, we used a pan-TGFβ-blocking antibody, 1D11, and Tg mice in which TGFβ receptor type 2 (Tgfbr2) is deleted from smooth muscle cells and myofibroblasts (α-SMA-Cre

Topics: Actins; Animals; Bone Morphogenetic Protein Receptors, Type II; Disease Models, Animal; Fos-Related Antigen-2; Gene Deletion; Granulocytes; Mice, Transgenic; Myocytes, Smooth Muscle; Pneumonia; Protein Serine-Threonine Kinases; Pulmonary Alveoli; Pulmonary Fibrosis; Receptor, Transforming Growth Factor-beta Type II; Receptors, Transforming Growth Factor beta; Scleroderma, Systemic; Signal Transduction; Transforming Growth Factor beta; Vascular Diseases; Vascular Remodeling

Myofibroblasts, the primary effector cells that mediate matrix remodeling during pulmonary fibrosis, rapidly assemble an extracellular fibronectin matrix. Tensin (TNS) 1 is a key component of specialized cellular adhesions (fibrillar adhesions) that bind to extracellular fibronectin fibrils. We hypothesized that TNS1 may play a role in modulating myofibroblast-mediated matrix formation. We found that TNS1 expression is increased in fibroblastic foci from lungs with idiopathic pulmonary fibrosis. Transforming growth factor (TGF)-β profoundly up-regulates TNS1 expression with kinetics that parallel the expression of the myofibroblast marker, smooth muscle α-actin. TGF-β-induced TNS1 expression is dependent on signaling through the TGF-β receptor 1 and is Rho coiled-coiled kinase/actin/megakaryoblastic leukemia-1/serum response factor dependent. Small interfering RNA-mediated knockdown of TNS1 disrupted TGF-β-induced myofibroblast differentiation, without affecting TGF-β/Smad signaling. In contrast, loss of TNS1 resulted in disruption of focal adhesion kinase phosphorylation, focal adhesion formation, and actin stress fiber development. Finally, TNS1 was essential for the formation of fibrillar adhesions and the assembly of nascent fibronectin and collagen matrix in myofibroblasts. In summary, our data show that TNS1 is a novel megakaryoblastic leukemia-1-dependent gene that is induced during pulmonary fibrosis. TNS1 plays an essential role in TGF-β-induced myofibroblast differentiation and myofibroblast-mediated formation of extracellular fibronectin and collagen matrix. Targeted disruption of TNS1 and associated signaling may provide an avenue to inhibit tissue fibrosis.

Topics: Actins; Cell Differentiation; Extracellular Matrix; Focal Adhesions; Humans; MAP Kinase Kinase Kinases; Myofibroblasts; Polymerization; Protein Serine-Threonine Kinases; Pulmonary Fibrosis; Receptor, Transforming Growth Factor-beta Type I; Receptors, Transforming Growth Factor beta; rho-Associated Kinases; Signal Transduction; Smad Proteins; Tensins; Transforming Growth Factor beta; Up-Regulation

Transforming growth factor β (TGFβ) plays an important role in regulating aberrant extracellular matrix (ECM) production from alveolar/epithelial cells (AECs) and fibroblasts in pulmonary fibrosis. Although the tumor suppressor gene phosphatase and tensin homologue deleted from chromosome 10 (PTEN) can negatively control many TGFβ-activated signaling pathways via the phosphatase activity, hyperactivation of the TGFβ-related signaling pathways is often observed in fibrosis. Loss of PTEN expression might cause TGFβ-induced ECM production. In addition, TGFβ was recently shown to induce loss of PTEN enzymatic activity by phosphorylating the PTEN C-terminus. Therefore, we hypothesized that exogenous transfer of unphosphorylated PTEN (PTEN4A) might lead to reduce TGFβ-induced ECM expression in not only epithelial cells but also fibroblasts. Adenovirus-based exogenous PTEN4A induction successfully reduced TGFβ-induced fibronectin expression and retained β-catenin at the cell membrane in human epithelial cells. Exogenous unphosphorylated PTEN also attenuated TGFβ-induced ECM production and inhibited TGFβ-induced β-catenin translocation in a human fibroblast cell line and in mouse primary isolated lung fibroblasts. Conversely, TGFβ-induced α-smooth muscle actin expression did not seem to be inhibited in these fibroblasts. Our data suggest that exogenous administration of unphosphorylated PTEN might be a promising strategy to restore TGFβ-induced loss of PTEN activity and reduce aberrant TGFβ-induced ECM production from epithelial cells and fibroblasts in lung fibrosis as compared with wild-type PTEN induction.

Topics: Animals; beta Catenin; Cell Line; Extracellular Matrix; Fibroblasts; Fibronectins; Humans; Lung; Mice; Phosphorylation; PTEN Phosphohydrolase; Pulmonary Fibrosis; Signal Transduction; Transforming Growth Factor beta

Wnt signalling has been implicated in activating a fibrogenic programme in fibroblasts in systemic sclerosis (SSc). Porcupine is an O-acyltransferase required for secretion of Wnt proteins in mammals. Here, we aimed to evaluate the antifibrotic effects of pharmacological inhibition of porcupine in preclinical models of SSc.. The porcupine inhibitor GNF6231 was evaluated in the mouse models of bleomycin-induced skin fibrosis, in tight-skin-1 mice, in murine sclerodermatous chronic-graft-versus-host disease (cGvHD) and in fibrosis induced by a constitutively active transforming growth factor-β-receptor I.. Treatment with pharmacologically relevant and well-tolerated doses of GNF6231 inhibited the activation of Wnt signalling in fibrotic murine skin. GNF6231 ameliorated skin fibrosis in all four models. Treatment with GNF6231 also reduced pulmonary fibrosis associated with murine cGvHD. Most importantly, GNF6231 prevented progression of fibrosis and showed evidence of reversal of established fibrosis.. These data suggest that targeting the Wnt pathway through inhibition of porcupine provides a potential therapeutic approach to fibrosis in SSc. This is of particular interest, as a close analogue of GNF6231 has already demonstrated robust pathway inhibition in humans and could be available for clinical trials.

Topics: Acyltransferases; Aminopyridines; Animals; Bleomycin; Disease Models, Animal; Disease Progression; Female; Fibrosis; Graft vs Host Disease; Membrane Proteins; Mice, Inbred BALB C; Piperazines; Protein Serine-Threonine Kinases; Pulmonary Fibrosis; Receptor, Transforming Growth Factor-beta Type I; Receptors, Transforming Growth Factor beta; Scleroderma, Localized; Scleroderma, Systemic; Skin; Transforming Growth Factor beta; Wnt Signaling Pathway

MicroRNA 29b (miR-29b) replacement therapy is effective for suppressing fibrosis in a mouse model. However, to develop clinical applications for miRNA mimics, the side effects of nucleic acid drugs have to be addressed. In this study, we focused on miRNA mimics in order to develop therapies for idiopathic pulmonary fibrosis. We developed a single-stranded RNA, termed "miR-29b Psh-match," that has a unique structure to avoid problems associated with the therapeutic uses of miRNAs. A comparison of miR-29b Psh-match and double-stranded one, termed "miR-29b mimic" indicated that the single-stranded form was significantly effective towards fibrosis according to both in vivo and in vitro experiments. This novel form of miR-29b may become the foundation for developing an effective therapeutic drug for pulmonary fibrosis.

Topics: Administration, Inhalation; Animals; Bleomycin; Fibroblasts; Hydroxyproline; Male; Mice; Mice, Inbred C57BL; MicroRNAs; NIH 3T3 Cells; Pulmonary Fibrosis; Signal Transduction; Toll-Like Receptors; Transforming Growth Factor beta

Allogeneic hematopoietic stem cell transplantation is hampered by chronic graft-versus-host disease (cGVHD), resulting in multiorgan fibrosis and diminished function. Fibrosis in lung and skin leads to progressive bronchiolitis obliterans (BO) and scleroderma, respectively, for which new treatments are needed. We evaluated pirfenidone, a Food and Drug Administration (FDA)-approved drug for idiopathic pulmonary fibrosis, for its therapeutic effect in cGVHD mouse models with distinct pathophysiology. In a full major histocompatibility complex (MHC)-mismatched, multiorgan system model with BO, donor T-cell responses that support pathogenic antibody production are required for cGVHD development. Pirfenidone treatment beginning one month post-transplant restored pulmonary function and reversed lung fibrosis, which was associated with reduced macrophage infiltration and transforming growth factor-β production. Pirfenidone dampened splenic germinal center B-cell and T-follicular helper cell frequencies that collaborate to produce antibody. In both a minor histocompatibility antigen-mismatched as well as a MHC-haploidentical model of sclerodermatous cGVHD, pirfenidone significantly reduced macrophages in the skin, although clinical improvement of scleroderma was only seen in one model. In vitro chemotaxis assays demonstrated that pirfenidone impaired macrophage migration to monocyte chemoattractant protein-1 (MCP-1) as well as IL-17A, which has been linked to cGVHD generation. Taken together, our data suggest that pirfenidone is a potential therapeutic agent to ameliorate fibrosis in cGVHD.

Topics: Allografts; Animals; B-Lymphocytes; Bronchiolitis Obliterans; Chemokine CCL2; Disease Models, Animal; Graft vs Host Disease; Hematopoietic Stem Cell Transplantation; Interleukin-17; Macrophages; Mice; Mice, Mutant Strains; Pulmonary Fibrosis; Pyridones; Skin Diseases; T-Lymphocytes, Helper-Inducer; Transforming Growth Factor beta

Pulmonary fibrosis is common in a variety of inflammatory lung diseases, such as interstitial pneumonia, chronic obstructive pulmonary disease, and silicosis. There is currently no effective clinical drug treatment. It has been reported that grape seed extracts (GSE) has extensive pharmacological effects with minimal toxicity. Although it has been found that GSE can improve the lung collagen deposition and fibrosis pathology induced by bleomycin in rat, its effects on pulmonary function, inflammation, growth factors, matrix metalloproteinases and epithelial-mesenchymal transition remain to be researched. In the present study, we studied whether GSE provided protection against bleomycin (BLM)-induced mouse pulmonary fibrosis. ICR strain mice were treated with BLM in order to establish pulmonary fibrosis models. GSE was given daily via intragastric administration for three weeks starting at one day after intratracheal instillation. GSE at 50 or 100mg/kg significantly reduced BLM-induced inflammatory cells infiltration, proinflammatory factor protein expression, and hydroxyproline in lung tissues, and improved pulmonary function in mice. Additionally, treatment with GSE also significantly impaired BLM-induced increases in lung fibrotic marker expression (collagen type I alpha 1 and fibronectin 1) and decreases in an anti-fibrotic marker (E-cadherin). Further investigation indicated that the possible molecular targets of GSE are matrix metalloproteinases-9 (MMP-9) and TGF-β1, given that treatment with GSE significantly prevented BLM-induced increases in MMP-9 and TGF-β1 expression in the lungs. Together, these results suggest that supplementation with GSE may improve the quality of life of lung fibrosis patients by inhibiting MMP-9 and TGF-β1 expression in the lungs.

Topics: Animals; Anti-Inflammatory Agents; Antibiotics, Antineoplastic; Bleomycin; Bronchoalveolar Lavage Fluid; Female; Grape Seed Extract; Lung; Matrix Metalloproteinase 9; Mice, Inbred ICR; Pulmonary Fibrosis; Respiratory Function Tests; Transforming Growth Factor beta

We aimed to investigate the preventive effect of Infliximab (IFX), a tumor necrosis factor (TNF)-α inhibitor, on bleomycin (BLC)-induced lung fibrosis in rats. Rats were assigned into four groups as follows: I-BLC group, a single intra-tracheal BLC (2.5 mg/kg) was installed; II-control group, a single intra-tracheal saline was installed; III-IFX + BLC group, a single-dose IFX (7 mg/kg) was administered intraperitoneally (i.p.), 72 h before the intra-tracheal BLC installation; IV-IFX group, IFX (7 mg/kg) was administered alone i.p. on the same day with IFX + BLC group. All animals were sacrificed on the 14th day of BLC installation. Levels of tumor necrosis factor (TNF)-α, transforming growth factor (TGF)-β, interleukin (IL)-6, periostin, YKL-40, nitric oxide (NO) in rat serum were measured, as well as, myeloperoxidase (MPO), superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) activity, and reduced glutathione (GSH), hydroxyproline, malondialdehyde (MDA) content in lung homogenates. Lung tissues were stained with hematoxylin and eosin (H&E) for quantitative histological evaluation. The inducible nitric oxide synthase (iNOS) expression and cell apoptosis in the lung tissues were determined quantitatively by immunohistochemical staining (INOS) and by TUNNEL staining, respectively. BLC installation worsened antioxidant status (such as SOD, CAT, GPx, GSH, MPO), while it increased the serum TNF-α, TGF-β, IL-6, periostin, YKL-40, and lipid peroxidation, and collagen deposition, measured by MDA and hydroxyproline, respectively. IFX pretreatment improved antioxidant status as well as BLC-induced lung pathological changes, while it decreased the TNF-α, TGF-β, IL-6, periostin, YKL-40, lipid peroxidation and collagen deposition. Finally, histological, immunohistochemical, and TUNNEL evidence also supported the ability of IFX to prevent BLC-induced lung fibrosis. The results of the present study indicate that IFX pretreatment can attenuate BLC-induced pulmonary fibrosis.

Topics: Animals; Antioxidants; Apoptosis; Bleomycin; Cell Adhesion Molecules; Glutathione; Glutathione Peroxidase; Infliximab; Interleukin-6; Lung; Male; Malondialdehyde; Nitric Oxide; Nitric Oxide Synthase Type II; Pulmonary Fibrosis; Random Allocation; Rats; Rats, Wistar; Superoxide Dismutase; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha

Carbon nanotubes (CNT) have been reported to induce lung inflammation and fibrosis in rodents. We investigated the direct and indirect cellular mechanisms mediating the fibrogenic activity of multi-wall (MW) CNT on fibroblasts. We showed that MWCNT indirectly stimulate lung fibroblast (MLg) differentiation, via epithelial cells and macrophages, whereas no direct effect of MWCNT on fibroblast differentiation or collagen production was detected. MWCNT directly stimulated the proliferation of fibroblasts primed with low concentrations of growth factors, such as PDGF, TGF-β or EGF. MWCNT prolonged ERK 1/2 phosphorylation induced by low concentrations of PDGF or TGF-β in fibroblasts. This phenomenon and the proliferative activity of MWCNT on fibroblasts was abrogated by the inhibitors of ERK 1/2, PDGF-, TGF-β- and EGF-receptors. This activity was also reduced by amiloride, an endocytosis inhibitor. Finally, the lung fibrotic response to several MWCNT samples (different in length and diameter) correlated with their in vitro capacity to stimulate the proliferation of fibroblasts and to prolong ERK 1/2 signaling in these cells. Our findings point to a crosstalk between MWCNT, kinase receptors, ERK 1/2 signaling and endocytosis which stimulates the proliferation of fibroblasts. The mechanisms of action identified in this study contribute to predict the fibrogenic potential of MWCNT.

Topics: Amiloride; Animals; Cell Differentiation; Cell Proliferation; Cells, Cultured; Collagen; Endocytosis; ErbB Receptors; Female; Fibroblasts; MAP Kinase Signaling System; Mice; Nanotubes, Carbon; Phosphorylation; Platelet-Derived Growth Factor; Pulmonary Fibrosis; Receptors, Platelet-Derived Growth Factor; Receptors, Transforming Growth Factor beta; Signal Transduction; Transforming Growth Factor beta

Transforming growth factor-β (TGF-β) is central during the pathogenesis of pulmonary fibrosis, in which the plasminogen activator inhibitor-1 (PAI-1) also has an established role. TGF-β is also known to be the strongest inducer of PAI-1. To investigate the link between PAI-1 and TGF-β in fibrotic processes, we evaluated the effect of SK-216, a PAI-1-specific inhibitor, in TGF-β-dependent epithelial-mesenchymal transition (EMT) and fibroblast to myofibroblast differentiation. In human alveolar epithelial A549 cells, treatment with TGF-β induced EMT, whereas co-treatment with SK-216 attenuated the occurrence of EMT. The inhibition of TGF-β-induced EMT by SK-216 was also confirmed in the experiment using murine epithelial LA-4 cells. Blocking EMT by SK-216 inhibited TGF-β-induced endogenous production of PAI-1 and TGF-β in A549 cells as well. These effects of SK-216 were not likely mediated by suppressing either Smad or ERK pathways. Using human lung fibroblast MRC-5 cells, we demonstrated that SK-216 inhibited TGF-β-dependent differentiation of fibroblasts to myofibroblasts. We also observed this inhibition by SK-216 in human primary lung fibroblasts. Following these in vitro results, we tested oral administration of SK-216 into mice injected intratracheally with bleomycin. We found that SK-216 reduced the degree of bleomycin-induced pulmonary fibrosis in mice. Although the precise mechanisms underlying the link between TGF-β and PAI-1 regarding fibrotic process were not determined, PAI-1 seems to act as a potent downstream effector on the pro-fibrotic property of TGF-β. In addition, inhibition of PAI-1 activity by a PAI-1 inhibitor exerts an antifibrotic effect even in vivo. These data suggest that targeting PAI-1 as a downstream effector of TGF-β could be a promising therapeutic strategy for pulmonary fibrosis.

Topics: Aged; Animals; Benzoxazoles; Bleomycin; Cell Differentiation; Cell Line; Dicarboxylic Acids; Epithelial-Mesenchymal Transition; Female; Fibroblasts; Humans; Male; Mice; Mice, Inbred C57BL; Middle Aged; Myofibroblasts; Plasminogen Activator Inhibitor 1; Pulmonary Alveoli; Pulmonary Fibrosis; Transforming Growth Factor beta

C/EBP homologous protein (Chop) has been shown to have altered expression in patients with idiopathic pulmonary fibrosis (IPF), but its exact role in IPF pathoaetiology has not been fully addressed. Studies conducted in patients with IPF and Chop(-/-) mice have dissected the role of Chop and endoplasmic reticulum (ER) stress in pulmonary fibrosis pathogenesis. The effect of Chop deficiency on macrophage polarization and related signalling pathways were investigated to identify the underlying mechanisms. Patients with IPF and mice with bleomycin (BLM)-induced pulmonary fibrosis were affected by the altered Chop expression and ER stress. In particular, Chop deficiency protected mice against BLM-induced lung injury and fibrosis. Loss of Chop significantly attenuated transforming growth factor β (TGF-β) production and reduced M2 macrophage infiltration in the lung following BLM induction. Mechanistic studies showed that Chop deficiency repressed the M2 program in macrophages, which then attenuated TGF-β secretion. Specifically, loss of Chop promoted the expression of suppressors of cytokine signaling 1 and suppressors of cytokine signaling 3, and through which Chop deficiency repressed signal transducer and activator of transcription 6/peroxisome proliferator-activated receptor gamma signaling, the essential pathway for the M2 program in macrophages. Together, our data support the idea that Chop and ER stress are implicated in IPF pathoaetiology, involving at least the induction and differentiation of M2 macrophages.

Topics: Aged; Animals; Bleomycin; Cell Differentiation; Disease Models, Animal; Endoplasmic Reticulum Stress; Female; Humans; Macrophages; Male; Mice; Middle Aged; Pulmonary Fibrosis; Signal Transduction; Transcription Factor CHOP; Transforming Growth Factor beta

Caffeine is a commonly used food additive found naturally in many products. In addition to potently stimulating the central nervous system caffeine is able to affect various systems within the body including the cardiovascular and respiratory systems. Importantly, caffeine is used clinically to treat apnoea and bronchopulmonary dysplasia in premature babies. Recently, caffeine has been shown to exhibit antifibrotic effects in the liver in part through reducing collagen expression and deposition, and reducing expression of the profibrotic cytokine TGFβ. The potential antifibrotic effects of caffeine in the lung have not previously been investigated. Using a combined in vitro and ex vivo approach we have demonstrated that caffeine can act as an antifibrotic agent in the lung by acting on two distinct cell types, namely epithelial cells and fibroblasts. Caffeine inhibited TGFβ activation by lung epithelial cells in a concentration-dependent manner but had no effect on TGFβ activation in fibroblasts. Importantly, however, caffeine abrogated profibrotic responses to TGFβ in lung fibroblasts. It inhibited basal expression of the α-smooth muscle actin gene and reduced TGFβ-induced increases in profibrotic genes. Finally, caffeine reduced established bleomycin-induced fibrosis after 5 days treatment in an ex vivo precision-cut lung slice model. Together, these findings suggest that there is merit in further investigating the potential use of caffeine, or its analogues, as antifibrotic agents in the lung.

Topics: Animals; Biomarkers; Caffeine; Cells, Cultured; Epithelial Cells; Fibroblasts; Gene Expression Regulation; Humans; Mice; Pulmonary Fibrosis; Signal Transduction; Transforming Growth Factor beta

Aging has been implicated in the development of pulmonary fibrosis, which has seen a sharp increase in incidence in those older than 50 years. Recent studies demonstrate a role for the nucleotide-binding domain and leucine rich repeat containing family, pyrin domain containing 3 (NLRP3) inflammasome and its regulated cytokines in experimental lung fibrosis. In this study, we tested the hypothesis that age-related NLRP3 inflammasome activation is an important predisposing factor in the development of pulmonary fibrosis. Briefly, young and aged wild-type and NLRP3(-/-) mice were subjected to bleomycin-induced lung injury. Pulmonary fibrosis was determined by histology and hydroxyproline accumulation. Bone marrow and alveolar macrophages were isolated from these mice. NLRP3 inflammasome activation was assessed by co-immunoprecipitation experiments. IL-1β and IL-18 production was measured by ELISA. The current study demonstrated that aged wild-type mice developed more lung fibrosis and exhibited increased morbidity and mortality after bleomycin-induced lung injury, when compared with young mice. Bleomycin-exposed aged NLRP3(-/-) mice had reduced fibrosis compared with their wild-type age-matched counterparts. Bone marrow-derived and alveolar macrophages from aged mice displayed higher levels of NLRP3 inflammasome activation and caspase-1-dependent IL-1β and IL-18 production, which was associated with altered mitochondrial function and increased production of reactive oxygen species. Our study demonstrated that age-dependent increases in alveolar macrophage mitochondrial reactive oxygen species production and NLRP3 inflammasome activation contribute to the development of experimental fibrosis.

Topics: Aging; Animals; Bleomycin; Disease Susceptibility; Inflammasomes; Instillation, Drug; Interleukin-18; Interleukin-1beta; Lung; Lung Injury; Macrophages; Male; Mice, Inbred C57BL; Mitochondria; NLR Family, Pyrin Domain-Containing 3 Protein; Pulmonary Fibrosis; Transforming Growth Factor beta

Fibrosis is a common pathological sequela of tissue injury or inflammation, and is a major cause of organ failure. Subsets of fibroblasts contribute to tissue fibrosis in multiple ways, including generating contractile force to activate integrin-bound, latent TGFβ and secreting excess amounts of collagens and other extracellular matrix proteins (ECM) that make up pathologic scar. However, the precise fibroblast subsets that drive fibrosis have been poorly understood. In the absence of well-characterized markers, α-smooth muscle actin (αSMA) is often used to identify pathologic fibroblasts, and some authors have equated αSMA(+) cells with contractile myofibroblasts and proposed that these cells are the major source of ECM. Here, we investigated how well αSMA expression describes fibroblast subsets responsible for TGFβ activation and collagen production in three commonly used models of organ fibrosis that we previously reported could be inhibited by loss of αv integrins on all fibroblasts (using PDGFRβ-Cre). Interestingly, αSMA-directed deletion of αv integrins protected mice from CCl4-induced hepatic fibrosis, but not bleomycin-induced pulmonary or unilateral ureteral obstruction-induced renal fibrosis. Using Col-EGFP/αSMA-RFP dual reporter mice, we found that only a minority of collagen-producing cells coexpress αSMA in the fibrotic lung and kidney. Notably, Col-EGFP(+)αSMA-RFP(-) cells isolated from the fibrotic lung and kidney were equally capable of activating TGFβ as were Col-EGFP(+)αSMA-RFP(+) cells from the same organ, and this TGFβ activation was blocked by a TGFβ-blocking antibody and an inhibitor of nonmuscle myosin, respectively. Taken together, our results suggest that αSMA is an inconsistent marker of contractile and collagen-producing fibroblasts in murine experimental models of organ fibrosis.

Topics: Actins; Animals; Biomarkers; Cells, Cultured; Collagen; Fibroblasts; Integrins; Kidney; Lung; Mice, Inbred C57BL; Mice, Transgenic; Pulmonary Fibrosis; Receptors, Platelet-Derived Growth Factor; Transforming Growth Factor beta

Complement activation, an integral arm of innate immunity, may be the critical link to the pathogenesis of idiopathic pulmonary fibrosis (IPF). Whereas we have previously reported elevated anaphylatoxins-complement component 3a (C3a) and complement component 5a (C5a)-in IPF, which interact with TGF-β and augment epithelial injury in vitro, their role in IPF pathogenesis remains unclear. The objective of the current study is to determine the mechanistic role of the binding of C3a/C5a to their respective receptors (C3aR and C5aR) in the progression of lung fibrosis. In normal primary human fetal lung fibroblasts, C3a and C5a induces mesenchymal activation, matrix synthesis, and the expression of their respective receptors. We investigated the role of C3aR and C5aR in lung fibrosis by using bleomycin-injured mice with fibrotic lungs, elevated local C3a and C5a, and overexpression of their receptors via pharmacologic and RNA interference interventions. Histopathologic examination revealed an arrest in disease progression and attenuated lung collagen deposition (Masson's trichrome, hydroxyproline, collagen type I α 1 chain, and collagen type I α 2 chain). Pharmacologic or RNA interference-specific interventions suppressed complement activation (C3a and C5a) and soluble terminal complement complex formation (C5b-9) locally and active TGF-β1 systemically. C3aR/C5aR antagonists suppressed local mRNA expressions of tgfb2, tgfbr1/2, ltbp1/2, serpine1, tsp1, bmp1/4, pdgfbb, igf1, but restored the proteoglycan, dcn Clinically, compared with pathologically normal human subjects, patients with IPF presented local induction of C5aR, local and systemic induction of soluble C5b-9, and amplified expression of C3aR/C5aR in lesions. The blockade of C3aR and C5aR arrested the progression of fibrosis by attenuating local complement activation and TGF-β/bone morphologic protein signaling as well as restoring decorin, which suggests a promising therapeutic strategy for patients with IPF.-Gu, H., Fisher, A. J., Mickler, E. A., Duerson, F., III, Cummings, O. W., Peters-Golden, M., Twigg, H. L., III, Woodruff, T. M., Wilkes, D. S., Vittal, R. Contribution of the anaphylatoxin receptors, C3aR and C5aR, to the pathogenesis of pulmonary fibrosis.

Topics: Aged; Aged, 80 and over; Animals; Antibiotics, Antineoplastic; Bleomycin; Cell Line; Collagen Type I, alpha 1 Chain; Complement Membrane Attack Complex; Down-Regulation; Fibroblasts; Gene Expression Regulation; Humans; Lung Injury; Mice; Mice, Inbred C57BL; Middle Aged; Pulmonary Fibrosis; Receptor, Anaphylatoxin C5a; Receptors, Complement; RNA Interference; Signal Transduction; Transforming Growth Factor beta; Up-Regulation

In this study, the bleomycin model of pulmonary fibrosis was utilized to investigate putative anti-fibrotic activity of Ac-SDKP in vivo. Male CD-1 mice received intra-tracheal bleomycin (BLEO, 1 mg/kg) instillation in the absence or presence of Ac-SDKP (a dose of 0.6 mg/kg delivered intra-peritoneally on the day of BLEO treatment, d0, followed by bi-weekly additional doses). To evaluate therapeutic effects in a subset of mice, Ac-SDKP was administered one week after BLEO instillation (d7). Animals were sacrificed at one, two, or three weeks later. Measurement of fluid and collagen content in the lung, Broncho Alveolar Lavage Fluid (BALF) analysis, lung histology, immunohistochemistry (IHC), and molecular analysis were performed. Compared to BLEO-treated mice, animals that received also Ac-SDKP (at both d0 and d7) had significantly decreased mortality, weight loss, inflammation (edema, and leukocyte lung infiltration), lung damage (histological evidence of lung injury), and fibrosis (collagen histological staining and soluble collagen content in the lung) at up to 21 days. Moreover, IHC and quantitative RT-PCR results demonstrated a significant decrease in BLEO-induced IL-17 and TGF-β expression in lung tissue. Importantly, α-SMA expression, the hallmark of myofibroblast differentiation, was also decreased. This is the first report showing not only a preventive protective role of Ac-SDKP but also its significant therapeutic effects in the bleomycin model of pulmonary fibrosis, thus supporting further preclinical and clinical studies.

Topics: Actins; Animals; Bleomycin; Chemotaxis, Leukocyte; Collagen; Cytoprotection; Disease Models, Animal; Interleukin-17; Lung; Male; Mice; Myofibroblasts; Oligopeptides; Protective Agents; Pulmonary Edema; Pulmonary Fibrosis; Time Factors; Transforming Growth Factor beta

Paeoniflorin has shown to attenuate bleomycin-induced pulmonary fibrosis (PF) in mice. Because the epithelial-mesenchymal transition (EMT) in type 2 lung endothelial cells contributes to excessive fibroblasts and myofibroblasts during multiple fibrosis of tissues, we investigated the effects of paeoniflorin on TGF-β mediated pulmonary EMT in bleomycin-induced PF mice.. PF was induced in mice by intratracheal instillation of bleomycin (5 mg/kg). The mice were orally treated with paeoniflorin or prednisone for 21 d. After the mice were sacrificed, lung tissues were collected for analysis. An in vitro EMT model was established in alveolar epithelial cells (A549 cells) incubated with TGF-β1 (2 ng/mL). EMT identification and the expression of related proteins were performed using immunohistochemistry, transwell assay, ELISA, Western blot and RT-qPCR.. In PF mice, paeoniflorin (50, 100 mg·kg(-1)·d(-1)) or prednisone (6 mg·kg(-1)·d(-1)) significantly decreased the expression of FSP-1 and α-SMA, and increased the expression of E-cadherin in lung tissues. In A549 cells, TGF-β1 stimulation induced EMT, as shown by the changes in cell morphology, the increased cell migration, and the increased vimentin and α-SMA expression as well as type I and type III collagen levels, and by the decreased E-cadherin expression. In contrast, effects of paeoniflorin on EMT disappeared when the A549 cells were pretreated with TGF-β1 for 24 h. TGF-β1 stimulation markedly increased the expression of Snail and activated Smad2/3, Akt, ERK, JNK and p38 MAPK in A549 cells. Co-incubation with paeoniflorin (1-30 μmol/L) dose-dependently attenuated TGF-β1-induced expression of Snail and activation of Smad2/3, but slightly affected TGF-β1-induced activation of Akt, ERK, JNK and p38 MAPK. Moreover, paeoniflorin markedly increased Smad7 level, and decreased ALK5 level in A549 cells.. Paeoniflorin suppresses the early stages of TGF-β mediated EMT in alveolar epithelial cells, likely by decreasing the expression of the transcription factors Snail via a Smad-dependent pathway involving the up-regulation of Smad7.

Topics: A549 Cells; Animals; Anti-Inflammatory Agents, Non-Steroidal; Bleomycin; Cell Survival; Epithelial Cells; Epithelial-Mesenchymal Transition; Glucosides; Humans; Lung; Male; Mice; Mice, Inbred ICR; Monoterpenes; Paeonia; Pulmonary Fibrosis; Signal Transduction; Smad Proteins; Transforming Growth Factor beta

Nano-scaled cerium oxide (nCeO2) is used in a variety of applications, including use as a fuel additive, catalyst, and polishing agent, yet potential adverse health effects associated with nCeO2 exposure remain incompletely understood. Given the increasing utility and demand for engineered nanomaterials (ENMs) such as nCeO2, "safety-by-design" approaches are currently being sought, meaning that the physicochemical properties (e.g., size and surface chemistry) of the ENMs are altered in an effort to maximize functionality while minimizing potential toxicity. In vivo studies have shown in a rat model that inhaled nCeO2 deposited deep in the lung and induced fibrosis. However, little is known about how the physicochemical properties of nCeO2, or the coating of the particles with a material such as amorphous silica (aSiO2), may affect the bio-activity of these particles. Thus, we hypothesized that the physicochemical properties of nCeO2 may explain its potential to induce fibrogenesis, and that a nano-thin aSiO2 coating on nCeO2 may counteract that effect.. Primary normal human lung fibroblasts were treated at occupationally relevant doses with nCeO2 that was either left uncoated or was coated with aSiO2 (amsCeO2). Subsequently, fibroblasts were analyzed for known hallmarks of fibrogenesis, including cell proliferation and collagen production, as well as the formation of fibroblastic nodules. The results of this study are consistent with this hypothesis, as we found that nCeO2 directly induced significant production of collagen I and increased cell proliferation in vitro, while amsCeO2 did not. Furthermore, treatment of fibroblasts with nCeO2, but not amsCeO2, significantly induced the formation of fibroblastic nodules, a clear indicator of fibrogenicity. Such in vitro data is consistent with recent in vivo observations using the same nCeO2 nanoparticles and relevant doses. This effect appeared to be mediated through TGFβ signaling since chemical inhibition of the TGFβ receptor abolished these responses.. These results indicate that differences in the physicochemical properties of nCeO2 may alter the fibrogenicity of this material, thus highlighting the potential benefits of "safety-by-design" strategies. In addition, this study provides an efficient in vitro method for testing the fibrogenicity of ENMs that strongly correlates with in vivo findings.

Topics: Air Pollutants; Cell Proliferation; Cells, Cultured; Cerium; Chemical Phenomena; Gene Expression Regulation; Humans; Lung; Metal Nanoparticles; Particle Size; Physical Phenomena; Pulmonary Fibrosis; Respiratory Mucosa; Signal Transduction; Silicon Dioxide; Surface Properties; Toxicity Tests, Acute; Transforming Growth Factor beta

Idiopathic pulmonary fibrosis (IPF) is a progressive, debilitating disease for which two medications, pirfenidone and nintedanib, have only recently been approved for treatment. The cytokine TGF-β has been shown to be a central mediator in the disease process. We investigated the role of a novel kinase, MAP3K19, upregulated in IPF tissue, in TGF-β-induced signal transduction and in bleomycin-induced pulmonary fibrosis. MAP3K19 has a very limited tissue expression, restricted primarily to the lungs and trachea. In pulmonary tissue, expression was predominantly localized to alveolar and interstitial macrophages, bronchial epithelial cells and type II pneumocytes of the epithelium. MAP3K19 was also found to be overexpressed in bronchoalveolar lavage macrophages from IPF patients compared to normal patients. Treatment of A549 or THP-1 cells with either MAP3K19 siRNA or a highly potent and specific inhibitor reduced phospho-Smad2 & 3 nuclear translocation following TGF-β stimulation. TGF-β-induced gene transcription was also strongly inhibited by both the MAP3K19 inhibitor and nintedanib, whereas pirfenidone had a much less pronounced effect. In combination, the MAP3K19 inhibitor appeared to act synergistically with either pirfenidone or nintedanib, at the level of target gene transcription or protein production. Finally, in an animal model of IPF, inhibition of MAP3K19 strongly attenuated bleomycin-induced pulmonary fibrosis when administered either prophylactically ortherapeutically. In summary, these results strongly suggest that inhibition of MAP3K19 may have a beneficial therapeutic effect in the treatment of IPF and represents a novel strategy to target this disease.

Topics: A549 Cells; Animals; Bleomycin; Bronchoalveolar Lavage; Cell Line, Tumor; Disease Models, Animal; Epithelial Cells; Female; HeLa Cells; Humans; Idiopathic Pulmonary Fibrosis; Indoles; Lung; Lung Injury; MAP Kinase Kinase Kinases; Mice; Mice, Inbred C57BL; Pulmonary Fibrosis; Pyridones; Signal Transduction; Transforming Growth Factor beta; Up-Regulation

Multi-walled carbon nanotubes (MWCNT) are currently under intense toxicological investigation due to concern on their potential health effects. Current in vitro and in vivo data indicate that MWCNT exposure is strongly associated with lung toxicity (inflammation, fibrosis, granuloma, cancer and airway injury) and their effects might be comparable to asbestos-induced carcinogenesis. Although fibrosis is a multi-origin disease, epithelial-mesenchymal transition (EMT) is recently recognized as an important pathway in cell transformation. It is known that MWCNT exposure induces EMT through the activation of the TGF-β/Smad signalling pathway thus promoting pulmonary fibrosis, but the molecular mechanisms involved are not fully understood. In the present work we propose a new mechanism involving a TGF-β-mediated signalling pathway.. Human bronchial epithelial cells were incubated with two different MWCNT samples at various concentrations for up to 96 h and several markers of EMT were investigated. Quantitative real time PCR, western blot, immunofluorescent staining and gelatin zymographies were performed to detect the marker protein alterations. ELISA was performed to evaluate TGF-β production. Experiments with neutralizing anti-TGF-β antibody, specific inhibitors of GSK-3β and Akt and siRNA were carried out in order to confirm their involvement in MWCNT-induced EMT. In vivo experiments of pharyngeal aspiration in C57BL/6 mice were also performed. Data were analyzed by a one-way ANOVA with Tukey's post-hoc test.. Fully characterized MWCNT (mean length < 5 μm) are able to induce EMT in an in vitro human model (BEAS-2B cells) after long-term incubation at sub-cytotoxic concentrations. MWCNT stimulate TGF-β secretion, Akt activation and GSK-3β inhibition, which induces nuclear accumulation of SNAIL-1 and its transcriptional activity, thus contributing to switch on the EMT program. Moreover, a significant increment of nuclear β-catenin - due to E-cadherin repression and following translocation to nucleus - likely reinforces signalling for EMT promotion. In vivo results supported the occurrence of pulmonary fibrosis following MWCNT exposure.. We demonstrate a new molecular mechanism of MWCNT-mediated EMT, which is Smad-independent and involves TGF-β and its intracellular effectors Akt/GSK-3β that activate the SNAIL-1 signalling pathway. This finding suggests potential novel targets in the development of therapeutic and preventive approaches.

Topics: Animals; Bronchi; Carcinogenicity Tests; Cell Line; Epithelial-Mesenchymal Transition; Glycogen Synthase Kinase 3 beta; Humans; Inhalation Exposure; Male; Mice, Inbred C57BL; Microscopy, Electron, Scanning; Microscopy, Electron, Transmission; Nanotubes, Carbon; Particle Size; Proto-Oncogene Proteins c-akt; Pulmonary Fibrosis; Respiratory Mucosa; Signal Transduction; Snail Family Transcription Factors; Surface Properties; Transforming Growth Factor beta

Pulmonary fibrosis is a progressive and fatal disorder. In our previous study, we found that the Yiqihuoxue formula (YQHX), a prescription of Traditional Chinese Medicine, had a curative effect on scleroderma, a typical fibrotic disease. The aim of this study was to determine the key ingredient mediating the therapeutic effects of YQHX and to examine its effect on pulmonary fibrosis, including its mechanism. Luciferase reporter assays showed that the most important anti-fibrotic component of the YQHX was Salviae miltiorrhiza (SM). Experiments performed using a bleomycin-instilled mouse model of pulmonary fibrosis showed that Salvianolic acid B (SAB), the major ingredient of SM, had strong anti-inflammatory and anti-fibrotic effects through its inhibition of inflammatory cell infiltration, alveolar structure disruption, and collagen deposition. Furthermore, SAB suppressed TGF-β-induced myofibroblastic differentiation of MRC-5 fibroblasts and TGF-β-mediated epithelial-to-mesenchymal transition of A549 cells by inhibiting both Smad-dependent signaling and the Smad-independent MAPK pathway. Taken together, our results suggest that SM is the key anti-fibrotic component of the YQHX and that SAB, the major ingredient of SM, alleviates experimental pulmonary fibrosis both in vivo and in vitro by inhibiting the TGF-β signaling pathway. Together, these results suggest that SAB potently inhibits pulmonary fibrosis.

Topics: A549 Cells; Animals; Benzofurans; Bleomycin; Bronchoalveolar Lavage Fluid; Cell Differentiation; Cell Proliferation; Disease Models, Animal; Drugs, Chinese Herbal; Fibroblasts; Humans; Medicine, Chinese Traditional; Mice; NIH 3T3 Cells; Pulmonary Fibrosis; Salvia; Signal Transduction; Transforming Growth Factor beta

TGF-β signaling regulates a variety of cellular processes, including proliferation, apoptosis, differentiation, immune responses, and fibrogenesis. Here, we describe a lysine methylation-mediated mechanism that controls the pro-fibrogenic activity of TGF-β. We find that the methyltransferase Set9 potentiates TGF-β signaling by targeting Smad7, an inhibitory downstream effector. Smad7 methylation promotes interaction with the E3 ligase Arkadia and, thus, ubiquitination-dependent degradation. Depletion or pharmacological inhibition of Set9 results in elevated Smad7 protein levels and inhibits TGF-β-dependent expression of genes encoding extracellular matrix components. The inhibitory effect of Set9 on TGF-β-mediated extracellular matrix production is further demonstrated in mouse models of pulmonary fibrosis. Lung fibrosis induced by bleomycin or Ad-TGF-β treatment was highly compromised in Set9-deficient mice. These results uncover a complex regulatory interplay among multiple Smad7 modifications and highlight the possibility that protein methyltransferases may represent promising therapeutic targets for treating lung fibrosis.

Topics: Acetylation; Animals; Bleomycin; Extracellular Matrix; Gene Expression Regulation; HeLa Cells; Histone-Lysine N-Methyltransferase; Humans; Lysine; Male; Methylation; Mice, Inbred C57BL; Mice, Knockout; Models, Biological; Nuclear Proteins; Protein Methyltransferases; Protein Stability; Pulmonary Fibrosis; Signal Transduction; Smad7 Protein; Transforming Growth Factor beta; Ubiquitin-Protein Ligases; Ubiquitination

Idiopathic pulmonary fibrosis (IPF) is a lethal human disease with short survival time and few treatment options. Herein, we demonstrated that discoidin domain receptor 2 (DDR2), a receptor tyrosine kinase that predominantly transduces signals from fibrillar collagens, plays a critical role in the induction of fibrosis and angiogenesis in the lung. In vitro cell studies showed that DDR2 can synergize the actions of both transforming growth factor (TGF)-β and fibrillar collagen to stimulate lung fibroblasts to undergo myofibroblastic changes and vascular endothelial growth factor (VEGF) expression. In addition, we confirmed that late treatment of the injured mice with specific siRNA against DDR2 or its kinase inhibitor exhibited therapeutic efficacy against lung fibrosis. Thus, this study not only elucidated novel mechanisms by which DDR2 controls the development of pulmonary fibrosis, but also provided candidate target for the intervention of this stubborn disease.

Topics: Animals; Cell Differentiation; Cells, Cultured; Collagen Type I; Discoidin Domain Receptor 2; Disease Models, Animal; Extracellular Matrix; Humans; Mice; Myofibroblasts; Protein Kinase Inhibitors; Pulmonary Fibrosis; RNA, Small Interfering; Transforming Growth Factor beta; Up-Regulation; Vascular Endothelial Growth Factor A

Idiopathic pulmonary fibrosis (IPF) is a chronic and ultimately fatal disease, characterized by excessive accumulation of fibroblasts, extensive deposition of extracellular matrix, and destruction of alveolar architecture. IPF is associated with an epithelial-dependent fibroblast-activated process, termed the epithelial-to-mesenchymal transition (EMT). However, there is still a lack of strategies to target EMT for the treatment of IPF. Sunitinib, a small-molecule multi-targeted tyrosine kinase inhibitor, targets multiple kinases that may play an important role in developing pulmonary fibrosis. Here, we explored the therapeutic potential of sunitinib using a mouse model of pulmonary fibrosis. Mice received intratracheal instillation of bleomycin (BLM). Then, the mice were intragastrically administrated with sunitinib or normal saline until the end of the experiment. Distinguished destruction of pulmonary architecture, conspicuous proliferation of fibroblasts and extensive deposition of collagen fibers were found in BLM mice. Sunitinib attenuated the pulmonary fibrosis and inhibited the accumulation of fibroblasts in the lung of BLM mice. To investigate if the inhibition of fibroblast accumulation in the lung by sunitinib was associated with EMT, we used human bronchial epithelial cells (HBEs) and W138 human lung fibroblasts. Sunitinib suppressed the degree of EMT induced by TGF-β, a profibrotic factor, in HBEs and the proliferation of WI38 fibroblasts. Moreover, sunitinib reduced the degree of phosphorylation of serine residues on Smad2/3 that was induced by TGF-β in HBEs. As EMT and accumulation of fibroblasts are critical for the development of pulmonary fibrosis, targeting multiple pro-fibrosis signaling pathways with sunitinib may be a novel strategy to treat pulmonary fibrosis.

Topics: Animals; Bleomycin; Bronchi; Cell Proliferation; Disease Models, Animal; Epithelial Cells; Extracellular Matrix; Female; Fibroblasts; Humans; Indoles; Mesoderm; Mice; Mice, Inbred C57BL; Phosphorylation; Platelet-Derived Growth Factor; Protein Kinase Inhibitors; Pulmonary Fibrosis; Pyrroles; Signal Transduction; Smad Proteins; Small Molecule Libraries; Sunitinib; Transforming Growth Factor beta; Vascular Endothelial Growth Factor A

Fibrosis of lung tissue is a disease where a chronic inflammatory process determines a pathological remodelling of lung parenchyma. The animal model obtained by intra-tracheal administration of bleomycin in C57BL/6 mice is one of the most validated murine model. Bleomycin stimulates oxidative stress and the production of pro-inflammatory mediators. Histamine H4R have recently been implicated in inflammation and immune diseases. This study was focused to investigate the effects of H4R ligands in the modulation of inflammation and in the reduction of lung fibrosis in C57BL/6 mice treated with bleomycin. C57BL/6 mice were treated with vehicle, JNJ7777120 (JNJ, selective H4R antagonist) or ST-1006 (partial H4R agonist), ST-994 (H4R neutral antagonist) and ST-1012 (inverse H4R agonist) at equimolar doses, released by micro-osmotic pumps for 21days. Airway resistance to inflation was assayed and lung samples were processed to measure malondialdehyde (TBARS); 8-hydroxy-2'-deoxyguanosine (8OHdG); myeloperoxidase (MPO); COX-2 expression and activity as markers of oxidative stress and inflammation. Fibrosis and airway remodelling were evaluated throughout transforming growth factor-β (TGF-β), percentage of positive Goblet cells, smooth muscle layer thickness determination. Our results indicated that JNJ, ST-994 and ST-1012 decreased inflammation and oxidative stress markers, i.e. the number of infiltrating leukocytes evaluated as lung tissue MPO, COX-2 expression and activity, TBARS and 8OHdG production. They also reduced the level of TGF-β, a pro-fibrotic cytokine, collagen deposition, thickness of smooth muscle layer, Goblet cells hyperplasia; resulting in a decrease of airway functional impairment. The results here reported clearly demonstrated that H4R ligands have a beneficial effect in a model of lung fibrosis in the mouse, thus indicating that H4R antagonists or inverse agonists could be a novel therapeutic strategy for lung inflammatory diseases.

Topics: Animals; Anti-Inflammatory Agents; Biomarkers; Bleomycin; Collagen; Cytoprotection; Disease Models, Animal; Drug Partial Agonism; Goblet Cells; Histamine Antagonists; Hyperplasia; Indoles; Inflammation Mediators; Ligands; Lung; Male; Mice, Inbred C57BL; Oxidative Stress; Piperazines; Pneumonia; Pulmonary Fibrosis; Pyrimidines; Receptors, Histamine H4; Signal Transduction; Transforming Growth Factor beta

Protein kinase Cζ (PKCζ) is highly expressed in the lung, where it plays several regulating roles in the pathogenesis of acute lung injury (ALI). Proliferation and differentiation of integrin β4

Topics: Acute Lung Injury; Animals; Bleomycin; Bronchoalveolar Lavage Fluid; Cell Death; Cell Movement; Cell Proliferation; Cell Separation; Compliance; Disease Models, Animal; Enzyme Activation; Epithelial Cells; Mice, Inbred C57BL; Models, Biological; Neutrophils; Permeability; Protein Kinase C; Protein Kinase Inhibitors; Pulmonary Fibrosis; Reactive Oxygen Species; Stem Cells; Transforming Growth Factor beta

The accumulation of fibroblasts/myofibroblasts in fibrotic foci is one of the characteristics of idiopathic pulmonary fibrosis (IPF). Enhancer of zeste homolog 2 (EZH2) is the catalytic component of a multiprotein complex, polycomb repressive complex 2, which is involved in the trimethylation of histone H3 at lysine 27. In this study, we investigated the role and mechanisms of EZH2 in the differentiation of fibroblasts into myofibroblasts. We found that EZH2 was upregulated in the lungs of patients with IPF and in mice with bleomycin-induced lung fibrosis. The upregulation of EZH2 occurred in myofibroblasts. The inhibition of EZH2 by its inhibitor 3-deazaneplanocin A (DZNep) or an shRNA reduced the TGF-β1-induced differentiation of human lung fibroblasts into myofibroblasts, as demonstrated by the expression of the myofibroblast markers α-smooth muscle actin and fibronectin, and contractility. DZNep inhibited Smad2/3 nuclear translocation without affecting Smad2/3 phosphorylation. DZNep treatment attenuated bleomycin-induced pulmonary fibrosis in mice. We conclude that EZH2 induces the differentiation of fibroblasts to myofibroblasts by enhancing Smad2/3 nuclear translocation.

Topics: Adenosine; Adult; Animals; Bleomycin; Cell Differentiation; Cells, Cultured; Enhancer of Zeste Homolog 2 Protein; Female; Fibroblasts; Humans; Idiopathic Pulmonary Fibrosis; Lung; Mice; Mice, Inbred C57BL; Myofibroblasts; Pulmonary Fibrosis; RNA, Small Interfering; Transforming Growth Factor beta; Up-Regulation

To investigate the effect of fibroblasts on regulating airway stem cell proliferation in idiopathic pulmonary fibrosis.. Lung cell suspension was prepared from β-actin-GFP mice. Airway stem cells were obtained by fluorescence activated cell sorting and co-cultured with lung fibroblasts. The fibroblasts were treated with TGF-β inhibitor SB43142. The expression of growth factors FGF1/2 and the effect of FGF1/2 on stem cell proliferation were observed.. The cloning efficiency of airway stem cells, when co-cultured with normal lung fibroblast cells for 8 days, was (3.5±1.1)%, while the cloning efficiency was reduced to (0.04±0.04)% when co-cultured with lung fibroblasts from idiopathic pulmonary fibrosis patients. The difference between the 2 groups was statistically significant(P=0.002 5). TGF-β receptor inhibitor SB431542 increased lung fibroblast growth factors FGF1/2 expression.FGF1 mRNA expression was increased to the experimental group 0.005 5 from 0.000 2 in the control group.FGF2 mRNA expression of the amount raised to the experimental group 0.000 15 from 0.000 8 in the control group.FGF1/2 promoted the growth of airway stem cells. After FGF1/2 was co-cultured with normal lung fibroblast cells for 8 days, the cloning efficiency of airway stem cells was (0.3±0.1)%.. During the development of idiopathic pulmonary fibrosis, fibroblast secreted FGF1/2 regulate airway stem cell proliferation.

Topics: Actins; Animals; Benzamides; Cell Movement; Cell Proliferation; Cells, Cultured; Dioxoles; Fibroblast Growth Factor 1; Fibroblast Growth Factor 2; Fibroblasts; Gene Expression Regulation; Humans; Idiopathic Pulmonary Fibrosis; Lung; Mice; MicroRNAs; Protein Serine-Threonine Kinases; Pulmonary Fibrosis; Receptor, Transforming Growth Factor-beta Type II; Receptors, Fibroblast Growth Factor; Receptors, Transforming Growth Factor beta; Transforming Growth Factor beta

Alveolar epithelial cell (AEC) dysfunction underlies the pathogenesis of pulmonary fibrosis in Hermansky-Pudlak syndrome (HPS) and other genetic syndromes associated with interstitial lung disease; however, mechanisms linking AEC dysfunction and fibrotic remodeling are incompletely understood. Since increased macrophage recruitment precedes pulmonary fibrosis in HPS, we investigated whether crosstalk between AECs and macrophages determines fibrotic susceptibility. We found that AECs from HPS mice produce excessive MCP-1, which was associated with increased macrophages in the lungs of unchallenged HPS mice. Blocking MCP-1/CCR2 signaling in HPS mice with genetic deficiency of CCR2 or targeted deletion of MCP-1 in AECs normalized macrophage recruitment, decreased AEC apoptosis, and reduced lung fibrosis in these mice following treatment with low-dose bleomycin. We observed increased TGF-β production by HPS macrophages, which was eliminated by CCR2 deletion. Selective deletion of TGF-β in myeloid cells or of TGF-β signaling in AECs through deletion of TGFBR2 protected HPS mice from AEC apoptosis and bleomycin-induced fibrosis. Together, these data reveal a feedback loop in which increased MCP-1 production by dysfunctional AECs results in recruitment and activation of lung macrophages that produce TGF-β, thus amplifying the fibrotic cascade through AEC apoptosis and stimulation of fibrotic remodeling.

Topics: Animals; Bleomycin; Chemokine CCL2; Disease Susceptibility; Epithelial Cells; Female; Hermanski-Pudlak Syndrome; Macrophages; Male; Mice; Mice, Inbred C57BL; Protein Serine-Threonine Kinases; Pulmonary Alveoli; Pulmonary Fibrosis; Receptor, Transforming Growth Factor-beta Type II; Receptors, CCR2; Receptors, Transforming Growth Factor beta; Transforming Growth Factor beta

Chronic epithelial injury triggers a TGF-β-mediated cellular transition from normal epithelium into a mesenchymal-like state that produces subepithelial fibrosis and airway remodeling. Here we examined how TGF-β induces the mesenchymal cell state and determined its mechanism. We observed that TGF-β stimulation activates an inflammatory gene program controlled by the NF-κB/RelA signaling pathway. In the mesenchymal state, NF-κB-dependent immediate-early genes accumulate euchromatin marks and processive RNA polymerase. This program of immediate-early genes is activated by enhanced expression, nuclear translocation, and activating phosphorylation of the NF-κB/RelA transcription factor on Ser276, mediated by a paracrine signal. Phospho-Ser276 RelA binds to the BRD4/CDK9 transcriptional elongation complex, activating the paused RNA Pol II by phosphorylation on Ser2 in its carboxy-terminal domain. RelA-initiated transcriptional elongation is required for expression of the core epithelial-mesenchymal transition transcriptional regulators SNAI1, TWIST1, and ZEB1 and mesenchymal genes. Finally, we observed that pharmacological inhibition of BRD4 can attenuate experimental lung fibrosis induced by repetitive TGF-β challenge in a mouse model. These data provide a detailed mechanism for how activated NF-κB and BRD4 control epithelial-mesenchymal transition initiation and transcriptional elongation in model airway epithelial cells in vitro and in a murine pulmonary fibrosis model in vivo. Our data validate BRD4 as an in vivo target for the treatment of pulmonary fibrosis associated with inflammation-coupled remodeling in chronic lung diseases.

Topics: Animals; Cell Cycle Proteins; Cell Nucleus; Chromatin Immunoprecipitation; Cyclin-Dependent Kinase 9; Epigenesis, Genetic; Epithelial Cells; Epithelial-Mesenchymal Transition; Genes, Immediate-Early; Humans; Immunity, Innate; Lung; Male; Mice, Inbred C57BL; Models, Biological; NF-kappa B; Nuclear Proteins; Phosphorylation; Protein Binding; Protein Stability; Protein Transport; Pulmonary Fibrosis; Signal Transduction; Transcription Elongation, Genetic; Transcription Factor RelA; Transcription Factors; Transforming Growth Factor beta

Upregulation of the intermediate filament protein nestin was identified in a subpopulation of fibroblasts during reactive and reparative fibrosis and directly contributed to the enhanced proliferative phenotype. The present study tested the hypothesis that nestin was expressed in lung fibroblasts and the pattern of expression represented a distinct marker of pulmonary remodeling secondary to myocardial infarction and type I diabetes. Nestin((+)) fibroblasts were detected in rat lungs and a subpopulation exhibited a myofibroblast phenotype delineated by the co-expression of smooth muscle α-actin. In the lungs of myocardial infarcted rats, interstitial collagen content and nestin mRNA/protein levels were significantly increased despite the absence of secondary pulmonary hypertension, whereas smooth muscle α-actin protein expression was unchanged. Exposure of rat pulmonary fibroblasts to pro-fibrotic stimuli angiotensin II and transforming growth factor-β significantly increased nestin protein levels. In the lungs of type I diabetic rats, the absence of a reactive fibrotic response was associated with a significant downregulation of nestin mRNA/protein expression. Nestin was reported a target of miR-125b, albeit miR-125b levels were unchanged in pulmonary fibroblasts treated with pro-fibrotic stimuli. Nestin((+)) cells lacking smooth muscle α-actin/collagen staining were also identified in rodent lungs and a transgenic approach revealed that expression of the intermediate filament protein was driven by intron 2 of the nestin gene. The disparate regulation of nestin characterized a distinct pattern of pulmonary remodeling secondary to myocardial infarction and type I diabetes and upregulation of the intermediate filament protein in lung fibroblasts may have facilitated in part the reactive fibrotic response.

Topics: Actins; Airway Remodeling; Angiotensin II; Animals; Biomarkers; Cell Differentiation; Collagen Type I; Diabetes Mellitus, Type 1; Fibroblasts; Heart Failure; Humans; Hypertension, Pulmonary; Hypertrophy, Right Ventricular; Lung; Male; MicroRNAs; Myocardial Contraction; Myocardial Infarction; Nestin; Pulmonary Fibrosis; Rats; Rats, Sprague-Dawley; RNA, Messenger; Streptozocin; Transforming Growth Factor beta

The aim of this work was to develop an innovative tool for the treatment of pulmonary fibrosis based on our previous findings, which demonstrated that intranasally administered soluble bovine hyaluronidase (HYAL) increases the numbers of mesenchymal (MSC)-like cells in the bronchoalveolar fluid (BALF) and thus reduces the bleomycin-induced fibrosis. To this end, we developed poly(D,L-lactide-co-glycolide) (PLGA) microparticles (MPs) loaded with HYAL (HYAL-MP) to preserve the enzyme's biological activity and to facilitate its delivery to the lung. Nonloaded MPs (Control-MPs) and HYAL-MPs were prepared using the emulsion and solvent evaporation methods and thoroughly characterized. The HYAL-MPs and Control-MPs exhibited an average diameter of 4.3±2.1 and 4.4±1.5 μm, respectively. The encapsulation efficiency of the HYAL-MPs was 68%, and encapsulation led to a reduced release rate. Additionally, the HYAL-MPs were efficiently phagocytosed by J-774.1 cells. Compared with the soluble HYAL, the HYAL-MPs increased the proportion of MSC-like cells in the BALF of C57BL6 mice 96 h after treatment. The efficacy of the HYAL-MPs was also tested in C57BL6 mice that were previously exposed to 4 U/kg of bleomycin to induce lung fibrosis. The results demonstrated that the HYAL-MPs reduced neutrophil recruitment after bleomycin treatment more effectively than did the soluble HYAL, whereas the Control-MPs did not exhibit any effect. The HYAL-MPs also reduced the bleomycin-induced fibrosis more efficiently, and 134% of the collagen deposition in the lung compared with the soluble HYAL and the Control-MPs. In summary, our data indicate that HYAL-MPs are an effective delivery system that could feasibly be used in the treatment of pulmonary fibrosis.

Topics: Animals; Cattle; Cell Count; Cell Line; Collagen; Cytoskeleton; Hyaluronoglucosaminidase; Lactic Acid; Mice, Inbred C57BL; Microscopy, Electron, Scanning; Microspheres; Particle Size; Phagocytes; Pneumonia; Polyglycolic Acid; Polylactic Acid-Polyglycolic Acid Copolymer; Pulmonary Fibrosis; RNA, Messenger; Solubility; Static Electricity; Transforming Growth Factor beta

Laminins are heterotrimeric proteins that are secreted by the alveolar epithelium into the basement membrane, and their expression is altered in extracellular matrices from patients with pulmonary fibrosis. In a small number of patients with pulmonary fibrosis, we found that the normal basement membrane distribution of the α3 laminin subunit was lost in fibrotic regions of the lung. To determine if these changes play a causal role in the development of fibrosis, we generated mice lacking the α3 laminin subunit specifically in the lung epithelium by crossing mice expressing Cre recombinase driven by the surfactant protein C promoter (SPC-Cre) with mice expressing floxed alleles encoding the α3 laminin gene (Lama3(fl/fl)). These mice exhibited no developmental abnormalities in the lungs up to 6 months of age, but, compared with control mice, had worsened mortality, increased inflammation, and increased fibrosis after the intratracheal administration of bleomycin. Similarly, the severity of fibrosis induced by an adenovirus encoding an active form of transforming growth factor-β was worse in mice deficient in α3 laminin in the lung. Taken together, our results suggest that the loss of α3 laminin in the lung epithelium does not affect lung development, but plays a causal role in the development of fibrosis in response to bleomycin or adenovirally delivered transforming growth factor-β. Thus, we speculate that the loss of the normal basement membrane organization of α3 laminin that we observe in fibrotic regions from the lungs of patients with pulmonary fibrosis contributes to their disease progression.

Topics: Animals; Bleomycin; Humans; Laminin; Lung; Mice, Transgenic; Pulmonary Alveoli; Pulmonary Fibrosis; Transforming Growth Factor beta

Inhalation of crystalline silica nanoparticles causes pulmonary damage resulting in progressive lung fibrosis. Currently, there is no effective treatment for silicosis. Tamoxifen citrate is a selective estrogen receptor modulator, which is one of the adjuvant treatment choices for breast cancer. It is also known with its inhibitory effect on the production of transforming growth factor-beta (TGF-β) and studied for the anti-fibrotic effect in some fibrotic diseases. The aim of the study was to determine the effect of tamoxifen citrate on the prevention of pulmonary fibrosis and the treatment of silicosis.. A total of 100 adult female Wistar Albino rats (200-250 g) were used in this study. The rats were divided into five groups including 20 rats in each. Rats were exposed to silica for 84 d in all groups. In group 1, rats were sacrificed on the day 84 without receiving treatment. In group 2, rats received 1 mg/kg tamoxifen (tmx1 + 1), from the first day of the study for the whole 114 d of the study. In group 3, (tmx10 + 10) rats were given 10 mg/kg tamoxifen from the first day of the study for the whole 114 d of the study. In group 4 (tmx1), rats were started 1 mg/kg of tamoxifen on day 84 and were given until day 114. In group 5 (tmx10), rats were fed with 10 mg/kg tamoxifen starting from day 84 to day 114. All rats except group 1 were sacrificed on 114 day of the study. Lung inflammation and fibrosis scores, serum TGF β levels, lung smooth muscle antigen and tissue transforming growth factor β (t-TGF-β) antibody staining levels, and number of silicotic rats were compared between groups.. Silicosis was caused successfully in all rats in group 1. There were six silicotic rats in group 3 and it was the lowest number of all groups. Plasma TGF-ß levels and fibrosis score were significantly lower in all groups when compared with the control group. Tamoxifen could have preventive or treating effects in silicosis and found that lung fibrosis score was significantly lower in rats treated with tamoxifen.. Tamoxifen treatment after and/or before induction of silicosis decreased lung fibrosis score with blood TGF-β levels. We hope that this study may introduce a new indication as prophylactic use of tamoxifen in high-risk groups for silicosis and for treatment of silicosis.

Topics: Animals; Disease Models, Animal; Female; Nanoparticles; Pulmonary Fibrosis; Rats, Wistar; Selective Estrogen Receptor Modulators; Silicon Dioxide; Silicosis; Tamoxifen; Transforming Growth Factor beta; Treatment Outcome

Alveolar type II epithelial (ATII) cell injury precedes development of pulmonary fibrosis. Mice lacking urokinase-type plasminogen activator (uPA) are highly susceptible, whereas those deficient in plasminogen activator inhibitor (PAI-1) are resistant to lung injury and pulmonary fibrosis. Epithelial-mesenchymal transition (EMT) has been considered, at least in part, as a source of myofibroblast formation during fibrogenesis. However, the contribution of altered expression of major components of the uPA system on ATII cell EMT during lung injury is not well understood. To investigate whether changes in uPA and PAI-1 by ATII cells contribute to EMT, ATII cells from patients with idiopathic pulmonary fibrosis and chronic obstructive pulmonary disease, and mice with bleomycin-, transforming growth factor β-, or passive cigarette smoke-induced lung injury were analyzed for uPA, PAI-1, and EMT markers. We found reduced expression of E-cadherin and zona occludens-1, whereas collagen-I and α-smooth muscle actin were increased in ATII cells isolated from injured lungs. These changes were associated with a parallel increase in PAI-1 and reduced uPA expression. Further, inhibition of Src kinase activity using caveolin-1 scaffolding domain peptide suppressed bleomycin-, transforming growth factor β-, or passive cigarette smoke-induced EMT and restored uPA expression while suppressing PAI-1. These studies show that induction of PAI-1 and inhibition of uPA during fibrosing lung injury lead to EMT in ATII cells.

Topics: Actins; Animals; Bleomycin; Cadherins; Collagen Type I; Disease Models, Animal; Epithelial-Mesenchymal Transition; Fibrinolysis; Gene Expression Regulation; Humans; Idiopathic Pulmonary Fibrosis; Lung; Lung Injury; Mice; Mice, Inbred C57BL; Phosphorylation; Plasminogen Activator Inhibitor 1; Pulmonary Disease, Chronic Obstructive; Pulmonary Fibrosis; Risk Factors; Serpin E2; Smoking; Transforming Growth Factor beta; Tumor Suppressor Protein p53; Urokinase-Type Plasminogen Activator; Zonula Occludens-1 Protein

Paracoccidioidomycosis (PCM) is a systemic mycosis in which the host response to the infectious agent typically consists of a chronic granulomatous inflammatory process. This condition causes lesions that impair lung function and lead to chronic pulmonary insufficiency resulting from fibrosis development, which is a sequel and disabling feature of the disease. The rPb27 protein has been studied for prophylactic and therapeutic treatment against PCM. Previous studies from our laboratory have shown a protective effect of rPb27 against PCM. However, these studies have not determined whether rPb27 immunization prevents lung fibrosis. We therefore conducted this study to investigate fibrosis resulting from infection by Paracoccidioides brasiliensis in the lungs of animals immunized with rPb27. Animals were immunized with rPb27 and subsequently infected with a virulent strain of P. brasiliensis. Fungal load was evaluated by counting colony-forming units, and Masson's trichrome staining was performed to evaluate fibrosis at 30 and 90 days post-infection. The levels of CCR7, active caspase 3, collagen and cytokines were analyzed. At the two time intervals mentioned, the rPb27 group showed lower levels of fibrosis on histology and reduced levels of collagen and the chemokine receptor CCR7 in the lungs. CCR7 was detected at higher levels in the control groups that developed very high levels of pulmonary fibrosis. Additionally, the immunized groups showed high levels of active caspase 3, IFN-γ, TGF-β and IL-10 in the early phase of P. brasiliensis infection. Immunization with Pb27, in addition to its protective effect, was shown to prevent pulmonary fibrosis.

Topics: Animals; Antifungal Agents; Antigens, Fungal; Caspase 3; Collagen; Fluconazole; Fungal Proteins; Fungal Vaccines; Immunization; Inflammation; Interferon-gamma; Interleukin-10; Lung; Male; Mice; Mice, Inbred BALB C; Paracoccidioides; Paracoccidioidomycosis; Propionibacterium acnes; Pulmonary Fibrosis; Receptors, CCR7; Recombinant Proteins; Transforming Growth Factor beta

Nuclear factor (NF)-κB signaling pathway contributes to the pathogenesis of lung fibrosis. However, the expression and roles of NF-κB p65 subunit in fibroblasts under fibrotic conditions have not been studied. We checked the expression of p65 in lung tissue and fibroblasts from bleomycin (BLM) challenged mice and investigated the roles of p65 in human lung fibroblast differentiation during transforming growth factor β (TGF-β) challenge.. A murine model of BLM-induced lung fibrosis and in vitro cultures of lung fibroblast from human and mouse were used to study the expression and role of p65 during fibrogenesis. The si-RNA knockdown strategy was used to check the mechanism of TGF-β induced p65 expression and roles of p65 during fibroblast differentiation.. We found that the expression of p65 was significantly increased in lung tissue, fibrotic foci and fibroblast from BLM-challenged mice. In vitro, TGF-β stimulated p65 expression, nuclear translocation and cell differentiation in human lung fibroblast; knockdown smad3 expression inhibited TGF-β-induced p65 expression and differentiation in human lung fibroblast. Additionally, knockdown the expression of p65 suppressed TGF-β-induced differentiation and p65 translocation to nuclei in human lung fibroblast.. These data suggested that p65 expression is up-regulated in lung fibroblast during differentiation through TGF-β/smad3 pathway and synergistically regulates TGF-β induced differentiation of lung fibroblasts.

Topics: Animals; Antibiotics, Antineoplastic; Bleomycin; Blotting, Western; Cell Differentiation; Cells, Cultured; Fibroblasts; Fluorescent Antibody Technique; Humans; Lung; Mice; Mice, Inbred C57BL; Pulmonary Fibrosis; Signal Transduction; Smad3 Protein; Transcription Factor RelA; Transforming Growth Factor beta; Up-Regulation

Idiopathic pulmonary fibrosis is a chronic pulmonary disease that is characterized by formation of scar tissue in lungs. Transforming growth factor-β (TGF-β) is considered an important cytokine in the pathogenesis of this disease. Hence, the antifibrotic effect of an inhibitor of the TGF-β type I receptor, namely, SB 431542, was investigated in our study. SB 431542 was used to treat TGF-β-treated IMR-90 cells; the expression of α-smooth muscle actin (α-SMA) was detected at the protein level by using an anti-α-SMA antibody, and at the gene level by reverse transcription-quantitative PCR. The effect of the inhibitor on cell proliferation was determined by a cell growth assay. The inhibitor was also administered into bleomycin-treated mice. Histopathological assessment and determination of total collagen levels were carried out to evaluate the severity of lung fibrosis in these mice. Our results demonstrated that treatment with SB 431542 inhibits TGF-β‑induced α-SMA expression in lung fibroblasts, at both the protein and the mRNA levels (P<0.05). However, the inhibitor did not significantly reduce lung fibroblast proliferation. In the bleomycin-induced pulmonary fibrosis mouse model, bleomycin treatment caused important morphological changes, accompanied by an increase in the collagen level of the lungs. Early treatment with SB 431542 prevented the manifestation of histopathological alterations, whereas delayed treatment significantly decreased the collagen level (P<0.05). These results suggest that inhibition of TGF-β signaling, via inhibition of the activin receptor-like kinase-5 (ALK-5) by SB 431542, may attenuate pulmonary fibrosis.

Topics: Actins; Animals; Benzamides; Cell Line; Cell Proliferation; Cell Survival; Dioxoles; Disease Models, Animal; Female; Fibroblasts; Humans; Hydroxyproline; Mice; Protein Serine-Threonine Kinases; Pulmonary Fibrosis; Receptor, Transforming Growth Factor-beta Type I; Receptors, Transforming Growth Factor beta; Transforming Growth Factor beta

Myofibroblasts have increased expression of contractile proteins and display augmented contractility. It is not known if the augmented contractile gene expression characterizing the myofibroblast phenotype impacts its intrinsic ability to assemble fibronectin (FN) and extracellular matrix. In this study we investigated whether myofibroblasts displayed increased rates of FN fibril assembly when compared with their undifferentiated counterparts. Freshly plated myofibroblasts assemble exogenous FN (488-FN) into a fibrillar matrix more rapidly than fibroblasts that have not undergone myofibroblast differentiation. The augmented rate of FN matrix formation by myofibroblasts was dependent on intact Rho/Rho kinase (ROCK) and myosin signals inasmuch as treatment with Y27632 or blebbistatin attenuated 488-FN assembly. Inhibiting contractile gene expression by pharmacologic disruption of the transcription factors megakaryoblastic leukemia-1 (MKL1)/serum response factor (SRF) during myofibroblast differentiation resulted in decreased contractile force generation and attenuated 488-FN incorporation although not FN expression. Furthermore, disruption of the MKL1/SRF target gene, smooth muscle α-actin (α-SMA) via siRNA knockdown resulted in attenuation of 488-FN assembly. In conclusion, this study demonstrates a linkage between increased contractile gene expression, most importantly α-SMA, and the intrinsic capacity of myofibroblasts to assemble exogenous FN into fibrillar extracellular matrix.

Topics: Actins; Cell Differentiation; Cells, Cultured; Extracellular Matrix; Fibroblasts; Fibronectins; Humans; Myofibroblasts; Pulmonary Fibrosis; Serum Response Factor; Trans-Activators; Transforming Growth Factor beta

Pulmonary fibrosis is a progressive and fatal disease that involves the remodeling of the distal airspace and the lung parenchyma, which results in compromised gas exchange. The median survival time once diagnosed is less than three years. Interleukin (IL)-13 has been shown to play a role in a number of inflammatory and fibrotic diseases. IL-13 modulates its effector functions via a complex receptor system that includes the IL-4 receptor (R) α, IL-13Rα1, and the IL-13Rα2. IL-13Rα1 binds IL-13 with low affinity, yet, when it forms a complex with IL-4α, it binds with much higher affinity, inducing the effector functions of IL-13. IL-13Rα2 binds IL-13 with high affinity but has a short cytoplasmic tail and has been shown to act as a nonsignaling decoy receptor. Transfection of fibroblasts and epithelial cells with IL-13Rα2 inhibited the IL-13 induction of soluble collagen, TGF-β, and CCL17. Adenoviral overexpression of IL-13Rα2 in the lung reduced bleomycin-induced fibrosis. Our work shows that overexpression of IL-13Rα2 inhibits the IL-13 induction of fibrotic markers in vitro and inhibits bleomycin-induced pulmonary fibrosis. In summary our study highlights the antifibrotic nature of IL-13Ra2.

Topics: Animals; Bleomycin; Chemokine CCL17; Collagen; HEK293 Cells; Humans; Interleukin-13; Interleukin-13 Receptor alpha2 Subunit; Mice; Mice, Inbred C57BL; NIH 3T3 Cells; Pulmonary Fibrosis; Transforming Growth Factor beta

Interleukin-27 (IL-27) is a multifunctional cytokine with both pro-inflammatory and immunoregulatory functions. At present, the role of IL-27 in pulmonary fibrosis remains unknown.. In this study, we observed the expression of IL-27/IL-27R in a mouse model of bleomycin (BLM)-induced pulmonary fibrosis. We verified the role of IL-27 using hematoxylin and eosin as well as Masson's staining methods and measuring the content of hydroxyproline as well as collagen I and III. We assessed the differentiation of T lymphocytes in the spleen and measured the concentration of cytokines in bronchoalveolar lavage fluid (BALF) and the expression level of relevant proteins in the JAK/STAT and TGF-ß/Smad signaling pathways in lung tissue.. Increased IL-27 expression in BLM-induced pulmonary fibrosis was noted. IL-27 treatment may alleviate pulmonary fibrosis and increase the survival of mice. IL-27 inhibited the development of CD4(+) IL-17(+), CD4(+) IL-4(+) T, and CD4(+) Foxp3(+) cells and the secretion of IL-17, IL-4, IL-6, and TGF-ß. IL-27 induced the production of CD4(+) IL-10(+) and CD4(+) INF-γ(+) T cells. IL-27 decreased the levels of phosphorylated STAT1, STAT3, STAT5, Smad1, and Smad3 but increased the level of SOCS3.. This study demonstrates that IL-27 potentially attenuates BLM-induced pulmonary fibrosis by regulating Th17 differentiation and cytokine secretion.

Topics: Animals; Antibiotics, Antineoplastic; Bleomycin; Blotting, Western; Bronchoalveolar Lavage Fluid; Cell Differentiation; Cytokines; Enzyme-Linked Immunosorbent Assay; Flow Cytometry; Interleukins; Janus Kinases; Lung; Male; Mice; Mice, Inbred C57BL; Pulmonary Fibrosis; Real-Time Polymerase Chain Reaction; Receptors, Cytokine; Receptors, Interleukin; Reverse Transcriptase Polymerase Chain Reaction; Signal Transduction; Smad Proteins; Spleen; STAT Transcription Factors; Suppressor of Cytokine Signaling 3 Protein; Suppressor of Cytokine Signaling Proteins; T-Lymphocytes; Th17 Cells; Transforming Growth Factor beta

Epithelial to mesenchymal transition (EMT) of alveolar epithelial cells occurs in lung fibrotic diseases. Tanshinone IIA (Tan IIA) has been reported to exert anti-inflammatory effects in pulmonary fibrosis. Nonetheless, whether Tan IIA affects lung fibrosis-related EMT remains unknown and requires for further investigations.. A single intratracheal instillation of saline containing bleomycin (BLM; 5 mg/kg body weight) was performed to induce pulmonary fibrosis in Sprague-Dawley rats. Rats receiving an instillation of equivoluminal normal saline served as controls. Then, these rats were given a daily intraperitoneal administration of Tan IIA (15 mg/kg body weight) for 28 d before sacrifice. In vitro, recombinant transforming growth factor-beta 1 (TGF-β1; 10 ng/mL) was used to treat human alveolar epithelial A549 cells for 48 h. Tan IIA (10 μM) or control DMSO was used to pretreat cells for 2 h before TGF-β1 stimulation. Rat lung tissue samples and A549 cells were then subjected to further assessments.. Tan IIA was noted to alleviate BLM-induced pulmonary collagen deposition and macrophage infiltration in rats. Epithelial-cadherin expression was decreased after BLM stimulation, whereas α-smooth muscle actin, fibronectin, and vimentin were increased. These expression alterations were partially reversed by Tan IIA. Moreover, Tan IIA suppressed BLM-induced increases in TGF-β1, phosphorylated Smad-2, and -3 in rats. Additionally, pretreatment of Tan IIA inhibited TGF-β1-triggered EMT, reduced collagen Ⅰ production, and blocked TGF-β signal transduction in A549 cells.. Our research suggests that Tan IIA mitigates BLM-induced pulmonary fibrosis and suppresses TGF-β-dependent EMT of lung alveolar epithelial cells.

Topics: Abietanes; Animals; Anti-Inflammatory Agents, Non-Steroidal; Biomarkers; Bleomycin; Blotting, Western; Cell Line; Enzyme-Linked Immunosorbent Assay; Epithelial Cells; Epithelial-Mesenchymal Transition; Humans; Pulmonary Alveoli; Pulmonary Fibrosis; Rats; Rats, Sprague-Dawley; Real-Time Polymerase Chain Reaction; Transforming Growth Factor beta; Transforming Growth Factor beta1

It has also been shown that the decreased expression of eukaryotic translation initiation factor 3a (eIF3a) by L-mimosine caused cell cycle arrest. Our previous study has found that eIF3a is involved in bleomycin-induced pulmonary fibrosis. Whether the eIF3a/p27 signal pathway is involved in the inhibitory effect of L-mimosine on bleomycin-induced pulmonary fibrosis remains unknown. Pulmonary fibrosis was induced by intratracheal instillation of bleomycin (5 mg/kg) in rats. Primary pulmonary fibroblasts were cultured to investigate the proliferation by BrdU incorporation method and flow cytometry. The expression of eIF3a, p27, α-SMA, collagen I and collagen III was analyzed by qPCR and Western blot. In vivo, L-mimosine treatment significantly ameliorated the bleomycin-mediated histological fibrosis alterations and blocked collagen deposition concomitantly with reversing bleomycin-induced expression up-regulation of eIF3a, α-SMA, collagen I and collagen III (both mRNA and protein) and expression down- regulation of p27. In vitro, L-mimosine remarkably attenuated proliferation of pulmonary fibroblasts and expression of α-SMA, collagen I and collagen III induced by TGF-β1, and this inhibitory effect of L-mimosine was accompanied by inhibiting eIF3a expression and increasing p27 expression. Knockdown of eIF3a gene expression reversed TGF-β1-induced proliferation of fibroblasts, down-regulation of p27 expression and up-regulation of α-SMA, collagen I, and collagen III expression. These results suggest that L-mimosine inhibited the progression of bleomycin-induced pulmonary fibrosis in rats via the eIF3a/p27 pathway.

Topics: Actins; Animals; Bleomycin; Cell Proliferation; Cells, Cultured; Collagen; Cyclin-Dependent Kinase Inhibitor p27; Eukaryotic Initiation Factor-3; Fibroblasts; Gene Expression Regulation; Humans; Lung; Male; Mimosine; Pulmonary Fibrosis; Rats; Rats, Sprague-Dawley; RNA, Small Interfering; Signal Transduction; Transforming Growth Factor beta

Integrins are transmembrane heterodimeric receptors that contribute to diverse biological functions and play critical roles in many human diseases. Studies using integrin subunit knockout mice and inhibitory antibodies have identified important roles for nearly every integrin heterodimer and led to the development of a number of potentially useful therapeutics. One notable exception is the αvβ1 integrin. αv and β1 subunits are individually present in numerous dimer pairs, making it challenging to infer specific roles for αvβ1 by genetic inactivation of individual subunits, and αvβ1 complex-specific blocking antibodies do not yet exist. We therefore developed a potent and highly specific small-molecule inhibitor of αvβ1 to probe the function of this understudied integrin. We found that αvβ1, which is highly expressed on activated fibroblasts, directly binds to the latency-associated peptide of transforming growth factor-β1 (TGFβ1) and mediates TGFβ1 activation. Therapeutic delivery of this αvβ1 inhibitor attenuated bleomycin-induced pulmonary fibrosis and carbon tetrachloride-induced liver fibrosis, suggesting that drugs based on this lead compound could be broadly useful for treatment of diseases characterized by excessive tissue fibrosis.

Topics: Animals; Bleomycin; Cells, Cultured; Chemical and Drug Induced Liver Injury; Drug Design; Fibroblasts; Hydrocarbons, Brominated; Liver; Liver Cirrhosis, Experimental; Lung; Mice; Mice, Inbred C57BL; Molecular Targeted Therapy; Pulmonary Fibrosis; Receptors, Vitronectin; Signal Transduction; Transforming Growth Factor beta

Aliskiren is a drug classified as a direct renin inhibitor. The renin-angiotensin system plays an important role in pulmonary fibrogeneses. This study aimed to investigate the impact of aliskiren on pulmonary fibrosis induced by bleomycin. Forty adult mice were divided into group I (control), group II (aliskiren 25mg/kg/day IP), group III (bleomycin 0.035U/g intraperitoneally twice weekly for 4 weeks) and group IV (aliskiren+bleomycin). Plasma renin activity (PRA), lung content of hydroxyproline and transforming growth factor-β1 (TGF-β1) were assayed. Lung paraffin sections were prepared for histological study and immunohistochemical detection of alpha smooth muscle actin (αSMA) as a marker for myofibroblasts activation and differentiation. Bleomycin induced a significant elevation of PRA with a significant increase in hydroxyproline and TGF-β1 in group III. Microscopically, pulmonary fibrosis was evident in the form of areas of collapsed alveoli, intense inflammatory cells infiltrations, excess accumulation of collagen, and excessively encountered αSMA positively immune-stained myofibroblasts, compared to a negative immune-reaction in groups I and II. In group IV, aliskiren resulted in a significant decrease in PRA, TGF-β1 and hydroxyproline, with an attenuation of pulmonary fibrosis and a decrease in αSMA positively immune-stained myofibroblasts. In conclusion, renin inhibition by aliskiren attenuated pulmonary fibrosis through decreasing TGF-β1 and myofibroblasts activation and differentiation.

Topics: Actins; Amides; Animals; Bleomycin; Cell Differentiation; Fumarates; Mice; Myofibroblasts; Pulmonary Fibrosis; Renin; Renin-Angiotensin System; Transforming Growth Factor beta

Interstitial lung disease accounts for a group of chronic and progressive disorders associated with severe pulmonary vascular remodelling, peripheral vascular rarefaction and fibrosis, thus limiting lung function. We have previously shown that Akt is necessary for myofibroblast differentiation, a critical event in organ fibrosis. However, the contributory role of the Akt-mTOR pathway in interstitial lung disease and the therapeutic benefits of targeting Akt and mTOR remain unclear.. We investigated the role of the Akt-mTOR pathway and its downstream molecular mechanisms in chronic hypoxia- and TGFβ-induced pulmonary vascular pruning and fibrosis in mice. We also determined the therapeutic benefits of the Akt inhibitor triciribine and the mTOR inhibitor rapamycin for the treatment of pulmonary fibrosis in mice.. Akt1(-) (/) (-) mice were protected from chronic hypoxia-induced peripheral vascular pruning. In contrast, hyperactivation of Akt1 induced focal fibrosis similar to TGFβ-induced fibrosis. Pharmacological inhibition of Akt, but not the Akt substrate mTOR, inhibited hypoxia- and TGFβ-induced pulmonary vascular rarefaction and fibrosis. Mechanistically, we found that Akt1 modulates pulmonary remodelling via regulation of thrombospondin1 (TSP1) expression. Hypoxic Akt1(-) (/) (-) mice lungs expressed less TSP1. Moreover, TSP1(-) (/) (-) mice were resistant to adMyrAkt1-induced pulmonary fibrosis.. Our study identified Akt1 as a novel target for the treatment of interstitial lung disease and provides preclinical data on the potential benefits of the Akt inhibitor triciribine for the treatment of interstitial lung disease.

Topics: Animals; Cells, Cultured; Fibroblasts; Humans; Hypoxia; Lung; Male; Mice, Knockout; Proto-Oncogene Proteins c-akt; Pulmonary Fibrosis; Ribonucleosides; Thrombospondin 1; Transforming Growth Factor beta

Thymosin β4 (Tβ4) and its amino-terminal fragment comprising N-acetyl-seryl-aspartyl-lysyl-proline (Ac-SDKP) have been reported to act as anti-inflammatory and anti-fibrotic agents in vitro and in vivo. In recent papers, we have shown that Tβ4 exerts a widely protective role in mice treated with bleomycin, and in particular, we have demonstrated its inhibitory effects on both inflammation and early fibrosis.. In this study, the putative anti-proliferative and anti-fibrogenic effects of Tβ4 and Ac-SDKP were evaluated in vitro. In addition, the effects of Tβ4 up to 21 days were evaluated in the bleomycin mouse model of lung fibrosis.. We utilized both control and TGF-β-stimulated primary human lung fibroblasts isolated from both idiopathic pulmonary fibrosis (IPF) and control tissues. The in vivo effects of Tβ4 were assessed in CD1 mice treated with bleomycin.. In the in vitro experiments, we observed significant anti-proliferative effects of Ac-SDKP in IPF fibroblasts. In those cells, Ac-SDKP significantly inhibited TGF-β-induced α-SMA and collagen expression, hallmarks of fibroblast differentiation into myofibroblasts triggered by TGF-β. In vivo, despite its previously described protective role in mice treated with bleomycin at 7 days, Tβ4 failed to prevent fibrosis induced by the drug at 14 and 21 days.. We conclude that, compared to Tβ4, Ac-SDKP may have greater potential as an anti-fibrotic agent in the lung. Further in vivo experiments are warranted.

Topics: Animals; Anti-Inflammatory Agents; Bleomycin; Cells, Cultured; Disease Models, Animal; Fibroblasts; Humans; Lung; Male; Mice; Oligopeptides; Pneumonia; Protein Structure, Tertiary; Pulmonary Fibrosis; Thymosin; Transforming Growth Factor beta

To examine the effects and possible mechanism of the immunosuppressant agent cyclophosphamide (CP) combined with bone marrow mesenchymal stromal cells (BM-MSCs) on bleomycin induced lung fibrosis in mice.. BM-MSCs labeled with red fluorescence protein (RFP) from male Friend virus B-type(FVB) mice were cultured in vitro. Pulmonary fibrosis(PF) was induced in female wild type FVB mice and mice were randomly divided into five groups: control, model, CP, BM-MSCs, and BM-MSCs+CP. Pathological changes and distribution of RFP (+) BMSC in lung tissue were observed and hydroxyproline (Hyp) content in the lungs was measured. Changes in TGF-β mRNA, PDGF mRNA, and SDF-1mRNA expression in lung tissue were measured.. PF and Hyp levels in the BM-MSCs and BM-MSCs+CP groups were significantly alleviated (p<0.01) compared to the model group. The RFP (+) cells were distributed in the periphery of the alveolar space and endomembrane of bronchus. Hyp levels were reduced in the BM-MSCs+CP group compared to the BM-MSCs group (p<0.05). TGF-β and PDGF levels in the BM-MSCs and BM-MSCs+CP groups were higher than in the control or model group (p<0.05). SDF-1 level in the CP group showed no significant differences compared to the control group, in the other groups were higher than in the control group (p<0.05) and in the BM-MSCs+CP group was lower than in the BM-MSCs group (p<0.05).. It was concluded that CP alone does not improve PF and may be harmful. In contrast, combined application of BM-MSCs with CP provided better protection against PF and may serve as an effective therapy.

Topics: Animals; Biological Assay; Bleomycin; Cell Differentiation; Chemokine CXCL12; Collagen; Combined Modality Therapy; Cyclophosphamide; Female; Gene Expression Regulation; Hydroxyproline; Immunohistochemistry; Lung; Male; Mesenchymal Stem Cell Transplantation; Mesenchymal Stem Cells; Mice; Platelet-Derived Growth Factor; Pulmonary Fibrosis; RNA, Messenger; Transforming Growth Factor beta

Interstitial lung disease (ILD) characterized by pulmonary fibrosis and inflammation poses a substantial biomedical challenge due to often negative disease outcomes combined with the need to develop better, more effective therapies. We assessed the in vivo effect of administration of a pharmacological inhibitor of S-nitrosoglutathione reductase, SPL-334 (4-{[2-[(2-cyanobenzyl)thio]-4-oxothieno[3,2-d]pyrimidin-3(4H)-yl]methyl}benzoic acid), in a mouse model of ILD induced by intratracheal instillation of bleomycin (BLM). Daily i.p. administration of SPL-334 alone at 0.3, 1.0, or 3.0 mg/kg had no effect on animal body weight, appearance, behavior, total and differential bronchoalveolar lavage (BAL) cell counts, or collagen accumulation in the lungs, showing no toxicity of our investigational compound. Similar administration of SPL-334 for 7 days before and for an additional 14 days after BLM instillation resulted in a preventive protective effect on the BLM challenge-induced decline in total body weight and changes in total and differential BAL cellularity. In the therapeutic treatment regimen, SPL-334 was administered at days 7-21 after BLM challenge. Such treatment attenuated the BLM challenge-induced decline in total body weight, changes in total and differential BAL cellularity, and magnitudes of histologic changes and collagen accumulation in the lungs. These changes were accompanied by an attenuation of BLM-induced elevations in pulmonary levels of profibrotic cytokines interleukin-6, monocyte chemoattractant protein-1, and transforming growth factor-β (TGF-β). Experiments in cell cultures of primary normal human lung fibroblast have demonstrated attenuation of TGF-β-induced upregulation in collagen by SPL-334. It was concluded that SPL-334 is a potential therapeutic agent for ILD.

Topics: Aldehyde Oxidoreductases; Animals; Benzoates; Benzoic Acid; Bleomycin; Collagen; Enzyme Inhibitors; Fibroblasts; Humans; Lung; Mice; Pneumonia; Pulmonary Fibrosis; Pyrimidinones; Transforming Growth Factor beta

Pulmonary fibrosis is a common and dose-limiting side-effect of ionizing radiation used to treat cancers of the thoracic region. Few effective therapies are available for this disease. Pulmonary fibrosis is characterized by an accumulation of myofibroblasts and excess deposition of extracellular matrix proteins. Although prior studies have reported that ionizing radiation induces fibroblast to myofibroblast differentiation and collagen production, the mechanism remains unclear. Transforming growth factor-β (TGF-β) is a key profibrotic cytokine that drives myofibroblast differentiation and extracellular matrix production. However, its activation and precise role in radiation-induced fibrosis are poorly understood. Recently, we reported that lactate activates latent TGF-β through a pH-dependent mechanism. Here, we wanted to test the hypothesis that ionizing radiation leads to excessive lactate production via expression of the enzyme lactate dehydrogenase-A (LDHA) to promote myofibroblast differentiation. We found that LDHA expression is increased in human and animal lung tissue exposed to ionizing radiation. We demonstrate that ionizing radiation induces LDHA, lactate production, and extracellular acidification in primary human lung fibroblasts in a dose-dependent manner. We also demonstrate that genetic and pharmacologic inhibition of LDHA protects against radiation-induced myofibroblast differentiation. Furthermore, LDHA inhibition protects from radiation-induced activation of TGF-β. We propose a profibrotic feed forward loop, in which radiation induces LDHA expression and lactate production, which can lead to further activation of TGF-β to drive the fibrotic process. These studies support the concept of LDHA as an important therapeutic target in radiation-induced pulmonary fibrosis.

Topics: Animals; Cell Differentiation; Cells, Cultured; Enzyme Inhibitors; Gossypol; Humans; Isoenzymes; L-Lactate Dehydrogenase; Lactate Dehydrogenase 5; Lactic Acid; Lung; Mice; Mice, Inbred C57BL; Models, Biological; Myofibroblasts; Pulmonary Fibrosis; Radiation Injuries; Transforming Growth Factor beta

Idiopathic pulmonary fibrosis (IPF) is characterised by accumulation of fibroblasts and myofibroblasts and deposition of extracellular matrix proteins. Sphingosine-1-phosphate (S1P) signalling plays a critical role in pulmonary fibrosis.. S1P lyase (S1PL) expression in peripheral blood mononuclear cells (PBMCs) was correlated with pulmonary functions and overall survival; used a murine model to check the role of S1PL on the fibrogenesis and a cell culture system to study the effect of S1PL expression on transforming growth factor (TGF)-β- and S1P-induced fibroblast differentiation.. S1PL expression was upregulated in fibrotic lung tissues and primary lung fibroblasts isolated from patients with IPF and bleomycin-challenged mice. TGF-β increased the expression of S1PL in human lung fibroblasts via activation and binding of Smad3 transcription factor to Sgpl1 promoter. Overexpression of S1PL attenuated TGF-β-induced and S1P-induced differentiation of human lung fibroblasts through regulation of the expression of LC3 and beclin 1. Knockdown of S1PL (Sgpl1(+/-)) in mice augmented bleomycin-induced pulmonary fibrosis, and patients with IPF reduced Sgpl1 mRNA expression in PBMCs exhibited higher severity of fibrosis and lower survival rate.. These studies suggest that S1PL is a novel endogenous suppressor of pulmonary fibrosis in human IPF and animal models.

Topics: Aldehyde-Lyases; Animals; Autophagy; Cell Differentiation; Disease Models, Animal; Fibroblasts; Humans; Immunohistochemistry; Leukocytes, Mononuclear; Lung; Mice; Pulmonary Fibrosis; Signal Transduction; Smad Proteins; Transforming Growth Factor beta; Up-Regulation

Pulmonary fibrosis is a progressive disease with only few treatment options available at the moment. Recently, the nucleoside uridine has been shown to exert anti-inflammatory effects in different animal models, e.g. in acute lung injury or bronchial asthma.. Therefore, we investigated the influence of uridine supplementation on inflammation and fibrosis in the classical bleomycin model. Male C57BL/6 mice received an intratracheal injection of bleomycin on day 0 and were treated intraperitoneally with uridine or vehicle. The degree of inflammation and fibrosis was assessed at different time points.. Uridine administration resulted in attenuated inflammation, as demonstrated by reduced leukocytes and pro-inflammatory cytokines in the broncho-alveolar lavage (BAL) fluid. Furthermore, collagen deposition in the lung interstitium was also reduced by uridine supplementation. Similar results were obtained in a model in which animals received repeated intraperitoneal bleomycin injections. In addition uridine inhibited collagen and TGF-ß synthesis by primary lung fibroblasts, the release of pro-inflammatory cytokines by human lung epithelial cells, as well as the production of reactive oxygen species by human neutrophils.. In summary, we were able to show that uridine has potent anti-inflammatory and anti-fibrotic properties. As uridine supplementation has been shown to be well tolerated and safe in humans, this might be a new therapeutic approach for the treatment of fibrotic lung diseases.

Topics: Animals; Anti-Inflammatory Agents; Bleomycin; Bronchoalveolar Lavage Fluid; Cell Line, Tumor; Collagen; Cytokines; Disease Models, Animal; Epithelial Cells; Fibroblasts; Humans; Inflammation Mediators; Leukocytes; Lung; Male; Mice, Inbred C57BL; Pneumonia; Pulmonary Fibrosis; Reactive Oxygen Species; Time Factors; Transforming Growth Factor beta; Uridine

Myofibroblasts are one of the primary cell types responsible for the accumulation of extracellular matrix in fibrosing diseases, and targeting myofibroblast differentiation is an important therapeutic strategy for the treatment of pulmonary fibrosis. Transforming growth factor-β (TGF-β) has been shown to be an important inducer of myofibroblast differentiation. We previously demonstrated that lactate dehydrogenase and its metabolic product lactic acid are important mediators of myofibroblast differentiation, via acid-induced activation of latent TGF-β. Here we explore whether pharmacologic inhibition of LDH activity can prevent TGF-β-induced myofibroblast differentiation. Primary human lung fibroblasts from healthy patients and those with pulmonary fibrosis were treated with TGF-β and or gossypol, an LDH inhibitor. Protein and RNA were analyzed for markers of myofibroblast differentiation and extracellular matrix generation. Gossypol inhibited TGF-β-induced expression of the myofibroblast marker α-smooth muscle actin (α-SMA) in a dose-dependent manner in both healthy and fibrotic human lung fibroblasts. Gossypol also inhibited expression of collagen 1, collagen 3, and fibronectin. Gossypol inhibited LDH activity, the generation of extracellular lactic acid, and the rate of extracellular acidification in a dose-dependent manner. Furthermore, gossypol inhibited TGF-β bioactivity in a dose-dependent manner. Concurrent treatment with an LDH siRNA increased the ability of gossypol to inhibit TGF-β-induced myofibroblast differentiation. Gossypol inhibits TGF-β-induced myofibroblast differentiation through inhibition of LDH, inhibition of extracellular accumulation of lactic acid, and inhibition of TGF-β bioactivity. These data support the hypothesis that pharmacologic inhibition of LDH may play an important role in the treatment of pulmonary fibrosis.

Topics: Animals; Cell Differentiation; Cell Line; Enzyme Inhibitors; Extracellular Matrix; Gene Knockdown Techniques; Gossypol; Humans; L-Lactate Dehydrogenase; Lactic Acid; Lung; Mink; Myofibroblasts; Pulmonary Fibrosis; Tissue Donors; Transforming Growth Factor beta

Interstitial lung disease (ILD) is a serious side-effect of epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitor (TKI) treatment. Therefore, it is necessary to study underlying mechanisms for the development of pulmonary fibrosis induced by EGFR-TKI and potential approaches to attenuate it. Metformin is a well-established and widely prescribed oral hypoglycemic drug, and has gained attention for its potential anticancer effects. Recent reports have also demonstrated its role in inhibiting epithelial-mesenchymal transition and fibrosis. However, it is unknown whether metformin attenuates EGFR-TKI-induced pulmonary fibrosis. The effect of metformin on EGFR-TKI-induced exacerbation of pulmonary fibrosis was examined in vitro and in vivo using MTT, Ki67 incorporation assay, flow cytometry, immunostaining, Western blot analysis, and a bleomycin-induced pulmonary fibrosis rat model. We found that in lung HFL-1 fibroblast cells, TGF-β or conditioned medium from TKI-treated lung cancer PC-9 cells or conditioned medium from TKI-resistant PC-9GR cells, induced significant fibrosis, as shown by increased expression of Collegen1a1 and α-actin, while metformin inhibited expression of fibrosis markers. Moreover, metformin decreased activation of TGF-β signaling as shown by decreased expression of pSMAD2 and pSMAD3. In vivo, oral administration of gefitinib exacerbated bleomycin-induced pulmonary fibrosis in rats, as demonstrated by HE staining and Masson staining. Significantly, oral co-administration of metformin suppressed exacerbation of bleomycin-induced pulmonary fibrosis by gefitinib. We have shown that metformin attenuates gefitinib-induced exacerbation of TGF-β or bleomycin-induced pulmonary fibrosis. These observations indicate metformin may be combined with EGFR-TKI to treat NSCLC patients.

Topics: Animals; Antineoplastic Agents; Bleomycin; Blotting, Western; Epithelial-Mesenchymal Transition; Flow Cytometry; Gefitinib; Humans; Immunohistochemistry; Lung Diseases, Interstitial; Male; Metformin; Pulmonary Fibrosis; Quinazolines; Rats; Rats, Sprague-Dawley; Signal Transduction; Transforming Growth Factor beta

Persistent myofibroblast differentiation is a hallmark of fibrotic diseases. Myofibroblasts are characterized by de novo expression of alpha smooth muscle actin (αSMA) and excess fibronectin assembly. Recent studies provide conflicting reports on the effects of tyrosine kinase inhibitor dasatinib on myofibroblast differentiation and fibrosis. Also, it is not fully understood whether dasatinib modulates myofibroblast differentiation by targeting Src kinase. Herein, we investigated the effect of dasatinib on cSrc and transforming growth factor-β (TGFβ)-induced myofibroblast differentiation in vitro. Our results indicated that selective Src kinase inhibition using PP2 mimicked the effect of dasatinib in attenuating myofibroblast differentiation as evident by blunted αSMA expression and modest, but significant inhibition of fibronectin assembly in both NIH 3T3 and fibrotic human lung fibroblasts. Mechanistically, our data showed that dasatinib modulates αSMA synthesis through Src kinase-mediated modulation of serum response factor expression. Collectively, our results demonstrate that dasatinib modulates myofibroblast differentiation through Src-SRF pathway. Thus, dasatinib could potentially be a therapeutic option in fibrotic diseases.

Topics: Actins; Animals; Cell Differentiation; Cell Line; Dasatinib; Fibronectins; Gene Expression Regulation; Humans; Lung; Mice; Myofibroblasts; Pulmonary Fibrosis; Serum Response Factor; Signal Transduction; src-Family Kinases; Transforming Growth Factor beta

Secretoglobin (SCGB) 3A2 is a novel lung-enriched cytokine, previously shown to exhibit anti-inflammatory, growth factor, and anti-fibrotic activities. The latter activity was demonstrated using exogenously-administered recombinant SCGB3A2 in the bleomycin (BLM)-induced pulmonary fibrosis model. Whether SCGB3A2 exhibits anti-fibrotic activity in vivo is not known.. Mice null for the Scgb3a2 gene were subjected to the BLM-induced pulmonary fibrosis model, and the severity of pulmonary fibrosis determined using histological and biochemical methods.. BLM treatment caused weight loss of both Scgb3a2-null and wild-type mice, however, the loss was far more pronounced in BLM-treated Scgb3a2-null than wild-type mice, and the weight of day 21 of BLM-treated Scgb3a2-null mice was about half of that of BLM-treated wild-type mice. Hematoxylin & Eosin, Masson Trichrome, and Sirius Red staining of lung sections, Ashcroft fibrosis scores, hydroxyproline contents, and the levels of mRNAs encoding various collagens demonstrated that BLM-treated Scgb3a2-null mouse lungs had more severe fibrosis than those of wild-type mouse lungs. Total and differential inflammatory cell numbers in bronchoalveolar lavage fluids, and levels of lung mRNAs including those encoding Th2 cytokines such as IL-4 and profibrotic cytokines such as TGFβ were higher in BLM-treated Scgb3a2-null mouse lungs as compared to those of wild-type mouse lungs. In contrast, mRNAs encoding surfactant proteins A, B, C, and D, and SCGB1A1 did not differ between BLM-treated Scgb3a2-null and wild-type mouse lungs.. The role of SCGB3A2 in fibrosis was revisited using Scgb3a2-null mice and littermate controls in the BLM-induced pulmonary fibrosis model. The pulmonary fibrosis in the Scgb3a2-null mice was more severe than the wild-type controls, thus establishing that SCGB3A2 has anti-fibrotic activity in vivo. Importantly, surfactant proteins and SCGB1A1 appear not to be involved in the susceptibility of Scgb3a2-null mice to BLM-induced pulmonary fibrosis.

Topics: Animals; Bleomycin; Bronchoalveolar Lavage Fluid; Collagen; Collectins; Cytokines; Disease Models, Animal; Hydroxyproline; Inflammation; Interleukin-4; Lung; Mice; Mice, Inbred C57BL; Mice, Knockout; Pulmonary Fibrosis; Real-Time Polymerase Chain Reaction; RNA, Messenger; Secretoglobins; Severity of Illness Index; Transforming Growth Factor beta

To investigate the effects of total flavonoids from astragalus complanatus (FAC) on paraquat poisoning-induced pulmonary fibrosis in rats.. The rats were divided into six groups randomly: control group, paraquat group, prednisolone group and FAC low-dose, middle-dose, high-dose group. Pulmonary fibrosis model was replicated by intratracheal injection of paraquat. In the mext day,the rats were treated by intragastric administration once a day. After 28 days, the rats were sacrificed. The lung index and the levels of HYP and T-AOC were measured, and the pathologic changes of the lung tissue were obtained by HE staining. The levels of TGF-β, Smad2, α-SMA protein were analyzed by Western blot.. FAC improved the activity of T-AOC in serum and reduced pulmonary index and the content of HYP as well (P<0.05 or P<0.01), the alveolitis and fibrosis extent were attenuated. The expression of Smad2 significantly decreased in groups of FAC low-dose, middle-dose and high-dose (0.31±0.11, 0.45±0.12 and 0.30±0.05) as compared with that of the PQ group (0.85±0.34) (P<0.05). The expression of α-SMA significantly decreased in groups of FAC low-dose, middle-dose and high-dose (0.31±0.11, 0.35±0.07 and 0.32±0.10) as compared with that of the PQ group (0.45±0.08) (P<0.05). The expression of TGF-β significantly decreased in groups of FAC low-dose, middle-dose and high-dose (0.35±0.04, 0.27±0.05 and 0.18±0.04)as compared with that of the PQ group (0.63±0.11) (P<0.05).. FAC can alleviate PQ-induced pulmonary fibrosis in rats through inhibiting TGF-β/Smad signaling pathway.

Topics: Actins; Animals; Astragalus Plant; Flavonoids; Lung; Paraquat; Phytochemicals; Pulmonary Fibrosis; Rats; Smad2 Protein; Transforming Growth Factor beta

Interstitial lung diseases (ILDs) are characterized by injury, inflammation, and scarring of alveoli, leading to impaired function. The etiology of idiopathic forms of ILD is not understood, making them particularly difficult to study due to the lack of appropriate animal models. Consequently, few effective therapies have emerged. We developed an inbred mouse model of ILD using vanadium pentoxide (V2O5), the most common form of a transition metal found in cigarette smoke, fuel ash, mineral ores, and steel alloys. Pulmonary responses to V2O5, including dose-dependent increases in lung permeability, inflammation, collagen content, and dysfunction, were significantly greater in DBA/2J mice compared to C57BL/6J mice. Inflammatory and fibrotic responses persisted for 4 mo in DBA/2J mice, while limited responses in C57BL/6J mice resolved. We investigated the genetic basis for differential responses through genetic mapping of V2O5-induced lung collagen content in BXD recombinant inbred (RI) strains and identified significant linkage on chromosome 4 with candidate genes that associate with V2O5-induced collagen content across the RI strains. Results suggest that V2O5 may induce pulmonary fibrosis through mechanisms distinct from those in other models of pulmonary fibrosis. These findings should further advance our understanding of mechanisms involved in ILD and thereby aid in identification of new therapeutic targets.

Topics: Animals; Bronchoalveolar Lavage Fluid; Dose-Response Relationship, Drug; Enzyme-Linked Immunosorbent Assay; Genetic Predisposition to Disease; Male; Mice; Mice, Inbred C57BL; Mice, Inbred DBA; Pulmonary Fibrosis; Real-Time Polymerase Chain Reaction; Transforming Growth Factor beta; Vanadium Compounds

MicroRNAs are small noncoding RNAs that inhibit protein expression. We have previously shown that the inhibition of the microRNA let-7d in epithelial cells caused changes consistent with epithelial-to-mesenchymal transition (EMT) both in vitro and in vivo. The aim of this study was to determine whether the introduction of let-7d into fibroblasts alters their mesenchymal properties. Transfection of primary fibroblasts with let-7d caused a decrease in expression of the mesenchymal markers α-smooth muscle actin, N-cadherin, fibroblast-specific protein-1, and fibronectin, as well as an increase in the epithelial markers tight junction protein-1 and keratin 19. Phenotypic changes were also present, including a delay in wound healing, reduced motility, and proliferation of fibroblasts following transfection. In addition, we examined the effects of transfection on fibroblast responsiveness to TGF-β, an important factor in many fibrotic processes such as lung fibrosis and found that let-7d transfection significantly attenuated high-mobility group-A2 protein induction by TGF-β. Our results indicate that administration of the epithelial microRNA let-7d can significantly alter the phenotype of primary fibroblasts.

Topics: Actins; Cadherins; Calcium-Binding Proteins; Cell Movement; Cell Proliferation; Cells, Cultured; Epithelial-Mesenchymal Transition; Fibroblasts; Fibronectins; HMGA2 Protein; HMGB2 Protein; Humans; Idiopathic Pulmonary Fibrosis; Keratin-19; Lung; MicroRNAs; Myofibroblasts; Pulmonary Alveoli; Pulmonary Fibrosis; S100 Calcium-Binding Protein A4; Snail Family Transcription Factors; Transcription Factors; Transfection; Transforming Growth Factor beta; Wound Healing; Zonula Occludens-1 Protein

Idiopathic pulmonary fibrosis has been associated with the reactivation of developmental pathways, notably the Hedgehog-Glioma-associated oncogene homolog (GLI) pathway. In this study, we determined whether the Hedgehog pathway was activated in bleomycin-induced lung injury in mice, and whether targeting the Hedgehog-Gli pathway could decrease bleomycin-induced lung fibrosis. After intratracheal injection of bleomycin on Day 0, C57Bl6 mice received GDC-0449 (an inhibitor of Smoothened, the transducer of the pathway), or 2,2'-[[Dihydro-2-(4-pyridinyl)-1,3(2H,4H)-pyrimidinediyl]bis(methylene)]bis[N,N dimethylbenzenamine (GANT61; an inhibitor of GLI transcription factors in the nucleus), from Day 7 to Day 13. At Day 14, whole-lung homogenates were obtained for morphological analysis, assessment of cell apoptosis and proliferation, collagen quantification, and evaluation of profibrotic (transforming growth factor-β, connective tissue growth factor, plasminogen activator inhibitor 1, vascular endothelial growth factor-A) and proinflammatory mediators (IL-1β) expression. We showed that the Hedgehog pathway was activated in bleomycin-induced lung fibrosis on Day 14 after injury, with an increased lung expression of the ligand, Sonic Hedgehog, and with increased messenger RNA expression and nuclear localization of GLI1 and GLI2. Inhibition of Smoothened with GDC-0449 did not influence the development of bleomycin-induced lung fibrosis. By contrast, the inhibition of GLI activity with GANT61 decreased lung fibrosis and lung collagen accumulation, and promoted an antifibrotic and anti-inflammatory environment. Our results identify the hedgehog-Gli pathway as a profibrotic pathway in experimental fibrosis. Inhibition of the Hedgehog-Gli pathway at the level of GLI transcriptional activity could be a therapeutic option in fibrotic lung diseases.

Topics: Anilides; Animals; Antibiotics, Antineoplastic; Apoptosis; Bleomycin; Blotting, Western; Cell Proliferation; Collagen; Fluorescent Antibody Technique; Glioma; Hedgehog Proteins; Immunoenzyme Techniques; Kruppel-Like Transcription Factors; Male; Mice; Mice, Inbred C57BL; Pulmonary Fibrosis; Pyridines; Pyrimidines; Real-Time Polymerase Chain Reaction; Receptors, G-Protein-Coupled; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Smoothened Receptor; Transforming Growth Factor beta; Zinc Finger Protein GLI1

The airway epithelium serves as a biological barrier essential for host defense against inhaled pollutants. While chronic epithelial injury, commonly associated with chronic obstructive pulmonary disease and bronchiolitis obliterans syndrome, often results in airway fibrosis, limited animal models of airway fibrosis have been established. Club cells (Clara cells) in the small airways represent an important population of epithelial progenitor cells and also the principal site of localization of the cytochrome P-450 monooxygenase system, which metabolically activates xenobiotic chemicals such as naphthalene by converting them to toxic epoxide intermediates. We hypothesized that repeated exposure to naphthalene may cause prolonged loss of club cells, triggering aberrant local epithelial repair mechanisms that lead to peribronchial fibrosis. We administered intraperitoneal injections of naphthalene to C57/BL6J mice once a week for 14 consecutive weeks. Repeated club cell injury caused by naphthalene triggered regional hyperproliferation of epithelial progenitor cells, while other regions remained denuded or squamated, resulting in fibroblast proliferation and peribronchial collagen deposition associated with upregulation of the fibrogenic cytokines transforming growth factor-β and connective tissue growth factor. The total collagen content of the lung assessed by measurement of the hydroxyproline content was also increased after repeated exposure to naphthalene. These results lend support to the relevance of repeated injury of airway epithelial cells as a trigger for resting fibroblast proliferation and airway fibrosis. This model of airway fibrosis is simple and easy to reproduce, and may be expected to advance our understanding of the pathogenesis and potential treatment of airway fibrotic disorders.

Topics: Animals; Cell Proliferation; Connective Tissue Growth Factor; Disease Models, Animal; Dose-Response Relationship, Drug; Environmental Pollutants; Fibroblasts; Injections, Intraperitoneal; Male; Mice; Mice, Inbred C57BL; Naphthalenes; Pulmonary Fibrosis; Respiratory Mucosa; Transforming Growth Factor beta

Fibrogenesis involves a pathological accumulation of activated fibroblasts and extensive matrix remodeling. Profibrotic cytokines, such as TGF-β, stimulate fibroblasts to overexpress fibrotic matrix proteins and induce further expression of profibrotic cytokines, resulting in progressive fibrosis. Connective tissue growth factor (CTGF) is a profibrotic cytokine that is indicative of fibroblast activation. Epithelial cells are abundant in the normal lung, but their contribution to fibrogenesis remains poorly defined. Profibrotic cytokines may activate epithelial cells with protein expression and functions that overlap with the functions of active fibroblasts. We found that alveolar epithelial cells undergoing TGF-β-mediated mesenchymal transition in vitro were also capable of activating lung fibroblasts through production of CTGF. Alveolar epithelial cell expression of CTGF was dramatically reduced by inhibition of Rho signaling. CTGF reporter mice demonstrated increased CTGF promoter activity by lung epithelial cells acutely after bleomycin in vivo. Furthermore, mice with lung epithelial cell-specific deletion of CTGF had an attenuated fibrotic response to bleomycin. These studies provide direct evidence that epithelial cell activation initiates a cycle of fibrogenic effector cell activation during progressive fibrosis. Therapy targeted at epithelial cell production of CTGF offers a novel pathway for abrogating this progressive cycle and limiting tissue fibrosis.

Topics: Animals; Antibiotics, Antineoplastic; Autocrine Communication; Bleomycin; Blotting, Western; Bronchoalveolar Lavage; Connective Tissue Growth Factor; Culture Media, Conditioned; Enzyme-Linked Immunosorbent Assay; Epithelial Cells; Extracellular Matrix Proteins; Fluorescent Antibody Technique; Hydroxyproline; Mice; Mice, Inbred C57BL; Mice, Knockout; Paracrine Communication; Pulmonary Alveoli; Pulmonary Fibrosis; Real-Time Polymerase Chain Reaction; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; RNA, Small Interfering; Transforming Growth Factor beta

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

Topics: Animals; Anti-Bacterial Agents; Antibodies, Neutralizing; Antigens, Neoplasm; Bronchoalveolar Lavage; Collagen; Doxycycline; Enzyme-Linked Immunosorbent Assay; Female; Humans; Immunoenzyme Techniques; Integrins; Male; Mice; Mice, Transgenic; Pulmonary Fibrosis; Real-Time Polymerase Chain Reaction; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Transforming Growth Factor alpha; Transforming Growth Factor beta; Uteroglobin

Pulmonary fibrosis is a lung disease wherein lung parenchyma is gradually and irreversibly replaced with collagen. The molecular pathogenesis of pulmonary fibrosis is not fully understood and the only effective treatment available is lung transplantation. To test if Del-1, an endogenous anti-inflammatory molecule, may be implicated in the development of pulmonary fibrosis, we induced pulmonary fibrosis in wild type (WT) and Del-1(-/-) mice by intratracheal administration of bleomycin. Del-1 expression in the lung was decreased in the WT mice treated with bleomycin compared to control mice. In addition, bleomycin-induced pulmonary fibrosis increased collagen deposition and TGF-β production in the lung of Del-1(-/-) mice. Finally, Del-1(-/-) mice treated with bleomycin displayed higher weight loss and greater mortality than did WT mice identically treated. These findings suggest that Del-1 may negatively regulate development of pulmonary fibrosis. Further delineation of a role for Del-1 in the development of pulmonary fibrosis will broaden our understanding of the molecular pathogenesis of this disease and hopefully help develop potential therapeutics.

Topics: Animals; Antibiotics, Antineoplastic; Bleomycin; Calcium-Binding Proteins; Carrier Proteins; Cell Adhesion Molecules; Collagen; Gene Deletion; Intercellular Signaling Peptides and Proteins; Lung; Male; Mice; Mice, Inbred C57BL; Pulmonary Fibrosis; Transforming Growth Factor beta

Systemic sclerosis (SSc)-related interstitial lung disease (ILD) is one of the leading causes of mortality. We undertook this study to analyze the gene expression of lung tissue in a prospective cohort of patients with SSc-related ILD and to compare it with that in control lungs and with 2 prospective clinical parameters in order to understand the molecular pathways implicated in progressive lung disease.. Lung tissue was obtained by open lung biopsy in 28 consecutive patients with SSc-related ILD and in 4 controls. High-resolution computed tomography (HRCT) and pulmonary function testing (PFT) were performed at baseline and 2-3 years after treatment based on lung histologic classification. Microarray analysis was performed, and the results were correlated with changes in the HRCT score (FibMax) and PFT values. Quantitative polymerase chain reaction (qPCR) and immunohistochemistry were used to confirm differential levels of messenger RNA and protein.. Lung microarray data distinguished patients with SSc-related ILD from healthy controls. In the lungs of patients with SSc-related ILD who had nonspecific interstitial pneumonia (NSIP), expressed genes included macrophage markers, chemokines, collagen, and transforming growth factor β (TGFβ)- and interferon (IFN)-regulated genes. Expression of these genes correlated with progressive lung fibrosis defined by the change in FibMax. Immunohistochemistry confirmed increased markers of collagen (COL1A1), IFN (OAS1 and IFI44), and macrophages (CCL18 and CD163), and the positive correlation with the change in FibMax was confirmed by qPCR in a larger group of SSc patients with NSIP. Several genes correlated with both the change in FibMax (r > 0.4) and the change in % predicted forced vital capacity (r < -0.1), including IFN and macrophage markers, chemokines, and heat-shock proteins.. These results highlight major pathogenic pathways relevant to progressive pulmonary fibrosis in SSc-related ILD: macrophage emigration and activation, and up-regulated expression of TGFβ- and IFN-regulated genes.

Topics: 2',5'-Oligoadenylate Synthetase; Adult; Antigens; Antigens, CD; Antigens, Differentiation, Myelomonocytic; Chemokines, CC; Collagen Type I; Collagen Type I, alpha 1 Chain; Cytoskeletal Proteins; Disease Progression; Female; Humans; Lung; Macrophage Activation; Male; Middle Aged; Pulmonary Fibrosis; Receptors, Cell Surface; Respiratory Function Tests; Scleroderma, Systemic; Transforming Growth Factor beta

Adipose stem cells (ASCs) are detectable in the parenchyma and large airways of lungs after systemic administration, and ameliorate inflammatory infiltration and cell death in animal models of emphysema. We evaluated whether ASC treatment could attenuate lung fibrosis induced by repetitive intratracheal bleomycin administration. Male 8-week-old C57BL/6J mice (control group, bleomycin-only group, and bleomycin-plus-ASC group) were used. Eight biweekly doses of bleomycin were injected intratracheally via an intubation procedure at a dose of 0.04 units in a total volume of 100 μL of sterile saline. During the latter 2 months of the 4-month bleomycin exposure, human ASCs (3 × 10(5) cells) were administered repeatedly via intraperitoneal injection at the same time as bleomycin. Lung tissues were evaluated for histology, collagen content, TUNEL staining, and TGF-β levels. Bronchoalveolar lavage (BAL) was performed for cell counting. Administrations of ASCs ameliorated the deleterious effects of repetitive intratracheal instillation of bleomycin, namely hyperplasia of Club cells (Clara cells) and cuboidal alveolar epithelial cells, infiltration of the perialveolar ducts by inflammatory cells, septal thickening, enlarged alveoli, and extensive fibrosis. Addition of ASC led to suppression of bleomycin-induced epithelial cell apoptosis and expression of TGF-β. These results suggest a useful therapeutic effect of ASCs on pulmonary fibrosis induced by repetitive bleomycin administration. Further studies will be required to evaluate the efficacy of ASC therapy for the treatment of idiopathic pulmonary fibrosis.

Topics: Adipose Tissue; Animals; Bleomycin; Bronchoalveolar Lavage Fluid; Hydroxyproline; In Situ Nick-End Labeling; Lung; Male; Mesenchymal Stem Cell Transplantation; Mice; Mice, Inbred C57BL; Pulmonary Fibrosis; Transforming Growth Factor beta

Idiopathic pulmonary fibrosis (IPF) is a progressive fibro-proliferative disorder refractory to current therapy commonly complicated by the development of pulmonary hypertension (PH); the associated morbidity and mortality are substantial. Natriuretic peptides possess vasodilator and anti-fibrotic actions, and pharmacological augmentation of their bioactivity ameliorates renal and myocardial fibrosis. Here, we investigated whether natriuretic peptides possess an intrinsic cytoprotective function preventing the development of pulmonary fibrosis and associated PH, and whether therapeutics targeting natriuretic peptide signalling demonstrate efficacy in this life-threatening disorder.. Pulmonary haemodynamics, right ventricular function and markers of lung fibrosis were determined in wild-type (WT) and natriuretic peptide receptor (NPR)-A knockout (KO) mice exposed to bleomycin (1 mg·kg(-1) ). Human myofibroblast differentiation was studied in vitro.. Exacerbated cardiac, vascular and fibrotic pathology was observed in NPR-A KO animals, compared with WT mice, exposed to bleomycin. Treatment with a drug combination that raised circulating natriuretic peptide levels (ecadotril) and potentiated natriuretic peptide-dependent signalling (sildenafil) reduced indices of disease progression, whether administered prophylactically or to animals with established lung disease. This positive pharmacodynamic effect was diminished in NPR-A KO mice. Atrial natriuretic peptide and sildenafil synergistically reduced TGFβ-induced human myofibroblast differentiation, a key driver of remodelling in IPF patients.. These data highlight an endogenous host-defence capacity of natriuretic peptides in lung fibrosis and PH. A combination of ecadotril and sildenafil reversed the pulmonary haemodynamic aberrations and remodelling that characterize the disease, advocating therapeutic manipulation of natriuretic peptide bioactivity in patients with IPF.

Topics: Animals; Atrial Natriuretic Factor; Bleomycin; Cell Differentiation; Dose-Response Relationship, Drug; Humans; Hypertension, Pulmonary; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Myofibroblasts; Natriuretic Peptide, C-Type; Natriuretic Peptides; Protein Precursors; Pulmonary Fibrosis; Structure-Activity Relationship; Transforming Growth Factor beta

Fibrotic lung diseases represent a diverse group of progressive and often fatal disorders with limited treatment options. Although the pathogenesis of these conditions remains incompletely understood, receptor type protein tyrosine phosphatase α (PTP-α encoded by PTPRA) has emerged as a key regulator of fibroblast signaling. We previously reported that PTP-α regulates cellular responses to cytokines and growth factors through integrin-mediated signaling and that PTP-α promotes fibroblast expression of matrix metalloproteinase 3, a matrix-degrading proteinase linked to pulmonary fibrosis. Here, we sought to determine more directly the role of PTP-α in pulmonary fibrosis. Mice genetically deficient in PTP-α (Ptpra(-/-)) were protected from pulmonary fibrosis induced by intratracheal bleomycin, with minimal alterations in the early inflammatory response or production of TGF-β. Ptpra(-/-) mice were also protected from pulmonary fibrosis induced by adenoviral-mediated expression of active TGF-β1. In reciprocal bone marrow chimera experiments, the protective phenotype tracked with lung parenchymal cells but not bone marrow-derived cells. Because fibroblasts are key contributors to tissue fibrosis, we compared profibrotic responses in wild-type and Ptpra(-/-) mouse embryonic and lung fibroblasts. Ptpra(-/-) fibroblasts exhibited hyporesponsiveness to TGF-β, manifested by diminished expression of αSMA, EDA-fibronectin, collagen 1A, and CTGF. Ptpra(-/-) fibroblasts exhibited markedly attenuated TGF-β-induced Smad2/3 transcriptional activity. We conclude that PTP-α promotes profibrotic signaling pathways in fibroblasts through control of cellular responsiveness to TGF-β.

Topics: Adenoviridae; Animals; Bleomycin; Cytokines; Fibroblasts; Gene Deletion; Genes, Reporter; Lung; Mice; Mice, Inbred C57BL; NIH 3T3 Cells; Pneumonia; Pulmonary Fibrosis; Receptor-Like Protein Tyrosine Phosphatases, Class 4; Receptors, Transforming Growth Factor beta; Signal Transduction; Smad Proteins; Transcription, Genetic; Transforming Growth Factor beta

The purpose of the study was to explore the effects of breast regression protein 39 (BRP-39) in bleomycin-induced pulmonary fibrosis and its mechanism in pulmonary fibrosis by studying change in BRP-39 to provide a novel direction for the treatment of idiopathic pulmonary fibrosis. SPF grade male C57BL/6 rats were randomly divided into three groups, including bleomycin group, bleomycin+ BRP-39 recombinant protein group and control group. HE and Masson staining were applied to test the change in lung tissue after being treated by BRP-39, ELISA was applied to test the expression of TGF-β1 in different groups, and Western blot was used to test the expression of BRP-39 in rat lung tissue. Expression of BRP-39 increased, the fibrosis was obvious, and lung tissue collagen increased in bleomycin-induced pulmonary fibrosis in rat lung tissue. Increasing BRP-39 protein level and intratracheal bleomycin medication to establish pulmonary fibrosis model can aggravate pulmonary fibrosis. Along with the increase in BRP-39 protein level, TGF-β1 expression level also increased in lung tissue. Western blot results showed the expression of BRP-39, and TGF-β1 had the same trend in different groups. BRP-39 has effects in bleomycin-induced rat pulmonary fibrosis. Change in BRP-39 can affect the process of bleomycin-induced pulmonary fibrosis. The mechanism of BRP-3 in pulmonary fibrosis may work by regulating TGF-β1.

Topics: Animals; Bleomycin; Chitinase-3-Like Protein 1; Gene Expression Regulation; Glycoproteins; Male; Mice; Mice, Inbred C57BL; Proline; Pulmonary Fibrosis; Transforming Growth Factor beta

This study was designed to investigate the effect of recombinant sTGFβ1RII and sIL13Rα2 receptor proteins on schistosomiasis japonica, hepatic fibrosis and the expression of SMAD3 and STAT6. The proteins sTGFβ1RII and sIL13Rα2 were expressed in Escherichiacoli, purified using affinity chromatography and characterized by Western blotting. Female BALB/C mice (48) were randomly divided into eight groups and infected with Schistosoma japonicum. Five weeks after infection, test groups were injected with the recombinant proteins at different doses. Eight weeks after infection, lung and hepatic tissue samples were obtained and stained with hematoxylin and eosin (HE) and Masson's trichrome. Immunohistochemical staining was used to detect the expression of SMAD3 and STAT6. The recombinant proteins sTGFβ1RII and sIL13Rα2 were successfully expressed, purified, and characterized. The granuloma area, hepatic hydroxyproline (HYP) level and hepatic fibrosis of the protein therapeutic groups were significantly smaller than those of the positive control group (P<0.01). Treatment with sTGFβ1RII was more effective when the protein was administered for 4weeks rather than 2 (P<0.01). Hepatic fibrosis in the groups using a low dose of protein sTGFβ1 was lower that of the combination group (P<0.05). The expression level of STAT6 was significantly lower in groups treated with sIL13Rα2 than in groups not treated with the protein (P<0.01). The recombinant proteins TGFβ1RII and sIL13Rα2 were able to decrease granuloma area and hepatic fibrosis in schistosomiasis japonica, and also reduced the expression of the signal transduction proteins SMAD3 and STAT6. The proteins were more effective when used in combination than when applied singly.

Topics: Animals; Disease Models, Animal; Eukaryotic Initiation Factors; Extracellular Matrix Proteins; Female; Granuloma; Hydroxyproline; Interleukin-13; Interleukin-13 Receptor alpha2 Subunit; Intracellular Signaling Peptides and Proteins; Liver; Liver Cirrhosis; Liver Diseases; Lung; Mice; Mice, Inbred BALB C; Pulmonary Fibrosis; Random Allocation; Real-Time Polymerase Chain Reaction; Recombinant Proteins; Schistosomiasis japonica; Signal Transduction; Smad Proteins; STAT6 Transcription Factor; Transforming Growth Factor beta

Pulmonary fibrosis (PF) is a common complication in those interstitial lung diseases patients, which will result in poor prognosis and short survival. Traditional therapeutic methods such as glucocorticoid and cytotoxic drugs are insufficient for treating PF and may cause severe side effects. Recent studies showed that traditional Chinese herbal abstraction such as Tanshinone IIA (TIIA) was displayed significant anti-PF effects in animal models. However, the exact mechanisms underlying the protective effects of TIIA were not fully understood. Here we further investigated the protective effects of TIIA and its mechanisms underlying. PF models of rat were induced by bleomycin (BLM); TIIA was administered subsequently. The PF changes were identified by histopathological analyses. The results showed that BLM resulted in severe PF and alveolar inflammation; together with significant elevation of transforming growth factor-β 1 (TGF-β1). Angiotensin-converting enzyme 2 (ACE-2) together with angiotensin-(1-7) [ANG-(1-7)] were both greatly reduced after BLM administration. TIIA treatment notably attenuated BLM induced PF and inflammation, decreased expression of TGF-β1 and reversed ACE-2 and ANG-(1-7) production in rat lungs. Thus we may draw the conclusion that TIIA may exert protective effects on BLM induced PF in rats, and the ACE-2/ANG-(1-7) axis may ascribe to those protective effects.

Topics: Abietanes; Angiotensin I; Angiotensin-Converting Enzyme 2; Animals; Bleomycin; Humans; Inflammation; Peptide Fragments; Peptidyl-Dipeptidase A; Pulmonary Fibrosis; Rats; Transforming Growth Factor beta

Given their extremely small size and light weight, carbon nanotubes (CNTs) can be readily inhaled by human lungs resulting in increased rates of pulmonary disorders, particularly fibrosis. Although the fibrogenic potential of CNTs is well established, there is a lack of consensus regarding the contribution of physicochemical attributes of CNTs on the underlying fibrotic outcome. We designed an experimentally validated in vitro fibroblast culture model aimed at investigating the effect of fiber length on single-walled CNT (SWCNT)-induced pulmonary fibrosis. The fibrogenic response to short and long SWCNTs was assessed via oxidative stress generation, collagen expression and transforming growth factor-beta (TGF-β) production as potential fibrosis biomarkers. Long SWCNTs were significantly more potent than short SWCNTs in terms of reactive oxygen species (ROS) response, collagen production and TGF-β release. Furthermore, our finding on the length-dependent in vitro fibrogenic response was validated by the in vivo lung fibrosis outcome, thus supporting the predictive value of the in vitro model. Our results also demonstrated the key role of ROS in SWCNT-induced collagen expression and TGF-β activation, indicating the potential mechanisms of length-dependent SWCNT-induced fibrosis. Together, our study provides new evidence for the role of fiber length in SWCNT-induced lung fibrosis and offers a rapid cell-based assay for fibrogenicity testing of nanomaterials with the ability to predict pulmonary fibrogenic response in vivo.

Topics: Cell Survival; Cells, Cultured; Collagen Type I; Cytotoxins; Fibroblasts; Humans; Nanotubes, Carbon; Oxidative Stress; Pulmonary Fibrosis; Reactive Oxygen Species; Transforming Growth Factor beta

Carbon monoxide (CO) has potent anti-inflammatory and anti-oxidant effects. We report herein on the preparation of a nanotechnology-based CO donor, CO-bound hemoglobin-vesicles (CO-HbV). We hypothesized that CO-HbV could have a therapeutic effect on idiopathic pulmonary fibrosis (IPF), an incurable lung fibrosis, that is thought to involve inflammation and the production of reactive oxygen species (ROS). Pulmonary fibril formation and respiratory function were quantitatively evaluated by measuring hydroxyproline levels and forced vital capacity, respectively, using a bleomycin-induced pulmonary fibrosis mice model. CO-HbV suppressed the progression of pulmonary fibril formation and improved respiratory function compared to saline and HbV. The suppressive effect of CO-HbV on pulmonary fibrosis can be attributed to a decrease in ROS generation by inflammatory cells, NADPH oxidase 4 and the production of inflammatory cells, cytokines and transforming growth factor-β in the lung. This is the first demonstration of the inhibitory effect of CO-HbV on the progression of pulmonary fibrosis via the anti-oxidative and anti-inflammatory effects of CO in the bleomycin-induced pulmonary fibrosis mice model. CO-HbV has the potential for use in the treatment of, not only IPF, but also a variety of other ROS and inflammation-related disorders.

Topics: Animals; Bleomycin; Bronchoalveolar Lavage Fluid; Carbon Monoxide; Chemokines; Dose-Response Relationship, Drug; Hemoglobins; Inflammation Mediators; Lipids; Lung; Male; Mice; Mice, Inbred ICR; Pulmonary Fibrosis; Reactive Oxygen Species; Transforming Growth Factor beta; Unilamellar Liposomes

Heme oxygenase-1 (HO-1) contributes to the pathogenesis of pulmonary fibrosis. However, the expression of HO-1 in fibroblasts under fibrotic conditions has not been studied.. This study was conducted to investigate the expression of HO-1 in lung fibroblasts from mice and humans under fibrotic conditions by Western blot.. We found that the expression of HO-1 was significantly decreased in lung fibroblasts isolated from bleomycin-challenged mice in comparison with control mice. Transforming growth factor-β (TGF-β) inhibited HO-1 expression and induced differentiation in human lung fibroblasts. Pretreatment with nuclear factor-κB (NF-κB) activation inhibitor or knockdown of the NF-κB p65 subunit attenuated TGF-β-induced inhibition of HO-1 expression and differentiation in human lung fibroblasts. Similarly, lysophosphatidic acid (LPA) induced TGF-β expression and decreased HO-1 expression in human lung fibroblasts. Interestingly, pretreatment with neutralized anti-TGF-β antibody attenuated LPA effects in human lung fibroblasts.. These data suggested that TGF-β inhibited HO-1 expression in human lung fibroblasts through activation of NF-κB.

Topics: Animals; Blotting, Western; Cell Differentiation; Fibroblasts; Gene Expression Regulation; Gene Knockdown Techniques; Heme Oxygenase-1; Humans; Lung; Lysophospholipids; Male; Mice; Mice, Inbred C57BL; NF-kappa B; Pulmonary Fibrosis; Transcription Factor RelA; Transforming Growth Factor beta

Paraquat (PQ) poisoning, with the lung as a primary target organ, is a devastating disease which irreversibly progresses to diffuse alveolitis followed by extensive lung fibrosis. In the present study, we aimed to investigate the effect of FTY720, an immune modulator, on PQ-induced lung injury in mice. C57BL/6 mice were randomized into four groups: 1) PQ group (n=12): mice was instilled with PQ (30 mg/kg, ip); 2) PQ+FTY720 group (n=12): animals received FTY720 (0.1mg/kg, ip) solution 2h after PQ exposure and twice a week for 4 consecutive weeks; 3) FTY720 group (n=5): FTY720 (0.1mg/kg, ip) was administrated twice a week for 4 consecutive weeks; and 4) Control group (n=10): same volumes of saline were injected. Mice were sacrificed on either day 3 or day 28 for histopathological, biochemical and immunohistochemical analyses of lung damage indicators. We found that FTY720 treatment attenuated PQ-induced acute lung injury and lung fibrosis as evaluated by histopathological changes and Ashcroft score. On day 3, FTY720 administration reduced PQ-induced increases in lung wet weight/body weight (LW/BW), total protein and cytokine levels including interleukin-1β (IL-1β), interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) in bronchoalceolar lavage fluid (BALF). On day 28, the expressions of alpha-smooth muscle actin (α-SMA), transforming growth factor-beta (TGF-β) and vascular endothelial growth factor (VEGF) detected by immunohistochemistry, as well as the mRNA levels of α-SMA, Type-I Collagen and Type-III Collagen examined by Real-time PCR were down-regulated after FTY720 treatment. These results indicate that FTY720 could attenuate PQ-induced lung injury, but further investigation is necessary.

Topics: Actins; Acute Lung Injury; Animals; Body Weight; Bronchoalveolar Lavage Fluid; Collagen Type I; Collagen Type III; Disease Models, Animal; Fingolimod Hydrochloride; Interleukin-1beta; Interleukin-6; Lung; Lung Injury; Mice; Mice, Inbred C57BL; Paraquat; Propylene Glycols; Pulmonary Edema; Pulmonary Fibrosis; Sphingosine; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha; Vascular Endothelial Growth Factor A

Myofibroblasts are effector cells in fibrotic disorders that synthesize and remodel the extracellular matrix (ECM). This study investigated the role of the Src kinase pathway in myofibroblast activation in vitro and fibrogenesis in vivo. The profibrotic cytokine, transforming growth factor β1 (TGF-β1), induced rapid activation of Src kinase, which led to myofibroblast differentiation of human lung fibroblasts. The Src kinase inhibitor AZD0530 (saracatinib) blocked TGF-β1-induced Src kinase activation in a dose-dependent manner. Inhibition of Src kinase significantly reduced α-smooth muscle actin (α-SMA) expression, a marker of myofibroblast differentiation, in TGF-β1-treated lung fibroblasts. In addition, the induced expression of collagen and fibronectin and three-dimensional collagen gel contraction were also significantly inhibited in AZD0530-treated fibroblasts. The therapeutic efficiency of Src kinase inhibition in vivo was tested in the bleomycin murine lung fibrosis model. Src kinase activation and collagen accumulation were significantly reduced in the lungs of AZD0530-treated mice when compared with controls. Furthermore, the total fibrotic area and expression of α-SMA and ECM proteins were significantly decreased in lungs of AZD0530-treated mice. These results indicate that Src kinase promotes myofibroblast differentiation and activation of lung fibroblasts. Additionally, these studies provide proof-of-concept for targeting the noncanonical TGF-β signaling pathway involving Src kinase as an effective therapeutic strategy for lung fibrosis.

Topics: Actins; Animals; Benzodioxoles; Cell Differentiation; Cell Line; Cells, Cultured; Collagen; Enzyme Inhibitors; Female; Humans; Male; Mice; Mice, Inbred C57BL; Myofibroblasts; Pulmonary Fibrosis; Quinazolines; src-Family Kinases; Transforming Growth Factor beta

A characteristic of dysregulated wound healing in IPF is fibroblastic-mediated damage to lung epithelial cells within fibroblastic foci. In these foci, TGF-β and other growth factors activate fibroblasts that secrete growth factors and matrix regulatory proteins, which activate a fibrotic cascade. Our studies and those of others have revealed that Akt is activated in IPF fibroblasts and it mediates the activation by TGF-β of pro-fibrotic pathways. Recent studies show that mTORC2, a component of the mTOR pathway, mediates the activation of Akt. In this study we set out to determine if blocking mTORC2 with MLN0128, an active site dual mTOR inhibitor, which blocks both mTORC1 and mTORC2, inhibits lung fibrosis. We examined the effect of MLN0128 on TGF-β-mediated induction of stromal proteins in IPF lung fibroblasts; also, we looked at its effect on TGF-β-mediated epithelial injury using a Transwell co-culture system. Additionally, we assessed MLN0128 in the murine bleomycin lung model. We found that TGF-β induces the Rictor component of mTORC2 in IPF lung fibroblasts, which led to Akt activation, and that MLN0128 exhibited potent anti-fibrotic activity in vitro and in vivo. Also, we observed that Rictor induction is Akt-mediated. MLN0128 displays multiple anti-fibrotic and lung epithelial-protective activities; it (1) inhibited the expression of pro-fibrotic matrix-regulatory proteins in TGF-β-stimulated IPF fibroblasts; (2) inhibited fibrosis in a murine bleomycin lung model; and (3) protected lung epithelial cells from injury caused by TGF-β-stimulated IPF fibroblasts. Our findings support a role for mTORC2 in the pathogenesis of lung fibrosis and for the potential of active site mTOR inhibitors in the treatment of IPF and other fibrotic lung diseases.

Topics: Animals; Benzoxazoles; Bleomycin; Carrier Proteins; Coculture Techniques; Epithelial Cells; Extracellular Matrix Proteins; Fibroblasts; Gene Expression Regulation; Mechanistic Target of Rapamycin Complex 1; Mechanistic Target of Rapamycin Complex 2; Mice; Multiprotein Complexes; Protective Agents; Proto-Oncogene Proteins c-akt; Pulmonary Fibrosis; Pyrimidines; Rapamycin-Insensitive Companion of mTOR Protein; Signal Transduction; TOR Serine-Threonine Kinases; Transforming Growth Factor beta

β-catenin contributes to the pathogenesis of lung fibrosis. However, the expression of β-catenin in fibroblasts under fibrotic conditions has not been studied. We investigated the expression of β-catenin in lung fibroblasts from bleomycin (BLM)‑challenged mice and human lung fibroblasts treated with transforming growth factor β (TGF-β) or lysophosphatidic acid (LPA) by western blot analysis. The result showed that the expression of β-catenin was significantly increased in lung fibrotic foci and lung fibroblasts from bleomycin‑challenged mice. TGF-β stimulated β-catenin expression and induced differentiation in human lung fibroblasts in vitro. Pretreatment of the NF-κB activation inhibitor attenuated the TGF-β‑induced expression of β-catenin and differentiation in human lung fibroblasts. Similarly, LPA induced β-catenin expression in human lung fibroblasts, and pre-treatment of the neutralized anti-TGF-β antibody attenuated the LPA‑induced expression of β-catenin and differentiation in human lung fibroblasts. The results suggested that β-catenin expression is upregulated in lung fibroblast during differentiation, and that TGF-β induced β-catenin expression in human lung fibroblasts through the activation of NF-κB.

Topics: Animals; beta Catenin; Bleomycin; Cell Differentiation; Cell Line; Fibroblasts; Humans; Lung; Lysophospholipids; Male; Mice; Mice, Inbred C57BL; NF-kappa B; Pulmonary Fibrosis; Transforming Growth Factor beta; Up-Regulation

Pulmonary fibrosis, a progressive and lethal lung disease characterized by inflammation and accumulation of extracellular matrix components, is a major therapeutic challenge for which new therapeutic strategies are warranted. Cyclooxygenase (COX) inhibitors have been previously utilized to reduce inflammation. Histamine H4 receptor (H4R), largely expressed in hematopoietic cells, has been identified as a novel target for inflammatory and immune disorders. The aim of this study was to evaluate the effect of JNJ7777120 (1-[(5-chloro-1H-indol-2-yl)carbonyl]-4-methylpiperazine), a selective H4R antagonist, and naproxen, a well known nonsteroidal anti-inflammatory drug, and their combination in a murine model of bleomycin-induced fibrosis. Bleomycin (0.05 IU) was instilled intratracheally to C57BL/6 mice, which were then treated by micro-osmotic pump with vehicle, JNJ7777120 (40 mg/kg b.wt.), naproxen (21 mg/kg b.wt.), or a combination of both. Airway resistance to inflation, an index of lung stiffness, was assessed, and lung specimens were processed for inflammation, oxidative stress, and fibrosis markers. Both drugs alone were able to reduce the airway resistance to inflation induced by bleomycin and the inflammatory response by decreasing COX-2 and myeloperoxidase expression and activity and thiobarbituric acid-reactive substance and 8-hydroxy-2'-deoxyguanosine production. Lung fibrosis was inhibited, as demonstrated by the reduction of tissue levels of transforming growth factor-β, collagen deposition, relative goblet cell number, and smooth muscle layer thickness. Our results demonstrate that both JNJ7777120 and naproxen exert an anti-inflammatory and antifibrotic effect that is increased by their combination, which could be an effective therapeutic strategy in the treatment of pulmonary fibrosis.

Topics: 8-Hydroxy-2'-Deoxyguanosine; Animals; Anti-Inflammatory Agents; Bleomycin; Collagen; Cyclooxygenase 2; Deoxyguanosine; Disease Models, Animal; Goblet Cells; Indoles; Lung; Mice; Muscle, Smooth; Naproxen; Oxidative Stress; Peroxidase; Piperazines; Pneumonia; Pulmonary Fibrosis; Thiobarbituric Acid Reactive Substances; Transforming Growth Factor beta

Idiopathic pulmonary fibrosis (IPF) is a progressive, fatal disease, thought to be largely transforming growth factor β (TGFβ) driven, for which there is no effective therapy. We assessed the potential benefits in IPF of nitrated fatty acids (NFAs), which are unique endogenous agonists of peroxisome proliferator-activated receptor γ (PPARγ), a nuclear hormone receptor that exhibits wound-healing and antifibrotic properties potentially useful for IPF therapy. We found that pulmonary PPARγ is down-regulated in patients with IPF. In vitro, knockdown or knockout of PPARγ expression in isolated human and mouse lung fibroblasts induced a profibrotic phenotype, whereas treating human fibroblasts with NFAs up-regulated PPARγ and blocked TGFβ signaling and actions. NFAs also converted TGFβ to inactive monomers in cell-free solution, suggesting an additional mechanism through which they may inhibit TGFβ. In vivo, treating mice bearing experimental pulmonary fibrosis with NFAs reduced disease severity. Also, NFAs up-regulated the collagen-targeting factor milk fat globule-EGF factor 8 (MFG-E8), stimulated collagen uptake and degradation by alveolar macrophages, and promoted myofibroblast dedifferentiation. Moreover, treating mice with established pulmonary fibrosis using NFAs reversed their existing myofibroblast differentiation and collagen deposition. These findings raise the prospect of treating IPF with NFAs to halt and perhaps even reverse the progress of IPF.

Topics: Animals; Bleomycin; Cell Dedifferentiation; Collagen; Fatty Acids; Humans; Macrophages, Alveolar; Male; Mice; Mice, Inbred C57BL; Nitrates; PPAR gamma; Pulmonary Fibrosis; Real-Time Polymerase Chain Reaction; Transforming Growth Factor beta

Silicosis is an occupational lung disease, characterized by irreversible and progressive fibrosis. Silica exposure leads to intense lung inflammation, reactive oxygen production, and extracellular ATP (eATP) release by macrophages. The P2X7 purinergic receptor is thought to be an important immunomodulator that responds to eATP in sites of inflammation and tissue damage. The present study investigates the role of P2X7 receptor in a murine model of silicosis. To that end wild-type (C57BL/6) and P2X7 receptor knockout mice received intratracheal injection of saline or silica particles. After 14 days, changes in lung mechanics were determined by the end-inflation occlusion method. Bronchoalveolar lavage and flow cytometry analyzes were performed. Lungs were harvested for histological and immunochemistry analysis of fibers content, inflammatory infiltration, apoptosis, as well as cytokine and oxidative stress expression. Silica particle effects on lung alveolar macrophages and fibroblasts were also evaluated in cell line cultures. Phagocytosis assay was performed in peritoneal macrophages. Silica exposure increased lung mechanical parameters in wild-type but not in P2X7 knockout mice. Inflammatory cell infiltration and collagen deposition in lung parenchyma, apoptosis, TGF-β and NF-κB activation, as well as nitric oxide, reactive oxygen species (ROS) and IL-1β secretion were higher in wild-type than knockout silica-exposed mice. In vitro studies suggested that P2X7 receptor participates in silica particle phagocytosis, IL-1β secretion, as well as reactive oxygen species and nitric oxide production. In conclusion, our data showed a significant role for P2X7 receptor in silica-induced lung changes, modulating lung inflammatory, fibrotic, and functional changes.

Topics: Animals; Apoptosis; Bronchoalveolar Lavage Fluid; Collagen; Fibroblasts; Immunophenotyping; Inflammation; Interleukin-1beta; Lung; Macrophages, Alveolar; Mice; Mice, Inbred C57BL; NF-kappa B; NIH 3T3 Cells; Nitric Oxide; Nitric Oxide Synthase Type II; Phagocytosis; Pulmonary Fibrosis; Purinergic P2X Receptor Antagonists; Reactive Oxygen Species; Receptors, Purinergic P2X7; Rosaniline Dyes; Signal Transduction; Silicon Dioxide; Smad2 Protein; Smad3 Protein; Transforming Growth Factor beta

Naja naja atra venom (NNAV) displays diverse pharmacological actions including analgesia, anti-inflammation and immune regulation.In this study, we investigated the effects of NNAV on pulmonary fibrosis and its mechanisms of action.. To determine if Naja naja atra venom (NNAV) can produce beneficial effects on pulmonary fibrosis, two marine models of pulmonary fibrosis were produced with bleomycin (BLM) and lipopolysaccharide (LPS). NNAV (30, 90, 270 μg/kg) was orally administered once a day started five days before BLM and LPS until to the end of experiment. The effects of NNAV treatment on pulmonary injury were evaluated with arterial blood gas analysis, hydroxyproline (HYP) content assessment and HE/Masson staining. The effects of NNAV treatment on inflammatory related cytokines, fibrosis related TGF-β/Smad signaling pathway and oxidative stress were examined.. The results showed that NNAV improved the lung gas-exchange function and attenuated the fibrotic lesions in lung. NNAV decreased IL-1β and TNF-α levels in serum in both pulmonary fibrosis models. NNAV inhibited the activation of NF-κB in LPS-induced and TGF-β/Smad pathway in BLM-induced pulmonary fibrosis. Additionally, NNAV also increased the levels of SOD and GSH and reduced the levels of MDA in BLM-induced pulmonary fibrosis model.. The present study indicates that NNAV attenuates LPS- and BLM-induced lung fibrosis. Its mechanisms of action are associated with inhibiting inflammatory response and oxidative stress. The study suggests that NNAV might be a potential therapeutic drug for treatment of pulmonary fibrosis.

Topics: Animals; Anti-Inflammatory Agents; Antioxidants; Bleomycin; Elapid Venoms; Elapidae; Female; Fibrosis; Hydroxyproline; Inflammation; Interleukin-1beta; Lung; Male; Mice; NF-kappa B; Oxidative Stress; Pulmonary Fibrosis; Rats, Sprague-Dawley; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha

Zinc oxide (ZnO) nanoparticles are widely used in various products, and the safety evaluation of this manufactured material is important. The present study investigated the inflammatory and fibrotic effects of pulmonary exposure to ZnO nanoparticles in a mouse model of pulmonary fibrosis. Pulmonary fibrosis was induced by constant subcutaneous infusion of bleomycin (BLM). Female C57BL/6Jcl mice were divided into BLM-treated and non-treated groups. In each treatment group, 0, 10, 20 or 30 µg of ZnO nanoparticles were delivered into the lungs through pharyngeal aspiration. Bronchoalveolar lavage fluid (BALF) and the lungs were sampled at Day 10 or 14 after administration. Pulmonary exposure by a single bolus of ZnO nanoparticles resulted in severe, but transient inflammatory infiltration and thickening of the alveolar septa in the lungs, along with the increase of total and differential cell counts in BLAF. The BALF level of interleukin (IL)-1β and transforming growth factor (TGF)-β was increased at Day 10 and 14, respectively. At Day 10, the synergistic effect of BLM and ZnO exposure was detected on IL-1β and monocyte chemotactic protein (MCP)-1 in BALF. The present study demonstrated the synergistic effect of pulmonary exposure to ZnO nanoparticles and subcutaneous infusion of BLM on the secretion of pro-fibrotic cytokines in the lungs.

Topics: Animals; Bleomycin; Bronchoalveolar Lavage Fluid; Chemokine CCL2; Cytokines; Disease Models, Animal; Drug Synergism; Female; Interleukin-1beta; Lung; Metal Nanoparticles; Mice; Mice, Inbred C57BL; Pulmonary Fibrosis; Transforming Growth Factor beta; Zinc Oxide

Dysfunctional pulmonary homeostasis and repair, including diseases such as pulmonary fibrosis, chronic obstructive pulmonary disease (COPD*), and tumorigenesis, have been increasing steadily over the past decade, a fact that heavily implicates environmental influences. Several investigations have suggested that the lung "precursor cell"--the alveolar type II (ATII) epithelial cell--is central in the initiation and progression of pulmonary fibrosis. Specifically, ATII cells have been shown (Iwano et al. 2002) to be capable of undergoing an epithelial-to-mesenchymal transition (EMT). EMT, the de-differentiation of an epithelial cell into a mesenchymal cell, has been theorized to increase the number of extracellular matrix (ECM)-secreting mesenchymal cells, perpetuating fibrotic conditions and resulting in increased lung tissue stiffness. In addition, increased exposure to pollution and inhalation of particulate matter (PM) have been shown to be highly correlated with an increased incidence of pulmonary fibrosis. Although both of these events are involved in the progression of pulmonary fibrosis, the relationship between tissue stiffness, exposure to PM, and the initiation and course of EMT remains unclear. The hypothesis of this study was twofold: 1. That alveolar epithelial cells cultured on increasingly stiff substrates become increasingly contractile, leading to enhanced transforming growth factor beta (TGF-β) activation and EMT; and 2. That exposure of alveolar epithelial cells to PM with an aerodynamic diameter ≤ 2.5 μm (PM2.5; also known as fine PM) results in enhanced cell contractility and EMT. Our study focused on the relationship between the micromechanical environment and external environmental stimuli on the phenotype of alveolar epithelial cells. This relationship was explored by first determining how increased tissue stiffness affects the regulation of fibronectin (Fn)-mediated EMT in ATII cells in vitro. We cultured ATII cells on substrates of increasing stiffness and evaluated changes in cell contractility and EMT. We found that stiff, but not soft, Fn substrates were able to induce EMT and that this event depended on a contractile phenotype of the cell and the subsequent activation of TGF-β. In addition, we were able to show that activation or suppression of cell contractility by way of exogenous factors was sufficient to overcome the effect of substrate stiffness. Pulse-chase experiments indicated that the effect on cell contractility is d

Topics: Animals; Cell Differentiation; Cell Line; Cells, Cultured; Disease Progression; Elastic Modulus; Epithelial Cells; Epithelial-Mesenchymal Transition; Extracellular Matrix; Immunoblotting; Mice; Mice, Inbred C57BL; Particulate Matter; Phenotype; Pulmonary Alveoli; Pulmonary Fibrosis; Transforming Growth Factor beta

Idiopathic pulmonary fibrosis (IPF) is thought to involve inflammatory cells and reactive oxygen species (ROS), such as superoxide anion radical (O2(·-)). There is currently no effective treatment of IPF. We previously developed a human serum albumin (HSA)-thioredoxin 1 (Trx) fusion protein (HSA-Trx) designed to overcome the unfavorable pharmacokinetic and short pharmacological properties of Trx, an antioxidative and anti-inflammatory protein. In this study, we examined the therapeutic effect of HSA-Trx on an IPF animal model of bleomycin (BLM)-induced pulmonary fibrosis. A pharmacokinetic study of HSA-Trx or Trx in BLM mice showed that the plasma retention and lung distribution of Trxc was markedly improved by fusion with HSA. A weekly intravenous administration of HSA-Trx, but not Trx, ameliorated BLM-induced fibrosis, as evidenced by a histopathological analysis and pulmonary hydroxyproline levels. HSA-Trx suppressed active-transforming growth factor (TGF)-β levels in the lung and inhibited the increase of inflammatory cells in bronchoalveolar lavage fluid, pulmonary inflammatory cytokines, and oxidative stress markers. An in vitro EPR experiment using phosphate-buffered saline-stimulated neutrophils confirmed the O2(·-) scavenging ability of HSA-Trx. Furthermore, post-treatment of HSA-Trx had a suppressive effect against BLM-induced fibrosis. These results suggest that HSA-Trx has potential as a novel therapeutic agent for IPF, because of its long-acting antioxidative and anti-inflammatory modulation effects.

Topics: Animals; Antibiotics, Antineoplastic; Bleomycin; Blotting, Western; Bronchoalveolar Lavage Fluid; Disease Progression; Hydroxyproline; Interleukin-6; Lung; Macrophage Migration-Inhibitory Factors; Macrophages; Malondialdehyde; Mice; Neutrophils; Pulmonary Fibrosis; Reactive Oxygen Species; Recombinant Fusion Proteins; Serum Albumin; Thioredoxins; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha

Mitogen-activated protein kinase-activated protein kinase-2 (MAPKAPK2, or MK2), a serine/threonine kinase downstream of p38 mitogen-activated protein kinase, has been implicated in inflammation and fibrosis. Compared with pathologically normal lung tissue, significantly higher concentrations of activated MK2 are evident in lung biopsies of patients with idiopathic pulmonary fibrosis (IPF). Expression is localized to fibroblasts and epithelial cells. In the murine bleomycin model of pulmonary fibrosis, we observed robust, activated MK2 expression on Day 7 (prefibrotic stage) and Day 14 (postfibrotic stage). To determine the effects of MK2 inhibition during the postinflammatory/prefibrotic and postfibrotic stages, C57BL/6 mice received intratracheal bleomycin instillation (0.025 U; Day 0), followed by PBS or the MK2 inhibitor (MK2i; 37.5 μg/kg), administered via either local (nebulized) or systemic (intraperitoneal) routes. MK2i or PBS was dosed daily for 14 days subsequent to bleomycin injury, beginning on either Day 7 or Day 14. Regardless of mode of administration or stage of intervention, MK2i significantly abrogated collagen deposition, myofibroblast differentiation and activated MK2 expression. MK2i also decreased circulating TNF-α and IL-6 concentrations, and modulated the local mRNA expression of profibrotic cytokine il-1β, matrix-related genes col1a2, col3a1, and lox, and transforming growth factor-β family members, including smad3, serpine1 (pai1), and smad6/7. In vitro, MK2i dose-dependently attenuated total MK2, myofibroblast differentiation, the secretion of collagen Type I, fibronectin, and the activation of focal adhesion kinase, whereas activated MK2 was attenuated at optimal doses. The peptide-mediated inhibition of MK2 affects both inflammatory and fibrotic responses, and thus may offer a promising therapeutic target for IPF.

Topics: Amino Acid Sequence; Animals; Anti-Inflammatory Agents, Non-Steroidal; Bleomycin; Cell Differentiation; Collagen Type I; Dose-Response Relationship, Drug; Enzyme Activation; Focal Adhesion Kinase 1; Gene Expression Regulation; Humans; Interleukin-1beta; Interleukin-6; Intracellular Signaling Peptides and Proteins; Lung; Mice; Mice, Inbred C57BL; Molecular Sequence Data; Myofibroblasts; Peptide Fragments; Peptides; Protein Serine-Threonine Kinases; Pulmonary Fibrosis; RNA, Messenger; Time Factors; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha

Idiopathic pulmonary fibrosis (IPF) is a progressive, debilitating and fatal lung disorder with high mortality rate. Unfortunately, to date the treatment for IPF remains unsatisfying and in severe cases lung transplantations are performed as a therapeutic measure. Thus, it becomes great interest to find novel agents to treat IPF. Berberine, a plant alkaloid known for its broad pharmacological activities remains a remedy against multiple diseases. This study was hypothesized to investigate the antifibrotic potential of berberine against bleomycin-induced lung injury and fibrosis, a tentative animal model. Male wistar rats were subjected to single intratracheal instillation of 2.5 U/kg of bleomycin on day 0. Berberine treatments were either provided in preventive or therapeutic mode respectively. Berberine administration significantly ameliorated the bleomycin mediated histological alterations and reduced the inflammatory cell infiltrate in BALF. Berberine significantly blocked collagen accumulations with parallel reduction in the hydroxyproline level. The immunological sign of bleomycin stimulated mast cell deposition and histamine release were considerably reduced by berberine. Berberine enhanced the antioxidant status, through upregulating the redox sensing transcription factor nuclear factor E2-related factor 2 (Nrf2). Berberine inhibited the bleomycin mediated activation of inflammatory mediator nuclear factor kappa B (NF-κB) and suppressed its downstream target inducible nitric oxide synthase (iNOS). Strikingly, berberine exhibited target attenuation of tumor necrosis factor alpha (TNF-α) and key pro-fibrotic mediator, transforming growth factor beta 1 (TGF-β1). Taken together, this study reveals the beneficial effects of berberine against bleomycin mediated fibrotic challenge through activating Nrf2 and suppressing NF-κB dependent inflammatory and TGF-β1 mediated fibrotic events.

Topics: Animals; Antimetabolites; Berberine; Bleomycin; Blotting, Western; Bronchoalveolar Lavage Fluid; Collagen; Histamine Release; Hydroxyproline; Immunohistochemistry; Lung Diseases; Male; Malondialdehyde; Microscopy, Confocal; NF-E2-Related Factor 2; NF-kappa B; Nitric Oxide Synthase Type II; Nitrites; Oxidative Stress; Peroxidase; Pulmonary Fibrosis; Rats; Rats, Wistar; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha; Weight Gain

Connective tissue growth factor (CTGF) plays an important role in the pathogenesis of chronic fibrotic diseases. However, the mechanism by which paracrine effects of CTGF control the cell fate of neighboring epithelial cells is not known. In this study, we investigated the paracrine effects of CTGF overexpressed in fibroblasts of Col1a2-CTGF transgenic mice on epithelial cells of skin and lung. The skin and lungs of Col1a2-CTGF transgenic mice were examined for phenotypic markers of epithelial activation and differentiation and stimulation of signal transduction pathways. In addition to an expansion of the dermal compartment in Col1a2-CTGF transgenic mice, the epidermis was characterized by focal hyperplasia, and basal cells stained positive for αSMA, Snail, S100A4 and Sox9, indicating that these cells had undergone a change in their genetic program. Activation of phosphorylated p38 and phosphorylated Erk1/2 was observed in the granular and cornified layers of the skin. Lung fibrosis was associated with a marked increase in cells co-expressing epithelial and mesenchymal markers in the lesional and unaffected lung tissue of Col1a2-CTGF mice. In epithelial cells treated with TGFβ, CTGF-specific siRNA-mediated knockdown suppressed Snail, Sox9, S100A4 protein levels and restored E-cadherin levels. Both adenoviral expression of CTGF in epithelial cells and treatment with recombinant CTGF induced EMT-like morphological changes and expression of α-SMA. Our in vivo and in vitro data supports the notion that CTGF expression in mesenchymal cells in the skin and lungs can cause changes in the differentiation program of adjacent epithelial cells. We speculate that these changes might contribute to fibrogenesis.

Topics: Animals; Biomarkers; Cell Differentiation; Cells, Cultured; Collagen Type I; Connective Tissue Growth Factor; Epithelial-Mesenchymal Transition; Fibroblasts; Focal Epithelial Hyperplasia; Lung; MAP Kinase Signaling System; Mice; Mice, Transgenic; Paracrine Communication; Pulmonary Fibrosis; RNA, Small Interfering; Signal Transduction; Skin; Transforming Growth Factor beta; Transgenes

Didecyldimethylammonium chloride (DDAC) is a representative dialkyl-quaternary ammonium compound that is used as a disinfectant against several pathogens and is also used in commercial, industrial, and residential settings. We previously investigated toxicity on air way system following single instillation of DDAC to the lungs in mice, and found that DDAC causes pulmonary injury, which is associated with altered antioxidant antimicrobial responses; the inflammatory phase is accompanied or followed by fibrotic response. The present study was conducted to monitor transforming growth factor-β (TGF-β) signaling in pulmonary fibrosis induced by DDAC. Mice were intratracheally instilled with DDAC and sacrificed 1, 3, or 7 days after treatment to measure TGF-β signaling. In order to further evaluate TGF-β signaling, we treated isolated mouse lung fibroblasts with DDAC. Fibrotic foci were observed in the lungs on day 3, and were widely extended on day 7, with evidence of increased α-smooth muscle actin-positive mesenchymal cells and upregulation of Type I procollagen mRNA. Developing fibrotic foci were likely associated with increased expression of Tgf-β1 mRNA, in addition to decreased expression of Bone morphogenetic protein-7 mRNA. In fibrotic lung samples, the expression of phosphorylated SMAD2/3 was considerably higher than that of phosphorylated SMAD1/5. In isolated lung fibroblasts, the mRNA levels of Tgf-β1 were specifically increased by DDAC treatment, which prolonged phosphorylation of SMAD2/3. These effects were abolished by treatment with SD208 - a TGF-βRI kinase inhibitor. The results suggest that DDAC induces pulmonary fibrosis in association with TGF-β signaling.

Topics: Animals; Blotting, Western; Disinfectants; Immunohistochemistry; Mice; Mice, Inbred C57BL; Pulmonary Fibrosis; Quaternary Ammonium Compounds; Reverse Transcriptase Polymerase Chain Reaction; Signal Transduction; Transforming Growth Factor beta

Myofibroblast differentiation induced by transforming growth factor-β (TGF-β) is characterized by the expression of smooth muscle α-actin (SMA) and extracellular matrix proteins. We and others have previously shown that these changes are regulated by protein kinase A (PKA). Adrenomedullin (ADM) is a vasodilator peptide that activates cAMP/PKA signaling through the calcitonin-receptor-like receptor (CRLR) and receptor-activity-modifying proteins (RAMP). In this study, we found that recombinant ADM had little effect on cAMP/PKA in quiescent human pulmonary fibroblasts, whereas it induced a profound activation of cAMP/PKA signaling in differentiated (by TGF-β) myofibroblasts. In contrast, the prostacyclin agonist iloprost was equally effective at activating PKA in both quiescent fibroblasts and differentiated myofibroblasts. TGF-β stimulated a profound expression of CRLR with a time course that mirrored the increased PKA responses to ADM. The TGF-β receptor kinase inhibitor SB431542 abolished expression of CRLR and attenuated the PKA responses of cells to ADM but not to iloprost. CRLR expression was also dramatically increased in lungs from bleomycin-treated mice. Functionally, ADM did not affect initial differentiation of quiescent fibroblasts in response to TGF-β but significantly attenuated the expression of SMA, collagen-1, and fibronectin in pre-differentiated myofibroblasts, which was accompanied by decreased contractility of myofibroblasts. Finally, sensitization of ADM signaling by transgenic overexpression of RAMP2 in myofibroblasts resulted in enhanced survival and reduced pulmonary fibrosis in the bleomycin model of the disease. In conclusion, differentiated pulmonary myofibroblasts gain responsiveness to ADM via increased CRLR expression, suggesting the possibility of using ADM for targeting pathological myofibroblasts without affecting normal fibroblasts.

Topics: Actins; Adrenomedullin; Animals; Bleomycin; Calcitonin Receptor-Like Protein; Cell Differentiation; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Humans; Iloprost; Mice; Myofibroblasts; Pulmonary Fibrosis; Receptor Activity-Modifying Protein 2; Signal Transduction; Transforming Growth Factor beta

The antifibrotic effect of simvastatin has been demonstrated in human lung fibroblasts. This study aimed to measure the effects of simvastatin in the development of pulmonary and cutaneous fibrosis in a murine model of SSc and to explore the mechanisms of these effects.. Chronic oxidant stress SSc was induced in BALB/c mice by daily s.c. injections of HOCl for 6 weeks. Mice were randomized in three arms: treatment with HOCl, HOCl plus simvastatin or vehicle alone. Statin treatment was initiated 30 min after HOCl s.c. injection and continued daily for 6 weeks. Skin and lung fibrosis were evaluated by histological methods. Immunohistochemical staining for α-smooth muscle actin in cutaneous and pulmonary tissues was performed to evaluate myofibroblast differentiation. Lung and skin concentrations of VEGF, extracellular signal-related kinase (ERK), rat sarcoma protein (Ras), Ras homologue gene family (Rho) and TGF-β were analysed by western blot.. Injections of HOCl induced cutaneous and lung fibrosis in BALB/c mice. Simvastatin treatment prevented both skin thickness and pulmonary fibrosis. Myofibroblast differentiation was also inhibited by simvastatin in the skin and in the lung. Increased cutaneous and pulmonary expression of VEGF, ERK, Ras and Rho in mice treated with HOCl was significantly lower in mice treated with HOCl plus simvastatin.. Simvastatin reduces the development of pulmonary fibrosis, potentially modulating adverse lung remodelling, as shown by the reduced deposition of collagen in alveolar septae. Simvastatin also reduces skin thickness in this model.

Topics: Animals; Biopsy, Needle; Blotting, Western; Disease Models, Animal; Dose-Response Relationship, Drug; Fibrosis; Immunohistochemistry; Mice; Mice, Inbred BALB C; Pulmonary Fibrosis; Random Allocation; Reactive Oxygen Species; Scleroderma, Systemic; Sensitivity and Specificity; Simvastatin; Skin Diseases; Transforming Growth Factor beta; Vascular Endothelial Growth Factor A

Bone morphogenetic protein-7 (BMP-7) has been shown to inhibit liver and renal fibrosis in in vivo and vitro studies. There is no study to investigate BMP-7's role in the development of pulmonary fibrosis induced by silica. In the current study, we used the rat model to explore the potential antifibrotic role of BMP-7 and its underlying mechanism in silica-induced pulmonary fibrosis. Sixty Wistar rats were randomly assigned into three groups. Control group received saline, silica group received silica and BMP-7 treated group received silica and BMP-7. BMP-7 was administered to silica-treated rats intraperitoneally at a dose of 300μg/kg/injection from day 8 to day 30 every other day. After the animals were sacrificed on day 15 and 30, hydroxyproline levels, the protein expressions of BMP/Smad and TGF-β/Smad signaling, and histopathology in lung tissues were analyzed. The hydroxyproline contents in BMP-7 treated groups were significantly lower than the silica groups (P<0.05). Histopathological results showed BMP-7 could reduce the progression of silica induced fibrosis. Furthermore, the expression of p-Smad1/5/8, a marker of BMP/Smad signaling, was significantly up-regulated in BMP-7 treated groups (P<0.05) compared with the silica groups. On the contrary, the expression of p-Smad2/3, a marker for TGF-β/Smad signaling, reduced significantly in BMP-7-treated groups compared with silica groups (P<0.05). In conclusion, the pulmonary fibrosis induced by silica in rats was significantly reduced with the therapeutic treatment of BMP-7. The antifibrotic effect of BMP-7 could be related to the activation of BMP/Smad signaling and inhibition of TGF-β/Smad pathways.

Topics: Animals; Bone Morphogenetic Protein 7; Dust; Humans; Hydroxyproline; Lung; Male; Pulmonary Fibrosis; Random Allocation; Rats; Rats, Wistar; Recombinant Proteins; Signal Transduction; Silicon Dioxide; Smad Proteins; Transforming Growth Factor beta

The inflammation and fibrosis induced by silica dust are considered to be substantial responses in silicosis progression. Interleukin-1 beta (IL-1β) plays an important role in silica-induced lung inflammation, but the mechanisms that underlie the influence of IL-1β on the progression of silicosis remain unclear. In this study, the role of IL-1β in silica-induced inflammation and fibrosis was evaluated by administering a suspension of 2.5-mg silica dust, either with or without 40 μg anti-mouse IL-1β monoclonal antibody (mAb), to the lungs of male C57BL/6 mice. Silica + anti-IL-1β mAb-treated mice showed the depletion of IL-1β as well as the attenuation of inflammation, as evaluated in the bronchoalveolar lavage fluid (BALF) and histological sections from 1 to 84 days after silica exposure. Further study of the BALF indicated that inhibition of IL-1β could reduce the contents of tumor necrosis factor-alpha and monocyte chemoattractant protein-1. The real-time PCR and pathology results showed that the neutralization of IL-1β attenuated silica-induced fibrosis by inhibiting the gene expression of transforming growth factor-beta 1, collagen I and fibronectin. The examination of Th1-cytokine and Th2-cytokine suggested that depletion of IL-1β decelerated the Th1/Th2 balance toward a Th2-dominant response. In conclusion, the present study suggests that the neutralization of IL-1β attenuates silica-induced inflammation and fibrosis by inhibiting other inflammatory and fibrogenic mediators and modulating the Th1/Th2 balance.

Topics: Animals; Antibodies, Blocking; Antibodies, Monoclonal; Bronchoalveolar Lavage Fluid; Cell Count; Collagen Type I; Fibronectins; In Vitro Techniques; Interleukin-1beta; Lung; Male; Mice; Mice, Inbred C57BL; Pulmonary Alveoli; Pulmonary Fibrosis; Silicon Dioxide; Silicosis; Th1-Th2 Balance; Transforming Growth Factor beta

Focal adhesion kinase (FAK) is a nonreceptor tyrosine kinase involved in various biological functions, including cell survival, proliferation, migration, and adhesion. FAK is an essential factor for transforming growth factor β to induce myofibroblast differentiation. In the present study, we investigated whether the targeted inhibition of FAK by using a specific inhibitor, TAE226, has the potential to regulate pulmonary fibrosis. TAE226 showed inhibitory activity of autophosphorylation of FAK at tyrosine 397 in lung fibroblasts. The addition of TAE226 inhibited the proliferation of lung fibroblasts in response to various growth factors, including platelet-derived growth factor and insulin-like growth factor I, in vitro. TAE226 strongly suppressed the production of type I collagen by lung fibroblasts. Furthermore, treatment of fibroblasts with TAE226 reduced the expression of α-smooth muscle actin induced by transforming growth factor β, indicating the inhibition of differentiation of fibroblasts to myofibroblasts. Administration of TAE226 ameliorated the pulmonary fibrosis induced by bleomycin in mice even when used late in the treatment. The number of proliferating mesenchymal cells was reduced in the lungs of TAE226-treated mice. These data suggest that FAK signal plays a significant role in the progression of pulmonary fibrosis and that it can become a promising target for therapeutic approaches to pulmonary fibrosis.

Topics: Actins; Animals; Bleomycin; Cell Differentiation; Cell Proliferation; Collagen Type I; DNA; Epithelial Cells; Female; Fibroblasts; Focal Adhesion Kinase 1; Insulin-Like Growth Factor I; Mesenchymal Stem Cells; Mice; Mice, Inbred C57BL; Morpholines; Myofibroblasts; Phosphorylation; Platelet-Derived Growth Factor; Protein Kinase Inhibitors; Pulmonary Fibrosis; Transforming Growth Factor beta; Tyrosine

Multiwall carbon nanotubes (MWCNTs) have been widely used in many disciplines due to their unique physical and chemical properties, but have also raised great concerns about their possible negative health impacts, especially through occupational exposure. Although recent studies have demonstrated that MWCNTs induce granuloma formation and/or fibrotic responses in the lungs of rats or mice, their cellular and molecular mechanisms remain largely unaddressed. Here, it is reported that the TGF-β/Smad signaling pathway can be activated by MWCNTs and play a critical role in MWCNT-induced pulmonary fibrosis. Firstly, in vivo data show that spontaneously hypertensive (SH) rats administered long MWCNTs (20-50 μm) but not short MWCNTs (0.5-2 μm) exhibit increased fibroblast proliferation, collagen deposition and granuloma formation in lung tissue. Secondly, the in vivo experiments also indicate that only long MWCNTs can significantly activate macrophages and increase the production of transforming growth factor (TGF)-β1, which induces the phosphorylation of Smad2 and then the expression of collagen I/III and extracellular matrix (ECM) protease inhibitors in lung tissues. Finally, the present in vitro studies further demonstrate that the TGF-β/Smad signaling pathway is indeed necessary for the expression of collagen III in fibroblast cells. Together, these data demonstrate that MWCNTs stimulate pulmonary fibrotic responses such as fibroblast proliferation and collagen deposition in a TGF-β/Smad-dependent manner. These observations also suggest that tube length acts as an important factor in MWCNT-induced macrophage activation and subsequent TGF-β1 secretion. These in vivo and in vitro studies further highlight the potential adverse health effects that may occur following MWCNT exposure and provide a better understanding of the cellular and molecular mechanisms by which MWCNTs induce pulmonary fibrotic reactions.

Topics: Animals; Macrophage Activation; Nanotubes, Carbon; Pulmonary Fibrosis; Rats; Rats, Inbred SHR; Signal Transduction; Smad Proteins; Transforming Growth Factor beta

Pulmonary Fibrosis (PF) is a devastating progressive disease in which normal lung structure and function is compromised by scarring. Lung fibrosis can be caused by thoracic radiation, injury from chemotherapy and systemic diseases such as rheumatoid arthritis that involve inflammatory responses. CDDO-Me (Methyl 2-cyano-3,12-dioxooleana-1,9(11)dien-28-oate, Bardoxolone methyl) is a novel triterpenoid with anti-fibrotic and anti-inflammatory properties as shown by our in vitro studies. Based on this evidence, we hypothesized that CDDO-Me would reduce lung inflammation, fibrosis and lung function impairment in a bleomycin model of lung injury and fibrosis. To test this hypothesis, mice received bleomycin via oropharyngeal aspiration (OA) on day zero and CDDO-Me during the inflammatory phase from days -1 to 9 every other day. Bronchoalveolar lavage fluid (BALF) and lung tissue were harvested on day 7 to evaluate inflammation, while fibrosis and lung function were evaluated on day 21. On day 7, CDDO-Me reduced total BALF protein by 50%, alveolar macrophage infiltration by 40%, neutrophil infiltration by 90% (p≤0.01), inhibited production of the inflammatory cytokines KC and IL-6 by over 90% (p≤0.001), and excess production of the pro-fibrotic cytokine TGFβ by 50%. CDDO-Me also inhibited α-smooth muscle actin and fibronectin mRNA by 50% (p≤0.05). On day 21, CDDO-Me treatment reduced histological fibrosis, collagen deposition and αSMA production. Lung function was significantly improved at day 21 by treatment with CDDO-Me, as demonstrated by respiratory rate and dynamic compliance. These new findings reveal that CDDO-Me exhibits potent anti-fibrotic and anti-inflammatory properties in vivo. CDDO-Me is a potential new class of drugs to arrest inflammation and ameliorate fibrosis in patients who are predisposed to lung injury and fibrosis incited by cancer treatments (e.g. chemotherapy and radiation) and by systemic autoimmune diseases.

Topics: Actins; Administration, Inhalation; Animals; Anti-Inflammatory Agents, Non-Steroidal; Bleomycin; Bronchoalveolar Lavage Fluid; Collagen; Fibronectins; Gene Expression; Humans; Macrophages, Alveolar; Male; Mice; Mice, Inbred C57BL; Neutrophil Infiltration; Oleanolic Acid; Pneumonia; Pulmonary Fibrosis; Respiratory Function Tests; Transforming Growth Factor beta

No successful therapies are available for pulmonary fibrosis, indicating the need for new treatments. Lipoxins and their 15-epimers, aspirin-triggered lipoxins (ATL), present potent antiinflammatory and proresolution effects (Martins et al., J Immunol 2009;182:5374-5381). We show that ATLa, an ATL synthetic analog, therapeutically reversed a well-established pulmonary fibrotic process induced by bleomycin (BLM) in mice. We investigated the mechanisms involved in its effect and found that systemic treatment with ATLa 1 week after BLM instillation considerably reversed the inflammatory response, total collagen and collagen type 1 deposition, vascular endothelial growth factor, and transforming growth factor (TGF)-β expression in the lung and restored surfactant protein C expression levels. ATLa also inhibited BLM-induced apoptosis and cellular accumulation in bronchoalveolar lavage fluid and in the lung parenchyma as evaluated by light microscopy and flow cytometry (Ly6G(+), F4/80(+), CD11c(+), CD4(+), and B220(+) cells) assays. Moreover, ATLa inhibited the lung production of IL-1β, IL-17, TNF-α, and TGF-β induced by BLM-challenged mice. ATLa restored the balance of inducible nitric oxide synthase-positive and arginase-positive cells in the lungs, suggesting a prevalence of M2 versus M1 macrophages. Together, these effects improved pulmonary mechanics because ATLa treatment brought to normal levels lung resistance and elastance, which were clearly altered at 7 days after BLM challenge. Our findings support ATLa as a promising therapeutic agent to treat lung fibrosis.

Topics: Animals; Arginase; Aspirin; Biomarkers; Bleomycin; Bronchoalveolar Lavage Fluid; Cell Count; Cytokines; Disease Models, Animal; Intercellular Signaling Peptides and Proteins; Lipoxins; Mice; Mice, Inbred C57BL; Nitric Oxide Synthase Type II; Peptides; Pulmonary Fibrosis; Pulmonary Surfactant-Associated Protein C; Respiratory Mechanics; Transforming Growth Factor beta; Vascular Endothelial Growth Factor A

Transforming growth factor β (TGFβ) has significant profibrotic activity both in vitro and in vivo. This reflects its capacity to stimulate fibrogenic mediators and induce the expression of other profibrotic cytokines such as platelet-derived growth factor (PDGF) and epidermal growth factor (EGF/ErbB) ligands. Here we address both the mechanisms by which TGFβ induced ErbB ligands and the physiological significance of inhibiting multiple TGFβ-regulated processes. The data document that ErbB ligand induction requires PDGF receptor (PDGFR) mediation and engages a positive autocrine/paracrine feedback loop via ErbB receptors. Whereas PDGFRs are essential for TGFβ-stimulated ErbB ligand up-regulation, TGFβ-specific signals are also required for ErbB receptor activation. Subsequent profibrotic responses are shown to involve the cooperative action of PDGF and ErbB signaling. Moreover, using a murine treatment model of bleomycin-induced pulmonary fibrosis we found that inhibition of TGFβ/PDGF and ErbB pathways with imatinib plus lapatinib, respectively, not only prevented myofibroblast gene expression to a greater extent than either drug alone, but also essentially stabilized gas exchange (oxygen saturation) as an overall measure of lung function. These observations provide important mechanistic insights into profibrotic TGFβ signaling and indicate that targeting multiple cytokines represents a possible strategy to ameliorate organ fibrosis dependent on TGFβ.

Topics: Animals; Benzamides; Bleomycin; Cell Line; Drug Interactions; Epidermal Growth Factor; ErbB Receptors; Feedback, Physiological; Imatinib Mesylate; Lapatinib; Lung; Mice; Myofibroblasts; Paracrine Communication; Piperazines; Platelet-Derived Growth Factor; Protein Kinase Inhibitors; Pulmonary Fibrosis; Pulmonary Gas Exchange; Pyrimidines; Quinazolines; Receptor, ErbB-2; Receptors, Platelet-Derived Growth Factor; Transforming Growth Factor beta; Up-Regulation

Topics: Amino Acid Oxidoreductases; Antibodies, Monoclonal; Apoptosis; Biomedical Research; Drug Delivery Systems; Humans; Pulmonary Fibrosis; Transforming Growth Factor beta

This study was designed to evaluate the role of interleukin (IL)-1β in the development of fibrosis in mice exposed to silica.. The total of 96 Male C57BL/6 mice were divided into four groups. (1) blank control group, (2) PBS group in which mice were instilled with PBS only, (3) silica + IL-1β mAb group in which mice were instilled with 2.5 mg silica dust and 40 µg anti-IL-1β mAb, (4) silica group in which mice were instilled with 2.5 mg silica dust and 40 µg IgG. The final volume of suspension or PBS instilled into the mouse was 50 µl. At 7, 28 and 84 days after treatment, 8 mice were sacrificed in each group. Then BALF was collected for the count of inflammatory cells and cytokines determination. The lung tissues were collected for the detecting of mRNA levels of fibrogenic molecules.. The collagen deposition induced by silica in the lung tissues was partly inhibited by anti-IL-1β. A intensely pulmonary cytokines such as IL-1β, TNF-α, MCP-1 were induced by crystalline silica exposure, and partly inhibited by anti-IL-1β. The levels of TGF-β and fibronectin in silica exposed mice were significantly elevated than those in control mice at days 28 and 84 after treatment (P < 0.01). And the mRNA levels of TGF-β, collagen I and fibronectin were significantly decreased in silica+IL-1β mAb group when compared with those in silica group at days 7, 28 and 84 (P < 0.01). There was a significant decrease of the ratios of IFN-γ/IL-4 in both silica+anti-IL-1β mAb and silica groups when compared with those in control mice at the above three time points (P < 0.01). However, the IFN-γ/IL-4 ratios in silica+anti-IL-1β group were significantly higher than those in silica group at 7, 28 and 84 days (P < 0.05 or P < 0.01).. IL-1β may promote the pulmonary fibrosis in mice exposed to silica.

Topics: Animals; Antibodies, Monoclonal; Bronchoalveolar Lavage Fluid; Collagen Type I; Disease Models, Animal; Fibronectins; Interferon-gamma; Interleukin-1beta; Interleukin-4; Lung; Male; Mice; Mice, Inbred C57BL; Pulmonary Fibrosis; Silicon Dioxide; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha

The excess and persistent accumulation of fibroblasts due to aberrant tissue repair results in fibrotic diseases such as idiopathic pulmonary fibrosis. Recent reports have revealed significant changes in microRNAs during idiopathic pulmonary fibrosis and evidence in support of a role for microRNAs in myofibroblast differentiation and the epithelial-mesenchymal transition in the context of fibrosis. It has been reported that microRNA-21 is up-regulated in myofibroblasts during fibrosis and promotes transforming growth factor-beta signaling by inhibiting Smad7. However, expression changes in microRNA-21 and the role of microRNA-21 in epithelial-mesenchymal transition during lung fibrosis have not yet been defined.. Lungs from saline- or bleomycin-treated C57BL/6 J mice and lung specimens from patients with idiopathic pulmonary fibrosis were analyzed. Enzymatic digestions were performed to isolate single lung cells. Lung epithelial cells were isolated by flow cytometric cell sorting. The expression of microRNA-21 was analyzed using both quantitative PCR and in situ hybridization. To induce epithelial-mesenchymal transition in culture, isolated mouse lung alveolar type II cells were cultured on fibronectin-coated chamber slides in the presence of transforming growth factor-β, thus generating conditions that enhance epithelial-mesenchymal transition. To investigate the role of microRNA-21 in epithelial-mesenchymal transition, we transfected cells with a microRNA-21 inhibitor. Total RNA was isolated from the freshly isolated and cultured cells. MicroRNA-21, as well as mRNAs of genes that are markers of alveolar epithelial or mesenchymal cell differentiation, were quantified using quantitative PCR.. The lung epithelial cells isolated from the bleomycin-induced lung fibrosis model system had decreased expression of epithelial marker genes, whereas the expression of mesenchymal marker genes was increased. MicroRNA-21 was significantly upregulated in isolated lung epithelial cells during bleomycin-induced lung fibrosis and human idiopathic pulmonary fibrosis. MicroRNA-21 was also upregulated in the cultured alveolar epithelial cells under the conditions that enhance epithelial-mesenchymal transition. Exogenous administration of a microRNA-21 inhibitor prevented the increased expression of vimentin and alpha-smooth muscle actin in cultured primary mouse alveolar type II cells under culture conditions that induce epithelial-mesenchymal transition.. Our experiments demonstrate that microRNA-21 is increased in lung epithelial cells during lung fibrosis and that it promotes epithelial-mesenchymal transition.

Topics: Actins; Animals; Bleomycin; Cells, Cultured; Disease Models, Animal; Epithelial Cells; Epithelial-Mesenchymal Transition; Humans; Lung; Male; Mice; Mice, Inbred C57BL; MicroRNAs; Pulmonary Fibrosis; RNA; Transfection; Transforming Growth Factor beta; Vimentin

Caveolar domains act as platforms for the organization of molecular complexes involved in signal transduction. Caveolin proteins, the principal structural components of caveolae, have been involved in many cellular processes. Caveolin-1 (Cav-1) and caveolin-2 (Cav-2) are highly expressed in the lung. Cav-1-deficient mice (Cav-1(-/-)) and Cav-2-deficient mice (Cav-2(-/-)) exhibit severe lung dysfunction attributed to a lack of Cav-2 expression. Recently, Cav-1 has been shown to regulate lung fibrosis in different models. Here, we show that Cav-2 is also involved in modulation of the fibrotic response, but through distinct mechanisms. Treatment of wild-type mice with the pulmonary fibrosis-inducer bleomycin reduced the expression of Cav-2 and its phosphorylation at tyrosine 19. Importantly, Cav-2(-/-) mice, but not Cav-1(-/-) mice, were more sensitive to bleomycin-induced lung injury in comparison to wild-type mice. Bleomycin-induced lung injury was characterized by alveolar thickening, increase in cell density, and extracellular matrix deposition. The lung injury observed in bleomycin-treated Cav-2(-/-) mice was not associated with alterations in the TGF-β signaling pathway and/or in the ability to produce collagen. However, apoptosis and proliferation were more prominent in lungs of bleomycin-treated Cav-2(-/-) mice. Since Cav-1(-/-) mice also lack Cav-2 expression and show a different outcome after bleomycin treatment, we conclude that Cav-1 and Cav-2 have distinct roles in bleomycin induced-lung fibrosis, and that the balance of both proteins determines the development of the fibrotic process.

Topics: Acute Lung Injury; Animals; Apoptosis; Bleomycin; Caveolin 1; Caveolin 2; Cell Survival; Collagen; Epithelial Cells; Extracellular Matrix; Gene Expression Regulation; Lung; Male; Mice; Mice, Knockout; Pulmonary Fibrosis; Respiratory Mucosa; Signal Transduction; Transforming Growth Factor beta

IGF-1 is elevated in pulmonary fibrosis and acute lung injury, where fibroblast activation is a prominent feature. We previously demonstrated that blockade of IGF pathway in murine model of lung fibrosis improved outcome and decreased fibrosis. We now expand that study to examine effects of IGF pathway on lung fibroblast behaviors that could contribute to fibrosis.. We first examined mice that express αSMA promoter upstream of GFP reporter treated with A12, a blocking antibody to IGF-1 receptor, after bleomycin induced lung injury. We then examined the effect of IGF-1 alone, or in combination with the pro-fibrotic cytokine TGFβ on expression of markers of myofibroblast activation in vitro, including αSMA, collagen α1, type 1, collagen α1, type III, and TGFβ expression.. After bleomycin injury, we found decreased number of αSMA-GFP + cells in A12 treated mice, validated by αSMA immunofluorescent staining. We found that IGF-1, alone or in combination with TGF-β, did not affect αSMA RNA expression, promoter activity, or protein levels when fibroblasts were cultured on stiff substrate. IGF-1 stimulated Col1a1 and Col3a1 expression on stiff substrate. In contrast, IGF-1 treatment on soft substrate resulted in upregulation of αSMA gene and protein expression, as well as Col1a1 and Col3a1 transcripts. In conclusion, IGF-1 stimulates differentiation of fibroblasts into a myofibroblast phenotype in a soft matrix environment and has a modest effect on αSMA stress fiber organization in mouse lung fibroblasts.

Topics: Actins; Animals; Antibodies, Monoclonal; Bleomycin; Cell Differentiation; Collagen; Disease Models, Animal; Fibroblasts; In Vitro Techniques; Insulin-Like Growth Factor I; Lung; Mice; Mice, Inbred C57BL; Mice, Transgenic; Myofibroblasts; Pulmonary Fibrosis; Receptor, IGF Type 1; Signal Transduction; Transforming Growth Factor beta

Idiopathic pulmonary fibrosis (IPF) is a fatal interstitial lung disease characterized by progressive scarring and matrix deposition. Recent reports highlight an autoimmune component in IPF pathogenesis. We have reported anti-col(V) immunity in IPF patients. The objective of our study was to determine the specificity of col(V) expression profile and anti-col(V) immunity relative to col(I) in clinical IPF and the efficacy of nebulized col(V) in pre-clinical IPF models.. Col(V) and col(I) expression profile was analyzed in normal human and IPF tissues. C57-BL6 mice were intratracheally instilled with bleomycin (0.025 U) followed by col(V) nebulization at pre-/post-fibrotic stage and analyzed for systemic and local responses.. Compared to normal lungs, IPF lungs had higher protein and transcript expression of the alpha 1 chain of col(V) and col(I). Systemic anti-col(V) antibody concentrations, but not of anti-col(I), were higher in IPF patients. Nebulized col(V), but not col(I), prevented bleomycin-induced fibrosis, collagen deposition, and myofibroblast differentiation. Col(V) treatment suppressed systemic levels of anti-col(V) antibodies, IL-6 and TNF-α; and local Il-17a transcripts. Compared to controls, nebulized col(V)-induced tolerance abrogated antigen-specific proliferation in mediastinal lymphocytes and production of IL-17A, IL-6, TNF-α and IFN-γ. In a clinically relevant established fibrosis model, nebulized col(V) decreased collagen deposition. mRNA array revealed downregulation of genes specific to fibrosis (Tgf-β, Il-1β, Pdgfb), matrix (Acta2, Col1a2, Col3a1, Lox, Itgb1/6, Itga2/3) and members of the TGF-β superfamily (Tgfbr1/2, Smad2/3, Ltbp1, Serpine1, Nfkb/Sp1/Cebpb).. Anti-col(V) immunity is pathogenic in IPF, and col(V)-induced tolerance abrogates bleomycin-induced fibrogenesis and down regulates TGF- β-related signaling pathways.

Topics: Animals; Autoantibodies; Bleomycin; Collagen Type I; Collagen Type V; Cytokines; Disease Models, Animal; Female; Gene Expression; Gene Expression Regulation; Humans; Idiopathic Pulmonary Fibrosis; Immune Tolerance; Inflammation Mediators; Lymphocyte Activation; Mice; Nebulizers and Vaporizers; Pulmonary Fibrosis; RNA, Messenger; T-Lymphocyte Subsets; Transcription, Genetic; Transforming Growth Factor beta

Idiopathic pulmonary fibrosis (IPF) is a chronic devastating disease with poor prognosis. Multiple pathological processes, including inflammation, epithelial mesenchymal transition (EMT), apoptosis, and oxidative stress, are involved in the pathogenesis of IPF. Recent findings suggested that nuclear factor-κB (NF-κB) is constitutively activated in IPF and acts as a central regulator in the pathogenesis of IPF. The aim of our study was to reveal the value of andrographolide on bleomycin-induced inflammation and fibrosis in mice. The indicated dosages of andrographolide were administered in mice with bleomycin-induced pulmonary fibrosis. On day 21, cell counts of total cells, macrophages, neutrophils and lymphocytes, alone with TNF-α in bronchoalveolar lavage fluid (BALF) were measured. HE staining and Masson's trichrome (MT) staining were used to observe the histological alterations of lungs. The Ashcroft score and hydroxyproline content of lungs were also measured. TGF-β1 and α-SMA mRNA and protein were analyzed. Activation of NF-κB was determined by western blotting and electrophoretic mobility shift assay (EMSA). On day 21 after bleomycin stimulation, andrographolide dose-dependently inhibited the inflammatory cells and TNF-α in BALF. Meanwhile, our data demonstrated that the Ashcroft score and hydroxyproline content of the bleomycin-stimulated lung were reduced by andrographolide administration. Furthermore, andrographloide suppressed TGF-β1 and α-SMA mRNA and protein expression in bleomycin-induced pulmonary fibrosis. Meanwhile, andrographolide significantly dose-dependently inhibited the ratio of phospho-NF-κB p65/total NF-κB p65 and NF-κB p65 DNA binding activities. Our findings indicate that andrographolide compromised bleomycin-induced pulmonary inflammation and fibrosis possibly through inactivation of NF-κB. Andrographolide holds promise as a novel drug to treat the devastating disease of pulmonary fibrosis.

Topics: Actins; Animals; Anti-Inflammatory Agents; Bleomycin; Bronchoalveolar Lavage Fluid; Diterpenes; Down-Regulation; Lung; Lymphocyte Count; Lymphocytes; Male; Mice; Mice, Inbred C57BL; NF-kappa B; Protein Binding; Pulmonary Fibrosis; RNA, Messenger; Transforming Growth Factor beta; Transforming Growth Factor beta1; Tumor Necrosis Factor-alpha

Owing to the development of new materials and technology, the pollutants in the environment are becoming more varied and complex over time. In our previous study using ICR mice, we suggested that a single intratracheal instillation of single-walled carbon nanotubes (SWCNTs) induced early lung fibrosis and subchronic tissue damage. In the present study, to investigate the role of CCR5 in inflammatory responses to the uptake of SWCNTs, we compared BAL (Bronchoalveolar lavage) cell composition, cell cycles, cytokines, cell phenotypes, inflammatory response-related proteins, cell surface receptors and histopathology using CCR5 knockout (KO) and wild-type mice. Results showed that the distribution of neutrophils in BAL fluid significantly decreased in KO mice. The expression of apoptosis-related proteins including caspase-3, p53, phospho-p53, p21 and cleaved PARP, TGF βl and mesothelin markedly increased in KO mice compared with wild-type mice. Histopathological lesions were also more frequently noted in KO mice. Moreover, the secretion of IL-13 and IL-17 with IL-6 significantly increased in KO mice compared with wild-type mice, whereas that of IL-12 significantly decreased in comparison to wild-type mice. The distribution of B cells and CD8+ T cells was predominant in the inflammatory responses in KO mice, whereas that of T cells and CD4+ T cells was predominant in the inflammatory responses in wild-type mice. Furthermore, the expression of CCR4 and CCR7 significantly increased in KO mice. Based on these results, we suggest that the absence of CCR5 delays the resolution of inflammatory responses triggered by SWCNTs inflowing into the lungs and shifts inflammatory response for SWCNTs clearance from Th1-type to Th2-type.

Topics: Animals; Apoptosis; Bronchoalveolar Lavage; Caspase 3; Cell Cycle; Immunoglobulin E; Inflammation; Interleukin-13; Interleukin-17; Interleukin-6; Lung; Mesothelin; Mice; Mice, Inbred C57BL; Mice, Knockout; Nanotubes, Carbon; Neutrophils; Phenotype; Pulmonary Fibrosis; Receptors, CCR4; Receptors, CCR5; Receptors, CCR7; Transforming Growth Factor beta; Tumor Suppressor Protein p53

Increased tissue stiffness and epithelial-to-mesenchymal transitions (EMTs) are two seemingly discrete hallmarks of fibrotic diseases. Despite recent findings highlighting the influence of tissue mechanical properties on cell phenotype, it remains unclear what role increased tissue stiffness has in the regulation of previously reported fibronectin-mediated EMTs associated with pulmonary fibrosis. Nano-indentation testing of lung interstitial spaces showed that in vivo cell-level Young's moduli increase with the onset of fibrosis from ∼2 to ∼17 kPa. In vitro, we found that stiff, but not soft, fibronectin substrates induce EMT, a response dependent on cell contraction-mediated integrin activation of TGFβ. Activation or suppression of cell contractility with exogenous factors was sufficient to overcome the effect of substrate stiffness. Pulse-chase experiments indicate that the effect of cell contractility is dose- and time-dependent. In response to low levels of TGFβ on soft surfaces, either added exogenously or produced through thrombin-induced contraction, cells will initiate the EMT programme, but upon removal revert to an epithelial phenotype. These results identify matrix stiffness and/or cell contractility as critical targets for novel therapeutics for fibrotic diseases.

Topics: Alveolar Epithelial Cells; Animals; Biomechanical Phenomena; Bleomycin; Cells, Cultured; Cellular Microenvironment; Coculture Techniques; Disease Models, Animal; Elastic Modulus; Epithelial-Mesenchymal Transition; Fibronectins; Integrins; Laminin; Lung; Male; Mechanotransduction, Cellular; Mice; Mice, Inbred C57BL; Microscopy, Atomic Force; Mink; Nanotechnology; Phenotype; Pulmonary Fibrosis; Rats; Rats, Sprague-Dawley; Thrombin; Time Factors; Transforming Growth Factor beta

Previous studies suggest that single-walled carbon nanotube (SWCNT) exposure causes pulmonary fibrosis. We investigated the contribution of epithelial-mesenchymal transition (EMT) during SWCNT-induced pulmonary fibrosis. C57BL6 female mice were intratracheally instilled with SWCNT at 80 μg/mouse for up to 56 days. SWCNT exposure caused pulmonary epithelial and mesenchymal injury, followed by granulomatous and fibrotic changes. Immunofluorescence staining demonstrated the increasing occurrence of epithelial-derived fibroblasts up to 42 days post-exposure. Flow cytometry analysis revealed that 42.60% of N-cadherin (N-cad)-positive fibroblasts were derived from pulmonary epithelial cells, and, in separate experiments, 30.68% of SPC positive cells were stained for N-cad at 42 days. These epithelial-derived fibroblasts were functional in collagen production. With the progression of fibrosis, there were increases in the number of hyperplastic epithelial cells stained positively for TGF-β/p-Smad2 or β-catenin. Therefore, EMT contributes significantly to fibroblast expansion. Aberrant activations of TGF-β/p-Smad2 and β-catenin are postulated to induce EMT during SWCNT-induced pathogenic fibrosis.

Topics: Animals; beta Catenin; Epithelial-Mesenchymal Transition; Female; Flow Cytometry; Lung; Matrix Metalloproteinases, Secreted; Mice; Mice, Inbred C57BL; Nanotubes, Carbon; Pulmonary Fibrosis; Pulmonary Surfactant-Associated Proteins; Smad2 Protein; Transforming Growth Factor beta

To determine the role of insulin-like growth factor binding protein 3 (IGFBP-3) in mediating the effects of transforming growth factor β (TGFβ) on tenascin-C (TN-C) production and to assess the levels of TN-C in vivo in patients with systemic sclerosis (SSc)-associated pulmonary fibrosis.. Human primary lung fibroblasts were stimulated with TGFβ or IGFBP-3 in the presence or absence of specific small interfering RNAs and chemical inhibitors of the signaling cascade. TN-C levels in lung tissue specimens obtained from patients with SSc-associated pulmonary fibrosis were assessed using immunohistochemical analysis and were compared with the levels in specimens obtained from normal donors. TN-C levels were quantified in sera from normal donors and patients with SSc with or without pulmonary fibrosis, using an enzyme-linked immunosorbent assay.. IGFBP-3 mediated the induction of TN-C by TGFβ. Direct induction of TN-C by IGFBP-3 occurred in a p38 MAP kinase-dependent manner. TN-C levels were abundant in lung tissues from patients with SSc and were localized to subepithelial layers of the distal airways. No TN-C was detectable around the proximal airways. Patients with SSc-associated pulmonary fibrosis had significantly higher levels of circulating TN-C compared with SSc patients without pulmonary fibrosis. Longitudinal samples obtained from patients with SSc before and after the onset of pulmonary fibrosis showed increased levels of TN-C after the onset of pulmonary fibrosis.. IGFBP-3, which is overexpressed in fibrotic lungs, induces production of TN-C by subepithelial fibroblasts. The increased lung tissue levels of TN-C parallel the levels detected in the sera of SSc patients with pulmonary fibrosis, suggesting that TN-C may be a useful biomarker for SSc-related pulmonary fibrosis.

Topics: Cells, Cultured; Culture Media, Conditioned; Fibroblasts; Humans; Insulin-Like Growth Factor Binding Protein 3; Lung; p38 Mitogen-Activated Protein Kinases; Pulmonary Fibrosis; Recombinant Proteins; RNA Interference; RNA, Small Interfering; Scleroderma, Systemic; Tenascin; Transforming Growth Factor beta

Pulmonary fibrosis, characterized by excess deposition of extracellular matrix by myofibroblasts, is a serious component of chronic lung diseases. Cadherin-11 (CDH11) is increased in wound healing and fibrotic skin. We hypothesized that CDH11 is increased in pulmonary fibrosis and contributes its development. CDH11 expression was assessed in lung tissue from idiopathic pulmonary fibrosis patients. The role of CDH11 in lung fibrosis was determined using the bleomycin model of pulmonary fibrosis, and in vitro analyses were performed on A549 cells during the process of epithelial to mesenchymal transition (EMT). Immunohistochemical studies demonstrated CDH11 expression on fibroblasts, epithelial cells, and alveolar macrophages of patients with pulmonary fibrosis and mice given bleomycin. Interestingly, CDH11-deficient mice had decreased fibrotic endpoints in the bleomycin model of pulmonary fibrosis compared to wild-type mice. Furthermore, anti-CDH11-neutralizing monoclonal antibodies successfully treated established pulmonary fibrosis induced by bleomycin. TGF-β levels were reduced in bronchoalveolar lavage (BAL) fluid, BAL cells, and primary alveolar macrophages from CDH11-deficient mice. Mechanistic studies demonstrated that TGF-β up-regulated CDH11 expression on A549 cells, and inhibition of CDH11 expression using siRNA reduced TGF-β-induced EMT. Together, these results identify CDH11 as a novel therapeutic target for pulmonary fibrosis.

Topics: Animals; Bleomycin; Cadherins; Cell Line; Disease Models, Animal; Epithelial-Mesenchymal Transition; Female; Humans; Idiopathic Pulmonary Fibrosis; Macrophages, Alveolar; Mice; Mice, 129 Strain; Mice, Inbred C57BL; Mice, Knockout; Pulmonary Fibrosis; Transforming Growth Factor beta

The dysfunction of alveolar barriers is a critical factor in the development of lung injury and subsequent fibrosis, but the underlying molecular mechanisms remain poorly understood. To clarify the pathogenic roles of tight junctions in lung injury and fibrosis, we examined the altered expression of claudins, the major components of tight junctions, in the lungs of disease models with pulmonary fibrosis. Among the 24 known claudins, claudin-1, claudin-3, claudin-4, claudin-7, and claudin-10 were identified as components of airway tight junctions. Claudin-5 and claudin-18 were identified as components of alveolar tight junctions and were expressed in endothelial and alveolar epithelial cells, respectively. In experimental bleomycin-induced lung injury, the levels of mRNA encoding tight junction proteins were reduced, particularly those of claudin-18. The integrity of the epithelial tight junctions was disturbed in the fibrotic lesions 14 days after the intraperitoneal instillation of bleomycin. These results suggest that bleomycin mainly injured alveolar epithelial cells and impaired alveolar barrier function. In addition, we analyzed the influence of transforming growth factor-β (TGF-β), a critical mediator of pulmonary fibrosis that is upregulated after bleomycin-induced lung injury, on tight junctions in vitro. The addition of TGF-β decreased the expression of claudin-5 in human umbilical vein endothelial cells and disrupted the tight junctions of epithelial cells (A549). These results suggest that bleomycin-induced lung injury causes pathogenic alterations in tight junctions and that such alterations seem to be induced by TGF-β.

Topics: Animals; Bleomycin; Blood-Air Barrier; Cell Line; Claudins; Epithelial Cells; Female; Human Umbilical Vein Endothelial Cells; Humans; Lung; Lung Injury; Mice; Mice, Inbred C57BL; Pulmonary Alveoli; Pulmonary Fibrosis; Rats; Rats, Wistar; Respiratory Mucosa; RNA, Messenger; Tight Junctions; Transforming Growth Factor beta

Young (4 month) and aged (15-18 months) mice were given intranasal saline or γ--herpesvirus-68 infection. After 21 days, aged, but not young mice, showed significant increases in collagen content and fibrosis. There were no differences in viral clearance or inflammatory cells (including fibrocytes) between infected aged and young mice. Enzyme-linked immunosorbent assays showed increased transforming growth factor-β in whole lung homogenates of infected aged mice compared with young mice. When fibroblasts from aged and young mice were infected in vitro, aged, but not young, fibroblasts upregulate alpha-smooth muscle actin and collagen I protein. Infection with virus in vivo also demonstrates increased alpha-smooth muscle actin and collagen I protein and collagen I, collagen III, and fibronectin messenger RNA in aged fibroblasts. Furthermore, evaluation revealed that aged fibroblasts at baseline have increased transforming growth factor-β receptor 1 and 2 levels compared with young fibroblasts and are resistant to apoptosis. Increased responsiveness to transforming growth factor-β was verified by increased collagen III and fibronectin messenger RNA after treatment in vitro with transforming growth factor-β.

Topics: Aging; Animals; Apoptosis; Collagen Type III; Cytokines; Fibroblasts; Fibronectins; Gammaherpesvirinae; Herpesviridae Infections; Male; Mice; Mice, Inbred C57BL; Pulmonary Fibrosis; Receptors, Transforming Growth Factor beta; Transforming Growth Factor beta; Virus Activation

The incidence of idiopathic pulmonary fibrosis (IPF) increases with age. The mechanisms that underlie the age-dependent risk for IPF are unknown. Based on studies that suggest an association of IPF and γherpesvirus infection, we infected young (2-3 mo) and old (≥18 mo) C57BL/6 mice with the murine γherpesvirus 68. Acute murine γherpesvirus 68 infection in aging mice resulted in severe pneumonitis and fibrosis compared with young animals. Progressive clinical deterioration and lung fibrosis in the late chronic phase of infection was observed exclusively in old mice with diminution of tidal volume. Infected aging mice showed higher expression of transforming growth factor-β during the acute phase of infection. In addition, aging, infected mice showed elevation of proinflammatory cytokines and the fibrocyte recruitment chemokine, CXCL12, in bronchoalveolar lavage. Analyses of lytic virus infection and virus reactivation indicate that old mice were able to control chronic infection and elicit antivirus immune responses. However, old, infected mice showed a significant increase in apoptotic responses determined by in situ terminal deoxynucleotidyl transferase dUTP nick end labeling assay, levels of caspase-3, and expression of the proapoptotitc molecule, Bcl-2 interacting mediator. Apoptosis of type II lung epithelial cells in aging lungs was accompanied by up-regulation of endoplasmic reticulum stress marker, binding immunoglobulin protein, and splicing of X-box-binding protein 1. These results indicate that the aging lung is more susceptible to injury and fibrosis associated with endoplasmic reticulum stress, apoptosis of type II lung epithelial cells, and activation of profibrotic pathways.

Topics: Animals; Apoptosis; Blotting, Western; Bronchoalveolar Lavage Fluid; Endoplasmic Reticulum; Immunohistochemistry; Mice; Mice, Inbred C57BL; Pulmonary Fibrosis; Real-Time Polymerase Chain Reaction; Stress, Physiological; Transforming Growth Factor beta

Much evidence suggests that immune imbalance in the lung plays a crucial role in the development of pulmonary fibrosis. Recently, all-trans retinoic acid (ATRA) shifting the regulatory T/T-helper 17 (Treg/Th17) profile had been proven in some diseases. However, to date, the effect of ARTA of pulmonary fibrosis has not been examined from this aspect. The objective of this study was to study the effect of ATRA on bleomycin-induced pulmonary fibrosis in mice and its possible mechanism. Pulmonary fibrosis was induced in C57BL/6 male mice by intratracheal instillation of bleomycin (5 mg.kg(-1)), which were randomly divided into control, bleomycin, and ATRA groups. Five mice in each group were sacrificed on day 28 after intratracheal instillation. Hemotoxylin and eosin (H&E) and Masson staining were used for pathological examination, and hydroxyproline in lung tissue was measured. Interleukin (IL)-17A protein expression was observed in lung with immunohistochemistry. The expression of IL-17A, IL-10, IL-6, and transforming growth factor (TGF)-β mRNAs were detected by reverse transcriptase-polymerase chain reaction (RT-PCR). Th17 and Treg expression in spleen lymphocytes were measured by flow cytometry. H&E and masson staining and expression of hydroxyproline showed that ATRA significantly alleviated lung fibrosis than in the bleomycin group. The expression of IL-17A, IL-10, IL-6, and TGF-β mRNAs were higher in the bleomycin group than in the normal group. ATRA can decrease these cytokines except for IL-10. CD4+CD25+ Treg cell ratio in the bleomycin group was significantly lower than normal, but CD4+IL-17+ T cells was higher; ARTA reversed this kind of expression. ATRA may ease the bleomycin-induced pulmonary fibrosis by inhibiting the expression of IL-6 and TGF-β, shifting the Treg/Th17 ratio and reducing the secretion of IL-17A.

Topics: Animals; Antibiotics, Antineoplastic; Bleomycin; CD4-Positive T-Lymphocytes; Cell Count; Disease Models, Animal; Drug Therapy, Combination; Gene Expression; Hydroxyproline; Interleukins; Lung; Male; Mice; Mice, Inbred C57BL; Pulmonary Fibrosis; T-Lymphocytes, Regulatory; Th17 Cells; Transforming Growth Factor beta; Tretinoin

Cyclooxygenase (COX)-inhibiting nitric oxide (NO) donors (CINODs) are designed to inhibit COX-1 and COX-2 while releasing NO. COX inhibition is responsible for anti-inflammatory and pain-relieving effects, whereas NO donation can improve microcirculation and exert anti-inflammatory and antioxidant actions. In an in vivo mouse model of bleomycin-induced lung fibrosis, we evaluated whether a prototype CINOD compound, (S)-(5S)-5,6-bis(nitrooxy)hexyl)2-(6-methoxynaphthalen-2-yl)propanoate (NCX 466), may show an advantage over naproxen, its congener drug not releasing NO. Bleomycin (0.05 IU) was instilled intratracheally to C57BL/6 mice, which were then treated orally with vehicle, NCX 466 (1.9 or 19 mg/kg), or an equimolar dose of naproxen (1 or 10 mg/kg) once daily for 14 days. Afterward, airway resistance, assumed as lung stiffness index, was assayed, and lung specimens were collected for analysis of lung inflammation and fibrosis. NCX 466 and naproxen dose-dependently prevented bleomycin-induced airway stiffness and collagen accumulation. NCX 466, at the highest dose, was significantly more effective than naproxen in reducing the levels of the profibrotic cytokine transforming growth factor-β and the oxidative stress markers thiobarbituric acid reactive substance and 8-hydroxy-2'-deoxyguanosine. NCX 466 also decreased myeloperoxidase activity, a leukocyte recruitment index, to a greater extent than naproxen. A similar inhibition of prostaglandin E₂ was achieved by both compounds. In conclusion, NCX 466 has shown a significantly higher efficacy than naproxen in reducing lung inflammation and preventing collagen accumulation. These findings suggest that COX inhibition along with NO donation may possess a therapeutic potential in lung inflammatory diseases with fibrotic outcome.

Topics: 8-Hydroxy-2'-Deoxyguanosine; Animals; Anti-Inflammatory Agents; Antibiotics, Antineoplastic; Bleomycin; Collagen; Cyclooxygenase Inhibitors; Deoxyguanosine; Dinoprostone; Inflammation; Lung; Male; Mice; Mice, Inbred C57BL; Naproxen; Nitrates; Nitric Oxide; Nitric Oxide Donors; Oxidative Stress; Peroxidase; Propionates; Prostaglandin-Endoperoxide Synthases; Pulmonary Fibrosis; Thiobarbiturates; Transforming Growth Factor beta

Oxymatrine (OM) is an alkaloid extracted from the Chinese herb Sophora flavescens Ait. with a variety of pharmacological activities. The aim of this study was to investigate the preventive effects of OM on bleomycin (BLM)-induced pulmonary fibrosis (PF) and to further explore the underlying mechanisms. C57BL/6 mice were randomly assigned to five groups: the saline sham group; the BLM group, in which mice were endotracheally instilled with BLM (3.0 mg/kg); and the BLM plus OM groups, in which OM was given to mice daily (10, 20 or 40 mg/kg) one day after BLM instillation for 21 days. The bronchoalveolar lavage fluid (BALF) and lung tissues were collected at 15 and 22 days post BLM administration, respectively. Lung tissues were stained with hematoxylin and eosin (H&E) for histological evaluation. Levels of tumor necrosis factor (TNF)-α, interleukin-6 (IL-6) and nitric oxide (NO) in mouse BALF were measured, as well as myeloperoxidase (MPO) activity and malondialdehyde (MDA) content in lung homogenates. The inducible nitric oxide synthase (iNOS) expression in the lung tissues was determined by immunohistochemical staining, quantitative real-time PCR and western blot analysis. Moreover, the expression of transforming growth factor (TGF)-β1, Smad2, Smad3, p-Smad2 and p-Smad3 were also detected. We found that OM improved BLM-induced lung pathological changes, inhibited MPO activity and reduced MDA levels in a dose-dependent manner. OM also dose-dependently inhibited the release of TNF-α and IL-6, and decreased the expression of iNOS in lung tissues and thus prevented NO release in response to BLM challenge. In addition, OM decreased the expression of TGF-β1, p-Smad2 and p-Smad3, which are all important members of the TGF-β/Smad signaling pathway. Our study provides evidence that OM significantly ameliorated BLM-induced PF in mice via the inhibition of iNOS expression and the TGF-β/Smad pathway.

Topics: Alkaloids; Animals; Antibiotics, Antineoplastic; Antiviral Agents; Bleomycin; Lung; Male; Mice; Mice, Inbred C57BL; Nitric Oxide Synthase Type II; Peroxidase; Pulmonary Fibrosis; Quinolizines; Signal Transduction; Smad Proteins; Sophora; Transforming Growth Factor beta

Loss of microRNA-29 (miR-29) is known to be a mechanism of transforming growth factor-β (TGF-β)-mediated pulmonary fibrosis, but the therapeutic implication of miR-29 for pulmonary fibrosis remains unexplored. The present study investigated whether miR-29 had therapeutic potential for lung disease induced by bleomycin in mice. In addition, the signaling mechanisms that regulated miR-29 expression were investigated in vivo and in vitro. We found that miR-29 was a downstream target gene of Smad3 and negatively regulated by TGF-β/Smad signaling in fibrosis. This was evidenced by the findings that mice or pulmonary fibroblasts null for Smad3 were protected against bleomycin or TGF-β1-induced loss of miR-29 along with fibrosis in vivo and in vitro. Interestingly, overexpression of miR-29 could in turn negatively regulated TGF-β and connective tissue growth factor (CTGF) expression and Smad3 signaling. Therefore, Sleeping Beauty (SB)-mediated miR-29 gene transfer into normal and diseased lung tissues was capable of preventing and treating pulmonary fibrosis including inflammatory macrophage infiltration induced by bleomycin in mice. In conclusion, miR-29 is negatively regulated by TGF-β/Smad3 and has a therapeutic potential for pulmonary fibrosis. SB-mediated miR-29 gene therapy is a non-invasive therapeutic strategy for lung disease associated with fibrosis.

Topics: Animals; Bleomycin; Cells, Cultured; Disease Models, Animal; DNA Transposable Elements; Female; Fibroblasts; Gene Expression; Gene Expression Regulation; Gene Transfer Techniques; Genetic Therapy; Mice; Mice, Inbred C57BL; Mice, Knockout; MicroRNAs; Pulmonary Fibrosis; Signal Transduction; Smad3 Protein; Time Factors; Transforming Growth Factor beta

The inhalation of asbestos fibers is considered to be highly harmful, and lead to fibrotic and/or malignant disease. Epithelial-to-mesenchymal transition (EMT) is a common pathogenic mechanism in asbestos associated fibrotic (asbestosis) and malignant lung diseases. The characterization of molecular pathways contributing to EMT may provide new possibilities for prognostic and therapeutic applications. The role of asbestos as an inducer of EMT has not been previously characterized. We exposed cultured human lung epithelial cells to crocidolite asbestos and analyzed alterations in the expression of epithelial and mesenchymal marker proteins and cell morphology. Asbestos was found to induce downregulation of E-cadherin protein levels in A549 lung carcinoma cells in 2-dimensional (2D) and 3D cultures. Similar findings were made in primary small airway epithelial cells cultured in 3D conditions where the cells retained alveolar type II cell phenotype. A549 cells also exhibited loss of cell-cell contacts, actin reorganization and expression of α-smooth muscle actin (α-SMA) in 2D cultures. These phenotypic changes were not associated with increased transforming growth factor (TGF)-β signaling activity. MAPK/Erk signaling pathway was found to mediate asbestos-induced downregulation of E-cadherin and alterations in cell morphology. Our results suggest that asbestos can induce epithelial plasticity, which can be interfered by blocking the MAPK/Erk kinase activity.

Topics: Actins; Alveolar Epithelial Cells; Asbestos, Crocidolite; Cadherins; Cell Line, Tumor; Epithelial Cells; Epithelial-Mesenchymal Transition; Extracellular Signal-Regulated MAP Kinases; Humans; JNK Mitogen-Activated Protein Kinases; MAP Kinase Signaling System; NF-kappa B; Pulmonary Alveoli; Pulmonary Fibrosis; Transforming Growth Factor beta

The enforcement of sphingosine-1-phosphate (S1P) signaling network protects from radiation-induced pneumonitis. We now demonstrate that, in contrast to early postirradiation period, late postirradiation sphingosine kinase-1 (SphK1) and sphingoid base-1-phosphates are associated with radiation-induced pulmonary fibrosis (RIF). Using the mouse model, we demonstrate that RIF is characterized by a marked upregulation of S1P and dihydrosphingosine-1-phosphate (DHS1P) levels in the lung tissue and in circulation accompanied by increased lung SphK1 expression and activity. Inhibition of sphingolipid de novo biosynthesis by targeting serine palmitoyltransferase (SPT) with myriocin reduced radiation-induced pulmonary inflammation and delayed the onset of RIF as evidenced by increased animal lifespan and decreased expression of markers of fibrogenesis, such as collagen and α-smooth muscle actin (α-SMA), in the lung. Long-term inhibition of SPT also decreased radiation-induced SphK activity in the lung and the levels of S1P-DHS1P in the lung tissue and in circulation. In vitro, inhibition or silencing of serine palmitoyltransferase attenuated transforming growth factor-β1 (TGF-β)-induced upregulation of α-SMA through the negative regulation of SphK1 expression in normal human lung fibroblasts. These data demonstrate a novel role for SPT in regulating TGF-β signaling and fibrogenesis that is linked to the regulation of SphK1 expression and S1P-DHS1P formation.

Topics: Animals; Enzyme Inhibitors; Fatty Acids, Monounsaturated; Female; Gene Expression Regulation, Enzymologic; Humans; Mice; Phosphotransferases (Alcohol Group Acceptor); Pulmonary Fibrosis; Radiation Injuries, Experimental; Serine C-Palmitoyltransferase; Signal Transduction; Sphingosine; Thorax; Time Factors; Transforming Growth Factor beta; Up-Regulation

Fibroproliferative disorders such as idiopathic pulmonary fibrosis and systemic sclerosis have no effective therapies and result in significant morbidity and mortality due to progressive organ fibrosis. We examined the effect of peptides derived from endostatin on existing fibrosis and fibrosis triggered by two potent mediators, transforming growth factor-β (TGF-β) and bleomycin, in human and mouse tissues in vitro, ex vivo, and in vivo. We identified one peptide, E4, with potent antifibrotic activity. E4 prevented TGF-β-induced dermal fibrosis in vivo in a mouse model, ex vivo in human skin, and in bleomycin-induced dermal and pulmonary fibrosis in vivo, demonstrating that E4 exerts potent antifibrotic effects. In addition, E4 significantly reduced existing fibrosis in these preclinical models. E4 amelioration of fibrosis was accompanied by reduced cell apoptosis and lower levels of lysyl oxidase, an enzyme that cross-links collagen, and Egr-1 (early growth response gene-1), a transcription factor that mediates the effects of several fibrotic triggers. Our findings identify E4 as a peptide with potent antifibrotic activity and a possible therapeutic agent for organ fibrosis.

Topics: Animals; Bleomycin; Endostatins; Fibrosis; Humans; In Vitro Techniques; Male; Mice; Mice, Inbred C57BL; Peptides; Protein-Lysine 6-Oxidase; Pulmonary Fibrosis; Skin; Transforming Growth Factor beta

Lung fibroblast functions such as matrix remodeling and activation of latent transforming growth factor-β1 (TGF-β1) are associated with expression of the myofibroblast phenotype and are directly linked to fibroblast capacity to generate force and deform the extracellular matrix. However, the study of fibroblast force-generating capacities through methods such as traction force microscopy is hindered by low throughput and time-consuming procedures. In this study, we improved at the detail level methods for higher-throughput traction measurements on polyacrylamide hydrogels using gel-surface-bound fluorescent beads to permit autofocusing and automated displacement mapping, and transduction of fibroblasts with a fluorescent label to streamline cell boundary identification. Together these advances substantially improve the throughput of traction microscopy and allow us to efficiently compute the forces exerted by lung fibroblasts on substrates spanning the stiffness range present in normal and fibrotic lung tissue. Our results reveal that lung fibroblasts dramatically alter the forces they transmit to the extracellular matrix as its stiffness changes, with very low forces generated on matrices as compliant as normal lung tissue. Moreover, exogenous TGF-β1 selectively accentuates tractions on stiff matrices, mimicking fibrotic lung, but not on physiological stiffness matrices, despite equivalent changes in Smad2/3 activation. Taken together, these results demonstrate a pivotal role for matrix mechanical properties in regulating baseline and TGF-β1-stimulated contraction of lung fibroblasts and suggest that stiff fibrotic lung tissue may promote myofibroblast activation through contractility-driven events, whereas normal lung tissue compliance may protect against such feedback amplification of fibroblast activation.

Topics: Cells, Cultured; Extracellular Matrix; Fetus; Fibroblasts; Fluorescent Antibody Technique; Humans; Lung; Microscopy; Phosphorylation; Pulmonary Fibrosis; Signal Transduction; Smad2 Protein; Smad3 Protein; Traction; Transforming Growth Factor beta

Lung fibrosis can affect the parenchyma and the airways, classically giving rise to idiopathic pulmonary fibrosis (IPF) in the parenchyma or airway remodeling in asthma and chronic obstructive pulmonary disease. TGF-β activation has been implicated in the fibrosis of both IPF and airway remodeling. However, the mechanisms of TGF-β activation appear to differ depending on the cellular and anatomical compartments, with implications on disease pathogenesis. Although it appears that epithelial cell activation of TGF-β by the αvβ6 integrin is central in IPF, mesenchymal activation of TGF-β by the αvβ5 and αvβ8 integrins appears to predominate in airway remodeling. Interestingly, the mechanism of TGF-β by the integrins αvβ6 and αvβ5 is shared, relying on cytoskeletal changes, whereas activation of TGF-β by the αvβ8 integrin is distinct, relying on proteolytic cleavage of the latency-associated peptide of TGF-β by matrix metalloproteinase 14. This article describes the mechanisms through which epithelial cells activate TGF-β by the αvβ6 integrin and mesenchymal cells activate TGF-β by the αvβ5 integrin, and highlights their roles in lung fibrosis.

Topics: Airway Remodeling; Asthma; Epithelial Cells; Epithelial-Mesenchymal Transition; Extracellular Matrix; Humans; Integrin alpha Chains; Integrin alphaV; Mesoderm; Pulmonary Alveoli; Pulmonary Fibrosis; Transforming Growth Factor beta

Transforming growth factor β (TGF-β) regulates inflammation, immunosuppression, and wound-healing cascades, but it remains unclear whether any of these functions involve regulation of myeloid cell function. The present study demonstrates that selective deletion of TGF-βRII expression in myeloid phagocytes i) impairs macrophage-mediated suppressor activity, ii) increases baseline mRNA expression of proinflammatory chemokines/cytokines in the lung, and iii) enhances type 2 immunity against the hookworm parasite Nippostrongylus brasiliensis. Strikingly, TGF-β-responsive myeloid cells promote repair of hookworm-damaged lung tissue, because LysM(Cre)TGF-βRII(flox/flox) mice develop emphysema more rapidly than wild-type littermate controls. Emphysematous pathology in LysM(Cre)TGF-βRII(flox/flox) mice is characterized by excessive matrix metalloprotease (MMP) activity, reduced lung elasticity, increased total lung capacity, and dysregulated respiration. Thus, TGF-β effects on myeloid cells suppress helminth immunity as a consequence of restoring lung function after infection.

Topics: Animals; Bone Marrow Cells; Emphysema; Hookworm Infections; Immunity; Lung; Lymphocyte Activation; Macrophages, Alveolar; Matrix Metalloproteinases; Mice; Mice, Inbred C57BL; Mice, Knockout; Myeloid Cells; Nippostrongylus; Pneumonia; Protein Serine-Threonine Kinases; Pulmonary Fibrosis; Receptor, Transforming Growth Factor-beta Type II; Receptors, Transforming Growth Factor beta; T-Lymphocytes; Transforming Growth Factor beta; Wound Healing

Previously, we found that catecholamine (CA) infusion in rats induced pulmonary injury with edema and inflammation resembling acute lung injury in humans. Here, we examined effects of norepinephrine (NE) and of selective α- and β-adrenergic agonists on the remodeling of pulmonary extracellular matrix.. Eighty rats were infused over 8-72 h with NE, phenylephrine (PE), isoproterenol (ISO) or NaCl solution. We investigated mRNA expression of collagen, matrix metalloproteinase (MMP)-2, its tissue inhibitor (TIMP-2) and transforming growth factor (TGF)-β isoforms in lung tissue. Additionally, lung histology, hemodynamic function and cardiac hypertrophy were evaluated.. After 72 h of infusion, lung histology showed beginning fibrosis and vascular hypertrophy. Collagen type I, MMP-2 and TIMP-2 mRNA expression were significantly elevated. All these effects were most pronounced with NE while PE and ISO induced weaker responses. TGF-β mRNA expression was also elevated after 72 h, predominantly after PE infusion. Cardiac hypertrophy was most pronounced after ISO infusion.. CA infusion over 72 h may induce pulmonary remodeling. Mainly α-adrenergic but also β-adrenergic mechanisms contribute to these processes. In contrast, cardiac hypertrophy is predominantly mediated by β-adrenergic stimulation and hence, is considered to be a direct adrenergic effect rather than a consequence of pulmonary fibrosis.

Topics: Animals; Extracellular Matrix; Female; Isoproterenol; Matrix Metalloproteinase 2; Norepinephrine; Phenylephrine; Pulmonary Fibrosis; Rats; Rats, Sprague-Dawley; Time Factors; Tissue Inhibitor of Metalloproteinase-2; Transforming Growth Factor beta

To investigate the therapeutic effect of C-phycocyanin (C-PC) from Spirulina platensis on paraquat (PQ)-induced pulmonary fibrosis in rats.. A total of 90 healthy Wistar rats were randomly and equally divided into normal control group, model group (PQ group), and C-PC treatment group (C-PC group). Each rat in the PQ group and C-PC group were orally administered with a single dose of PQ (50 mg/kg) to establish a rat model of PQ poisoning. Then, the rats in the normal control group and PQ group were orally given saline solution (1 ml/100 g) every day, and the rats in the C-PC group were orally given C-PC (50 mg/kg) every day. Six rats were randomly selected from each group on days 1, 3, 7, 14, and 28. The inferior lobe of each rat's right lung was homogenized for the measurement of hydroxyproline (HYP) and maleic dialdehyde (MDA) levels and superoxide dismutase (SOD) activity. Parts of each rat's left lung were subject to HE staining and Masson staining for pathological observation, and the expression of transforming growth factor-β(1) (TGF-β(1)), nuclear factor-kappa B p65 (NF-κB p65), and tumor necrosis factor-α (TNF-α) in lung tissue was measured by immunohistochemistry.. The HYP levels on days 1, 3, 7, 14, and 28 and MDA levels on days 14 and 28 were significantly lower in the C-PC group than in the PQ group (P < 0.05, P < 0.01). The SOD activity was significantly higher in the C-PC group than in the PQ group on days 1, 7, 14, and 28 (P < 0.05, P < 0.01). The protein content of TGF-β(1) and the activities of NF-κB p65 and TNF-α in the PQ group and C-PC group were significantly higher than those in the normal control group, while the indices in the C-PC group were significantly lower than those in the PQ group (P < 0.05, P < 0.01). The pathological observation showed that C-PC could alleviate pulmonary alveolitis and fibrosis in rats with PQ poisoning.. C-PC can significantly inhibit PQ-induced pulmonary alveolitis and fibrosis in rats.

Topics: Animals; Lung; Male; NF-kappa B; Paraquat; Phycocyanin; Pulmonary Fibrosis; Rats; Rats, Wistar; Transcription Factor RelA; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha

Interstitial lung disease is a devastating disease in humans that can be further complicated by the development of secondary pulmonary hypertension. Accumulating evidence indicates that the oxidant superoxide can contribute to the pathogenesis of both interstitial lung disease and pulmonary hypertension. We used a model of pulmonary hypertension secondary to bleomycin-induced pulmonary fibrosis to test the hypothesis that an imbalance in extracellular superoxide and its antioxidant defense, extracellular superoxide dismutase, will promote pulmonary vascular remodeling and pulmonary hypertension. We exposed transgenic mice overexpressing lung extracellular superoxide dismutase and wild-type littermates to a single dose of intratracheal bleomycin, and evaluated the mice weekly for up to 35 days. We assessed pulmonary vascular remodeling and the expression of several genes critical to lung fibrosis, as well as pulmonary hypertension and mortality. The overexpression of extracellular superoxide dismutase protected against late remodeling within the medial, adventitial, and intimal layers of the vessel wall after the administration of bleomycin, and attenuated pulmonary hypertension at the same late time point. The overexpression of extracellular superoxide dismutase also blocked the early up-regulation of two key genes in the lung known to be critical in pulmonary fibrosis and vascular remodeling, the transcription factor early growth response-1 and transforming growth factor-β. The overexpression of extracellular superoxide dismutase attenuated late pulmonary hypertension and significantly improved survival after exposure to bleomycin. These data indicate an important role for an extracellular oxidant/antioxidant imbalance in the pathogenesis of pulmonary vascular remodeling associated with secondary pulmonary hypertension attributable to bleomycin-induced lung fibrosis.

Topics: Animals; Bleomycin; Cell Proliferation; Early Growth Response Protein 1; Extracellular Space; Gene Expression Regulation; Humans; Hypertension, Pulmonary; Lung; Mice; Mice, Inbred C57BL; Mice, Transgenic; Nitric Oxide Synthase Type III; Nitrosation; Oxidation-Reduction; Pulmonary Artery; Pulmonary Fibrosis; Stress, Physiological; Superoxide Dismutase; Transforming Growth Factor beta; Tyrosine

Thrombospondin-1 (TSP-1) is an extracellular protein critical to normal lung homeostasis, and is reported to activate latent transforming growth factor-β (TGF-β). Because active TGF-β is causally involved in lung fibrosis after bleomycin challenge, alterations in TSP-1 may be relevant to pulmonary fibrosis. We sought to determine the effects of TSP-1 deficiency on the susceptibility to bleomycin-induced pulmonary fibrosis in a murine model. Age-matched and sex-matched C57BL/6 wild-type (WT) and TSP-1-deficient mice were treated twice weekly for 4 weeks with intraperitoneal bleomycin (0.035 U/g) or PBS, and were allowed to rest 1 week before being killed. Their lungs were inflated with PBS, fixed in formalin, paraffin-embedded, and sectioned. A certified veterinary pathologist blindly scored each slide for inflammation and fibrosis. Lungs were homogenized to obtain RNA and protein for the real-time RT-PCR analysis of connective tissue growth factor (CTGF) and collagen I, and for Western blotting to detect phospho-Smad2, or total Smad2/3, respectively. In response to bleomycin treatment, measures of fibrosis and inflammation, along with CTGF and collagen I mRNA concentrations, were increased in TSP-1-deficient mice compared with WT mice. Notably, Smad 2/3 signaling was of equal strength in WT and TSP-1 knockout mice treated with bleomycin, suggesting that TSP-1 is not required for the activation of TGF-β. These results demonstrate that TSP-1 deficiency does not protect mice from systemic bleomycin challenge, and that TSP-1 deficiency is associated with increased expression of lung collagen and CTGF.

Topics: Animals; Bleomycin; Collagen; Connective Tissue Growth Factor; Gene Expression Regulation; Lipopolysaccharides; Macrophage Activation; Macrophages; Mice; Mice, Inbred C57BL; Phosphorylation; Pneumonia; Pulmonary Fibrosis; RNA, Messenger; Smad2 Protein; Thrombospondin 1; Transforming Growth Factor beta

This study tests the hypothesis that bone marrow-derived mononuclear cell (BMDMC) therapy may reduce lung inflammation and fibrosis leading to an improvement in respiratory mechanics in a murine model of silicosis. 52 female C57BL/6 mice were randomly assigned into four groups. In the silica group (SIL), silica suspension (20 mg/50 μL in saline) was intratracheally instilled. In the control animals, 50 μL saline was administered intratracheally. At 1 h, the control and SIL groups were further randomised, receiving BMDMC (2×10⁶ i.v. control-cell and SIL-cell) or saline (50 μL i.v. control and SIL). BMDMC were obtained from male donor mice. At day 15, lung mechanics, histology, and the presence of Y chromosome, interleukin (IL)-1β, IL-1α, IL-1 receptor antagonist (IL-1RN), IL-1 receptor type 1, transforming growth factor (TGF)-β and caspase-3 mRNA expressions in lung tissue were analysed. In the SIL-cell group, the fraction area of granuloma, the number of macrophages and the collagen fibre content were reduced, yielding improved lung mechanics. The presence of male donor cells in lung tissue was not confirmed using detection of Y chromosome DNA. Nevertheless, caspase-3, IL-1β, IL-1α, IL-1RN and TGF-β mRNA expression diminished after cell therapy. In conclusion, BMDMC acted on inflammatory and fibrogenic processes improving lung function through paracrine effects.

Topics: Animals; Caspase 3; Female; Interleukin-1alpha; Interleukin-1beta; Male; Mice; Mice, Inbred C57BL; Monocytes; Pulmonary Fibrosis; Receptors, Interleukin-1; Silicon Dioxide; Silicosis; Transforming Growth Factor beta; Y Chromosome

CC chemokines play an important role in the pathogenesis of idiopathic pulmonary fibrosis. Few studies have evaluated the efficacy of therapeutically targeting CC chemokines and their receptors during interstitial lung diseases. In the present study, the therapeutic effects of Evasin-1, a tick-derived chemokine-binding protein that has high affinity for CCL3/microphage inflammatory protein (MIP)-1α, was investigated in a murine model of bleomycin-induced lung fibrosis. CCL3/MIP-1α concentrations in lung homogenates increased significantly with time after bleomycin challenge, and this was accompanied by increased number of leukocytes and elevated levels of CCL2/monocyte chemoattractant protein (MCP)-1, CCL5/regulated upon activation, normal T cell expressed and secreted, TNF-α and transforming growth factor-β(1), and pulmonary fibrosis. Administration of evasin-1 on a preventive (from the day of bleomycin administration) or therapeutic (from Day 8 after bleomycin) schedule decreased number of leukocytes in the lung, reduced levels of TNF-α and transforming growth factor-β(1), and attenuated lung fibrosis. These protective effects were similar to those observed in CCL3/MIP-1α-deficient mice. In conclusion, targeting CCL3/MIP-1α by treatment with evasin-1 is beneficial in the context of bleomycin-induced lung injury, even when treatment is started after the fibrogenic insult. Mechanistically, evasin-1 treatment was associated with decreased recruitment of leukocytes and production of fibrogenic cytokines. Modulation of CCL3/MIP-1α function by evasin-1 could be useful for the treatment of idiopathic pulmonary fibrosis.

Topics: Animals; Antibiotics, Antineoplastic; Bleomycin; Chemokine CCL3; Disease Models, Animal; Gene Expression Regulation; Leukocytes; Male; Mice; Mice, Knockout; Pulmonary Fibrosis; Receptors, Chemokine; Rhipicephalus sanguineus; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha

Various anti-inflammatory agents have been used to treat acute or chronic lung injury-induced pulmonary fibrosis (PF). However, the efficacy of the available treatments is disappointing, and new therapies are urgently needed. In the current study, we investigated the effect of a novel α-melanocyte-stimulating hormone analog, STY39, on bleomycin (BLM)-induced pulmonary inflammation and fibrosis in mice. C57BL/6 mice received an intratracheal injection of BLM before being treated with STY39 (0.625, 1.25, or 2.5 mg/kg, i.p.) once a day for 14 consecutive days. Various parameters, reflecting the inflammatory reaction, metabolism of extracellular matrix, myofibroblast proliferation, and degree of fibrosis in the lung, were evaluated. We found that STY39 significantly improved the survival of mice with lethal BLM-induced lung injury, limited body weight loss and the increase in the lung index, reduced the mRNA expression of types I and III procollagen and the production of hydroxyproline in the lung, diminished myofibroblast proliferation, and ultimately reduced BLM-induced lung damage. Further investigation revealed that, in a dose-dependent manner, STY39 treatment inhibited leukocyte migration into the lung; reduced the production of TNF-α, IL-6, macrophage inflammatory protein 2, and transforming growth factor β1 in the lung; and altered the ratio of matrix metalloproteinase 1 to tissue inhibitors of metalloproteinase 1. These findings suggest that STY39 attenuates BLM-induced experimental PF by limiting the inflammatory reaction through the inhibition of proinflammatory and profibrosis cytokines and by accelerating the metabolism of extracellular matrix. Therefore, STY39 may be an effective therapy for preventing PF.

Topics: alpha-MSH; Animals; Anti-Inflammatory Agents; Bleomycin; Immunohistochemistry; Interleukin-6; Matrix Metalloproteinase 1; Mice; Pneumonia; Polymerase Chain Reaction; Pulmonary Fibrosis; Tissue Inhibitor of Metalloproteinase-1; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha

The pleiotropic growth factor TGFβ(1) promotes many of the pathogenic mechanisms observed in lung fibrosis and airway remodeling, such as aberrant extracellular matrix deposition due to both fibroblast activation and fibroblast to myofibroblast differentiation. Serum amyloid P (SAP), a member of the pentraxin family of proteins inhibits bleomycin-induced lung fibrosis through an inhibition of pulmonary fibrocyte and pro-fibrotic alternative (M2) macrophage accumulation. It is unknown if SAP has effects downstream of TGFβ(1), a major mediator of pulmonary fibrosis. Using the lung specific TGFβ(1) transgenic mouse model, we determined that SAP inhibits all of the pathologies driven by TGFβ(1) including apoptosis, airway inflammation, pulmonary fibrocyte accumulation and collagen deposition, without affecting levels of TGFβ(1). To explore the role of monocyte derived cells in this model we used liposomal clodronate to deplete pulmonary macrophages. This led to pronounced anti-fibrotic effects that were independent of fibrocyte accumulation. Administration of SAP mirrored these effects and reduced both pulmonary M2 macrophages and increased chemokine IP10/CXCL10 expression in a SMAD 3-independent manner. Interestingly, SAP concentrations were reduced in the circulation of IPF patients and correlated with disease severity. Last, SAP directly inhibited M2 macrophage differentiation of monocytes obtained from these patients. These data suggest that the beneficial anti-fibrotic effects of SAP in TGFβ(1)-induced lung disease are via modulating monocyte responses.

Topics: Airway Remodeling; Animals; Bleomycin; Cells, Cultured; Clodronic Acid; Humans; Intercellular Signaling Peptides and Proteins; Lung; Macrophages, Alveolar; Mice; Mice, Transgenic; Monocytes; Pulmonary Fibrosis; Serum Amyloid P-Component; Signal Transduction; Transforming Growth Factor beta

Human amnion epithelial cells (hAECs) have attracted recent attention as a promising source of cells for regenerative therapies, with reports that cells derived from human term amnion possess multipotent differentiation ability, low immunogenicity, and anti-inflammatory properties. Specifically, in animal models of lung disease characterized by significant loss of lung tissue secondary to chronic inflammation and fibrosis, the transplantation of hAECs has been shown to reduce both inflammation and subsequent fibrosis. To further explore the mechanisms by which hAECs reduce pulmonary fibrosis and enhance lung regeneration, we utilized a bleomycin-induced model of pulmonary fibrosis and investigated the ability of hAECs to reduce fibrosis and thereby improve pulmonary function. We aimed to determine if hAECs, injected into the peritoneal cavity could migrate to the lung, engraft, and form functional lung epithelium, and whether hAECs could modulate the inflammatory environment in the bleomycin-injured lung. We demonstrated that, compared to bleomycin alone, IP administration of hAECs 24 h after bleomcyin, decreased gene expression of the proinflammatory cytokines TNF-α, TGF-β, IFN-γ, and IL-6 and decreased subsequent pulmonary fibrosis with less pulmonary collagen deposition, reduced levels of α-smooth muscle actin and decreased inflammatory cell infiltrate. We also showed that hAECs are able to prevent a decline in pulmonary function associated with bleomycin-induced lung damage. We were unable to detect any significant engraftment of hAECs in injured, or uninjured, lung after administration. The findings from this study support the further investigation of hAECs as a potential cell therapy for inflammatory and fibrogenic diseases.

Topics: Amnion; Animals; Bleomycin; Epithelial Cells; Female; Humans; Injections, Intraperitoneal; Interferon-gamma; Interleukin-6; Lung; Lung Injury; Mice; Mice, Inbred C57BL; Pulmonary Fibrosis; Respiratory Function Tests; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha

Idiopathic pulmonary fibrosis (IPF) is a chronic fibroproliferative pulmonary disorder for which there are currently no treatments. Although the etiology of IPF is unknown, dysregulated TGF-β signaling has been implicated in its pathogenesis. Recent studies also suggest a central role for abnormal epithelial repair. In this study, we sought to elucidate the function of epithelial TGF-β signaling via TGF-β receptor II (TβRII) and its contribution to fibrosis by generating mice in which TβRII was specifically inactivated in mouse lung epithelium. These mice, which are referred to herein as TβRIINkx2.1-cre mice, were used to determine the impact of TβRII inactivation on (a) embryonic lung morphogenesis in vivo; and (b) the epithelial cell response to TGF-β signaling in vitro and in a bleomycin-induced, TGF-β-mediated mouse model of pulmonary fibrosis. Although postnatally viable with no discernible abnormalities in lung morphogenesis and epithelial cell differentiation, TβRIINkx2.1-cre mice developed emphysema, suggesting a requirement for epithelial TβRII in alveolar homeostasis. Absence of TβRII increased phosphorylation of Smad2 and decreased, but did not entirely block, phosphorylation of Smad3 in response to endogenous/physiologic TGF-β. However, TβRIINkx2.1-cre mice exhibited increased survival and resistance to bleomycin-induced pulmonary fibrosis. To our knowledge, these findings are the first to demonstrate a specific role for TGF-β signaling in the lung epithelium in the pathogenesis of pulmonary fibrosis.

Topics: Animals; Bleomycin; Cell Differentiation; Disease Models, Animal; Epithelium; Female; Humans; Lung; Male; Mice; Mice, Knockout; Mice, Transgenic; Pregnancy; Protein Serine-Threonine Kinases; Pulmonary Fibrosis; Receptor, Transforming Growth Factor-beta Type II; Receptors, Transforming Growth Factor beta; Signal Transduction; Transforming Growth Factor beta

The differentiation of fibroblasts into myofibroblasts is a cardinal feature of idiopathic pulmonary fibrosis (IPF). The transcription factor Yin Yang 1 (YY1) plays a role in the proliferation and differentiation of diverse cell types, but its role in fibrotic lung diseases is not known.. To elucidate the mechanism by which YY1 regulates fibroblast differentiation and lung fibrosis.. Lung fibroblasts were cultured with transforming growth factor (TGF)-β or tumor necrosis factor-α. Nuclear factor (NF)-κB, YY1, and α-smooth muscle actin (SMA) were determined in protein, mRNA, and promoter reporter level. Lung fibroblasts and lung fibrosis were assessed in a partial YY1-deficient mouse and a YY1(f/f) conditional knockout mouse after being exposed to silica or bleomycin.. TGF-β and tumor necrosis factor-α up-regulated YY1 expression in lung fibroblasts. TGF-β-induced YY1 expression was dramatically decreased by an inhibitor of NF-κB, which blocked I-κB degradation. YY1 is significantly overexpressed in both human IPF and murine models of lung fibrosis, including in the aggregated pulmonary fibroblasts of fibrotic foci. Furthermore, the mechanism of fibrogenesis is that YY1 can up-regulate α-SMA expression in pulmonary fibroblasts. YY1-deficient (YY1(+/-)) mice were significantly protected from lung fibrosis, which was associated with attenuated α-SMA and collagen expression. Finally, decreasing YY1 expression through instilled adenovirus-cre in floxed-YY1(f/f) mice reduced lung fibrosis.. YY1 is overexpressed in fibroblasts in both human IPF and murine models in a NF-κB-dependent manner, and YY1 regulates fibrogenesis at least in part by increasing α-SMA and collagen expression. Decreasing YY1 expression may provide a new therapeutic strategy for pulmonary fibrosis.

Topics: Actins; Animals; Cell Differentiation; Cell Proliferation; Cells, Cultured; Fibroblasts; Fluorescent Antibody Technique; Humans; Lung; Mice; NF-kappa B; Pulmonary Fibrosis; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha; Up-Regulation; YY1 Transcription Factor

Idiopathic pulmonary fibrosis (IPF) is a progressive fibrotic lung disease and characterized by abnormal growth of fibroblasts and lung scarring. While the pathogenesis of IPF is not clearly understood, activation of transforming growth factor-β (TGF-β) and disruption of alveolar basement membrane seem to play important roles in leading to excess disruption of the matrix, which is associated with activated matrix metalloproteinase (MMP) and aberrant proliferation of myofibroblasts. The Wnt/β-catenin pathway is an important regulator of cellular proliferation and differentiation and abnormal activation of Wnt/β-catenin signal was observed in IPF. We examined whether inhibition of the Wnt/β-catenin pathway could attenuate pulmonary fibrosis in a bleomycin-induced murine model of pulmonary fibrosis. Pulmonary fibrosis was induced in C57BL/6N mice by intratracheal instillation of bleomycin. To inhibit the Wnt/β-catenin pathway, small interfering RNA (siRNA) for β-catenin was administered into trachea 2 h before bleomycin instillation and every 48 h afterward until sacrifice on day 14. The level of β-catenin expression was increased in the epithelial cells of bleomycin-administered mice. Intratracheal treatment with β-catenin siRNA significantly reduced β-catenin expression, pulmonary fibrosis and collagen synthesis in bleomycin-administered mice compared with controls, with no significant effect on the inflammatory response. The β-catenin-targeted siRNA also significantly decreased the levels of MMP-2 (P<0.01) and TGF-β (P<0.01) expression in the lung tissue. Blockade of the Wnt/β-catenin pathway by β-catenin siRNA decreased bleomycin-induced pulmonary fibrosis in the murine model. These findings suggest that targeting Wnt/β-catenin signaling may be an effective therapeutic approach in the treatment of IPF.

Topics: Animals; beta Catenin; Bleomycin; Bronchi; Cell Line; Epithelium; Female; Matrix Metalloproteinase 2; Matrix Metalloproteinase Inhibitors; Mice; Mice, Inbred C57BL; Pneumonia; Pulmonary Fibrosis; RNA, Small Interfering; Signal Transduction; Transforming Growth Factor beta; Wnt Proteins

Quercetin is a flavonoid with a wide variety of cytoprotective and modulatory functions. Heme oxygenase-1 (HO-1) is an inducible enzyme. Its reaction product, carbon monoxide (CO), confers cellular protection in a number of conditions and diseases associated with oxidative or inflammatory lung injury. Furthermore, quercetin was reported to be a potent inducer of HO-1 in several cell types. We hypothesized that quercetin suppresses the production of collagen in fibroblasts via the induction of HO-1. Here, we showed that quercetin suppresses transforming growth factor-β (TGF-β)-induced collagen production in NIH3T3 cells and in normal human lung fibroblasts. This suppressive effect of quercetin was mediated by quercetin-induced HO-1. The suppression of collagen production was conferred by the reaction product of HO-1, CO, but not by bilirubin. Furthermore, the translocation of the nuclear factor E2-related factor-2 (Nrf2), an important transcription factor that regulates the expression of HO-1 from the cytoplasm to the nuclei, was demonstrated in NIH3T3 cells by exposure to quercetin. Assessment of the signal transduction pathway involved in TGF-β signaling showed that quercetin stimulated the Smad and mitogen-activated protein kinase pathway to varying degrees. Our results demonstrate that quercetin exerts suppressive effects on the expression of collagen by the induction of HO-1. Idiopathic pulmonary fibrosis is the most lethal diffuse fibrosing lung disease, and is characterized by the deposition of extracellular matrix. Given that HO-1 is one of the important molecules emerging as a central player in diseases, quercetin or its derivatives, which effectively induced HO-1, will lead to new therapeutic strategies for promoting antifibrotic therapy in respiratory diseases.

Topics: Animals; Antioxidants; Cell Nucleus; Collagen; Cytoplasm; Enzyme Inhibitors; Fibroblasts; Gene Expression Regulation, Enzymologic; Heme Oxygenase-1; Humans; Immunohistochemistry; Mice; NIH 3T3 Cells; Pulmonary Fibrosis; Quercetin; Transforming Growth Factor beta

Hypoxia-inducible factor-1α (HIF-1α), a transcription factor that functions as a master regulator of oxygen homeostasis, has been implicated in fibrinogenesis. Here, we explore the role of HIF-1α in transforming growth factor-β (TGF-β) signaling by examining the effects of TGF-β(1) on the expression of plasminogen activator inhibitor-1 (PAI-1). Immunohistochemistry of lung tissue from a mouse bleomycin (BLM)-induced pulmonary fibrosis model revealed that expression of HIF-1α and PAI-1 was predominantly induced in alveolar macrophages. Real-time RT-PCR and ELISA analysis showed that PAI-1 mRNA and activated PAI-1 protein level were strongly induced 7 days after BLM instillation. Stimulation of cultured mouse alveolar macrophages (MH-S cells) with TGF-β(1) induced PAI-1 production, which was associated with HIF-1α protein accumulation. This accumulation of HIF-1α protein was inhibited by SB431542 (type I TGF-β receptor/ALK receptor inhibitor) but not by PD98059 (MEK1 inhibitor) and SB203580 (p38 MAP kinase inhibitor). Expression of prolyl-hydroxylase domain (PHD)-2, which is essential for HIF-1α degradation, was inhibited by TGF-β(1), and this decrease was abolished by SB431542. TGF-β(1) induction of PAI-1 mRNA and its protein expression were significantly attenuated by HIF-1α silencing. Transcriptome analysis by cDNA microarray of MH-S cells after HIF-1α silencing uncovered several pro-fibrotic genes whose regulation by TGF-β(1) required HIF-1α, including platelet-derived growth factor-A. Taken together, these findings expand our concept of the role of HIF-1α in pulmonary fibrosis in mediating the effects of TGF-β(1) on the expression of the pro-fibrotic genes in activated alveolar macrophages.

Topics: Animals; Benzamides; Bleomycin; Cell Hypoxia; Dioxoles; Hypoxia-Inducible Factor 1, alpha Subunit; Macrophages, Alveolar; Male; Mice; Mice, Inbred C57BL; Plasminogen Activator Inhibitor 1; Pulmonary Fibrosis; Transforming Growth Factor beta

The molecular mechanisms of lung injury and fibrosis are incompletely understood. MicroRNAs (miRNAs) are crucial biological regulators that act by suppressing their target genes and are involved in a variety of pathophysiological processes. To gain insight into miRNAs in the regulation of lung fibrosis, total RNA was isolated from mouse lungs harvested at different days after bleomycin treatment, and miRNA array with 1,810 miRNA probes was performed thereafter. MiRNAs expressed in lungs with bleomycin treatment at different time points were compared with miRNAs expressed in lungs without bleomycin treatment, resulting in 161 miRNAs differentially expressed. Furthermore, miRNA expression patterns regulated in initial and late periods after bleomycin were identified. Target genes were predicted in silico for differentially expressed miRNAs, including let-7f, let-7g, miR-196b, miR-16, miR-195, miR-25, miR-144, miR-351, miR-153, miR-468, miR-449b, miR-361, miR-700, miR-704, miR-717, miR-10a, miR-211, miR-34a, miR-367, and miR-21. Target genes were then cross-referenced to the molecular pathways, suggesting that the differentially expressed miRNAs regulate apoptosis, Wnt, Toll-like receptor, and TGF-β signaling. Our study demonstrated a relative abundance of miRNA levels in bleomycin-induced lung fibrosis. The miRNAs and their potential target genes identified may contribute to the understanding of the complex transcriptional program of lung fibrosis.

Topics: Animals; Bleomycin; Gene Expression Profiling; Gene Expression Regulation; Mice; Mice, Inbred C57BL; MicroRNAs; Oligonucleotide Array Sequence Analysis; Pulmonary Fibrosis; Signal Transduction; Transforming Growth Factor beta

The authors investigated the role of resveratrol (RV), a natural poliphenolic molecule with several biological activities, in transforming growth factor-β (TGF-β)-induced proliferation and differentiation of ex vivo human pulmonary fibroblasts into myofibroblasts. The effects of RV treatment were evaluated by analyzing TGF-β-induced α-smooth muscle actin (α-SMA) expression and collagen production, as well as cell proliferation of both normal and idiopathic pulmonary fibrosis (IPF) lung fibroblasts. Results demonstrate that RV inhibits TGF-β-induced cell proliferation of both normal and pathological lung fibroblasts, attenuates α-SMA expression at both the mRNA and protein levels, and also inhibits intracellular collagen deposition. In order to understand the molecular mechanisms, the authors also investigated the effects of RV treatment on signaling pathways involved in TGF-β-induced fibrosis. The authors show that RV inhibited TGF-β-induced phosphorylation of both extracellular signal-regulated kinases (ERK1/2) and the serine/threonine kinase, Akt. Moreover, RV treatment blocked the TGF-β-induced decrease in phosphatase and tensin homolog (PTEN) expression levels.

Topics: Actins; Cell Differentiation; Cell Line; Cell Proliferation; Collagen; Extracellular Signal-Regulated MAP Kinases; Fibroblasts; Gene Expression; Humans; Lung; Myofibroblasts; Proto-Oncogene Proteins c-akt; PTEN Phosphohydrolase; Pulmonary Fibrosis; Resveratrol; RNA, Messenger; Signal Transduction; Stilbenes; Transforming Growth Factor beta

Previous studies in rats have suggested a causal relationship between progressive pulmonary inflammation and lung fibrosis induced by crystalline silica particles. We report here that, in NMRI mice, the lung response to silica particles is accompanied by a mild and non progressive pulmonary inflammation which is dispensable for the development of lung fibrosis. We found that glucocorticoid (dexamethasone) dramatically reduced lung injury, cellular inflammation and pro-inflammatory cytokine expression (TNF-α, IL-1β and KC) but had no significant effect on silica-induced lung fibrosis and expression of the fibrogenic and suppressive cytokines TGF-β and IL-10 in mice. Other anti-inflammatory molecules such as the COX inhibitor piroxicam or the phosphodiesterase 5 inhibitor sildenafil also reduced lung inflammation without modifying collagen, TGF-β or IL-10 lung content. Our findings indicate that the development of lung fibrosis in silica-treated NMRI mice is not driven by inflammatory lung responses and suggest that suppressive cytokines may represent critical fibrotic factors and potential therapeutic targets in silicosis.

Topics: Animals; Bronchoalveolar Lavage Fluid; Cell Count; Collagen; Cyclooxygenase Inhibitors; Female; Interleukin-10; L-Lactate Dehydrogenase; Mice; Mice, Inbred C57BL; Mice, Inbred DBA; Phosphodiesterase 5 Inhibitors; Piperazines; Piroxicam; Pneumonia; Pulmonary Fibrosis; Purines; Sildenafil Citrate; Silicon Dioxide; Sulfones; Transforming Growth Factor beta

The response of alveolar epithelial cells (AECs) to lung injury plays a central role in the pathogenesis of pulmonary fibrosis, but the mechanisms by which AECs regulate fibrotic processes are not well defined. We aimed to elucidate how transforming growth factor-β (TGFβ) signaling in lung epithelium impacts lung fibrosis in the intratracheal bleomycin model. Mice with selective deficiency of TGFβ receptor 2 (TGFβR2) in lung epithelium were generated and crossed to cell fate reporter mice that express β-galactosidase (β-gal) in cells of lung epithelial lineage. Mice were given intratracheal bleomycin (0.08 U), and the following parameters were assessed: AEC death by terminal deoxynucleotidyl transferase dUTP-mediated nick-end labeling assay, inflammation by total and differential cell counts from bronchoalveolar lavage, fibrosis by scoring of trichrome-stained lung sections, and total lung collagen content. Mice with lung epithelial deficiency of TGFβR2 had improved AEC survival, despite greater lung inflammation, after bleomycin administration. At 3 wk after bleomycin administration, mice with epithelial TGFβR2 deficiency showed a significantly attenuated fibrotic response in the lungs, as determined by semiquantitatve scoring and total collagen content. The reduction in lung fibrosis in these mice was associated with a marked decrease in the lung fibroblast population, both total lung fibroblasts and epithelial-to-mesenchymal transition-derived (S100A4(+)/β-gal(+)) fibroblasts. Attenuation of TGFβ signaling in lung epithelium provides protection from bleomycin-induced fibrosis, indicating a critical role for the epithelium in transducing the profibrotic effects of this cytokine.

Topics: Animals; Antibiotics, Antineoplastic; beta-Galactosidase; Bleomycin; Blotting, Western; Cell Proliferation; Cells, Cultured; Disease Models, Animal; Epithelium; Fibroblasts; Fluorescent Antibody Technique; Lung Injury; Mice; Mice, Transgenic; Pneumonia; Protein Serine-Threonine Kinases; Pulmonary Alveoli; Pulmonary Fibrosis; Receptor, Transforming Growth Factor-beta Type II; Receptors, Transforming Growth Factor beta; Signal Transduction; Transforming Growth Factor beta

Pulmonary fibrosis is a relentlessly progressive disease for which the etiology can be idiopathic or associated with environmental or occupational exposures. There is not a clear explanation for the chronic and progressive nature of the disease, leaving treatment and prevention options limited. However, there is increasing evidence of an autoimmune component, since fibrotic diseases are often accompanied by production of autoantibodies. Because exposure to silicates such as silica and asbestos can lead to both autoantibodies and pulmonary/pleural fibrosis, these exposures provide an excellent tool for examining the relationship between these outcomes. This study explored the possibility that autoantibodies induced by asbestos exposure in mice would affect fibroblast phenotype. L929 fibroblasts and primary lung fibroblasts were treated with serum IgG from asbestos- or saline-treated mice, and tested for binding using cell-based ELISA, and for phenotypic changes using immunofluorescence, laser scanning cytometry and Sirius Red collagen assay. Autoantibodies in the serum of C57Bl/6 mice exposed to asbestos (but not sera from untreated mice) bound to mouse fibroblasts. The autoantibodies induced differentiation to a myofibroblast phenotype, as demonstrated by increased expression of smooth muscle α-actin (SMA), which was lost when the serum was cleared of IgG. Cells treated with purified IgG of exposed mice produced excess collagen. Using ELISA, we tested serum antibody binding to DNA topoisomerase (Topo) I, vimentin, TGFβ-R, and PDGF-Rα. Antibodies to DNA Topo I and to PDGF-Rα were detected, both of which have been shown by others to be able to affect fibroblast phenotype. The anti-fibroblast antibodies (AFA) also induced STAT-1 activation, implicating the PDGF-R pathway as part of the response to AFA binding. These data support the hypothesis that asbestos induces AFA that modify fibroblast phenotype, and suggest a mechanism whereby autoantibodies may mediate some of the fibrotic manifestations of asbestos exposure.

Topics: Animals; Asbestos; Autoantibodies; Carcinogens; Cell Line; DNA Topoisomerases, Type I; Female; Fibroblasts; Humans; Immunoglobulin G; Lung; Mice; Occupational Exposure; Pulmonary Fibrosis; Receptor, Platelet-Derived Growth Factor beta; STAT1 Transcription Factor; Transforming Growth Factor beta

Adrenomedullin (AM), a 52-amino acid ringed-structure peptide with C-terminal amidation, was originally isolated from human pheochromocytoma. AM are widely distributed in various tissues and acts as a local vasoactive hormone in various conditions.. In the present study, we investigated the efficacy of AM on the animal model of bleomycin (BLM)-induced lung injury. Mice were subjected to intratracheal administration of BLM and were assigned to receive AM daily by an intraperitoneal injection of 200 ngr/kg.. Myeloperoxidase activity, lung histology, immunohistochemical analyses for cytokines and adhesion molecules expression, inducible nitric oxide synthase (iNOS), nitrotyrosine, and poly (ADP-ribose) polymerase (PARP) were performed one week after fibrosis induction. Lung histology and transforming growth factor beta (TGF-β) were performed 14 and 21 days after treatments. After bleomycin administration, AM-treated mice exhibited a reduced degree of lung damage and inflammation compared with BLM-treated mice, as shown by the reduction of (1) myeloperoxidase activity (MPO), (2) cytokines and adhesion molecules expression, (3) nitric oxide synthase expression, (4) the nitration of tyrosine residues, (5) poly (ADP-ribose) (PAR) formation, a product of the nuclear enzyme poly (ADP-ribose) polymerase (PARP) (6) transforming growth factor beta (TGF-β) (7)and the degree of lung injury.. Our results indicate that AM administration is able to prevent bleomycin induced lung injury through the down regulation of proinflammatory factors.

Topics: Adrenomedullin; Animals; Anti-Inflammatory Agents; Bleomycin; Cell Adhesion Molecules; Cytokines; Disease Models, Animal; Inflammation Mediators; Injections, Intraperitoneal; Lung; Male; Mice; Mice, Inbred ICR; Nitric Oxide Synthase Type II; Peroxidase; Pneumonia; Poly(ADP-ribose) Polymerases; Pulmonary Fibrosis; Severity of Illness Index; Time Factors; Transforming Growth Factor beta; Tyrosine

With increasing worldwide rates of morbidity and mortality of pulmonary fibrosis, the development of effective therapeutics for this disease is of great interest. Secretoglobin (SCGB) 3A2, a novel cytokine-like molecule predominantly expressed in pulmonary airways epithelium, exhibits anti-inflammatory and growth factor activities. In the current study SCGB3A2 was found to inhibit TGFβ-induced differentiation of fibroblasts to myofibroblasts, a hallmark of the fibrogenic process, using pulmonary fibroblasts isolated from adult mice. This induction was through increased phosphorylation of STAT1 and expression of SMAD7 and decreased phosphorylation of SMAD2 and SMAD3. To demonstrate the effect of SCGB3A2 on the TGFβ signaling in vivo, a bleomycin-induced pulmonary fibrosis mouse model was used. Mice were administered bleomycin intratracheally followed by intravenous injection of recombinant SCGB3A2. Histological examination in conjunction with inflammatory cell counts in bronchoalveolar lavage fluids demonstrated that SCGB3A2 suppressed bleomycin-induced pulmonary fibrosis. Microarray analysis was carried out using RNAs from lungs of bleomycin-treated mice with or without SCGB3A2 and normal mice treated with SCGB3A2. The results demonstrated that SCGB3A2 affects TGFβ signaling and reduces the expression of genes involved in fibrosis. This study suggests the potential utility of SCGB3A2 for targeting TGFβ signaling in the treatment of pulmonary fibrosis.

Topics: Animals; Antibiotics, Antineoplastic; Bleomycin; Cell Differentiation; Down-Regulation; Female; Fibroblasts; Gene Expression Profiling; Humans; Mice; Oligonucleotide Array Sequence Analysis; Phosphorylation; Proteins; Pulmonary Fibrosis; Secretoglobins; Signal Transduction; Smad Proteins; STAT1 Transcription Factor; Transforming Growth Factor beta

Lung fibroblast proliferation and differentiation into myofibroblasts are pathological key events during development of lung fibrosis. Cyclic nucleotide signaling is described as a negative modulator of these cellular processes, and cyclic nucleotide degrading type 4 phosphodiesterases (PDE4) are important regulators of these pathways. In this study, we elucidated expression and the role of individual subtypes of PDE4 in primary normal human lung fibroblast (NHLF) in controlling cytokines-induced proliferation and conversion to myofibroblasts by short-interfering RNAs (siRNAs) induced knockdown. We verified the expression of PDE4A, B, and D, while PDE4C was only minor or even not expressed in NHLF. An efficient liposome-mediated transfection method for mRNA silencing and a knockdown of the expressed PDE4 subtypes was achieved in these cells. This knockdown was further validated by PDE4 protein expression analysis and PDE4 activity measurements. Functionally, the knockdown of PDE4A and PDE4B inhibited proliferation induced by the cytokine combination of bFGF and IL-1β, whereas knockdown of PDE4D was ineffective. In contrast, TGF-β induced differentiation into myofibroblasts was affected by knockdown of PDE4B and PDE4D, but not by PDE4A knockdown. In summary, our data allow to assign different PDE4 subtypes to distinct functions of human lung fibroblasts and highlight the predominant role of PDE4B in controlling pathophysiological processes of human lung fibroblasts. This provides a scientific rationale for focused therapeutic targeting of PDE4B to treat respiratory diseases with fibrotic lesions in the lung.

Topics: Aminopyridines; Benzamides; Cell Differentiation; Cell Proliferation; Cells, Cultured; Cyclic Nucleotide Phosphodiesterases, Type 4; Cyclopropanes; Cytokines; Fibroblast Growth Factor 2; Fibroblasts; Gene Expression Profiling; Humans; Interleukin-1beta; Isoenzymes; Lung; Myofibroblasts; Phosphodiesterase 4 Inhibitors; Pulmonary Fibrosis; Pyridines; RNA, Small Interfering; Transfection; Transforming Growth Factor beta

Lung fibrosis is a common and serious complication of radiation therapy for lung cancer, for which there are no efficient treatments. Emerging evidence indicates that lysophosphatidic acid (LPA) and its receptors (LPARs) are involved in the pathogenesis of fibrosis. Here, we reported that thoracic radiation with 16Gy in mice induced development of radiation lung fibrosis (RLF) accompanied by obvious increases in LPA release and LPAR1 and LPAR3 (LPAR1/3) transcripts. RLF was significantly alleviated in mice treated with the dual LPAR1/3 antagonist, VPC12249. VPC12249 administration effectively prolonged animal survival, restored lung structure, inhibited fibroblast accumulation and reduced collagen deposition. Moreover, profibrotic cytokines in radiation-challenged lungs obviously decreased following administration of VPC12249, including transforming growth factor β1 (TGFβ1) and connective tissue growth factor (CTGF). In vitro, LPA induced both fibroblast proliferation and CTGF expression in a dose-dependent manner, and both were suppressed by blockade of LPAR1/3. The pro-proliferative activity of LPA on fibroblasts was inhibited by siRNA directed against CTGF. Together, our data suggest that the LPA-LPAR1/3 signaling system is involved in the development of RLF through promoting fibroblast proliferation in a CTGF-dependent manner. The LPA-LPAR1/3-CTGF pathway may be a potential target for RLF therapy.

Topics: Animals; Cell Proliferation; Connective Tissue Growth Factor; Down-Regulation; Fibroblasts; Mice; Mice, Inbred C57BL; Oleic Acids; Organophosphates; Pulmonary Fibrosis; Radiation Injuries, Experimental; Receptors, Lysophosphatidic Acid; Transforming Growth Factor beta

To investigate whether occurrence of early radiation effects in lung tissue depends on local dose only.. Twenty-five percent, 50%, 66%, 88%, or 100% of the rat lung was irradiated using single fractions of 150-MeV protons. For all volumes, in-field and out-of-field dose-response curves were obtained 8 weeks after irradiation. The pathohistology of parenchymal inflammation, infiltrates, fibrosis, and vascular damage and the relative expression of proinflammatory cytokines interleukin (IL)-1α, transforming growth factor-β, IL-6, and tumor necrosis factor-α were assessed.. For all histologic endpoints, irradiated dose- and volume-dependent in-field and out-of-field effects were observed, albeit with different dynamics. Of note, the out-of-field effects for vascular damage were very similar to the in-field effects. Interestingly, only IL-6 showed a clear dose-dependent increase in expression both in-field and out-of-field, whereas the expression levels of IL-1α, transforming growth factor-β, and tumor necrosis factor-α were either very low or without a clear dose-volume relation. As such, none of the radiation effects studied depended only on local dose to the tissue.. The effects of radiation to lung tissue do not only depend on local dose to that tissue. Especially at high-volume irradiation, lung damage seems to present globally rather than locally. The accuracy of predictive modeling may be improved by including nonlocal effects.

Topics: Animals; Dose-Response Relationship, Radiation; Interleukin-1alpha; Interleukin-6; Lung; Male; Models, Biological; Muscle, Smooth, Vascular; Myocytes, Smooth Muscle; Protons; Pulmonary Fibrosis; Radiation Injuries, Experimental; Rats; Rats, Wistar; Time Factors; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha

To study the curative effects of pirfenidone (PF) on pulmonary fibrosis induced by paraquat (PQ) in mice and to provide the theoretical basis for clinical treatment.. Ninety adult healthy male ICR mice were randomly divided into six groups: control group, PQ group, 2 mg/kg Dexamethasone group, 25 mg/kg PF group, 50 mg/kg PF group and 100 mg/kg PF group, there were 15 mice in each group. The corresponding volume of normal saline was given to the each mouse in control group according to the weight, after 2 h 0.1% CMC was given to the each mouse of control group one time by intragastric administration, then the CMC was administrated at regular time until sacrifice. All mice for other 5 groups were exposed to 100 mg/kg PQ by intragastric administration. At 2 h after exposure to PQ, 0.02 ml/10 g dexamethasone and 25, 50, 100 mg/kg PF were given to mice for dexamethasone group and for 3 PF groups by intragastric administration each day for 49 days, respectively. The lung coefficient was calculated and pathological changes of lung tissue were observed by HE staining for each mouse. The hydroxyproline (HYP) level in lung tissue was measured for each mouse. The mRNA level of and the protein level of TGF-β(1) in lung tissue for each mouse were determined, and the protein level of TGF-β(1) in the bronchus-alveolus lavage fluid (BALF) of each mouse was detected.. The survival rates on the 3rd day in PQ group, 3 PF groups and dexamethasone group were 53.33%, 46.67%, 73.33%, 86.67% and 80%, respectively. The survival rates on the 3rd day in dexamethasone group, 50 mg/kg and 100 mg/kg PF groups were significantly higher than those of PQ group and 25 mg/kg PF group (P < 0.05). The lung coefficients of 3 PF groups were significantly lower than that of the PQ group (P < 0.05). The lung tissue HYP levels of dexamethasone group and 3 PF groups were 50.95 ± 11.65, 44.52 ± 9.48, 43.27 ± 6.01 and 40.82 ± 5.90 mg/g respectively, which were significantly lower than that (74.27 ± 3.68) of PQ group (P < 0.01). The TGF-β(1) protein levels of BALF in dexamethasone group, 50 and 100 mg/kg PF groups were 22.03 ± 7.27, 27.75 ± 5.84 and 21.31 ± 6.82 ng/ml respectively, which were significantly lower than that (52.52 ± 15.51) ng/ml of PQ group (P < 0.01) The expression level of TGF-β(1) mRNA in 100 mg/kg PF group decreased significantly, as compared with PQ group (P < 0.01).. PF could reduce the collagen deposition and pulmonary fibrosis induced by PQ in mice lungs.

Topics: Animals; Disease Models, Animal; Lung; Male; Mice; Mice, Inbred ICR; Paraquat; Pulmonary Fibrosis; Pyridones; Transforming Growth Factor beta

To investigate the effects of andrographolide on the concentration of TNF-α and TGF-β1 in bronchoalveolar lavage fluid (BALF) and the expressions of type I and III collagen mRNA in Lung tissue in bleomycin (BLM)-induced pulmonary fibrosis in rats.. 90 healthy SD male rats were randomly divided into 6 groups with 15 rats each group: normal saline (NS) group, BLM group, prednisone (Pred) group and different doses of andrographolide groups (andrographolide group A 62.5 mg/kg, andrographolide group B 125 mg/kg and andrographolide group C 250 mg/kg). BLM was given to BLM group, Pred group and andrographolide group A, B, C by intratracheal instillation, and same volume of NS was given to NS group in the same way. And then NS was given to NS group and BLM group, Pred was given to Pred group and different does of andrographolide were given to andrograoholide group A, B, C by gavage every day. Five rats of each group were killed respectively at day 7, 14, 28 after intratracheal instillation. Alveolitis and fibrosis were observed by HE and Masson staining. Real-time fluorescent quantitative reverse transcription- polymerase chain reaction (FQ RT-PCR) was performed to detect the expressions of type I and III collagen mRNA in lung tissue, and the concentration of TNF-α and TGF-β1 in BALF was determined by enzyme-linked immunosorbnent assay. Blood urea nitrogen (BUN), creatinine (Cr), alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were also examined.. (1) The AST, ALT, BUN and Cr in every group had no significant diference(P>0.05). (2) Alveolar septal edema, inflammatory cell infiltration and fibrosis were not found in NS group. In the BLM group, lots of inflammatory cells infiltration were observed in the alveolar at day 7; the alveolitis was still existed, but inflammatory cells were significantly reduced, and the number of the fibroblasts and matrix in alveolar septum were obviously increased at day 14, at the same time, alveolar structure was damaged and alveolar septum widened; the inflammation cells infiltration of the alveolar was relieved, pulmonary fibrosis was increased, and parts of alveolar space was disappeared , severe fibrosis was found at day 28. It was similar between andrographolide group A and BLM group in pathomorphology. A lot of inflammatory cells infiltration and local accumulation were observed at day 7 in andrographolide group B, C and Pred group. However, compared with andrographolide group A and BLM group, the fibrosis at day 14, 28 was significantly reduced.(3) The concentration of TGF-β1, TNF-α in BALF of NS group was significantly lower than that of Pred group, BLM group, andrographolide group A, B, C at each time point(P<0.05). The concentration of TGF-β1 and TNF-α in BALF of BLM group at day 7, 14, 28 was higher than that of Pred group, andrographolide group B and andrographolide group C (P<0.05). Compared with BLM group, the concentration of TGF-β1 and TNF-α in BALF of andrographolide group A had no significant difference. (4) The expression of type I and III collagen mRNA in lung tissue of NS group was significantly lower than that of group Pred, BLM, andrographolide group A, B, C at each time point (P<0.05). The expression of type I and III collagen mRNA in lung tissue of BLM group at day 7, 14, 28 was higher than that of Pred group, andrographolide group B and andrographolide group C (P<0.05). Compared with BLM group, the expression of type I and III collagen mRNA in lung tissue of andrographolide group A had no significant difference.. In BLM-induced rat pulmonary fibrosis, andrographolide could attenuate alveolitis and fibrosis, decrease mRNA expression of collagen I and III in lung tissue and decrease the concentration of TNF-α and TGF-β1 in BALF. It had no side effects on liver and kidney function.

Topics: Animals; Bleomycin; Bronchoalveolar Lavage Fluid; Collagen Type I; Collagen Type III; Cytokines; Diterpenes; Dose-Response Relationship, Drug; Gene Expression Regulation; Lung; Male; Pulmonary Fibrosis; Rats; Rats, Sprague-Dawley; RNA, Messenger; Transforming Growth Factor beta; Transforming Growth Factor beta1

Fibrotic interstitial pneumonias are more prevalent in males of advancing age, although little is known about the underlying mechanisms. To evaluate the contributions of age and sex to the development of pulmonary fibrosis, we intratracheally instilled young (8-12 wk) and aged (52-54 wk) male and female mice with bleomycin and assessed the development and severity of fibrotic lung disease by measurements of lung collagen levels, static compliance, leukocyte infiltration, and stereological quantification of fibrotic areas in histological sections. We also quantified proinflammatory and profibrotic chemokine and cytokine levels in the bronchoalveolar lavage fluid. Aged male mice developed more severe lung disease, indicated by increased mortality, increased collagen deposition, and neutrophilic alveolitis compared with aged female mice or young mice of either sex. Aged male mice also exhibited increased levels of transforming growth factor-β, IL-17A, and CXCL1 in their bronchoalveolar lavage fluid. Young male mice developed a more fibrotic disease after bleomycin instillation compared with female mice, regardless of age. There was no difference in fibrosis between young and aged female mice. Taken together, these findings suggest that the variables of advanced age and male sex contribute to the severity of pulmonary fibrosis in this model. Our findings also emphasize the importance of stratifying experimental groups on the basis of age and sex in experimental and epidemiological studies of this nature.

Topics: Age Factors; Animals; Bleomycin; Bronchoalveolar Lavage Fluid; Chemokine CXCL1; Collagen; Enzyme-Linked Immunosorbent Assay; Female; Lung; Lung Compliance; Lung Injury; Male; Mice; Mice, Inbred C57BL; Pulmonary Fibrosis; Sex Characteristics; Sex Factors; Survival Rate; Transforming Growth Factor beta; Trauma Severity Indices

Idiopathic pulmonary fibrosis (IPF) may be triggered by epithelial injury that results in aberrant production of growth factors, cytokines, and proteinases, leading to proliferation of myofibroblasts, excess deposition of collagen, and destruction of the lung architecture. The precise mechanisms and key signaling mediators responsible for this aberrant repair process remain unclear. We assessed the importance of matrix metalloproteinase-3 (MMP-3) in the pathogenesis of IPF through i) determination of MMP-3 expression in patients with IPF, ii) in vivo experiments examining the relevance of MMP-3 in experimental models of fibrosis, and iii) in vitro experiments to elucidate possible mechanisms of action. Gene expression analysis, quantitative RT-PCR, and Western blot analysis of explanted human lungs revealed enhanced expression of MMP-3 in IPF, compared with control. Transient adenoviral vector-mediated expression of recombinant MMP-3 in rat lung resulted in accumulation of myofibroblasts and pulmonary fibrosis. Conversely, MMP-3-null mice were protected against bleomycin-induced pulmonary fibrosis. In vitro treatment of cultured lung epithelial cells with purified MMP-3 resulted in activation of the β-catenin signaling pathway, via cleavage of E-cadherin, and induction of epithelial-mesenchymal transition. These processes were inhibited in bleomycin-treated MMP-3-null mice, as assessed by cytosolic translocation of β-catenin and cyclin D1 expression. These observations support a novel role for MMP-3 in the pathogenesis of IPF, through activation of β-catenin signaling and induction of epithelial-mesenchymal transition.

Topics: Adenoviridae; Animals; beta Catenin; Bleomycin; Cadherins; Cyclin D1; Disease Models, Animal; Epithelial Cells; Epithelial-Mesenchymal Transition; Female; Gene Expression Regulation, Enzymologic; Genetic Vectors; Humans; Lung; Matrix Metalloproteinase 2; Matrix Metalloproteinase 3; Matrix Metalloproteinase 9; Mice; Mice, Inbred C57BL; Protein Transport; Pulmonary Fibrosis; Rats; Rats, Sprague-Dawley; RNA, Messenger; Signal Transduction; Transforming Growth Factor beta

Pulmonary infections and pneumonitis occur frequently after hematopoietic stem cell transplantation. Using a syngeneic mouse model of bone marrow transplantation (BMT), we have previously demonstrated that BMT mice are more susceptible to acute gammaherpesvirus 68 (MHV-68) replication at day 7 after infection. By day 21, the virus is latent in lungs of BMT and control mice, and there is no difference in viral load. Despite similar latent viral load, BMT mice develop severe pneumonitis associated with reduced oxygen saturation, fibrosis, peripheral inflammation, hyaline membranes, and foamy alveolar macrophages, a phenotype that persists for 7 weeks after infection. BMT mice demonstrate increased bronchoalveolar lavage (BAL) cells, and this population is enriched in neutrophils and T cells. Alternatively, activated macrophages appear earlier than do classically activated macrophages. BAL fluid from BMT mice at day 21 after infection contains increased levels of hydrogen peroxide, nitrite, and transforming growth factor-β (TGF-β). Mice expressing the dominant-negative transgene dn-TGFβRII in multiple cell types were used as BMT donors. BMT mice with T-cell dnTGFβRII are largely protected from the pneumonitis phenotype, whereas mice with CD11c-dnTGFβRII BMT mice are only modestly protected from pneumonitis. Protection in BMT mice with T-cell dnTGFβRII is associated with decreased TGF-β derived from parenchymal cells in the BAL fluid, lower nitrite levels, and reduced apoptosis, whereas alternatively activated macrophage markers are unchanged.

Topics: Animals; Apoptosis; Bone Marrow Transplantation; Bronchoalveolar Lavage Fluid; Gammaherpesvirinae; Herpesviridae Infections; Macrophage Activation; Mice; Mice, Inbred C57BL; Neutrophil Infiltration; Pneumonia; Pulmonary Fibrosis; Signal Transduction; T-Lymphocytes, Regulatory; Transforming Growth Factor beta; Transgenes; Viral Load

Fibrosis results from inflammatory tissue damage and impaired regeneration. In the context of bleomycin-induced pulmonary fibrosis, we demonstrated that the matricellular protein termed secreted protein acidic and rich in cysteine (SPARC) distinctly regulates inflammation and collagen deposition, depending on its cellular origin. Reciprocal Sparc(-/-) and wild-type (WT) bone marrow chimeras revealed that SPARC expression in host fibroblasts is required and sufficient to induce collagen fibrosis in a proper inflammatory environment. Accordingly, Sparc(-/-) >WT chimeras showed exacerbated inflammation and fibrosis due to the inability of Sparc(-/-) macrophages to down-regulate tumor necrosis factor production because of impaired responses to tumor growth factor-β. Hence, the use of bone marrow cells expressing a dominant-negative form of tumor growth factor-β receptor type II under the monocyte-specific CD68 promoter, as a decoy, phenocopied Sparc(-/-) donor chimeras. Our results point to an unexpected dual role of SPARC in oppositely influencing the outcome of fibrosis.

Topics: Animals; Bleomycin; Bone Marrow Cells; Chimera; Collagen; Down-Regulation; Fibroblasts; Leukocytes; Macrophages; Mice; Mice, Inbred BALB C; Osteonectin; Pneumonia; Pulmonary Fibrosis; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha

As passive environmental tobacco smoke (ETS) exposure in nonsmokers can increase both asthma symptoms and the frequency of asthma exacerbations, we utilized a mouse model, in which ovalbumin (OVA) + ETS induce significantly increased levels of eosinophilic airway inflammation and remodeling compared to either stimulus alone, to determine whether a Toll-like receptor-9 (TLR-9) agonist could reduce levels of airway inflammation, airway remodeling and airway hyperreactivity (AHR).. Mice treated with or without a TLR-9 agonist were sensitized to OVA and challenged with OVA + ETS for 1 month. AHR to methacholine was assessed in intubated and ventilated mice. Lung Th2 cytokines and TGF-beta(1) were measured by ELISA. Lungs were processed for histology and immunohistology to quantify eosinophils, mucus, peribronchial fibrosis and smooth muscle changes using image analysis.. Administration of a TLR-9 agonist to mice coexposed to chronic ETS and chronic OVA allergen significantly reduced levels of eosinophilic airway inflammation, mucus production, peribronchial fibrosis, the thickness of the peribronchial smooth muscle layer, and AHR. The reduced airway remodeling in mice treated with the TLR-9 agonist was associated with significantly reduced numbers of peribronchial MBP+ and peribronchial TGF-beta(1)+ cells, and with significantly reduced levels of lung Th2 cytokines [interleukin-5 and interleukin-13] and TGF-beta(1).. These studies demonstrate that TLR-9-based therapies inhibit airway inflammation, remodeling and AHR in mice coexposed to ETS and allergen who exhibit enhanced airway inflammation and remodeling.

Topics: Air Pollutants; Airway Remodeling; Allergens; Animals; Asthma; Cell Movement; Eosinophils; Interleukin-13; Interleukin-5; Lung; Mice; Mice, Inbred BALB C; Oligodeoxyribonucleotides; Ovalbumin; Pulmonary Fibrosis; Smoking; Toll-Like Receptor 9; Transforming Growth Factor beta

Topics: Aged; Alleles; Arthritis, Rheumatoid; Female; Genotype; HLA-DR Antigens; HLA-DRB1 Chains; Humans; Interleukin-4; Male; Middle Aged; Pulmonary Fibrosis; Risk Factors; Transforming Growth Factor beta

Connective tissue growth factor (CTGF) is a cysteine-rich secreted matricellular protein involved in wound healing and tissue repair. Enhanced and prolonged expression of CTGF has been associated with tissue fibrosis in humans. However, questions remain as to whether CTGF expression alone is sufficient to drive fibrosis. This study was undertaken to investigate whether CTGF alone is sufficient to cause fibrosis in intact animals and whether its effects are mediated through activation of transforming growth factor beta (TGFbeta) signaling or through distinct signal transduction pathways.. We generated mice overexpressing CTGF in fibroblasts under the control of the fibroblast-specific collagen alpha2(I) promoter enhancer. Tissues such as skin, lung, and kidney were harvested for histologic analysis. Mouse embryonic fibroblasts were prepared from embryos (14.5 days postcoitum) for biochemical analysis.. Mice overexpressing CTGF in fibroblasts were susceptible to accelerated tissue fibrosis affecting the skin, lung, kidney, and vasculature, most notably the small arteries. We identified a marked expansion of the myofibroblast cell population in the dermis. RNA analysis of transgenic dermal fibroblasts revealed elevated expression of key matrix genes, consistent with a fibrogenic response. CTGF induced phosphorylation of p38, ERK-1/2, JNK, and Akt, but not Smad3, in transgenic mouse fibroblasts compared with wild-type mouse fibroblasts. Transfection experiments showed significantly increased basal activity of the CTGF and serum response element promoters, and enhanced induction of the CTGF promoter in the presence of TGFbeta.. These results demonstrate that selective expression of CTGF in fibroblasts alone causes tissue fibrosis in vivo through specific signaling pathways, integrating cues from the extracellular matrix into signal transduction pathways to orchestrate pivotal biologic responses relevant to tissue repair and fibrosis.

Topics: Animals; Basement Membrane; Blood Vessels; Cell Differentiation; Collagen; Collagen Type I; Connective Tissue Growth Factor; Fibroblasts; Gene Expression; Glomerulosclerosis, Focal Segmental; Kidney; Lac Operon; Liver Cirrhosis; Lymphokines; Mice; Mice, Inbred C57BL; Mice, Inbred CBA; Mice, Transgenic; Promoter Regions, Genetic; Pulmonary Fibrosis; Signal Transduction; Skin; Transforming Growth Factor beta

This study aimed to analyze whether Ulinastatin, a urinary trypsin inhibitor (UTI), inhibits the TGF-beta signaling pathway and lung fibrosis induced by thoracic irradiation in a lung injury mouse model. The thoraces of 9-week-old female fibrosis-sensitive C57BL/6 mice were irradiated with a single X-ray dose of 12 Gy or 24 Gy. UTI was administrated intraperitoneally at a dose of 200,000 units/kg concurrently with radiation (concurrent UTI) or daily during the post-irradiation period for 8-14 days (post-RT UTI). Mice were sacrificed at 16 weeks after irradiation to assess the histological grade of lung fibrosis and immunohistochemical TGF-beta expression. Survival rates of mice given 24 Gy to the whole lung +/- UTI were also compared. Post-RT UTI reduced the score of lung fibrosis in mice, but concurrent UTI had no beneficial effects in irradiated mice. The fibrosis score in post-RT UTI mice was 3.2 +/- 1.0, which was significantly smaller than that of irradiated mice without UTI treatment (RT alone; 6.0 +/- 1.3; p < 0.01). The rates of TGF-beta positive cells in post-RT UTI and the RT alone mice were 0.18 +/- 0.03 and 0.23 +/- 0.04, respectively (p < 0.01). There was a significantly positive correlation between the fibrosis score and the TGF-beta positive rate (R(2) = 0.26, p < 0.01). The survival rate at 30 weeks for post-RT UTI mice was significantly better than that of RT alone mice (33% vs. 10%, p < 0.05). The administration of post-RT UTI suppressed TGF-beta expression and radiation-induced lung fibrosis, which resulted in significant survival prolongation of the irradiated mice.

Topics: Animals; Dose-Response Relationship, Radiation; Female; Fibrosis; Glycoproteins; Immunohistochemistry; Lung; Mice; Mice, Inbred C57BL; Pulmonary Fibrosis; Radiation Pneumonitis; Time Factors; Transforming Growth Factor beta; Treatment Outcome

The role of the receptor for advanced glycation end-products (RAGE) has been shown to differ in two different mouse models of asbestos and bleomycin induced pulmonary fibrosis. RAGE knockout (KO) mice get worse fibrosis when challenged with asbestos, whereas in the bleomycin model they are largely protected against fibrosis. In the current study the role of RAGE in a mouse model of silica induced pulmonary fibrosis was investigated.. Wild type (WT) and RAGE KO mice received a single intratracheal (i.t.) instillation of silica in saline or saline alone as vehicle control. Fourteen days after treatment mice were subjected to a lung mechanistic study and the lungs were lavaged and inflammatory cells, protein and TGF-beta levels in lavage fluid determined. Lungs were subsequently either fixed for histology or excised for biochemical assessment of fibrosis and determination of RAGE protein- and mRNA levels. There was no difference in the inflammatory response or degree of fibrosis (hydroxyproline levels) in the lungs between WT and RAGE KO mice after silica injury. However, histologically the fibrotic lesions in the RAGE KO mice had a more diffuse alveolar septal fibrosis compared to the nodular fibrosis in WT mice. Furthermore, RAGE KO mice had a significantly higher histologic score, a measure of affected areas of the lung, compared to WT silica treated mice. A lung mechanistic study revealed a significant decrease in lung function after silica compared to control, but no difference between WT and RAGE KO. While a dose response study showed similar degrees of fibrosis after silica treatment in the two strains, the RAGE KO mice had some differences in the inflammatory response compared to WT mice.. Aside from the difference in the fibrotic pattern, these studies showed no indicators of RAGE having an effect on the severity of pulmonary fibrosis following silica injury.

Topics: Animals; Bronchoalveolar Lavage Fluid; Disease Models, Animal; Gene Expression Regulation; Glycation End Products, Advanced; Hydroxyproline; Inflammation; Lung; Mice; Mice, Inbred C57BL; Mice, Knockout; Pulmonary Fibrosis; Receptor for Advanced Glycation End Products; Receptors, Immunologic; Silicosis; Transforming Growth Factor beta

Transforming growth factor-beta (TGF-β) is a multifunctional cytokine that regulates cell proliferation, death, development or differentiation. In addition, TGF-β is considered a key mediator in fibrogenic processes, and signals either directly or indirectly through types I, II and III (TβRI, II, and III) receptor complexes. The type III TGF-β (TβRIII or betaglycan) is a transmembrane proteoglycan without a functional kinase domain, and is considered as a coreceptor to increase the affinity of ligand binding to TβRII. Little is studied on TGF-β and TβRIII (or betaglycan) signaling, while it is well known about TGF-β ligand and TβRII signaling. In this study, we investigated the effects of TβRIII expression on TGF-β induced differentiation, in view of the finding that TβRIII is significantly downregulated during TGF-β-induced differentiation in fibroblasts. TGF-beta induced alpha-SMA and Procollagen Type I expression were markedly inhibited in fibroblasts stably expressing TβRIII. Endogenous TβRIII expression did not alter the TβRI or TβRII levels, but inhibited Smad 2/3, Akt and ERK phosphorylation. The molecular mechanism of TβRIII action in TGF-β-induced differentiation is associated with both Smad-dependent and Smad-independent pathways. Our results suggest that TβRIII is a novel molecular target for regulation of TGF-β signaling in myofibroblast differentiation.

Topics: Animals; Cell Differentiation; Cell Line; Cell Proliferation; Collagen Type I; Down-Regulation; Fibroblasts; Lung; Mice; Myofibroblasts; NIH 3T3 Cells; Proteoglycans; Pulmonary Fibrosis; Receptors, Transforming Growth Factor beta; Signal Transduction; Smad Proteins; Transforming Growth Factor beta

There is a growing body of evidence demonstrating that plasminogen activator inhibitor-1 (PAI-1) is involved in the progression of pulmonary fibrosis. In fact, PAI-1 knockout mice are protected from bleomycin-induced pulmonary fibrosis. This study was conducted to determine whether the intrapulmonary administration of small interfering RNA (siRNA) targeting PAI-1 (PAI-1-siRNA) limits the development of bleomycin-induced pulmonary fibrosis.. Lung biopsies from patients with idiopathic pulmonary fibrosis (IPF) were stained for PAI-1. The distribution of siRNA in the lung, the PAI-1 level in bronchoalveolar (BAL) fluid and the extent of fibrotic changes in the lung were evaluated following the intranasal administration of PAI-1-siRNA in a mouse model of bleomycin-induced pulmonary fibrosis. The effect of PAI-1-siRNA on the epithelial to mesenchymal transition (EMT) was also evaluated using a mouse lung epithelial cell line, LA-4.. PAI-1 was overexpressed in the hyperplastic type 2 pneumocytes lining the honeycomb lesions of patients with IPF. The single intranasal instillation of PAI-1-siRNA resulted in the diffuse uptake of siRNA into the epithelial cells lining the dense fibrotic lesions. The repeated administration of PAI-1-siRNA initiated during either the inflammatory or the fibrotic phase into bleomycin-injured mice reduced the PAI-1 level in BAL fluid and limited the accumulation of collagen in the lungs. EMT induced by transforming growth factor beta (TGFbeta) in LA-4 cells was inhibited by transfection with PAI-1-siRNA.. The direct suppression of PAI-1 in the lung by the intrapulmonary administration of PAI-1-siRNA attenuated the development and progression of pulmonary fibrosis. The inhibition of EMT may be, at least in part, involved in this effect.

Topics: Animals; Apoptosis; Bleomycin; Bronchoalveolar Lavage Fluid; Cell Differentiation; Cell Line; Cell Proliferation; Disease Models, Animal; Genetic Therapy; Humans; Lung; Mice; Mice, Inbred C57BL; Plasminogen Activator Inhibitor 1; Pulmonary Fibrosis; Respiratory Mucosa; RNA Interference; Transfection; Transforming Growth Factor beta

Drug-induced interstitial lung disease (ILD), particularly pulmonary fibrosis, is a serious clinical concern and myofibroblasts have been suggested to have a major role, with it recently being revealed that some of these myofibroblasts are derived from lung epithelial cells through epithelial-mesenchymal transition (EMT). In this study, we examined the EMT-inducing abilities of drugs known to induce ILD clinically. EMT-like phenotypes were induced by A771726, an active metabolite of leflunomide having an inhibitory effect on dihydroorotate dehydrogenase (DHODH). Smad-interacting protein 1 (a transcription factor regulating EMT) and the Notch-signaling pathway but not transforming growth factor-β was shown to be involved in A771726-induced EMT-like phenotypes. When the cultures were supplemented with exogenous uridine, the A771726-induced EMT-like phenotypes and activation of the Notch-signaling pathway disappeared. Similarly, an A771726 analog without inhibitory activity on DHODH produced no induction, suggesting that this process is mediated through the inhibition of DHODH. In vivo, administration of leflunomide stimulated bleomycin-induced EMT-like phenomenon in pulmonary tissue, and exacerbated bleomycin-induced pulmonary fibrosis, both of which were suppressed by coadministration of uridine. Taken together, these findings suggest that leflunomide-dependent exacerbation of bleomycin-induced pulmonary fibrosis is mediated by stimulation of EMT of lung epithelial cells, providing the first evidence that drug-induced pulmonary fibrosis involves EMT of these cells.

Topics: Aniline Compounds; Animals; Bleomycin; Cells, Cultured; Crotonates; Dihydroorotate Dehydrogenase; Enzyme Inhibitors; Epithelial-Mesenchymal Transition; Homeodomain Proteins; Humans; Hydroxybutyrates; Hydroxyproline; Mice; Nitriles; Oxidoreductases Acting on CH-CH Group Donors; Phenotype; Pulmonary Fibrosis; Rats; Receptors, Notch; Repressor Proteins; RNA Interference; RNA, Small Interfering; Signal Transduction; Toluidines; Transforming Growth Factor beta; Uridine; Zinc Finger E-box Binding Homeobox 2

Breast cancer that recurs as metastatic disease many years after primary tumor resection and adjuvant therapy seems to arise from tumor cells that disseminated early in the course of disease but did not develop into clinically apparent lesions. These long-term surviving, disseminated tumor cells maintain a state of dormancy, but may be triggered to proliferate through largely unknown factors. We now show that the induction of fibrosis, associated with deposition of type I collagen (Col-I) in the in vivo metastatic microenvironment, induces dormant D2.0R cells to form proliferative metastatic lesions through beta1-integrin signaling. In vitro studies using a three-dimensional culture system modeling dormancy showed that Col-I induces quiescent D2.0R cells to proliferate through beta1-integrin activation of SRC and focal adhesion kinase, leading to extracellular signal-regulated kinase (ERK)-dependent myosin light chain phosphorylation by myosin light chain kinase and actin stress fiber formation. Blocking beta1-integrin, Src, ERK, or myosin light chain kinase by short hairpin RNA or pharmacologic approaches inhibited Col-I-induced activation of this signaling cascade, cytoskeletal reorganization, and proliferation. These findings show that fibrosis with Col-I enrichment at the metastatic site may be a critical determinant of cytoskeletal reorganization in dormant tumor cells, leading to their transition from dormancy to metastatic growth. Thus, inhibiting Col-I production, its interaction with beta1-integrin, and downstream signaling of beta1-integrin may be important strategies for preventing or treating recurrent metastatic disease.

Topics: Actins; Animals; Cell Growth Processes; Collagen Type I; Extracellular Matrix; Extracellular Signal-Regulated MAP Kinases; Female; Focal Adhesion Kinase 1; Integrin beta1; Lung Neoplasms; Mammary Neoplasms, Experimental; Mice; Mice, Inbred BALB C; Mice, Nude; Myosin-Light-Chain Kinase; Phosphorylation; Pulmonary Fibrosis; Rats; Signal Transduction; src-Family Kinases; Transforming Growth Factor beta

Topics: Animals; Bone Marrow Cells; Cytoskeleton; Disease Progression; Endothelium; Fibroblasts; Fibrosis; Gene Expression Regulation; Humans; Mesoderm; Models, Biological; Pulmonary Fibrosis; ras Proteins; Signal Transduction; Stem Cells; Transforming Growth Factor beta

Pulmonary fibrosis is a type of interstitial lung disease that causes progressive scarring in lung tissues. Although there have been many studies on fibrosis, there is no standard treatment for fibrotic disease. Thus, there is an urgent need for the development of effective anti-fibrotic drugs. Transforming growth factor beta (TGF-beta) is a major fibrotic mediator known to stimulate fibrosis. To identify small molecules that inhibit TGF-beta responses, we performed cell-based chemical screening using genetically engineered HEK293 reporter cells. Among 8000 chemical compounds containing biologically active natural products and synthetic or clinically used compounds, we found that 3-(2-chlorobenzyl)-1,7-dimethyl-1H-imidazo[2,1-f]purine-2,4(3H,8H)-dione (IM-412) significantly decreased TGF-beta stimulated reporter activity in a dose-dependent manner. In addition, IM-412 inhibited TGF-beta-induced expression of the fibrotic markers alpha-smooth muscle actin (alpha-SMA) and fibronectin, and collagen accumulation in CCD-18Lu human normal lung fibroblasts without cell cytotoxicity. IM-412 decreased Smad2 and -3 phosphorylation as well as JNK and ERK activity. Moreover, expression levels of TGF-beta receptor I (TbetaRI) and receptor II (TbetaRII) were down-regulated by IM-412 in a dose-dependent manner. Thus, our findings indicate that the small molecule IM-412 attenuated TGF-beta-mediated fibroblast differentiation through inhibition of the overall TGF-beta response and may be a promising novel agent for the treatment of pathological fibrotic conditions.

Topics: Cell Differentiation; Cell Line; Fibroblasts; Humans; Imidazoles; Lung; Pulmonary Fibrosis; Purines; Transforming Growth Factor beta

Prostaglandin (PG) D(2) exerts contrasting activities in the inflamed lung via two receptors, the D prostanoid receptor (DP) and the chemoattractant receptor-homologous molecule expressed on T helper 2 lymphocytes. DP activation is known mainly to inhibit proinflammatory cell functions. We tested the effect of a DP-specific agonist, (4S)-(3-[(3R,S)-3-cyclohexyl-3-hydroxypropyl]-2,5-dioxo)-4-imidazolidineheptanoic acid (BW245C), on pulmonary fibroblast functions in vitro and in a mouse model of lung fibrosis induced by bleomycin. DP mRNA expression was detected in cultured mouse lung primary fibroblasts and human fetal lung fibroblasts and found to be up- and down-regulated by interleukin-13 and transforming growth factor (TGF)-β, respectively. Although micromolar concentrations of BW245C and PGD(2) did not affect mouse fibroblast collagen synthesis or differentiation in myofibroblasts, they both inhibited fibroblast basal and TGF-β-induced proliferation in vitro. The repeated administration of BW245C (500 nmol/kg body weight instilled transorally in the lungs 2 days before and three times per week for 3 weeks) in bleomycin-treated mice significantly decreased both inflammatory cell recruitment and collagen accumulation in the lung (21 days). Our results indicate that BW245C can reduce lung fibrosis in part via its activity on fibroblast proliferation and suggest that DP activation should be considered as a new therapeutic target in fibroproliferative lung diseases.

Topics: Animals; Bleomycin; Bronchoalveolar Lavage Fluid; Cell Proliferation; Cells, Cultured; Collagen; Female; Fibroblasts; Humans; Hydantoins; Interleukin-13; Mice; Mice, Inbred C57BL; Pulmonary Fibrosis; Receptors, Prostaglandin; Transforming Growth Factor beta

Micro-computed tomography (micro-CT) is a novel tool for monitoring acute and chronic disease states in small laboratory animals. Its value for assessing progressive lung fibrosis in mice has not been reported so far. Here we examined the importance of in vivo micro-CT as non-invasive tool to assess progression of pulmonary fibrosis in mice over time.. Pulmonary fibrosis was induced in mice by intratracheal delivery of an adenoviral gene vector encoding biologically active TGF-β1 (AdTGF-β1). Respiratory gated and ungated micro-CT scans were performed at 1, 2, 3, and 4 weeks post pulmonary adenoviral gene or control vector delivery, and were then correlated with respective histopathology-based Ashcroft scoring of pulmonary fibrosis in mice. Visual assessment of image quality and consolidation was performed by 3 observers and a semi-automated quantification algorithm was applied to quantify aerated pulmonary volume as an inverse surrogate marker for pulmonary fibrosis.. We found a significant correlation between classical Ashcroft scoring and micro-CT assessment using both visual assessment and the semi-automated quantification algorithm. Pulmonary fibrosis could be clearly detected in micro-CT, image quality values were higher for respiratory gated exams, although differences were not significant. For assessment of fibrosis no significant difference between respiratory gated and ungated exams was observed.. Together, we show that micro-CT is a powerful tool to assess pulmonary fibrosis in mice, using both visual assessment and semi-automated quantification algorithms. These data may be important in view of pre-clinical pharmacologic interventions for the treatment of lung fibrosis in small laboratory animals.

Topics: Adenoviridae; Animals; Female; Lung; Mice; Mice, Inbred C57BL; Pulmonary Fibrosis; Sensitivity and Specificity; Tomography, X-Ray Computed; Transfection; Transforming Growth Factor beta

Dipeptidyl peptidase IV (DP IV)-related proteases and aminopeptidase N (APN) are drug targets in various diseases. Here we investigated for the first time the effects of DP-IV-related protease inhibitors and APN inhibitors on chronic inflammatory lung diseases.. A murine model of silica (SiO2)-induced lung fibrosis and in vitro cultures of human lung epithelial cells and monocytes have been used and the influence of silica-treatment and inhibitors on inflammation and fibrosis has been measured.. We found increased inflammation and secretion of the chemokines IL-6, MCP-1 and MIP-alpha 2 weeks after SiO2 application, and increased lung fibrosis after 3 months. Treatment with the APN inhibitor actinonin reduced chemokine secretion in the lung and bronchoalveolar lavage fluid, and in cell culture, and decreased the level of fibrosis after 3 months. Treatment with inhibitors of DP-IV-related proteases, or a combination of DP IV inhibitors and APN inhibitors, had no significant effect. We found no obvious side effects of long-term treatment with inhibitors of APN and DP IV.. Overall, our findings show that actinonin, an inhibitor of aminopeptidase N, might modulate chemokine secretion in the lung and thus attenuate the development of lung fibrosis. Additional targeting of DP-IV-related proteases had no significant effect on these processes.

Topics: Animals; CD13 Antigens; Cell Line; Cytokines; Dipeptidyl-Peptidase IV Inhibitors; Humans; Hydroxamic Acids; Interleukin-6; Interleukin-8; Lung; Mice; Mice, Inbred C57BL; Protease Inhibitors; Pulmonary Fibrosis; Silicon Dioxide; Transforming Growth Factor beta

Idiopathic pulmonary fibrosis is a lethal parenchymal lung disease characterized by denudation of the lung epithelium, fibroblast proliferation, and collagen deposition. Cellular changes underlying disease progression involve injury to alveolar epithelial cells, epithelial to mesenchymal transition, proliferation of alpha-smooth muscle actin (alpha-SMA)-expressing myofibroblasts and of fibroblasts resulting in enhanced deposition of extracellular matrix proteins. Hepatocyte growth factor (HGF) inhibits progression of bleomycin-induced pulmonary fibrosis in mice. The mechanism underlying the inhibitory effect of HGF was investigated in an in vitro model. We show that HGF markedly antagonizes basal and transforming growth factor (TGF)-beta-induced expression of myofibroblast markers such as alpha-SMA, collagen type 1, and fibronectin in rat alveolar epithelial cells. HGF also inhibited TGF-beta-induced alpha-SMA expression in primary murine alveolar epithelial cells. Since TGF-beta is known to regulate alpha-SMA expression, the effect of HGF on components of TGF-beta signaling was investigated. HGF induced expression of Smad7, an inhibitor of TGF-beta signaling, in a mitogen-activated protein kinase-dependent manner. HGF also induced the nuclear export of Smad7 and Smad ubiquitin regulatory factor 1 (Smurf1) to the cytoplasm. HGF-dependent decrease in alpha-SMA was abolished with specific siRNAs targeted to Smad7. Thus, induction of Smad7 by HGF serves to limit acquisition of the myofibroblast phenotype in alveolar epithelial cells.

Topics: Actins; Animals; Bleomycin; Epithelium; Fibroblasts; Hepatocyte Growth Factor; Lung; MAP Kinase Signaling System; Mice; Muscle, Smooth; Phenotype; Pulmonary Fibrosis; Rats; Smad7 Protein; Transforming Growth Factor beta

Transforming growth factor-beta (TGF-beta) plays a pivotal role in the fibrogenic action involved in the induction of connective tissue growth factor (CTGF), extracellular matrix and fibroblast transformation. Smad3 mediates TGF-beta signaling related to the fibrotic response. In human lung fibroblasts or bronchial smooth muscle cells, we demonstrated that an increase in the intracellular glutathione level suppressed TGF-beta1-induced phosphorylation of Smad3, while inhibiting TGF-beta1-induced expressions of CTGF, collagen type1, fibronectin and transformation into myofibroblasts, which are characterized by the expression of alpha-smooth muscle actin. These data indicate that the intracellular glutathione redox status regulates TGF-beta-induced fibrogenic effects through Smad3 activation.

Topics: Actins; Cell Line; Collagen Type I; Connective Tissue Growth Factor; Dexamethasone; Fibroblasts; Fibronectins; Glutathione; Humans; Myoblasts; Phosphorylation; Pulmonary Fibrosis; Smad3 Protein; Transforming Growth Factor beta

Previous study showed that aerosolized signal transducer and activator of transcription-1 (STAT1) antisense oligodeoxynucleotide (ASON) inhibited the expression of STAT1 and ICAM-1 mRNA and protein in alveolar macrophages (AMs) and decreased the concentrations of TGF-beta, PDGF and TNF-alpha in bronchioalveolar lavage fluid (BALF) in bleomycin (BLM)-induced rat pulmonary fibrosis. Administration of STAT1 ASON ameliorated alveolitis in rat pulmonary fibrosis. However, further investigations are needed to determine whether there is an effect from administration of STAT1 ASON on fibrosis. This study investigated the effect of aerosolized STAT1 ASON on the expressions of inflammatory mediators, hydroxyproline and type I and type III collagen mRNA in BLM-induced rat pulmonary fibrosis. The results showed that STAT1 ASON applied by aerosolization could ameliorate alveolitis and fibrosis, inhibit the expressions of inflammatory mediators, decrease the content of hydroxyproline, and suppress the expressions of type I and type III collagen mRNA in lung tissue in BLM-induced rat pulmonary fibrosis. These results suggest that aerosolized STAT1 ASON might be considered as a promising new strategy in the treatment of pulmonary fibrosis.

Topics: Administration, Inhalation; Aerosols; Animals; Bleomycin; Bronchoalveolar Lavage Fluid; Collagen Type I; Collagen Type III; Female; Intercellular Adhesion Molecule-1; Interferon-gamma; Liposomes; Lung; Oligodeoxyribonucleotides, Antisense; Platelet-Derived Growth Factor; Pulmonary Fibrosis; Rats; Rats, Wistar; Smad4 Protein; STAT1 Transcription Factor; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha

Topics: Epithelium; Extracellular Matrix; Fibroblasts; Humans; Hyaluronic Acid; Hypertension, Pulmonary; Idiopathic Interstitial Pneumonias; Prognosis; Pulmonary Fibrosis; Randomized Controlled Trials as Topic; Scleroderma, Systemic; Transforming Growth Factor beta

Pulmonary fibrosis is a progressive scarring disease with no effective treatment. Transforming growth factor (TGF)-beta is up-regulated in fibrotic diseases, where it stimulates differentiation of fibroblasts to myofibroblasts and production of excess extracellular matrix. Peroxisome proliferator-activated receptor (PPAR) gamma is a transcription factor that regulates adipogenesis, insulin sensitization, and inflammation. We report here that a novel PPARgamma ligand, 2-cyano-3,12-dioxoolean-1,9-dien-28-oic acid (CDDO), is a potent inhibitor of TGF-beta-stimulated differentiation of human lung fibroblasts to myofibroblasts, and suppresses up-regulation of alpha-smooth muscle actin, fibronectin, collagen, and the novel myofibroblast marker, calponin. The inhibitory concentration causing a 50% decrease in aSMA for CDDO was 20-fold lower than the endogenous PPARgamma ligand, 15-deoxy-Delta(12,14)-prostaglandin J(2) (15 d-PGJ(2)), and 400-fold lower than the synthetic ligand, rosiglitazone. Pharmacologic and genetic approaches were used to demonstrate that CDDO mediates its activity via a PPARgamma-independent pathway. CDDO and 15 d-PGJ(2) contain an alpha/beta unsaturated ketone, which acts as an electrophilic center that can form covalent bonds with cellular proteins. Prostaglandin A(1) and diphenyl diselenide, both strong electrophiles, also inhibit myofibroblast differentiation, but a structural analog of 15 d-PGJ(2) lacking the electrophilic center is much less potent. CDDO does not alter TGF-beta-induced Smad or AP-1 signaling, but does inhibit acetylation of CREB binding protein/p300, a critical coactivator in the transcriptional regulation of TGF-beta-responsive genes. Overall, these data indicate that certain PPARgamma ligands, and other small molecules with electrophilic centers, are potent inhibitors of critical TGF-beta-mediated profibrogenic activities through pathways independent of PPARgamma. As the inhibitory concentration causing a 50% decrease in aSMA for CDDO is 400-fold lower than that in rosiglitazone, the translational potential of CDDO for treatment of fibrotic diseases is high.

Topics: Acetylation; Actins; Base Sequence; Cell Differentiation; Cells, Cultured; DNA Primers; Fibroblasts; Humans; Ligands; Lung; Oleanolic Acid; p300-CBP Transcription Factors; PPAR gamma; Prostaglandin D2; Pulmonary Fibrosis; Rosiglitazone; Signal Transduction; Smad Proteins; Thiazolidinediones; Transcription Factor AP-1; Transforming Growth Factor beta

Acute lung injury involving extremely immature lungs often heals without excessive fibrosis unlike later in gestation and in adults. Several factors may be involved, but fibroblast contraction of collagen has been linked to the level of wound fibrosis. To assess whether human lung fibroblasts of fetal versus adult origin differ in ability to contract collagen and define the molecular underpinnings, we performed three-dimensional collagen contraction assay, analyzed their differential mRNA profile, specifically for transforming growth factor-beta (TGF-beta) signaling pathway and extracellular matrix components, studied the cell response to TGF-beta in culture, and used two-dimensional gel electrophoresis followed by mass spectrometry to identify differences in their overall proteomes. Human lung fetal fibroblasts contracted the collagen matrix less than the adults. Smooth muscle actin expression did not differ. TGF-beta stimulation resulted in greater Smad3 phosphorylation in fetal compared with adults. mRNA and proteomic profiling reveal a number of TGF-beta pathways, ECM components, and cytoskeletal regulatory molecules are differentially expressed between the cell types. Of note is TGF-beta receptor interacting protein 1 (TRIP-1), which we show inhibits fibroblast collagen contraction and is higher in fetal than adult fibroblasts. We conclude that human lung fetal fibroblasts are less able to contract collagen than adult lung fibroblasts. The diminished ability is not due to impediment of Smad3 activation but rather, at least in part, due to their higher level of TRIP-1 expression. TRIP-1 is a novel modulator of fibroblast collagen contraction.

Topics: Adult; Cells, Cultured; Collagen Type I; Electrophoresis, Gel, Two-Dimensional; Eukaryotic Initiation Factor-3; Fetus; Fibroblasts; Gene Expression Regulation, Developmental; Humans; Lung; Proteomics; Pulmonary Fibrosis; Reverse Transcriptase Polymerase Chain Reaction; Smad3 Protein; Transforming Growth Factor beta; Wound Healing

Pulmonary diseases associated with fibrosis, including scleroderma lung disease, are characterized by the accumulation of T cells in the lungs. These cells are thought to facilitate lung fibrosis, but the exact mechanisms of their profibrotic action are not clear. Several alphaV-containing integrins, including alphaVbeta3 and alphaVbeta5, have been shown to directly activate transforming growth factor beta (TGFbeta) and promote collagen accumulation. The aim of this study was to investigate whether pulmonary T cells express profibrotic integrins and regulate collagen accumulation.. Expression of integrins was assessed by immunohistochemical analysis of lung tissue, by flow cytometry using bronchoalveolar lavage fluid from patients with systemic sclerosis (SSc), and in a CCL18 overexpression animal model of pulmonary T cell infiltration. Experiments in cell cultures were performed to determine whether integrin-expressing T cells are profibrotic in cocultures with pulmonary fibroblasts and, if so, through what possible mechanism.. Lymphocytes and integrin-positive cells were present in the lungs, and pulmonary T cells expressed integrins alphaVbeta3 and alphaVbeta5 in patients with SSc and in the animal model. Systemic administration of neutralizing anti-integrin alphaV antibody or a genetic deficiency of integrin beta3 in the CCL18 overexpression model significantly attenuated CCL18-driven pulmonary lymphocytic infiltration and collagen accumulation. Jurkat T cells overexpressing integrin alphaVbeta3 or integrin alphaVbeta5 in cocultures with primary pulmonary fibroblasts stimulated collagen accumulation and Smad2 nuclear translocation. Neutralizing anti-TGFbeta antibody attenuated the profibrotic effect of integrin-expressing T cells.. Pulmonary infiltrating T lymphocytes may express integrins alphaVbeta3 and alphaVbeta5 that are necessary for lymphocytic infiltration and T cell-associated TGFbeta activation and collagen accumulation.

Topics: Animals; Antibodies; Bronchoalveolar Lavage Fluid; Cells, Cultured; Collagen; Flow Cytometry; Humans; Immunohistochemistry; Integrin alphaV; Integrin alphaVbeta3; Jurkat Cells; Lung; Mice; Mice, Inbred C57BL; Pulmonary Fibrosis; Receptors, Vitronectin; Scleroderma, Systemic; T-Lymphocytes; Transforming Growth Factor beta

Idiopathic pulmonary fibrosis (IPF) is a devastating disease with poor prognosis. Leukotrienes play an important role in IPF, and leukotriene (LT)B(4) is one of the key eicosanoids in IPF. In this study, we investigated whether ONO-4057, a LTB(4) receptor (BLTR) antagonist is capable of preventing bleomycin-induced pulmonary fibrosis. On day 1, C57BL/6 male mice were given a single intratracheal injection of bleomycin (2.5 mg x kg(-1)), and ONO-4057 (1.0 mg x kg(-1)) or vehicle alone, administered by intraperitoneal injection on days 1-5 each week for 3 weeks after the bleomycin injection. ONO-4057 reduced the total cell count in bronchoalveolar lavage fluid (BALF) on days 7, 14 and 21 and the Ashcroft score and the lung hydroxyproline content on days 14 and 21. The LTB(4), interleukin (IL)-6, IL-13, transforming growth factor (TGF)-beta levels in BALF and the TGF-beta expression in lung tissue, assessed by immunohistochemistry were decreased on day 7, whereas interferon (IFN)-gamma level in BALF was increased on day 14. The results of this study indicated that the BLTR antagonist inhibited the development of bleomycin-induced pulmonary fibrosis in mice by decreasing inflammation and altering TGF-beta, IL-6, IL-13 and IFN-gamma.

Topics: Animals; Antibiotics, Antineoplastic; Bleomycin; Bronchoalveolar Lavage Fluid; Immunohistochemistry; Immunosuppressive Agents; Interferon-gamma; Interleukin-13; Interleukin-6; Male; Mice; Mice, Inbred C57BL; Phenylpropionates; Prognosis; Pulmonary Fibrosis; Receptors, Leukotriene B4; Transforming Growth Factor beta

Acute respiratory distress syndrome is characterized by loss of lung tissue as a result of inflammation and fibrosis. Augmenting tissue repair by the use of mesenchymal stem cells may be an important advance in treating this condition. We evaluated the role of term human umbilical cord cells derived from Wharton's jelly with a phenotype consistent with mesenchymal stem cells (uMSCs) in the treatment of a bleomycin-induced mouse model of lung injury. uMSCs were administered systemically, and lungs were harvested at 7, 14, and 28 days post-bleomycin. Injected uMSCs were located in the lung 2 weeks later only in areas of inflammation and fibrosis but not in healthy lung tissue. The administration of uMSCs reduced inflammation and inhibited the expression of transforming growth factor-beta, interferon-gamma, and the proinflammatory cytokines macrophage migratory inhibitory factor and tumor necrosis factor-alpha. Collagen concentration in the lung was significantly reduced by uMSC treatment, which may have been a consequence of the simultaneous reduction in Smad2 phosphorylation (transforming growth factor-beta activity). uMSCs also increased matrix metalloproteinase-2 levels and reduced their endogenous inhibitors, tissue inhibitors of matrix metalloproteinases, favoring a pro-degradative milieu following collagen deposition. Notably, injected human lung fibroblasts did not influence either collagen or matrix metalloproteinase levels in the lung. The results of this study suggest that uMSCs have antifibrotic properties and may augment lung repair if used to treat acute respiratory distress syndrome.

Topics: Animals; Antibiotics, Antineoplastic; Bleomycin; Blotting, Western; Collagen; Cord Blood Stem Cell Transplantation; Disease Models, Animal; Enzyme-Linked Immunosorbent Assay; Fetal Blood; Humans; Immunohistochemistry; Inflammation; Lung Injury; Macrophage Migration-Inhibitory Factors; Matrix Metalloproteinase 2; Mesenchymal Stem Cell Transplantation; Mesenchymal Stem Cells; Mice; Mice, SCID; Polymerase Chain Reaction; Pulmonary Fibrosis; Respiratory Distress Syndrome; RNA, Messenger; Tissue Inhibitor of Metalloproteinases; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha

Advanced glycation end products (AGEs) have been proposed to be involved in pulmonary fibrosis, but its role in this process has not been fully understood. To investigate the role of AGE formation in pulmonary fibrosis, we used a bleomycin (BLM)-stimulated rat model treated with aminoguanidine (AG), a crosslink inhibitor of AGE formation.. Rats were intratracheally instilled with BLM (5 mg/kg) and orally administered with AG (40, 80, 120 mg/kg) once daily for two weeks. AGEs level in lung tissue was determined by ELISA and pulmonary fibrosis was evaluated by Ashcroft score and hydroxyproline assay. The expression of heat shock protein 47 (HSP47), a collagen specific molecular chaperone, was measured with RT-PCR and Western blot. Moreover, TGFbeta1 and its downstream Smad proteins were analyzed by Western blot.. AGEs level in rat lungs, as well as lung hydroxyproline content and Ashcroft score, was significantly enhanced by BLM stimulation, which was abrogated by AG treatment. BLM significantly increased the expression of HSP47 mRNA and protein in lung tissues, and AG treatment markedly decreased BLM-induced HSP47 expression in a dose-dependent manner (p < 0.05). In addition, AG dose-dependently downregulated BLM-stimulated overexpressions of TGFbeta1, phosphorylated (p)-Smad2 and p-Smad3 protein in lung tissues.. These findings suggest AGE formation may participate in the process of BLM-induced pulmonary fibrosis, and blockade of AGE formation by AG treatment attenuates BLM-induced pulmonary fibrosis in rats, which is implicated in inhibition of HSP47 expression and TGFbeta/Smads signaling.

Topics: Animals; Antimetabolites, Antineoplastic; Bleomycin; Blotting, Western; Enzyme Inhibitors; Enzyme-Linked Immunosorbent Assay; Glycation End Products, Advanced; Guanidines; HSP47 Heat-Shock Proteins; Hydroxyproline; Lung; Male; Pulmonary Fibrosis; Rats; Rats, Sprague-Dawley; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Signal Transduction; Smad2 Protein; Smad3 Protein; Transforming Growth Factor beta

To investigate the curative effects of inhaling signal transducer and activator of transcription 1 (STAT1) antisense oligonucleotide (ASON) on alveolitis and pulmonary fibrosis and the best administration time.. Twenty-five adult female Wistar rats were randomly divided into 5 equal groups: BLM group, undergoing intra-tracheal perfusion of BLM so as to establish animal models of alveolitis and pulmonary fibrosis and then inhaling aerosolized normal saline (NS); NS group undergoing intra-tracheal perfusion of NS and then inhaling aerosolized NS; ASON 0 d group, undergoing intra-tracheal perfusion of BLM and then inhaling aerosolized STAT1 ASON 3 ml immediately; ASON 7 d group, undergoing intra-tracheal perfusion of BLM and then inhaling STAT1 ASON 3 ml 7 days later; and ASON 14 d group undergoing intra-tracheal perfusion of BLM and then inhaling aerosolized STAT1 ASON 3 ml 14 days later. Aerosolized inhalation was repeated once every other day for 4 times. Twenty-eight days after intra-tracheal perfusion the rats were sacrificed with their lungs taken out to undergo pathological examination. NS was infused into the right lungs to get bronchoalveolar lavage fluid (BALF). ELISA was used to examine the concentrations of transforming growth factor-beta (TGF-beta) and tumor necrosis factor-alpha (TNF-alpha) in the BALF.. The pathology result of the lung tissues showed that compared with the BLM and ASON 14 d groups, the alveolitis and pulmonary fibrosis of the ASON 0 d group were obviously milder. The scores of alveolitis and pulmonary fibrosis of the ASON 0 d group were (1.80 +/- 0.84) and (2.60 +/- 0.55) respectively, both significantly lower than those of the BLM group [(2.40 +/- 0.55) and (4.40 +/- 0.55) respectively] and those of the ASON 7 d group [(2.20 +/- 0.45) and (3.00 +/- 0.71) respectively] (all P < 0.05). The scores of pulmonary fibrosis of the ASON 7 d group was significantly lower than those of the BLM and ASON 14 d groups (both P < 0.05). The concentrations of TGF-beta and TNF-alpha in BALF of the ASON 0 d group were (48.11 +/- 3.46) pg/ml and (1.93 +/- 0.14) ng/ml respectively, both significantly lower than those of the BLM group [(57.67 +/- 2.46) pg/ml and (2.45 +/- 0.25) ng/ml respectively, both P < 0.05]. The concentration of TGF-beta in BALF of the ASON 0 d group was significantly lower than those of the ASON 7 d and ASON 14 d groups [(51.42 +/- 3.57) pg/ml and (55.8 3 +/- 1.79) pg/ml respectively, both P < 0.05]. The concentration of TGF-beta in BALF of the ASON7 d group was significantly lower than those of the BLM and ASON 14 d groups (both P < 0.05).. STAT1 ASON administered in the early stage helps depress the pulmonary fibrosis procedure, and the earlier the drug is administrated the better effect would be obtained. Aerosolized STAT1 ASON can be used as a therapeutic method for pulmonary fibrosis.

Topics: Administration, Inhalation; Animals; Bleomycin; Chronotherapy; Female; Oligonucleotides, Antisense; Pulmonary Fibrosis; Rats; Rats, Wistar; STAT1 Transcription Factor; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha

Uncontrolled activation of the coagulation cascade contributes to the pathophysiology of several conditions, including acute and chronic lung diseases. Coagulation zymogens are considered to be largely derived from the circulation and locally activated in response to tissue injury and microvascular leak. Here we report that expression of coagulation factor X (FX) is locally increased in human and murine fibrotic lung tissue, with marked immunostaining associated with bronchial and alveolar epithelia. FXa was a potent inducer of the myofibroblast differentiation program in cultured primary human adult lung fibroblasts via TGF-beta activation that was mediated by proteinase-activated receptor-1 (PAR1) and integrin alphavbeta5. PAR1, alphavbeta5, and alpha-SMA colocalized to fibrotic foci in lung biopsy specimens from individuals with idiopathic pulmonary fibrosis. Moreover, we demonstrated a causal link between FXa and fibrosis development by showing that a direct FXa inhibitor attenuated bleomycin-induced pulmonary fibrosis in mice. These data support what we believe to be a novel pathogenetic mechanism by which FXa, a central proteinase of the coagulation cascade, is locally expressed and drives the fibrotic response to lung injury. These findings herald a shift in our understanding of the origins of excessive procoagulant activity and place PAR1 central to the cross-talk between local procoagulant signaling and tissue remodeling.

Topics: Actins; Adult; Aged; Animals; Base Sequence; Bleomycin; Case-Control Studies; Cell Differentiation; Cells, Cultured; Factor Xa; Factor Xa Inhibitors; Female; Fibroblasts; Gene Expression; Humans; Idiopathic Pulmonary Fibrosis; Lung Injury; Male; Mice; Mice, Inbred C57BL; Middle Aged; Models, Biological; Pulmonary Fibrosis; Receptor, PAR-1; Receptors, Vitronectin; RNA, Messenger; Transforming Growth Factor beta; Up-Regulation

Extracorporeal photopheresis has anti-inflammatory properties. The development of pulmonary fibrosis includes inflammatory episodes. This study evaluates effects of extracorporeal photopheresis in experimental pulmonary fibrosis. The bleomycin model of pulmonary fibrosis was used. Two groups of 4 rats received intratracheal bleomycin to induce fibrosis. The treatment group received infusions of photochemically treated leukocytes harvested from syngeneic animals. All animals were sacrificed at day 21 after fibrosis induction and analyzed with respect to lung histology and hydroxyproline content, cellular composition of bronchoalveolar lavages, serum and lavage concentrations of transforming growth factor-beta, interferon-gamma, and interleukin-10, and expression of selected genes in the lung. Interleukin-10 and transforming growth factor-beta protein concentrations increased in the plasma of treated animals, whereas the interferon-gamma protein concentration was higher in bronchoalveolar lavages. Interferon-gamma gene expression was up-regulated in the lung tissue of treated animals. No significant differences between treated and untreated animals were found with respect to hydroxyproline, histology, and lavage cell count. To conclude, extracorporeal photopheresis has positive molecular effects but does not attenuate experimental lung fibrosis with respect to histology, hydroxyproline, and lavage cell count in the applied treatment regimen. Further investigations of extracorporeal photopheresis in experimental pulmonary fibrosis are justified.

Topics: Animals; Bleomycin; Bronchoalveolar Lavage Fluid; Disease Models, Animal; Gene Expression Regulation; Hydroxyproline; Interferon-gamma; Interleukin-10; Lung; Photopheresis; Pulmonary Fibrosis; Rats; Rats, Inbred Lew; Severity of Illness Index; Time Factors; Transforming Growth Factor beta

Idiopathic pulmonary fibrosis (IPF) is a fatal interstitial lung disease characterised by accumulation of activated (myo)fibroblasts and excessive extracellular matrix deposition. The enhanced accumulation of (myo)fibroblasts may be attributed, in part, to the process of transforming growth factor beta1 (TGFbeta1)-induced epithelial-mesenchymal transition (EMT), the phenotypic switching of epithelial to fibroblast-like cells. Although alveolar epithelial type II (ATII) cells have been shown to undergo EMT, the precise mediators and mechanisms remain to be resolved. The objective of this study is to investigate the role of SNAI transcription factors in the process of EMT and in IPF.. Using quantitative reverse transcription-PCR (RT-PCR), immunofluorescence, immunohistochemistry, western blotting, as well as gain- and loss-of-function studies and functional assays, the role of SNAI1 and SNAI2 in TGFbeta1-induced EMT in ATII cells in vitro was assessed; and the expression of SNAI transcription factors was analysed in experimental and human IPF in vivo.. TGFbeta1 treatment increased the expression and nuclear accumulation of SNAI1 and SNAI2, in concert with induction of EMT in ATII cells. SNAI overexpression was sufficient to induce EMT, and small interfering RNA (siRNA)-mediated SNAI depletion attenuated TGFbeta1-induced ATII cell migration and EMT. SNAI expression was elevated in experimental and human IPF and localised to hyperplastic ATII cells in vivo.. The results demonstrate that TGFbeta1-induced EMT in ATII cells is essentially controlled by the expression and nuclear translocation of SNAI transcription factors. Increased SNAI1 and SNAI2 expression in experimental and human IPF in vivo suggests that SNAI-mediated EMT may contribute to the fibroblast pool in idiopathic pulmonary fibrosis.

Topics: Adult; Animals; Cell Differentiation; Cell Line; Cell Movement; Cells, Cultured; Disease Models, Animal; Epithelial Cells; Extracellular Matrix Proteins; Female; Gene Silencing; Humans; Male; Mesenchymal Stem Cells; Mice; Mice, Inbred C57BL; Middle Aged; Pulmonary Fibrosis; Reverse Transcriptase Polymerase Chain Reaction; RNA, Small Interfering; Snail Family Transcription Factors; Transcription Factors; Transforming Growth Factor beta

Tissue fibrosis is a major cause of morbidity and mortality in systemic sclerosis (SSc), and an increasing number of promising molecular targets for antifibrotic therapies have been described recently. Transforming growth factor beta (TGFbeta) is well known to be the principal factor that leads to tissue fibrosis. The present study was undertaken to investigate the ability of HSc025, a novel small compound that antagonizes TGFbeta/Smad signaling through the activation of nuclear translocation of Y-box binding protein 1, to prevent tissue fibrosis in vitro or in mouse models of SSc.. Human dermal fibroblasts were exposed to HSc025 at various concentrations in the presence of TGFbeta, and levels of collagen or fibronectin expression were determined. HSc025 (15 mg/kg/day for 14 days) was administered orally to tight skin mice and to mice with bleomycin-induced pulmonary fibrosis. Improvement of tissue fibrosis was evaluated by histologic or biochemical examination in each model.. Pretreatment with HSc025 prevented Smad-dependent promoter activation, in a dose-dependent manner; however, HSc025 had no effect on TGFbeta-induced phosphorylation of Smad3. The inhibitory effects of HSc025 on TGFbeta-induced collagen or fibronectin expression were also confirmed in vitro. Orally administered HSc025 significantly reduced hypodermal thickness and hydroxyproline content in tight skin mice, and markedly decreased the histologic score and hydroxyproline content in the lungs of bleomycin-treated mice.. These results demonstrate that HSc025 is a novel inhibitor of TGFbeta/Smad signaling, resulting in the improvement of skin and pulmonary fibrosis. Orally available HSc025 might therefore be useful in the treatment of SSc.

Topics: Alkadienes; Animals; Bleomycin; Cells, Cultured; Collagen; Disease Models, Animal; Dose-Response Relationship, Drug; Female; Fibroblasts; Fibronectins; Fibrosis; Humans; Mice; Mice, Inbred C57BL; Plant Extracts; Pulmonary Fibrosis; Scleroderma, Systemic; Signal Transduction; Skin; Smad Proteins; Transcriptional Activation; Transforming Growth Factor beta; Zanthoxylum

Topics: Animals; Dinoprost; Mice; Pulmonary Fibrosis; Transforming Growth Factor beta

Idiopathic pulmonary fibrosis (IPF) is a progressive disease characterized by fibroblast proliferation and excess deposition of collagen and other extracellular matrix (ECM) proteins, which lead to distorted lung architecture and function. Given that anti-inflammatory or immunosuppressive therapy currently used for IPF does not improve disease progression therapies targeted to blocking the mechanisms of fibrogenesis are needed. Although transforming growth factor-beta (TGF-beta) functions are crucial in fibrosis, antagonizing this pathway in bleomycin-induced pulmonary fibrosis, an animal model of IPF, does not prevent fibrosis completely, indicating an additional pathway also has a key role in fibrogenesis. Given that the loss of cytosolic phospholipase A(2) (cPLA(2)) suppresses bleomycin-induced pulmonary fibrosis, we examined the roles of prostaglandins using mice lacking each prostoaglandin receptor. Here we show that loss of prostaglandin F (PGF) receptor (FP) selectively attenuates pulmonary fibrosis while maintaining similar levels of alveolar inflammation and TGF-beta stimulation as compared to wild-type (WT) mice, and that FP deficiency and inhibition of TGF-beta signaling additively decrease fibrosis. Furthermore, PGF(2alpha) is abundant in bronchoalveolar lavage fluid (BALF) of subjects with IPF and stimulates proliferation and collagen production of lung fibroblasts via FP, independently of TGF-beta. These findings show that PGF(2alpha)-FP signaling facilitates pulmonary fibrosis independently of TGF-beta and suggests this signaling pathway as a therapeutic target for IPF.

Topics: Animals; Arachidonic Acid; Bleomycin; Bronchoalveolar Lavage Fluid; Humans; Mice; Pulmonary Fibrosis; Receptors, Prostaglandin; Signal Transduction; Transforming Growth Factor beta

To investigate whether the effect of N-acetyl-seryl-aspartyl-lysyl-proline (AcSDKP) on transforming growth factor beta (TGF-beta1) and connective tissues growth factor (CTGF) was involved in AcSDKP's antifibrotic effect on the rats with silicosis.. Rats were divided into 6 groups randomly, 10 rats in each group: Control of silicotic model: 1.0 ml normal sodium and was killed after 4 or 8 weeks; Silicotic model 1: 50 mg/ml silica suspension and was killed after 4 weeks; Silicotic model 2: 50 mg/ml silica suspension and was killed after 8 weeks; Anti-fibrosis treatment of AcSDKP: after each rat was intratracheally instilled with 50 mg/ml silica suspension for 4 weeks, AcSDKP 800 microg/(kg x d) was administered into every rat and rats were killed at the 8 weeks; Preventing fibrosis treatment of AcSDKP: after AcSDKP [800 microg/(kg x d)] was administered into every rat for 48 hours, each rat was intratracheally instilled with 50 mg/ml silica suspension and rats were killed at the 8 weeks. Lung fibrosis in morphology was observed by HE staining. The expressions of TGF-beta1 and CTGF in lung were observed by immunohistochemistry. The mRNA expressions of TGF-beta1 and CTGF in lung were observed by real-time PCR.. In anti-fibrosis treatment of AcSDKP group, protein expression of TGF-beta1 and CTGF were (0.244 +/- 0.016) and (0.241 +/- 0.017) respectively, and significantly lower that those in the silicotic model 1 and 2 groups; mRNA expressions of TGF-beta1 and CTGF decreased, mRNA expressions of CTGF were significantly lower that those in the silicotic model 1 and 2 groups (P < 0.05); In preventing fibrosis treatment of AcSDKP group, protein expression and mRNA expression of TGF-beta1 were significantly lower that those in the silicotic model 2 group (P < 0.05).. AcSDKP can decrease the expressions of TGF-beta1 and CTGF in lung tissues of the rats with experimentally induced pulmonary fibrosis.

Topics: Animals; Connective Tissue Growth Factor; Disease Models, Animal; Lung; Male; Oligopeptides; Pulmonary Fibrosis; Rats; Rats, Sprague-Dawley; Silicosis; Transforming Growth Factor beta

Tissue damage following injury leads to inflammation and fibrosis. To understand the molecular mechanisms and the proteins involved in the fibrotic process, we used the well-established unilateral ureteric obstruction rat model and we analyzed the alterations at early and late time intervals using a classical proteomic approach. Data analysis demonstrates a correlation between calreticulin up-regulation and progression of fibrosis. Calreticulin is involved in Ca++ homeostasis but has not been previously implicated in animal models of fibrosis. Proteomic analysis consistently revealed up-regulation of calreticulin in both early and late time intervals. These findings were further confirmed by biochemical and morphological approaches. Next, animal models of lung fibrosis (bleomycin-induced) and heart fibrosis (desmin-null) were examined. In the lung model, calreticulin expression was up-regulated from early time intervals, whereas in the heart model no change in the expression of calreticulin was observed. In addition, TGF-beta, a well known major contributing factor in several fibrotic processes, was found to up-regulate calreticulin in cultured human proximal tubule epithelial cells. The above observations suggest that calreticulin might be involved in fibrotic processes; however the mechanism(s) underlying its possible involvement are yet unresolved.

Topics: Animals; Bleomycin; Calreticulin; Cell Line, Transformed; Cells, Cultured; Collagen; Desmin; Disease Models, Animal; Epithelial Cells; Female; Fibrosis; Gene Expression Regulation; Humans; Immunohistochemistry; Kidney Tubules, Proximal; Male; Mice; Mice, Inbred C57BL; Mice, Mutant Strains; Models, Biological; Proteomics; Pulmonary Fibrosis; Rats; Rats, Wistar; Time Factors; Transforming Growth Factor beta

It has been demonstrated that alveolar macrophages (AMs) play a key role in the pathogenesis of pulmonary fibrosis by releasing a variety of cytokines and inflammatory mediators. In addition, abnormal signal transducer and activator of transcription-1 (STAT1) activation in AMs may play a pivotal role in the process of alveolitis and pulmonary fibrosis. In this study, we transfected STAT1 antisense oligodeoxynucleotide (ASON) into rats by aerosolization, and then investigated the effect of STAT1 ASON on inflammatory mediators such as TGF-beta, PDGF and TNF-alpha in bronchoalveolar lavage fluid (BALF) from rats with bleomycin (BLM)-induced rat pulmonary fibrosis. Our results showed that STAT1 ASON by aerosolization could enter into lung tissues and AMs. STAT1 ASON could inhibit mRNA and protein expressions of STAT1 and ICAM-1 in AMs of rat with pulmonary fibrosis, and had no toxic side effect on liver and kidney. Aerosolized STAT1 ASON could ameliorate the alveolitis through inhibiting the secretion of inflammatory mediators in BLM-induced rat pulmonary fibrosis. These results suggest that aerosolized STAT1 ASON might be considered as a promising new strategy in the treatment of pulmonary fibrosis.

Topics: Aerosols; Animals; Bleomycin; Bronchoalveolar Lavage Fluid; Female; Inflammation Mediators; Intercellular Adhesion Molecule-1; Liposomes; Lung; Macrophages, Alveolar; Oligonucleotides, Antisense; Platelet-Derived Growth Factor; Pulmonary Fibrosis; Rats; Rats, Wistar; STAT1 Transcription Factor; Transfection; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha

The presence of activated fibroblasts or myofibroblasts represents a hallmark of progressive lung fibrosis. Because the transcriptional response of fibroblasts to transforming growth factor-beta(1) (TGF-beta(1)) is a determinant of disease progression, we investigated the role of the transcriptional regulator inhibitor of differentiation-1 (Id1) in the setting of lung fibrosis. Mice lacking the gene for Id1 had increased susceptibility to bleomycin-induced lung fibrosis, and fibroblasts lacking Id1 exhibited enhanced responses to TGF-beta(1). Because the effect of Id1 on fibrosis could not be explained by known mechanisms, we performed protein interaction screening and identified a novel binding partner for Id1, known as dead ringer-like-1 (Dril1). Dril1 shares structural similarities with Id1 and was recently implicated in TGF-beta(1) signaling during embryogenesis. To date, little is known about the function of Dril1 in humans. Although it has not been previously implicated in fibrotic disease, we found that Dril1 was highly expressed in lungs from patients with idiopathic pulmonary fibrosis and was regulated by TGF-beta(1) in human fibroblasts. Dril1 enhanced activation of TGF-beta(1) target genes, whereas Id1 decreased expression of these same molecules. Id1 inhibited DNA binding by Dril1, and the two proteins co-localized in vitro and in vivo, providing a potential mechanism for suppression of fibrosis by Id1 through inhibition of the profibrotic function of Dril1.

Topics: Animals; Bleomycin; Cells, Cultured; DNA-Binding Proteins; Fibroblasts; Humans; Inhibitor of Differentiation Protein 1; Lung; Mice; Mice, Knockout; Oncogenes; Protein Binding; Pulmonary Fibrosis; Trans-Activators; Transcription Factors; Transforming Growth Factor beta

Exposure to allergens or air pollutants often leads to asthma exacerbations associated with aggravation of airway inflammation. Although, repeated allergen challenge often induces chronic allergic airway inflammation (CAAI) and airway remodelling, yet, the effects of brief exposure to air pollutants such as SO(2) on development of CAAI and airway remodelling remain to be clarified.. The aim of the experiment was to investigate the effects of acute neutrophilic airway inflammation induced by brief exposure to SO(2) on development of CAAI and subepithelial fibrosis (SEF) in a murine model of asthma.. Acute airway inflammation was induced by brief exposure to 50 p.p.m. SO(2) (1 h/d, 3 days). CAAI and SEF in BALB/c mice were induced by repeated challenge with ovalbumin (OVA) for 5 or 9 weeks with or without prior exposure to SO(2). Bronchoalveolar lavage fluid (BALF) eosinophilia as index of CAAI, BALF endothelin-1 (ET-1) and TGF-beta1 levels, morphometric evaluation of fibrotic area beneath subbasement membrane and lung hydroxyproline content (Hyp) as indexes of SEF were monitored.. Exposure to SO(2) led to acute neutrophilic inflammation and epithelial sloughing with profound elevation of BALF ET-1. Repeated OVA challenge resulted in CAAI and SEF along with elevation of Hyp, increase of fibrotic area beneath subbasement membrane and elevation of BALF TGF-beta1. Preceding SO(2) exposure exaggerated BALF eosinophilia, facilitated and enhanced SEF with more significant elevation of BALF ET-1 and TGF-beta1 levels compared with OVA-challenged mice without prior exposure to SO(2). The increase of Hyp was positively correlated with elevation of BALF TGF-beta1 during CAAI (r=0.842, P<0.01).. This data demonstrated that SEF developed in parallel with severity and time course of CAAI following repeated OVA challenge. SO(2)-induced acute epithelial injury and neutrophilic inflammation could enhance CAAI and promote SEF, probably through overexpression of ET-1 and TGF-beta1.

Topics: Air Pollutants; Allergens; Animals; Bronchitis; Bronchoalveolar Lavage Fluid; Chronic Disease; Endothelin-1; Female; Lung; Mice; Mice, Inbred BALB C; Neutrophils; Ovalbumin; Pulmonary Fibrosis; Respiratory Mucosa; Sulfur Dioxide; Transforming Growth Factor beta

Topics: Extracellular Matrix; Fibrosis; Humans; Kidney Diseases; Protein Kinase Inhibitors; Protein-Tyrosine Kinases; Pulmonary Fibrosis; Scleroderma, Systemic; Signal Transduction; Transforming Growth Factor beta

Recent studies have implicated caveolin 1 in the regulation of transforming growth factor beta (TGFbeta) downstream signaling. Given the crucial role of TGFbeta in the pathogenesis of systemic sclerosis (SSc), we sought to determine whether caveolin 1 is also involved in the pathogenesis of tissue fibrosis in SSc. We analyzed the expression of CAV1 in affected SSc tissues, studied the effects of lack of expression of CAV1 in vitro and in vivo, and analyzed the effects of restoration of caveolin 1 function on the fibrotic phenotype of SSc fibroblasts in vitro.. CAV1 expression in tissues was analyzed by immunofluorescence and confocal microscopy. The extent of tissue fibrosis in Cav1-knockout mice was assessed by histologic/histochemical analyses and quantified by hydroxyproline assays. Cav1-null and SSc fibroblast phenotypes and protein production were analyzed by real-time polymerase chain reaction, immunofluorescence, Western blot, and multiplexed enzyme-linked immunosorbent assay techniques. The effects of restoration of caveolin 1 function in SSc fibroblasts in vitro were also examined using a cell-permeable recombinant CAV1 peptide.. CAV1 was markedly decreased in the affected lungs and skin of SSc patients. Cav1-knockout mice developed pulmonary and skin fibrosis. Down-regulation of caveolin 1 was maintained in cultured SSc fibroblasts, and restoration of caveolin 1 function in vitro normalized their phenotype and abrogated TGFbeta stimulation through inhibition of Smad3 activation.. Caveolin 1 appears to participate in the pathogenesis of tissue fibrosis in SSc. Restoration of caveolin 1 function by treatment with a cell-permeable peptide corresponding to the CAV1 scaffolding domain may be a novel therapeutic approach in SSc.

Topics: Animals; Blotting, Western; Caveolin 1; Cell Survival; Cells, Cultured; Collagen Type I; Collagen Type I, alpha 1 Chain; Connective Tissue Growth Factor; Down-Regulation; Enzyme-Linked Immunosorbent Assay; Fibroblasts; Fibrosis; Fluorescent Antibody Technique; Humans; Lung; Mice; Mice, Knockout; Microscopy, Confocal; Pulmonary Fibrosis; Recombinant Proteins; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Scleroderma, Systemic; Signal Transduction; Skin; Smad3 Protein; Transforming Growth Factor beta; Up-Regulation

As a group, fibroproliferative disorders of the lung, liver, kidney, heart, vasculature and integument are common, progressive and refractory to therapy. They can emerge following toxic insults, but are frequently idiopathic. Their enigmatic propensity to resist therapy and progress to organ failure has focused attention on the myofibroblast-the primary effector of the fibroproliferative response. We have recently shown that aberrant beta 1 integrin signaling in fibrotic fibroblasts results in defective PTEN function, unrestrained Akt signaling and subsequent activation of the translation initiation machinery. How this pathological integrin signaling alters the gene expression pathway has not been elucidated.. Using a systems approach to study this question in a prototype fibrotic disease, Idiopathic Pulmonary Fibrosis (IPF); here we show organized changes in the gene expression pathway of primary lung myofibroblasts that persist for up to 9 sub-cultivations in vitro. When comparing IPF and control myofibroblasts in a 3-dimensional type I collagen matrix, more genes differed at the level of ribosome recruitment than at the level of transcript abundance, indicating pathological translational control as a major characteristic of IPF myofibroblasts. To determine the effect of matrix state on translational control, myofibroblasts were permitted to contract the matrix. Ribosome recruitment in control myofibroblasts was relatively stable. In contrast, IPF cells manifested large alterations in the ribosome recruitment pattern. Pathological studies suggest an epithelial origin for IPF myofibroblasts through the epithelial to mesenchymal transition (EMT). In accord with this, we found systems-level indications for TGF-beta -driven EMT as one source of IPF myofibroblasts.. These findings establish the power of systems level genome-wide analysis to provide mechanistic insights into fibrotic disorders such as IPF. Our data point to derangements of translational control downstream of aberrant beta 1 integrin signaling as a fundamental component of IPF pathobiology and indicates that TGF-beta -driven EMT is one source for IPF myofibroblasts.

Topics: Aged; Aged, 80 and over; Female; Fibroblasts; Genome, Human; Humans; Integrin beta1; Male; Middle Aged; Models, Biological; Protein Biosynthesis; Pulmonary Fibrosis; Ribosomes; Signal Transduction; Transforming Growth Factor beta

Topics: Bone Marrow Transplantation; Clinical Trials as Topic; Fibrosis; Genetic Predisposition to Disease; Humans; Hypertension, Pulmonary; Kidney Diseases; Oligonucleotide Array Sequence Analysis; Pulmonary Fibrosis; Scleroderma, Systemic; Skin; Stem Cell Transplantation; Transforming Growth Factor beta

In experimental models, lung fibrosis is dependent on transforming growth factor (TGF)-beta signaling. TGF-beta is secreted in a latent complex with its propeptide, and TGF-beta activators release TGF-beta from this complex. Because the integrin alpha(v)beta6 is a major TGF-beta activator in the lung, inhibition of alpha(v)beta6-mediated TGF-beta activation is a logical strategy to treat lung fibrosis.. To determine, by genetic and pharmacologic approaches, whether murine radiation-induced lung fibrosis is dependent on alpha(v)beta6.. Wild-type mice, alpha(v)beta6-deficient (Itgb6-/-) mice, and mice heterozygous for a Tgfb1 mutation that eliminates integrin-mediated activation (Tgfb1(+/RGE)) were exposed to 14 Gy thoracic radiation. Some mice were treated with an anti-alpha(v)beta6 monoclonal antibody or a soluble TGF-beta receptor fusion protein. Alpha(v)beta6 expression was determined by immunohistochemistry. Fibrosis, inflammation, and gene expression patterns were assessed 20-32 weeks postirradiation.. Beta6 integrin expression increased within the alveolar epithelium 18 weeks postirradiation, just before onset of fibrosis. Itgb6-/- mice were completely protected from fibrosis, but not from late radiation-induced mortality. Anti-alpha(v)beta6 therapy (1-10 mg/kg/wk) prevented fibrosis, but only higher doses (6-10 mg/kg/wk) caused lung inflammation similar to that in Itgb6-/- mice. Tgfb1-haploinsufficient mice were also protected from fibrosis.. Alpha(v)beta6-mediated TGF-beta activation is required for radiation-induced lung fibrosis. Together with previous data, our results demonstrate a robust requirement for alpha(v)beta6 in distinct fibrosis models. Inhibition of alphavbeta6-mediated TGF-beta activation is a promising new approach for antifibrosis therapy.

Topics: Animals; Antibodies, Monoclonal; Antigens, Neoplasm; Disease Models, Animal; Dose-Response Relationship, Drug; Female; Gene Expression Regulation; Haplotypes; Heterozygote; Integrins; Mice; Mice, Inbred C57BL; Mice, Knockout; Pulmonary Alveoli; Pulmonary Fibrosis; Radiation Pneumonitis; Signal Transduction; Transforming Growth Factor beta; Transforming Growth Factor beta1

Transforming growth factor (TGF)-beta has a central role in driving many of the pathological processes that characterize pulmonary fibrosis. Inhibition of the integrin alpha(v)beta6, a key activator of TGF-beta in lung, is an attractive therapeutic strategy, as it may be possible to inhibit TGF-beta at sites of alpha(v)beta6 up-regulation without affecting other homeostatic roles of TGF-beta.. To analyze the expression of alpha(v)beta6 in human pulmonary fibrosis, and to functionally test the efficacy of therapeutic inhibition of alpha(v)beta6-mediated TGF-beta activation in murine bleomycin-induced pulmonary fibrosis.. Lung biopsies from patients with a diagnosis of systemic sclerosis or idiopathic pulmonary fibrosis were stained for alpha(v)beta6 expression. A range of concentrations of a monoclonal antibody that blocks alpha(v)beta6-mediated TGF-beta activation was evaluated in murine bleomycin-induced lung fibrosis.. Alpha(v)beta6 is overexpressed in human lung fibrosis within pneumocytes lining the alveolar ducts and alveoli. In the bleomycin model, alpha(v)beta6 antibody was effective in blocking pulmonary fibrosis. At high doses, there was increased expression of markers of inflammation and macrophage activation, consistent with the effects of TGF-beta inhibition in the lung. Low doses of antibody attenuated collagen expression without increasing alveolar inflammatory cell populations or macrophage activation markers.. Partial inhibition of TGF-beta using alpha(v)beta6 integrin antibodies is effective in blocking murine pulmonary fibrosis without exacerbating inflammation. In addition, the elevated expression of alpha(v)beta6, an activator of the fibrogenic cytokine, TGF-beta, in human pulmonary fibrosis suggests that alpha(v)beta6 monoclonal antibodies could represent a promising new therapeutic strategy for treating pulmonary fibrosis.

Topics: Animals; Antibodies, Monoclonal; Antigens, Neoplasm; Collagen; Disease Models, Animal; Dose-Response Relationship, Drug; Integrins; Mice; Mice, Inbred BALB C; Pulmonary Alveoli; Pulmonary Fibrosis; Scleroderma, Systemic; Transforming Growth Factor beta

Members of the transforming growth factor (TGF)-beta superfamily, including TGF-betas and bone morphogenetic proteins (BMPs), are essential for the maintenance of tissue homeostasis and regeneration after injury. We have observed that the BMP antagonist, gremlin, is highly up-regulated in idiopathic pulmonary fibrosis (IPF).. To investigate the role of gremlin in the regulation of BMP signaling in pulmonary fibrosis.. Progressive asbestos-induced fibrosis in the mouse was used as a model of human IPF. TGF-beta and BMP expression and signaling activities were measured from murine and human fibrotic lungs. The mechanism of gremlin induction was analyzed in cultured lung epithelial cells. In addition, the possible therapeutic role of gremlin inhibition was tested by administration of BMP-7 to mice after asbestos exposure.. Gremlin mRNA levels were up-regulated in the asbestos-exposed mouse lungs, which is in agreement with the human IPF biopsy data. Down-regulation of BMP signaling was demonstrated by reduced levels of Smad1/5/8 and enhanced Smad2 phosphorylation in asbestos-treated lungs. Accordingly, analyses of cultured human bronchial epithelial cells indicated that asbestos-induced gremlin expression could be prevented by inhibitors of the TGF-beta receptor and also by inhibitors of the mitogen-activated protein kinase kinase/extracellular signal-regulated protein kinase pathways. BMP-7 treatment significantly reduced hydroxyproline contents in the asbestos-treated mice.. The TGF-beta and BMP signaling balance is important for lung regenerative events and is significantly perturbed in pulmonary fibrosis. Rescue of BMP signaling activity may represent a potential beneficial strategy for treating human pulmonary fibrosis.

Topics: Animals; Asbestos; Bone Morphogenetic Protein 7; Bone Morphogenetic Proteins; Cell Line, Tumor; Disease Models, Animal; Down-Regulation; Humans; Intercellular Signaling Peptides and Proteins; Mice; Pulmonary Fibrosis; Signal Transduction; Transforming Growth Factor beta; Transforming Growth Factor beta1; Up-Regulation

Topics: Extracellular Matrix; Homeostasis; Humans; Oxidation-Reduction; Pulmonary Fibrosis; Transforming Growth Factor beta

Asthma can progress to subepithelial airway fibrosis, mediated in large part by transforming growth factor-beta (TGF-beta). The scaffolding protein caveolin-1 (cav1) can inhibit the activity of TGF-beta, perhaps by forming membrane invaginations that enfold TGF-beta receptors. The study goals were 1) to evaluate how allergen challenge affects lung expression of cav1 and the density of caveolae in vivo 2) to determine whether reduced cav1 expression is mediated by interleukin (IL)-4 and 3) to measure the effects of decreased expression of cav1 on TGF-beta signaling. C57BL/6J, IL-4-deficient mice, and cav1-deficient mice, sensitized by intraperitoneal injections of phosphate-buffered saline or ovalbumin (OVA) at days 0 and 12, received intranasal phosphate-buffered saline or OVA challenges at days 24, 26, and 28. Additionally, another group of C57BL/6J mice received IL-4 by intratracheal instillation for 7 days. We confirmed that the OVA-allergen challenge increased eosinophilia and T-helper type 2-related cytokine levels (IL-4, IL-5, and IL-13) in bronchoalveolar lavage. Allergen challenge reduced lung cav1 mRNA abundance by 40%, cav1 protein by 30%, and the number of lung fibroblast caveolae by 50%. Administration of IL-4 in vivo also substantially decreased cav1 expression. In contrast, the allergen challenge did not decrease cav1 expression in IL-4-deficient mice. The reduced expression of cav1 was associated with activation of TGF-beta signaling that was further enhanced in OVA-sensitized and challenged cav1-deficient mice. This study demonstrates a previously unknown modulation of TGF-beta signaling by IL-4, via cav1, suggesting novel therapeutic targets for controlling the effects of TGF-beta and thereby ameliorating pathological airway remodeling.

Topics: Allergens; Animals; Asthma; Bronchoalveolar Lavage; Caveolin 1; Down-Regulation; Interleukins; Mice; Mice, Knockout; Pulmonary Fibrosis; Signal Transduction; Th2 Cells; Time Factors; Transforming Growth Factor beta

Topics: Animals; Bleomycin; Disease Models, Animal; Disease Progression; Humans; Mice; Pulmonary Fibrosis; Rats; Transforming Growth Factor beta

Enhanced transforming growth factor (TGF) -beta signaling contributes to idiopathic pulmonary fibrosis (IPF), a progressive and fatal disease characterized by alveolar epithelial type II (ATII) cell hyperplasia, (myo)fibroblast accumulation, and excessive extracellular matrix deposition. TGF-beta is a potent inducer of lung fibrosis, and it regulates the ATII cell phenotype; however, direct TGF-beta target genes controlling the ATII cell phenotype remain elusive. Here, we identified the transgelin (tagln) gene as a novel immediate target of TGF-beta/Smad3-dependent gene expression in ATII cells using a Smad3 chromatin immunoprecipitation (ChIP) screen. Direct ChIP confirmed the rapid and specific binding of Smad3 to the tagln promoter. Luciferase assays demonstrated transactivation of the tagln promoter by activin-like kinase (Alk) 5-mediated TGF-beta signaling. TGF-beta treatment resulted in rapid up-regulation of tagln, but not tagln2, mRNA and protein expression, assessed by reverse transcription-polymerase chain reaction (RT-PCR), Western blotting, and immunofluorescence. In vivo, tagln expression was significantly increased in ATII cells of mice during bleomycin-induced lung fibrosis, as well as in lung specimen obtained from IPF patients, as assessed by RT-PCR and immunohistochemistry. Knockdown of tagln using siRNA inhibited TGF-beta-induced migration of lung epithelial A549 cells, as well as primary ATII cells. We thus identified tagln as a novel target of TGF-beta/Smad3-dependent gene expression in ATII cells. Increased ATII cell expression of tagln in experimental and idiopathic pulmonary fibrosis may contribute to TGF-beta-dependent ATII cell injury, repair, and migration in lung fibrosis.

Topics: Cell Line; Cell Movement; Epithelial Cells; Humans; Microfilament Proteins; Muscle Proteins; Promoter Regions, Genetic; Pulmonary Fibrosis; Receptors, Transforming Growth Factor beta; Smad3 Protein; Transforming Growth Factor beta

Idiopathic pulmonary fibrosis (IPF) is a chronic progressive disorder with a poor prognosis. Epithelial instability is a crucial step in the development and progression of the disease, including neoplastic transformation. Few tissue markers for epithelial instability have been reported in IPF. Squamous cell carcinoma antigen (SCCA) is a serine protease inhibitor typically expressed by dysplastic and neoplastic cells of epithelial origin, more often in squamous cell tumours. At present, no information is available on its expression in IPF.. SCCA and transforming growth factor beta (TGFbeta) expression in surgical lung biopsies from 22 patients with IPF and 20 control cases was examined. An in vitro study using A549 pneumocytes was also conducted to investigate the relationship between SCCA and TGFbeta expression. SCCA and TGFbeta epithelial expression was evaluated by immunohistochemistry and reverse transcription-PCR (RT-PCR). SCCA values were correlated with different pathological and clinical parameters. Time course analysis of TGFbeta expression in A549 pneumocytes incubated with different SCCA concentrations was assessed by real time RT-PCR.. SCCA was expressed in many metaplastic alveolar epithelial cells in all IPF cases with a mean value of 24.9% while it was seen in only two control patients in up to 5% of metaplastic cells. In patients with IPF, SCCA correlated positively with extension of fibroblastic foci (r = 0.49, p = 0.02), expression of TGFbeta (r = 0.78, p<0.0001) and with carbon monoxide transfer factor decline after 9 months of follow-up (r = 0.59, p = 0.01). In vitro experiments showed that incubation of cultured cells with SCCA induced TGFbeta expression, with a peak at 24 h.. Our findings provide for the first time a potential mechanism by which SCCA secreted from metaplastic epithelial cells may exert a profibrotic effect in IPF. SCCA could be an important biomarker in this incurable disease.

Topics: Adult; Antigens, Neoplasm; Biopsy; Case-Control Studies; Cells, Cultured; Female; Humans; Immunohistochemistry; Lung; Male; Middle Aged; Pulmonary Fibrosis; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Serpins; Transforming Growth Factor beta

To explore increased susceptibility to fibrosis following experimental injury to alveolar epithelial cells (AECs) in a novel transgenic mouse model of scleroderma with fibroblast-specific perturbation of transforming growth factor beta (TGFbeta) signaling (TbetaRIIDeltak-fib mice).. Wild-type (WT) and transgenic mice were injured with intratracheally administered saline or bleomycin, and the lungs were harvested for biochemical, histologic, and electron microscopic analysis.. Electron microscopy revealed AEC abnormalities in the lungs of untreated transgenic mice and bleomycin-treated WT mice; the lungs of transgenic mice treated with bleomycin showed severe epithelial damage. Compared with lungs from bleomycin-treated WT mice, lungs from bleomycin-treated transgenic mice demonstrated increased fibroproliferation, myofibroblast persistence, and impaired hyperplasia and increased apoptosis of type II AECs. The lungs from saline-treated transgenic mice and those from bleomycin-treated WT mice had phenotypic similarities, suggesting enhanced susceptibility to minor epithelial injury in the transgenic strain. The level of collagen was increased in the lungs from transgenic mice compared with that in the lungs from WT mice after treatment with either bleomycin or saline. Persistent fibrosis in bleomycin-treated transgenic mice was independent of ongoing neutrophil inflammation but was associated with impaired alveolar epithelial repair.. These results suggest that in the context of fibroblast-specific perturbation of TGFbeta signaling, even minor epithelial injury induces significant fibrosis. The model supports a central role for TGFbeta in determining fibrosis and demonstrates that lung fibroblasts may regulate the response of AECs to injury. Our findings provide insight into likely pathogenic mechanisms in scleroderma-associated pulmonary fibrosis.

Topics: Animals; Bleomycin; Cells, Cultured; Disease Models, Animal; Epithelial Cells; Fibroblasts; Irritants; Mice; Mice, Transgenic; Pulmonary Alveoli; Pulmonary Fibrosis; Scleroderma, Systemic; Signal Transduction; Sodium Chloride; Transforming Growth Factor beta

There has been ongoing controversy related to what differentiates normal lung repair and fibrosis. For example, the current prevailing concept has been that idiopathic forms of pulmonary fibrosis are due only to epithelial injury in response to some unknown cause that results in persistent evolving fibrosis without preceding inflammation. This concept would suggest that the lung responds to injury in a different manner than other organs, such as the liver, kidney, and heart. However, that would seem to contradict known established pathological concepts. To address this controversy, concepts were presented as follows: (1) loss of basement membrane integrity is critical in determining the "point of no return," and contributes to the inability to reestablish normal lung architecture with promotion of fibrosis; (2) loss of epithelial cells, endothelial cells, and basement membrane integrity in usual interstitial pneumonia associated with idiopathic pulmonary fibrosis leads to destroyed lung architecture and perpetual fibrosis; (3) transforming growth factor-beta is necessary, but not entirely sufficient, to promote permanent fibrosis; (4) persistent injury/antigen/irritant is critical for the propagation of fibrosis; (5) idiopathic pulmonary fibrosis is an example of a process related to the persistence of an "antigen(s)," chronic inflammation, and fibrosis; and (6) unique cells are critical cellular players in the regulation of fibrosis. In keeping with the theme of the Aspen Lung Conference, it is hoped that more questions are raised than answered in this presentation, in support of the continued need for research in this area to address these important concepts.

Topics: Basement Membrane; Endothelial Cells; Humans; Inflammation; Lung; Lung Diseases, Interstitial; Macrophages, Alveolar; Pulmonary Fibrosis; Respiratory Distress Syndrome; Respiratory Mucosa; Transforming Growth Factor beta; Wound Healing

In systemic sclerosis (SSc), a disease characterized by fibrosis of the skin and internal organs, the occurrence of interstitial lung disease is responsible for high morbidity and mortality. We previously demonstrated that proteasome inhibitors (PI) show anti-fibrotic properties in vitro by reducing collagen production and favoring collagen degradation in a c-jun N-terminal kinase (JNK)-dependent manner in human fibroblasts. Therefore, we tested whether PI could control fibrosis development in bleomycin-induced lung injury, which is preceded by massive inflammation. We extended the study to test PI in TSK-1/+ mice, where skin fibrosis develops in the absence of overt inflammation. C57Bl/6 mice received bleomycin intratracheally and were treated or not with PI. Lung inflammation and fibrosis were assessed by histology and quantification of hydroxyproline content, type I collagen mRNA, and TGF-beta at Days 7, 15, and 21, respectively. Histology was used to detect skin fibrosis in TSK-1/+mice. The chymotryptic activity of 20S proteasome was assessed in mice blood. JNK and Smad2 phosphorylation were evaluated by Western blot on lung protein extracts. PI reduced collagen mRNA levels in murine lung fibroblasts, without affecting their viability in vitro. In addition, PI inhibited the chymotryptic activity of proteasome and enhanced JNK and TGF-beta signaling in vivo. PI failed to prevent bleomycin-induced lung inflammation and fibrosis and to attenuate skin fibrosis in TSK-1/+mice. In conclusion, our results provide direct evidence that, despite promising in vitro results, proteasome blockade may not be a strategy easily applicable to control fibrosis development in diseases such as lung fibrosis and scleroderma.

Topics: Animals; Bleomycin; Boronic Acids; Bortezomib; Cells, Cultured; Collagen Type I; Fibrosis; Hydroxyproline; Leupeptins; Lung; Male; Mice; Mice, Inbred C57BL; Mice, Transgenic; Proteasome Inhibitors; Protein Serine-Threonine Kinases; Pulmonary Fibrosis; Pyrazines; Scleroderma, Systemic; Signal Transduction; Skin; Transforming Growth Factor beta

In the healthy lung, airway epithelial cells (AEC) regulate fibroblast proliferation through release of soluble factors, such as prostaglandins and proteins. Fibroproliferative diseases and airway remodelling may result from an inadequate generation of suppressive factors by AEC or the inability of fibroblasts to respond to them appropriately.. The aim of this study was to study the effect of primary human AEC on the proliferation of fibroblasts obtained from healthy and fibrotic lungs in an interactive cell culture model.. Conditioned medium (CM) from 14 out of 16 AEC lines significantly inhibited proliferation of normal human lung fibroblasts by 51.2+/-6.0%. The proliferation of fibroblasts derived from patients with lung fibrosis was equally inhibited by CM of AEC. The inhibitory effect of AEC-CM was completely reversed when fibroblasts were pre-incubated with 2.5 microm indomethacin. Furthermore, primary human AEC, but not fibroblasts, secrete TGF-beta, and the inhibitory effect of the AEC-CM was blocked by neutralizing anti-TGF-beta antibodies.. These results demonstrate that AEC actively inhibit the proliferation of both normal and fibrotic fibroblasts via TGF-beta, which induces the prostaglandin E(2) synthesis in fibroblasts. The data indicate that proliferative lung diseases may be treated using the epithelial cell as the target of medication.

Topics: Blotting, Western; Cell Proliferation; Cells, Cultured; Enzyme-Linked Immunosorbent Assay; Fibroblasts; Humans; Pulmonary Fibrosis; Respiratory Mucosa; Transforming Growth Factor beta

Recent advances in fibrosis biology have identified transforming growth factor (TGF)-beta type I receptor-mediated activation of Smads as playing a central part in the development of fibrosis. However, to date, there have been few studies that examined the localisation and distribution of receptor-activated Smads protein (R-Smads: Smad2 and 3) during the fibrosis progression.. To histopathologically assess the time-course change of the localisation and distribution of the Smads protein in pulmonary fibrosis.. Pulmonary fibrosis was induced by intranasal injection of bleomycin (0.3 U/mouse). Lungs were isolated 2, 5, 7, 9 and 14 days after bleomycin treatment. Histological changes in the lungs were evaluated by haematoxylin-eosin stain or Masson's trichrome stain, and scored. TGF-beta1, Smad3 and phosphorylated Smad2 localisations in lung tissues were determined by immunohistochemistry.. The bleomycin treatment led to considerable pulmonary fibrotic changes accompanied by marked increase in TGF-beta1 expression in infiltrating macrophages. With the progression in fibrosis (day 7-14), marked increases in Smad3-positive and pSmad2-positive cells were observed. There were intense Smad3-positive and pSmad2-positive signals localised to the nuclei of the infiltrating macrophages and to type II epithelial cells, and less intense signals in fibroblasts and hyperplastic alveolar/bronchiolar epithelial cells.. The time-course data of TGF-beta1 and R-Smads indicate that progressive enhancement of TGF-beta1 signalling via R-Smad is activated in the process of fibrosis progression.

Topics: Animals; Antibiotics, Antineoplastic; Bleomycin; Disease Models, Animal; Disease Progression; Immunoenzyme Techniques; Male; Mice; Mice, Inbred BALB C; Pulmonary Fibrosis; Signal Transduction; Smad Proteins, Receptor-Regulated; Smad2 Protein; Smad3 Protein; Transforming Growth Factor beta

Transforming growth factor beta 1 (TGF-beta1) plays an important role in the development of radiation- and drug-induced organ diseases. Proteinases-activated receptor 1 (PAR-1) is involved in many pathophysiologic processes after its activation by serine proteases. The aim of the present study was to determine messenger RNA (mRNA) production of TGF-beta1 and PAR-1 in the lungs after local irradiation. Mice of C57BL/6 and C3H/J strains with different susceptibility to fibrosis development were exposed to a of 15Gy. Non-irradiated mice of both strains were used as negative controls. Control (irradiated) and irradiated angiotensin-converting enzyme (ACE) inhibitor-treated animals were examined simultaneously. The ACE inhibitor group was given butylaminiperindopril for 9 days after irradiation (15Gy) at a daily dose of 0.1 or 0.2mg/kg per rectum. On day 9, all mice were sacrificed, and the production of mRNA TGF-beta1 and PAR-1 in lung tissue was determined semiquantitatively using reverse transcriptase polymerase chain reaction, and immunohistochemical analysis of PAR-1 expression in pulmonary tissue was performed. In the fibrosing murine strain C57Bl/6, there was an increase in the mRNA TGF-beta1 and PAR-1 levels in lungs 9 days after irradiation as compared with non-irradiated controls and non-fibrosing murine strain C3H/J. In butylaminiperindopril-treated mice, a decrease in transcript of TGF-beta1 and PAR-1 was observed. Thus, PAR-1 is involved in radiation-induced lung fibrosis in correlation with TGF-beta1 production. Administration of ACEI influences PAR-1 and TGF-beta1 expression.

Topics: Angiotensin-Converting Enzyme Inhibitors; Animals; Female; Gamma Rays; Immunoenzyme Techniques; Lung; Mice; Mice, Inbred C3H; Mice, Inbred C57BL; Perindopril; Pulmonary Fibrosis; Radiation Injuries, Experimental; Radiation-Protective Agents; Receptor, PAR-1; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Transforming Growth Factor beta

Neutrophil elastase is a serine protease stored in the azurophilic granules of leukocytes. It has been implicated in the pathology of several lung diseases and is generally presumed to contribute to the tissue destruction and extracellular matrix damage associated with these conditions. To delineate the role of neutrophil elastase in pulmonary inflammation and fibrosis, neutrophil elastase-null mice were intratracheally instilled with bleomycin. In neutrophil elastase-null mice, biochemical and morphological characteristics of pulmonary fibrosis were attenuated for at least 60 days after bleomycin administration despite a typical response to bleomycin as evidenced by assessment of indices of DNA and cell damage. Neutrophil burden of bleomycin-treated wild-type and neutrophil elastase-null mice was comparable, and marked neutrophilic alveolitis was manifest in bleomycin-treated neutrophil elastase-null mice. An absence of immunostaining for active transforming growth factor (TGF)-beta in lung tissue from bleomycin-treated neutrophil elastase-null mice suggested a defect in TGF-beta activation, which was confirmed by biochemical assessment of TGF-beta levels in bronchoalveolar lavage fluid and lung tissue. These data point to novel and unexpected fibrogenic consequences of neutrophil elastase activity in the inflamed lung.

Topics: Animals; Bleomycin; Bronchoalveolar Lavage Fluid; DNA Damage; Drug Resistance; Leukocyte Elastase; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Mice, Knockout; Pulmonary Fibrosis; Transforming Growth Factor beta

Idiopathic pulmonary fibrosis (IPF) is a chronic progressive fibrotic lung disorder of unknown cause. Several studies suggest an association between Epstein-Barr virus pulmonary infection and the development of IPF.. To determine whether reduction of gamma-herpesvirus reactivation from latency would alter progressive lung fibrogenesis in an animal model of virus-induced pulmonary fibrosis.. IFN-gamma receptor-deficient (IFN-gammaR(-/-)) mice infected intranasally with murine gamma-herpesvirus 68 (MHV68) develop lung fibrosis that progresses for up to at least 180 days after initial infection. Viral replication during the chronic phase of infection was controlled by two methods: the administration of cidofovir, an antiviral drug effective at clearing lytic but not latent virus, and by using a mutant gamma-herpesvirus defective in virus reactivation from latency.. Ten percent of the asymptomatic MHV68-infected animals that received antiviral treatment beginning on Day 45 postinfection had severe pulmonary fibrosis compared with 40% of the control saline-treated animals. Absence of severe fibrosis was also observed in IFN-gammaR(-/-) mice infected with the defective reactivation mutant MHV68 v-cyclin stop. Decreased fibrosis was associated with lower levels of transforming growth factor-beta, vascular endothelial growth factor, and markers of macrophage alternative activation. When antiviral treatment was administered on Day 60 in symptomatic animals, survival improved from 20 to 80% compared with untreated symptomatic animals, but lung fibrosis persisted in 60% of the mice.. MHV68-induced fibrosis is a result of viral lytic replication during chronic lung herpesvirus infection in mice. We speculate that antiviral therapy might help to control lung fibrosis in humans with IPF and associated herpesvirus infection.

Topics: Animals; Antigens, Viral; Arginase; beta-N-Acetylhexosaminidases; Biomarkers; Bronchoalveolar Lavage Fluid; Cytokines; Disease Models, Animal; Disease Progression; Fluorescent Antibody Technique, Indirect; Gene Expression Regulation, Viral; Herpesviridae Infections; Hydroxyproline; Immunoassay; Intercellular Signaling Peptides and Proteins; Interferon gamma Receptor; Lectins; Mice; Nerve Growth Factor; Proteins; Pulmonary Fibrosis; Receptors, Interferon; Reverse Transcriptase Polymerase Chain Reaction; Rhadinovirus; RNA, Viral; Transforming Growth Factor beta; Tumor Virus Infections; Vascular Endothelial Growth Factor A; Viral Envelope Proteins; Virus Activation

To investigate the therapeutic effect and mechanism of Qidan granule on blemycinA5-induced pulmonary fibrosis in rats.. A total of 70 SD rats were randomly divided into normal group, the model group, Qidan group and hydrocortisone group and observed for 28 days and 42 days, respectively. Rat pulmonary fibrosis was induced by intrabronchial injection of blemycinA5. Treatment started from day 14 to day 42 with Qidan granule and Hydrocortisone for 14 days (day 28 group) and for 28 days (day 42 group), respectively. The lung pathological grades were observed by hematoxylin and eosin (HE) staining and expressions of transforming growth factor beta (TGF-beta(1)) protein and tumor necrosis factor alpha (TNF-alpha) protein were tested by the immunohistochemical technique.. (1) Lung pathobiology fibrosis were alleviated was alleviated significantly in Qidan granule group compared with those in model group and hydrocortisone group (p < 0.01). (2) In Qidan group and hydrocortisone group, the expression of TGF-beta(1) protein was 1.71 +/- 0.17 and 1, 78 +/- 0.17 in day 28 group and day 42 group, respectively. The expression of TNF-aprotein was 2.16 +/- 0.40 and 1.98 +/- 0.33 in day 28 group and day 42 group, respectively. The expression of TGF-beta(1) and TNF-alpha protein was significantly difference from those in the model group and the hydrocortisone group (p < 0.01).. Qidan granule ameliorate the pulmonary fibrosis by decreasing expressions of TGF-beta(1) and TNF-alpha proteins in lung tissue.

Topics: Animals; Drugs, Chinese Herbal; Lung; Male; Phytotherapy; Pulmonary Fibrosis; Rats; Rats, Sprague-Dawley; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha

Leukotrienes are lipid mediators of inflammation derived from the 5-lipoxygenase pathway of arachidonic acid metabolism, and recent evidence suggests that they play an important role in pulmonary fibrosis. Montelukast is a cysteinyl-leukotriene 1 receptor antagonist that has been found to reduce airway remodeling, including subepithelial fibrosis, in a murine model of asthma, but the therapeutic effect of montelukast on pulmonary fibrosis remains unclear. In this study, we investigated whether montelukast is capable of preventing bleomycin-induced pulmonary fibrosis in mice. On day 1, C57BL/6 mice were given a single intratracheal injection of bleomycin (2.5 mg/kg), and montelukast (1.0 mg/kg) or vehicle alone subcutaneously 2 h later and on days 1-5 of each week for two weeks. The total number of cells in bronchoalveolar lavage fluid (BALF) was reduced in the montelukast group on day 7 and on day 14, and cellular inflammation and fibrosis were attenuated on day 14 as indicated by significant decrease in the Ashcroft score and lung hydroxyproline content. Although cysteinyl-leukotriene level in BALF was not significantly different, transforming growth factor beta (TGFbeta) level in BALF by ELISA and TGFbeta expression in lung tissue by immunohistochemistry was reduced on day 14 in the montelukast group. The results of this study show that montelukast inhibits the inflammatory process and development of bleomycin-induced pulmonary fibrosis in mice and that these effects may be associated with a decrease in TGFbeta expression. They also suggest that montelukast may serve as a new therapy for patients with interstitial pulmonary fibrosis.

Topics: Animals; Bleomycin; Bronchoalveolar Lavage Fluid; Cysteine; Disease Models, Animal; Hydroxyproline; Leukotriene Antagonists; Leukotrienes; Male; Mice; Mice, Inbred C57BL; Pulmonary Fibrosis; Receptors, Leukotriene; Transforming Growth Factor beta

Progressive, irreversible fibrosis is one of the most clinically significant consequences of ionizing radiation on normal tissue. When applied to lungs, it leads to a complication described as idiopathic pneumonia syndrome (IPS) and eventually to organ fibrosis. For its high mortality, the condition precludes treatment with high doses of radiation. There is widespread interest to understand the pathogenetic mechanisms of IPS and to find drugs effective in the prevention of its development. This report summarizes our experience with the protective effects of L 158,809, an angiotensin II (ANG II) receptor blocker, and two angiotensin converting enzyme (ACE) inhibitors in the development of IPS and the role of transforming growth factor beta (TGF-beta) and of alpha-actomyosin (alpha SMA) in pathogenesis of radiation induced pulmonary fibrosis in an experimental model of bone marrow transplant (BMT). Male WAG/Riji/MCV rats received total body irradiation and a regimen of cyclophosphamide (CTX) in preparation for bone marrow transplant. While one group of animals remained untreated, the remainders were subdivided into three groups, each of them receiving either the ANG II receptor blocker or one of the two ACE inhibitors (Captopril or Enalapril). Each of the three drugs was administered orally from 11 days before the transplant up to 56 days post transplant. At sacrifice time the irradiated rats receiving only CTX showed a chronic pneumonitis with septal fibrosis and vasculitis affecting, in particular, small caliber pulmonary arteries and arterioles. Their lung content of hydroxyproline was also markedly elevated in association with the lung concentrations of thromboxane (TXA2) and prostaglandin (PGI(2)), (two markers of pulmonary endothelial damage). A significant increase of alpha actomyosin staining was observed in vessels, septa and macrophages of the same animals which also overexpressed TGF-beta. When L 158,809, Captopril and Enalapril were added to the radiation and cytoxan treatment, a significant amelioration of the histological damage as well as the overexpression of alpha SMA was observed. Lung concentrations of hydroxyproline, PGI(2), TXA2 and TGF-beta were also observed in these animals so that the values of these compounds were closer to those measured in untreated control rats than to their irradiated and cytoxan treated counterparts. Angiotensin II plays an important role in the regulation of TGF-beta and alpha SMA, two proteins involved in th

Topics: Actomyosin; Angiotensin Receptor Antagonists; Angiotensin-Converting Enzyme Inhibitors; Animals; Lung; Male; Peptidyl-Dipeptidase A; Pulmonary Fibrosis; Radiation Injuries, Experimental; Rats; Receptors, Angiotensin; Transforming Growth Factor beta

To observe the effect and mechanism of curcumin on pulmonary fibrosis induced by bleomycin in rats.. Fifty-four SD rats were randomly divided into 3 groups, 18 rats each group. The control group received a single intratracheal dose of 2 ml/kg of sterile physiological saline at day 0, and from 14 d, 0.5 ml/kg of sterile physiological saline intraperitoneally every day. The bleomycin group received a single intratracheal dose of 5 mg/kg of bleomycin A(5), and from 14 d, 0.5 ml/kg of suspension of 6% alcohol and 6% polyethylene glycol intraperitoneally every day. The curcumin group received a single intratracheal dose of 5 mg/kg of bleomycin A(5), and from 14 d, 50 mg/kg of curcumin (suspended in 6% alcohol and 6% polyethylene glycol) intraperitoneally every day. Six rats in each group were killed at day 17, 21, 28 in batches. The sections of lungs were stained with hematoxylin-eosin (HE) and Masson's trichrome to evaluate the severity of alveolitis and pulmonary fibrosis. The content of hydroxyproline and the expression of transforming growth factor-beta(1) (TGF-beta(1)) mRNA, interferon-gamma (IFN-gamma) mRNA in lung tissues were analyzed. The concentration of TGF-beta(1) and IFN-gamma in bronchoalveolar lavage fluid (BALF) were measured.. (1) The scores of alveolitis in the curcumin group and the bleomycin group at day 28 were 1.3 +/- 0.5, 2.0 +/- 0.9, respectively, the difference being significant (q = 3.26, P < 0.05). (2) The scores of pulmonary fibrosis in the curcumin group and the bleomycin group were 1.3 +/- 0.5, 1.8 +/- 0.4 at day 21, and 1.2 +/- 0.4, 2.2 +/- 1.0 at day 28, the difference being significant between the two groups (q = 3.33, 4.00, all P < 0.05). (3) The content of hydroxyproline in lung tissues in the curcumin group and the bleomycin group were (1.75 +/- 0.36) microg/g, (2.47 +/- 0.24) microg/g at day 28, the difference being significant (q = 7.20, P < 0.01). (4) The concentration of TGF-beta(1) in BALF in the curcumin group and the bleomycin group were (20 +/- 3) ng/L, (39 +/- 7) ng/L at day 21, and (24 +/- 4) ng/L, (40 +/- 7) ng/L at day 28, all being statistically different between the two groups (q = 5.30, 6.27, all P < 0.05). (5) The expression of TGF-beta(1) mRNA in lung tissues in the curcumin group and the bleomycin group were 0.51 +/- 0.11, 0.59 +/- 0.13 at day 21, and 0.50 +/- 0.07, 0.64 +/- 0.11 at day 28, all being not statistically different between the two groups (q = 1.55, 3.13, all P > 0.05). (6) The concentrations of IFN-gamma in BALF in the curcumin group and the bleomycin group were 0.49 +/- 0.17, 0.50 +/- 0.08 at day 21, and 0.52 +/- 0.15, 0.52 +/- 0.11 at day 28, all being not statistically different between the two groups (q = 1.85, 2.03, all P > 0.05). (7) The expression of IFN-gamma mRNA in the curcumin group and the bleomycin group were (28 +/- 5) ng/L, (35 +/- 13) ng/L at day 21, and (30 +/- 11) ng/L, (39 +/- 13) ng/L at day 28, no significant difference between the two groups (q = 0.17, 0.00, all P > 0.05).. Curcumin can alleviate alveolitis and pulmonary fibrosis induced by bleomycin in rats, possibly through its inhibition of TGF-beta(1).

Topics: Animals; Bleomycin; Curcumin; Lung; Male; Pulmonary Fibrosis; Rats; Rats, Sprague-Dawley; Transforming Growth Factor beta

Unc119 is an adaptor protein that is involved in the development of the vertebrate nervous system. We have shown that Unc119 stimulates the induction of alpha-smooth muscle actin (alpha-SMA) and myofibroblast differentiation by TGF-beta in human lung fibroblasts. Unc119 increases the kinase activity of Fyn and associates with it in coprecipitation and colocalization studies. Phosphorylation and activation of Fyn in response to TGF-beta and platelet-derived growth factor is delayed in Unc119-deficient cells. This delay translates into suppressed cell proliferation. In Src family kinase-deficient (SYF) cells, Unc119 knockdown does not affect cell proliferation. The result suggests that Unc119 interacts with Fyn in the early stages of signal generation and its presence is essential for conducive signal transduction. Unc119 overexpression does not stimulate alpha-SMA in SYF cells and this defect is restored upon reconstitution with Fyn indicating that Unc119 stimulation of alpha-SMA requires at least Fyn. Unc119 overexpression stimulated p38, but not JNK, phosphorylation. Blocking p38 MAPK resulted in reduced alpha-SMA expression by Unc119 suggesting that the p38 pathway regulates Unc119-induced myofibroblast differentiation. Unc119 stimulates the production of TGF-beta and IL-6, known inducers of myofibroblast differentiation. Thus, Unc119 regulates receptor-mediated signal transduction and myofibroblast differentiation by activating Fyn and the p38 MAPK pathway. Using primary lung fibroblasts from patients with fibrotic lung diseases and control subjects, we show that the expression of alpha-smooth muscle actin is highly correlated with that of Unc119. Taken together, our results suggest that Unc119 plays an important role in fibrotic processes through myofibroblast differentiation.

Topics: Actins; Adaptor Proteins, Signal Transducing; Animals; Cell Differentiation; Cell Line; Cell Line, Tumor; Enzyme Activation; Eye Proteins; Fibroblasts; Humans; Intracellular Signaling Peptides and Proteins; MAP Kinase Signaling System; Mice; Microtubule Proteins; NIH 3T3 Cells; p38 Mitogen-Activated Protein Kinases; Proto-Oncogene Proteins c-fyn; Pulmonary Fibrosis; src-Family Kinases; Transforming Growth Factor beta; Up-Regulation

The status of the patient's associated disease can generally affect the onset or healing of acquired reactive perforating collagenosis (ARPC). We treated eight cases of ARPC and noted that the patients had similar findings. However, it was not clear why ARPC developed in the patients with these diseases. Nevertheless, several factors related to the diseases associated with ARPC could affect the degeneration of collagen fibers or the production of dermal products. Some patients had diseases that were characterized by fibrosis and an increased amount of reticular fibers. Factors related to tissue remodeling might act not only in diseases associated with ARPC but also in ARPC itself.

Topics: Adolescent; Adult; Aged; Carcinoma, Hepatocellular; Collagen; Collagen Diseases; Diabetes Mellitus, Type 2; Female; Humans; Liver Cirrhosis; Liver Neoplasms; Male; Middle Aged; Pulmonary Fibrosis; Reticulin; Skin Diseases; Transforming Growth Factor beta

To investigate the anti-fibrotic effects of Qidan granule in rats.. The rats were randomly divided into six experimental groups: normal group, model group, Qidan group, Tetrandrine group. All rats except normal group were treated with silicon dioxide (50 mg/rat) by intratracheal instillation to induce silicosis. Qidan group and Tetrandrine group were treated with Qidan granule (3125 mg/kg) or treated with Tetrandrine (22 mg/kg) respectively. All the rats were sacrificed after 5 months. Calculate Lung/body coefficient by weighting the lung wet weight and the body weight of rats. Content of Hydroxyproline was measured by alkaline hydrolysis. The gene expression of transforming growth factor-beta1 was examined by using enzyme-linked immunosorbent assay (ELISA). Paraffin embedded lung sections with HE staining, VG staining and Gomori staining were observed under light microscope.. In Qidan group and Tetrandrine group, Lung/body coefficient and content of Hydroxyproline and expression of transforming growth factor-beta1 were lower as compared with model group (P < 0.05). Model group mainly showed III approximately IV grade silicotic nodule, which contained thick collagen and sparse reticulum fibe; Qidan group and Tetrandrine group appeared with II grade silicotic nodule, which contained tiny collagen and intensive reticulum fibe. Tetrandrine group showed injury of kidney, and others were normal.. Qidan granule extract should prevent and from inhibit the remarkably silicotic fibrosis in rats.

Topics: Animals; Disease Models, Animal; Drugs, Chinese Herbal; Phytotherapy; Pulmonary Fibrosis; Rats; Rats, Wistar; Silicosis; Transforming Growth Factor beta

Fibrosis is excessive scarring caused by the accumulation and contraction of extracellular matrix proteins and is a common end pathway in many chronic diseases, including scleroderma (systemic sclerosis [SSc]). Indeed, pulmonary fibrosis is a major cause of death in SSc. Transforming growth factor beta (TGFbeta) induces endothelin 1 (ET-1) in human lung fibroblasts by a Smad-independent, JNK-dependent mechanism. The goal of this study was to assess whether ET-1 is a downstream mediator of the profibrotic effects of TGFbeta in lung fibroblasts.. We used a specific endothelin receptor antagonist to determine whether ET-1 is a downstream mediator of TGFbeta responses in lung fibroblasts, using microarray technology, real-time polymerase chain reaction, and Western blot analyses.. The ability of TGFbeta to induce the expression of a cohort of profibrotic genes, including type I collagen, fibronectin, and CCN2, and to contract a collagen gel matrix, depends on ET-1.. ET-1 contributes to the ability of TGFbeta to promote a profibrotic phenotype in human lung fibroblasts, consistent with the notion that endothelin receptor antagonism may be beneficial in controlling fibrogenic responses in lung fibroblasts.

Topics: Bosentan; Cells, Cultured; Collagen Type I; Connective Tissue Growth Factor; Endothelin Receptor Antagonists; Endothelin-1; Extracellular Matrix; Fibroblasts; Fibronectins; Gene Expression Profiling; Humans; Immediate-Early Proteins; Intercellular Signaling Peptides and Proteins; Lung; Phenotype; Pulmonary Fibrosis; Sulfonamides; Transforming Growth Factor beta

Myofibroblasts are primary effector cells in idiopathic pulmonary fibrosis (IPF). Defining mechanisms of myofibroblast differentiation may be critical to the development of novel therapeutic agents.. To show that myofibroblast differentiation is regulated by phosphatase and tensin homolog deleted on chromosome 10 (PTEN) activity in vivo, and to identify a potential mechanism by which this occurs.. We used tissue sections of surgical lung biopsies from patients with IPF to localize expression of PTEN and alpha-smooth muscle actin (alpha-SMA). We used cell culture of pten(-/-) and wild-type fibroblasts, as well as adenoviral strategies and pharmacologic inhibitors, to determine the mechanism by which PTEN inhibits alpha-SMA, fibroblast proliferation, and collagen production.. In human lung specimens of IPF, myofibroblasts within fibroblastic foci demonstrated diminished PTEN expression. Furthermore, inhibition of PTEN in mice worsened bleomycin-induced fibrosis. In pten(-/-) fibroblasts, and in normal fibroblasts in which PTEN was inhibited, alpha-SMA, proliferation, and collagen production was upregulated. Addition of transforming growth factor-beta to wild-type cells, but not pten(-/-) cells, resulted in increased alpha-SMA expression in a time-dependent fashion. In pten(-/-) cells, reconstitution of PTEN decreased alpha-SMA expression, proliferation, and collagen production, whereas overexpression of PTEN in wild-type cells inhibited transforming growth factor-beta-induced myofibroblast differentiation. It was observed that both the protein and lipid phosphatase actions of PTEN were capable of modulating the myofibroblast phenotype.. The results indicate that in IPF, myofibroblasts have diminished PTEN expression. Inhibition of PTEN in vivo promotes fibrosis, and PTEN inhibits myofibroblast differentiation in vitro.

Topics: Actins; Animals; Antibiotics, Antineoplastic; Bleomycin; Cell Culture Techniques; Cell Differentiation; Fibroblasts; Fibrosis; Humans; Lung Diseases, Interstitial; Mice; Mice, Inbred C57BL; PTEN Phosphohydrolase; Pulmonary Fibrosis; Transforming Growth Factor beta

Adenosine, a signaling nucleoside, exhibits tissue-protective and tissue-destructive effects. Adenosine levels in tissues are controlled in part by the enzyme adenosine deaminase (ADA). ADA-deficient mice accumulate adenosine levels in multiple tissues, including the lung, where adenosine contributes to the development of pulmonary inflammation and chronic airway remodeling. The present study describes the development of pulmonary fibrosis in mice that have been genetically engineered to possess partial ADA enzyme activity and, thus, accumulate adenosine over a prolonged period of time. These partially ADA-deficient mice live for up to 5 mo and die from apparent respiratory distress. Detailed investigations of the lung histopathology of partially ADA-deficient mice revealed progressive pulmonary fibrosis marked by an increase in the number of pulmonary myofibroblasts and an increase in collagen deposition. In addition, in regions of the distal airways that did not exhibit fibrosis, an increase in the number of large foamy macrophages and a substantial enlargement of the alveolar air spaces suggest emphysemic changes. Furthermore, important proinflammatory and profibrotic signaling pathways, including IL-13 and transforming growth factor-beta1, were activated. Increases in tissue fibrosis were also seen in the liver and kidneys of these mice. These changes occurred in association with pronounced elevations of lung adenosine concentrations and alterations in lung adenosine receptor levels, supporting the hypothesis that elevation of endogenous adenosine is a proinflammatory and profibrotic signal in this model.

Topics: Adenosine; Adenosine Deaminase; Animals; Collagen; Fibroblasts; Homozygote; Interleukin-13; Macrophages; Mice; Mice, Inbred C57BL; Mice, Knockout; Myocytes, Smooth Muscle; Pulmonary Alveoli; Pulmonary Fibrosis; Respiratory System; Signal Transduction; Transforming Growth Factor beta; Transforming Growth Factor beta1

In addition to regulating blood pressure and body fluid homeostasis, the renin-angiotensin system is also involved in lung fibrogenesis.. To study the effect of losartan, an angiotensin II antagonist, on bleomycin-induced pulmonary fibrosis in rats and its possible mechanism.. Pulmonary fibrosis was induced in SD rats by intratracheal instillation of bleomycin (5 mg x kg(-1)). Subsequently, the rats received daily losartan (3, 9 and 27 mg x kg(-1)) or prednisone (20 mg x kg(-1)) orally. Six rats in each group were sacrificed 14 and 21 days after intratracheal instillation. Hydroxyproline, superoxide dismutase (SOD), and malondialdehyde (MDA) levels in lung tissues were determined by spectroscopy. The levels of TGF-beta1 in serum were measured by ELISA. Histological changes in the lungs were evaluated by hematoxylin-eosin stain, and scored.. Rat body weight evidently decreased while the indices of lung and hydroxyproline contents in lung tissue were significantly increased 14 and 21 days after intratracheal bleomycin instillation. Inflammatory cell infiltration and fibrotic scores were more prominent in the model group compared to the sham group. Losartan (3, 9 and 27 mg.kg(-1), i.g.) apparently attenuated the degree of pulmonary fibrosis. Further study showed that losartan significantly increased SOD levels while it decreased MDA contents in lung homogenates. Serum TGF-beta1 levels of pulmonary fibrosis rats were also decreased by losartan.. Losartan had an inhibitory effect on bleomycin-induced pulmonary fibrosis, and its effect may be associated with its anti-free radicals and the reduction in TGF-beta1.

Topics: Angiotensin II Type 1 Receptor Blockers; Animals; Antibiotics, Antineoplastic; Bleomycin; Body Weight; Glucocorticoids; Hydroxyproline; Losartan; Lung; Male; Malondialdehyde; Prednisone; Pulmonary Fibrosis; Rats; Rats, Sprague-Dawley; Superoxide Dismutase; Transforming Growth Factor beta; Transforming Growth Factor beta1; Weight Gain

Topics: Actins; Cell Differentiation; Fibroblasts; Gene Expression; Humans; Lung; PTEN Phosphohydrolase; Pulmonary Fibrosis; Transforming Growth Factor beta

Eotaxin/CCL11 plays an important role in asthma. It acts through the chemokine receptor CCR3 expressed on hematopoietic and nonhematopoietic cells in the lung.. To determine whether eotaxin/CCL11 modulates lung and bronchial fibroblast properties and thereby might contribute to airway remodeling.. CCR3 expression was characterized on a lung fibroblast line (MRC-5; flow cytometry, fluorescent microscopy, RT-PCR, and Northern blotting), on primary bronchial fibroblasts (flow cytometry), and on fibroblasts in human lung tissue (confocal laser microscopy). The effects of eotaxin/CCL11 on lung fibroblast migration (Boyden chamber), proliferation (tritiated thymidine incorporation), alpha-smooth muscle actin expression (ELISA), 3-dimensional collagen gel contraction (floating gel), pro-alpha1(I) collagen mRNA (Northern blotting), total collagen synthesis (tritiated proline incorporation), matrix metalloproteinase activity (gelatin zymography), and TGF-beta(1) release (ELISA) were evaluated. The contribution of eotaxin/CCL11/CCR3 binding on lung fibroblasts was also investigated by neutralizing experiments.. CCR3 is constitutively expressed in cultured lung and primary bronchial fibroblasts and colocalizes with specific surface markers for human fibroblasts in lung tissue. Eotaxin/CCL11 selectively modulates fibroblast activities by increasing their proliferation, matrix metalloproteinase 2 activity, and collagen synthesis but not their differentiation into myofibroblasts, contractility in collagen gel, or TGF-beta(1) release. Eotaxin/CCL11 enhances migration of lung fibroblasts in response to nonspecific chemoattractants, and this effect is completely inhibited by anti-CCR3-neutralizing antibodies.. These data demonstrate that eotaxin/CCL11 has a direct and selective profibrogenic effect on lung and bronchial fibroblasts, providing a novel mechanism whereby eotaxin/CCL11 can participate in airway remodeling in asthma.

Topics: Cell Differentiation; Cell Proliferation; Chemokine CCL11; Chemokines, CC; Chemotaxis; Collagen; Collagen Type I; Collagen Type I, alpha 1 Chain; Female; Fibroblasts; Humans; Male; Matrix Metalloproteinase 2; Middle Aged; Protein Precursors; Pulmonary Fibrosis; Receptors, CCR3; Receptors, Chemokine; RNA, Messenger; Transforming Growth Factor beta; Transforming Growth Factor beta1

The model most often used to study the pathogenesis of pulmonary fibroses is the bleomycin (BLM)-induced lung fibrosis model. Several treatments have been efficacious in this model, but not in the clinic.. To describe the time course of inflammation and fibrosis in the BLM model and to study the effect of timing of antiinflammatory and antifibrotic treatments on efficacy.. Rats were given single intratracheal injections of BLM on Day 0. At specified time points, 10 rats were killed and their lungs studied for proinflammatory cytokines and for profibrotic growth factor mRNA. After a single intratracheal injection of BLM on Day 0, rats were treated from Day 1 or 10 daily with oral prednisolone (10 mg/kg) or oral imatinib mesylate (50 mg/kg) for 21 d.. After BLM administration, the expression of inflammatory cytokines was elevated and returned to background levels at later time points. Profibrotic gene expression peaked between Days 9 and 14 and remained elevated till the end of the experiment, suggesting a "switch" between inflammation and fibrosis in this interval. Antiinflammatory treatment (oral prednisolone) was beneficial when commenced at Day 1, but had no effect if administered from Day 10 onward. However, imatinib mesylate was effective independently of the dosing regime.. The response of the BLM model to antifibrotic or antiinflammatory interventions is critically dependent on timing after the initial injury.

Topics: Animals; Benzamides; Biomarkers; Bleomycin; Drug Therapy, Combination; Follow-Up Studies; Gene Expression; Glucocorticoids; Imatinib Mesylate; Lung; Male; Piperazines; Pneumonia; Prednisolone; Protein Kinase Inhibitors; Protein-Tyrosine Kinases; Pulmonary Fibrosis; Pyrimidines; Rats; RNA, Messenger; Transforming Growth Factor beta; Transforming Growth Factor beta1; Treatment Outcome

Fourteen-membered ring macrolides have been effective in reducing chronic airway inflammation and also preventing lung injury and fibrosis in bleomycin-challenged mice via anti-inflammatory effects. EM703 is a new derivative of erythromycin (EM) without the bactericidal effects. We investigated the anti-inflammatory and antifibrotic effects of EM703 in an experimental model of bleomycin-induced lung injury and subsequent fibrosis in mice.. Seven-week-old male ICR mice were used. All experiments used eight mice/group, unless otherwise noted in the figure legends. Bleomycin was administered intravenously to the mice on day 0. EM703 was orally administered daily to mice. All groups were examined for cell populations in the bronchoalveolar lavage (BAL) fluid and for induction of messenger RNA (mRNA) of Smad3 and Smad4 in the lung tissues by reverse transcriptase (RT)-polymerase chainreaction (PCR) on day 7. Fibroblastic foci were assessed histologically, and the hydroxyproline content was chemically determined in the lung tissues on day 28. We performed assay of proliferation and soluble collagen production, and examined the induction of mRNA of Smad3 and Smad4 by RT-PCR in murine lung fibroblast cell line MLg2908. We also examined Smad3, Smad4 and phosphorylated Smad2/3 (p-Smad2/3) protein assay by western blotting in MLg2908.. Bleomycin-induced lung fibrosis, and the infiltration of macrophages and neutrophils into the airspace were inhibited by EM703. The expression of Smad3 and Smad4 mRNA was clearly attenuated by bleomycin, but was recovered by EM703. EM703 also inhibited fibroblast proliferation and the collagen production in lung fibroblasts induced by Transforming growth factor-beta (TGF-beta). The expression of Smad3 and Smad4 mRNA in murine lung fibroblasts disappeared due to TGF-beta, but was recovered by EM703. EM703 inhibited the expression of p-Smad2/3 and Smad4 protein in murine lung fibroblasts induced by TGF-beta.. These findings suggest that EM703 improves bleomycin-induced pulmonary fibrosis in mice by actions of anti-inflammation and regulation of TGF-beta signaling in lung fibroblasts.

Topics: Animals; Anti-Inflammatory Agents; Base Sequence; Bleomycin; Cell Line; Cell Proliferation; Collagen; Dose-Response Relationship, Drug; Erythromycin; Fibroblasts; Gene Expression Regulation; Hydroxyproline; Lung; Male; Mice; Mice, Inbred ICR; Molecular Sequence Data; Pulmonary Fibrosis; Smad2 Protein; Smad3 Protein; Smad4 Protein; Transforming Growth Factor beta

The molecular mechanisms of pulmonary fibrosis are poorly understood. Previous reports indicate that activation of TGF-beta1 is essential for the development of pulmonary fibrosis. Here, we report that the proapoptotic Bcl-2 family member Bid is required for the development of pulmonary fibrosis after the intratracheal instillation of bleomycin. Mice lacking Bid exhibited significantly less pulmonary fibrosis in response to bleomycin compared with WT mice. The attenuation in pulmonary fibrosis was observed despite similar levels of inflammation, lung injury, and active TGF-beta1 in bronchoalveolar lavage fluid 5 days after the administration of bleomycin in mice lacking Bid and in WT controls. Bleomycin induced similar levels cell death in vitro in alveolar epithelial cells isolated from WT and bid(-/-) mice. By contrast, alveolar epithelial cells from bid(-/-) mice were resistant to TGF-beta1-induced cell death. These results indicate that Bcl-2 family members are critical regulators for the development of pulmonary fibrosis downstream of TGF-beta1 activation.

Topics: Animals; Apoptosis; BH3 Interacting Domain Death Agonist Protein; Bleomycin; Bronchoalveolar Lavage Fluid; Epithelial Cells; Fibroblasts; Lung; Mice; Mice, Mutant Strains; Pulmonary Fibrosis; Transforming Growth Factor beta; Transforming Growth Factor beta1

This study examined effects of S-allyl cysteine (SAC) on carbon tetrachloride (CCl4)-induced interstitial pulmonary fibrosis in Wistar rats. CCl4 (0.5 ml/kg) was intraperitoneally injected into rats twice a week for 8 weeks, and SAC (50, 100, or 200 mg/kg), N-acetyl cysteine (NAC, 200 or 600 mg/kg), or L-cysteine (CYS, 600 mg/kg) were orally administrated to rats everyday for 8 weeks. SAC significantly reduced the increases of transforming growth factor beta, lipid peroxides, AST, and ALT in plasma, induced by CCl4. Although CCl4 is mainly metabolized by hepatic cytochrome P450, CCl4 induced systemic inflammation and some organ fibrosis. SAC dose-dependently and significantly attenuated CCl4-induced systemic inflammation and fibrosis of lung. SAC also inhibited the decrease of thiol levels, the increase of inducible nitric oxide synthase expression, the infiltration of leukocytes, and the generation of reactive oxygen species in lungs. Although NAC and CYS attenuated CCl4-induced pulmonary inflammation and fibrosis, the order of preventive potency was SAC > NAC > CYS according to their applied doses. These results indicate that SAC is more effective than other cysteine compounds in reducing CCl4-induced lung injury, and might be useful in prevention of interstitial pulmonary fibrosis.

Topics: Animals; Bronchoalveolar Lavage Fluid; Carbon Tetrachloride; Cysteine; Enzyme Precursors; Glutathione; Hydroxyproline; Liver; Lung; Male; Matrix Metalloproteinase 9; Nitric Oxide; Nitric Oxide Synthase Type II; Oxidative Stress; Pulmonary Fibrosis; Rats; Rats, Wistar; Reactive Oxygen Species; Sulfhydryl Compounds; Transforming Growth Factor beta; Transforming Growth Factor beta1

The reasons for variable sensitivity among and within species to lung injury and fibrosis caused by bleomycin (BLM) are unknown. Because T helper (Th) 1 and 2 (Th1 and Th2) polarization of CD4+ T lymphocytes is one of the factors that affects the BLM response, we hypothesized that preventing expression of the Th1 transcription factor T-bet would render BLM-resistant BALB/c mice sensitive to BLM. Wild-type and T-bet-deficient (T-bet-/-) BALB/c mice were treated with BLM or saline solution intratracheally. After BLM treatment, collagen content in the lung increased twofold by day 14 in lungs from T-bet-/- mice but was unaffected in lungs from wild-type BALB/c mice. These findings were confirmed by collagen staining of histopathological sections. BLM treatment significantly increased respiratory frequency and decreased tidal volume by day 14 in T-bet-/- mice but had no effect in wild-type mice. Lung fibrosis in BLM-treated T-bet-/- mice was associated with increased circulating levels of Th2 cytokines and increased expression of the profibrotic factor transforming growth factor-beta1. Depletion of CD4+, but not CD8+, T cells in T-bet-/- mice diminished BLM-induced lung fibrosis and the expression of transforming growth factor-beta1. These data suggest that the T-bet pathway in CD4+ T lymphocytes can confer resistance to BLM-induced lung fibrosis in BALB/c mice.

Topics: Animals; Bleomycin; CD4-Positive T-Lymphocytes; CD8-Positive T-Lymphocytes; Cytokines; Disease Susceptibility; Growth Substances; Leukocyte Reduction Procedures; Lung; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Mice, Knockout; Pulmonary Fibrosis; Severity of Illness Index; T-Box Domain Proteins; Transforming Growth Factor beta; Transforming Growth Factor beta1

Transforming growth factor-beta (TGFbeta) is a major mediator of normal wound healing and of pathological conditions involving fibrosis, such as idiopathic pulmonary fibrosis. TGFbeta also stimulates the differentiation of myofibroblasts, a hallmark of fibrotic diseases. In this study, we examined the underlying processes of TGFbetaRI kinase activity in myofibroblast conversion of human lung fibroblasts using specific inhibitors of TGFbetaRI (SD-208) and p38 mitogen-activated kinase (SD-282). We demonstrated that SD-208, but not SD-282, inhibited TGFbeta-induced SMAD signaling, myofibroblast transformation, and collagen gel contraction. Furthermore, we extended our findings to a rat bleomycin-induced lung fibrosis model, demonstrating a significant decrease in the number of myofibroblasts at fibroblastic foci in animals treated with SD-208 but not those treated with SD-282. SD-208 also reduced collagen deposition in this in vivo model. Microarray analysis of human lung fibroblasts identified molecular fingerprints of these processes and showed that SD-208 had global effects on reversing TGFbeta-induced genes involved in fibrosis, inflammation, cell proliferation, cytoskeletal organization, and apoptosis. These studies also revealed that although the p38 pathway may not be needed for appearance or disappearance of the myofibroblast, it can mediate a subset of inflammatory and fibrogenic events of the myofibroblast during the process of tissue repair and fibrosis. Our findings suggest that inhibitors such as SD-208 may be therapeutically useful in human interstitial lung diseases and pulmonary fibrosis.

Topics: Activin Receptors, Type I; Cell Differentiation; Cells, Cultured; Collagen; Connective Tissue Growth Factor; Cytoskeleton; Fibroblasts; Gene Expression Regulation; Humans; Immediate-Early Proteins; Inflammation; Intercellular Signaling Peptides and Proteins; Lung; MAP Kinase Signaling System; Oligonucleotide Array Sequence Analysis; p38 Mitogen-Activated Protein Kinases; Protein Serine-Threonine Kinases; Pteridines; Pulmonary Fibrosis; Receptor, Transforming Growth Factor-beta Type I; Receptors, Transforming Growth Factor beta; Reverse Transcriptase Polymerase Chain Reaction; Signal Transduction; Smad Proteins; Transforming Growth Factor beta; Wound Healing

Idiopathic Pulmonary Fibrosis (IPF) is a debilitating disease characterized by exaggerated extracellular matrix deposition and aggressive lung structural remodeling. Disease pathogenesis is driven by fibroblastic foci formation, consequent on growth factor overexpression and myofibroblast proliferation. We have previously shown that both CTGF overexpression and myofibroblast formation in IPF cell lines are dependent on RhoA signaling. As RhoA-mediated regulation is also involved in cell cycle progression, we hypothesise that this pathway is key to lung fibroblast turnover through modulation of cyclin D1 kinetic expression.. Cyclin D1 expression was compared in primary IPF patient-derived fibroblasts and equivalent normal control cells. Quantitative real time PCR was employed to examine relative expression levels of cyclin D1 mRNA; protein expression was confirmed by western blotting. Effects of Rho signaling were investigated using transient transfection of constitutively active and dominant negative RhoA constructs as well as pharmacological inhibitors. Cellular proliferation of lung fibroblasts was determined by BrdU incorporation ELISA. To further explore RhoA regulation of cyclin D1 in lung fibroblasts and associated cell cycle progression, an established Rho inhibitor, Simvastatin, was incorporated in our studies.. Cyclin D1 expression was upregulated in IPF compared to normal lung fibroblasts under exponential growth conditions (p < 0.05). Serum deprivation inhibited cyclin D1 expression, which was restored following treatment with fibrogenic growth factors (TGF-beta1 and CTGF). RhoA inhibition, using a dominant negative mutant and a pharmacological inhibitor (C3 exotoxin), suppressed levels of cyclin D1 mRNA and protein in IPF fibroblasts, with significant abrogation of cell turnover (p < 0.05). Furthermore, Simvastatin dose-dependently inhibited fibroblast cyclin D1 gene and protein expression, inducing G1 cell cycle arrest. Similar trends were observed in control experiments using normal lung fibroblasts, though exhibited responses were lower in magnitude.. These findings report for the first time that cyclin D1 expression is deregulated in IPF through a RhoA dependent mechanism that influences lung fibroblast proliferation. This potentially unravels new molecular targets for future anti-IPF strategies; accordingly, Simvastatin inhibition of Rho-mediated cyclin D1 expression in IPF fibroblasts merits further exploitation.

Topics: ADP Ribose Transferases; Botulinum Toxins; Cell Division; Cell Line; Connective Tissue Growth Factor; Cyclin D1; Fibroblasts; G1 Phase; Gene Expression; Humans; Hydroxymethylglutaryl-CoA Reductase Inhibitors; Immediate-Early Proteins; Intercellular Signaling Peptides and Proteins; Lung; Pulmonary Fibrosis; rhoA GTP-Binding Protein; Signal Transduction; Simvastatin; Transforming Growth Factor beta; Transforming Growth Factor beta1; Up-Regulation

Pulmonary fibrosis develops in approximately 25% of patients with chronic sarcoidosis. Transforming growth factor (TGF)-beta1 plays a central role in fibrosis, and accruing reports address the implication of TGF-beta2 and TGF-beta3 in this process. We determined whether single-nucleotide polymorphisms (SNPs) in the TGF-beta1, TGF-beta2, and TGF-beta3 genes might contribute to pulmonary fibrosis in sarcoidosis patients.. A hospital in the Netherlands.. Five SNPs per TGF-beta gene were investigated.. Patients with either acute/self-remitting sarcoidosis (n = 50) and Löfgren syndrome (n = 46) or chronic disease with fibrosis (n = 24) and without fibrosis (n = 34) were assessed over a 4-year follow-up period. The control subjects included 315 individuals.. Polymorphism frequencies were not discordant between the patients and control subjects. The TGF-beta3 4875 A allele was significantly higher in fibrotic patients (carrier frequency, 0.29) than in patients with acute/self-remitting (0.06) and chronic (0.03) sarcoidosis combined (corrected p = 0.01; odds ratio [OR], 7.9). The TGF-beta3 17369 C allele carrier frequency was significantly higher in fibrotic patients (0.29) compared to acute/self-remitting (0.08) and chronic (0.06) patients combined (corrected p = 0.05; OR, 5.1). Although not significant after correction, the TGF-beta3 15101 G allele carrier frequency was lower in fibrotic patients (0.79) compared to acute/self-remitting (0.94) and chronic (1.00) patients combined (p = 0.02; corrected p = 0.1; OR, 0.15). The TGF-beta2 59941 G allele was more abundant in fibrotic patients (carrier frequency, 0.62) compared to patients with acute/self-remitting (0.41) and chronic sarcoidosis combined (0.28) [p = 0.04; corrected p = 0.2; OR, 2.9]. TGF-beta1 gene polymorphisms were not associated with fibrosis.. This study is the first to suggest the implication of genetic variation of TGF-beta3 in the predilection for pulmonary fibrosis developing in sarcoidosis patients.

Topics: Adolescent; Adult; Aged; Aged, 80 and over; Case-Control Studies; Female; Follow-Up Studies; Genetic Predisposition to Disease; Haplotypes; Humans; Male; Middle Aged; Polymorphism, Single Nucleotide; Pulmonary Fibrosis; Radiography; Sarcoidosis, Pulmonary; Transforming Growth Factor beta

Idiopathic pulmonary fibrosis is a devastating disorder for which there is no effective treatment. Transforming growth factor (TGF)-beta plays a critical role in provoking fibrosis. Interleukin (IL)-10 is a potent immunosuppressive cytokine but its effect on the fibrosing process is unclear. A study was undertaken to examine whether IL-10 affects the production and activation of TGF-beta and thus can attenuate the fibrosis.. Mice were given an intratracheal injection of bleomycin. On day 1 or 14, IL-10 gene was delivered by rapid intravenous injection of Ringer's solution containing plasmid. Two weeks after the plasmid injection the mice were examined for fibrosis. The effect of IL-10 on TGF-beta production by alveolar macrophages was assessed.. Even when delivered during the fibrosing phase, IL-10 gene significantly suppressed the pathological findings, hydroxyproline content, and production of both active and total forms of TGF-beta1 in the lung. Immunohistochemical analyses showed that alveolar macrophages were one of the major sources of TGF-beta1 and IL-10 diminished the intensity of the staining. IL-10 also suppressed the expression of alphaV beta6 integrin, a molecule that plays an important role in TGF-beta activation, on lung epithelial cells. Alveolar macrophages from bleomycin injected mice produced TGF-beta1 spontaneously ex vivo, which was significantly suppressed by treatment of the mice in vivo or by treatment of the explanted macrophages ex vivo with IL-10.. IL-10 suppresses the production and activation of TGF-beta in the lung and thus attenuates pulmonary fibrosis, even when delivered in the chronic phase.

Topics: Animals; Bleomycin; Bronchoalveolar Lavage Fluid; Cell Line; Gene Transfer Techniques; Genetic Therapy; Immunohistochemistry; Integrin alpha6; Interleukin-10; Macrophages, Alveolar; Male; Mice; Mice, Inbred C57BL; Plasmids; Pulmonary Fibrosis; Transforming Growth Factor beta

The signal transduction mechanisms generating pathological fibrosis are almost wholly unknown. Endothelin-1 (ET-1), which is up-regulated during tissue repair and fibrosis, induces lung fibroblasts to produce and contract extracellular matrix. Lung fibroblasts isolated from scleroderma patients with chronic pulmonary fibrosis produce elevated levels of ET-1, which contribute to the persistent fibrotic phenotype of these cells. Transforming growth factor beta (TGF-beta) induces fibroblasts to produce and contract matrix. In this report, we show that TGF-beta induces ET-1 in normal and fibrotic lung fibroblasts in a Smad-independent ALK5/c-Jun N-terminal kinase (JNK)/Ap-1-dependent fashion. ET-1 induces JNK through TAK1. Fibrotic lung fibroblasts display constitutive JNK activation, which was reduced by the dual ETA/ETB receptor inhibitor, bosentan, providing evidence of an autocrine endothelin loop. Thus, ET-1 and TGF-beta are likely to cooperate in the pathogenesis of pulmonary fibrosis. As elevated JNK activation in fibrotic lung fibroblasts contributes to the persistence of the myofibroblast phenotype in pulmonary fibrosis by promoting an autocrine ET-1 loop, targeting the ETA and ETB receptors or constitutive JNK activation by fibrotic lung fibroblasts is likely to be of benefit in combating chronic pulmonary fibrosis.

Topics: Actins; Activin Receptors, Type I; Animals; Cells, Cultured; Endothelin A Receptor Antagonists; Endothelin B Receptor Antagonists; Endothelin-1; Enzyme Activation; Fibroblasts; Humans; JNK Mitogen-Activated Protein Kinases; Mice; Models, Biological; Protein Serine-Threonine Kinases; Pulmonary Fibrosis; Receptor, Endothelin A; Receptor, Endothelin B; Receptor, Transforming Growth Factor-beta Type I; Receptors, Transforming Growth Factor beta; Smad Proteins; Transcription Factor AP-1; Transforming Growth Factor beta

Idiopathic pulmonary fibrosis (IPF), ie, usual interstitial pneumonia in histopathology, is a disease characterized by tissue destruction and active areas of fibroproliferation in the lung. Gremlin (Drm), a member of the cysteine knot family of bone morphogenetic protein (BMP) inhibitors, functions to antagonize BMP-4-mediated signals during lung development. We describe here consistent overexpression of gremlin in the lung interstitium of IPF patients. Quantitative real-time reverse transcriptase-polymerase chain reaction analyses revealed considerably higher levels of gremlin mRNA in lung biopsies from IPF patients, the highest level being 35-fold higher compared to controls. Lung fibroblasts isolated from IPF patients also expressed elevated levels of gremlin, which was associated with impaired responsiveness to endogenous and exogenous BMP-4. Transforming growth factor-beta-induced epithelial-to-mesenchymal transition of A549 lung epithelial cells in culture was also associated with induction of gremlin mRNA expression. In addition, A549 cells transfected to overexpress gremlin were more susceptible to transforming growth factor-beta-induced epithelial-to-mesenchymal transition. Gremlin-mediated inhibition of BMP-4 signaling pathways is likely to enhance the fibrotic response and reduce epithelial regeneration in the lung. The overexpression of this developmental gene in IPF may be a key event in the persistence of myofibroblasts in the lung interstitium and provides a potential target for therapeutic intervention.

Topics: Blotting, Northern; Blotting, Western; Bone Morphogenetic Protein 4; Bone Morphogenetic Proteins; Cell Differentiation; Cells, Cultured; Electrophoresis, Polyacrylamide Gel; Fibroblasts; Fluorescent Antibody Technique; Humans; Intercellular Signaling Peptides and Proteins; Pulmonary Fibrosis; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Signal Transduction; Transfection; Transforming Growth Factor beta

Recent studies have demonstrated that Th2 cytokines, such as interleukin-4 and interleukin-13, enhance fibrotic processes by activating fibroblast proliferation and collagen production, whereas interferon-gamma, a Th1 cytokine, inhibits these processes. Th1 and Th2 cells both differentiate from common T precursor cells, with transcription factor GATA-3 a key regulator of Th2 differentiation. In the present study, therefore, we examined the effects of GATA-3 overexpression on the development of pulmonary fibrosis in a mouse model. Wild-type C57BL/6 mice and GATA-3-overexpressing (GATA-3-tg) mice of the same background were intratracheally treated with bleomycin. The survival rate after bleomycin was significantly decreased in GATA-3-tg mice compared with wild-type mice. The degree of pulmonary fibrosis was much greater in GATA-3-tg mice than in wild-type mice 28 days after bleomycin treatment. Lung interferon-gamma concentration was significantly decreased in GATA-3-tg mice compared with wild-type mice by 7 days after either saline or bleomycin treatment. The concentration of transforming growth factor-beta, a fibrogenic cytokine, was significantly higher in GATA-3-tg mice than in wild-type mice. Exogenous administration of interferon-gamma to GATA-3-tg mice improved the degree of pulmonary fibrosis and thus increased survival. These results indicate that overexpression of GATA-3 enhances the development of pulmonary fibrosis, possibly by reducing interferon-gamma levels in the lung.

Topics: Animals; Antimetabolites, Antineoplastic; Bleomycin; Bronchoalveolar Lavage Fluid; CD4-Positive T-Lymphocytes; Flow Cytometry; GATA3 Transcription Factor; Interferon-gamma; Mice; Mice, Transgenic; Pulmonary Fibrosis; Transforming Growth Factor beta

Exaggerated levels of IL-13 and leukotriene (LT) pathway activation frequently coexist at sites of Th2 inflammation and in tissue fibrotic responses. However, the relationship(s) between the IL-13 and LTs in these responses have not been defined. We hypothesized that the 5-lipoxygenase (5-LO) pathway of LT metabolism plays an important role in the pathogenesis of IL-13-induced chronic inflammation and remodeling. To test this hypothesis, we evaluated the effects of IL-13 on components of the 5-LO metabolic and activation pathways. We also compared the effects of transgenic IL-13 in C57BL/6 mice with wild-type and null 5-LO genetic loci. These studies demonstrate that IL-13 increases the levels of mRNA encoding cytosolic phospholipase A(2), LTA(4) hydrolase, and 5-LO-activating protein without altering the expression of 5-LO, LTC(4) synthase, LTB(4) receptors 1 and 2, and cysteinyl-LT receptors 1 and 2. They also demonstrate that this activation is associated with the enhanced accumulation of LTB(4) but not of cysteinyl-LTs. Furthermore, they demonstrate that this stimulation plays a critical role in the pathogenesis of IL-13-induced inflammation, tissue fibrosis, and respiratory failure-induced death while inhibiting alveolar remodeling. Lastly, mechanistic insights are provided by demonstrating that IL-13-induced 5-LO activation is required for optimal stimulation and activation of TGF-beta(1) and the inhibition of matrix metalloproteinase-12. When viewed in combination, these studies demonstrate that 5-LO plays an important role in IL-13-induced inflammation and remodeling.

Topics: Animals; Arachidonate 5-Lipoxygenase; Chronic Disease; Dinoprostone; Inflammation; Interleukin-13; Leukotrienes; Lung; Matrix Metalloproteinase 12; Metalloendopeptidases; Mice; Mice, Inbred C57BL; Mice, Knockout; Mice, Transgenic; Pulmonary Alveoli; Pulmonary Fibrosis; Signal Transduction; Transforming Growth Factor beta; Transforming Growth Factor beta1

Chronic inflammation and fibrosis are hallmarks of lung pathology of newborn Ureaplasma infection. We hypothesized that antenatally acquired Ureaplasma stimulates a chronic inflammatory, profibrotic immune response that contributes to lung injury, altered developmental signaling, and fibrosis. Lung specimens from 125-d gestation baboon newborns ventilated for 14 d that were either infected antenatally with Ureaplasma serovar 1 or noninfected, and 125-d and 140-d gestational controls were obtained from the Baboon BPD Resource Center (San Antonio, TX). Trichrome stain to assess fibrosis and immunohistochemistry for alpha-smooth muscle actin (alpha-SMA) and transforming growth factor beta1 (TGFbeta1) were performed. Lung homogenates were analyzed by enzyme-linked immunosorbent assay (ELISA) for cytokines [tumor necrosis factor alpha (TNFalpha), interleukin (IL)-1beta, TGFbeta1, oncostatin M (OSM), IL-10, and interferon gamma (IFNgamma)] and the chemokine MCP-1 and by Western blot for Smad2, Smad3, and Smad7. Compared with noninfected ventilated and gestational controls, Ureaplasma-infected lungs demonstrated more extensive fibrosis, increased alpha-SMA and TGFbeta1 immunostaining, and higher concentrations of active TGFbeta1, IL-1beta, and OSM, but no difference in IL-10 levels. There was a trend toward higher Smad2/Smad7 and Smad3/Smad7 ratios in Ureaplasma lung homogenates, consistent with up-regulation of TGFbeta1 signaling. Collectively, these data suggest that a prolonged proinflammatory response initiated by intrauterine Ureaplasma infection contributes to early fibrosis and altered developmental signaling in the immature lung.

Topics: Actins; Animals; Animals, Newborn; Chemokine CCL2; Cytokines; In Vitro Techniques; Inflammation Mediators; Lung; Muscle, Smooth; Papio; Premature Birth; Pulmonary Fibrosis; Respiration, Artificial; Respiratory Tract Infections; Smad Proteins; Transforming Growth Factor beta; Ureaplasma Infections; Ureaplasma urealyticum

Idiopathic pulmonary fibrosis (IPF) is characterized by fibroblast expansion and extracellular matrix accumulation. However, the mechanisms involved in matrix remodeling have not been elucidated. In this study, the authors aimed to evaluate the expression of the tissue inhibitors of matrix metalloproteinases (TIMPs) in human fibroblasts and whole tissues from IPF and normal lungs. They also determined the role of mitogen-activated protein kinase (MAPK) in TIMP3 expression. TIMP1, TIMP2, and TIMP3 were highly expressed in lung fibroblasts. Transforming growth factor (TGF)-beta1, a profibrotic mediator, induced strong up-regulation of TIMP3 at the mRNA and protein levels. The authors examined whether the MAPK pathway was involved in TGF-beta1-induced TIMP3 expression. TGF-beta1 induced the phosphorylation of p38 and extracellular signal-regulated kinase (ERK)1/2. Biochemical blockade of p38 by SB203580, but not of the ERK MAPK pathway, inhibited the effect of this factor. The effect was also blocked by the tyrosine kinase inhibitor genistein and by antagonizing TGF-beta1 receptor type I (activin-linked kinase [ALK5]). In IPF tissues TIMP3 gene expression was significantly increased and the protein was localized to fibroblastic foci and extracellular matrix. Our findings suggest that TGF-beta1-induced TIMP3 may be an important mediator in lung fibrogenesis.

Topics: Cells, Cultured; Endothelial Cells; Enzyme Activation; Enzyme Inhibitors; Fibroblasts; Genistein; Humans; Imidazoles; Interferon-gamma; Mitogen-Activated Protein Kinase 3; Mitogen-Activated Protein Kinase Kinases; p38 Mitogen-Activated Protein Kinases; Pulmonary Fibrosis; Pyridines; RNA, Messenger; Tissue Inhibitor of Metalloproteinase-1; Tissue Inhibitor of Metalloproteinase-3; Transforming Growth Factor beta; Transforming Growth Factor beta1; Up-Regulation

Mechanisms leading to fibroblast accumulation during pulmonary fibrogenesis remain unclear. Although there is in vitro evidence of lung alveolar epithelial-to-mesenchymal transition (EMT), whether EMT occurs within the lung is currently unknown. Biopsies from fibrotic human lungs demonstrate epithelial cells with mesenchymal features, suggesting EMT. To more definitively test the capacity of alveolar epithelial cells for EMT, mice expressing beta-galactosidase (beta-gal) exclusively in lung epithelial cells were generated, and their fates were followed in an established model of pulmonary fibrosis, overexpression of active TGF-beta1. beta-gal-positive cells expressing mesenchymal markers accumulated within 3 weeks of in vivo TGF-beta1 expression. The increase in vimentin-positive cells within injured lungs was nearly all beta-gal-positive, indicating epithelial cells as the main source of mesenchymal expansion in this model. Ex vivo, primary alveolar epithelial cells cultured on provisional matrix components, fibronectin or fibrin, undergo robust EMT via integrin-dependent activation of endogenous latent TGF-beta1. In contrast, primary cells cultured on laminin/collagen mixtures do not activate the TGF-beta1 pathway and, if exposed to active TGF-beta1, undergo apoptosis rather than EMT. These data reveal alveolar epithelial cells as progenitors for fibroblasts in vivo and implicate the provisional extracellular matrix as a key regulator of epithelial transdifferentiation during fibrogenesis.

Topics: Animals; Apoptosis; beta-Galactosidase; Cells, Cultured; Collagen; Drug Combinations; Epithelial Cells; Extracellular Matrix; Fibronectins; Genes, Reporter; Humans; Laminin; Mesoderm; Mice; Mice, Transgenic; Proteoglycans; Pulmonary Alveoli; Pulmonary Fibrosis; Transforming Growth Factor beta; Transforming Growth Factor beta1

The molecular mechanisms of Idiopathic Pulmonary Fibrosis (IPF) remain elusive. Transforming Growth Factor beta 1(TGF-beta1) is a key effector cytokine in the development of lung fibrosis. We used microarray and computational biology strategies to identify genes whose expression is significantly altered in alveolar epithelial cells (A549) in response to TGF-beta1, IL-4 and IL-13 and Epstein Barr virus. A549 cells were exposed to 10 ng/ml TGF-beta1, IL-4 and IL-13 at serial time points. Total RNA was used for hybridisation to Affymetrix Human Genome U133A microarrays. Each in vitro time-point was studied in duplicate and an average RMA value computed. Expression data for each time point was compared to control and a signal log ratio of 0.6 or greater taken to identify significant differential regulation. Using normalised RMA values and unsupervised Average Linkage Hierarchical Cluster Analysis, a list of 312 extracellular matrix (ECM) proteins or modulators of matrix turnover was curated via Onto-Compare and Gene-Ontology (GO) databases for baited cluster analysis of ECM associated genes. Interrogation of the dataset using ontological classification focused cluster analysis revealed coordinate differential expression of a large cohort of extracellular matrix associated genes. Of this grouping members of the ADAM (A disintegrin and Metalloproteinase domain containing) family of genes were differentially expressed. ADAM gene expression was also identified in EBV infected A549 cells as well as IL-13 and IL-4 stimulated cells. We probed pathologenomic activities (activation and functional activity) of ADAM19 and ADAMTS9 using siRNA and collagen assays. Knockdown of these genes resulted in diminished production of collagen in A549 cells exposed to TGF-beta1, suggesting a potential role for these molecules in ECM accumulation in IPF.

Topics: ADAM Proteins; Adenocarcinoma; Cell Line, Tumor; Coculture Techniques; Humans; Oligonucleotide Array Sequence Analysis; Pulmonary Alveoli; Pulmonary Fibrosis; Respiratory Mucosa; RNA; Transforming Growth Factor beta

Lung fibrosis is the ultimate outcome of hyperoxia-induced chronic lung diseases (CLD) and connective tissue growth factor (CTGF) is correlated with fibrosis. This study investigated the role of CTGF in hyperoxia-induced CLD.. Fifty premature rats were randomly exposed to hyperoxia (Model group) and to room air (Control group) (n = 25 each). CLD was induced by hyperoxia exposure. The expression of CTGF was detected by immunohistochemical method at 1, 3, 7, 14 and 21 days after exposure. The severity of pulmonary fibrosis was evaluated.. In the Control group there was a slight expression of CTGF in the bronchial epithelial cells and vascular endothelial cells. The intensity and range of CTGF expression in the Model group were similar to the Control group on days 1, 3 and 7 of exposure. On the 14th day, CTGF was expressed in some alveolar epithelial cells, fibroblasts and interstitial cells, and the intensity of CTGF expression increased significantly compared with the Control group, with the IODT of CTGF of 10.53 +/- 4.24 vs 5.58 +/- 1.18 (P < 0.01). On day 21, the expression intensity and range of CTGF in the Model group (IODT: 16.61 +/- 5.39) increased compared with that of Control group (P < 0.01). The expression of CTGF was correlated with the degree of fibrosis in the Model group on days 14 and 21 (r = 0.903, r = 0.926 respectively, P < 0.01).. The CTGF expression increased with the time of hyperoxia exposure and the development of fibrosis. CTGF is closely related to the development of hyperoxia-induced pulmonary fibrosis.

Topics: Animals; Chronic Disease; Connective Tissue Growth Factor; Female; Hyperoxia; Immediate-Early Proteins; Immunohistochemistry; Intercellular Signaling Peptides and Proteins; Lung; Pulmonary Fibrosis; Rats; Rats, Sprague-Dawley; Transforming Growth Factor beta

To investigate the effects of gene therapy with replication-defective adenovirus enclosing Egr-1 promoter and Smad7 cDNA on irradiation-induced pulmonary fibrosis.. The recombinant replication-defective adenovirus AD. Egr-Smad7 enclosing Egr-1 promoter and Smad7 cDNA was constructed. 288 C57BL mice were randomly divided into 6 groups: AD. Egr-Smad7 group (Group RA, receiving intratracheal instillation of AD. Egr-Smad7 of the dose of 10(9) pfu/0.1 ml), AD. Egr-Smad7 + radiation group (Group RAR, receiving intratracheal instillation of AD. Egr-Smad7 of the dose of 10(9) pfu/0.1 ml and then radiation to the chest 14 h later), replication-defective adenovirus group (Group AV, receiving intratracheal instillation of replication-defective adenovirus of the dose of 10(9) pfu/0.1 ml), replication-defective adenovirus + irradiation group (Group AVR, receiving intratracheal instillation of replication-defective adenovirus of the dose of 10(9) pfu/0.1 ml and then radiation to the chest 14 h later), blank control group (Group C), and pure irradiation group (Group R), each group was re-divided into 6 subgroups of 8 mice to be observed 0, 1, 2, 4, 8, and 12 weeks after the treatment. The mice were killed at different time points and their lungs were taken out. The levels of type I collagen, type III collagen, connective tissue growth factor (CTGF), and transforming growth factor-beta1 (TGF-beta1) were detected by ELISA. The level of hydroxyproline was examined by alkaline hydrolysis method. The lung tissues were stained with HE to undergo pathological examination.. The TGF-beta1 levels of the irradiation groups all increased, peaking in the second week (all P < 0.05), all significantly higher than those of Group C. However, the TGF-beta1 levels at different time points of Group RAR were all significantly lower then those of the other irradiation groups. The CTGF levels of different groups at different time points were all significantly higher than those of Group C (P < 0.05 or P < 0.01), and the CTGF levels of Groups RA and AV were decreased to almost normal 12 weeks after the irradiation. The levels of type I collagen and type III collagen of the 1 and 2-week subgroups of Group RAR were significantly lower than those of Group C (all P < 0.01), then gradually increased, and were slightly higher than those Group C 12 weeks later. The levels of type I collagen and type III collagen at different time points of the other groups were all significantly higher than those of Group C (P < 0.05 or P < 0.01). However, The levels of type I collagen at different time points of Group RAR were all lower than those of the other groups except Group C, and the levels of type III collagen in the first to eighth weeks after irradiation of Group RAR were all lower than those of the other groups except Group C. The hydroxyproline level of the 1 and 2 week subgroups of Group RAR were significantly lower than those of Group C (all P < 0.01), and then gradually increased. The hydroxyproline levels of the other irradiation groups all gradually increased significantly, peaking at the 12 th week (all P < 0.01). 1 approximately 2 weeks after irradiation Groups RAR, RA, and AV showed remarkable pulmonary congestion changes, even more remarkable then those in Group R, 8 approximately 12 week later, fibrosis changes were found in Group R and AVR, and 12 weeks later the histological structure of lung of Group AV, RAR, and RA returned almost normal.. Radioactive rays induce Egr-1 promoter to regulated the expression of exogenous Smad7 gene that blocks the signal transduction of TGF-beta. Thus use of AD. Egr-Smad7 may become a novel strategy of gene therapy in prevention and treatment of pulmonary fibrosis.

Topics: Adenoviridae; Animals; Defective Viruses; DNA, Complementary; Early Growth Response Protein 1; Gene Expression; Genetic Therapy; Genetic Vectors; Male; Mice; Mice, Inbred C57BL; Promoter Regions, Genetic; Pulmonary Fibrosis; Radiation Injuries, Experimental; Random Allocation; Smad7 Protein; Transfection; Transforming Growth Factor beta; Virus Replication

Pulmonary fibrosis is the result of abnormal processes of repair that occur after lung injury. Transforming growth factor (TGF)-beta is a key molecule in the progression of pulmonary fibrosis. Although clinical use of interferon (IFN)-beta did not improve survival in patients with idiopathic pulmonary fibrosis, because some preclinical studies have suggested that IFN-beta is a potent inhibitor of fibrogenesis, beneficial effects of IFN-beta have been expected. We therefore attempted to determine effects of IFN-beta and investigated the mechanism of action of IFN-beta in bleomycin-induced pulmonary fibrosis. Bleomycin at Day 0 and IFN-beta for 4 wk were administered intravenously to ICR mice. At 28 d after bleomycin injection, histologic and chemical analysis was performed for evaluation of effects of IFN-beta. Tissue distribution and amounts of TGF-beta1 and thrombospondin (TSP)-1/2 were analyzed. IFN-beta attenuated prolylhydroxylase activity, resulting in inhibition of pulmonary fibrosis. Bleomycin-induced increase in TGF-beta1 in epithelial cells and extracellular matrix was attenuated by IFN-beta. TSP-1/2 was limited in platelets of control mice, but was present in foamy cells in fibrotic regions induced by bleomycin. These findings suggest that the antifibrotic effect of IFN-beta is inhibition of TGF-beta and its activation via decrease in TSP-1/2 in lung tissue and change in location of TSP-1/2 from platelets to foamy cells.

Topics: Animals; Antibiotics, Antineoplastic; Bleomycin; Epithelial Cells; Extracellular Matrix; Foam Cells; Immunohistochemistry; Interferon-beta; Lung; Male; Mice; Mice, Inbred ICR; Procollagen-Proline Dioxygenase; Pulmonary Fibrosis; Thrombospondin 1; Thrombospondins; Transforming Growth Factor beta; Transforming Growth Factor beta1

Pulmonary fibrosis is characterized by chronic scar formation and deposition of extracellular matrix, resulting in impaired lung function and respiratory failure. Idiopathic pulmonary fibrosis (IPF) is associated with pronounced morbidity and mortality and responds poorly to known therapeutic interventions; there are no known drugs that effectively block or reverse progressive fibrosis. Transforming growth factor beta (TGF-beta) is known to mediate extracellular matrix gene regulation and appears to be a major player in both the initiation and progression of IPF. TGF-beta mediates its biological effects through members of a family of activin receptor-like kinases (ALK). We have used a gene transfer model of progressive TGF-beta1-induced pulmonary fibrosis in rats to study a newly described orally active small molecular weight drug that is a potent and selective inhibitor of the kinase activity of ALK5, the specific TGF-beta receptor. We show that the drug inhibits the induction of fibrosis when administered at the time of initiation of fibrogenesis and, most important, blocks progressive fibrosis when administered transiently to animals with established fibrosis. These data show promise of the development of an effective therapeutic intervention for IPF and that inhibition of chronic progressive fibrosis may be achieved by blocking TGF-beta receptor activation.

Topics: Activin Receptors, Type I; Administration, Oral; Animals; Cells, Cultured; Down-Regulation; Female; Fibroblasts; Lung; Oligonucleotide Array Sequence Analysis; Protein Kinase Inhibitors; Protein Serine-Threonine Kinases; Pteridines; Pulmonary Fibrosis; Rats; Rats, Sprague-Dawley; Receptor, Transforming Growth Factor-beta Type I; Receptors, Transforming Growth Factor beta; Transforming Growth Factor beta; Transforming Growth Factor beta1; Up-Regulation

Chronic inflammation and proinflammatory cytokines as well as T helper type 2 (Th2) cytokines have been involved in the pathogenesis of pulmonary injury and lung fibrosis. The actual role of IL-10 in lung fibrosis is still unclear because this cytokine has been identified as Th2 but possesses strong anti-inflammatory properties. To better dissect the potential role of IL-10 in silica-induced lung fibrosis, IL-10 was overexpressed in the lung of mice by adenoviral gene transfer during the inflammatory (administered at day -1) or the fibrotic (administered at day +30) stages of the disease. Pulmonary overexpression of IL-10 during both silica-induced lung inflammation and fibrosis exacerbated the fibrotic lesions as estimated by the measurement of hydroxyproline and other biochemical and histological markers. Increased expression of IL-10 significantly enhanced the number of lung lymphocytes and bronchoalveolar lavage fluid IgG1 but not IgG2a levels, indicating the induction of a Th2-like immune response. In addition, the production of the profibrotic Th2 cytokines IL-4 and IL-13 was also significantly increased upon IL-10 overexpression. No difference in transforming growth factor-beta or PGE(2) production was noted after adenoviral IL-10 treatment of silica-treated mice. Together, these data indicate that the increased expression of IL-10 significantly contributed to silica-induced lung fibrosis by exacerbating the Th2 response and the production of the profibrotic cytokines IL-4 and IL-13.

Topics: Adenoviridae; Animals; Dinoprostone; Female; Gene Expression; Interleukin-10; Interleukin-13; Interleukin-4; Lung; Mice; Mice, Inbred C57BL; Pulmonary Fibrosis; Silicon Dioxide; Silicosis; Th2 Cells; Transforming Growth Factor beta

This study presents the antifibrotic effect of interferon gamma (IFN-gamma) in silicosis of rats. Wistar rats were received saline or 20 mg of quartz by intratracheal instillation. The next day after instillation, silicosis model of rats were daily treated IFN-gamma (1.0 x 10(5) units/rat) or saline by intramuscular route. At 1 month or 2 months after instillation, rats were sacrificed to determine hydroxyproline content and observe the change of fibrosis and expression of interleukin-4 (IL-4) and transforming growth factor-beta1 (TGF-beta1) in the lung of rats. The hydroxyproline content of IFN-gamma treated groups was significantly lower than that of quartz groups (P<0.05). At 1 month after instillation, there were Stage II and II+ of silicotic nodules in quartz group; however, in IFN-gamma treated group only cellular nodules (Stage I) were observed. At 2 months after instillation, there was Stage II+ and III of silicotic nodules in quartz group. In IFN-gamma treated group Stage II was found. The expressions of IL-4 (2.19+/-0.90, 0.61+/-0.22, respectively) and TGF-beta1 (1.37+/-0.31, 1.76+/-0.72, respectively) in IFN-gamma treated groups were significantly lower than those in quartz groups (IL-4: 3.33+/-1.27, 1.99+/-0.80; TGF-beta1: 3.67+/-0.63, 4.90+/-1.11, respectively; P<0.05 or P<0.01). It may be concluded that the decreased expression of IL-4 and TGF-beta1 may be associated with decreased hydroxyproline synthesis, which then results in decreased lung fibrosis.

Topics: Animals; Disease Models, Animal; Female; Hydroxyproline; Immunohistochemistry; Interferon-gamma; Interleukin-4; Lung; Male; Pulmonary Fibrosis; Quartz; Rats; Rats, Wistar; Silicosis; Transforming Growth Factor beta

Simvastatin is best known for its antilipidemic action and use in cardiovascular disease due to its inhibition of 3-hydroxy-3-methylglutaryl CoenzymeA (HMG CoA) reductase, a key enzyme in the cholesterol synthesis pathway. Inhibition of biological precursors in this pathway also enables pleiotrophic immunomodulatory and anti-inflammatory capabilities, including modulation of growth factor expression. Connective tissue growth factor (CTGF) and persistent myofibroblast formation are major determinants of the aggressive fibrotic disease, idiopathic pulmonary fibrosis (IPF). In this study we used human lung fibroblasts derived from healthy and IPF lungs to examine Simvastatin effects on CTGF gene and protein expression, analyzed by RT-PCR and ELISA, respectively. Simvastatin significantly inhibited (P < 0.05) CTGF gene and protein expression, overriding the induction by transforming growth factor-beta1, a known potent inducer of CTGF. Such Simvastatin suppressor action on growth factor interaction was reflected functionally on recognized phenotypes of fibrosis. alpha-smooth muscle actin expression was downregulated and collagen gel contraction reduced by 4.94- and 7.58-fold in IMR90 and HIPF lung fibroblasts, respectively, when preconditioned with 10 microM Simvastatin compared with transforming growth factor-beta1 treatment alone after 24 h. Our data suggest that Simvastatin can modify critical determinants of the profibrogenic machinery responsible for the aggressive clinical profile of IPF, and potentially prevents adverse lung parenchymal remodeling associated with persistent myofibroblast formation.

Topics: Actins; ADP Ribose Transferases; Biomarkers; Botulinum Toxins; Cell Line; Collagen; Connective Tissue Growth Factor; Fibroblasts; Gels; Gene Expression; Humans; Immediate-Early Proteins; Intercellular Signaling Peptides and Proteins; Lung; Pulmonary Fibrosis; Recombinant Proteins; rho GTP-Binding Proteins; Simvastatin; Transforming Growth Factor beta; Transforming Growth Factor beta1

IL-13 is a major stimulator of inflammation and tissue remodeling at sites of Th2 inflammation. In Th2-dominant inflammatory disorders such as asthma, IL-11 is simultaneously induced. However, the relationship(s) between IL-11 and IL-13 in these responses has not been defined, and the role(s) of IL-11 in the genesis of the tissue effects of IL-13 has not been evaluated. We hypothesized that IL-11, signaling via the IL-11Ralpha-gp130 receptor complex, plays a key role in IL-13-induced tissue responses. To test this hypothesis we compared the expression of IL-11, IL-11Ralpha, and gp130 in lungs from wild-type mice and transgenic mice in which IL-13 was overexpressed in a lung-specific fashion. We simultaneously characterized the effects of a null mutation of IL-11Ralpha on the tissue effects of transgenic IL-13. These studies demonstrate that IL-13 is a potent stimulator of IL-11 and IL-11Ralpha. They also demonstrate that IL-13 is a potent stimulator of inflammation, fibrosis, hyaluronic acid accumulation, myofibroblast accumulation, alveolar remodeling, mucus metaplasia, and respiratory failure and death in mice with wild-type IL-11Ralpha loci and that these alterations are ameliorated in the absence of IL-11Ralpha. Lastly, they provide insight into the mechanisms of these processes by demonstrating that IL-13 stimulates CC chemokines, matrix metalloproteinases, mucin genes, and gob-5 and stimulates and activates TGF-beta1 via IL-11Ralpha-dependent pathways. When viewed in combination, these studies demonstrate that IL-11Ralpha plays a key role in the pathogenesis of IL-13-induced inflammation and remodeling.

Topics: Animals; Chemokines, CC; Fibroblasts; Hyaluronic Acid; Hyperoxia; Inflammation; Interleukin-11; Interleukin-11 Receptor alpha Subunit; Interleukin-13; Interleukin-13 Receptor alpha1 Subunit; Lung; Matrix Metalloproteinases; Metaplasia; Mice; Mice, Inbred C57BL; Mice, Knockout; Mice, Transgenic; Mucins; Protein Subunits; Pulmonary Alveoli; Pulmonary Fibrosis; Receptors, Interleukin; Receptors, Interleukin-11; Receptors, Interleukin-13; Respiratory Insufficiency; Signal Transduction; Transforming Growth Factor beta; Transforming Growth Factor beta1

Pulmonary fibrosis is a progressive life-threatening disease for which no effective therapy exists. Myofibroblasts are one of the key effector cells in pulmonary fibrosis and are the primary source of extracellular matrix production. Drugs that inhibit the differentiation of fibroblasts to myofibroblasts have potential as antifibrotic therapies. Peroxisome proliferator-activated receptor (PPAR)-gamma is a transcription factor that upon ligation with PPARgamma agonists activates target genes containing PPAR response elements. PPARgamma agonists have anti-inflammatory activities and may have potential as antifibrotic agents. In this study, we examined the abilities of PPARgamma agonists to block two of the most important profibrotic activities of TGF-beta on pulmonary fibroblasts: myofibroblast differentiation and production of excess collagen. Both natural (15d-PGJ2) and synthetic (ciglitazone and rosiglitazone) PPARgamma agonists inhibited TGF-beta-driven myofibroblast differentiation, as determined by alpha-smooth muscle actin-specific immunocytochemistry and Western blot analysis. PPARgamma agonists also potently attenuated TGF-beta-driven type I collagen protein production. A dominant-negative PPARgamma partially reversed the inhibition of myofibroblast differentiation by 15d-PGJ2 and rosiglitazone, but the irreversible PPARgamma antagonist GW-9662 did not, suggesting that the antifibrotic effects of the PPARgamma agonists are mediated through both PPARgamma-dependent and independent mechanisms. Thus PPARgamma agonists have novel and potent antifibrotic effects in human lung fibroblasts and may have potential for therapy of fibrotic diseases in the lung and other tissues.

Topics: Actins; Anilides; Cell Differentiation; Collagen Type I; Fibroblasts; Humans; Hypoglycemic Agents; Lung; Muscle, Smooth; PPAR gamma; Prostaglandin D2; Pulmonary Fibrosis; Rosiglitazone; Thiazolidinediones; Transforming Growth Factor beta

The effects of isoliensinine (IL), a bisbenzylisoquinoline alkaloid extracted from the Chinese traditional medicine seed embryo of Nelumbo nucifera Gaertn., on bleomycin (BLM)-induced pulmonary fibrosis in mice were investigated. Seventy-two male Kungming mice were divided randomly into eight groups as BLM-IL10, BLM-IL20, BLM-IL40, BLM-Sal, Sal-IL10, Sal-IL20, Sal-IL40 and Sal-Sal groups. BLM (0.1 mg in 0.05 ml saline per animal, once) or saline (0.05 ml per animal, once) was applied intratracheally, and IL (10, 20, 40 mg/kg) or saline was administered orally 3 times per day in the appropriate groups. Animals were sacrificed 14 days after intratracheal treatment. Lung tissue and serum superoxide dismutase (SOD) activity and malondialdehyde (MDA) levels, tumor necrosis factor alpha (TNF-alpha) and transforming growth factor beta 1 (TGF-beta (1)) were determined by biochemical measurements and immunohistochemistry. BLM treatment resulted in a significant increase of the hydroxyproline content and an obvious lung histological injury as compared to the Sal-Sal group. Administration of IL remarkably suppressed the increase in hydroxyproline content and abated the lung histological injury induced by BLM. There was a decrease in SOD activity and an increase in MDA level in lung tissue and serum in the BLM-Sal group (p < 0.01 , p < 0.01, vs. Sal-Sal group, respectively). And IL could obviously enhance the SOD activity and decrease the MDA level in a concentration-dependent manner. Moreover, IL also significantly inhibited the overexpression of TNF-alpha and TGF-beta (1) induced by BLM. These results indicated that IL possessed a significant inhibitory effect on BLM-induced pulmonary fibrosis, probably due to its antioxidant and/or anti-inflammatory activities and inhibitory overexpressing TNF-alpha and TGF-beta (1) induced by BLM.

Topics: Administration, Oral; Animals; Anti-Inflammatory Agents, Non-Steroidal; Bleomycin; Dose-Response Relationship, Drug; Immunohistochemistry; Isomerism; Isoquinolines; Lung; Male; Mice; Nelumbo; Phenols; Phytotherapy; Plant Extracts; Pulmonary Fibrosis; Seeds; Trachea; Transforming Growth Factor beta; Transforming Growth Factor beta1; Tumor Necrosis Factor-alpha

To investigate the contribution of chemokine receptor-CXCR3 to the fibrotic disease process induced by bleomycin in CXCR3 gene deficient mice.. Sixty-two CXCR3 knock-out mice with C57Bl/6 background were established by gene targeting. Forty-eight sex-, age- and weight-matched C57Bl/6 mice were challenged by injection of bleomycin via trachea. Lung tissue was stained with HE and Masson-trichrome methods. Lung hydroxyproline content was evaluated. Bronchoalveolar lavage was performed using PBS, and the cell number and differentials were counted by Diff-Quick staining. TNF-alpha, IL-5 and TGF-beta in the lung homogenate were measured by ELISA. T lymphocytes were analyzed by FACScan. Unpaired t test was explored to compare the difference between two groups.. On day 14 after bleomycin injection, CXCR3 knockout mice were protected from BLM-induced lung injury and fibrosis as evidenced by less inflammatory cells in the lung interstitium and hydroxyproline content in the lung tissue compared to the wild type littermates [3.92 +/- 0.37 vs 5.33 +/- 0.34, P < 0.05; (67.0 +/- 24.2) microg/left lung vs (211.5 +/- 24.2) microg/left lung, P < 0.01]. On day 7 after bleomycin administration, there was less infiltration of neutrophils, lymphocytes and CD(4)(+) T lymphocytes into lung tissue in CXCR3 knockout mice than those in the wild-type mice [(3.25 +/- 0.61) x 10(5) vs (6.13 +/- 0.86) x 10(5), P < 0.05]; [(8.15 +/- 1.96) x 10(5) vs (13.48 +/- 1.47) x 10(5), P < 0.05]; [(9.00 +/- 1.00) x 10(4) vs (15.60 +/- 2.00) x 10(4), P < 0.05]. Lower level of fibrogenic cytokine release, including the altered production of TNF-alpha and IL-5, was seen in CXCR3 knockout mice compared to the wild type mice [(1,023 +/- 113) microg/L vs (1,530 +/- 178) microg/L, P < 0.05; (403 +/- 50) microg/L vs (755 +/- 95) microg/L, P < 0.05] on day 7 postbleomycin injection, whereas TGF-beta level was decreased significantly in CXCR3 knockout mice compared to the wild-type mice on day 14 postbleomycin administration [(2,211 +/- 289) microg/L vs (5,388 +/- 1,071) microg/L, P < 0.05].. These data imply that CXCR3 signaling promotes CD(4)(+) T-cell recruiting and initiates fibrogenic cytokine cascade following intratracheal bleomycin administration and indicate that CXCR3 might be a therapeutic target for pulmonary fibrosis treatment.

Topics: Animals; Bleomycin; Bronchoalveolar Lavage Fluid; Disease Models, Animal; Interleukin-5; Mice; Mice, Inbred C57BL; Mice, Knockout; Pulmonary Fibrosis; Receptors, Chemokine; Receptors, CXCR3; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha

The hallmark of idiopathic pulmonary fibrosis (IPF) is the myofibroblast, the cellular origin of which in the lung is unknown. We hypothesized that alveolar epithelial cells (AECs) may serve as a source of myofibroblasts through epithelial-mesenchymal transition (EMT). Effects of chronic exposure to transforming growth factor (TGF)-beta1 on the phenotype of isolated rat AECs in primary culture and a rat type II cell line (RLE-6TN) were evaluated. Additionally, tissue samples from patients with IPF were evaluated for cells co-expressing epithelial (thyroid transcription factor (TTF)-1 and pro-surfactant protein-B (pro-SP-B), and mesenchymal (alpha-smooth muscle actin (alpha-SMA)) markers. RLE-6TN cells exposed to TGF-beta1 for 6 days demonstrated increased expression of mesenchymal cell markers and a fibroblast-like morphology, an effect augmented by tumor necrosis factor-alpha (TNF-alpha). Exposure of rat AECs to TGF-beta1 (100 pmol/L) resulted in increased expression of alpha-SMA, type I collagen, vimentin, and desmin, with concurrent transition to a fibroblast-like morphology and decreased expression of TTF-1, aquaporin-5 (AQP5), zonula occludens-1 (ZO-1), and cytokeratins. Cells co-expressing epithelial markers and alpha-SMA were abundant in lung tissue from IPF patients. These results suggest that AECs undergo EMT when chronically exposed to TGF-beta1, raising the possibility that epithelial cells may serve as a novel source of myofibroblasts in IPF.

Topics: Actins; Animals; Aquaporin 5; Aquaporins; Biomarkers; Cell Line; Epithelial Cells; Fibroblasts; Humans; Male; Membrane Proteins; Mesoderm; Myocytes, Smooth Muscle; Nuclear Proteins; Phenotype; Pulmonary Alveoli; Pulmonary Fibrosis; Rats; Rats, Sprague-Dawley; Thyroid Nuclear Factor 1; Transcription Factors; Transforming Growth Factor beta; Transforming Growth Factor beta1

Activation of the coagulation cascade is commonly observed in the lungs of patients with both acute and chronic inflammatory and fibrotic lung disorders, as well as in animal models of these disorders. The aim of this study was to examine the contribution of the major thrombin receptor, proteinase-activated receptor-1 (PAR-1), during the acute inflammatory and chronic fibrotic phases of lung injury induced by intratracheal instillation of bleomycin in mice. Inflammatory cell recruitment and increases in bronchoalveolar lavage fluid (BALF) protein were attenuated by 56 +/- 10% (P < 0.05) and 53 +/- 12% (P < 0.05), respectively, in PAR-1-deficient (PAR-1-/-) mice compared with wild-type (WT) mice. PAR-1-/- mice were also protected from bleomycin-induced pulmonary fibrosis with total lung collagen accumulation reduced by 59 +/- 5% (P < 0.05). The protection afforded by PAR-1 deficiency was accompanied by significant reductions in pulmonary levels of the potent PAR-1-inducible proinflammatory and profibrotic mediators, monocyte chemoattractant protein-1 (MCP-1), transforming growth factor-beta-1 (TGF-beta1), and connective tissue growth factor/fibroblast-inducible secreted protein-12 (CTGF/FISP12). In addition, PAR-1 was highly expressed in inflammatory and fibroproliferative lesions in lung sections obtained from patients with fibrotic lung disease. These data show for the first time that PAR-1 signaling plays a key role in experimentally induced lung injury, and they further identify PAR-1 as one of the critical receptors involved in orchestrating the interplay between coagulation, inflammation, and remodeling in response to tissue injury.

Topics: Animals; Biopsy; Bleomycin; Bronchoalveolar Lavage Fluid; Capillary Permeability; Cell Count; Connective Tissue Growth Factor; Cytoprotection; Humans; Immediate-Early Proteins; Immunohistochemistry; Intercellular Signaling Peptides and Proteins; Lung; Mice; Mice, Inbred C57BL; Mice, Knockout; Pneumonia; Pulmonary Fibrosis; Receptor, PAR-1; Signal Transduction; Transforming Growth Factor beta; Transforming Growth Factor beta1

Cell-cell signaling roles for reactive oxygen species (ROS) generated in response to growth factors/cytokines in nonphagocytic cells are not well defined. In this study, we show that fibroblasts isolated from lungs of patients with idiopathic pulmonary fibrosis (IPF) generate extracellular hydrogen peroxide (H2O2) in response to the multifunctional cytokine, transforming growth factor-beta1 (TGF-beta1). In contrast, TGF-beta1 stimulation of small airway epithelial cells (SAECs) does not result in detectable levels of extracellular H2O2. IPF fibroblasts independently stimulated with TGF-beta1 induce loss of viability and death of overlying SAECs when cocultured in a compartmentalized Transwell system. These effects on SAECs are inhibited by the addition of catalase to the coculture system or by the selective enzymatic blockade of H2O2 production by IPF fibroblasts. IPF fibroblasts heterogeneously express alpha-smooth muscle actin stress fibers, a marker of myofibroblast differentiation. Cellular localization of H2O2 by a fluorescent-labeling strategy demonstrated that extracellular secretion of H2O2 is specific to the myofibroblast phenotype. Thus, myofibroblast secretion of H2O2 functions as a diffusible death signal for lung epithelial cells. This novel mechanism for intercellular ROS signaling may be important in physiological/pathophysiological processes characterized by regenerating epithelial cells and activated myofibroblasts.

Topics: Catalase; Cell Death; Cell Division; Cells, Cultured; Coculture Techniques; Diffusion; Epithelial Cells; Fibroblasts; Fluorescent Antibody Technique; Humans; Hydrogen Peroxide; Muscle Cells; Oxidative Stress; Pulmonary Fibrosis; Signal Transduction; Transforming Growth Factor beta; Transforming Growth Factor beta1

Transplant-related lung fibrosis is characterized by excessive fibro-collagenous deposition. Induction of arginase, an enzyme that metabolizes L-arginine to urea and L-ornithine, is vital for collagen synthesis. Pirfenidone is an investigational anti-fibrotic agent shown to be effective in blocking pulmonary fibrosis. The purpose of this study was to determine if pirfenidone was protective against the development of fibro-collagenous injury in rat lung orthotopic transplants through altering L-arginine-arginase metabolic pathways. Lung transplants were performed using Lewis donors and Sprague-Dawley recipients (allografts) or the same strain (isografts). Recipients were given pirfenidone (0.5% chow) 1-21-day post-transplantation. A significantly increased peak airway pressure (PawP) with excessive collagen deposition was found in untreated lung allografts. Pirfenidone treatment decreased PawP and collagen content in lung allografts. The beneficial effects were associated with downregulation of arginase protein expression and activity. In addition, pirfenidone decreased endogenous transforming growth factor (TGF)-beta level in lung allografts, and TGF-beta stimulated arginase activity in a dose-dependent manner in both lung tissue and fibroblasts. These results suggest that pirfenidone inhibits local arginase activity possibly through suppression of endogenous TGF-beta, hence, limiting the development of fibrosis in lung allografts.

Topics: Animals; Anti-Inflammatory Agents, Non-Steroidal; Arginase; Arginine; Cells, Cultured; Collagen; Down-Regulation; Gene Expression Regulation; Graft Rejection; Lung Transplantation; Male; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Pulmonary Fibrosis; Pyridones; Rats; Rats, Inbred Lew; Rats, Sprague-Dawley; Signal Transduction; Transforming Growth Factor beta; Transforming Growth Factor beta1; Transplantation, Homologous; Tumor Necrosis Factor-alpha

Activins are members of the transforming growth factor-beta superfamily thought to be involved in repair processes after tissue injury.. The aim of this study was to clarify whether activin and its antagonist, follistatin, played a significant role in lung injury and fibrosis.. In bleomycin (BLM)-treated rat lung, mRNA for the beta(A) subunit of activin was upregulated on Days 3 and 7 and decreased gradually thereafter. Immunoreactive activin A was abundantly expressed in macrophages infiltrated in the lung, and was detected in fibroblasts accumulated in the fibrotic area on Day 28. We then administered follistatin, an activin antagonist, to BLM-treated rats. Follistatin significantly reduced the number of macrophages and neutrophils in bronchoalveolar lavage and reduced the protein content. Histologically, follistatin markedly reduced the number of infiltrating cells, ameliorated the destruction of lung architecture on Day 7, and attenuated lung fibrosis on Day 28. The hydroxyproline content was significantly lower in follistatin-treated rats. In cultured lung fibroblasts, production of activin A was augmented by transforming growth factor-beta, and activin antagonist follistatin significantly inhibited transforming growth factor-beta-induced fibroblast activation. These results suggest that activin A was produced in the lung after BLM treatment and promoted acute inflammation and subsequent fibrosis.. Follistatin is effective in treating acute lung injury and BLM-induced fibrosis by blocking the actions of activin and transforming growth factor-beta.

Topics: Activin Receptors; Activins; Animals; Bleomycin; Cell Differentiation; Cells, Cultured; Collagen; Fibroblasts; Follistatin; Humans; Inhibin-beta Subunits; Lung; Pulmonary Fibrosis; Rats; Rats, Wistar; Recombinant Proteins; Transforming Growth Factor beta; Up-Regulation

The mechanisms of idiopathic pulmonary fibrosis pathogenesis, a chronic and progressive interstitial lung disease, remain elusive. The complement system, a crucial arm of the innate immune response, plays a pivotal role in several pathological disorders; however, the contribution of individual complement components to lung fibrosis has not yet been examined. Complement factor 5 (C5) and its cleavage product C5a are critical mediators in inflammatory diseases. Thus, to evaluate the role of C5 in lung fibrosis, we compared congenic C5-sufficient and C5-deficient mice in a well-characterized murine model of bleomycin-induced pulmonary fibrosis. C5-deficient mice had an exaggerated inflammatory phenotype compared with C5-sufficient mice during acute bleomycin-induced lung injury. These findings suggest a protective and anti-inflammatory role for C5, which was linked to the regulation of matrix metalloproteinases involved in cell migration. In contrast, C5 had a detrimental effect during chronic stages of bleomycin-induced injury, indicating a profibrotic role for C5. This deleterious activity for C5 was associated with expression of the fibrogenic cytokine TGF-beta1 and matrix metalloproteinase-3, an important mediator in fibroblast contraction. Altogether, our data reveal novel and opposing roles for C5 in both inflammation and tissue repair. Furthermore, these findings provide insight into the development of new therapeutic strategies for idiopathic pulmonary fibrosis patients.

Topics: Animals; Antibodies, Blocking; Antibodies, Monoclonal; Bleomycin; Chronic Disease; Collagen; Complement C5; Complement C5a; Disease Models, Animal; Fibroblasts; Lung; Matrix Metalloproteinase 3; Matrix Metalloproteinase Inhibitors; Matrix Metalloproteinases; Mice; Mice, Congenic; Mice, Inbred C57BL; NIH 3T3 Cells; Pulmonary Fibrosis; RNA, Messenger; Transforming Growth Factor beta; Transforming Growth Factor beta1

Lung fibrosis is characterized by tissue remodeling resulting from an imbalance between synthesis and degradation of extracellular organic matrices. To examine whether cathepsin(s) (Cat) are important in the development of pulmonary fibrosis, we assessed the expression of four Cat known for their collagenolytic activity in a model of silica-induced lung fibrosis.. Different strains of mice were transorally instilled with 2.5 mg crystalline silica or other particles. Cat expression (Cat K, S, L and B) was quantified in lung tissue and isolated pulmonary cells by quantitative RT-PCR. In vitro, we assessed the effect of different cytokines, involved in lung inflammatory and fibrotic responses, on the expression of Cat K by alveolar macrophages and fibroblasts.. In lung tissue, Cat K transcript was the most strongly upregulated in response to silica, and this upregulation was intimately related to the fibrotic process. In mouse strains known for their differential response to silica, we showed that the level of Cat K expression following silica treatment was inversely related to the level of TGF-beta expression and the susceptibility of these strains to develop fibrosis. Pulmonary macrophages and fibroblasts were identified as Cat K overproducing cells in the lung of silicotic mice. In vitro, Cat K was downregulated in mouse and human lung fibroblasts by the profibrotic growth factor TGF-beta1.. Altogether, these data suggest that while Cat K may contribute to control lung fibrosis, TGF-beta appears to limit its overexpression in response to silica particles.

Topics: Animals; Cathepsin K; Cathepsins; Cells, Cultured; Enzyme Activation; Female; Fibroblasts; Humans; Mice; Mice, Inbred C57BL; Pulmonary Fibrosis; Silicon Dioxide; Silicosis; Transforming Growth Factor beta; Transforming Growth Factor beta1; Up-Regulation

Fibroblasts consist of heterogeneous subpopulations that have distinct roles in fibrotic responses. Previously we reported enhanced proliferation in response to fibrogenic growth factors and selective activation of latent transforming growth factor (TGF)-beta in fibroblasts lacking cell surface expression of Thy-1 glycoprotein, suggesting that Thy-1 modulates the fibrogenic potential of fibroblasts. Here we report that compared to controls Thy-1-/- C57BL/6 mice displayed more severe histopathological lung fibrosis, greater accumulation of lung collagen, and increased TGF-beta activation in the lungs 14 days after intratracheal bleomycin. The majority of cells demonstrating TGF-beta activation and myofibroblast differentiation in bleomycin-induced lesions were Thy-1-negative. Histological sections from patients with idiopathic pulmonary fibrosis demonstrated absent Thy-1 staining within fibroblastic foci. Normal lung fibroblasts, in both mice and humans, were predominantly Thy-1-positive. The fibrogenic cytokines interleukin-1 and tumor necrosis factor-alpha induced loss of fibroblast Thy-1 surface expression in vitro, which was associated with Thy-1 shedding, Smad phosphorylation, and myofibroblast differentiation. These results suggest that fibrogenic injury promotes loss of lung fibroblast Thy-1 expression, resulting in enhanced fibrogenesis.

Topics: Animals; Antimetabolites, Antineoplastic; Bleomycin; Cell Differentiation; DNA-Binding Proteins; Female; Fibroblasts; Humans; Interleukin-1; Lung; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Muscle, Smooth; Phosphorylation; Pulmonary Fibrosis; Smad3 Protein; Thy-1 Antigens; Trans-Activators; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha

Pulmonary fibroblasts regulate extracellular matrix production and degradation; thus, they are critical for maintenance of lung structure, function, and repair. In pulmonary fibrosis, fibroblasts produce excess collagen and form fibrotic foci that eventually impair lung function, but the mechanisms responsible for these alterations are not known. Receptors coupled to the stimulation of cAMP production can inhibit activation of fibroblasts and thereby are antifibrotic. To test whether this signaling pathway is altered in pulmonary fibrosis, we compared the ability of normal adult human pulmonary fibroblasts to generate and respond to cAMP with that of cells isolated from lungs with idiopathic pulmonary fibrosis. Serum- and transforming growth factor (TGF)-beta-stimulated cell proliferation was inhibited approximately 50% by forskolin and approximately 100% by prostaglandin (PG) E(2) in the normal cells but substantially less in the diseased cells. Collagen synthesis was also inhibited >50% by the same drugs in the normal cells but significantly less so in the diseased cells, despite responding with similar increases in cAMP production. Although expression of protein kinase A (PKA) and cAMP-stimulated PKA activity were similar in both the normal and diseased cell types, forskolin- and PGE(2)-stimulated cAMP response element-binding protein (CREB) phosphorylation was decreased in the diseased cell lines compared with the normal cells. cAMP-mediated activation and TGF-beta-mediated inhibition of CREB DNA binding was also diminished in the diseased cells. Thus, pulmonary fibroblasts derived from patients with pulmonary fibrosis are refractory to the inhibition by cAMP due to altered activity of components distal to the activity of PKA, in particular the phosphorylation of CREB.

Topics: Blotting, Western; Cell Line; Cell Proliferation; Colforsin; Collagen; Cyclic AMP; Cyclic AMP Response Element-Binding Protein; Cyclic AMP-Dependent Protein Kinases; Dinoprostone; DNA; Electrophoretic Mobility Shift Assay; Fibroblasts; Humans; Phosphorylation; Pulmonary Fibrosis; Reverse Transcriptase Polymerase Chain Reaction; Smad Proteins; Transforming Growth Factor beta

To study the effect of valsartan on bleomycin-induced pulmonary fibrosis in rats and its possible mechanism.. Sixty Wistar rats were divided into three groups. In the valsartan group and the model groups, pulmonary fibrosis was induced by intratracheal instillation of bleomycin (BLM), and then the former group received valsartan 16 mg/kg daily, but the latter group received oral normal saline. In the control group, normal saline was given both intratracheally and orally. Five rats in each group were sacrificed 7, 14, 28, and 42 days after intratracheal instillation. Collagen content of the lung tissue was assessed by hydroxyproline concentration. Histological changes of the lungs were evaluated by HE stain and Masson's trichrome stain. Lung expression of transforming growth factor-beta (TGF-beta) protein was assessed by immunohistochemistry. The mRNA of hepatocyte growth factor (HGF) was detected by in situ hybridization, and the level of HGF protein was further measured by ELISA.. In the valsartan group, alveolitis [(0.88 +/- 0.12) points, (0.79 +/- 0.21) points, (0.75 +/- 0.17) points] decreased significantly 14 days after treatment with BLM and was significantly lower as compared with the model group [(2.05 +/- 0.25) points, (1.38 +/- 0.12) points, (1.19 +/- 0.11) points, P < 0.01]. Pulmonary fibrosis on day 7, 28, 42 [(0.28 +/- 0.03) points vs (0.45 +/- 0.10) points, (1.74 +/- 0.18) points vs (2.08 +/- 0.32) points, (1.91 +/- 0.09) points vs (2.77 +/- 0.15) points, P < 0.05], hydroxyproline concentrations on day 14, 42 [(1.08 +/- 0.13) microg/mg x pro vs (1.45 +/- 0.19) microg/mg x pro, (1.39 +/- 0.20) microg/mg x pro vs (2.19 +/- 0.37) microg/mg x pro, P < 0.01] and protein expression of TGF-beta on day 7, 42 [2.27 +/- 0.30 vs 3.99 +/- 0.43, 2.05 +/- 0.18 vs 2.71 +/- 0.46, P < 0.05] in the valsartan group all reduced significantly as compared with that of the model group. HGF mRNA [1.61 +/- 0.20 vs 1.98 +/- 0.23, 0.52 +/- 0.19 vs 0.28 +/- 0.14, P < 0.05] and its protein expression (204 +/- 18) pg/ml vs (260 +/- 21) pg/ml, (129 +/- 20) pg/ml vs (100 +/- 20) pg/ml, P < 0.05) on day 7 and day 42 in the valsartan group were increased as compared with those of the model group.. Valsartan alleviates BLM-induced pulmonary fibrosis in rats. Inhibiting the expressions of TGF-beta and stimulating the expression of HGF in lung tissues may be one of the mechanisms.

Topics: Animals; Hepatocyte Growth Factor; Lung; Male; Pulmonary Fibrosis; Rats; Rats, Wistar; RNA, Messenger; Tetrazoles; Transforming Growth Factor beta; Valine; Valsartan

Transforming growth factor-beta1 (TGF-beta1) contributes to the fibrosis of injured organs. Angiotensin II (Ang II) is an inducer of TGF-beta1 in cells of the heart and kidneys, and the regulation of TGF-beta1 by Ang II has not yet been confirmed in lung tissue. We evaluated the role of TGF-beta1 and its relationship with Ang II in paraquat-induced lung fibrosis. Adult male Sprague-Dawley rats were treated intraperitoneally with paraquat (20mg/kg) or saline in the control group. On days 1, 3, 7, and 21 after paraquat treatment, TGF-beta1 and collagen gene expressions, TGF-beta1 protein, angiotensin-converting enzyme (ACE) activity, Ang II, and hydroxyproline contents were measured in lung tissue. Lung TGF-beta1 mRNA expression progressively increased and reached a peak on day 7 after paraquat treatment. Increases in TGF-beta1 mRNA expression and TGF-beta1 levels preceded the onset of increased collagen I mRNA expression and hydroxyproline contents. c-myc mRNA expressions were inversely correlated with TGF-beta1 protein levels in paraquat-treated lungs. Lung ACE activity decreased after paraquat administration and the decrement was maximal on day 7. Lung Ang II concentrations immediately decreased after paraquat administration and the values were not related to TGF-beta1 levels. We conclude that TGF-beta1 is upregulated and contribute to the paraquat-induced lung fibrosis and this effect is independent of the renin-angiotensin system.

Topics: Actins; Angiotensin II; Animals; Collagen Type I; Collagen Type III; Disease Models, Animal; Hydroxyproline; Lung; Male; Microscopy, Polarization; Paraquat; Peptidyl-Dipeptidase A; Proto-Oncogene Proteins c-myc; Pulmonary Fibrosis; Rats; Rats, Sprague-Dawley; RNA, Messenger; Time Factors; Toxicity Tests; Transforming Growth Factor beta; Transforming Growth Factor beta1; Up-Regulation

Transient adenovirus-mediated gene transfer of IL-1beta (AdIL-1beta), a proinflammatory cytokine, induces marked inflammation and severe and progressive fibrosis in rat lungs. This is associated with an increase in TGF-beta1 concentration in bronchoalveolar lavage (BAL) fluid. TGF-beta1 is a key cytokine in the process of fibrogenesis, using intracellular signaling pathways involving Smad2 and Smad3. In this study we investigate whether inflammation induced by IL-1beta is able to independently induce lung fibrosis in mice deficient in the Smad3 gene. Seven days after AdIL-1beta administration, similar levels of IL-1beta transgene are seen in BAL in both wild-type (WT) and knockout (KO) mice, and BAL cell profiles demonstrated a similar marked neutrophilic inflammation. Phospho-Smad2 staining was positive in areas of inflammation in both WT and KO mice at day 7. By day 35 after transient IL-1beta expression, WT mice showed marked fibrosis in peribronchial areas, quantified by picrosirius red staining and morphometry. However, there was no evidence of fibrosis or collagen accumulation in IL-1beta-treated KO mice, and peribronchial areas were not different from KO mice treated with the control adenovector. TGF-beta1 and phospho-Smad2 were strongly positive at day 35 in fibrotic areas observed in WT mice, but no such staining was detectable in KO mice. The IL-1beta-induced chronic fibrotic response in mouse lungs is dependent on Smad3. KO and WT animals demonstrated a similar inflammatory response to overexpression of IL-1beta indicating that inflammation must link to the Smad3 pathway, likely through TGF-beta, to induce progressive fibrosis.

Topics: Activin Receptors, Type I; Animals; Bronchoalveolar Lavage Fluid; Chronic Disease; Disease Models, Animal; Disease Progression; Humans; Inflammation; Interleukin-1; Lung; Mice; Mice, Inbred C57BL; Mice, Knockout; Protein Serine-Threonine Kinases; Pulmonary Fibrosis; Rats; Receptor, Transforming Growth Factor-beta Type I; Receptors, Transforming Growth Factor beta; Signal Transduction; Smad3 Protein; Transforming Growth Factor beta; Up-Regulation

Pulmonary fibrosis is a progressive illness characterized by interstitial fibrosis. Although the precise mechanism for pulmonary fibrosis is not completely understood, an immune response involving interferon (IFN)-gamma appears to play a role. Therefore, we examined the functional roles of natural killer T (NKT) cells, which produce IFN-gamma and interleukin-4 on activation, in bleomycin-induced pulmonary fibrosis. In NKT cell-deficient mice, pulmonary fibrosis was worse in terms of histology, hydroxyproline levels, and mortality than in control mice. The transforming growth factor (TGF)-beta1 levels were higher in the lung after injecting bleomycin, and blockade of TGF-beta1 by neutralizing monoclonal antibody attenuated the pulmonary fibrosis in CD1d-/- mice. In contrast, the production of IFN-gamma was reduced in lungs from CD1d-/- mice. Moreover, the adoptive transfer of NKT cells into CD1d-/- mice increased IFN-gamma and reduced TGF-beta1 production, attenuating pulmonary fibrosis. An in vitro assay demonstrated that IFN-gamma was involved in suppressing TGF-beta1 production in cells collected from bronchoalveolar lavage. The adoptive transfer of NKT cells from IFN-gamma-/- mice did not reverse pulmonary fibrosis or TGF-beta1 production in lungs of CD1d-/- mice whereas NKT cells from B6 control mice attenuated fibrosis and reduced TGF-beta1 production. In conclusion, IFN-gamma-producing NKT cells play a novel anti-fibrotic role in pulmonary fibrosis by regulating TGF-beta1 production.

Topics: Adoptive Transfer; Animals; Bleomycin; Body Weight; Cells, Cultured; Disease Models, Animal; Hydroxyproline; Interferon-gamma; Killer Cells, Natural; Lung; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Pulmonary Fibrosis; T-Lymphocyte Subsets; Transforming Growth Factor beta; Transforming Growth Factor beta1

Feitai, a Chinese medicine formulation, has been shown to protect against lung fibrosis induced by bleomycin (BLM). In the present study, we investigated the effect of Feitai on transforming growth factor (TGF)-beta1 and monocyte chemoattractant protein-1 (MCP-1), which play important roles in the pathogenesis of BLM-induced lung fibrosis. The results demonstrated that Feitai could significantly attenuate BLM-induced acute lung inflammation and subsequent lung fibrosis. Meanwhile, the expression of MCP-1 and TGF-beta1 mRNA in the lungs increased in the BLM-treated group compared with the saline-instilled control group and Feitai treatment significantly decreased cytokine expression in BLM-treated mice. In addition, Feitai diminished the accumulation of MCP-1- and TGF-beta1-positive cells in lung tissues at the time of peak mRNA levels. In summary, the results of the present study indicate that treatment with Feitai ameliorates BLM-induced lung fibrosis, at least in part via the inhibition of MCP-1 and TGF-beta1 expression.

Topics: Animals; Anti-Bacterial Agents; Bleomycin; Cell Differentiation; Chemokine CCL2; Drugs, Chinese Herbal; Female; Fibroblasts; Hydroxyproline; Immunohistochemistry; Lung; Mice; Mice, Inbred C57BL; Peroxidase; Pulmonary Fibrosis; Reverse Transcriptase Polymerase Chain Reaction; Transforming Growth Factor beta; Transforming Growth Factor beta1

Angiotensin II (ANG II), generated by activation of local renin-angiotensin systems, is believed to play an important role in tissue repair and remodeling, in part via transforming growth factor-beta (TGF-beta). Angiotensin-converting enzyme (ACE) inhibitors have been shown to abrogate experimental lung injury via a number of potential mechanisms; however, the potentially fibroproliferative role for ANG II in the lung has not been characterized. We hypothesized that, after lung injury, ANG II would stimulate fibroblast procollagen synthesis and promote lung collagen deposition in rats. In vitro, ANG II was a potent inducer of procollagen production in human lung fibroblasts via activation of the type 1 receptor and, at least in part, via the autocrine action of TGF-beta. After bleomycin-induced lung injury, an increase in lung ANG II concentration was observed by day 3 that preceded increases in lung collagen and was maintained until death at day 21. Administration of an ACE inhibitor (ramipril) reduced ACE activity, ANG II concentration, TGF-beta expression, and collagen deposition. Losartan (an ANG II type 1 receptor antagonist) also attenuated the increase in TGF-beta expression and lung collagen deposition. These observations suggest that ANG II, possibly generated locally within the lung, may play an important role in the fibrotic response to acute lung injury, at least in part via the action of TGF-beta. ACE inhibitors and receptor antagonists, already widely used clinically, should be assessed as potential new therapies for fibrotic lung disease.

Topics: Acute Disease; Angiotensin II; Angiotensin-Converting Enzyme Inhibitors; Antibiotics, Antineoplastic; Antihypertensive Agents; Bleomycin; Cell Division; Cells, Cultured; Collagen; Fibroblasts; Humans; Losartan; Lung; Peptidyl-Dipeptidase A; Procollagen; Pulmonary Fibrosis; Ramipril; Renin-Angiotensin System; Transforming Growth Factor beta

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.

Topics: Age Factors; Animals; Anti-Bacterial Agents; Cell Division; Collagen; Disease Progression; Doxycycline; Extracellular Matrix Proteins; Gene Expression; Mice; Mice, Transgenic; Pneumonia; Pulmonary Fibrosis; Transforming Growth Factor alpha; Transforming Growth Factor beta

Transforming growth factor-beta2-treated Ag-pulsed APC mimic APC from the immune privileged eye, and provide signals that generate regulatory T (Tr) cells and mediate peripheral tolerance. We postulated that TGF-beta2-treated Ag-pulsed APC (tolerogenic APC (tol-APC)) might also orchestrate regulation of immune mediated pathogenesis in nonimmune privileged tissues such as the lung. We used an adoptive transfer model of autoimmune pulmonary interstitial fibrosis called hapten immune pulmonary interstitial fibrosis (ADT-HIPIF) in this study. Mice that received 2,4,6-trinitrobenzene sulfonic acid-sensitized cells and challenged (intratracheally) with the hapten developed pulmonary interstitial fibrosis. However, transfer (i.v.) of TGF-beta2-treated 2,4,6-trinitrobenzene sulfonic acid-pulsed bone marrow-derived APC (tol-APC) to experimental mice 1 day after intratracheal challenge reduced the collagen deposition in the interstitium of the lung that usually follows challenge. Furthermore, ADT-HIPIF mice that received tol-APC developed Ag-specific efferent CD8+ Tr cells. Adoptive transfer of the Tr cells to another set of presensitized mice mediated suppression of the efferent phase of Th1 immune response and the subsequent immune dependent pulmonary interstitial fibrosis. Thus, tol-APC induced efferent CD8+ Tr cells in immune mice, and the regulation of the immune response limited the development of autoimmune pulmonary fibrosis in sensitized and pulmonary-challenged mice. Because ADT-HIPIF shares etiological and pathological characteristics with a variety of human immune inflammatory conditions in the lung that eventuate into interstitial fibrosis, these studies provide insight into potential therapy to alter the course of pulmonary fibrosis in humans.

Topics: Adoptive Transfer; Animals; Antigen-Presenting Cells; Bone Marrow Cells; CD8-Positive T-Lymphocytes; Cell Division; Cells, Cultured; Dermatitis, Contact; Disease Models, Animal; Female; Haptens; Immune Tolerance; Lymphocyte Activation; Mice; Mice, Inbred BALB C; Pulmonary Fibrosis; T-Lymphocyte Subsets; Transforming Growth Factor beta; Transforming Growth Factor beta2; Trinitrobenzenesulfonic Acid

Signalling of angiotensin II via angiotensin II type 1 receptor (AT1) promotes cardiac and renal fibrosis, but its role in lung fibrosis is little understood. Using a rat bleomycin (BLM) induced model of pulmonary fibrosis, we examined the expression of AT1 in the lung and the effect of an AT1 antagonist on pulmonary fibrosis.. Adult male Sprague-Dawley rats were given 0.3 mg/kg BLM intratracheally. Two days earlier they had received 10 mg/kg/day of the AT1 antagonist candesartan cilexetil mixed in the drinking water. AT1 expression in the lungs was examined by immunohistochemistry and immunoblot methods. The effect of the AT1 antagonist on pulmonary fibrosis was studied by analysis of bronchoalveolar lavage (BAL) fluid, histopathology, and hydroxyproline assay.. Immunohistochemical studies showed overexpression of AT1 in inflammatory immune cells, alveolar type II cells, and fibroblasts. A quantitative assay for AT1 showed that AT1 expression was significantly upregulated in cells from BAL fluid after day 3 and in the lung homogenates after day 21. Candesartan cilexetil significantly inhibited the increase in total protein and albumin, as well as the increase in total cells and neutrophils in BAL fluid. On day 21 candesartan cilexetil also ameliorated morphological changes and an increased amount of hydroxyproline in lung homogenates. In addition, BLM increased the expression of transforming growth factor (TGF)-beta1 in BAL fluid on day 7; this increase was significantly reduced by candesartan cilexetil.. AT1 expression is upregulated in fibrotic lungs. Angiotensin II promotes lung fibrosis via AT1 and, presumably, in part via TGF-beta1.

Topics: Albumins; Angiotensin II Type 1 Receptor Blockers; Animals; Antibiotics, Antineoplastic; Benzimidazoles; Biphenyl Compounds; Bleomycin; Blotting, Western; Bronchoalveolar Lavage Fluid; Hydroxyproline; Immunohistochemistry; Male; Proteins; Pulmonary Fibrosis; Rats; Rats, Sprague-Dawley; Receptor, Angiotensin, Type 1; Tetrazoles; Transforming Growth Factor beta; Transforming Growth Factor beta1

Topics: Humans; Interferon-gamma; Pulmonary Fibrosis; Recombinant Proteins; Transcription, Genetic; Transforming Growth Factor beta; Treatment Failure

The origin of fibroblasts in pulmonary fibrosis is assumed to be intrapulmonary, but their extrapulmonary origin and especially derivation from bone marrow (BM) progenitor cells has not been ruled out. To examine this possibility directly, adult mice were durably engrafted with BM isolated from transgenic mice expressing enhanced GFP. Induction of pulmonary fibrosis in such chimera mice by endotracheal bleomycin (BLM) injection caused large numbers of GFP(+) cells to appear in active fibrotic lesions, while only a few GFP(+) cells could be identified in control lungs. Flow-cytometric analysis of lung cells confirmed the BLM-induced increase in GFP(+) cells in chimera mice and revealed a significant increase in GFP(+) cells that also express type I collagen. GFP(+) lung fibroblasts isolated from chimera mice expressed collagen and telomerase reverse transcriptase but not alpha-smooth muscle actin. Treatment of isolated GFP(+) fibroblasts with TGF-beta failed to induce myofibroblast differentiation. Cultured lung fibroblasts expressed the chemokine receptors CXCR4 and CCR7 and responded chemotactically to their cognate ligands, stromal cell-derived factor-1 alpha and secondary lymphoid chemokine, respectively. Thus the collagen-producing lung fibroblasts in pulmonary fibrosis can also be derived from BM progenitor cells.

Topics: Actins; Animals; Antimetabolites, Antineoplastic; Bleomycin; Bone Marrow Cells; Bone Marrow Transplantation; Cell Differentiation; Chemokine CXCL12; Chemokines; Chemokines, CXC; Collagen; Fibroblasts; Fibrosis; Flow Cytometry; Green Fluorescent Proteins; Immunohistochemistry; Ligands; Luminescent Proteins; Lung; Mice; Muscle, Smooth; Phenotype; Polymerase Chain Reaction; Pulmonary Fibrosis; Receptors, CCR7; Receptors, Chemokine; Receptors, CXCR4; RNA; Spleen; Stem Cells; Time Factors; Transforming Growth Factor beta

Precision-cut rat lung slices have been employed in combination with an extensive immunohistochemistry of paraffin-embedded slices for monitoring of early pathohistological changes after exposure to CdCl(2)/TGF-beta(1). Three days of CdCl(2) exposure in combination with TGF-beta(1) seem to be sufficient to induce lung injury with alterations similar to changes observed in early lung fibrogenesis: (1) extracellular matrix accumulation and myofibroblast transdifferentiation (Sirius red staining, collagen type IV, alpha-smooth muscle actin), (2) type I cell injury with loss of type I cell antigens (T1alpha antigen, aquaporin-5, RAGE), (3) increased apoptosis of pulmonary cells (active caspase-3, vimentin cleavage product V1 of caspase-9), and (4) activation of microvascular endothelial cells (podocalyxin, caveolin-1). Western blot analysis confirmed the increasing amount of alpha-smooth muscle actin, the loss of T1alpha antigen, and the increase in caveolin-1 immunoreactivity. The explant culture using CdCl(2)/TGF-beta(1) provides a suitable tool for the study of other factors involved in pulmonary pathology including transcription factors, cytokines, and other metabolites involved in early stages of fibrogenesis.

Topics: Actins; Animals; Apoptosis; Biomarkers; Cadmium Chloride; Caveolin 1; Caveolins; Disease Models, Animal; Drug Combinations; Immunohistochemistry; Lung; Membrane Glycoproteins; Membrane Proteins; Organ Culture Techniques; Pulmonary Fibrosis; Rats; Rats, Wistar; Transforming Growth Factor beta; Transforming Growth Factor beta1

To determine the role of IL-5 in airway remodeling, IL-5-deficient and WT mice were sensitized to OVA and challenged by repetitive administration of OVA for 3 months. IL-5-deficient mice had significantly less peribronchial fibrosis (total lung collagen content, peribronchial collagens III and V) and significantly less peribronchial smooth muscle (thickness of peribronchial smooth muscle layer, alpha-smooth muscle actin immunostaining) compared with WT mice challenged with OVA. WT mice had a significant increase in the number of peribronchial cells staining positive for major basic protein and TGF-beta. In contrast, IL-5-deficient mice had a significant reduction in the number of peribronchial cells staining positive for major basic protein, which was paralleled by a similar reduction in the number of cells staining positive for TGF-beta, suggesting that eosinophils are a significant source of TGF-beta in the remodeled airway. OVA challenge induced significantly higher levels of airway epithelial alphaVbeta6 integrin expression, as well as significantly higher levels of bioactive lung TGF-beta in WT compared with IL-5-deficient mice. Increased airway epithelial expression of alphaVbeta6 integrin may contribute to the increased activation of latent TGF-beta. These results suggest an important role for IL-5, eosinophils, alphaVbeta6, and TGF-beta in airway remodeling.

Topics: Animals; Antigens, Neoplasm; Bronchial Provocation Tests; Bronchoalveolar Lavage Fluid; Eosinophils; Humans; Integrins; Interleukin-5; Mice; Mice, Knockout; Ovalbumin; Pulmonary Fibrosis; Respiratory Mucosa; Respiratory System; Transforming Growth Factor beta

We previously described a reduction of silica-induced lung fibrosis in interleukin-10-deficient mice (IL-10-/-) (Huaux and colleagues; Am. J. Respir. Cell Mol. Biol. 1998;18:51-59). In the present study, we further dissect the exact functions of IL-10 in experimental silicosis. The reduced lung fibrotic response to silica in IL-10-/- mice was accompanied by a marked recruitment of TH1 CD4+ lymphocytes. However, treatment with anti-CD4 antibodies reduced silica-induced lung fibrosis in both IL-10-/- and IL-10+/+ mice, suggesting that this T cell population actually contributes to the extension of the fibrotic lesions in a manner that is independent of IL-10. In IL-10-/- mice, silica-induced lung production of the profibrotic mediator transforming growth factor (TGF)-beta1 and the antifibrotic eicosanoid PGE2 were reduced and increased, respectively, relative to that in IL-10+/+ mice. In addition, in vitro experiments indicated that recombinant IL-10 upregulated TGF-beta1 expression in alveolar macrophages while in contrast it downregulated PGE2 production and cyclooxygenase-2 expression in both lung fibroblasts and macrophages. Thus the net profibrotic activity of IL-10 in vivo appears to be mediated by its ability to stimulate the expression of the profibrotic cytokine TGF-beta1 while suppressing the expression of cyclooxygenase-2 and thus production of the antifibrotic eicosanoid PGE2. These effects appear to be independent of the enhanced lung CD4+ T-lymphocytosis observed in IL-10-deficient mice.

Topics: Animals; Antibodies; CD4-Positive T-Lymphocytes; Chemotaxis, Leukocyte; Cyclooxygenase 2; Dinoprostone; Disease Models, Animal; Down-Regulation; Female; Fibroblasts; Interleukin-10; Isoenzymes; Lung; Macrophages; Mice; Mice, Inbred C57BL; Mice, Knockout; Prostaglandin-Endoperoxide Synthases; Pulmonary Fibrosis; Receptors, Interleukin; Receptors, Interleukin-10; RNA, Messenger; Silicon Dioxide; Th1 Cells; Transforming Growth Factor beta; Transforming Growth Factor beta1

Asthma is a chronic inflammatory disease characterized by variable bronchial obstruction, hyperresponsiveness, and by tissue damage known as airway remodeling. In the present study we demonstrate that interleukin (IL)-5 plays an obligatory role in the airway remodeling observed in experimental asthma. BALB/c mice sensitized by intraperitoneal injections of ovalbumin and exposed daily to aerosol of ovalbumin for up to 3 wk, develop eosinophilic infiltration of the bronchi and subepithelial and peribronchial fibrosis. The lesions are associated with increased amounts of hydroxyproline in the lungs and elevated levels of eosinophils and transforming growth factor (TGF)-beta1 in the bronchoalveolar lavage fluid. After 1 wk of allergen challenge, TGF-beta is mainly produced by eosinophils accumulated in the peribronchial and perivascular lesions. At a later stage of the disease, the main source of TGF-beta is myofibroblasts, identified by alpha-smooth muscle actin mAb. We show that all these lesions, including fibrosis, are abolished in sensitized and allergen-exposed IL-5 receptor-null mice, whereas they are markedly accentuated in IL-5 transgenic animals. More importantly, treatment of wild-type mice with neutralizing anti-IL-5 antibody, administered before each allergen challenge, almost completely prevented subepithelial and peribronchial fibrosis. These findings demonstrated that eosinophils are involved in allergen-induced subepithelial and peribronchial fibrosis probably by producing a fibrogenic factor, TGF-beta1.

Topics: Actins; Allergens; Animals; Antibodies; Asthma; Bronchi; Chemotaxis, Leukocyte; Collagen; Disease Models, Animal; Disease Progression; Eosinophils; Female; Fibroblasts; Hydroxyproline; Interleukin-5; Mice; Mice, Knockout; Ovalbumin; Pulmonary Fibrosis; Receptors, Interleukin; Receptors, Interleukin-5; Transforming Growth Factor beta; Transforming Growth Factor beta1; Up-Regulation

Osteopontin is a multifunctional matricellular protein abundantly expressed during inflammation and repair. Osteopontin deficiency is associated with abnormal wound repair characterized by aberrant collagen fibrillogenesis in the heart and skin. Recent gene microarray studies found that osteopontin is abundantly expressed in both human and mouse lung fibrosis. Macrophages and T cells are known to be major sources of osteopontin. During lung fibrosis, however, osteopontin expression continues to increase when inflammation has receded, suggesting alternative sources of ostepontin during this response. In this study, we demonstrate immunoreactivity for osteopontin in lung epithelial and inflammatory cells in human usual interstitial pneumonitis and murine bleomycin-induced lung fibrosis. After treatment with bleomycin, osteopontin-null mice develop lung fibrosis characterized by dilated distal air spaces and reduced type I collagen expression compared with wild-type controls. There is also a significant decrease in levels of active transforming growth factor-beta(1) and matrix metalloproteinase-2 in osteopontin null mice. Type III collagen expression and total collagenase activity are similar in both groups. These results demonstrate that osteopontin expression is associated with important fibrogenic signals in the lung and that the epithelium may be an important source of osteopontin during lung fibrosis.

Topics: Animals; Antibiotics, Antineoplastic; Bleomycin; Collagen Type I; Female; Gene Expression; Male; Matrix Metalloproteinase 2; Mice; Mice, Mutant Strains; Osteopontin; Pneumonia; Pulmonary Fibrosis; Sialoglycoproteins; Signal Transduction; Transforming Growth Factor beta; Transforming Growth Factor beta1

Pulmonary fibrosis is associated with a poor prognosis. The pathogenesis of fibrotic lung disorders remains unclear, but the extent of tissue damage due to the persistent presence of oxidants or proteases is believed to be important. The heme degrading enzyme heme oxygenase (HO) has been found to be expressed in experimental fibrosis, and generation of free iron and carbon monoxide (CO) by HO has been implicated in oxidant induced lung damage. A study was undertaken to examine the effects of the HO inhibitor Zn-deuteroporphyrin-IX-2,4-bisethylene glycol (Zndtp) on the development of pulmonary fibrosis in the bleomycin model of lung injury and repair.. Zndtp (10 micro mol/kg) was administered subcutaneously twice daily to mice 1 week following the intratracheal instillation of 0.025 U bleomycin. Animals were killed 10 or 21 days after bleomycin instillation and indices of lung damage and fibrosis were evaluated.. Bleomycin treatment induced pulmonary cytotoxicity, increased levels of active transforming growth factor beta (TGF-beta), enhanced lung collagen accumulation, and decreased glutathione content. Zndtp administration significantly attenuated these indices.. Administration of Zndtp in the bleomycin model resulted in appreciable alveolar cytoprotection and amelioration of pulmonary fibrosis. This molecule and its analogues may warrant further consideration in the treatment of acute lung injury and fibrotic lung disorders.

Topics: Animals; Antimetabolites, Antineoplastic; Bilirubin; Bleomycin; Bronchoalveolar Lavage Fluid; Deuteroporphyrins; Glutathione; Heme Oxygenase (Decyclizing); L-Lactate Dehydrogenase; Leukocytes; Male; Malondialdehyde; Mice; Mice, Inbred C57BL; Pulmonary Fibrosis; Transforming Growth Factor beta

Pro- and anti-fibrotic cytokine gene polymorphisms may affect expression of idiopathic pulmonary fibrosis (IPF). The aims of the present case-control study were to examine polymorphisms in the IL-6, transforming growth factor (TGF)-beta 1, tumour necrosis factor (TNF)-alpha and interleukin-1 (IL-1)Ra genes in patients with IPF (n = 22) -compared to healthy controls (n = 140). Genotyping was performed on DNA extracted from peripheral blood lymphocytes, using polymerase chain reaction - restriction fragment length polymorphism with gene polymorphisms determined according to -published techniques. The following sites were examined: (i) IL-1Ra*1-5 (86 bp variable tandem repeat intron 2), (ii) IL-6 (-174G > C), (iii) TNF-alpha (-308G > A) and (iv) TGF-beta 1 (Arg25Pro). The TNF-alpha (-308 A) allele was over-represented in the IPF (p(corr) = 0.004) group compared to controls. Risk of IPF was significant for heterozygotes for: (i) the TNF-alpha (-308 A) allele (A/G) (odds ratio (OR) 2.9; 95% confidence interval (CI) 1.2-7.2; P = 0.02), (ii) homozygotes (A/A) (OR 13.9; 95%CI 1.2-160; P = 0.04) and (iii) carriage of the allele (A/A + A/G) (OR 4; 95%CI 1.6-10.2; P = 0.003). The distribution of alleles and genotypes for IL-6, TGF-beta 1 and IL-1Ra between the two groups was not significantly different. This is the third study to independently confirm that there is a significant association of the TNF-alpha (-308 A) allele with IPF. Further research is needed to assess the utility of cytokine gene polymorphisms as markers of disease -susceptibility.

Topics: Aged; Case-Control Studies; Cytokines; Female; Genetic Predisposition to Disease; Genotype; Humans; Interleukin 1 Receptor Antagonist Protein; Interleukin-1; Interleukin-6; Male; Middle Aged; Polymorphism, Genetic; Pulmonary Fibrosis; Queensland; Sialoglycoproteins; Transforming Growth Factor beta; Transforming Growth Factor beta1; Tumor Necrosis Factor-alpha; White People

Intratracheal injection of FITC results in acute lung injury and progresses to fibrosis by day 21 postchallenge. In response to FITC, BALB/c mice produce IL-4 and IL-13 in the lung. To investigate whether IL-4 and/or IL-13 were important profibrotic mediators in this model, we examined the fibrotic response to FITC in mice that were genetically deficient in IL-4 (IL-4(-/-)), IL-13 (IL-13(-/-)), or IL-4 and IL-13 combined (IL-4/13(-/-)). Baseline levels of collagen were similar in all mice. In response to FITC, both BALB/c and IL-4(-/-) mice developed fibrosis, whereas the IL-13(-/-) and IL-4/13(-/-) mice were significantly protected, as measured by total lung collagen levels and histology. Total leukocyte recruitment to the lung was similar in all four strains of mice when measured on days 7, 14, and 21 post-FITC. BALB/c mice showed prominent eosinophilia on day 7 that was absent in IL-4(-/-), IL-13(-/-), and IL-4/13(-/-) mice, suggesting that eosinophilia is not necessary for development of a fibrotic response. There were no significant differences in the percentages of any other leukocytes analyzed between the genotypes. Similarly, protection in IL-13(-/-) mice was not associated with alterations in cytokine or eicosanoid profiles. Interestingly, TGF-beta1 production was not reduced in IL-13(-/-) mice. Analyses of fibroblasts isolated from the four genotypes demonstrated that although there were similar numbers of fibroblasts present in cultures of lung minces, fibroblasts from IL-13-deficient strains have reduced basal and stimulated levels of collagen production. IL-13Ralpha1 expression increases on fibroblasts during fibrotic responses in vivo, and IL-13 increases collagen synthesis in fibroblasts. Thus, IL-13 mediates its profibrotic actions through direct effects on fibroblast production of extracellular matrix.

Topics: Animals; Cell Count; Collagen; Cytokines; Down-Regulation; Fibroblasts; Fluorescein-5-isothiocyanate; Genetic Predisposition to Disease; Genotype; Inflammation; Interleukin-13; Interleukin-13 Receptor alpha1 Subunit; Interleukin-4; Mice; Mice, Inbred BALB C; Mice, Knockout; Pulmonary Eosinophilia; Pulmonary Fibrosis; Receptors, Interleukin; Receptors, Interleukin-13; Transforming Growth Factor beta; Transforming Growth Factor beta1

Pigment epithelium-derived factor (PEDF) is a 50-kD protein with angiostatic and neurotrophic activities that regulates vascular development within the eye. PEDF expression was increased in the lungs of patients with idiopathic pulmonary fibrosis (IPF) based on microarray analyses. Angiogenesis has been implicated in the pathogenesis of fibrotic lung diseases, we therefore hypothesized that regional abnormalities in vascularization occur in IPF as a result of an imbalance between PEDF and vascular endothelial growth factor. We demonstrated that vascular density is regionally decreased in IPF within the fibroblastic foci, and that within these areas PEDF was increased, whereas vascular endothelial growth factor was decreased. PEDF colocalized with the fibrogenic cytokine, transforming growth factor (TGF)-beta 1, particularly within the fibrotic interstitium and the fibroblastic focus, and prominently within the epithelium directly overlying the fibroblastic focus. This suggested that TGF-beta 1 might regulate PEDF expression. Using 3T3-L1 fibroblasts and human lung fibroblasts, we showed that PEDF was indeed a TGF-beta 1 target gene. Collectively, our findings implicate PEDF as a regulator of pulmonary angiogenesis and an important mediator in IPF.

Topics: Biopsy; Bronchoalveolar Lavage Fluid; Eye Proteins; Fibroblasts; Humans; Lung; Lung Diseases, Interstitial; Neovascularization, Pathologic; Nerve Growth Factors; Proteins; Pulmonary Fibrosis; Serpins; Transforming Growth Factor beta; Transforming Growth Factor beta1; Vascular Endothelial Growth Factor A

Transforming growth factor-beta 1 plays a key role in the pathogenesis of pulmonary fibrosis, mediating extracellular matrix (ECM) gene expression through a series of intracellular signaling molecules, including Smad2 and Smad3. We show that Smad3 null mice (knockout (KO)) develop progressive age-related increases in the size of alveolar spaces, associated with high spontaneous presence of matrix metalloproteinases (MMP-9 and MMP-12) in the lung. Moreover, transient overexpression of active TGF-beta 1 in lungs, using adenoviral vector-mediated gene transfer, resulted in progressive pulmonary fibrosis in wild-type mice, whereas no fibrosis was seen in the lungs of Smad3 KO mice up to 28 days. Significantly higher levels of matrix components (procollagen 3A1, connective tissue growth factor) and antiproteinases (plasminogen activator inhibitor-1, tissue inhibitor of metalloproteinase-1) were detected in wild-type lungs 4 days after TGF-beta 1 administration, while no such changes were seen in KO lungs. These data suggest a pivotal role of the Smad3 pathway in ECM metabolism. Basal activity of the pathway is required to maintain alveolar integrity and ECM homeostasis, but excessive signaling through the pathway results in fibrosis characterized by inhibited degradation and enhanced ECM deposition. The Smad3 pathway is involved in pathogenic mechanisms mediating tissue destruction (lack of repair) and fibrogenesis (excessive repair).

Topics: Animals; Cells, Cultured; Collagen; Crosses, Genetic; DNA-Binding Proteins; Extracellular Matrix; Extracellular Matrix Proteins; Fibroblasts; Fibrosis; Gene Expression Regulation; Immunity, Innate; Lung; Matrix Metalloproteinases; Mice; Mice, Inbred C57BL; Mice, Knockout; Pulmonary Alveoli; Pulmonary Emphysema; Pulmonary Fibrosis; Recombinant Fusion Proteins; Signal Transduction; Smad3 Protein; Specific Pathogen-Free Organisms; Tissue Inhibitor of Metalloproteinases; Trans-Activators; Transforming Growth Factor beta; Transforming Growth Factor beta1

Distinct subpopulations of fibroblasts contribute to lung fibrosis, although the mechanisms underlying fibrogenesis in these subpopulations are not clear. Differential expression of the glycophosphatidylinositol-linked protein Thy-1 affects proliferation and myofibroblast differentiation. Lung fibroblast populations selected on the basis of Thy-1 expression by cell sorting were examined for responses to fibrogenic stimuli. Thy-1 (-) and Thy-1 (+) fibroblast populations were treated with platelet-derived growth factor-BB, interleukin-1beta, interleukin-4, or bleomycin and assessed for activation of transforming growth factor (TGF)-beta, Smad3 phosphorylation, and alpha-smooth muscle actin and fibronectin expression. Thy-1 (-) fibroblasts responded to these stimuli with increased TGF-beta activity, Smad3 phosphorylation, and expression of alpha-smooth muscle actin and fibronectin, whereas Thy-1 (+) fibroblasts resisted stimulation. The unresponsiveness of Thy-1 (+) cells is not because of defective TGF-beta signaling because both subsets respond to exogenous active TGF-beta. Rather, Thy-1 (-) fibroblasts activate latent TGF-beta in response to fibrogenic stimuli, whereas Thy-1 (+) cells fail to do so. Defective activation is common to multiple mechanisms of TGF-beta activation, including thrombospondin 1, matrix metalloproteinase, or plasmin. Thy-1 (-) lung fibroblasts transfected with Thy-1 also become resistant to fibrogenic stimulation, indicating that Thy-1 is a critical biological response modifier that protects against fibrotic progression by controlling TGF-beta activation. These studies provide a molecular basis for understanding the differential roles of fibroblast subpopulations in fibrotic lung disease through control of latent TGF-beta activation.

Topics: Actins; Animals; Anticoagulants; Antineoplastic Agents; Becaplermin; Bleomycin; Cells, Cultured; DNA-Binding Proteins; Fibroblasts; Fibronectins; Gene Expression Regulation; Interleukin-1; Interleukin-4; Lung; Muscle, Smooth; Phosphorylation; Platelet-Derived Growth Factor; Proto-Oncogene Proteins c-sis; Pulmonary Fibrosis; Rats; Rats, Inbred Lew; Recombinant Proteins; Signal Transduction; Smad3 Protein; Thy-1 Antigens; Trans-Activators; Transforming Growth Factor beta

Pulmonary fibrosis is a severe complication associated with bis-chloronitrosourea (BCNU) therapy. However, the pathogenetic mechanism has never been well investigated. We report here a 26-year-old female with diffuse large B-cell lymphoma who died of severe pulmonary fibrosis 81 days after the administration of high-dose BCNU (600 mg/m2). Thoracoscopic wedge resection of left upper lung performed 10 days before patient's death showed severe pulmonary fibrosis with prominent hyperplasia of alveolar macrophages and type II pneumocytes. We further used immunohistochemistry (IHC) to examine the relative role of platelet-derived growth factor-B (PDGF-B), insulin-like growth factor I (IGF-I), transforming growth factor-beta1 (TGF-beta1) and cyclooxygenase-2 (COX-2) in the pathogenesis of BCNU-related pulmonary fibrosis. Strong expressions of PDGF-B and IGF-1 on alveolar macrophages and type II pneumocytes were clearly demonstrated, but in contrast, the expressions of TGF-beta1 and COX-2 were almost undetectable. In conclusion, pulmonary fibrosis can develop early and progress rapidly after the administration of high-dose BCNU. The markedly increased expression of fibrogenic factors PDGF-B and IGF-1 on hyperplastic alveolar macrophages and hyperplastic type II pneumocytes may play an important role in the fibrogenesis of this disease. These novel findings may offer specific therapeutic targets in the treatment of BCNU-associated pulmonary fibrosis.

Topics: Adult; Antineoplastic Agents, Alkylating; Carmustine; Cyclooxygenase 2; Fatal Outcome; Female; Humans; Insulin-Like Growth Factor I; Isoenzymes; Lung; Lymphoma, B-Cell; Lymphoma, Large B-Cell, Diffuse; Membrane Proteins; Prostaglandin-Endoperoxide Synthases; Proto-Oncogene Proteins c-sis; Pulmonary Fibrosis; Transforming Growth Factor beta; Transforming Growth Factor beta1

Fibrosis and apoptosis are juxtaposed in pulmonary disorders such as asthma and the interstitial diseases, and transforming growth factor (TGF)-beta(1) has been implicated in the pathogenesis of these responses. However, the in vivo effector functions of TGF-beta(1) in the lung and its roles in the pathogenesis of these responses are not completely understood. In addition, the relationships between apoptosis and other TGF-beta(1)-induced responses have not been defined. To address these issues, we targeted bioactive TGF-beta(1) to the murine lung using a novel externally regulatable, triple transgenic system. TGF-beta(1) produced a transient wave of epithelial apoptosis that was followed by mononuclear-rich inflammation, tissue fibrosis, myofibroblast and myocyte hyperplasia, and septal rupture with honeycombing. Studies of these mice highlighted the reversibility of this fibrotic response. They also demonstrated that a null mutation of early growth response gene (Egr)-1 or caspase inhibition blocked TGF-beta(1)-induced apoptosis. Interestingly, both interventions markedly ameliorated TGF-beta(1)-induced fibrosis and alveolar remodeling. These studies illustrate the complex effects of TGF-beta(1) in vivo and define the critical role of Egr-1 in the TGF-beta(1) phenotype. They also demonstrate that Egr-1-mediated apoptosis is a prerequisite for TGF-beta(1)-induced fibrosis and remodeling.

Topics: Actins; Animals; Apoptosis; DNA-Binding Proteins; Early Growth Response Protein 1; Immediate-Early Proteins; Mice; Mice, Inbred C57BL; Mice, Transgenic; Pulmonary Fibrosis; Transcription Factors; Transforming Growth Factor beta; Transforming Growth Factor beta1

The aim of this study was to investigate whether transforming growth factor-beta1 (TGF-beta1) could induce alveolar epithelial to mesenchymal transition (EMT) in vitro. Alveolar epithelial cells (AECs) from SD rats were isolated by elastase cell dispersion and IgG panning. Expression of alpha-smooth muscle actin (alpha-SMA) was assayed using Western blotting and immunostaining analysis. Morphological changes, the markers of epithelial cell (E-cadherin), and stress fiber by actin reorganization were detected by an indirect immunostaining. The contents of collagen I were determined by spectrophotometry. The levels of endogenous TGF-beta1 were measured with ELISA. Incubation of AECs with TGF-beta1 (0.1 approximately 10 ng/mL) induced abundant expression of alpha-SMA protein, and alpha-SMA expression in AECs reached a plateau when TGF-beta1 was > 3 ng/mL. Furthermore, we found that TGF-beta1 (3 ng/mL) exposure of AECs induced an authentic EMT characterized by abundant expression of alpha-smooth muscle actin, transformation of myofibroblastic morphology, increased formation of stress fiber by actin reorganization, and loss of epithelial marker E-cadherin. Meanwhile, significant increase in the levels of collagen I from 32.0 +/- 6.6 mg/g in control to 98 +/- 10.8 mg/g in TGF-beta1-treated group was found over a 72 h incubation period. Moreover, following stimulated by TGF-beta1 (3 ng/mL), a marked and time-dependent increase in endogenous TGF-beta1 released from AECs was observed. At time points 72 h, TGF-beta1 release mounted to 3451 pg/ml, which was much enough to induce EMT in vitro. These results demonstrated that AECs, under stimulation of TGF-beta1, underwent a conversion process into myofibroblasts in vitro.

Topics: Actins; Animals; Cadherins; Cells, Cultured; Collagen Type I; Enzyme-Linked Immunosorbent Assay; Epithelium; Immunoblotting; Immunohistochemistry; In Vitro Techniques; Male; Mesoderm; Muscle, Smooth; Pulmonary Alveoli; Pulmonary Fibrosis; Rats; Rats, Sprague-Dawley; Spectrophotometry; Time Factors; Transforming Growth Factor beta

To observe Smads protein expression in lung tissue of quartz exposed mice and to explore its association with pulmonary fibrosis in silicosis.. The experimental mice were divided into control and quartz groups. 0.2 g/kg weight of quartz was injected intratracheally in quartz group. Samples were collected at the 1st, 3rd, 5th, 7th, 14th and 28th day after injection. Immunohistochemical methods with quantitative image analysis were used to assay the protein expression of transforming growth factor beta(1) (TGF-beta(1)), Smad 2/3, Smad 4, and Smad 7 protein levels. Protein expression level is presented by positive unit (PU).. Smad 2/3 protein expression increased from day 3, reaching its peak level in day 14 [(42.2 +/- 2.4) PU], and decreased gradually. The elevation of Smad 4 protein level began from day 5, and the highest degree came into day 14 [(40.0 +/- 1.8) PU], decreased thereafter. The expression of Smad 7 presented a decreasing tendency at the beginning and reaching the lowest level in day 14 [(33.5 +/- 3.3) PU]. It seemed to elevate in day 28, but was still lower than the controls. There were positive correlation between Smad 2/3, Smad 4 and TGF-beta(1) (r = 0.91, r = 0.71, respectively, P < 0.05) and also between Smad 2/3 and hydroxyproline contents of lung tissue (r = 0.85, P < 0.05) except Smad 7.. Smad protein may have certain association with pulmonary fibrosis in silicosis.

Topics: Animals; DNA-Binding Proteins; Lung; Male; Mice; Mice, Inbred Strains; Pulmonary Fibrosis; Quartz; Smad2 Protein; Smad3 Protein; Smad4 Protein; Smad7 Protein; Trans-Activators; Transforming Growth Factor beta

Transforming growth factor beta (TGFbeta), a multifunctional cytokine, plays a crucial role in the accumulation of extracellular matrix components in lung fibrosis, where lung fibroblasts are considered to play a major role. Even though the effects of TGFbeta on the gene expression of several proteins have been investigated in several lung fibroblast cell lines, the global pattern of response to this cytokine in adult lung fibroblasts is still unknown.. We used Affymetrix oligonucleotide microarrays U95v2, containing approximately 12,000 human genes, to study the transcriptional profile in response to a four hour treatment with TGFbeta in control lung fibroblasts and in fibroblasts from patients with idiopathic and scleroderma-associated pulmonary fibrosis. A combination of the Affymetrix change algorithm (Microarray Suite 5) and of analysis of variance models was used to identify TGFbeta-regulated genes. Additional criteria were an average up- or down- regulation of at least two fold.. Exposure of fibroblasts to TGFbeta had a profound impact on gene expression, resulting in regulation of 129 transcripts. We focused on genes not previously found to be regulated by TGFbeta in lung fibroblasts or other cell types, including nuclear co-repressor 2, SMAD specific E3 ubiquitin protein ligase 2 (SMURF2), bone morphogenetic protein 4, and angiotensin II receptor type 1 (AGTR1), and confirmed the microarray results by real time-PCR. Western Blotting confirmed induction at the protein level of AGTR1, the most highly induced gene in both control and fibrotic lung fibroblasts among genes encoding for signal transduction molecules. Upregulation of AGTR1 occurred through the MKK1/MKK2 signalling pathway. Immunohistochemical staining showed AGTR1 expression by lung fibroblasts in fibroblastic foci within biopsies of idiopathic pulmonary fibrosis.. This study identifies several novel TGFbeta targets in lung fibroblasts, and confirms with independent methods the induction of angiotensin II receptor type 1, underlining a potential role for angiotensin II receptor 1 antagonism in the treatment of lung fibrosis.

Topics: Cells, Cultured; Drug Delivery Systems; Fibroblasts; Gene Expression Profiling; Humans; Pulmonary Fibrosis; Receptor, Angiotensin, Type 1; Transforming Growth Factor beta

Reactive perforating collagenosis (RPC) is a rare disorder characterized by the transepidermal elimination of altered collagen. The inherited form of RPC begins in early childhood, but acquired reactive perforating collagenosis (ARPC) begins in adult life. ARPC is associated with diabetes mellitus, renal disease, and malignancy. ARPC with lung fibrosis has not previously been reported in the literature, and the relationship between ARPC and lung fibrosis has not been studied. The etiological relationship between the two disorders appears to be uncertain. Although their association in this case could be due to chance, it may be due to the transforming growth factor beta abnormalities seen in both diseases. In this report, we describe a case of ARPC with lung fibrosis and propose an etiological association between the two diseases.

Topics: Collagen; Collagen Diseases; Elastic Tissue; Epidermis; Humans; Male; Middle Aged; Pulmonary Fibrosis; Skin Diseases, Papulosquamous; Transforming Growth Factor beta; Transforming Growth Factor beta1

This study compares the effect of nanosized silicon dioxide (nanosized SiO2) and microsized silicon dioxide (microsized SiO2) particles on fibrogenesis in rats. Wistar rats were instilled intratracheally with saline, 20 mg of nanosized SiO2 or 20 mg of microsized SiO2, and were sacrificed at 1 and 2 months after instillation. The lungs of rats were analysed for the changes of lung/body coefficient and hydroxyproline content. Changes in pathology and fibrotic grade were observed by use of hematoxylin and eosin and Van Gieson dyeing. The expression of interleukin-4 (IL-4) and transforming growth factor-beta1 (TGF-beta1) was observed by use of immunohistochemical technique, and protein expression quantitatively analysed by image analysis. The lung/body coefficient and hydroxyproline content of nanosized SiO2 groups were significantly lower than those of microsized SiO2 groups at both 1 and 2 months after instillation (P<0.05 or P<0.01), but without significant differences from those of saline control groups. At 1 month after instillation, there were mainly cellular nodules (Stage I) in nanosized SiO2 group, while in microsized SiO2 group Stage II, II+ of silicotic nodules were observed. At 2 months after instillation, there were still Stage I of silicotic nodules in nanosized SiO2 group. In microsized SiO2 group mainly Stage II+, III of silicotic nodules were found. Quantity image analysis showed that the expressions of IL-4 and TGF-beta1 in nanosized SiO2 groups were significantly lower than those in microsized SiO2 groups (P<0.01), but without significant difference from those of saline control groups. Our experiment revealed that the effect of fibrogenesis of nanosized SiO2 might be milder than that of microsized SiO2 in rats, potentially resulting from nanoparticals tending to be diffused and easily translocated due to their ultrafine particle size compared to microsized particles.

Topics: Animals; Female; Hydroxyproline; Image Processing, Computer-Assisted; Immunoenzyme Techniques; Inhalation Exposure; Interleukin-4; Intubation, Intratracheal; Male; Microchemistry; Nanostructures; Particle Size; Pulmonary Fibrosis; Rats; Rats, Wistar; Silicon Dioxide; Silicosis; Transforming Growth Factor beta; Transforming Growth Factor beta1

Amiodarone (AM) is an efficacious antidysrhythmic agent that can cause numerous adverse effects, including potentially life-threatening pulmonary fibrosis. The current study was undertaken to investigate potential protective mechanisms of vitamin E against AM-induced pulmonary toxicity (AIPT) in the hamster. Three weeks after intratracheal administration of AM (1.83 micromol), increased pulmonary hydroxyproline content and histological damage were observed, indicative of fibrosis. These effects were preceded by increased pulmonary levels of transforming growth factor (TGF)-beta1 mRNA at 1 week post-AM, which remained elevated 3 weeks post-AM. Dietary supplementation with vitamin E resulted in rapid pulmonary accumulation of the vitamin, and prevention of AM-induced increases in TGF-beta1, hydroxyproline, and histological damage. Although dietary supplementation also markedly elevated lung mitochondrial vitamin E content, it did not attenuate AM-induced inhibition of mitochondrial respiration or disruption of mitochondrial membrane potential in vitro, or lung mitochondrial respiratory inhibition resulting from in vivo AM administration. These results suggest that vitamin E reduces the extent of pulmonary damage after AM administration via down-regulating TGF-beta1 overexpression but that it does not modify AM-induced mitochondrial dysfunction, a potential initiating event in AIPT.

Topics: Amiodarone; Animals; Anti-Arrhythmia Agents; Antioxidants; Cricetinae; Diet; DNA Probes; Gene Expression; Hydroxyproline; In Situ Hybridization; Lung; Male; Membrane Potentials; Mesocricetus; Mitochondria; Oxygen Consumption; Polarography; Pulmonary Fibrosis; RNA, Messenger; Transforming Growth Factor beta; Transforming Growth Factor beta1; Vitamin E

Decorin, a small leucin-rich proteoglycan, is a negative regulator of transforming growth factor-beta, but the antifibrotic effect of decorin gene transfer has not been examined in a mouse model of usual interstitial pneumonia (UIP). We constructed a replication-defective recombinant adenovirus harboring human decorin gene (AdCMV.DC) and administered 1 x l0(9) plaque-forming units of AdCMV.DC intratracheally or intravenously to C57BL/6 mice with intraperitoneal injection of bleomycin, which induces a subpleural fibroproliferation, mimicking UIP, by day 28. Only intratracheal administration of AdCMV.DC increased decorin mRNA expression in the lung and decreased the hydroxyproline content augmented in bleomycin-induced pulmonary fibrosis (1.13 +/- 0.02 to 0.96 +/- 0.02, P = 0.006). In contrast, intravenous administration of AdCMV.DC increased the decorin expression only in the liver, but not in the lung, and without reducing lung fibrosis. These results indicate that adenoviral decorin gene transfer is effective only by direct administration to fibrosing lungs.

Topics: Adenoviridae; Animals; Antimetabolites, Antineoplastic; Bleomycin; Cell Line; Decorin; Disease Models, Animal; Extracellular Matrix Proteins; Female; Gene Transfer Techniques; Genes, Reporter; Genetic Therapy; Genetic Vectors; Humans; Injections, Intravenous; Instillation, Drug; Liver; Lung; Lung Diseases, Interstitial; Mice; Mink; Proteoglycans; Pulmonary Fibrosis; RNA, Messenger; Trachea; Transfection; Transforming Growth Factor beta

Idiopathic pulmonary fibrosis (IPF) is a progressive fatal fibrotic lung disease. Transforming growth factor (TGF)-beta1 is present in a biologically active conformation in the epithelial cells lining lesions with advanced IPF. To determine the role of aberrant expression of biologically active TGF-beta1 by alveolar epithelial cells (AECs), the AECs of explanted normal rat lungs were transfected with the TGF-beta1 gene using the retrovirus pMX-L-s223,225-TGF-beta1. In situ hybridization using a digoxigenin-labeled cDNA of the puromycin resistance gene contained in the pMX demonstrated that pMX-L-s233,225-TGF-beta1 was selectively transfected into AECs of the explants. Conditioned media overlying explants obtained 7 days after being treated with pMX-L-s223,225-TGF-beta1 contained 14.5 +/- 3.15 pg/ml of active TGF-beta1. With the use of Masson's trichrome staining of explant sections obtained 14 days after transfection, there were lesions similar to those in IPF, characterized by type II AEC hyperplasia, interstitial thickening, extensive increase in interstitial and subepithelial collagen, an increase in the number of fibroblasts, and areas resembling fibroblast buds. Collagens I, III, IV, and V and fibronectin were increased in explants treated with pMX-L-s223,225-TGF-beta1. The findings in the current study suggest that IPF may be a disorder of epithelial cells and not inflammatory cells.

Topics: Animals; Blotting, Western; Cells, Cultured; Collagen Type I; Collagen Type III; Collagen Type IV; Collagen Type V; Connective Tissue Growth Factor; Culture Media, Conditioned; DNA-Binding Proteins; Female; Fibroblasts; Fibronectins; Immediate-Early Proteins; Intercellular Signaling Peptides and Proteins; Phosphorylation; Pulmonary Alveoli; Pulmonary Fibrosis; Rats; Rats, Sprague-Dawley; Respiratory Mucosa; Smad2 Protein; Trans-Activators; Transfection; Transforming Growth Factor beta; Transforming Growth Factor beta1

During pulmonary development, Sonic hedgehog (Shh) and transforming growth factor beta1 (TGF-beta1) signalling both contribute to branching morphogenesis. In interstitial lung disease, the complex alveolar structure of the lung is disrupted and remodelled, which leads to fibrosis, loss of respiratory surface, morbidity, and mortality. It is well documented that TGF-beta1 is involved in fibrosis. However, little is known about Shh signalling in damaged epithelia. This study examined whether or not components of the Shh signalling pathway, as well as TGF-beta1, are expressed in human fibrotic lung disease (cryptogenic fibrosing alveolitis and bronchiectasis) and in murine experimental models of fibrotic and non-fibrotic chronic pulmonary inflammation. Using immunohistochemistry, it was observed that Shh, like TGF-beta1, is up-regulated in epithelial cells at sites of fibrotic disease but not non-fibrotic inflammation. The Shh receptor patched was detected in infiltrating mononuclear cells and alveolar macrophages, as well as normal resting peripheral blood T lymphocytes. Neither Shh nor patched is expressed by hyperproliferative goblet cells in inflammatory epithelium. This study demonstrates that patched is present in human peripheral CD4 and CD8 lymphocytes at both protein and mRNA levels. Taken together, these results suggest that components of the highly conserved Shh signalling pathway may play a role in the remodelling of damaged pulmonary epithelium and that damaged epithelium and cells of the immune system may communicate via this pathway.

Topics: Animals; Antigens, Dermatophagoides; Arthropod Proteins; Bronchiectasis; Chronic Disease; Cysteine Endopeptidases; Female; Hedgehog Proteins; Humans; Intracellular Signaling Peptides and Proteins; Membrane Proteins; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Patched Receptors; Patched-1 Receptor; Pneumonia; Pulmonary Fibrosis; Receptors, Cell Surface; Respiratory Hypersensitivity; RNA, Messenger; Signal Transduction; T-Lymphocyte Subsets; Trans-Activators; Transforming Growth Factor beta; Transforming Growth Factor beta1; Up-Regulation

Transforming growth factor-beta1 (TGF-beta1) is a cytokine that plays a key role in the development of idiopathic pulmonary fibrosis. There have been reports on the presence of two genetic polymorphisms in the DNA sequence encoding the leader sequence of the TGF-beta1 protein, located in codons 10 and 25. The objective of this study was to investigate the association between TGF-beta1 gene polymorphisms in codons 10 and 25 and the susceptibility to idiopathic pulmonary fibrosis and the progression of the disease. Compared with healthy control subjects (n = 140), patients with idiopathic pulmonary fibrosis (n = 128) showed no significant deviations in genotype or allele frequencies. One hundred and ten patients with idiopathic pulmonary fibrosis were followed up for 30.3 +/- 25 months. The presence of a proline allele at codon 10 was independently associated with a significant increase in alveolar arterial oxygen tension difference during follow-up, after controlling for the effect of treatment (coefficient = 0.59; 95% confidence intervals, 0.23 to 0.96; p = 0.002). These findings suggest that (1) TGF-beta1 gene polymorphisms in codons 10 and 25 do not predispose to the development of idiopathic pulmonary fibrosis; and (2) TGF-beta1 gene polymorphisms may affect disease progression in patients with idiopathic pulmonary fibrosis.

Topics: Adult; Aged; Base Sequence; Chi-Square Distribution; Codon; Confidence Intervals; Disease Progression; Female; Follow-Up Studies; Gene Frequency; Genotype; Humans; Linear Models; Male; Oxygen; Polymorphism, Genetic; Proline; Pulmonary Alveoli; Pulmonary Fibrosis; Transforming Growth Factor beta; Transforming Growth Factor beta1

Topics: Allergens; Animals; Asthma; Disease Models, Animal; Mast Cells; Mice; Pulmonary Fibrosis; Transforming Growth Factor beta

Pulmonary toxicity, including fibrosis, is a serious adverse effect associated with the antidysrhythmic drug amiodarone (AM). We tested the potential usefulness of pirfenidone against AM-induced pulmonary toxicity in the hamster model. Intratracheal AM administration resulted in pulmonary fibrosis 21 days posttreatment, as evidenced by an increased hydroxyproline content and histological damage. Dietary pirfenidone administration (0.5% w/w in chow), for 3 days prior to and continuously after AM, prevented fibrosis and suppressed elevation of pulmonary transforming growth factor (TGF)-beta1 mRNA content at 7 and 21 days post-AM. Protection against AM-induced lung damage was not observed when supplementation with pirfenidone was delayed until 7 days following AM administration, suggesting that alteration of early events in AM lung toxicity is necessary for the protective effect of pirfenidone. Both AM and bleomycin, another pulmonary fibrogen, caused inflammation 24 h after intratracheal dosing, measured as increased lactate dehydrogenase activity, protein content, and cellular alterations in bronchoalveolar lavage fluid, with the response to AM markedly greater than that to bleomycin. Administration of AM, but not bleomycin, also caused whole lung mitochondrial dysfunction, alveolar macrophage death, and an influx of eosinophils into the lung, of which pirfenidone was able to decrease only the latter. We conclude that: (1) AM induces alveolar macrophage death and severe, acute pulmonary inflammation with associated eosinophilia following intratracheal administration; (2) mitochondrial dysfunction may play an early role in AM pulmonary injury; and (3) pirfenidone decreases AM-induced pulmonary fibrosis in the hamster, probably through suppression of TGF-beta1 gene expression.

Topics: Acute Disease; Amiodarone; Animals; Anti-Arrhythmia Agents; Anti-Inflammatory Agents, Non-Steroidal; Bleomycin; Cricetinae; Disease Models, Animal; Hydroxyproline; Male; Membrane Potentials; Mitochondria; Oxygen Consumption; Pulmonary Fibrosis; Pyridones; RNA, Messenger; Time Factors; Transforming Growth Factor beta; Transforming Growth Factor beta1

To investigate the expression of platelet-derived growth factor(PDGF) and transforming growth factor-beta (TGF-beta) in transbronchial lung biopsy (TBLB) from patients with idiopathic pulmonary fibrosis(IPF), and study the potential role of cytokines in the development of IPF.. The immunohistochemical methods were used to determine the expression of PDGF, TGF-beta in TBLB from patients with IPF.. In IPF patients, TGF-beta mainly existed at tiny bronchial epithelial cells, alveolar epithelial type-II cells and alveolar macrophages, showing strong expression compared with controls (P<0.01). PDGF mainly existed at fibroblast-like cells surrounding pulmonary vessels, fibroblasts, tiny bronchial epithelial cells, alveolar epithelial type-II cells and alveolar macrophages, showing strong expression compared with controls (P<0.01).. PDGF and TGF-beta, which interact with pulmonary mesenchymal cells, are involved in the formation of pulmonary fibrosis.

Topics: Adult; Blood Vessels; Cytokines; Female; Humans; Immunohistochemistry; Lung; Male; Middle Aged; Platelet-Derived Growth Factor; Pulmonary Fibrosis; Transforming Growth Factor beta

Modulation of cytokine expression represents a potentially useful approach for the treatment of idiopathic pulmonary fibrosis (IPF). To identify potential targets for such intervention, semi-quantitative reverse transcriptase-polymerase chain reaction was used to compare the expression of messenger ribonucleic acids (mRNAs) coding for 17 cytokines in lung tissue obtained from patients with IPF at the time of diagnosis and control subjects. Some cytokines were also studied at the protein level by immunohistochemical techniques. mRNAs coding for all of the cytokines evaluated were detected in both control and fibrotic lung samples. Only transforming growth factor (TGF)-beta and interleukin (IL)-10 mRNAs were quantitatively increased in lung biopsies from patients with IPF compared with those of controls, results confirmed at the protein level by immunohistochemistry. Although mRNAs for platelet-derived growth factor (PDGF)-BB and keratinocyte growth factor (KGF) were expressed in similar amounts in lungs from patients with IPF and controls, localised accumulation of both factors was also observed in IPF. Hyperplastic alveolar epithelial cells were a prominent source of cytokines, where IL-10, PDGF-BB and KGF were present in increased amounts, although increased accumulation in fibroblasts, smooth-muscle cells and matrix components was also observed (PDGF-BB, TGF-beta). These results offer new insights into the cytokines produced in the lung in idiopathic pulmonary fibrosis and suggest that modulation of the production of transforming growth factor-beta and interleukin-10 may represent a potentially useful therapeutic strategy for this disabling disease.

Topics: Female; Humans; Immunohistochemistry; Interleukin-10; Lung; Male; Middle Aged; Pulmonary Fibrosis; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Transforming Growth Factor beta

In this study, we examined the sequential expression of several matrix metalloproteinases (MMPs), tissue inhibitors of metalloproteinases (TIMPs), and growth factors as well as the presence of apoptosis in a model of pulmonary fibrosis induced in rats with paraquat and hyperoxia. Animals showing neither clinical nor morphological changes with this double aggression were classified as "resistant". Rats were killed at 1, 2, 3, and 6 wk, and lungs were used for collagen content, gene expression by real-time PCR, gelatinolytic activity by zymography, apoptosis by in situ DNA fragmentation, and protein localization by immunohistochemistry. Our results showed a significant decrease of collagenases MMP-8 and MMP-13, with an increase of TIMP-1 and transforming growth factor-beta. Immunoreactive TIMP-1 was increased in experimental rats and primarily localized in alveolar macrophages. Expression of gelatinases MMP-2 and MMP-9 mRNAs was not affected, but lung zymography revealed an increase in progelatinase B, progelatinase A, and its active form. Epithelial apoptosis was evident from the first week, whereas at later periods, interstitial cell apoptosis was also noticed. Resistant animals behave as controls. These findings suggest that an imbalance between collagenases and TIMPs, excessive gelatinolytic activity, and epithelial apoptosis participate in the fibrotic response in this experimental model.

Topics: Animals; Apoptosis; Base Sequence; Biomarkers; Blotting, Northern; Collagenases; Disease Models, Animal; DNA Primers; Female; Gene Expression Regulation; Hydroxyproline; Male; Polymerase Chain Reaction; Pulmonary Fibrosis; Rats; Rats, Wistar; Respiratory Mucosa; Time Factors; Tissue Inhibitor of Metalloproteinase-1; Transforming Growth Factor beta

(1) We have studied whether curcumin prevents amiodarone-induced lung fibrosis in rats. Intratracheal instillation of amiodarone (6.25 mg kg(-1) on days 0 and 2, and then killed on day 3, day 5, week 1, week 3 and week 5 after amiodarone administration) induced increases in total protein and lactate dehydrogenase (LDH) activity on days 3 and 5 in bronchoalveolar lavage fluid (BALF). Total cell counts, alveolar macrophages, neutrophils and eosinophils recovered by BAL, and lung myeloperoxidase (MPO) activity were significantly higher in amiodarone rats. (2) Tumor necrosis factor-alpha (TNF-alpha) release after lipopolysaccharide (LPS) stimulation and superoxide anion generation after phorbol myristate acetate (PMA) stimulation were higher in the alveolar macrophages of amiodarone rats at 3 and 5 weeks postamiodarone instillation than in controls. Amiodarone also induced increases in transforming growth factor-beta1 (TGF-beta1) expression, collagen deposition, type I collagen expression and c-Jun protein in lungs. (3) Curcumin (200 mg kg(-1) body weight after first amiodarone instillation and daily thereafter for 5 weeks)-treated amiodarone rats had reduced levels of protein, LDH activity, total cell numbers and differential cell counts in BALF. LPS-stimulated TNF-alpha release and PMA-stimulated superoxide generation were significantly suppressed by curcumin. Furthermore, curcumin inhibited the increases in lung MPO activity, TGF-beta1 expression, lung hydroxyproline content, expression of type I collagen and c-Jun protein in amiodarone rats. Our results have important implications for the treatment of amiodarone-induced lung fibrosis.

Topics: Amiodarone; Animals; Bronchoalveolar Lavage Fluid; Collagen Type I; Curcumin; Dose-Response Relationship, Drug; Instillation, Drug; Intubation, Gastrointestinal; L-Lactate Dehydrogenase; Lipopolysaccharides; Lung; Macrophages, Alveolar; Male; Peroxidase; Proto-Oncogene Proteins c-jun; Pulmonary Fibrosis; Rats; Rats, Inbred F344; Superoxides; Time Factors; Trachea; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha; Up-Regulation

Pulmonary fibrosis is a major cause of death in scleroderma patients. Previous studies have shown an increase in CD8+ T cells in the lungs of scleroderma patients. In the present study, we sought to determine whether activated CD8+ T cells contribute to pulmonary fibrosis in scleroderma patients through the production and activation of profibrotic mediators.. CD8+ cells were isolated from bronchoalveolar lavage fluid obtained from 19 scleroderma patients and 7 healthy subjects. The phenotype of these cells was determined using DNA array technology. Expression of selected genes was confirmed in real-time polymerase chain reaction and enzyme-linked immunosorbent assay experiments.. Hierarchical clustering of gene expression profiles revealed 2 groups of subjects. Group 1 consisted of 11 patients (8 with and 3 without lung inflammation). Group 2 consisted of 15 subjects (7 healthy controls and 2 patients with and 6 without lung inflammation). Gene expression in group 1 indicated T cell activation, a type 2 phenotype, production of profibrotic factors and matrix metalloproteinases, and reduced activation-induced cell death. Increased expression of beta6 integrin messenger RNA by CD8+ T cells in group 1 suggested the possibility that these T cells might induce cell-contact-dependent activation of latent transforming growth factor beta (TGFbeta).. A subset of scleroderma patients at higher risk of progressive lung disease have activated, long-lived CD8+ T cells in their lungs that could promote fibrosis directly, through production of profibrotic factors such as interleukin-4 and oncostatin M, as well as indirectly, through activation of TGFbeta.

Topics: Adult; Bronchoalveolar Lavage Fluid; CD8-Positive T-Lymphocytes; Cell Adhesion Molecules; Cell Survival; Cluster Analysis; Ephrins; Extracellular Matrix; Female; Gene Expression; Humans; Integrins; Lung; Lymphocyte Activation; Male; Middle Aged; Oligonucleotide Array Sequence Analysis; Phenotype; Pulmonary Fibrosis; Scleroderma, Systemic; Transforming Growth Factor beta

Topics: Animals; Codon; Disease Models, Animal; Disease Progression; Exons; Humans; Polymorphism, Genetic; Proline; Pulmonary Fibrosis; Transforming Growth Factor beta; Transforming Growth Factor beta1

Subepithelial collagen and extracellular matrix protein deposition are important pathophysiological components of airway remodelling in chronic asthma. Animal models based on the local reaction to antigens show structural alterations in the airway submucosal region and provide important information regarding disease pathophysiology. We describe a murine model of peribronchial fibrosis using intratracheally instilled transforming growth factor (TGF)-beta(1) in BALB/C mice that facilitates a mechanistic approach to understanding the cellular and molecular pathways leading to airway fibrosis.. BALB/C mice were intratracheally instilled with either TGF-beta(1) or buffered saline. Airway fibrosis was assessed by light microscopy, hydroxyproline content, and polymerase chain reaction (PCR) for collagen I and III on microdissected airway samples. The lysyl oxidase inhibitor beta-aminoproprionitrile (BAPN) was administered to TGF-beta(1) treated mice to block airway collagen deposition. Airway hyperresponsiveness was also measured after treatment with TGF-beta(1).. During the 7 days after administration of TGF-beta(1) the mice developed increased subepithelial collagen which could be blocked by BAPN. Increased mRNAs for collagen types I and III were seen in microdissected airways 1 week after TGF-beta(1), and significantly increased total collagen was found in the airways 4 weeks after TGF-beta(1). A detectable increase in airway hyperreactivity occurred.. This new model should facilitate detailed study of airway remodelling that occurs in the absence of detectable cellular inflammation, and allow examination of the functional consequences of a major structural alteration in the conducting airways uncomplicated by inflammatory cell influx.

Topics: Aminopropionitrile; Animals; Bronchi; Bronchial Diseases; Bronchoalveolar Lavage Fluid; Collagen Type I; Collagen Type III; Immunohistochemistry; Mice; Mice, Inbred BALB C; Pulmonary Fibrosis; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Transforming Growth Factor beta; Transforming Growth Factor beta1

Topics: Animals; Connective Tissue Growth Factor; Humans; Immediate-Early Proteins; Intercellular Signaling Peptides and Proteins; Pulmonary Fibrosis; Rats; Transforming Growth Factor beta

The possible role of mast cell chymase in organ fibrosis was examined using a bleomycin-induced pulmonary fibrosis model in mice. Intratracheal injection of bleomycin to mice significantly increased not only hydroxyproline content but also chymase activity in the lung. Administration of a chymase inhibitor SUN C8077 (7-chloro-3-(3-amynophenyl) quinazoline-2, 4-dione methanesulfonate) dose-dependently reversed the bleomycin-induced increase in hydroxyproline content as well as chymase activity in the lung. Human chymase digested latent transforming growth factor-beta1 (TGF-beta1) to form mature TGF-beta1 in vitro, which was inhibited by SUN C8077. Human chymase, on the other hand, failed to stimulate DNA synthesis of human lung fibroblasts CCD-8Lu and LL97A. Taken together, it is suggested that mast cell chymase might participate in the pathogenesis of pulmonary fibrosis, and that the chymase-induced fibrosis might be mediated at least in part by TGF-beta1. Chymase inhibitor may be promising for treatment of pulmonary fibrosis in humans.

Topics: Animals; Antibiotics, Antineoplastic; Biotransformation; Bleomycin; Chymases; Dose-Response Relationship, Drug; Fibroblasts; Humans; Hydroxyproline; Lung; Male; Mast Cells; Mice; Mice, Inbred ICR; Mitogens; Pulmonary Fibrosis; Recombinant Proteins; Serine Endopeptidases; Transforming Growth Factor beta

The purpose of this study was to determine if radiation-induced lung injury is associated with prolonged oxidative stress, and whether chronic overexpression of extracellular superoxide dismutase (EC-SOD) in the lung of transgenic mice protects against radiation-induced lung injury.. Whole-lung radiation was delivered to EC-SOD overexpressing B6C3 transgenic (XRT-TG) mice and wild-type littermates (XRT-WT). Pulmonary function was assessed by breathing frequency. Right lung wet weight was used as a gross indicator of lung damage. Histopathology was used to assess collagen deposition and tissue fibrosis according to an established grading system. Immunohistochemistry was used to stain and quantify the number of macrophages. ELISA was used to measure activated TGF-beta1. Oxidative stress was assessed by measuring lipid oxidation products (malondialic acid) by HPLC.. Four of six XRT-WT mice required euthanasia at 15-19 weeks postradiation because of respiratory distress, whereas no XRT-TG mouse developed distress. All assessments of lung damage at 15-20 weeks postradiation were higher for XRT-WT mice compared with the XRT-TG mice, including breathing frequency (380 vs. 286 bpm, p

Topics: Animals; Collagen; Lipid Metabolism; Lung; Lung Diseases; Macrophages; Mice; Mice, Inbred C3H; Mice, Inbred C57BL; Mice, Transgenic; Organ Size; Oxidation-Reduction; Oxidative Stress; Pulmonary Fibrosis; Radiation Injuries; Respiration; Superoxide Dismutase; Transforming Growth Factor beta; Transforming Growth Factor beta1

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)-beta 1 (AdTGF beta 1(223/225)) 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-beta1 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 AdTGF beta 1(223/225), 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-beta 1 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-beta in this disorder.

Topics: Animals; Animals, Newborn; Bronchopulmonary Dysplasia; Disease Models, Animal; Gene Transfer Techniques; Humans; Infant, Newborn; Lung; Pulmonary Fibrosis; Rats; Rats, Sprague-Dawley; Transforming Growth Factor beta; Transforming Growth Factor beta1

To investigate the protein expression of transforming growth factor-beta(1) (TGF-beta(1)) in lung tissues of silica-treated mice.. The experimental mice were divided into control and silica groups. 0.2 g/kg body weight of silica was injected intratracheally in mice of silica group. Samples of lung tissue were collected 1, 3, 5, 7, 14 and 28 d after injection. The immunohistochemical method was used to analyze the protein expression of TGF-beta(1).. In control mice, the expression of TGF-beta(1) in lung tissue was slightly positive while it was markedly increased in silica-treated mice. The expression was significantly elevated from the 7th day to 14th day. The expression in alveolar macrophages reached the peak on the 5th day [(93.4% +/- 2.8%) vs (42.2% +/- 12.0%), P < 0.01].. TGF-beta(1) may play an important role in early development of silicosis.

Topics: Animals; Immunohistochemistry; Lung; Male; Mice; Pulmonary Fibrosis; Silicon Dioxide; Silicosis; Time Factors; Transforming Growth Factor beta

To investigate the gene expression of transforming growth factor-beta(1) (TGF-beta(1)) in lung tissues of silica-treated mice.. The experimental mice were divided into control and silica group. 0.2 g/kg body weight of silica was injected intratracheally in silica group. Samples of lung tissue were collected 1, 3, 5, 7, 14 and 28 d after injection. RT-PCR method was used to analyze the gene expression of TGF-beta(1) in lung tissue of silica-treated mice.. The expression of TGF-beta(1) gene in lung tissue elevated from the 3rd day (1.20 +/- 0.15) and the peak value was on the 7th day (1.74 +/- 0.19). Then the expression decreased from the 14th to 28th day. But there was still higher than control until the 28th day.. TGF-beta(1) may play an important role in silica-induced pulmonary fibrosis.

Topics: Animals; Gene Expression Regulation; Lung; Male; Mice; Pulmonary Fibrosis; Reverse Transcriptase Polymerase Chain Reaction; Silicon Dioxide; Time Factors; Transforming Growth Factor beta

Halofuginone, a coccidiostatic alkaloid, has anti-fibrotic properties, and may be useful as a therapeutic agent in lung fibrosis. To test this hypothesis we investigated the effect of halofuginone on bleomycin-induced lung fibrosis in Sprague-Dawley rats. Treatment groups included: (1) a single intratracheal (IT) instillation of 1.2U bleomycin, and intraperitoneal (IP) injection of halofuginone (0.5 mg/dose), every other day; (2) IT 1.2U bleomycin and IP distilled water (D.W.), every other day; (3) IT 0.8U bleomycin and daily IP halofuginone (0.5 mg/dose); (4) IT 0.8U bleomycin and daily IP D.W.; (5) IT saline and IP halofuginone, every other day; (6) IT saline and daily IP D.W.; (7) IT 0.625U bleomycin and oral halofuginone (10 mg/kg rodent lab chow); (8) IT 0.625U bleomycin and standard lab chow. Animals were studied 14 days after IT instillation. Lung injury was evaluated by total and differential cell count in bronchoalveolar lavage fluid, by a semi-quantitative morphological index of lung injury, and by biochemical analysis of lung hydroxyproline content. Overt signs of lung injury were apparent in bleomycin-treated rats by all measures. These changes were not affected by treatment with halofuginone, irrespective of the treatment regimen used. This study does not support the use of halofuginone to prevent or ameliorate lung fibrosis.

Topics: Angiogenesis Inhibitors; Animals; Antimetabolites, Antineoplastic; Bleomycin; Blotting, Northern; Bronchoalveolar Lavage Fluid; Cell Count; Collagen; Fibroblasts; Gene Expression Regulation; Hydroxyproline; Intubation, Intratracheal; Lung; Male; Piperidines; Pulmonary Fibrosis; Quinazolines; Quinazolinones; Rats; Rats, Sprague-Dawley; RNA; Transforming Growth Factor beta

Topics: Animals; Extracellular Matrix; Homeostasis; Humans; Hyaluronan Receptors; Mice; Mice, Inbred Strains; Protease Inhibitors; Pulmonary Fibrosis; Tissue Inhibitor of Metalloproteinases; Transforming Growth Factor beta; Transforming Growth Factor beta1

Pulmonary fibrosis is characterized by excessive deposition of extracellular matrix in the interstitium, resulting in impaired lung function and respiratory failure. Investigation of the differences in individual susceptibility to the development of fibrosis may help to detect patients that are at risk to fibrosis when exposed to fibrogenic stimuli. In this study we used adenoviral gene transfer to transiently expose a fibrosis-prone (C57BL/6) and a fibrosis-resistant (Balb/c) mouse strain to high levels of active transforming growth factor (TGF)-beta1, a key profibrotic cytokine. Balb/c mice developed significantly less fibrosis compared with C57BL/6 mice in response to active TGF-beta1 despite higher levels of the transgene protein in the lung. This was not due to a general unresponsiveness of cells to TGF-beta1, because primary fibroblasts of both strains increased collagen synthesis upon stimulation with TGF-beta1 in vitro to the same degree. However, TGF-beta1 induced a strong upregulation of tissue inhibitor of metalloprotease-1 gene in pulmonary fibroblasts as well as in lungs of C57BL/6 mice, in contrast to a weak induction in Balb/c mice. These findings suggest that the differences in susceptibility to pulmonary fibrosis are downstream from TGF-beta1 and that fibrosis-prone individuals may have an altered collagen metabolism in the lungs that is balanced toward a "nondegrading" environment.

Topics: Adenoviridae; Animals; Bronchoalveolar Lavage Fluid; Cell Line; Collagen; Female; Fibroblasts; Gene Transfer Techniques; Genetic Vectors; Humans; Hydroxyproline; Inflammation; Lung; Mice; Mice, Inbred BALB C; Mice, Inbred Strains; Protease Inhibitors; Pulmonary Fibrosis; Tissue Inhibitor of Metalloproteinase-1; Transforming Growth Factor beta; Transforming Growth Factor beta1

Bleomycin (BM)-induced pulmonary fibrosis involves excess production of proteoglycans (PGs). Because transforming growth factor-beta(1) (TGF-beta(1)) promotes fibrosis, and interferon-gamma (IFN-gamma) inhibits it, we hypothesized that TGF-beta(1) treatment would upregulate PG production in fibrotic lung fibroblasts, and IFN-gamma would abrogate this effect. Primary lung fibroblast cultures were established from rats 14 days after intratracheal instillation of saline (control) or BM (1.5 units). PGs were extracted and subjected to Western blot analysis. Bleomycin-exposed lung fibroblasts (BLF) exhibited increased production of versican (VS), heparan sulfate proteoglycan (HSPG), and biglycan (BG) compared with normal lung fibroblasts (NLF). Compared with NLF, BLF released significantly increased amounts of TGF-beta(1). TGF-beta(1) (5 ng/ml for 48 h) upregulated PG expression in both BLF and NLF. Incubation of BLF with anti-TGF-beta antibody (1, 5, and 10 microg/ml) inhibited PG expression in a dose-dependent manner. Treatment of BLF with IFN-gamma (500 U. ml(-1) x 48 h) reduced VS, HSPG, and BG expression. Furthermore, IFN-gamma inhibited TGF-beta(1)-induced increases in PG expression by these fibroblasts. Activation of fibroblasts by TGF-beta(1) promotes abnormal deposition of PGs in fibrotic lungs; downregulation of TGF-beta(1) by IFN-gamma may have potential therapeutic benefits in this disease.

Topics: Animals; Antibodies; Antimetabolites, Antineoplastic; Biglycan; Bleomycin; Cell Division; Cell Survival; Cells, Cultured; Chondroitin Sulfate Proteoglycans; Extracellular Matrix Proteins; Fibroblasts; Heparan Sulfate Proteoglycans; In Vitro Techniques; Interferon-gamma; Lectins, C-Type; Male; Proteoglycans; Pulmonary Fibrosis; Rats; Rats, Sprague-Dawley; Transforming Growth Factor beta; Transforming Growth Factor beta1; Up-Regulation; Versicans

The lysosomal enzymes N-acetylglucosaminidase (N-ACGA) and beta-galactosidase (beta-gal) are involved in cellular collagen metabolism and may, therefore, be markers of fibrosis in idiopathic interstitial pneumonias, such as idiopathic pulmonary fibrosis (IPF). N-ACGA and beta-gal were analyzed in the bronchoalveolar lavage fluid (BALF) of patients with the histologic pattern of usual interstitial pneumonia (UIP, n=10) and controls (n=9). Cellular distribution in BALF as well as the concentration of TGF-beta a well-known mediator of fibroblast matrix deposition were correlated to the enzyme activities in both groups of patients. We found that both, N-ACGA (UIP: 25.2 nmol/l s +/- 3.4; controls: 73 nmol/l s +/- 1.3) and beta-gal (UIP: 4.7 nmol/l s +/- 0.5; controls: 2.4 nmol/l s +/- 0.3) were elevated significantly in BALF of patients with IPF compared to that of control patients (P<0.003). This increase was paralleled by an increase in neutrophils (IPF: 17.9% +/- 21.8; controls: 5.4% +/- 6.3; P=0.03) and eosinophils (IPF: 2.0% +/- 1.5; controls: 0.2% +/- 0.45; P=0.002) in BALF fluid. In addition, N-ACGA activity correlated closely with lung function (FVC, TLC, and DLCO), transforming growth factor-beta (TGF-beta) in BALF (r=0.77, P=0.008) and activated lymphocytes (r=0.66, P=0.0021). Our findings suggest that measurement of lysosomal enzymes such as N-ACGA may represent a useful indicator of fibrotic activity in IPF.

Topics: Acetylglucosaminidase; beta-Galactosidase; Biomarkers; Bronchoalveolar Lavage Fluid; Case-Control Studies; Forced Expiratory Volume; Humans; Leukocyte Count; Middle Aged; Monitoring, Physiologic; Pulmonary Fibrosis; Statistics, Nonparametric; Transforming Growth Factor beta; Vital Capacity

Pulmonary fibrosis (PF) is caused by a number of inhaled agents, as well as by some drugs and toxic particles. The elaboration of certain peptide growth factors is thought to be key to the development of this disease process. In addition, genetic susceptibility plays a role in the development of PF. For instance, we have previously shown that the 129J strain of mice is resistant, whereas the C57BL/6 strain is highly susceptible, to asbestos-induced fibrosis. To pursue this further, in one mouse model, we crossed the 129J strain to the C57BL/6 strain to produce an F1 generation and subsequently backcrossed the F1 mice to the inbred founders. This backcross to the 129 inbred strain produced reverse similar 25% of the offspring with a phenotype that was protected from the fibrogenic effects of inhaled asbestos fibers. In the second model, both strains of mice were treated intratracheally with an adenovirus vector (AdV), which transduces expression of active transforming growth factor (TGF)-beta(1) in the lungs, producing fibroproliferative lung disease. Compared with C57 mice, a significant number of 129 strain mice exhibited at least a 1-wk delay in the fibroproliferative response to TGF-beta(1) expression at three concentrations of virus. These findings suggest that certain sequences in a gene or a cluster of genes in the 129 mouse strain impart a phenotype in which there is a delay in, or protection from, the development of lung fibrogenesis.

Topics: Adenoviridae; Animals; Asbestos; beta-Galactosidase; Bromodeoxyuridine; Bronchoalveolar Lavage Fluid; Carcinogens; Cell Division; Gene Expression; Genetic Predisposition to Disease; Mice; Mice, Inbred C57BL; Phenotype; Pulmonary Fibrosis; Species Specificity; Transforming Growth Factor beta; Transforming Growth Factor beta1

Investigators have shown that interstitial pulmonary fibrosis (IPF) can be induced in rats by overexpressing transforming growth factor beta1 (TGF-beta1) through a replication-deficient recombinant adenovirus vector instilled into the lungs (Sime et al. 1997). We have shown that this vector induces IPF in fibrogenic-resistant tumour necrosis factor alpha-receptor knockout (TNF-alphaRKO) mice (Liu et al. 2001). The object of our studies is to understand how peptide growth factors, such as TGF-beta1, mediate interstitial lung disease (ILD). To do so, we must be able to manipulate the dose of the factor and sort out its effects on multiple other mediators in the lung parenchyma. As a step in this complex process, in the studies reported here, we have determined the concentrations of the recombinant adenovirus vector carrying the gene for porcine active TGF-beta1 (AVTGFbeta1) that have little apparent effect, cause clear induction of disease, or severe disease. The disease largely resolves by 28 days in all cases, thus providing a valuable model to understand the mechanisms of the IPF that is mediated, at least in part, by TGF-beta1. The findings here show that 10(6) plaque-forming units (pfu) of AVTGFbeta1, provide essentially a 'no-effect' dose, but even this amount of TGF-beta1 causes a significant increase in whole-lung collagen by day 28 after treatment. In contrast, 10(8) and 10(9) pfu cause severe IPF in 4 days, whereas 10(7) and 5 x 10(7) are intermediate for all parameters studied, i.e. TGF-beta protein, inflammatory cells, cell proliferation, pro-alpha 1(I) collagen gene expression and whole-lung collagen accumulation, and expression of growth factors such as TGF-beta1, TNF-alpha and PDGF-A and -B. Interestingly enough, TGF-beta1, as a potent blocker of epithelial cell proliferation, appears to suppress airway epithelial cell growth that would be expected during the inflammatory phase of IPF. Thus, this model system helps us to understand some quantitative aspects of TGF-beta1 biological activity and allows us to manipulate this potent factor as a mediator of interstitial fibrogenesis.

Topics: Adenoviridae; Animals; Bronchoalveolar Lavage Fluid; Gene Expression; Gene Transfer Techniques; Genetic Vectors; Male; Mice; Mice, Inbred C57BL; Pneumonia; Pulmonary Fibrosis; Titrimetry; Transduction, Genetic; Transforming Growth Factor beta; Transforming Growth Factor beta1

Signal transducer and activator of transcription 6 (Stat6) is critical for Th2-mediated responses during allergic airway disease. To investigate the role of Stat6 in fungus-induced airway hyperresponsiveness and remodeling, Stat6-deficient (Stat6-/-) and Stat6-wildtype (Stat6+/+) mice were sensitized to Aspergillus fumigatus and airway disease was subsequently assessed in both groups at days 21, 30, 38, and 44 after an intratracheal challenge with live A. fumigatus conidia. At all times after conidia, histological analysis revealed an absence of goblet cell hyperplasia and markedly diminished peribronchial inflammation in Stat6-/- mice in contrast to Stat6+/+ mice. Airway hyperresponsiveness and peribronchial fibrosis in Stat6-/- mice were significantly reduced at day 21 after conidia compared with Stat6+/+ mice, but both groups exhibited significant, similar increases in these parameters at all subsequent times after conidia. In separate experiments, IL-13-responsive cells in Stat6-/- mice were targeted via the daily intranasal administration of 200 ng of IL-13-PE38QQR (IL13-PE), comprised of human IL-13 and a derivative of Pseudomonas exotoxin, from days 38 to 44 after the conidia challenge. IL13-PE treatment abolished airway hyperresponsiveness, but not peribronchial fibrosis in Stat6-/- mice. Taken together, these data demonstrate that the chronic development of airway hyperresponsiveness during fungal asthma is IL-13-dependent but Stat6-independent.

Topics: Animals; Aspergillosis, Allergic Bronchopulmonary; Aspergillus fumigatus; Asthma; Bronchial Hyperreactivity; Bronchoalveolar Lavage Fluid; Chemokine CCL11; Chemokine CCL2; Chemokine CCL5; Chemokines, CC; Chronic Disease; Collagen; Humans; Immunoglobulin E; Interleukin-13; Interleukin-4; Leukocytes; Lung; Mice; Mice, Inbred BALB C; Mice, Transgenic; Pulmonary Fibrosis; STAT6 Transcription Factor; T-Lymphocytes; Trans-Activators; Transforming Growth Factor beta

IL-13 stimulates inflammatory and remodeling responses and contributes to the pathogenesis of human airways disorders. To further understand the cellular and molecular events that mediate these responses, we characterized the effects of IL-13 on monocyte chemotactic proteins (MCPs) and compared the tissue effects of transgenic IL-13 in mice with wild-type (+/+) and null (-/-) CCR2 loci. Transgenic IL-13 was a potent stimulator of MCP-1, -2, -3, and -5. This stimulation was not specific for MCPs because macrophage-inflammatory protein (MIP)-1alpha, MIP-1beta, MIP-2, MIP-3alpha, thymus- and activation-regulated chemokine, thymus-expressed chemokine, eotaxin, eotaxin 2, macrophage-derived chemokines, and C10 were also induced. The ability of IL-13 to increase lung size, alveolar size, and lung compliance, to stimulate pulmonary inflammation, hyaluronic acid accumulation, and tissue fibrosis, and to cause respiratory failure and death were markedly decreased, whereas mucus metaplasia was not altered in CCR2(-/-) mice. CCR2 deficiency did not decrease the basal or IL-13-stimulated expression of target matrix metalloproteinases or cathepsins but did increase the levels of mRNA encoding alpha1-antitrypsin, tissue inhibitor of metalloproteinase-1, -2, and -4, and secretory leukocyte proteinase inhibitor. In addition, the levels of bioactive and total TGF-beta(1) were decreased in lavage fluids from IL-13 transgenic mice with -/- CCR2 loci. These studies demonstrate that IL-13 is a potent stimulator of MCPs and other CC chemokines and document the importance of MCP-CCR2 signaling in the pathogenesis of the IL-13-induced pulmonary phenotype.

Topics: Animals; Bronchoalveolar Lavage Fluid; Cells, Cultured; Chemokine CCL2; Chemokines, CC; Endopeptidases; Hyaluronic Acid; Inflammation; Interleukin-13; Lung; Lung Compliance; Metaplasia; Mice; Mice, Inbred C57BL; Mice, Knockout; Mice, Transgenic; Mink; Mucus; Phenotype; Protease Inhibitors; Pulmonary Alveoli; Pulmonary Fibrosis; Receptors, CCR2; Receptors, Chemokine; Respiratory Insufficiency; Respiratory Mucosa; RNA, Messenger; Total Lung Capacity; Transforming Growth Factor beta; Transforming Growth Factor beta1

Expression microarrays are a powerful tool that could provide new information about the molecular pathways regulating common lung diseases. To exemplify how this tool can be useful, selected examples of informative experiments are reviewed. In studies relevant to asthma, the cytokine interleukin-13 has been shown to produce many of the phenotypic features of this disease, but the cellular targets in the airways and the molecular pathways activated are largely unknown. We have used microarrays to begin to dissect the different transcriptional responses of primary lung cells to this cytokine. In experiments designed to identify global transcriptional programs responsible for regulating lung inflammation and pulmonary fibrosis, we performed microarray experiments on lung tissue from wild-type mice and mice lacking a member of the integrin family know to be involved in activation of latent transforming growth factor (TGF)-beta. In addition to identifying distinct cluster of genes involved in each of these processes, these studies led to the identification of novel pathways by which TGF-beta can regulate acute lung injury and emphysema. Together, these examples demonstrate how careful application and thorough analysis of expression microarrays can facilitate the discovery of novel molecular targets for intervening in common lung diseases.

Topics: Animals; Antigens, Neoplasm; Asthma; Gene Expression; Genes, Regulator; Genetic Predisposition to Disease; Humans; In Vitro Techniques; Integrins; Interleukin-13; Lung; Matrix Metalloproteinase 12; Matrix Metalloproteinase 7; Metalloendopeptidases; Mice; Mice, Knockout; Oligonucleotide Array Sequence Analysis; Pulmonary Emphysema; Pulmonary Fibrosis; Respiratory Distress Syndrome; Transcriptional Activation; Transforming Growth Factor beta

Topics: Animals; Asbestos; Crosses, Genetic; Genetic Predisposition to Disease; Lung; Mice; Mice, Inbred C57BL; Mice, Inbred Strains; Oligonucleotide Array Sequence Analysis; Phenotype; Pulmonary Fibrosis; Receptor, Platelet-Derived Growth Factor alpha; RNA, Messenger; Transforming Growth Factor beta

Topics: Animals; Decorin; Extracellular Matrix Proteins; Gene Transfer Techniques; Genetic Therapy; Hepatocyte Growth Factor; Lung; Mice; Mice, Inbred C57BL; Proteoglycans; Pulmonary Fibrosis; Regeneration; Transforming Growth Factor beta

Based on studies by our group and others, we hypothesized that IL-7 may possess antifibrotic activities in an IFN-gamma-dependent and independent manner. Here, we have evaluated the antifibrotic therapeutic potential of IL-7 in both in vitro and in vivo pulmonary fibrosis models. IL-7 inhibited both TGF-beta production and signaling in fibroblasts and required an intact JAK1/STAT1 signal transduction pathway. IL-7-mediated inhibition of TGF-beta signaling was found to be associated with an increase in Smad7, a major inhibitory regulator in the SMAD family. In the presence of IL-7, Smad7 dominant negative fibroblasts restored TGF-beta-induced collagen synthesis, indicating that an IL-7-mediated increase in Smad7 suppressed TGF-beta signaling. Consistent with these in vitro findings, recombinant IL-7 decreased bleomycin-induced pulmonary fibrosis in vivo, independent of IFN-gamma. The antifibrotic activities of IL-7 merit further basic and clinical investigation for the treatment of pulmonary fibrosis.

Topics: Animals; Bleomycin; Cells, Cultured; Collagen; DNA-Binding Proteins; Female; Fibroblasts; Gene Expression; Humans; Interleukin-7; Janus Kinase 1; Mice; Mice, Inbred C57BL; Mice, SCID; Protein-Tyrosine Kinases; Pulmonary Fibrosis; Recombinant Proteins; Signal Transduction; Smad7 Protein; STAT1 Transcription Factor; Trans-Activators; Transforming Growth Factor beta; Transforming Growth Factor beta1; Transforming Growth Factor beta2; Transforming Growth Factor beta3

Transforming growth factor-beta (TGF-beta) is a multifunctional cytokine involved in controlling critical cellular activities including proliferation, differentiation, extracellular matrix production, and apoptosis. TGF-beta signals are mediated by a family of Smad proteins, of which Smad2 and Smad3 are downstream intracellular targets of serine/threonine kinase receptors of TGF-beta. Although Smad2 and Smad3 are crucial for TGF-beta signaling, little is known about the regulation of their expression. In this study, we investigated the expression of Smad2 and Smad3 in an in vivo animal model of lung fibrosis induced by bleomycin. We found that the expression of Smad3 was regulated in lungs during bleomycin-induced pulmonary fibrosis. The decline of Smad3 mRNA was evident at day three of post-bleomycin instillation and the expression of Smad3 continually decreased during the reparative phase of lung injury (days 8 and 12), whereas the expression of Smad2 showed little change after bleomycin administration. We further investigated whether the expression of Smad3 was regulated by TGF-beta in an in vitro lung fibroblast culture system. Our results show an immediate translocation of Smad3 protein from the cytoplasm to the nucleus and a delayed down-regulation of Smad3 mRNA by TGF-beta in lung fibroblasts. These studies provide direct evidence for a differential regulation of Smad3 expression that is distinct from that of Smad2 during bleomycin-induced pulmonary fibrosis and suggest a ligand-induced negative feedback loop that modulates cellular TGF-beta signaling.

Topics: Active Transport, Cell Nucleus; Animals; Bleomycin; Cell Nucleus; Cells, Cultured; Cytoplasm; Disease Progression; DNA-Binding Proteins; Down-Regulation; Feedback, Physiological; Fibroblasts; Gene Expression Regulation; Pulmonary Fibrosis; Rats; Rats, Sprague-Dawley; RNA, Messenger; Signal Transduction; Smad2 Protein; Smad3 Protein; Trans-Activators; Transforming Growth Factor beta

To elucidate the pathophysiology of pulmonary fibrosis, we investigated the involvement of p38 mitogen-activated protein kinase (MAPK), which is one of the major signal transduction pathways of proinflammatory cytokines, in a murine model of bleomycin-induced lung fibrosis. p38 MAPK and its substrate, activating transcription factor (ATF)-2, in bronchoalveolar lavage fluid cells were phosphorylated by intratracheal exposure of bleomycin, and the phosphorylation of ATF-2 was inhibited by subcutaneous administration of a specific inhibitor of p38 MAPK, FR-167653. FR-167653 also inhibited augmented expression of tumor necrosis factor -alpha, connective tissue growth factor, and apoptosis of lung cells induced by bleomycin administration. Moreover, daily subcutaneous administration of FR-167653 (from 1 day before to 14 days after bleomycin administration) ameliorated pulmonary fibrosis and pulmonary cachexia induced by bleomycin. These findings demonstrated that p38 MAPK is involved in bleomycin-induced pulmonary fibrosis, and its inhibitor, FR-167653, may be a feasible therapeutic agent.

Topics: Activating Transcription Factor 2; Animals; Antibiotics, Antineoplastic; Bleomycin; Bronchoalveolar Lavage Fluid; Connective Tissue Growth Factor; Cyclic AMP Response Element-Binding Protein; Disease Models, Animal; Gene Expression; Growth Inhibitors; Growth Substances; Hydroxyproline; Immediate-Early Proteins; In Situ Nick-End Labeling; Injections, Intravenous; Intercellular Signaling Peptides and Proteins; Lung; Male; Mice; Mice, Inbred ICR; Mitogen-Activated Protein Kinases; p38 Mitogen-Activated Protein Kinases; Phosphorylation; Pulmonary Fibrosis; Pyrazoles; Pyridines; RNA, Messenger; Transcription Factors; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha; Weight Gain

The excessive matrix deposition in lung fibrosis is thought to be due to enhanced formation and activity of TGFbeta1, which stimulates synthesis and inhibits degradation of matrix proteins. The cellular mechanisms triggered by TGFbeta1 are still incompletely understood. Recently, a novel transcriptional target of TGFbeta1 has been identified, i.e. the human serum and glucocorticoid dependent kinase hSGK1. The present study has been performed to explore whether TGFbeta1 stimulates hSGK1 transcription in lung fibroblasts and whether lung fibrosis is associated with enhanced hSGK1 expression. As evident from Northern Blotting, TGFbeta1 strongly upregulates hSGK1 in human lung fibroblasts, an effect partially reversed by p38-kinase inhibitor SB203580. In situ hybridization experiments reveal that in intact lung tissue hSGK1 is expressed in single type II alveolar pneumocytes and macrophages. In contrast, in fibrotic lung tissue a dramatic upregulation of hSGK1 mRNA as well as a strong expression of hSGK1 protein is observed in epithelial cells and interstitial cells comprising macrophages and fibroblasts. In conclusion, in lung fibrosis, the serine/threonine kinase hSGK1 is upregulated, an effect at least partially accounted for by TGFbeta1. The full effect of TGFbeta1 requires the activation of p38 kinase.

Topics: Cell Line; Enzyme Induction; Gene Expression Regulation, Enzymologic; Humans; Immediate-Early Proteins; Immunohistochemistry; In Situ Hybridization; Lung; Mitogen-Activated Protein Kinases; Nuclear Proteins; p38 Mitogen-Activated Protein Kinases; Protein Serine-Threonine Kinases; Pulmonary Fibrosis; RNA, Messenger; Transcription, Genetic; Transforming Growth Factor beta; Transforming Growth Factor beta1

On the basis of its fibroblast growth factor (FGF) inhibitory effect we assessed the possible inhibitory anti-inflammatory role of Pygeum Africanum extract (Tadenan) on FGF and transforming growth factor beta (TGF beta 1/LAP) expression of macrophages and neutrophils in broncho-alveolar lavage fluid (BAL) of rats in a bleomycin-induced acute inflammation model. The rats were divided into three groups: 17 untreated controls, 10 bleomycin-instilled rats, receiving NaCl (0.9%), and 10 rats receiving Pygeum Africanum extract. On the 12th (and 15th day) we performed BAL and after labelling of cells expression of FGF and TGF beta 1 (LAP) was measured by flow-cytometry. We made a quantitative analysis of BAL cells as well. One-way ANOVA was used for statistical analysis. We found in Pygeum Africanum extract treated group 1, a significantly decreased number of neutrophil granulocytes (p < 0.05) compared with other groups 2, there was a considerable decrease (not significant) in expression of TGF beta 1(LAP) on BAL macrophages, but not in case of FGF.. our results show the possible 1. inhibitory effect of this drug on TGF beta 1 (LAP) expression, 2. anti-inflammatory role on neutrophil granulocytes.

Topics: Animals; Anti-Inflammatory Agents, Non-Steroidal; Bleomycin; Bronchoalveolar Lavage Fluid; Fatty Alcohols; Fibroblast Growth Factors; Macrophages, Alveolar; Male; Neutrophils; Plant Extracts; Pulmonary Fibrosis; Rats; Rats, Wistar; Transforming Growth Factor beta

In this study we evaluate the antifibrotic properties of PG-490-88, a water-soluble derivative of triptolide. Triptolide is an oxygenated diterpene that is derived from a traditional Chinese herb that has potent immunosuppressive and antitumor activity. We used the intratracheal bleomycin mouse model and found that PG490-88 inhibits fibrosis in the bleomycin group when given the same day or 5 days after bleomycin. PG490-88 also markedly reduced the number of myofibroblasts in the bleomycin treatment group. An enzyme-linked immunosorbent assay of transforming growth factor (TGF)-beta in the bronchoalveolar lavage fluid showed a significant decrease in TGF-beta in the PG490-88-treated groups compared to the bleomycin-treated group. Additionally, triptolide blocked bleomycin-induced increase in TGF-beta mRNA in cultured normal human lung fibroblasts. The efficacy of PG490-88 when administered late after bleomycin installation suggests a potential role in the treatment of idiopathic pulmonary fibrosis.

Topics: Animals; Bleomycin; Bronchoalveolar Lavage Fluid; Cell Survival; Diterpenes; Drugs, Chinese Herbal; Epoxy Compounds; Fibroblasts; Immunosuppressive Agents; Inflammation; Lung; Male; Mice; Mice, Inbred C57BL; Phenanthrenes; Pulmonary Fibrosis; Transcription, Genetic; Transforming Growth Factor beta

Pulmonary fibrosis is a chronic progressive disease with no effective therapy. Transforming growth factor beta (TGF-beta) is thought to be a key profibrotic mediator and blocking its activity is therefore one of the targets of new treatment strategies for fibrosis. Decorin is an endogenous proteoglycan and one of the known inhibitors of TGF-beta. The short half-life of peptide-based therapeutics makes gene transfer a promising approach to achieve prolonged protein levels in the lung. Replication-deficient adenovirus was used to deliver decorin transgene (AdDec) to the airways by a single intranasal injection in a murine bleomycin model of lung fibrosis. The ability of vector-derived decorin to inhibit TGF-beta was examined in a bioassay and its effect on bleomycin-induced pulmonary fibrosis was determined by histomorphology and lung hydroxyproline. In vitro, supernatant from cells infected with AdDec abrogated the bioactivity of TGF-beta in a dose-dependent manner whereas control virus (AdDL70) had no effect. In vivo, treatment of bleomycin-injected mice with AdDec substantially reduced the fibrogenic response compared with control virus (hydroxyproline: bleomycin/AdDec, 1.96 microg/mg; bleomycin/AdDL70, 3.05 microg/mg; p = 0.0005). These results suggest that a single administration of AdDec was able to generate a local pulmonary environment that effectively blocked the fibrogenic response to bleomycin by inhibition of TGF-beta.

Topics: Adenoviridae; Animals; Antibiotics, Antineoplastic; Bleomycin; Decorin; Extracellular Matrix Proteins; Female; Hydroxyproline; Lung; Mice; Mice, Inbred C57BL; Proteoglycans; Pulmonary Fibrosis; Transforming Growth Factor beta; Transgenes; Tumor Cells, Cultured

Lung fibrosis is a fatal condition of excess extracellular matrix (ECM) deposition associated with increased transforming growth factor beta (TGF-beta) activity. Although much is known about its pathological features, our understanding of the signal transduction pathways resulting in increased ECM and collagen deposition in response to TGF-beta is still incompletely defined. We have previously reported that a JunD homodimer of the transcription factor AP-1 is specifically activated by TGF-beta in lung fibroblasts. Here we demonstrate that JunD is also specifically required for TGF-beta-induced effects. Antisense against JunD, but not c-fos or c-jun, significantly inhibited collagen deposition in response to TGF-beta in primary human lung fibroblasts. We then investigated the ability of pharmacological agents to inhibit TGF-beta-induced signaling and collagen deposition. Cs-A and IFN-gamma, but not glucocorticoids, cyclophosphamide, or azathioprine, inhibited TGF-beta-induced signaling, as assessed by luciferase reporter gene assays, and collagen deposition. TGF-beta antagonism by Cs-A was associated with direct inhibition of JunD activation, as demonstrated by electrophoretic mobility shift analyses. In contrast, the effects of IFN-gamma required signal transducer and activator of transcription (STAT)-1. We thus identify the JunD isoform of AP-1 as an essential mediator of TGF-beta-induced effects in lung fibroblasts. TGF-beta-induced signaling and collagen deposition are efficiently antagonized by Cs-A and IFN-gamma treatment, both of which exhibit distinct molecular mechanisms of action. These observations therefore offer novel targets for future therapy of fibrotic lung disease.

Topics: Cells, Cultured; Collagen; Cyclosporine; DNA-Binding Proteins; Extracellular Matrix; Fibroblasts; Genes, Reporter; Humans; Interferon-gamma; Lung; Models, Biological; Oligonucleotides, Antisense; Proto-Oncogene Proteins c-jun; Pulmonary Fibrosis; Signal Transduction; STAT1 Transcription Factor; Trans-Activators; Transcription Factor AP-1; Transforming Growth Factor beta

Topics: Animals; Disease Models, Animal; Endomyocardial Fibrosis; Endothelium, Vascular; Fibroblasts; Gene Targeting; Humans; Oxidative Stress; Pulmonary Fibrosis; Scleroderma, Systemic; Signal Transduction; Transforming Growth Factor beta

Prostaglandin E(2) (PGE(2)) inhibits fibroblast proliferation and collagen production. Its synthesis by fibroblasts is induced by profibrotic mediators including transforming growth factor (TGF)-beta(1). However, in patients with pulmonary fibrosis, PGE(2) levels are decreased. In this study we examined the effect of TGF-beta(1) on PGE(2) synthesis, proliferation, collagen production, and cyclooxygenase (COX) mRNA levels in fibroblasts derived from fibrotic and nonfibrotic human lung. In addition, we examined the effect of bleomycin-induced pulmonary fibrosis in COX-2-deficient mice. We demonstrate that basal and TGF-beta(1)-induced PGE(2) synthesis is limited in fibroblasts from fibrotic lung. Functionally, this correlates with a loss of the anti-proliferative response to TGF-beta(1). This failure to induce PGE(2) synthesis is because of an inability to up-regulate COX-2 mRNA levels in these fibroblasts. Furthermore, mice deficient in COX-2 exhibit an enhanced response to bleomycin. We conclude that a decreased capacity to up-regulate COX-2 expression and COX-2-derived PGE(2) synthesis in the presence of increasing levels of profibrotic mediators such as TGF-beta(1) may lead to unopposed fibroblast proliferation and collagen synthesis and contribute to the pathogenesis of pulmonary fibrosis.

Topics: Bleomycin; Cell Division; Cell Line; Cyclooxygenase 1; Cyclooxygenase 2; Cyclooxygenase 2 Inhibitors; Cyclooxygenase Inhibitors; Dinoprostone; Fibroblasts; Humans; Indomethacin; Isoenzymes; Membrane Proteins; Procollagen; Prostaglandin-Endoperoxide Synthases; Pulmonary Fibrosis; RNA, Messenger; Transforming Growth Factor beta

To investigate the role of iron and active oxygen species (AOS) in asbestos-induced fibrosis, we loaded increasing amounts of Fe(II)/Fe(III) onto the surface of amosite asbestos fibers and then applied the fibers to rat tracheal explants. Explants were harvested after 7 d in air organ culture. Asbestos by itself doubled procollagen gene expression, and a further increase was seen with increasing iron loading; actual collagen content measured as hydroxyproline was increased in a similar pattern. Iron loading also increased gene expression of platelet-derived growth factor (PDGF)-A and transforming growth factor (TGF)-beta(1). Neither asbestos alone nor iron-loaded asbestos affected gene expression of PDGF-B, tumor necrosis factor-alpha, or TGF-alpha. The AOS scavenger tetramethylthiourea or treatment of fibers with the iron chelator deferoxamine prevented asbestos-induced increases in procollagen, PDGF-A, and TGF-beta gene expression, whereas glutathione had no effect. The proteasome inhibitor MG-132 abolished asbestos-induced increases in procollagen gene expression but did not affect increases in PDGF-A or TGF-beta(1) expression, whereas the extracellular signal-regulated protein kinase (ERK) inhibitor PD98059 had exactly the opposite effect. We conclude that surface iron as well as the iron-catalyzed generation of AOS play a role in asbestos-induced matrix (procollagen) production and that this process is driven in part through oxidant-induced nuclear factor kappa B activation. Surface iron and AOS also play a role in PDGF-A and TGF-beta gene expression, but through an ERK-dependent mechanism.

Topics: Animals; Asbestos, Amosite; Cells, Cultured; Cysteine Proteinase Inhibitors; Enzyme Inhibitors; Flavonoids; Gene Expression; Iron; Leupeptins; Male; MAP Kinase Signaling System; NF-kappa B; Platelet-Derived Growth Factor; Procollagen; Pulmonary Fibrosis; Rats; Rats, Sprague-Dawley; Reactive Oxygen Species; RNA, Messenger; Trachea; Transforming Growth Factor beta; Transforming Growth Factor beta1; Tumor Necrosis Factor-alpha

The aim of the presented study was to observe acute and subacute discrete TGF-beta1 production after a low-dose whole-body radiation stimulus, known to induce thrombocytopenia. TGF-beta1 mRNA production and the number of thrombocytes was followed up in two mouse strains with different tendencies to the origination of fibroses. Mice of the C57BL/6 and C3H/J strains were exposed to a whole-body dose of 7 Gy. Non-irradiated mice of both strains were used as negative controls. The relative number of thrombocytes recorded in lung capillaries was significantly lower in both strains on day 9 after irradiation in comparison with controls. This finding was in accordance with a decrease in the number of thrombocytes in the peripheral blood in irradiated animals of both strains. On day 56 relative platelet counts reached physiological numbers in comparison to controls. On the other hand, TGF-beta1 mRNA production was higher in the C57BL/6 strain (on day 9) contrary to minimal production in the C3H/J strain (on day 9) or no production in both groups on day 56 and in controls. Thus, TGF-beta1 production without increased thrombocyte trapping in lung vessels in acute stage suggests that an additional mechanism is involved in low-dose radiation-induced cytokine synthesis in lung tissue besides the release of growth factors from thrombocytes.

Topics: Animals; Blood Platelets; Capillaries; Endothelium, Vascular; Female; Gene Expression Regulation; Lung; Mice; Mice, Inbred C3H; Mice, Inbred C57BL; Platelet Count; Pulmonary Circulation; Pulmonary Fibrosis; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Species Specificity; Transcription, Genetic; Transforming Growth Factor beta; Whole-Body Irradiation

Transforming growth factor (TGF)-beta is a key cytokine in the pathogenesis of pulmonary fibrosis, and pharmacological interference with TGF-beta can ameliorate the fibrotic tissue response. The small proteoglycans decorin and biglycan are able to bind and inhibit TGF-beta activity in vitro. Although decorin has anti-TGF-beta properties in vivo, little is known about the physiological role of biglycan in vivo. Adenoviral gene transfer was used to overexpress active TGF-beta, decorin, and biglycan in cell culture and in murine lungs. Both proteoglycans were able to interfere with TGF-beta bioactivity in vitro in a dose-dependant manner. In vivo, overexpression of TGF-beta resulted in marked lung fibrosis, which was significantly reduced by concomitant overexpression of decorin. Biglycan, however, had no significant effect on lung fibrosis induced by TGF-beta. The data suggest that differences in tissue distribution are responsible for the different effects on TGF-beta bioactivity in vivo, indicating that decorin, but not biglycan, has potential therapeutic value in fibrotic disorders of the lung.

Topics: Adenoviridae; Animals; Biglycan; Bronchoalveolar Lavage Fluid; Cell Line; Decorin; Dose-Response Relationship, Drug; Extracellular Matrix Proteins; Female; Fibroblasts; Gene Transfer Techniques; Genetic Vectors; Humans; Hydroxyproline; Immunohistochemistry; Lung; Mice; Mice, Inbred C57BL; Proteoglycans; Pulmonary Fibrosis; Tissue Distribution; Transforming Growth Factor beta; Treatment Outcome

Idiopathic pulmonary fibrosis (IPF) is a chronic lung disorder characterized by fibroblast proliferation and extracellular matrix accumulation. However, studies on fibroblast growth rate and collagen synthesis have given contradictory results. Here we analyzed fibroblast growth rate by a formazan-based chromogenic assay; fibroblast apoptosis by in situ end labeling (ISEL) and propidium iodide staining; percent of alpha-smooth muscle actin (alpha-SMA) positive cells by fluorescence-activated cell sorter; and alpha1-(I) collagen, transforming growth factor (TGF)-beta1, collagenase-1, gelatinases A and B, and tissue inhibitor of metalloproteinase (TIMP)-1, -2, -3, and -4 expression by reverse transcriptase/polymerase chain reaction in fibroblasts derived from IPF and control lungs. Growth rate was significantly lower in IPF fibroblasts compared with controls (13.3 +/- 38.5% versus 294.6 +/- 57%, P < 0.0001 at 13 d). Conversely, a significantly higher percentage of apoptotic cells was observed in IPF-derived fibroblasts (ISEL: 31.9 +/- 7.0% versus 15.5 +/- 7.6% from controls; P < 0.008). alpha-SMA analysis revealed a significantly higher percentage of myofibroblasts in IPF samples (62.8 +/- 25.2% versus 14.8 +/- 11.7% from controls; P < 0.01). IPF fibroblasts were characterized by an increase in pro-alpha1-(I) collagen, TGF-beta1, gelatinase B, and all TIMPs' gene expression, whereas collagenase-1 and gelatinase A expression showed no differences. These results suggest that fibroblasts from IPF exhibit a profibrotic secretory phenotype, with lower growth rate and increased spontaneous apoptosis.

Topics: Apoptosis; Cell Division; Cells, Cultured; Collagen; Female; Fibroblasts; Gene Expression Profiling; Humans; Lung; Male; Matrix Metalloproteinase 1; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; Middle Aged; Pulmonary Fibrosis; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Tissue Inhibitor of Metalloproteinases; Transforming Growth Factor beta; Transforming Growth Factor beta1

Inhalation of numerous fibrogenic agents causes interstitial pulmonary fibrosis (IPF) in humans and in a number of animal models. Several of these models provide evidence that certain peptide growth factors (GF) are playing a role in the disease process. Transforming growth factor beta 1 (TGF-beta1) is a potent inducer of extracellular matrix production by mesenchymal cells, and we have shown that this peptide is produced in the lung after asbestos exposure. We used in situ hybridization to demonstrate that the mRNA for TGF-beta1 is rapidly expressed post-exposure at sites of initial asbestos-induced lung injury in both rats and mice. The TGF-beta1 is expressed by bronchiolar-alveolar epithelial cells as well as by mesenchymal cells and lung macrophages in exposed animals. Normal rats and mice express little TGF-beta1, as we have demonstrated previously for PDGF-A and -B, TGF-alpha, and TNF-alpha. TGF-beta1 expression is accompanied by collagen and fibronectin production in asbestos-exposed animals. Most interesting, TGF-beta1 expression is largely absent in the lungs of TNF-alpha receptor knockout mice that fail to develop asbestos-induced IPE We have shown previously that the mRNAs and cognate peptides of PDGF-A and -B and TGF-alpha, but not TNF-alpha, are reduced in the fibrosis-resistant knockout mice. In this article, we show that TGF-beta1 is included in this group of cytokines, supporting the postulate that TNF-alpha is necessary for the expression of other, more downstream growth factors, and the consequent development of idiopathic pulmonary fibrosis (IPF).

Topics: Administration, Inhalation; Animals; Asbestos; Asbestosis; Collagen; Fibronectins; Immunoenzyme Techniques; In Situ Hybridization; Lung; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Pulmonary Fibrosis; Rats; Rats, Sprague-Dawley; Receptors, Tumor Necrosis Factor; RNA, Messenger; Transforming Growth Factor beta; Transforming Growth Factor beta1

Topics: Animals; Inflammation; Lung; Models, Biological; Paracrine Communication; Pulmonary Fibrosis; Transforming Growth Factor beta; Transforming Growth Factor beta1; Wound Healing

IL-1beta is one of a family of proinflammatory cytokines thought to be involved in many acute and chronic diseases. Although it is considered to participate in wound repair, no major role has been attributed to IL-1beta in tissue fibrosis. We used adenoviral gene transfer to transiently overexpress IL-1beta in rat lungs after intratracheal administration. The high expression of IL-1beta in the first week after injection was accompanied by local increase of the proinflammatory cytokines IL-6 and TNF-alpha and a vigorous acute inflammatory tissue response with evidence of tissue injury. The profibrotic cytokines PDGF and TGF-beta1 were increased in lung fluid samples 1 week after peak expression of IL-1beta. Although PDGF returned to baseline in the third week, TGF-beta1 showed increased concentrations in bronchoalveolar lavage fluid for up to 60 days. This was associated with severe progressive tissue fibrosis in the lung, as shown by the presence of myofibroblasts, fibroblast foci, and significant extracellular accumulations of collagen and fibronectin. These data directly demonstrate how acute tissue injury in the lung, initiated by a highly proinflammatory cytokine, IL-1beta, converts to progressive fibrotic changes. IL-1beta should be considered a valid target for therapeutic intervention in diseases associated with fibrosis and tissue remodeling.

Topics: Acute-Phase Reaction; Adenoviridae; Animals; Bronchoalveolar Lavage Fluid; Disease Progression; Female; Genetic Vectors; Interleukin-1; Interleukin-6; Lung; Platelet-Derived Growth Factor; Pulmonary Fibrosis; Rats; Rats, Sprague-Dawley; Transforming Growth Factor beta; Transforming Growth Factor beta1; Transgenes; Tumor Necrosis Factor-alpha

Transforming growth factor beta1 is implicated in the pathogenesis of lung fibrosis. It promotes extracellular matrix accumulation by increasing procollagen synthesis and reducing degradation. TGFbeta1 gene and protein expression increase in experimental lung fibrosis, and TGFbeta1 antibodies attenuate fibrosis in mice. The role of other TGFbeta isoforms is unclear. This study aimed to localise TGFbeta1 and TGFbeta3 gene expression in fibrotic human lung and compare it with that in normal human lung.. Lung tissue from patients with cryptogenic fibrosing alveolitis and fibrosis associated with systemic sclerosis was examined by in situ hybridisation. Macroscopically normal lung from carcinoma resections was used as control tissue. Digoxigenin labelled riboprobes were synthesised from TGFbeta isoform specific cDNA templates.. The digoxigenin labelled riboprobes were sensitive and permitted precise cellular localisation of mRNA transcripts. TGFbeta1 and TGFbeta3 mRNA transcripts were widespread in normal lung and localised to alveolar macrophages and bronchiolar epithelium. TGFbeta1 but not TGFbeta3 mRNA was detected in mesenchymal and endothelial cells. In fibrotic lung tissue mRNA transcripts for both isoforms were also detected in metaplastic type II cells. TGFbeta1 gene expression was enhanced in some patients. TGFbeta3 was expressed in fibrotic lung but was not consistently altered compared with controls.. TGFbeta1 mRNA transcripts were localised in normal and fibrotic human lung and TGFbeta3 gene expression in human lung fibrosis was shown for the first time. The results suggest that TGFbeta1 may play the predominant role in pathogenesis. It is suggested that TGFbeta1 should be the primary target of anticytokine treatments for pulmonary fibrosis.

Topics: Adult; Biopsy; Bronchoalveolar Lavage Fluid; Case-Control Studies; Female; Gene Expression; Humans; In Situ Hybridization; Male; Middle Aged; Pulmonary Fibrosis; RNA, Messenger; Transforming Growth Factor beta

Topics: Bronchoalveolar Lavage Fluid; Humans; Pulmonary Fibrosis; Receptors, Transforming Growth Factor beta; Transforming Growth Factor beta; Transforming Growth Factor beta1

Transforming growth factor (TGF)-beta plays a central role in fibrosis, contributing both to the influx and activation of inflammatory cells, as well as to activation of fibroblasts to elaborate extracellular matrix. In the past few years, new insight has been gained into signal transduction pathways downstream of the TGF-beta receptor serine-threonine kinases with the identification of a family of evolutionarily conserved Smad proteins. Two receptor-activated Smad proteins, Smad2 and Smad3, are phosphorylated by the activated TGF-beta type I receptor kinase, after which they partner with the common mediator, Smad4, and are translocated to the nucleus to where they participate in transcriptional complexes to control expression of target genes. We have shown in wound healing studies of mice null for Smad3, that loss of this key signaling intermediate interferes with the chemotaxis of inflammatory cells to TGF-beta as well as with their ability to autoinduce TGF-beta. Moreover, studies with mouse embryo fibroblasts null for Smad3 show that TGF-beta-dependent induction of c-Jun and c-Fos, important in induction of collagen as well as in autoinduction of TGF-beta, is mediated by Smad3. Based on these observations, we hypothesize that loss of Smad3 will confer resistance to fibrosis and result in reduced inflammatory cell infiltrates, reduced autoinduction of TGF-beta, important to sustain the process, and reduced elaboration of collagen. Preliminary observations in a model of radiation-induced fibrosis confirm this hypothesis and suggest that inhibitors of Smad3 might have clinical application both to improve wound healing and to reduce fibrosis.

Topics: DNA-Binding Proteins; Humans; MAP Kinase Signaling System; Phosphorylation; Pulmonary Fibrosis; Signal Transduction; Smad3 Protein; Trans-Activators; Transforming Growth Factor beta; Wound Healing

The integrin alphavbeta6 is restricted to epithelial cells and is dramatically induced in response to injury and inflammation. Mice expressing a null mutation of this integrin develop exaggerated inflammation of the lungs and skin, but are dramatically protected from bleomycin-induced pulmonary fibrosis. This phenotype led to the identification of a unique role for this integrin in binding to and activating latent extracellular complexes of the anti-inflammatory, profibrotic cytokine, transforming growth factor-beta(1). This integrin-mediated activation is tightly spatially restricted and appears to require direct presentation of the activated cytokine to receptors on adjacent cells. The process also requires distinct regions of the beta6-subunit cytoplasmic domain and an intact actin cytoskeleton, suggesting the existence of additional cellular mechanisms to regulate this process. If this mechanism is found to be as important in humans as it is in mice, the integrin and as yet to be identified pathways for cellular regulation of this process could be exciting new targets for intervention in fibrotic diseases of the lung and other epithelial organs.

Topics: Animals; Antigens, Neoplasm; Integrins; Mice; Mice, Knockout; Models, Animal; Mutation; Pulmonary Fibrosis; Signal Transduction; Transforming Growth Factor beta; Transforming Growth Factor beta1

Topics: Animals; Immunohistochemistry; Lung; Mice; Mice, Inbred C57BL; Mice, Knockout; Pulmonary Fibrosis; Transforming Growth Factor beta

Topics: Biomarkers; Glucocorticoids; Humans; Pulmonary Fibrosis; Transforming Growth Factor beta; Transforming Growth Factor beta1

To investigate whether the results of intratracheal instillation studies on mineral fibers reflect the findings obtained by long-term inhalation data on mineral fibers, we have examined gene expression of cytokines and pathological features in lungs induced by intratracheal instillation and inhalation of mineral fibers. Male Wistar rats were given a single intratracheal instillation of 2 mg alumina silicate refractory fiber (RF1) or potassium octatitanate whisker (PT1), and were sacrificed 4 wk after the fiber instillation. Long-term inhalation studies were also performed. In these, animals were exposed to fiber aerosol of RF1 or PT1 for 5 days/wk for 1 yr, and sacrificed after 1 yr of inhalation. Expression of tumor necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6), and transforming growth factor-beta1 (TGF-beta1) from lungs was observed by reverse-transcription polymerase chain reaction (RT-PCR). The expression of TNF-alpha, IL-6, and TGF-beta1 mRNA in PT1-exposed lung was significantly higher than for those exposed to RF1 in both intratracheal instillation and inhalation studies. Pathological findings revealed that mild pulmonary fibrosis was seen in the lungs after intratracheal instillation and inhalation of PT1 but not RF1. Similarities were observed not only in gene expression of cytokines but in pathological features between both studies. These data suggested that the results of intratracheal instillation reflect the findings obtained from long-term inhalation data.

Topics: Aluminum Silicates; Animals; Cytokines; DNA Primers; Gene Expression; Interleukin-6; Intubation, Intratracheal; Lung; Male; Mineral Fibers; Pulmonary Fibrosis; Rats; Rats, Wistar; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Titanium; Transforming Growth Factor beta; Transforming Growth Factor beta1; Tumor Necrosis Factor-alpha

Tumor necrosis factor-alpha receptor knockout (TNF-alphaRKO) mice have homozygous deletions of the genes that code for both the 55- and 75-kD receptors. The mice are protected from the fibrogenic effects of bleomycin, silica, and inhaled asbestos. The asbestos-exposed animals exhibit reduced expression of other peptide growth factors such as transforming growth factor (TGF)-alpha, platelet-derived growth factors, and TGF-beta. In normal animals, these and other cytokines are elaborated at high levels during the development of fibroproliferative lung disease, but there is little information available that has allowed investigators to establish the role of the individual growth factors in disease pathogenesis. Here, we show that overexpression of TGF-beta(1) by means of a replication-deficient adenovirus vector induces fibrogenesis in the lungs of the fibrogenic-resistant TNF-alphaRKO mice. The fibrogenic lesions developed in both the KO and background controls within 7 d, and both types of animals exhibited similar incorporation of bromodeoxyuridine. Interestingly, airway epithelial cell proliferation appeared to be suppressed, perhaps due to the presence of the TGF-beta(1), a well-known inhibitor of epithelial mitogenesis. Before these experiments, there was no information available that would provide a basis for predicting whether or not TGF-beta(1) expression induces fibroproliferative lung disease in fibrogenic-resistant TNF-alphaRKO mice, an increasingly popular animal model.

Topics: Adenoviridae; Animals; Bromodeoxyuridine; Genetic Vectors; Mice; Mice, Inbred C57BL; Mice, Knockout; Pulmonary Fibrosis; Receptors, Tumor Necrosis Factor; Transduction, Genetic; Transforming Growth Factor beta

To analyse the mechanism by which a bleomycin derivative, peplomycin (PLM) induces pulmonary fibrosis, we investigated differentiation of rat pulmonary fibroblasts to myofibroblasts (MF). In intraperitoneally PLM (5 mg/kg/day)-injected rats, the peripheries of lungs adjacent to the pleura revealed advanced fibrosis with a small number of alpha-smooth muscle actin (alpha-SMA)-positive MF, which ultrastructurally possessed abundant microfilaments and cellular organelles. In the fibrotic tissue, the expression of alpha-SMA-mRNA was detected by in situ reverse transcription-polymerase (RT-PCR). The message was strong just after a 2-week administration of PLM then decreased thereafter, although fibrosis advanced. When pulmonary fibroblasts were separated from saline-injected rats (N-Fib) and cultivated for 7 days in the presence of 5 mg/mL PLM, alpha-SMA protein was weakly expressed, while the majority of pulmonary fibroblasts separated from PLM-injected rats (P-Fib) became positive for alpha-SMA in 7-day cultivation and the expression of alpha-SMA in P-Fib was strongly increased by cultivation in the presence of PLM and transforming growth factor-beta (TGF-beta), but not basic fibroblast growth factor (bFGF) or platelet-derived growth factor (PDGF), although the cell proliferation was most strongly enhanced by bFGF and only slightly by PLM and TGF-beta. The alpha-SMA-positive cells expressed vimentin, but only weakly expressed desmin. Additionally, P-Fib generated larger amounts of TGF-beta and bFGF than were generated by N-Fib. These results indicate that PLM induces pulmonary fibrosis by differentiating fibroblasts to alpha-SMA-positive MF, and that bFGF and TGF-beta play each critical role in the different phases of PLM-induced pulmonary fibrosis by inducing fibroblast proliferation and transformation, respectively.

Topics: Actins; Animals; Antibiotics, Antineoplastic; Blotting, Northern; Blotting, Western; Cell Culture Techniques; Cell Differentiation; Cell Division; Fibroblast Growth Factor 2; Fibroblasts; Gene Expression Regulation; Male; Peplomycin; Pulmonary Fibrosis; Rats; Rats, Inbred F344; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Transforming Growth Factor beta

Interleukin (IL)-13 is a key mediator of tissue fibrosis caused by T helper cell type 2 inflammation. We hypothesized that the fibrogenic effects of IL-13 are mediated by transforming growth factor (TGF)-beta. To test this hypothesis we compared the regulation of TGF-beta in lungs from wild-type mice and CC10-IL-13 mice in which IL-13 overexpression causes pulmonary fibrosis. IL-13 selectively stimulated TGF-beta(1) production in transgenic animals and macrophages were the major site of TGF-beta(1) production and deposition in these tissues. IL-13 also activated TGF-beta(1) in vivo. This activation was associated with decreased levels of mRNA encoding latent TGF-beta-binding protein-1 and increased mRNA encoding urinary plasminogen activator, matrix metalloproteinase (MMP)-9, and CD44. TGF-beta(1) activation was abrogated by the plasmin/serine protease antagonist aprotinin. It was also decreased in progeny of crosses of CC10-IL-13 mice and MMP-9 null mice but was not altered in crosses with CD44 null animals. IL-13-induced fibrosis was also significantly ameliorated by treatment with the TGF-beta antagonist soluble TGFbetaR-Fc (sTGFbetaR-Fc). These studies demonstrate that IL-13 is a potent stimulator and activator of TGF-beta(1) in vivo. They also demonstrate that this activation is mediated by a plasmin/serine protease- and MMP-9-dependent and CD44-independent mechanism(s) and that the fibrogenic effects of IL-13 are mediated, in great extent, by this TGF-beta pathway.

Topics: Animals; Hyaluronan Receptors; Interleukin-13; Matrix Metalloproteinase 9; Mice; Mice, Knockout; Mice, Transgenic; Pulmonary Fibrosis; Transforming Growth Factor beta; Transforming Growth Factor beta1; Urokinase-Type Plasminogen Activator

Amiodarone (AM) is an antidysrhythmic agent with a propensity to cause pulmonary toxicity, including potentially fatal fibrosis. In the present study, the potential roles of c-Jun and transforming growth factor (TGF)-beta 1 in AM-induced inflammation and fibrogenesis were examined after intratracheal administration of AM (1.83 micromol/day on days 0 and 2) or an equivalent volume (0.4 ml) of distilled water to male Fischer 344 rats. Northern and immunoblot analyses demonstrated that lung TGF-beta 1 (mRNA and protein) expression was increased 1.5- to 1.8-fold relative to control during the early inflammation period and 1 day, 1 wk, and 2 wk post-AM treatment. Lung c-Jun protein expression was increased concomitantly with evidence of AM-induced fibrosis; at 5 wk post-AM treatment, c-Jun protein was increased 3.3-fold relative to control. The results indicate a role for induction of c-jun and TGF-beta 1 expression in the development of AM-induced pulmonary fibrosis in the Fischer 344 rat and provide potential targets for therapeutic intervention.

Topics: Amiodarone; Animals; Gene Expression Regulation; Humans; Hydroxyproline; Immunohistochemistry; Lung; Male; Proto-Oncogene Proteins c-jun; Pulmonary Fibrosis; Rats; Rats, Inbred F344; RNA, Messenger; Severity of Illness Index; Transforming Growth Factor beta; Transforming Growth Factor beta1

Idiopathic pulmonary fibrosis (IPF) is characterised by subpleural fibrosis that progresses to involve all areas of the lung. The expression of transforming growth factor-beta1 (TGF-beta 1), a potent regulator of connective tissue synthesis, is increased in lung sections of patients with IPF. TGF-beta 1 is generally released in a biologically latent form (L-TGF-beta 1). Before being biologically active, TGF-beta must be converted to its active form and interact with both TGF-beta receptors type I and II (T beta R-I and T beta R-II). TGF-beta latency binding protein 1 (LTBP-1), which facilitates the release and activation of L-TGF-beta 1, is also important in the biology of TGF-beta 1.. Open lung biopsy samples from patients with IPF and normal controls were examined to localise T beta R-I, T beta R-II, and LTBP-1. Alveolar macrophages (AM) and bronchoalveolar lavage (BAL) fluid were examined using the CCL-64 bioassay to determine if TGF-beta is present in its active form in the lungs of patients with IPF.. Immunoreactive L-TGF-beta 1 was present in all lung cells of patients with IPF except for fibroblasts in the subepithelial regions of honeycomb cysts. LTBP-1 was detected primarily in AM and epithelial cells lining honeycomb cysts in areas of advanced IPF. In normal lungs LTBP-1 immunoreactivity was observed in a few AM. AM from the upper and lower lobes of patients with IPF secreted 1.6 (0.6) fmol and 4.1 (1.9) fmol active TGF-beta, respectively, while AM from the lower lobes of control patients secreted no active TGF-beta (p< or =0.01 for TGF-beta in the conditioned media from AM obtained from the lower lobes of IPF patients v normal controls). The difference in percentage active TGF-beta secreted by AM from the lower lobes of patients with IPF and the lower lobes of control patients was significant (p< or =0.01), but the difference between the total TGF-beta secreted from these lobes was not significant. The difference in active TGF-beta in conditioned media of AM from the upper and lower lobes of patients with IPF was also not statistically significant. BAL fluid from the upper and lower lobes of patients with IPF contained 0.7 (0.2) fmol and 2.9 (1.2) fmol active TGF-beta, respectively (p< or =0.03). The percentage of active TGF-beta in the upper and lower lobes was 17.6 (1.0)% and 78.4 (1.6)%, respectively (p< or =0.03). In contrast, BAL fluid from control patients contained small amounts of L-TGF-beta. Using immunostaining, both T beta R-I and T beta R-II were present on all cells of normal lungs but T beta R-I was markedly reduced in most cells in areas of honeycomb cysts except for interstitial myofibroblasts in lungs of patients with IPF. TGF-beta 1 inhibits epithelial cell proliferation and a lack of T beta R-I expression by epithelial cells lining honeycomb cysts would facilitate repair of the alveoli by epithelial cell proliferation. However, the presence of both T beta Rs on fibroblasts is likely to result in a response to TGF-beta 1 for synthesis of connective tissue proteins. Our findings show that biologically active TGF-beta 1 is only present in the lungs of patients with IPF. In addition, the effects of TGF-beta 1 on cells may be further regulated by the expression of T beta Rs.. Activation of L-TGF-beta 1 and the differential expression of T beta Rs may be important in the pathogenesis of remodelling and fibrosis in IPF.

Topics: Bronchoalveolar Lavage Fluid; Case-Control Studies; Culture Media, Conditioned; Humans; Macrophages, Alveolar; Protein Serine-Threonine Kinases; Pulmonary Fibrosis; Receptor, Transforming Growth Factor-beta Type II; Receptors, Transforming Growth Factor beta; Transforming Growth Factor beta

To study the role of matrix metalloproteinase-2 (MMP(2)) in rat pulmonary fibrosis.. (1) Dynamic changes of MMP(2) expression were observed in bleomycin-induced rat pulmonary fibrosis by use of immunohistochemical technique. (2) RT-PCR was carried out to show the dynamic changes of MMP(2) mRNA expression of isolated pulmonary fibroblasts (PFbs) during rat pulmonary fibrosis. (3) Northern blot analysis and immunocyto chemistry techniques were performed to detect the expression of MMP(2) mRNA and their relevant protein in cultured rat PFbs after treatment with transforming growth factor 1 (TGF beta(1)).. (1) On day 1-7th after receiving bleomycin, the infiltrated pulmonary macrophages and septa mesenchymal cells showed positive reaction with anti-MMP(2) and an increase in number. After 14 days and on 28 th of bleomycin treatment, the number of positive staining macrophages and mesenchymal cells were decreased respectively. (2) The gene expression of MMP(2) mRNA of PFbs were enhanced after bleomycin treatment from the Day 1 and decreased by Day 28. (3) The expression of MMP(2) mRNA and their relevant protein were enhanced in rat PFbs after TGF beta(1) treatment.. Over expression of MMP(2) in pulmonary tissue may be an important factor in the initiation of pulmonary fibrogenesis.

Topics: Animals; Cells, Cultured; Disease Models, Animal; Fibroblasts; Gene Expression; Male; Matrix Metalloproteinase 2; Pulmonary Fibrosis; Rats; Rats, Sprague-Dawley; Transcription, Genetic; Transforming Growth Factor beta; Transforming Growth Factor beta1

The effects of taurine (T) and niacin (N) on bleomycin (BL)-induced increased production of tumor necrosis factor-alpha (TNF-alpha), interleukin (IL)-1alpha, IL-6, and transforming growth factor-beta (TGF-beta) levels in the bronchoalveolar lavage fluid (BALF), and increased collagen content and nuclear factor-kappaB (NF-kappaB) activation in the lungs were investigated in mice. The mice were intratracheally instilled with saline (SA) or BL (0.1 U/mouse/50 microliter) under ketamine and xylazine anesthesia. They had ad libitum access to diet containing 2.5% niacin (w/w) or the same control diet (CD) and water with and without taurine (1%) 3 days before intratracheal instillation and throughout the study. The mice were sacrificed at different times for collecting BALF and lungs, which were appropriately processed for various measurements. Treatment with taurine and niacin attenuated the BL-induced increases in proinflammatory cytokines such as IL-1alpha, TNF-alpha, IL-6, and TGF-beta in BALF and lung hydroxyproline content of the mice in BL + TN groups. Reverse transcription-polymerase chain reaction analysis of total RNA from whole lung was performed to assess the induction of TNF-alpha and IL-1 mRNAs as markers of NF-kappaB activation. The NF-kappaB DNA-binding activity in whole-lung extract was evaluated by electrophoretic mobility shift assay. This revealed a progressive increase in NF-kappaB activation and IkBalpha depletion in lungs from mice in BL + CD groups from day 1 through day 21 compared with the corresponding SA + CD control groups. Treatment with taurine and niacin generally inhibited the BL-induced increases in the nuclear localization of NF-kappaB and preserved IkappaBalpha protein in BL + TN groups. This may be one of the mechanisms for the antifibrotic effect of taurine and niacin.

Topics: Animals; Anti-Bacterial Agents; Bleomycin; Blotting, Western; Bronchoalveolar Lavage Fluid; Down-Regulation; Hydroxyproline; Interleukin-1; Interleukin-6; Lung Diseases; Male; Mice; Mice, Inbred C57BL; NF-kappa B; Niacin; Pulmonary Fibrosis; Reactive Oxygen Species; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Taurine; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha

Interleukin (IL)-10 has been shown to reduce many inflammatory reactions. We investigated the in vivo effects of IL-10 on a bleomycin-induced lung injury model. Hemagglutinating virus of Japan (HVJ)-liposomes containing a human IL-10 expression vector (hIL10-HVJ) or a balanced salt solution as a control (Cont-HVJ) was intraperitoneally injected into mice on day -3. This was followed by intratracheal instillation of bleomycin (0.8 mg/kg) on day 0. Myeloperoxidase activity of bronchoalveolar lavage fluid and tumor necrosis factor-alpha mRNA expression in bronchoalveolar lavage fluid cells on day 7 and hydroxyproline content of the whole lung on day 21 were inhibited significantly by hIL10-HVJ treatment. However, Cont-HVJ treatment could not suppress any of these parameters. We also examined the in vitro effects of IL-10 on the human lung fibroblast cell line WI-38. IL-10 significantly reduced constitutive and transforming growth factor-beta-stimulated type I collagen mRNA expression. However, IL-10 did not affect the proliferation of WI-38 cells induced by platelet-derived growth factor. These data suggested that exogenous IL-10 may be useful in the treatment of pulmonary fibrosis.

Topics: Animals; Antibiotics, Antineoplastic; Bleomycin; Cell Division; Cells, Cultured; Collagen; Fibroblasts; Gene Expression; Gene Transfer Techniques; Humans; Interleukin-10; Mice; Mice, Inbred C57BL; Platelet-Derived Growth Factor; Pneumonia; Pulmonary Fibrosis; Respirovirus; RNA, Messenger; Specific Pathogen-Free Organisms; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha

Topics: Animals; Bleomycin; Disease Models, Animal; fas Receptor; Humans; Lung; Pulmonary Fibrosis; Transforming Growth Factor beta

Topics: Animals; Dose-Response Relationship, Radiation; Humans; Lung; Pulmonary Fibrosis; Radiation Pneumonitis; Radiotherapy; Transforming Growth Factor beta

To study the influence of TGF-beta1 and TGF-beta-neutralizing antibodies on the clonogenic activity and terminal differentiation of rat lung fibroblasts following radiation exposure.. Early passage rat lung fibroblasts were used in this study. Colony formation assays were applied to determine the radiation sensitivity as well as radiation-induced alterations in differentiation pattern. Based on a TGF-beta1-specific ELISA system, the amount of TGF-beta1 in the culture medium of sham-irradiated and irradiated cultures was determined.. Applying immediate (ip) and delayed plating (dp) procedures for cells irradiated in the subconfluent state, distinct differences in the radiation dose-response curves could be observed (SF2ip 0.20+/-0.025 versus SF2dp 0.51+/-0.0077). Upon irradiation with a single dose of 4Gy the level of TGF-beta1 found in the culture medium increased by about 60%. Radiation-induced terminal differentiation of progenitor fibroblasts (MF) to postmitotic fibrocytes (PMF) was expressed by the change of the ratio of PMF:MF (0.8 at 0 Gy versus 3.0 at 4 Gy). Neutralizing antibodies directed against TGF-beta inhibited both the radiation-induced reduction in clonogenic activity of rat lung fibroblasts as well as the radiation-induced terminal differentiation of MF progenitor fibroblasts to PMF postmitotic fibrocytes.. The results indicate the important role of TGF-beta1 in triggering radiation-induced inhibition of clonogenic activity as well as terminal differentiation of rat lung fibroblasts. The data presented support the hypothesis that terminal differentiation is an essential cellular process in the development of radiation-induced fibrosis in the lung.

Topics: Animals; Cell Differentiation; Cyclin-Dependent Kinase Inhibitor p21; Cyclins; Extracellular Matrix Proteins; Fibroblasts; Phenotype; Pulmonary Fibrosis; Radiation Injuries, Experimental; Rats; Transforming Growth Factor beta

The lung is the major dose-limiting organ for radiotherapy of cancer in the thoracic region. The pathogenesis of radiation-induced lung injury at the molecular level is still unclear. Immediate cellular damage after irradiation is supposed to result in cytokine-mediated multicellular interactions with induction and progression of fibrotic tissue reactions. The purpose of this investigation was to evaluate the acute and long-term effects of radiation on the gene expression of transforming growth factor beta (TGF-beta) in a model of lung injury using fibrosis-sensitive C57BL/6 mice.. The thoraces of C57BL/6 mice were irradiated with 6 and 12 Gy, respectively. Treated and sham-irradiated control mice were sacrificed at times corresponding to the latent period (1, 3, 6, 12, 24, 48, 72 hours and 1 week postirradiation), the pneumonic phase (2, 4, 8, and 16 weeks postirradiation), and the beginning of the fibrotic phase (24 weeks postirradiation). The lung tissue from three different mice per dosage and time point was analyzed by a combination of polymerase chain reaction (PCR), immunohistochemistry, and light microscopy. The mRNA expression of TGF-beta was quantified by competitive reverse transcriptase/polymerase chain reaction (RT-PCR); the cellular origin of the TGF-beta protein was identified by immunohistochemical staining (alkaline phosphatase-anti-alkaline phosphatase [APAAP]). The cytokine expression on mRNA and protein level was correlated with the histopathological alterations.. Following thoracic irradiation with a single dose of 12 Gy, radiation-induced TGF-beta release in lung tissue was appreciable already within the first hours (1, 3, and 6 hours postirradiation) and reached a significant increase after 12 hours; subsequently (48 hours, 72 hours, and 1 week postirradiation) the TGF-beta expression declined to basal levels. At the beginning of the pneumonic phase, irradiation-mediated stimulation of TGF-beta release reached maximal values at 2 and 4 weeks. The elevated levels of TGF-beta mRNA during the latent phase have been found to correlate with immunohistochemical staining of alveolar macrophages. The most striking increase in TGF-beta immunoreactivity was seen during the acute phase of pneumonitis. Throughout this observation period, type II pneumocytes and fibroblasts (apart from inflammatory cells) served as important sources of TGF-beta expression. Increased TGF-beta expression was detected prominently in regions of histopathologic radiation injury. After exposure to a single radiation dose of 6 Gy, the lung tissue revealed only a minor radiation-mediated TGF-beta mRNA response. The modest upregulation ranged from 6 hours to 48 hours after irradiation. Corresponding to the only minor histopathologic changes after thoracic irradiation with 6 Gy, measurement of TGF-beta mRNA levels during the later time points revealed no significant alterations in comparison to untreated control mice.. This study demonstrates an acute and long-lasting increase in the expression of TGF-beta in lung tissue following thoracic irradiation with 12 Gy. The predominant localization of TGF-beta in areas of inflammatory cell infiltrates and fibrosis suggests involvement of this cytokine in the pathogenesis of radiation-induced pulmonal fibrosis. Further studies should be performed to explore the role of other cytokines in the development of radiation injury. An improved understanding of the underlying mechanisms of pulmonary fibrosis may eventually lead to modulatory intervention at the molecular level to modify the fibrotic process.

Topics: Animals; Dose-Response Relationship, Radiation; Female; Gene Expression; Lung; Mice; Mice, Inbred C57BL; Polymerase Chain Reaction; Pulmonary Fibrosis; Radiation Pneumonitis; Radiobiology; RNA, Messenger; Time Factors; Transforming Growth Factor beta

We have previously reported the antifibrotic effects of pirfenidone (PD) in the bleomycin (BL)-hamster model of lung fibrosis. Since the development of fibrosis is generally preceded by acute lung inflammation, the present study was conducted to find out if dietary intake of PD (0.5%) has any effects on BL-induced lung inflammation. In this regard, we evaluated the effects of PD on BL-induced increased pulmonary vascular permeability, increased influx of inflammatory cells and increased levels of TGF-beta in the bronchoalveolar lavage fluid (BALF). Hamsters were intratracheally (IT) instilled with saline (SA) or BL (5.5 units/kg/5 ml). The animals were fed the control diet (CD) or the same diet containing 0.5% PD 2 days prior to IT instillation and throughout the study. The bronchoalveolar lavage was carried out at different times after IT instillation. Lavage fluid was used for total and differential cell counts and BALF-supernatant for measurement of total protein and TGF-beta. IT instillation of BL caused significant increases in total cells, neutrophils, macrophages and lymphocytes and in the levels of total protein and TGF-beta in BALF from hamsters in the BL + CD groups as compared to the corresponding SA + CD control groups. In contrast, treatment with pirfenidone in general, suppressed the BL-induced increases in the levels of proteins and TGF-beta and in the influx of neutrophils, macrophages and lymphocytes in BALF at the early time points in BL + PD groups. Based on the data reported in this study, we conclude that the anti-inflammatory effects of pirfenidone as evident by suppressions of BL-induced increased pulmonary vascular permeability and increased influx of inflammatory cells in the lung contribute additionally to its inherent anti-fibrotic effect.

Topics: Administration, Oral; Animal Feed; Animals; Anti-Inflammatory Agents, Non-Steroidal; Bleomycin; Bronchoalveolar Lavage Fluid; Capillary Permeability; Cricetinae; Inflammation; Macrophages, Alveolar; Male; Mesocricetus; Neutrophil Infiltration; Pulmonary Fibrosis; Pyridones; Transforming Growth Factor beta

Fibroblast growth factor (FGF)-2, which stimulates DNA synthesis by type II cells in the lung, has been shown to be regulated by transforming growth factor (TGF)-beta1, an important inflammatory cytokine, in vascular epithelium. The goal of this study was to determine if FGF-2 production by alveolar type II cells is modulated by TGF-beta1 or FGF-1, which also stimulates DNA synthesis by type II cells. Isolated rat type II cells were exposed to 0-40 ng/ml of TGF-beta1 or 0-500 ng/ml of FGF-1 in serum-free medium for 1-5 days. With a specific immunoassay, significant increases of FGF-2 protein in type II cell lysates to levels above those in control cells were achieved after 1 day of exposure to 100 ng/ml of FGF-1 and after 3 days of treatment with 8 ng/ml of TGF-beta1. Similarly, transcripts for FGF-2 were dramatically increased above those in control cells with TGF-beta1 or FGF-1, as were those for FGF receptor-1. These results demonstrate important regulatory links between FGF-2 and both TGF-beta1 and FGF-1 in the alveolar epithelium that could contribute to the regulation of normal cell turnover, development, and the repair processes after injury in the lung.

Topics: Animals; Cells, Cultured; Dose-Response Relationship, Drug; Fibroblast Growth Factor 1; Fibroblast Growth Factor 2; Gene Expression; Pulmonary Alveoli; Pulmonary Fibrosis; Rats; Rats, Inbred F344; Receptor Protein-Tyrosine Kinases; Receptor, Fibroblast Growth Factor, Type 1; Receptors, Fibroblast Growth Factor; RNA, Messenger; Specific Pathogen-Free Organisms; Transforming Growth Factor beta

Since transforming growth factor beta (TGF-beta) is presumed to play a role in lung fibrosis, we evaluated the effect of suramin (Sur), a substance with an anti-TGF-beta effect, in vivo on bleomycin (Bleo)-induced pulmonary injury in mice and in vitro on human lung fibroblasts. Four groups of C57BL/6 mice each received one of four treatments: (1) intratracheal (i.t.) instillation of Bleo and intraperitoneal (i.p.) injections of Sur, every other day, starting one day before i.t. instillation of Bleo (Bleo-Sur); (2) i.t. Bleo and i.p. injections of saline (Bleo-Sal); (3) i.t. saline and i.p. Sur (Sal-Sur); and (4) i.t. and i.p. saline (Sal-Sal). Animals were sacrificed 14 days after i.t. treatment. Lung injury was evaluated by analysis of bronchoalveolar lavage (BAL) fluid, histologically by the semiquantitative morphological index, and biochemically by analysis of lung hydroxyproline content. In vitro, Sur did not affect TGF-beta induced increase of alpha1 (I) collagen mRNA in human lung fibroblasts. In vivo treatment of mice with Sur did not affect Bleo-induced lung injury. These results indicate that despite its potential anti TGF-beta and lymphocytotoxic effects, Sur is not a therapeutic candidate drug for rescue of lung fibrosis.

Topics: Animals; Bleomycin; Bronchoalveolar Lavage Fluid; Cell Line; Collagen; Fibroblasts; Humans; Injections, Intraperitoneal; Instillation, Drug; Luciferases; Lung; Lymphocytes; Macrophages; Male; Mice; Mice, Inbred C57BL; Neutrophils; Promoter Regions, Genetic; Pulmonary Fibrosis; Suramin; Transfection; Transforming Growth Factor beta

To identify the role of cytokines involved in the development of lung fibrosis in patients with idiopathic-pulmonary fibrosis (IPF).. Proteins and gene expression of platelet-derived growth factor (PDGF)-A and -B, insulin-like growth factor 1 (IGF-1), and transforming growth factor beta (TGF-beta) were measured in alveolar macrophages and open lung biopsies from patients with IPF using immunohistochemistry (IHC) and in situ hybridization (ISH).. In specimens of bronchoalveolar lavage fluid (BALF), PDGF-A, PDGF-B, IGF-1, TGF-beta were localized in alveolar macrophages. Evaluation of open lung biopsies from patients with IPF showed that IGF-1 was prominently present in pulmonary vessel walls in fibrotic lesions. PDGF and TGF-beta proteins were localized to hyperplastic bronchio-alveolar epithelial cells, alveolar macrophages, fibroblasts, vascular smooth muscle and endothelial cells. Our in situ hybridization results were consistent with that of immunohistochemistry except that PDGF-A and TGF-beta mRNA transcripts were not detected in bronchoalveolar epithelial cells.. These observations suggest that (1) alveolar macrophages play key roles not only in inflammation but also in the fibrotic process by releasing PDGF, IGF-1 and TGF-beta; (2) IGF-1 could be responsible for angiogenesis in IPF; (3) PDGF, TGF-beta are associated with fibroplasia and the deposition of extracellular matrix, as well as vessel remodeling and epithelial cell repopularization.

Topics: Adult; Female; Gene Expression; Growth Substances; Humans; Immunohistochemistry; In Situ Hybridization; Insulin-Like Growth Factor I; Lung; Macrophages, Alveolar; Male; Middle Aged; Platelet-Derived Growth Factor; Pulmonary Fibrosis; RNA, Messenger; Transforming Growth Factor beta

To evaluate the therapeutic effect and mechanism of folium Ginkgo biloba (FGB) in treating pulmonary interstitial fibrosis (PIF) induced by bleomycin in rats.. PIF models of bleomycin-A5-induced pulmonary fibrosis were established in rats, they were treated by Bailuda, a preparation of FGB, and the pathological changes, collagen protein level, nuclear factor kB(NF-kB) activity, transforming growth factor beta (TGF-beta) mRNA expression and protein level of the lung tissue were measured.. In the Bailuda treated group after treatment, amelioration of the pulmonary alveolitis and fibrosis were shown in pathological section (P < 0.05) and collagen protein content was lesser (P < 0.01) as compared with those in the model control group. After 1 week of Bailuda treatment, the NF-kB activity of pulmonary alveola macrophage lowered by 47.3%, and levels of TGF-beta mRNA expression and protein were all decreased (P < 0.05).. Bailuda has definite effect in treating PIF. The mechanism may be through inhibiting the activity of NF-kB, decreasing TGF-beta mRNA expression and protein, so as to ameliorate the inflammation and fibrosis.

Topics: Animals; Bleomycin; Collagen; Drugs, Chinese Herbal; Ginkgo biloba; Lung; Male; NF-kappa B; Phytotherapy; Plant Leaves; Pulmonary Fibrosis; Rats; Rats, Wistar; Transforming Growth Factor beta

Transforming growth factor beta (TGF beta) family members are secreted in inactive complexes with a latency-associated peptide (LAP), a protein derived from the N-terminal region of the TGF beta gene product. Extracellular activation of these complexes is a critical but incompletely understood step in regulation of TGF beta function in vivo. We show that TGF beta 1 LAP is a ligand for the integrin alpha v beta 6 and that alpha v beta 6-expressing cells induce spatially restricted activation of TGF beta 1. This finding explains why mice lacking this integrin develop exaggerated inflammation and, as we show, are protected from pulmonary fibrosis. These data identify a novel mechanism for locally regulating TGF beta 1 function in vivo by regulating expression of the alpha v beta 6 integrin.

Topics: 3T3 Cells; Animals; Antigens, Neoplasm; Bleomycin; CHO Cells; Cricetinae; Epithelial Cells; Esophagus; Humans; Integrins; Keratinocytes; Ligands; Mice; Mice, Knockout; Peptide Fragments; Protein Binding; Protein Precursors; Proteins; Pulmonary Fibrosis; Transforming Growth Factor beta; Transforming Growth Factor beta1; Tumor Cells, Cultured

Transforming growth factor-beta1 (TGF-beta1) is known as the growth factor that stimulates the synthesis of extracellular matrix. Recently, TGF-beta has been found to control the growth of cancer cells. Small chondroitin-dermatan sulfate (decorin) is an abundant extracellular matrix component. TGF-beta1 stimulates the synthesis of decorin, and decorin is considered to bind TGF-beta1. The activity of decorin in neutralizing TGF-beta1 activity suggests that decorin serves as a negative-feedback regulator of TGF-beta1 activity. To investigate the role and relationship of TGF-beta1 and decorin in the formation of central fibrosis in pulmonary adenocarcinoma, we performed an immunohistochemical study of TGF-beta1 and decorin in 61 cases of T1 pulmonary adenocarcinoma. Positive stainings for TGF-beta1 were shown in 40 cases and negative in 21 cases. Twenty-seven of 32 cases with central fibrosis were positive for TGF-beta1. Positive staining for TGF-beta1 was significantly related to the appearance of central fibrosis in pulmonary adenocarcinoma. When central fibrosis was composed of proliferative connective tissue with loose staining for decorin, cancer cells showed intense staining for TGF-beta1. When central fibrosis was composed of old fibrotic tissue with dense staining for decorin, cancer cells showed weak staining for TGF-beta1. Our results suggest that TGF-beta1 has an important role in the formation of central fibrosis in pulmonary adenocarcinoma, and decorin may play a role as a negative feedback regulator in the production of TGF-beta1 in pulmonary adenocarcinoma.

Topics: Adenocarcinoma; Decorin; Extracellular Matrix Proteins; Humans; Immunohistochemistry; Lung Neoplasms; Proteoglycans; Pulmonary Fibrosis; Survival Rate; Transforming Growth Factor beta

Tumor necrosis factor (TNF)-alpha and transforming growth factor (TGF)-beta mRNA and protein expression and the degree of fibroproliferative response to inhaled asbestos fibers are clearly reduced in the 129 inbred mouse strain as compared with typical fibrogenesis observed in the C57BL/6 inbred strain. The C57BL/6 mice showed prominent lesions at bronchiolar-alveolar duct (BAD) junctions where asbestos fibers deposit and responding macrophages accumulate. The 129 mice, however, were generally indistinguishable from controls even though the numbers of asbestos fibers deposited in the lungs of all exposed animals were the same. Quantitative morphometry of H&E-stained lung sections comparing the C57BL/6 and 129 mice showed significantly less mean cross-sectional area of the BAD junctions in the 129 animals, apparent at both 48 hours and 4 weeks after exposure. In addition, fewer macrophages had accumulated at these sites in the 129 mice. Nuclear bromodeoxyuridine immunostaining demonstrated that the number of proliferating cells at first alveolar duct bifurcations and in adjacent terminal bronchioles was significantly reduced in the 129 strain compared with C57BL/6 mice at 48 hours after exposure (P < 0.01). TNF-alpha and TGF-beta1 gene expression, as measured by in situ hybridization, was reduced in the 129 mice at 48 hours after exposure, and expression of TNF-alpha and TGF-beta1 protein, as measured by immunohistochemistry, was similarly reduced or absent in the 129 animals. We postulate that the protection afforded the 129 mice is related to reduction of growth factor expression by the bronchiolar-alveolar epithelium and lung macrophages.

Topics: Animals; Asbestos; Cell Count; Cell Division; Lung; Macrophages; Male; Mice; Mice, Inbred C57BL; Microscopy, Electron, Scanning; Pulmonary Fibrosis; RNA, Messenger; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha

The expression of alphaEbeta7 integrin has been related to the selective retention of lymphocytes in mucosal tissues of gut, urogenital tract and lung. To identify potential disease-associated alphaEbeta7 expression patterns on cells accounting for lymphocytic alveolitis in interstitial lung disease (ILD), alphaE expression on CD4+ and CD8+ T cell subsets was evaluated by dual-colour flow cytometry in peripheral blood and bronchoalveolar lavage fluid (BALF) of patients with idiopathic pulmonary fibrosis (IPF; n = 18), hypersensitivity pneumonitis (HP; n = 20) and sarcoidosis (n = 44) in comparison with healthy controls (n = 15). In both healthy individuals and all patient groups the proportion of alphaE-bearing T cells in peripheral blood was < 2%, whereas the vast majority of alveolar CD8+ T cells consistently co-expressed alphaE. Absolute alveolar CD8+alphaE+ cell numbers/ml were up to 30-fold increased in HP patients. Proportions of alphaE-bearing CD4+ cells in BALF were significantly elevated in IPF (74.0 +/- 2.7%) and HP (70.0 +/- 2.4%) compared with normals (30.0 +/- 1.8%) (mean +/- s.e.m.; P < 0.01). In sarcoidosis, the alphaE expression on BALF CD4+ cells displayed subgroup dependency: proportions significantly lower than normal were noted in chest radiographic stage I (14.3 +/- 1.5%), but increased proportions in stages II (50.0 +/- 3.8%) and III (64.0 +/- 4.8%). Correlations between common markers of T cell activation or BALF transforming growth factor-beta (TGF-beta ) bioactivity and alphaE expression were not noted. We conclude that the vast majority of alveolar CD8+ T cells consistently express alphaEbeta7 and that distinct patterns of alphaEbeta7 expression on alveolar CD4+ lymphocytes in sarcoidosis are related to the diverse manifestations of the sarcoid inflammatory process in the lung.

Topics: Adolescent; Adult; Aged; Alveolitis, Extrinsic Allergic; Bronchoalveolar Lavage Fluid; CD4-Positive T-Lymphocytes; CD8-Positive T-Lymphocytes; Female; Humans; Integrins; Male; Middle Aged; Pulmonary Fibrosis; Sarcoidosis; Transforming Growth Factor beta

TGF-beta plays an important role in lung fibrosis, which is a major cause of suffering and death seen in pulmonary disease. Smad7 has been recently identified as an antagonist of TGF-beta signaling. To investigate whether this novel molecule can be exploited for therapy of lung fibrosis, we determined the effect of exogenous Smad7, introduced by a recombinant human type 5 adenovirus vector, on bleomycin-induced lung fibrosis in mice. C57BL/6 mice with bleomycin-induced lungs received an intratracheal injection of a recombinant adenovirus carrying mice Smad7 cDNA. These mice demonstrated suppression of type I precollagen mRNA, reduced hydroxyproline content, and no morphological fibrotic responses in the lungs when compared with mice administered adenovirus carrying Smad6 cDNA. In addition, we found that expression of Smad7 transgene blocked Smad2 phosphorylation induced by bleomycin in mouse lungs. These data indicated that gene transfer of Smad7 (but not Smad6) prevented bleomycin-induced lung fibrosis, suggesting that Smad7 may have applicability in the treatment of pulmonary fibrosis.

Topics: Adenoviridae; Animals; Bleomycin; Bronchoalveolar Lavage Fluid; Collagen; Cytomegalovirus; DNA-Binding Proteins; DNA, Complementary; Female; Gene Expression Regulation; Genetic Therapy; Genetic Vectors; Mice; Mice, Inbred C57BL; Pulmonary Fibrosis; Recombinant Fusion Proteins; Smad6 Protein; Smad7 Protein; Trans-Activators; Transfection; Transforming Growth Factor beta

TGF-beta1 is a prosclerotic cytokine implicated in fibrotic processes. Fibrosis of the pulmonary parenchyma and airways is a frequent presentation in lung transplant recipients before and after transplantation. There are two genetic polymorphisms in the DNA sequence encoding the leader sequence of the TGF-beta1 protein, located at codon 10 (either leucine or proline) and at codon 25 (either arginine or proline). The codon 25 arginine allele is associated with higher TGF-beta1 production by cells activated in vitro. We tested the hypothesis that inheritance of alleles of the TGF-beta1 gene conferring higher production of TGF-beta1 may be responsible for over-expression of TGF-beta1 in transplant recipients resulting in lung allograft fibrosis.. We extracted DNA from leukocytes collected from 91 pulmonary transplants performed at our centre and 96 normal healthy volunteers between May 1990 and September 1995. Part of the first exon was amplified by PCR. Samples were genotyped by using sequence specific oligonucleotide probes.. The distribution of codon 10 alleles was similar in a normal healthy control group and in lung transplant recipients, regardless of their pretransplant lung pathology. By contrast, there was a significant difference in the frequency of codon 25 alleles between the control and transplant groups. In the normal control group 81% were codon 25 arginine/arginine (A/A) homozygotes, 19% were arginine/proline (A/P) heterozygotes and none were proline/proline (P/P) homozygotes. The distribution of codon 25 alleles was similar in lung transplant recipients who did not have a significant fibrosis in pretransplant pathology, but in transplant recipients who came to transplantation with lung fibrosis 98% (41 of 42 patients) were homozygous for the codon 25 A/A allele (p < .05). After lung transplantation 39 of 91 patients developed lung allograft fibrosis, and of these 92.3% (36 of 39 recipients) were of homozygous codon 25 A/A high TGF-beta1 producer genotype (p < .001). Lung transplant recipients who were homozygous for both codon 10 L/L and codon 25 A/A showed poor survival compared with all other TGF-beta1 genotypes (p < .03).. Homozygosity for arginine at codon 25 of the leader sequence of TGF-beta1 that correlates with higher TGF-b production in vitro, is associated with fibrotic lung pathology before lung transplantation and with the development of fibrosis in the graft. In combination with the codon 10 leucine allele, homozygosity for the codon 25arginine allele is a marker for poor post-transplant prognosis and recipient survival.

Topics: Adolescent; Adult; Alleles; Amino Acid Sequence; Codon; Exons; Female; Gene Expression Regulation; Genotype; Graft Rejection; Humans; Lung; Lung Transplantation; Male; Middle Aged; Oligonucleotide Probes; Polymerase Chain Reaction; Promoter Regions, Genetic; Pulmonary Fibrosis; Reference Values; Survival Rate; Transforming Growth Factor beta

Animals exposed to silica or bleomycin (BLM) develop pulmonary fibrosis. Tetrandrine (TT) has been shown to inhibit stimulant-induced macrophage respiratory burst and effectively reduce silica-induced lung injury. The present study employed TT as a probe to assess the differences in mechanisms involved in silica- and BLM-induced pulmonary responses. Rats received a single intratracheal instillation of silica (40 mg/rat, sacrificed 4 wk postexposure) or BLM (1 mg/kg or approximately 0.25 mg/rat, sacrificed up to 2 wk postexposure). TT was administered orally at 18 mg/kg, 3 times/wk for desired time periods beginning 5 d before silica or BLM exposure. Both the silica and BLM exposures resulted in a significant increase in lung weight, total protein, lactate dehydrogenase (LDH), and phospholipids (PL) content in the acellular fluid from the first lavage, and hydroxyproline content in the lung tissue. Alveolar macrophages (AM) isolated from rats exposed to silica or BLM exhibited significant increases in secretion of interleukin-1 (IL-1), tumor necrosis factor alpha (TNF-alpha), and transforming growth factor beta (TGF-beta). TT treatment significantly lowered the silica- or BLM-induced increase in lung weight, while marginally reducing the release of IL-1 and TNF-alpha by AM. TT, however, markedly inhibited the silica-induced increase in the acellular protein, LDH and PL, hydroxyproline content, and the production of TGF-beta by AM but had no marked effect on these same parameters in BLM-exposed rats. Histological examination of rats exposed to BLM for 14 d showed pulmonary inflammation and fibrosis. TT treatment had only a small effect on limiting the extent of these lesions and did not significantly affect their severity. In summary, data indicate that many inflammatory and fibrotic effects of in vivo silica exposure are substantially attenuated by TT, whereas the stimulation by BLM is only marginally affected by this drug. Since TT acts to attenuate AM-mediated reactions, these results suggest that AM may play a pivotal role in silica-induced fibrotic development and may be less involved in the pathogenesis of BLM-induced fibrosis.

Topics: Alkaloids; Animals; Anti-Inflammatory Agents, Non-Steroidal; Antibiotics, Antineoplastic; Benzylisoquinolines; Bleomycin; Bronchoalveolar Lavage Fluid; Cell Count; Hydroxyproline; Interleukin-1; L-Lactate Dehydrogenase; Luminescent Measurements; Lung; Macrophages, Alveolar; Male; Organ Size; Phospholipids; Proteins; Pulmonary Fibrosis; Rats; Rats, Sprague-Dawley; Silicon Dioxide; Time Factors; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha

Eosinophils have been associated with fibrosis. To investigate their direct role in fibrosis, human peripheral blood eosinophil sonicate was added to human lung or dermal fibroblasts, and proliferation ([(3)H]thymidine) and collagen synthesis ([(3)H]proline) were evaluated. Proliferation was enhanced significantly in the monolayers in a dose-dependent manner. The activity of the eosinophil fibrogenic factor(s) remained unaltered when heated (56 degrees C, 30 min). Supernatants of cultured eosinophils (20 min or 18 hr) also enhanced lung fibroblast proliferation, indicating that the preformed mitogenic factor(s) can be released both promptly and with a long kinetic. Eosinophils significantly decreased collagen production in lung fibroblasts while increasing it in dermal fibroblasts. However, eosinophils containing matrix metalloproteinase 9 (zymography) in latent form and tissue inhibitors of metalloproteinases 1 and 2 (reverse zymography) did not influence either fibroblast matrix metalloproteinases or tissue inhibitors of metalloproteinases. Eosinophil sonicate added to skin and lung fibroblasts in tridimensional collagen lattices significantly enhanced lattice contraction. Transforming growth factor beta (TGF-beta) is a major fibrogenic cytokine produced by eosinophils. Therefore, to assess its role, eosinophil sonicate was preincubated with anti-TGF-beta neutralizing antibodies. This treatment partially inhibited proliferation of lung and collagen synthesis of dermal fibroblasts and suppressed the stimulation of lattice contraction, indicating the fibrogenic role of eosinophil-associated TGF-beta. In conclusion, we have shown that eosinophils act as direct modulatory cells in fibroblast proliferation, collagen synthesis, and lattice contraction, in part, through TGF-beta. These data corroborate the importance of eosinophils in skin and lung fibrosis.

Topics: Cell Division; Cells, Cultured; Collagen; Eosinophils; Fibroblasts; Fibrosis; Humans; Lung; Pulmonary Fibrosis; Skin; Skin Diseases; Transforming Growth Factor beta

Radiation pneumonitis remains a critical dose-limiting toxicity of total body irradiation (TBI) for use in bone marrow transplantation. The acute and chronic phases of radiation damage in the mouse lung have been shown to correlate with mouse strain genotype and are dependent on fraction size, total dose, and total lung volume. Our prior studies demonstrated effective prevention of irradiation-induced lung damage and improved survival in C57BL/6J mice by MnSOD plasmid/liposome gene therapy. In the present studies, we investigated the kinetics of irradiation-induced upregulation of mRNA for acute phase cytokines interleukin (IL)-1 and tumor necrosis factor (TNF)-alpha, and fibrosis-associated transforming growth factor (TGF)-beta and isoforms (TGF-beta1, TGF-beta2 and TGF-beta3) in 2000 cGy whole-lung irradiated C57BL/6J mice, a strain known to develop dose and volume-dependent organizing alveolitis/fibrosis. The results demonstrate increase in mRNA for IL-1 between days 1 and 14 after irradiation with return to baseline levels out to 120 days. TNF-alpha mRNA levels were not initially elevated but increased between 80 and 100 days and then decreased by 120 days. The mRNA levels for TGF-beta1 demonstrated an initial increase within the first 14 days after total lung irradiation with a decrease to baseline levels out to 100 days. Then, in striking contrast to the other two cytokines, an increase in TGF-beta2 mRNA occurred at around 120 days and correlated with the detection of organizing alveolitis/radiation fibrosis and mortality. These results are consistent with a two-phase mechanism in the molecular pathology of irradiation lung injury, in which IL-1 cytokine mRNA levels correlated with the acute pneumonitis phase and delayed elevation of TNF-alpha (80-100 days), TGF-beta1 (100 days), and TGF-beta2 (120 days) were associated with the fibrosis phase. Insight into the cell-specific and tissue-specific molecular mechanisms of ionizing irradiation induction of mRNA for pulmonary cytokines may provide new strategies for treatment of radiation pneumonitis in TBI patients.

Topics: Animals; Cytokines; Female; Genetic Therapy; Interleukin-1; Liposomes; Lung; Lung Diseases; Lung Injury; Mice; Mice, Inbred C57BL; Plasmids; Pulmonary Alveoli; Pulmonary Emphysema; Pulmonary Fibrosis; Radiation-Protective Agents; RNA, Messenger; Superoxide Dismutase; Time Factors; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha

This study was undertaken to investigate whether treatment with the antifibrotic drug pirfenidone (PD) down-regulates the bleomycin (BL)-induced overexpression of transforming growth factor (TGF)-beta gene in the lungs. Hamsters were intratracheally instilled with SA or BL (6.5 U/kg/4 ml) under anesthesia. They were fed a diet containing 0.5% PD or the same control diet (CD) without the drug 2 days before and throughout the study. After the animals were sacrificed, their lungs were appropriately processed. The BL treatment elevated the total influx of inflammatory cells, including macrophages, by severalfold at different days in bronchoalveolar lavage fluid (BALF) from hamsters in BL + CD groups, relative to the corresponding SA + CD control groups. Treatment with PD significantly (P

Topics: Animals; Anti-Inflammatory Agents, Non-Steroidal; Bleomycin; Bronchoalveolar Lavage Fluid; Cricetinae; Down-Regulation; Lung; Macrophages; Male; Mesocricetus; Pulmonary Fibrosis; Pyridones; Transcription, Genetic; Transforming Growth Factor beta

Obliterative bronchiolitis (OB) characterised by small-airway fibrosis is a major cause of morbidity and mortality after lung transplantation. TGF-beta has been implicated in the pathogenesis of fibrosis.. We immunohistochemically examined 380 transbronchial biopsies (from 91 pulmonary transplants) using TGF-beta polyclonal antibodies. OB and interstitial fibrosis were diagnosed and graded in all biopsies. Other potential histologic and clinical risk factors for OB were analysed.. Procedures were heart and lung (n = 32), bilateral sequential lung (n = 18), and single lung transplantation (n = 41). The incidence of OB in this group was 28.5%. In all patients with OB, TGF-beta was immunolocalized in the airways and lung parenchyma. TGF-beta expression was greater in OB patients (median score 8, range 5-12) in comparison to patients without OB (median score 4, range 1-13), p < .0001. Positive TGF-beta staining preceded the histologic confirmation of OB by 6 to 18 months. The development of OB was associated with two HLA mismatches at the A locus (p = .02); recurrent acute rejection episodes (p < .0005); lymphocytic bronchiolitis (p = .0001); and tissue eosinophilia, regardless of the rejection grade (p < .0001).. Increased expression of TGF-beta is a risk factor for the development of OB. Other risk factors are recurrent acute rejection, lymphocytic bronchiolitis, tissue eosinophilia, and two mismatches at the HLA-A locus. This suggests that the pathogenesis of progressive small airway fibrosis characteristic of OB may be inflammatory damage, followed by an aberrant repair process due to excessive TGF-beta production following allograft injury. Hence, modulation of TGF-beta levels or function by antagonists may represent an important approach to control OB.

Topics: Adult; Bronchiolitis Obliterans; Histocompatibility; HLA Antigens; Humans; Immunohistochemistry; Lung; Lung Transplantation; Middle Aged; Pulmonary Fibrosis; Retrospective Studies; Risk Factors; Transforming Growth Factor beta

Scleroderma, a debilitating acquired connective tissue disease, is characterized by fibrosis, particularly of the skin and lungs. Monocyte-produced TGF-beta1, a potent stimulus for collagen synthesis, is thought to drive the fibrosis. Here, we thoroughly characterize a murine sclerodermatous graft-vs-host disease (Scl GVHD) model for scleroderma that reproduces important features of scleroderma including skin thickening, lung fibrosis, and up-regulation of cutaneous collagen mRNA, which is preceded by monocyte infiltration and the up-regulation of cutaneous TGF-beta1 mRNA. Most importantly, we can prevent fibrosis in both the skin and lungs of mice with Scl GVHD by inhibiting TGF-beta with neutralizing Abs. The murine Scl GVHD model provides the unique opportunity to study basic immunologic mechanisms that drive fibrosing diseases and GVHD itself and will be useful for testing new therapies for these diseases.

Topics: Animals; Bone Marrow Transplantation; Cell Movement; Collagen; Disease Models, Animal; Female; Fibrosis; Graft vs Host Disease; Immune Sera; Injections, Intravenous; Leukocytes, Mononuclear; Macrophage-1 Antigen; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Pulmonary Fibrosis; RNA, Messenger; Scleroderma, Systemic; Skin; Transforming Growth Factor beta; Up-Regulation

Accumulation of monocytes and neutrophils and fibrous distortion of the airway are characteristics of airway disease secondary to smoking. The presence of inflammatory cells and fibrosis correlate, and, therefore, we postulated that lung fibroblasts might release chemotactic activity for neutrophils and monocytes in response to smoke extract. To test this hypothesis, human fetal lung (HFL1) fibroblasts were cultured, and the supernatant fluid was evaluated for neutrophil (NCA) and monocyte (MCA) chemotactic activities with a blind well chamber technique. HFL1 fibroblasts released chemotactic activity in response to smoke extract in a dose- and time-dependent manner (P < 0.05). Checkerboard analysis showed that the activity was predominantly chemotactic. Partial characterization of the released chemotactic activity revealed that the activity was partly heat labile, trypsin sensitive, and ethyl acetate extractable. Lipoxygenase inhibitors and cycloheximide inhibited the release of both NCA and MCA. Molecular-sieve chromatography revealed that NCA and MCA were heterogeneous. NCA was inhibited by anti-human interleukin (IL)-8 and anti-granulocyte colony-stimulating factor antibodies and a leukotriene (LT) B(4)-receptor antagonist. Anti-granulocyte-macrophage colony-stimulating factor (GM-CSF) and anti-monocyte chemoattractant protein (MCP)-1 antibodies and an LTB(4)-receptor antagonist inhibited MCA. Immunoreactive IL-8, granulocyte colony-stimulating factor, GM-CSF, and MCP-1 significantly increased in culture supernatant fluid in response to smoke extract. Finally, smoke extract augmented the expression of mRNAs of IL-8, GM-CSF, and MCP-1. These data demonstrate that lung fibroblasts release NCA and MCA in response to smoke extract and suggest that lung fibroblasts may modulate the inflammatory cell recruitment into the lung.

Topics: Cells, Cultured; Chemokine CCL2; Chemokine CCL5; Chemotaxis; Chromatography; Cycloheximide; Fibroblasts; Gene Expression; Granulocyte Colony-Stimulating Factor; Granulocyte-Macrophage Colony-Stimulating Factor; Humans; Immunosuppressive Agents; Interleukin-8; Isoquinolines; Leukotriene B4; Lung; Monocytes; Neutrophils; Nicotiana; Phenylpropionates; Plants, Toxic; Platelet Aggregation Inhibitors; Platelet Membrane Glycoproteins; Protein Synthesis Inhibitors; Pulmonary Fibrosis; Pyridinium Compounds; Receptors, Cell Surface; Receptors, G-Protein-Coupled; Smoking; Tetrahydroisoquinolines; Transforming Growth Factor beta

We previously observed increased expression of interleukin-1beta, platelet-derived growth factor-A, and insulin-like growth factor-I in bronchoalveolar lavage (BAL) cells during the development of pulmonary fibrosis after an intraperitoneal administration of bleomycin in mice. The purpose of this study was to investigate the roles of tumour necrosis factor (TNF)-alpha and transforming growth factor (TGF)-beta1 in this model.. We investigated the mRNA expression levels of TNF-alpha and TGF-beta1 in BAL cells of Institute for Cancer Research mice after 10 days of the intraperitoneal administration of bleomycin with or without treatment with a specific neutrophil elastase inhibitor, ONO-5046 x Na.. On day 1 but not on days 15 and 29, the relative amount of TGF-beta1 mRNA in the bleomycin-treated mice was significantly decreased compared with control mice. In the mice treated with both bleomycin and ONO-5046 x Na intermediate values for TGF-beta1 were obtained. No significant differences in TNF-alpha expression were observed in any of the treatment groups.. These results suggest that a reduced expression of TGF-beta1 in BAL cells in the early phase may be important during the development of murine pulmonary fibrosis induced by an intraperitoneal administration of bleomycin.

Topics: Animals; Biomarkers; Bleomycin; Bronchoalveolar Lavage Fluid; Disease Models, Animal; Injections, Intraperitoneal; Mice; Probability; Pulmonary Fibrosis; Reverse Transcriptase Polymerase Chain Reaction; Sensitivity and Specificity; Statistics, Nonparametric; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha

To evaluate the effect of erythromycin (EM) on nuclear factor-kappa B (NF-kappa B) activation and cytokines mRNA expression in bleomycin-induced pulmonary fibrosis in rats.. Wistar rats were divided into three groups of 27 as follow: BLM-group received intratracheal instillation of single dose BLM 5 mg/kg. EM treated-group received intratracheal instillation of BLM 5 mg/kg and an oral instillation EM 100 mg.kg-1.d-1. Control group received intratracheal and oral instillation of normal saline. Animals of all three groups were sacrificed on the 4th, 7th and the 28th day separately. NF-kappa B activation in alveolar macrophages (AM) was investigated by electrophoretic mobility shift assay. The expressions of IL-1 beta and TGF-beta mRNA in lung were evaluated by Northern blot analysis. The pathological changes of lung tissue were analyzed quantitatively by computer gray scan.. On the 4th and 7th day, the activities of NF-kappa B in AM were significantly increased in BLM-group compared with those of control group (P < 0.05), while they were significantly decreased in EM treated-group compared with BLM-group (P < 0.05). On the 7th day, the expressions of IL-1 beta and TGF-beta mRNA in lung were significantly decreased in EM treated-group compared with those in BLM-group. Pathologically, EM decreased exudation of inflammatory cells in the early response as well as degree of fibrosis in the late stage in the BLM-induced pulmonary fibrosis.. These results indicate that NF-kappa B is involved in the inflammatory response in BLM-induced pulmonary fibrosis in rats, EM has inhibitory effect on NF-kappa B activation, IL-1 beta and TGF-beta mRNA expression, by which it ameliorated acute lung injury and fibrosis those tested rats.

Topics: Animals; Anti-Bacterial Agents; Bleomycin; Cytokines; Disease Models, Animal; Erythromycin; Gene Expression; Interleukin-1; Male; NF-kappa B; Pulmonary Fibrosis; Rats; Rats, Wistar; RNA, Messenger; Transforming Growth Factor beta

To evaluate the therapeutic effects and mechanism of azithromycin treatment in bleomycin-induced pulmonary fibrosis of rats.. Animal model of bleomycin-A5-induced pulmonary fibrosis was established in rats.36 animal models were divided into two groups: a bleomycin-induced pulmonary fibrosis group and a azithromycin group in which the animal models were treated with azithromycin (80 mg/kg once a day for three continuous days in a week). The animals of the two groups were killed at the first,second and fourth week respectively. Another six rats constituted a normal control group, instillated withsaline intratracheally and killed at the first week. Pathological changes activity of nuclear factor kappaB (NF-kappaB) of alveolar macrophage, cytokine tumor necrosis factor (TNF) alpha, transforming growth factor-beta (TGF-beta) mRNA expression and protein levels of alveolar macrophage and lung tissue were studied or measured.. Amelioration of alveolitis and lung fibrosis after treatment with azithromycin was shown in pathological section (P < 0.05). The activity of NF-kappaB was significantly higher in one-week pulmonary fibrosis model than that in normal control and its level in alveolar macrophage reduced (67.2%) after treatment with azithromycin. The level of protein and mRNA of TNFalpha, TGF-beta in lung tissue and alveolar macrophage was increased in the early stage of pulmonary fibrosis and reduced after treatment with azithromycin (P < 0.05).. It is suggested that azithromycin might be a therapeutic drug for pulmonary fibrosis in the future. Azithromycin reduced the degree of alveolitis and fibrosis through inhibition of the activity of NF-kappaB and the expression of TNFalpha, TGF-beta mRNA and lowering the level of protein in alveolar macrophage and lung tissue in the early stage of pulmonary fibrosis. This might be one of the mechanisms of azithromycin treatment in pulmonary fibrosis.

Topics: Animals; Anti-Bacterial Agents; Bleomycin; Erythromycin; Male; NF-kappa B; Pulmonary Fibrosis; Rats; Rats, Wistar; RNA, Messenger; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha

To investigate the protein and gene expression in bronchoalveolar lavage cells and open lung biopsies from patients with IPF.. The immunohistochemical methods were used to analyze the expression of PDGF, TGF-beta in bronchoalveolar lavage cells of patients with IPF. For open lung biopsies, both immunohistochemistry and in situ hybridization were used.. In specimens of bronchoalveolar lavage fluid, PDGF-AA, PDGF-BB, TGF-beta were localized to alveolar macrophages in patients with IPF. Though there were no differences between IPF and sarcoidosis in terms of the staining intensity (2.5 - 3.0) or number of positive cells 81% - 90%, the number of such cells were less in the control (0 - 1.7, 25% - 32% respectively). Evaluation of open lung biopsies from patients with IPF showed that PDGF, TGF-beta proteins were localized to hyperplastic bronchio-alveolar epithelial cells (2.4 - 3.7, in control 0), alveolar macrophages (2.9 - 3.7, in control 0.8 - 1.3), fibroblasts (3.0 - 3.6, in control 2.7 - 2.8), vascular smooth muscle cells, vascular endothelial cells, and fibroblast-like cells surrounding pulmonary vessels (2.7 - 4.0, in control 2.1 - 2.9). In situ hybridization results were consistent with that of immunohistochemistry except that PDGF and TGF-beta mRNA transcripts were not detected in bronchio-alveolar epithelial cells. In control lungs, however, both ISH and IHC revealed that PDGF and TGF-beta were only present in the pleura and in fibroblast-like cells surrounding pulmonary vessels.. PDGF and TGF-beta, which interplay with pulmonary mesenchymal cells, are involved in fibroplasia and deposition of extracellular matrix as well as angiogenesis and epithelial cell repopularization.

Topics: Becaplermin; Bronchoalveolar Lavage Fluid; Humans; Hyaluronan Receptors; Immunohistochemistry; Platelet-Derived Growth Factor; Proto-Oncogene Proteins c-sis; Pulmonary Fibrosis; RNA, Messenger; Transforming Growth Factor beta

Using anti-transforming growth factor-beta(1) antibody binding to transforming growth factor-beta(1) (TGF-beta(1)) in conditioned supernatant obtained from alveolar macrophages (AM) in broncho-alveolar lavage fluid (BALF) in bleomycin-induced rats, we investigated the effect of binding of TGF beta(1) to anti-TGF beta(1) antibody on proliferation and synthesis of collagen by fibroblast in pulmonary fibrosis.. Bleomycin (0.75 mg/100 BW) was instilled intratracheally into wistar rats, then broncho-alveolar lavage (BAL) was performed 7 days later. AMs (5 x 10(5)/ml) were cultured in 0.2% FCS RPMI-1640 medium in vitro for 24 hours and conditioned medium was obtained. L929 fibroblast (7 x 10(3)/well) was cultured in 96-well microtiter plate with conditioned medium and in different doses of anti- TGF beta(1) antibody (1, 5, 10, 20 microg/ml) and IgG for 24 hours respectively. (3)H-Tdr was added 1 microCi per well for 6 hours before culture finished. The proliferation of fibroblast was studied by (3)H-Tdr incorporation rate and synthesis of collagen type IV was tested by ELISA.. (1) Fibroblast proliferation which was induced by AM conditioned medium could be significantly depressed by anti-TGF beta(1) antibody in vitro (P < 0.01), and it appeared in a dose-dependent manner. (2) Synthesis of collagen type IV was depressed about 32% by 10 microg/ml anti-TGF beta(1) antibody.. Fibroblast proliferation and collagen synthesis could be depressed by anti-TGF beta(1) antibody in vitro. It seems possible to provide a new way for the treatment of pulmonary fibrosis.

Topics: Animals; Antibodies, Monoclonal; Cell Division; Collagen Type IV; Female; Fibroblasts; Macrophages, Alveolar; Pulmonary Fibrosis; Rats; Rats, Wistar; Transforming Growth Factor beta; Transforming Growth Factor beta1

We have reported that taurine (T) and niacin (N) inhibit the expression of procollagen type I and type III genes at the level of gene transcription in the bleomycin (BL) hamster model of lung fibrosis. In the present study, we have investigated the effects of TN in diet on the temporal expression of transforming growth factor-beta1 (TGF-beta1) mRNA and TGF-beta1 protein production in the same model of lung fibrosis to determine whether the decreased transcription of procollagen genes is associated with downregulation of TGF-beta1 mRNA. Our results demonstrate that expression of TGF-beta1 mRNA in lungs is increased in BL-treated hamsters in the BL + control diet (CD) group, compared to saline controls in the saline-instilled (SA) + CD group, by 3.5-, 2.5-, 4-, and 2-fold at 3, 7, 14, and 21 d, respectively, and TN treatment caused significant decreases in TGF-beta1 mRNA expression in BL-treated animals in the BL + TN group from Day 3 through Day 21. In addition, TN treatment also reduced TGF-beta1 protein in bronchoalveolar lavage fluid (BALF) from BL-treated animals in the BL + TN group. These decreases in TGF-beta1 mRNA and TGF-beta1 protein correlated with decreased lung collagen content in hamsters in the BL + TN group as demonstrated in our earlier study. To confirm that the TGF-beta1 activity observed in BALF is reflected at the transcriptional level, total RNA was isolated from lavaged cells. Reverse transcriptase-polymerase chain reaction analysis demonstrated maximal expression of TGF-beta1 mRNA transcripts in BL-treated lavaged cells from animals in the BL + CD group and only low levels were detected in both saline control groups, and in BL + TN-treated lavaged cells. Nuclear runoff analysis indicated that TN-mediated reduction of TGF-beta1 mRNA steady-state levels was a result of decreased gene transcription, suggesting a transcriptional downregulation mechanism. Our results indicate that the combined treatment with TN ameliorates BL-induced lung fibrosis, at least in part, via inhibition of TGF-beta1 mRNA expression.

Topics: Animals; Bleomycin; Bronchoalveolar Lavage Fluid; Collagen; Cricetinae; Diet; Disease Models, Animal; Gene Expression Regulation; Male; Mesocricetus; Niacin; Pulmonary Fibrosis; Taurine; Transforming Growth Factor beta

Pulmonary fibrosis was induced following inoculation of Paracoccidioides brasiliensis conidia intranasally in BALB/c mice. Fibrosis was associated with formation of granulomas, increase in lung hydroxyproline, and sustained increases in tissue tumor necrosis factor-alpha and transforming growth factor-beta. This study suggests a role for these cytokines in generation of pulmonary fibrosis associated with chronic granulomatous infectious diseases.

Topics: Animals; Bronchoalveolar Lavage Fluid; Disease Models, Animal; Female; Granuloma; Hydroxyproline; Lung; Lung Diseases, Fungal; Male; Mice; Mice, Inbred BALB C; Paracoccidioides; Paracoccidioidomycosis; Pulmonary Fibrosis; Specific Pathogen-Free Organisms; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha

Topics: Cyclosporine; Enzyme-Linked Immunosorbent Assay; Humans; Immunosuppressive Agents; Lung Transplantation; Lymphocyte Activation; Pulmonary Fibrosis; Reference Values; Regression Analysis; Transforming Growth Factor beta

Cadmium chloride (CdCl2) exposure has been reported to induce pulmonary fibrosis in rats. Accumulating evidence has shown that cytokines play a pivotal role in the excessive production of connective tissue components in pulmonary fibrosis. In this report, rat lung slice cultures were used to study the synergistic involvement of transforming growth factor-beta1 (TGF-beta1) in CdCl2-induced alveolar fibrosis. Rat lung slices were maintained at the interphase of air and medium on a polyester mesh stretched on a plastic scaffold. Treatment of lung slices with 2.5, 5 or 10 microM CdCl2 for 7 days resulted in 85, 40 and 6% respectively for relative survival. Under these culture conditions, CdCl2 alone did not induce alveolar fibrosis in rat lung slices. However, in the presence of 0.5 ng/ml TGF-beta1, CdCl2 at a dose ranging from 1 to 5 microM increased the thickness of alveolar septa. Furthermore, the thickness of alveolar septa in lung slices treated with CdCl2 was dose-dependently increased by the presence of TGF-beta1. The thickened alveolar septa were apparently due to the deposition of excessive extracellular matrix, as revealed by trichrome stain and ultrastructural examination. Our results also show that fibrogenic activity induced by the combined treatment with CdCl2 and TGF-beta1 can be reduced by co-treatment with 200 microg/ml lambda-carrageenan, a TGF-beta1 inhibitor. Therefore, the present results indicate that TGF-beta1 can synergistically stimulate the fibrogenic activity in lung tissue subsequent to CdCl2 injury.

Topics: Animals; Azo Compounds; Cadmium Chloride; Carrageenan; Coloring Agents; Drug Synergism; Eosine Yellowish-(YS); Extracellular Matrix; Lung; Methyl Green; Organ Culture Techniques; Pulmonary Alveoli; Pulmonary Fibrosis; Rats; Rats, Sprague-Dawley; Tissue Fixation; Transforming Growth Factor beta

Connective tissue growth factor (CTGF) is a newly described 38-kDa peptide mitogen for fibroblasts and a promoter of connective tissue deposition in the skin. The CTGF gene promotor contains a transforming growth factor-beta1 (TGF-beta1) response element. Because TGF-beta1 expression is upregulated in several models of fibroproliferative lung disease, we asked whether CTGF is also upregulated in a murine lung fibrosis model and whether CTGF could mediate some of the fibrogenic effects associated with TGF-beta1. A portion of the rat CTGF gene was cloned and used to show that primary isolates of both murine and human lung fibroblasts express CTGF mRNA in vitro. There was a greater than twofold increase in CTGF expression in both human and murine lung fibroblasts 2, 4, and 24 h after the addition of TGF-beta1 in vitro. A bleomycin-sensitive mouse strain (C57BL/6) and a bleomycin-resistant mouse strain (BALB/c) were given bleomycin, a known lung fibrogenic agent. CTGF mRNA expression was upregulated in the sensitive, but not in the resistant, mouse strain after administration of bleomycin. In vivo differences in the CTGF expression between the two mouse strains were not due to an inherent inability of BALB/c lung fibroblasts to respond to TGF-beta1 because fibroblasts from untreated BALB/c mouse lung upregulated their CTGF message when treated with TGF-beta1 in vitro. These data demonstrate that CTGF is expressed in lung fibroblasts and may play a role in the pathogenesis of lung fibrosis.

Topics: Animals; Base Sequence; Bleomycin; Cloning, Molecular; Connective Tissue Growth Factor; Cytokines; DNA, Complementary; Fibroblasts; Gene Expression Regulation; Growth Substances; Humans; Immediate-Early Proteins; Intercellular Signaling Peptides and Proteins; Lung; Male; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Mitogens; Pulmonary Fibrosis; Rats; Rats, Sprague-Dawley; Recombinant Fusion Proteins; RNA, Messenger; Sequence Alignment; Sequence Homology, Nucleic Acid; Time Factors; Transcription, Genetic; Transforming Growth Factor beta

Tumor necrosis factor-alpha is up-regulated in a variety of different human immune-inflammatory and fibrotic pulmonary pathologies. However, its precise role in these pathologies and, in particular, the mechanism(s) by which it may induce fibrogenesis are not yet elucidated. Using a replication-deficient adenovirus to transfer the cDNA of tumor necrosis factor-alpha to rat lung, we have been able to study the effect of transient but prolonged (7 to 10 days) overexpression of tumor necrosis factor-alpha in normal adult pulmonary tissue. We have demonstrated that local overexpression resulted in severe pulmonary inflammation with significant increases in neutrophils, macrophages, and lymphocytes and, to a lesser extent, eosinophils, with a peak at day 7. By day 14, the inflammatory cell accumulation had declined, and fibrogenesis became evident, with fibroblast accumulation and deposition of extracellular matrix proteins. Fibrotic changes were patchy but persisted to beyond day 64. To elucidate the mechanism underlying this fibrogenesis, we examined bronchoalveolar fluids for the presence of the fibrogenic cytokine transforming growth factor-beta1 and tissues for induction of alpha-smooth muscle actin-rich myofibroblasts. Transforming growth factor-beta1 was transiently elevated from day 7 (peak at day 14) immediately preceding the onset of fibrogenesis. Furthermore, there was a striking accumulation of myofibroblasts from day 7, with the most extensive and intense immunostaining at day 14, ie, coincident with the up-regulation of transforming growth factor-beta1 and onset of fibrogenesis. Thus, we have provided a model of tumor necrosis factor-alpha-mediated pulmonary inflammation and fibrosis in normal adult lung, and we suggest that the fibrogenesis may be mediated by the secondary up-regulation of transforming growth factor-beta1 and induction of pulmonary myofibroblasts.

Topics: Adenoviridae; Animals; Bronchoalveolar Lavage Fluid; Extracellular Matrix Proteins; Fibroblasts; Gene Expression; Gene Transfer Techniques; Genetic Vectors; Immunoenzyme Techniques; Lung; Pneumonia; Pulmonary Fibrosis; Rats; Rats, Sprague-Dawley; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha; Up-Regulation

Transforming growth factor (TGF)-beta1 is a profibrogenetic cytokine that has been implicated in the development of fibrosis in transplanted tissues. In this study, we have analyzed the genetic regulation of TGF-beta1 production in lung transplant recipients.. A polymerase chain reaction-single-stranded conformational polymorphism technique was used to detect polymorphisms in the TGF-beta1 gene from genomic DNA. Polymorphisms were shown to correlate with in vitro TGF-beta1 production by stimulated lymphocytes. A single-specific oligonucleotide probe hybridization method was devised to screen for these polymorphisms in lung transplant groups and controls.. We have identified five polymorphisms in the TGF-beta1 gene: two in the promoter region at positions -800 and -509, one at position +72 in a nontranslated region, and two in the signal sequence at positions +869 and +915. The polymorphism at position +915 in the signal sequence, which changes codon 25 (arginine-->proline), is associated with interindividual variation in levels of TGF-beta1 production. Stimulated lymphocytes of homozygous genotype (arginine/arginine) from control individuals produced significantly more TGF-beta1 in vitro (10037+/-745 pg/ml) compared with heterozygous (arginine/proline) individuals (6729+/-883 pg/ml; P<0.02). In patients requiring lung transplantation for a fibrotic lung condition, there was an increase in the frequency of the high-producer TGF-beta1 allele (arginine). This allele was significantly associated with pretransplant fibrotic pathology (P<0.02) (n=45) when compared with controls (n=107) and with pretransplant nonfibrotic pathology (P<0.004) (n=50). This allele was also associated with allograft fibrosis in transbronchial biopsies when compared with controls (P<0.03) and with nonallograft fibrosis (P<0.01).. The production of TGF-beta1 is under genetic control, and this in turn influences the development of lung fibrosis. Hence, the TGF-beta1 genotype has prognostic significance in transplant recipients.

Topics: Alleles; Genetic Variation; Genotype; Humans; Lung Transplantation; Polymorphism, Genetic; Postoperative Complications; Pulmonary Fibrosis; Transforming Growth Factor beta

Since tumor growth factor beta (TGF-beta) and its receptor are ubiquitously expressed and because latent TGF-beta cannot bind to the cell surface receptor, the ability of a cell to activate latent TGF-beta upon secretion represents an important regulatory mechanism of TGF-beta action. In vivo, the protease plasmin is considered to be one of the main enzymes operative in the proteolytic cleavage of the latency-associated peptide moiety from TGF-beta, which converts it into the biologically active form. The TGF-beta response was characterized in alveolar macrophages during pulmonary inflammation (d 3) and fibrosis (d 120) induced by a single intratracheal instillation of silica particles (5 mg/mouse). To appreciate the role of urokinase-type plasminogen activator (uPA) in the activation of TGF-beta, the production of total, active and latent TGF-beta by explanted alveolar macrophages was compared in uPA-deficient (uPA-/-) mice and their normal counterparts (uPA+/+). At d 3 and 120 after silica treatment, a significant increase in cell-associated PA activity was found in uPA+/+ mice compared to that of saline controls. As expected, this response was almost totally absent in uPA-/- mice. Alveolar macrophages from uPA+/+ controls were found to release TGF-beta mainly expressed in a biologically active form. In response to silica treatment, inflammatory cells were found to upregulate, especially at the fibrotic stage, their secretion of total and bioactive TGF-beta. No significant difference was found between uPA-/- and uPA+/+ silica-treated animals for the expression of total, active, or latent TGF-beta. Although it has previously been reported that macrophage surface activation of TGF-beta is dependent on both plasmin generation and uPA cell surface receptor, no evidence was found to support this hypothesis in the present study.

Topics: Animals; Biomarkers; Cells, Cultured; Female; Macrophages, Alveolar; Male; Mice; Mice, Knockout; Pulmonary Fibrosis; Silicon Dioxide; Tissue Plasminogen Activator; Transforming Growth Factor beta; Urokinase-Type Plasminogen Activator

Exposure to mineral dusts is associated with the development of chronic airflow obstruction, probably mediated in part by dust-induced fibrosis of the small airways. To investigate the mechanism of fibrosis, we exposed rat tracheal explants to amosite asbestos, iron oxide, or titanium dioxide. Explants were then maintained in air organ culture, and the expression of genes encoding for various mediators and matrix components assessed by reverse transcriptase-polymerase chain reaction (RT-PCR). At 7 d, all dusts produced significant increases in platelet-derived growth factor-A (PDGF-A) and transforming growth factor-beta1 (TGF-beta1) gene expression compared with control; asbestos and titanium dioxide produced increases in PDGF-B, and titanium dioxide increased TGF-alpha expression. Only asbestos caused increases in procollagen expression. No dust increased expression of tumor necrosis factor-alpha (TNF-alpha), fibronectin, or tropoelastin. Elevations in these factors coincided temporally with transport of particles into the epithelium and then to the subepithelial space. By in situ hybridization, TGF-beta gene expression was found in both the epithelium and subepithelial (interstitial) space, and PDGF-B and procollagen gene expression in the subepithelial space. Chemical analysis showed a small increase in hydroxyproline, a measure of collagen content, in asbestos-treated explants. We conclude that mineral dusts can induce airway wall fibrosis by directly upregulating proliferative and fibrogenic mediators as well as matrix components in the airway epithelium and interstitium, and that neither airspace nor circulating inflammatory cells are required for these effects. Different mineral dusts produce different patterns of reaction.

Topics: Airway Obstruction; Animals; Asbestos, Amosite; Culture Techniques; Dust; Epithelium; Extracellular Matrix Proteins; Ferric Compounds; Fibronectins; Gene Expression Regulation; Hydroxyproline; Inflammation Mediators; Irritants; Lung; Male; Mineral Fibers; Minerals; Platelet-Derived Growth Factor; Procollagen; Protein-Tyrosine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-sis; Pulmonary Fibrosis; Rats; Rats, Sprague-Dawley; Titanium; Transforming Growth Factor beta; Tropoelastin; Tumor Necrosis Factor-alpha; Up-Regulation

Topical administration of granulocyte-macrophage colony-stimulating factor (GM-CSF) into the subcutaneous tissue or in the pulmonary alveoli of the rat induces a fibrotic reaction characterized by the presence of alpha-smooth muscle actin-rich myofibroblasts, suggesting that GM-CSF plays a role in the development of fibrotic changes. A high level of expression of GM-CSF also has been demonstrated in epidermal cells during human atopic dermatitis. It is accepted that transforming growth factor beta1 (TGF-beta1) plays a key role in the modulation from fibroblast into myofibroblast, although it is not known how TGF-beta1 activity is stimulated. Up until now, no evidence of early GM-CSF expression during development of fibrosis has been reported. Herein we have studied, using RT-competitive PCR, the expression of GM-CSF mRNA during the early steps of pulmonary fibrosis development after intra-alveolar instillation of bleomycin, a well-established experimental model of this lesion. GM-CSF mRNA was already increased in the total lung at 6 hours and maximal at 12 hours after bleomycin instillation and returned to basal levels at 24 hours. This was followed by an increase of TGF-beta1 and TGF-beta receptor type II (but not of types I and III) mRNAs. Analyses of macrophages and polymorphonuclear neutrophils isolated by bronchoalveolar lavage 12 hours after bleomycin instillation indicated that they were responsible, at least in part, for the accumulation of GM-CSF mRNA. Our results show for the first time that GM-CSF is expressed, very early and temporarily, by inflammatory cells accumulating in the alveolus after bleomycin administration and before the appearance of TGF-beta1. Moreover, we have shown that GM-CSF induces the expression of TGF-beta1 mRNA by alveolar macrophages. Our data support the possibility that GM-CSF participates in the initial steps of the chain of events leading to fibrosis, perhaps through a stimulation of TGF-beta1 production.

Topics: Actins; Animals; Bleomycin; Dermatitis, Atopic; Epidermis; Female; Gene Expression Regulation; Granulocyte-Macrophage Colony-Stimulating Factor; Humans; Instillation, Drug; Muscle, Smooth; Protein Serine-Threonine Kinases; Pulmonary Alveoli; Pulmonary Fibrosis; Rats; Rats, Wistar; Receptor, Transforming Growth Factor-beta Type II; Receptors, Transforming Growth Factor beta; Reverse Transcriptase Polymerase Chain Reaction; Transcription, Genetic; Transforming Growth Factor beta

Despite abundant evidence documenting the importance of TNF-alpha in the pathogenesis of pulmonary fibrosis, its actual role has not been fully elucidated. Recent observations also indicate that eosinophils found in fibrotic lung express elevated levels of cytokines known to be important in lung fibrosis. These findings suggest a possible role for TNF-alpha in eosinophil recruitment and cytokine expression in this disease. To examine this hypothesis, pulmonary fibrosis was induced in mice by endotracheal bleomycin treatment, and separate groups of animals were also treated with either anti-TNF-alpha Ab or control serum. On days 7 and 14 post-bleomycin treatment, lungs were harvested and analyzed for fibrosis, cytokine expression, and eosinophil influx. Anti-TNF-alpha caused a significant reduction in lung fibrosis, as indicated by a reduction in hydroxyproline content, which was accompanied by suppression of lung TGF-beta1, IL-5, and JE mRNA expression. Examination of tissue sections revealed a significant reduction in lung eosinophils and overall cellularity by anti-TNF-alpha treatment without a significant effect on the number of lung macrophages. The number of IL-5-expressing cells was also significantly reduced by anti-TNF-alpha treatment. Since IL-5 is important in eosinophil differentiation, activation, and recruitment, these findings suggest a novel mechanism by which TNF-alpha could mediate pulmonary fibrosis via induction of IL-5-mediated eosinophil recruitment and fibrogenic cytokine production. Since these eosinophil-derived cytokines include JE/monocyte chemotactic factor-1 and TGF-beta1, this cytokine networking orchestrated by TNF-alpha could, in turn, amplify the inflammatory response and drive the progression to fibrosis and end-stage lung disease.

Topics: Animals; Bleomycin; Chemokine CCL2; Chemotaxis, Leukocyte; Cytokines; Eosinophils; Female; Interleukin-5; Mice; Mice, Inbred CBA; Pulmonary Fibrosis; Signal Transduction; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha

We have previously reported that transfer to rat lung of the granulocyte-macrophage colony-stimulating factor (GM-CSF) gene leads to high expression of GM-CSF between days 1 and 4 and granulation tissue formation followed by an irreversible fibrotic response starting from day 12 onward. In the current study, we investigated the underlying mechanisms. We found that GM-CSF overexpression did not enhance production of tumor necrosis factor-alpha in a significant manner at any time after GM-CSF gene transfer. However, the content of transforming growth factor-beta 1 in bronchoalveolar lavage fluid was markedly induced at day 4 and appeared to be maximal around day 7 and remained high at day 12. Macrophages purified from bronchoalveolar lavage fluid 7 days after GM-CSF gene transfer spontaneously released significant quantities of transforming growth factor-beta 1 protein in vitro. After peak transforming growth factor-beta 1 production was the emergence of alpha-smooth muscle actin-rich myofibroblasts. Accumulation of these cells was most prominent at day 12 within the granulation tissues and they were still present in fibrotic areas between days 12 and 24 and diminished markedly afterward. Thus, we provide the first in vivo evidence that tumor necrosis factor-alpha may be dissociated from participation in a fibrotic process in the lung and GM-CSF may play a more direct role in pulmonary fibrogenesis at least in part through its capability to induce transforming growth factor-beta 1 in macrophages and the subsequent emergence of myofibroblast phenotypes. This GM-CSF transgene lung model is useful for a stepwise dissection of both cellular and molecular events involved in pulmonary fibrosis.

Topics: Actins; Animals; Fibroblasts; Gene Transfer Techniques; Granulocyte-Macrophage Colony-Stimulating Factor; Granuloma; Immunohistochemistry; Lung; Macrophages; Muscle, Smooth; Pulmonary Fibrosis; Rats; Rats, Sprague-Dawley; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha

To evaluate the possibility that TGF-beta and TNF-alpha are involved in fibrosis induced in mouse lung by irradiation, the proportion of cells immunoreactive for each was compared in two strains of mice. C3HeB/FeJ mice develop only classical pneumonitis during the early phase, whereas C57L/J mice develop small, tightly packed areas of inflammation which undergo fibrosis during the latent period, and exhibit progressive fibrosis of large regions of intense inflammation during the early phase. Very few cells were immunoreactive for an antibody to the latency-associated peptide (LAP) of TGF-beta during the latent period in C3HeB/FeJ mice, and no cells were positive during the early phase. In contrast, between 0.7 and 10% of cells were positive in C57L/J mice in lesions without fibrosis and in lesions in the early stages of fibrosis. Fibroblasts positive for LAP were seen only in lesions containing fibrosis. A similar pattern of immunoreactivity was seen in C57L/J mice using an antibody which recognizes active TGF-beta, with the exception that positive fibroblasts were observed within areas of inflammation without fibrosis. Thus the association of active TGF-beta with fibroblasts might be a characteristic of the initiation of fibrosis in this model. TNF-alpha was detected in macrophages in all classes of lesions, and minor differences between the strains did not appear to be biologically meaningful.

Topics: Animals; Disease Progression; Fibrosis; Gene Expression Regulation; Immunohistochemistry; Inflammation; Lung; Mice; Mice, Inbred C3H; Mice, Inbred Strains; Pulmonary Fibrosis; Radiation Injuries, Experimental; Thorax; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha

Asbestosis is characterized by increased collagen deposition along the walls of terminal respiratory bronchioles that extends into the alveolar ducts and septae. Alveolar macrophages are activated and release growth factors that stimulate mesenchymal cell proliferation and enhanced formation of extracellular matrix. Both insulin-like growth factor-I (IGF-I), and transforming growth factor beta (TGF-beta) regulate cellular growth and promote matrix accumulation and are hypothesized to play important roles in asbestosis. We performed immunohistochemistry using polyclonal antibodies to specific synthetic peptides of the three mammalian isoforms of TGF-beta (TGF-beta 1, -beta 2, -beta 3) and to IGF-I on lungs of sheep treated intratracheally with chrysotile asbestos. All three TGF-beta isoforms were found in bronchial and bronchiolar epithelium, macrophages, and bronchial and vascular smooth muscle in control lungs. The distribution of TGF-beta was increased in these lung constituents as fibrotic lesions developed. Fibrotic lesions additionally demonstrated intense immunostaining of all three TGF-beta isoforms that localized to the extracellular matrix zones with little staining of interstitial cells. In the control sheep lungs, IGF-I staining was detected in bronchial and bronchiolar epithelium, bronchial glands, bronchial and vascular smooth muscle, endothelium, and macrophages. IGF-I immunostaining was detected in macrophages in peribronchial fibrosis and in fibroblasts along the periphery of and within lesions, but not in the extracellular matrix. Metaplastic proliferating epithelium and macrophages were strongly immunoreactive for IGF-I in advanced lesions. Our data demonstrate different immunostaining patterns for IGF-I and TGF-beta in asbestosis, with IGF-I in the cellular periphery and TGF-beta in the extracellular matrix consistent with a complementary role in stimulating interstitial fibroblast proliferation and new collagen deposition in areas of active fibrosis.

Topics: Animals; Asbestos, Serpentine; Asbestosis; Disease Models, Animal; Immunohistochemistry; Insulin-Like Growth Factor I; Pulmonary Fibrosis; Sheep; Transforming Growth Factor beta

Transforming growth factor (TGF)-beta 1 may potentiate wound healing and fibrosis by stimulating fibroblast collagen deposition. TGF-beta 1 is implicated in the pathogenesis of pulmonary fibrosis, but the role of TGF-beta 2 and TGF-beta 3 remains unclear. We examined their effects on lung fibroblast procollagen metabolism in vitro and localized their gene expression during bleomycin-induced lung fibrosis using in situ hybridization with digoxigenin-labeled riboprobes. All three isoforms stimulated fibroblast procollagen production. TGF-beta 3 was the most potent and also reduced procollagen degradation. In normal mouse lung, TGF-beta 1 and TGF-beta 3 mRNA transcripts were abundant in bronchiolar epithelium. After bleomycin, TGF-beta 1 gene expression was maximally enhanced at 10 days, with the signal being predominant in macrophages. Signal was also enhanced in mesenchymal, pulmonary endothelial, and mesothelial cells. After 35 days, the pattern of TGF-beta 1 gene expression returned to that of control lung. TGF-beta 3 gene expression remained unchanged throughout compared with controls. TGF-beta 2 mRNA was not detected with the antisense probe, but signal obtained with the sense probe suggests the presence of a naturally occurring antisense. This study demonstrates that TGF-beta 1, -beta 2, and -beta 3 all exert profibrotic effects in vitro. However, TGF-beta isoform gene expression is differentially controlled during experimental pulmonary fibrosis with TGF-beta 1 the predominant isoform expressed during pathogenesis.

Topics: Animals; Bleomycin; Carrier Proteins; Cells, Cultured; Fibroblasts; Fibrosis; Gene Expression; Gene Expression Regulation; In Situ Hybridization; Intracellular Signaling Peptides and Proteins; Isomerism; Latent TGF-beta Binding Proteins; Lung; Mice; Procollagen; Pulmonary Fibrosis; Transforming Growth Factor beta

The prognosis of pulmonary fibrosis is poor and current therapies inadequate. Recent progress in understanding the mechanisms underlying the pathogenesis of this disease leads us to expect that inhibitors of cytokine and polypeptide growth factor will provide novel therapeutic agents. This paper outlines the role of cytokines in the pathogenesis of pulmonary fibrosis and concludes that there are compelling reasons to explore anticytokine therapeutic approaches. It also proposes criteria that will enable us to evaluate such agents in vivo and suggests approaches which might overcome the obstacles presented by control mechanisms which recruit a multiplicity of factors.

Topics: Antibodies; Antisense Elements (Genetics); Cytokines; Growth Inhibitors; Humans; Mannosephosphates; Pulmonary Fibrosis; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha

Although both procollagen III aminopeptide (P-III-P) and transforming growth factor-beta (TGF-beta) are reported to be present in lung tissue and/or elevated in bronchoalveolar lavage fluid (BALF) from idiopathic pulmonary fibrosis (IPF) patients, we have little knowledge concerning the clinical significance of elevated P-III-P and TGF-beta levels in BALF. Using a radioimmunoassay, we measured P-III-P and TGF-beta in BALF from 48 IPF patients (16F and 32M, 59 +/- 2 years, mean +/- S.E.) who received BAL in our clinic over the past 13 years before glucocorticosteroid treatment. Among them, we could detect a significant amount of P-III-P (2.2 +/- 1.0 U/ml; range 0.03 to 16.5 U/ml) in BALF in 18 of the patients (5F and 13M, 58 +/- 3 years) (group B). but not (0.03 U/ml or less) in the other 30 patients (11F and 19M, 59 +/- 2 years) (group A). Lymphocyte (%) and basophil (%) in BALF from group B was much larger than that from group A (33% vs. 8%, p < 0.01). Group B showed a longer duration of onset to BAL (36 months vs. 23 months, p < 0.05). TGF-beta levels were obtained using an ELISA system kit from the same BALF samples. TGF-beta was not detected in 10 patients (100 pg/ml or less) (3F and 7M, 59 +/- 4 years) (group I), while the remaining 38 patients showed a significant amount of TGF-beta (329 +/- 44 pg/ml, range 100 to 1,360 pg/ml). The latter patients were further divided into two groups; group II 100 to 300 pg/ml (10F and 14M, 56 +/- 3 years) and group III 350 or more (3F and 11M, 63 +/- 2 years). Group III showed significantly better values in PaO2, Aa-DO2, %VC and %DLco, and smaller percentage of basophils in BALF than did groups I and/or II, whereas survival after BAL in group III was significantly shorter than in group I (31 vs. 19 months, p < 0.05). There was no significant relationship between P-III-P and TGF-beta levels in BALF. These findings suggest that elevated P-III-P level is accompanied by an increase in lymphocyte population in BALF from IPF patients, resulting in a longer duration of the disease, while elevated TGF-beta level reflects alveolar inflammation at an earlier stage of the disease which induces a progression of the disease, resulting in a shorter survival in IPF patients.

Topics: Adult; Aged; Aged, 80 and over; Biomarkers; Bronchoalveolar Lavage Fluid; Enzyme-Linked Immunosorbent Assay; Female; Humans; Male; Middle Aged; Peptide Fragments; Procollagen; Pulmonary Fibrosis; Radioimmunoassay; Respiratory Function Tests; Transforming Growth Factor beta

Transforming growth factor (TGF)-beta1 has been implicated in the pathogenesis of fibrosis based upon its matrix-inducing effects on stromal cells in vitro, and studies demonstrating increased expression of total TGF-beta1 in fibrotic tissues from a variety of organs. The precise role in vivo of this cytokine in both its latent and active forms, however, remains unclear. Using replication-deficient adenovirus vectors to transfer the cDNA of porcine TGF-beta1 to rat lung, we have been able to study the effect of TGF-beta1 protein in the respiratory tract directly. We have demonstrated that transient overexpression of active, but not latent, TGF-beta1 resulted in prolonged and severe interstitial and pleural fibrosis characterized by extensive deposition of the extracellular matrix (ECM) proteins collagen, fibronectin, and elastin, and by emergence of cells with the myofibroblast phenotype. These results illustrate the role of TGF-beta1 and the importance of its activation in the pulmonary fibrotic process, and suggest that targeting active TGF-beta1 and steps involved in TGF-beta1 activation are likely to be valuable antifibrogenic therapeutic strategies. This new and versatile model of pulmonary fibrosis can be used to study such therapies.

Topics: Actins; Adenoviridae; Animals; Collagen; Elastin; Fibronectins; Gene Transfer Techniques; Genetic Vectors; Hydroxyproline; Lung; Male; Peptide Fragments; Protein Precursors; Proteins; Pulmonary Fibrosis; Rats; Rats, Sprague-Dawley; Time Factors; Transforming Growth Factor beta; Transforming Growth Factor beta1

The histopathology of bronchial asthma is associated with structural changes within the airways, including subepithelial fibrosis, as well as chronic eosinophilic inflammation. The mechanisms responsible for this tissue remodeling, and in particular the role of inflammatory cells, remain to be established. Transforming growth factor-beta (TGF-beta) is a potent profibrotic cytokine which may contribute to the thickening of the reticular lamina by the deposition of collagen fibers. To investigate the molecular mechanisms underlying these structural changes, we have investigated the expression of TGF-beta1 mRNA and immunoreactivity within the bronchial mucosa of mild to severe asthmatic individuals and normal control subjects using the techniques of in situ hybridization and immunocytochemistry. As eosinophils are prominent within the asthmatic airway and are known to synthesize pro-inflammatory cytokines, the presence of TGF-beta1 mRNA and immunoreactive protein in eosinophils was also examined. Asthmatic individuals exhibited a greater expression of TGF-beta1 mRNA and immunoreactivity in the airways submucosa than normal control subjects (P < 0.05), and these increases were directly related to the severity of the disorder. The extent of airways fibrosis, as detected histochemically, was also increased in asthmatics compared with normal control subjects (P < 0.005). In asthmatic subjects, the presence of subepithelial fibrosis was associated with the severity of the disease and correlated with the decline in forced expiratory volume in 1 s (r2 = 0.78; P < 0.05). Within the asthmatic airways, EG2-positive eosinophils represented the major source of TGF-beta1 mRNA and immunoreactivity. These results provide evidence that TGF-beta1 may play a role in the fibrotic changes occurring within asthmatic airways and that activated eosinophils are a major source of this cytokine.

Topics: Adult; Asthma; Bronchi; Eosinophils; Female; Gene Expression; Histocytochemistry; Humans; In Situ Hybridization; Male; Pulmonary Fibrosis; RNA, Messenger; Transforming Growth Factor beta

To examine the mechanism of steroid resistance in lung fibrosis, cytokines expressed in the lung tissue of mice with bleomycin-induced lung fibrosis were studied. 1. Glucocorticoid administration (1 mg/kg/day) did not affect the grade of lung fibrosis induced by intratracheal injection of bleomycin (3.76 micrograms/g). 2. Cytokines expressed in the lung tissue were studied with the reverse-transcriptase polymerase chain reaction. Levels of promotor cytokines, such as TNF alpha, TGF beta, INF gamma, and IL-2, were significantly higher in lung tissue from the bleomycin group. The expression of these cytokines in the glucocorticoid group was low, especially the peak value. Expression of IL-4 was high in the bleomycin group, and was not inhibited in the glucocorticoid group. Expression of the down-regulator cytokine IL-10 was also high in the bleomycin group and very low in the glucocorticoid group. 3. The non-selectivity of glucocorticoids with respect to promotor and suppressive cytokines may account in part for steroid resistance in bleomycin-induced pulmonary fibrosis.

Topics: Animals; Bleomycin; Cytokines; Drug Resistance; Glucocorticoids; Interferon-gamma; Interleukins; Male; Mice; Mice, Inbred C57BL; Pulmonary Fibrosis; RNA, Messenger; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha

Topics: Child; Cytokines; Fatal Outcome; Female; Humans; Infant, Newborn; Lymph Nodes; Lymphatic Diseases; Lymphoma, Non-Hodgkin; Pulmonary Fibrosis; Respiratory Distress Syndrome, Newborn; Transforming Growth Factor beta

Transforming growth factor-beta (TGF-beta) plays an important role in the pathogenesis of interstitial lung fibrosis. It can stimulate indirectly the mitosis of lung fibroblasts (LFb) as well as the synthesis and disposition of extracellular matrix. The purpose of this study was to evaluate the effects of TGF-beta monoclonal antibody on bleomycin-induced interstitial pulmonary fibrosis.. The effect of TGF-beta monoclonal antibody to bleomycin-induced pulmonary fibrosis in rat was studied either in vivo or in vitro. The proliferation of lung fibroblasts was studied by measuring the incorporation rate of 3H-TdR. Northern hybridization was used to detect mRNA level of procollagen A1 (I) and procollagen A1 (II) in cultured LFbs and lung tissue.. The results showed that the incubation supernatant of alveolar macrophages from rats with pulmonary fibrosis had the ability to stimulate fibroblast proliferation as well as their procollagen expression. The monoclonal antibody could inhibit the proliferation of LFbs in a concentration-dependent manner. The highest concentration, 100 micrograms/ml, of TGF-beta antibody could inhibit incorporation rate from 1749 +/- 322 of the fibrosis group to only 833 +/- 277 (P < 0.01). The mRNA level of procollagen I and II was decreased by 44% and 28% respectively after the antibody treatment. In vivo, procollagen I and II mRNA level were decreased by 40% and 12% after the administration of TGF-beta antibody though it could only slightly alleviate the extent of fibrosis and alveolitis in lung tissue.. TGF-beta is a key factor stimulating the proliferation and collagen synthesis. TGF-beta antibody can partially neutralize its action. This may suggest that the antibody might become a new therapeutic choice for pulmonary fibrosis in the future.

Topics: Animals; Antibodies, Monoclonal; Bleomycin; Cells, Cultured; Fibroblasts; Male; Procollagen; Pulmonary Fibrosis; Rats; Rats, Wistar; RNA, Messenger; Transforming Growth Factor beta

We have used the silica-induced model of pulmonary injury in the rat to study the pattern of collagen expression in granulomatous lung inflammation. A single intratracheal instillation of silica into adult rats resulted in granulomatous inflammation leading to fibrosis and alveolar proteinosis. The development of disease in these animals was characterized over a 27-day period after treatment by means of histological, biochemical, and molecular analyses. Biochemical analyses indicated that significant increases in the weights of silicotic lungs were due to elevated amounts of DNA and total protein. Analysis of hydroxyproline content showed a 15-fold increase in this amino acid in silicotic lungs, confirming the development of a fibrotic reaction. In situ hybridization for type I procollagen mRNA displayed increased gene expression in the parenchyma, conducting airways, and vasculature of silicotic rats. Within the parenchyma, type I procollagen was expressed uniquely within granulomatous lesions. Immunohistochemistry indicated type I procollagen was being expressed by an alpha-smooth muscle actin-negative population of cells. Immunolocalization of extra-cellular transforming growth factor-beta showed coincident temporal and spatial overlap with type I procollagen expression, implicating this cytokine as a mediator of collagen gene expression in this model.

Topics: Animals; Disease Models, Animal; DNA; Gene Expression Regulation; Granuloma; Lung; Male; Organ Size; Procollagen; Pulmonary Fibrosis; Rats; Rats, Sprague-Dawley; Silicosis; Time Factors; Transforming Growth Factor beta

The emergence of the myofibroblast phenotype (characterized by alpha-smooth muscle actin expression) in wound healing and in tissues undergoing fibrosis is thought to be responsible for the increased contractility of the affected tissues. In bleomycin-induced pulmonary fibrosis, the myofibroblast is also responsible for the observed increase in collagen gene expression. To evaluate further these phenotypic changes in lung fibroblasts, contractile and other phenotypic properties of fibroblasts isolated from lungs of rats with bleomycin-induced fibrosis were compared with those of normal rats using in vitro models. Pulmonary fibrosis was induced in rats by endotracheal injection on day 0, and 7 and 14 days later the animals were sacrificed and lung fibroblasts isolated. Using immunofluorescence, < 10% of fibroblasts from control animals express alpha-smooth muscle actin when cultured as a monolayer. In contrast, 19% and 21% of cells from day 7 and day 14 bleomycin-treated animals, respectively, expressed this actin and with greater intensity than in control lung cells. This increase in actin expression was associated with enhanced contractility when evaluated using a three-dimensional cell culture model consisting of fibroblast-populated collagen gels. This enhanced contractility was abolished by treatment with antibody to transforming growth factor-beta (TGF-beta), whereas exogenous TGF-beta 1 and serum-stimulated contraction of control lung fibroblasts. TGF-beta 1 gene expression was greater in cells from bleomycin-treated animals than those from control lungs. These results show that cells with the myofibroblast phenotype are more abundant in fibrotic lung, and that these cells possess greater contractile capacity in vitro at least partly by virtue of their enhanced endogenous TGF-beta 1 gene expression.

Topics: Actins; Animals; Base Sequence; Bleomycin; Cell Count; Cells, Cultured; Collagen; Culture Media; Fibroblasts; Gels; Lung; Male; Molecular Sequence Data; Muscle, Smooth; Phenotype; Pulmonary Fibrosis; Rats; Rats, Inbred F344; Time Factors; Transforming Growth Factor beta

Taurine (T) and niacin (N) have previously been found to block the accumulation of collagen in the bleomycin (BL) model of interstitial pulmonary fibrosis. The present study was designed to evaluate whether the mechanism for the antifibrotic effect of combined treatment with taurine and niacin involves the down-regulation of BL-induced overexpression of procollagen I and III messenger ribonucleic acid (mRNA) levels in lungs. Hamsters were intratracheally instilled with three consecutive doses of saline or BL at weekly intervals (2.5, 2.0, 1.5 units/5 ml/kg). Four groups of animals were fed a diet throughout the experiment containing either 2.5% taurine and 2.5% niacin or the same diet without the drugs. The four groups were saline-instilled with the control diet (SA + CD), saline-instilled with TN in the diet (SA + TN), BL-instilled with the control diet (BL + CD), and BL-instilled with the TN diet (BL + TN). Steady state transcript levels in total RNA prepared from lungs of all four groups were determined at 0, 3, 7, 14 and 21 days after the last BL instillation by slot blot and Northern blot analyses. Results indicate that procollagen I mRNA levels are elevated compared with saline control by 2.5-, 2.4- and 2.0-fold at 7, 14, and 21 days after the last dose of BL instillation, respectively. Dietary treatment with taurine and niacin decreased the steady state level of BL-induced increases of procollagen I mRNA from day 0 through 21. We observed a similar pattern of procollagen III inhibition by taurine and niacin from day 3 through day 21. Transcription of procollagen I and III genes was readily detected in nuclei prepared from BL-treated lung samples at 14 days after treatment. In contrast, transcription of procollagen I and III genes was barely detectable in nuclei prepared at the same time point from BL + TN treated lungs. Our results suggest that procollagen I and III gene expression in BL-induced lung fibrosis in hamsters is transcriptionally down-regulated by combined treatment with taurine and niacin.

Topics: Animals; Bleomycin; Collagen; Cricetinae; Gene Expression Regulation; Male; Mesocricetus; Niacin; Procollagen; Pulmonary Fibrosis; RNA, Messenger; Taurine; Transcription, Genetic; Transforming Growth Factor beta

Although it is recognized that three isoforms of transforming growth factor-beta (TGF-beta) exist in mammals, their expression, distribution, and function in injury and repair are not well characterized. Using immunohistochemistry and antibodies to synthetic peptides of TGF-beta 1, TGF-beta 2, and TGF-beta 3, we determined the distribution of TGF-beta isoforms in lung sections with acute and chronic lesions of idiopathic pulmonary fibrosis (IPF), chronic asbestosis and hypersensitivity pneumonitis, as well as non-specific pneumonitis. In lung sections with advanced pulmonary fibrosis and honeycombing, irrespective of the diagnosis, TGF-beta 1 was prominently expressed in epithelial cells and macrophages and was found to be associated with the extracellular matrix. In lungs with early lesions of IPF and only inflammatory changes, TGF-beta 1 was present in alveolar macrophages but TGF-beta 1 was not present in epithelial cells. Small amounts of matrix-associated TGF-beta 1 were present subepithelially in areas of lung sections from patients with IPF with minimal inflammation and no fibrosis. In normal lungs with no evidence of inflammation or fibrosis TGF-beta 1 was not seen in alveolar macrophages, epithelial cells, or extracellularly. TGF-beta 2 and TGF-beta 3 were expressed in alveolar macrophages, epithelial cells, and smooth muscle cells of vessels and bronchi of normal lungs and lungs with both inflammatory and fibrotic changes. Our findings suggest that while TGF-beta 2 and TGF-beta 3 are ubiquitously expressed in the lung, TGF-beta 1 is expressed in epithelial cells of fibrotic lungs where the presence of TGF-beta 1 is not disease-specific but an indication of the chronicity of the injury.

Topics: Adult; Aged; Antibody Specificity; Asbestosis; Biopsy; Cysts; Epithelium; Humans; Immunohistochemistry; Lung; Lung Diseases, Interstitial; Lung Neoplasms; Macrophages; Middle Aged; Muscle, Smooth, Vascular; Pulmonary Fibrosis; Transforming Growth Factor beta

Recent studies indicate potential roles of monocyte chemotactic protein-1 (MCP-1) in recruitment of monocytes to sites of inflammation. However, their increased expression does not always correlate with monocyte influx, suggesting other possible biological activities for this member of the C-C chemokine family. In view of its potential role in regulating extracellular matrix expression in fibrotic disorders, the effects of MCP-1 on lung fibroblast collagen expression were evaluated. Isolated rat lung fibroblasts were treated with increasing doses of MCP-1 for variable periods of time and examined for effects on collagen synthesis and expression of procollagen alpha1(I) mRNA expression. The results show that MCP-1 was able to stimulate collagen expression in these cells in a dose-dependent manner but required over 24 h for significant elevation to occur. In view of this delayed time course, the possibility of mediation via endogenous transforming growth factor beta (TGFbeta) was tested by the ability of anti-TGFbeta antibody to inhibit this MCP-1 stimulation of collagen expression. Significant but incomplete inhibition by this antibody was observed. Pretreatment of the cells with antisense but not by sense or missense TGFbeta1 oligodeoxyribonucleotides caused essentially complete inhibition of this MCP-1 stimulatory effect. Furthermore, MCP-1 treatment was found to also stimulate TGFbeta secretion and mRNA expression, which was also abolished by pretreatment with antisense TGFbeta1 oligodeoxyribonucleotides. The kinetics of TGFbeta expression indicates that significant increase preceded that for collagen expression. Binding studies using 125I-labeled MCP-1 indicated the presence of specific and saturable binding sites with a dissociation constant consistent with the dose response curves for stimulation of fibroblast collagen synthesis and TGFbeta activity by MCP-1. These results taken together suggest that MCP-1 stimulates fibroblast collagen expression via specific receptors and endogenous up-regulation of TGFbeta expression. The latter then results in autocrine and/or juxtacrine stimulation of collagen gene expression.

Topics: Animals; Base Sequence; Chemokine CCL2; Collagen; Dose-Response Relationship, Drug; Fibroblasts; Gene Expression; Kinetics; Lung; Molecular Sequence Data; Protein Binding; Pulmonary Fibrosis; Rats; Receptors, CCR2; Receptors, Chemokine; Receptors, Cytokine; RNA, Messenger; Transforming Growth Factor beta

Binding of urokinase-type plasminogen activator (uPA) to a specific receptor (uPAR) on human lung fibroblasts enables it to regulate cellular proteolysis and remodeling of the extracellular matrix. Binding studies with radiolabeled uPA indicated that both normal and fibrotic lung fibroblasts express the receptor, but cells from fibrotic tissues bound significantly more uPA (P < 0.001). Phorbol myristate acetate, lipopolysaccharide, transforming growth factor-beta (TGF-beta), and tumor necrosis factor-alpha (TNF-alpha) increased uPA binding and plasminogen activation at the cell surface, with a greater maximal effect on fibrotic than on normal fibroblasts. Excess unlabeled uPA, specific antibody, or antisense oligonucleotides inhibited uPA binding. Ribonuclease (RNase) protection assays showed higher levels of uPAR messenger ribonuleic acid (mRNA) in each of the five fibrotic cell lines than in normal fibroblasts. uPA was mitogenic for normal as well as fibrotic fibroblasts, indicating that receptor binding concurrently localizes cellular proteolytic activity and stimulates mitogenesis. Morphometry and immunohistochemical analysis showed that uPAR, as well as uPA, was increased in fibroblasts in fibrotic lung tissue. Increased expression of uPAR by fibrotic lung fibroblasts and enhanced urokinase binding induced by proinflammatory cytokines suggest a novel mechanism by which fibroblast-mediated matrix remodeling and proliferation may be regulated in interstitial lung diseases.

Topics: Base Sequence; Binding, Competitive; Cell Division; Cells, Cultured; DNA; Electrophoresis, Polyacrylamide Gel; Fibroblasts; Gene Expression Regulation; Humans; Immunohistochemistry; Iodine Radioisotopes; Ligands; Lipopolysaccharides; Lung; Molecular Sequence Data; Plasminogen Activators; Pulmonary Fibrosis; Receptors, Cell Surface; Receptors, Urokinase Plasminogen Activator; RNA, Messenger; Tetradecanoylphorbol Acetate; Time Factors; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha; Urokinase-Type Plasminogen Activator

Fibrosis is a common sequela of various exogenous insults to a variety of parenchymal tissues. The underlying mechanisms of the induction and progression of fibrosis both at the molecular and cellular level have not been clarified so far. In the present study the cellular processes that ultimately may lead to interstitial fibrosis are described using the model of radiation-induced terminal differentiation in the fibroblast/fibrocyte cell system. The data reported herein will provide evidence that exogenously induced changes in the proliferation and differentiation pattern of the fibroblast/fibrocyte cell system based on either autocrine and/or paracrine mediators represent the underlying cellular mechanism of fibrosis. Using co-culture systems of parenchymal cells (fibroblasts and type II pneumocytes), the intercellular communication via cytokines, which may lead to fibrosis have been studied. TGF beta 1 could be described as one key modulator of these cellular processes.

Topics: Animals; Cell Differentiation; Fibroblasts; Humans; Lung; Mitosis; Phenotype; Pulmonary Fibrosis; Rats; Stem Cells; Transforming Growth Factor beta

We have investigated whether enhanced secretion of transforming growth factor-beta (TGF-beta) by distal respiratory epithelial cells was associated with the development of bleomycin-induced pulmonary fibrosis. Type 2 pneumocyte-enriched preparations of bronchioloalveolar epithelial cells from normal mouse lung tissue released latent TGF-beta when cultured in serum-free medium. TGF-beta in culture supernatants could be detected using a sensitive enzyme immunoassay which employed enzyme complex amplification as a reporter system, as well as by a radiolabelled receptor competition assay. Exposure to bleomycin and other potentially fibrogenic stimuli in vitro did not stimulate production of TGF-beta by the epithelial cells but release was enhanced by treatment of the cells with interferon-gamma. Type 2 pneumocyte-enriched cell preparations obtained following induction of a pulmonary inflammatory response by administration of intratracheal bleomycin to susceptible C57BL/6 mice did not demonstrate increased release of TGF-beta in culture. However, the concentration of TGF-beta in bronchoalveolar lavage (BAL) fluids was significantly elevated compared to controls at 1 and 2 weeks after bleomycin-induced injury in these mice. No such increase was detected in BAL fluids from BALB/c mice, which are resistant to the effects of bleomycin. These results provide no support for a pathogenetic role of alveolar epithelial cell-derived TGF-beta in bleomycin-induced pulmonary fibrosis. Nevertheless, elevated levels of TGF-beta in BAL fluids may provide a marker of the progression of pulmonary injury to fibrosis.

Topics: Animals; Bleomycin; Bronchoalveolar Lavage Fluid; Culture Media, Serum-Free; Epithelium; Female; Immunoenzyme Techniques; Lung; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Pulmonary Fibrosis; Transforming Growth Factor beta

Cytokine release from irradiated cells has been postulated to start soon after irradiation preceding detectable clinical and pathological manifestation of lung injury. The expression of transforming growth factor beta (TGF beta), a fibrogenic and radiation-inducible cytokine, was studied from 1-16 weeks after the 15 and 30 Gray (Gy) of thoracic irradiation to rats. Thoracic irradiation caused an increase in TGF beta protein in bronchoalveolar lavage (BAL) fluid peaking at 3-6 weeks as compared to sham-irradiated control rats. Steady state TGF beta mRNA expression as shown by whole lung northern blot assay paralleled the TGF beta protein expression in BAL fluid. The peak of TGF beta protein increase in BAL fluid between 3 and 6 weeks coincided with the initial influx of inflammatory cells in BAL fluid, but preceded histologically discernable pulmonary fibrosis that was not apparent until 8-10 weeks after irradiation. In conclusion. TGF beta and mRNA and protein upregulation preceded the radiation-induced pulmonary fibrosis, suggesting a pathogenetic role in the development of radiation fibrosis.

Topics: Animals; Bronchoalveolar Lavage Fluid; Gene Expression Regulation; Inflammation; Lung; Male; Pulmonary Edema; Pulmonary Fibrosis; Radiation Injuries, Experimental; Rats; Rats, Sprague-Dawley; RNA, Messenger; Transforming Growth Factor beta

Silicosis is characterized by fibrosing nodular lesions that may eventually develop into progressive massive fibrosis (PMF). Cytokines (interleukin-1beta [IL-1beta], tumor necrosis factor-alpha [TNF-alpha] and growth factors insulin-like growth factor-1 [IGF-1] platelet-derived growth factor [PDGF]) have been implicated in the formation of these lesions. TGF-beta promotes extracellular matrix accumulation by upregulating collagen and fibronectin gene expression, and inhibits matrix degradation by decreasing secretion of proteases and increasing secretion of protease inhibitors. We hypothesized that TGF-beta is associated with matrix deposition and fibrosis in silicosis. To test this hypothesis we studied early and late nodular lesions and PMF (11 cases and two controls) with immunohistochemistry, using rabbit polyclonal antibody to the purified whole molecule of TGF-beta in Bouin's fixed lung tissue. This antibody is reactive with both intra- and extracellular forms of TGF-beta. In the control lungs, small amounts of TGF-beta were present in the bronchial epithelium, macrophages, bronchial and vascular smooth muscle, and bronchial glands. There was minimal to moderate staining in the early silicotic peribronchiolar lesions. In the nodular lesions of silicosis, central hyalinized areas contained the maximum staining for TGF-beta. Fibroblasts in the periphery of the nodular lesions were also positive. In acute silicosis, there was marked staining of hyperplastic alveolar epithelium. Macrophages were markedly positive. In the PMF lesions, large areas of scar tissue contained TGF-beta. These data suggest a major role for TGF-beta in silicosis, particularly in the formation of silicotic nodules and the development of PMF.

Topics: Adult; Aged; Animals; Case-Control Studies; Extracellular Matrix; Humans; Immunoenzyme Techniques; Lung; Macrophages, Alveolar; Male; Middle Aged; Pulmonary Fibrosis; Rabbits; Silicosis; Smoking; Transforming Growth Factor beta

T lymphocytes expressing the alpha E beta 7 integrin are localized and selectively retained in mucosal tissues. To investigate a potential relationship between alpha E beta 7 expression and pulmonary inflammation, the distribution of alpha E beta 7-bearing CD4+ and CD8+ T cells in peripheral blood and bronchoalveolar lavage (BAL) fluids obtained from patients with allergic asthma, sarcoidosis, hypersensitivity pneumonitis, and idiopathic pulmonary fibrosis (IPF) was determined. In contrast to the distribution in peripheral blood, BAL fluid from these patients contained high number of cells expressing alpha E beta 7 with markedly different expression patterns on CD4 or CD8 cells as well as among the various diseases. Despite similar numbers of activated CD4 cells, alpha E beta 7+CD4+ T cells ranged from 15% in asthmatics to 70% in IPF. In contrast, even in normal individuals, 60% to 90% of BAL fluid CD8+ T cells express alpha E beta 7, suggesting differential induction mechanisms on CD4 and CD8 cells. In vitro experiments revealed that a substantial proportion of peripheral blood CD+ T cells express alpha E beta 7 after stimulation with anti-CD3 antibodies, and up to 80% positive cells were found after the addition of TGF-beta. In contrast, less than 10% of CD4 cells express this particular integrin after in vitro stimulation, and the presence of TGF-beta only increased the number to 30%. Supernatants from in vitro-activated BAL cells as well as concentrated BAL fluid from patients with high alpha E beta 7 expression had no further enhancing effect. However, crosslinking of alpha 4 beta 1-, but not beta 2-integrins, significantly increased the number of alpha E beta 7 expressing CD4+ and CD8+ T cells, even in the absence of TGF-beta. These data indicate that in addition to TGF-beta, the interaction of particular T-cell subsets with specific endothelial cell and extracellular matrix proteins may upregulate alpha E beta 7 integrin expression and thereby contribute to the selective accumulation of these cells in inflammatory lung diseases.

Topics: Adult; Alveolitis, Extrinsic Allergic; Antibodies; Asthma; Bronchoalveolar Lavage Fluid; Cells, Cultured; Cross-Linking Reagents; Eosinophils; Female; HLA-DR Antigens; Humans; Integrin alpha4beta1; Integrins; Leukocyte Common Antigens; Lung Diseases, Interstitial; Lymphocyte Activation; Lymphocyte Subsets; Macrophages, Alveolar; Male; Middle Aged; Neutrophils; Pulmonary Fibrosis; Receptors, Interleukin-2; Receptors, Lymphocyte Homing; Sarcoidosis; T-Lymphocytes; Transforming Growth Factor beta

Pulmonary fibrosis is the common end stage of a number of pneumopathies. In this study, we examined the ability of the human cytokine, relaxin, to block extracellular matrix deposition by human lung fibroblasts in vitro, and to inhibit lung fibrosis in a bleomycin-induced murine model. In vitro, relaxin (1-100 ng/ml) inhibited the transforming growth factor-beta-mediated over-expression of interstitial collagen types I and III by human lung fibroblasts by up to 45% in a dose-dependent manner. Relaxin did not affect basal levels of collagen expression in the absence of TGF-beta-induced stimulation. Relaxin also blocked transforming growth factor-beta-induced upregulation of fibronectin by 80% at the highest relaxin dose tested (100 ng/ml). The expression of matrix metalloproteinase-1, or procollagenase, was stimulated in a biphasic, dose-dependent manner by relaxin. In vivo, relaxin, at a steady state circulating concentration of approximately 50 ng/ml, inhibited bleomycin-mediated alveolar thickening compared with the vehicle only control group (P < 0.05). Relaxin also restored bleomycin-induced collagen accumulation, as measured by lung hydroxyproline content, to normal levels (P < 0.05). In summary, relaxin induced a matrix degradative phenotype in human lung fibroblasts in vitro and inhibited bleomycin-induced fibrosis in a murine model in vivo. These data indicate that relaxin may be efficacious in the treatment of pathologies characterized by lung fibrosis.

Topics: Animals; Bleomycin; Blotting, Western; Collagen; Collagenases; Disease Models, Animal; Electrophoresis, Polyacrylamide Gel; Enzyme Precursors; Fibronectins; Gene Expression Regulation; Histocytochemistry; Humans; Lung; Lung Injury; Mice; Procollagen; Pulmonary Fibrosis; Recombinant Proteins; Relaxin; Transforming Growth Factor beta

To investigate the influence of alveolar macrophages (AMs), fibroblasts and interstitial cells on development of lung fibrosis, and the interactions among TGF-beta 1 PDGF and IGF-1 and these cytokines-effects on lung fibrosis.. Expressions of TGF-beta 1, PDGF and IGF-1 mRNA in the lung cells and lung tissues in different stages of Bleomycin-A5-induced pulmonary fibrosis in rats were studied through Northern hybridization.. The expressions of TGF-beta 1 and PDGF mRNA reached their peaks in AMs of pulmonary fibrosis in rats on the 7th day after Bleomycin-A5 instillation. It was similar with that in the lung tissues. IGF-1 mRNA remained relatively stable in AMs during the course. PDGF and IGF-1 mRNA increased gradually in fibroblasts, and reached the highest expressions in the interstitial cells. There was almost no TGF-beta 1 mRNA expression in all groups of fibroblasts.. AMs are the main sources of TGF-beta 1 and PDGF in the lung tissues with fibrosis induced by Bleomycin-A5 AMs are activated in the first weekend and secrete TGF-beta 1 and PDGF to promote fibroblasts proliferation and fibrosis. As fibrosis developed, fibroblasts have established PDGF and IGF-1 autocrine and these three cytokines paracrine nets combined with the interstitial cells to promote lung fibrosis.

Topics: Animals; Bleomycin; Insulin-Like Growth Factor I; Lung; Male; Platelet-Derived Growth Factor; Pulmonary Fibrosis; Rats; Rats, Wistar; RNA, Messenger; Transforming Growth Factor beta

Evidence suggests that transforming growth factor beta (TGF-beta) may play a central role in a variety of fibroproliferative disorders via the induction of extracellular matrix accumulation. The three mammalian TGF-beta isoforms are present in the normal lung, but very little is known about their expression during lung injury and repair. To more fully understand the role of TGF-beta in lung repair, we investigated the expression of the TGF-beta 1, TGF-beta 2, and TGF-beta 3 isoforms in a bleomycin-induced model of pulmonary fibrosis using immunohistochemical and in situ hybridization techniques. We found expression of the three TGF-beta isoforms, in an identical pattern, widely distributed throughout the normal rat lung: in airways, blood vessels, lung parenchyma, and alveolar macrophages. In general, the distribution of TGF-beta mRNA and protein coincided; however, bronchial epithelial cells were a notable exception, exhibiting immunoreactivity but no mRNA expression. During the "inflammatory" phase (days 1 and 3) of bleomycin-induced injury there was an increase in the mRNA and protein expression of all three TGF-beta isoforms in the injured areas, most prominently in parenchymal cells and alveolar macrophages. There was a further increase in TGF-beta isoform expression in the areas of developing fibrosis during the later reparative phase (days 7 and 14), and the bronchial epithelium, previously not expressing TGF-beta mRNA, showed strong expression of mRNA for the three isoforms concomitant with increased immunoreactivity. These findings implicate the three mammalian TGF-beta isoforms in the dysregulated repair process that results in pulmonary fibrosis. Furthermore, the pattern of TGF-beta mRNA and protein expression by the bronchial epithelium suggests that a transition may occur at this site from a paracrine mode of action in the normal lung to an autocrine mode of action during the "reparative" phase of fibrosis.

Topics: Animals; Bleomycin; Cell Adhesion Molecules, Neuronal; Extracellular Matrix Proteins; Fibronectins; Immunohistochemistry; In Situ Hybridization; Male; Pulmonary Fibrosis; Rats; Rats, Sprague-Dawley; RNA Probes; RNA, Messenger; Tenascin; Tissue Distribution; Transforming Growth Factor beta

The murine TNF-alpha gene was expressed under the control of the human surfactant protein SP-C promoter in transgenic mice. A number of the SP-C TNF-alpha mice died at birth or after a few weeks with very severe lung lesions. Surviving mice transmitted a pulmonary disease to their offspring, the severity and evolution of which was related to the level of TNF-alpha mRNA in the lung; TNF-alpha RNA was detected in alveolar epithelium, presumably in type II epithelial cells. In a longitudinal study of two independent mouse lines, pulmonary pathology, at 1-2 mo of age, consisted of a leukocytic alveolitis with a predominance of T lymphocytes. Leukocyte infiltration was associated with endothelial changes and increased levels of mRNA for the endothelial adhesion molecule VCAM-1. In the following months, alveolar spaces enlarged in association with thickening of the alveolar walls due to an accumulation of desmin-containing fibroblasts, collagen fibers, and lymphocytes. Alveolar surfaces were lined by regenerating type II epithelial cells, and alveolar spaces contained desquamating epithelial cells in places. Platelet trapping in the damaged alveolar capillaries was observed. Pulmonary pathology in the SP-C TNF-alpha mice bears a striking resemblance to human idiopathic pulmonary fibrosis, in which increased expression of TNF-alpha in type II epithelial cells has also been noted. These mice provide a valuable animal model for understanding the pathogenesis of pulmonary fibrosis and exploring possible therapeutic approaches.

Topics: Animals; Base Sequence; Cell Adhesion Molecules; Disease Models, Animal; Female; Immunohistochemistry; Lung; Male; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Mice, Inbred DBA; Mice, Transgenic; Molecular Sequence Data; Pulmonary Fibrosis; RNA, Messenger; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha; Vascular Cell Adhesion Molecule-1

Bleomycin-induced pulmonary fibrosis is associated with increased lung transforming growth factor-beta (TGF-beta) gene expression, but cellular localization of the source of this expression has not been unequivocally established. In this study, lung fibrosis was induced in rats by endotracheal bleomycin injection on day 0 and, on selected days afterwards, lungs were harvested for in situ hybridization, immunohistochemical and histochemical analyses for TGF-beta 1 mRNA and protein expression, and cell identification. The results show that control lungs express essentially no detectable TGF-beta 1 mRNA or protein in the parenchyma. Before day 3 after bleomycin treatment, scattered bronchiolar epithelial cells, mononuclear cells, and eosinophils expressed elevated levels of TGF-beta 1. Between days 3 and 14, there was a major increase in the number of eosinophils, myofibroblasts, and fibroblasts strongly expressing TGF-beta 1 mRNA and protein. TGF-beta 1-producing cells were predominantly localized within areas of injury and active fibrosis. After day 14, the intensity and number of TGF-beta 1-expressing cells significantly declined and were predominantly found in fibroblasts in fibrotic areas. The expression of TGF-beta 1 protein was generally coincident with that for mRNA with the exception of bronchiolar epithelial cells in which strong protein expression was unaccompanied by a commensurate increase in mRNA. The study demonstrates that myofibroblasts, fibroblasts, and eosinophils represent the major sources of increased lung TGF-beta 1 expression in this model of pulmonary fibrosis.

Topics: Animals; Base Sequence; Bleomycin; Histocytochemistry; Immunochemistry; In Situ Hybridization; Lung; Male; Molecular Sequence Data; Oligonucleotide Probes; Pulmonary Fibrosis; Rats; Rats, Inbred F344; RNA, Messenger; Time Factors; Tissue Distribution; Transforming Growth Factor beta

Interstitial pneumonia is characterized by alveolitis with resulting fibrosis of the interstitium. To determine the relevance of humoral factors in the pathogenesis of interstitial pneumonia, we introduced expression vectors into Wistar rats via the trachea to locally overexpress humoral factors in the lungs. Human interleukin (IL) 6 and IL-6 receptor genes induced lymphocytic alveolitis without marked fibroblast proliferation. In contrast, overexpression of human transforming growth factor beta 1 or human platelet-derived growth factor B gene induced only mild or apparent cellular infiltration in the alveoli, respectively. However, both factors induced significant proliferation of fibroblasts and deposition of collagen fibrils. These histopathologic changes induced by the transforming growth factor beta 1 and platelet-derived growth factor B gene are partly akin to those changes seen in lung tissues from patients with pulmonary fibrosis and markedly contrast with the changes induced by overexpression of the IL-6 and IL-6 receptor genes that mimics lymphocytic interstitial pneumonia.

Topics: Adenoviridae; Animals; Antigens, CD; Becaplermin; Genetic Vectors; Humans; Interleukin-6; Lung; Lung Diseases, Interstitial; Male; Platelet-Derived Growth Factor; Proto-Oncogene Proteins c-sis; Pulmonary Fibrosis; Rats; Rats, Wistar; Receptors, Interleukin; Receptors, Interleukin-6; Recombinant Proteins; Transforming Growth Factor beta

Fibrosis, characterized by the accumulation of collagen, is a late result of thoracic irradiation. The purpose of this study was to determine if extracellular matrix protein and transforming growth factor beta mRNA expression are altered late in the course of pulmonary fibrosis after irradiation, and then to determine if these changes differ between two strains of mice which vary in their sensitivity to radiation. Radiation-sensitive (C57BL/6) and radiation-resistant (C3H/HeJ) mice were irradiated with a single dose of 5 or 12.5 Gy to the thorax. Total lung RNA was prepared and immobilized by Northern and slot blotting and hybridized with radiolabeled cDNA probes for collagens I, III and IV, fibronectin, and transforming growth factor beta 1 and beta 3. Autoradiographic data were quantified by video densitometry and results normalized to a control probe encoding for glyceraldehyde-3-phosphate dehydrogenase. Alterations in mRNA abundance were observed in the sensitive mice at all times, while levels in the resistant mice were unaffected until 26 weeks after irradiation. The relationship between extracellular matrix protein per se and increased mRNA abundance suggests that late matrix protein accumulation may be a function of gene expression. Differences in levels of transforming growth factor beta mRNA may lead to strain-dependent variation in fibrotic response and may also contribute to the radiation-induced component of pulmonary fibrosis.

Topics: Animals; Collagen; Female; Fibronectins; Gene Expression Regulation; Lung; Mice; Mice, Inbred C3H; Mice, Inbred C57BL; Pulmonary Fibrosis; Radiation Tolerance; RNA, Messenger; Species Specificity; Transforming Growth Factor beta

Idiopathic pulmonary fibrosis (IPF) is a poorly understood interstitial disease that usually proves refractory to therapy and results in irreversible tissue scarring and pulmonary dysfunction. Previous investigations have suggested a number of possible mediators of inflammation and fibrosis that typify IPF. We report increases in lung interleukin-1 receptor antagonist protein (IRAP) content in patients with IPF, as compared with normal control subjects. Importantly, this increase in IRAP was not accompanied by concomitant increases in interleukin-1 beta (IL-1 beta), resulting in a local environment that may be profibrotic. Tissue homogenates and bronchoalveolar lavage fluid from patients with IPF both demonstrate elevated IRAP content compared with that in normal subjects. Immunohistochemical staining and in situ hybridization localize IRAP to hyperplastic type II pneumocytes, macrophages, and local stromal cells. Finally, in vitro studies utilizing fibroblasts isolated from patients with IPF demonstrated no difference in constitutive IRAP production compared with that in normal subjects, but they revealed an exaggerated response to stimulation with transforming growth factor-beta (TGF-beta). These findings suggest that the fibrotic tissue changes of IPF and possibly other chronic interstitial lung diseases may result in part from the local effects of IRAP, and they also demonstrate that pulmonary nonimmune cells may influence local tissue changes through the elaboration of IRAP.

Topics: Bronchoalveolar Lavage Fluid; Case-Control Studies; Female; Fibroblasts; Gene Expression; Humans; Immunohistochemistry; In Situ Hybridization; Interleukin 1 Receptor Antagonist Protein; Interleukin-1; Lung; Male; Middle Aged; Pulmonary Fibrosis; Receptors, Interleukin-1; RNA, Messenger; Sialoglycoproteins; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha

Pulmonary biopsy specimens from ten cases of idiopathic pulmonary fibrosis (IPF) were examined using routine histological stains, including toluidine blue, and immunohistochemistry by means of specific antibodies against alpha-smooth muscle (alpha-SM) actin, desmin, keratin, TGF beta 1, and TNF alpha. The sections were compared with two cases of normal lung. As shown previously, normal alveolar interstitium did not contain alpha-SM actin positive myofibroblasts nor did the alveolar lining contain any significant number of TGF beta 1 or TNF alpha laden epithelial cells. In IPF, during the inflammatory stage, the alveolar myofibroblasts expressed alpha-SM actin and the regenerating type II alveolar epithelium staining strongly with TGF beta 1 and TNF alpha antibodies. The former cytokine was also detected in the interstitial matrix and fibroblastic cells as well as in the wall of vessels. At this stage, a manifest mast cell infiltration was noted. In very fibrotic and cystic alveolar tissue, i.e., at end stage fibrosis, the number of alpha-SM actin positive myofibroblasts as well as that of TNF alpha laden type II epithelial cells diminished, while TGF beta 1 positive cells persisted. Our findings demonstrate that during IPF alveolar type II epithelium constitutes, if not the site of synthesis, at least the main reservoir for TGF beta 1 and TNF alpha. These cytokines, besides their involvement in fibrogenesis, play probably an important role in the expression of alpha-SM actin by alveolar myofibroblasts. Our study suggests the possibility of an interaction between interstitial cells and alveolar epithelium, during IPF.

Topics: Actins; Adult; Aged; Bronchi; Cytoskeletal Proteins; Female; Fibroblasts; Humans; Immunohistochemistry; Male; Middle Aged; Pulmonary Alveoli; Pulmonary Fibrosis; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha

Transforming growth factor-beta (TGF-beta) is a cytokine with diverse biological activity. It can regulate the metabolic function of extracellular matrix (ECM) and play an important role in the development of pulmonary fibrosis. Collagen are major ECM components that are responsible for normal lung structure and function. Collagen accumulation is a major feature of pulmonary fibrosis. In order to study the mechanism of pulmonary fibrosis, we examined the effects of TGF-beta on the production, mRNA expression of type I collagen and type IV collagen by cultures of human embryonic lung fibroblasts. The results indicated that TGF-beta can induce collagen formation and type I collagen, type IV collagen mRNA expression without affecting cell proliferation. We concluded that TGF-beta plays a role in collagen accumulation of pulmonary fibrosis and the changes in collagen production appear to be mediated on collagen mRNA level.

Topics: Collagen; Fetus; Fibroblasts; Humans; Lung; Pulmonary Fibrosis; RNA, Messenger; Transforming Growth Factor beta

We examined the contribution of the cytokine transforming growth factor beta 1 (TGF-beta 1) in the inflammatory response and fibrotic reaction in a mouse model of immune-induced lung fibrosis caused by repeated intranasal exposure to heat-killed bacillus Calmette-Guérin (BCG). Mice received 200 micrograms of BCG 3 days/week for 4 weeks, and simultaneous intraperitoneal injections of a monospecific rabbit antiserum against mouse TGF-beta 1 or a preimmune serum (normal rabbit globulin). BCG instillations generated a copious release of antigenic TGF-beta 1 in the lungs at 1, 2, 3 and 4 weeks (up to 15 ng/lungs/mouse). Treatment with anti-TGF-beta 1 antiserum significantly diminished the number of free lung cells recovered by bronchoalveolar lavage (BAL), although the BAL cellular profile was not affected. Moreover, anti-TGF-beta 1 treatment of challenged mice diminished very significantly the total levels of interleukin-1 beta (IL-1 beta) and tumour necrosis factor-alpha (TNF-alpha) in the lungs of animals challenged with BCG. Histological examination and morphometric analysis of Masson's Trichrome-stained sections and measurements of total lung hydroxyproline levels showed a substantial decrease in lung fibrosis and granulomatous response of challenged mice given anti-TGF-beta 1. These data argue for a role for TGF-beta 1 in inducing inflammation and lung fibrosis in response to an immune stimulus.

Topics: Animals; Antigens, Bacterial; Cytokines; Disease Models, Animal; Female; Immunization, Passive; Lung; Mice; Mice, Inbred C57BL; Mycobacterium bovis; Pulmonary Fibrosis; Transforming Growth Factor beta

To identify the clinical relevance of cytokines involved in the development of lung fibrosis observed in patients with coal workers' pneumoconiosis (CWP), we investigated the BAL fluid contents and AM secretions of three mediators that modulate fibroblast growth: platelet-derived growth factor (PDGF), Type I insulin-like growth factor (IGF-I), and transforming growth factor Type beta (TGF-beta). Our study population consisted of 25 patients with CWP (16 simple pneumoconiosis, SP, 9 progressive massive fibrosis, PMF, 9 control subjects, and 6 patients with idiopathic pulmonary fibrosis (IPF). The fibrotic potency of AM supernatants was also tested for their ability to promote the growth of a human lung fibroblast cell line appreciated by [3H]-thymidine incorporation. PDGF and IGF-I concentrations were increased in BAL fluids of patients with PMF compared with SP and control subjects, whereas TGF-beta concentration was significantly higher in BAL fluid of patients with SP compared with PMF and control subjects. PDGF, IGF-I, and TGF-beta concentrations in AM supernatants followed the same profile observed in BAL fluids, suggesting that AM is one of the main cell sources of PDGF, IGF-I, and TGF-beta in the lung of pneumoconiotic patients. After treatment by acidification, which activated the latent form of TGF-beta, AM from patients with SP induced an inhibition of [3H]-thymidine incorporation and fibroblast growth was restored after neutralization of TGF-beta by specific antibodies. In contrast, AM supernatants from patients with PMF and IPF promoted the proliferation of fibroblasts and treatment by acidification did not modify this effect.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Aged; Analysis of Variance; Bronchoalveolar Lavage Fluid; Cells, Cultured; Coal Mining; Dust; France; Humans; Insulin-Like Growth Factor I; Macrophages, Alveolar; Middle Aged; Minerals; Platelet-Derived Growth Factor; Pneumoconiosis; Pulmonary Fibrosis; Severity of Illness Index; Statistics, Nonparametric; Transforming Growth Factor beta

We are studying the development of fibrogenic lesions in the lungs of rats exposed briefly to an aerosol of chrysotile asbestos fibers. This model of asbestosis has enabled us to establish very early cellular events at the specific locations where interstitial fibrosis will develop. These sites, the first alveolar duct bifurcations, are where the fibers are initially deposited and where macrophages first accumulate. In the studies presented here, we used immunohistochemical techniques to show that these macrophages exhibit strong localization of transforming growth factor-beta. In the adjacent developing fibrogenic lesions a clear increase in fibronectin staining was demonstrated and morphological analysis indicated a significant increase in amounts of smooth muscle actin. Such studies are essential in furthering our understanding of the distribution of potential mediators of the fibrogenic process and the cellular responses they elicit during the pathogenesis of disease.

Topics: Actins; Analysis of Variance; Animals; Asbestos, Serpentine; Fibronectins; Immunohistochemistry; Male; Muscle, Smooth; Pulmonary Fibrosis; Rats; Rats, Inbred Strains; Transforming Growth Factor beta

Sarcoidosis is a chronic inflammatory disease of unknown cause characterized by the formation of nonnecrotizing granulomas in affected tissues, most notably the lungs. Granuloma healing may result in pulmonary fibrosis and respiratory impairment in some patients. Transforming growth factor-beta 1 (TGF-beta 1) is a potent cytokine that promotes fibrosis by enhancing the synthesis of extracellular matrix components, including fibronectin and the alpha 5 beta 1 fibronectin receptor. The role of TGF-beta 1 in promoting lung fibrosis in the setting of pulmonary sarcoidosis has not yet been investigated. Accordingly, we determined the extent and distribution of TGF-beta 1 in lung tissue obtained from seven patients with clinical and histologic features of pulmonary sarcoidosis. The tissue distributions of TGF-beta 1, the TGF-beta 1 binding proteoglycan decorin, fibronectin, and the alpha 5 beta 1 fibronectin receptor were assessed immunohistochemically. In all cases, the epithelioid histiocytes comprising nonnecrotizing granulomas of pulmonary sarcoidosis contained abundant TGF-beta 1. We further demonstrated decorin, fibronectin, and the alpha 5 beta 1 fibronectin receptor within nonnecrotizing granulomas and in the fibrous tissue surrounding the lesions. TGF-beta 1 staining was also observed in bronchiolar epithelial cells, hyperplastic Type II pneumocytes, and occasional alveolar macrophages. This study demonstrates enhanced tissue localization of TGF-beta 1 and related extracellular matrix proteins associated with the nonnecrotizing granulomas of pulmonary sarcoidosis. Through its actions on matrix protein synthesis, TGF-beta 1 may modulate the fibrotic repair process accompanying granuloma healing in sarcoidosis.

Topics: Adult; Aged; Bronchi; Decorin; Epithelioid Cells; Epithelium; Extracellular Matrix; Extracellular Matrix Proteins; Fibronectins; Histiocytes; Humans; Immunohistochemistry; Macrophages, Alveolar; Middle Aged; Proteoglycans; Pulmonary Fibrosis; Radiography; Receptors, Fibronectin; Sarcoidosis, Pulmonary; Severity of Illness Index; Transforming Growth Factor beta; Wound Healing

Fibrosis, characterized by the accumulation of collagen, is a late result of thoracic irradiation. The expression of late radiation injury can be found immediately after irradiation by measuring messenger RNA (mRNA) abundance.. To determine if extracellular matrix mRNA and transforming growth factor beta abundance was affected acutely after irradiation, we measured mRNA levels of collagen I (CI), collagen III (CIII), collagen IV (CIV), fibronectin (FN), and transforming growth factor beta (TGF beta 1,2&3) in mouse lungs on day 1 and day 14 after graded doses of radiation. C57BL/6 female mice were irradiated with a single dose to the thorax of 5 or 12.5 Gy. Total lung RNA was prepared and immobilized by Northern and slot blotting and hybridized with radiolabelled cDNA probes for CI, CIII, CIV, FN, TGF beta 1,2&3 and a control probe encoding for glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Autoradiographic data were quantified by video densitometry and results normalized to GAPDH.. Changes in the expression of CI, CIII, CIV, FN and TGF beta 1,2&3 were observed as early as 1 day after exposure. Through 14 days, changes in mRNA up to 5-fold were seen for any one dose. Dose related changes as high as 10-fold were also evident. The CI:CIII ratio increased gradually for the 5 Gy dose at 14 days postirradiation while the CI:CII ratio for the 12.5 Gy dose decreased by approximately 4-fold as compared to the control.. These studies suggest that alterations in expression of extracellular matrix and TGF beta mRNA occur very early after radiation injury even at low doses and may play a role in the development of chronic fibrosis.

Topics: Animals; Extracellular Matrix; Female; Gene Expression; Lung; Mice; Mice, Inbred C57BL; Pulmonary Fibrosis; Radiation Genetics; Transforming Growth Factor beta

Fibroblast heterogeneity is known to exist in chronically inflamed tissue such as pulmonary fibrosis (IPF) and scleroderma. We have previously shown differences in proliferation rates in primary lines and cloned lines of fibroblasts derived from IPF tissue compared with normal lung. In this study, we report that cell lines derived from fibrotic tissue demonstrate anchorage-independent growth in soft agarose culture whereas normal lung fibroblast lines do not. We also show that fibroblast lines derived from neonatal lung tissue form colonies at about the same frequency as the fibrotic cells. Colonies from both fibrotic and neonatal lines were shown to be positive for vimentin, laminin, fibronectin, fibronectin receptor, beta-actin, and tropomyosin by immunohistochemistry but were negative for desmin, keratin, Factor VIII, alpha-smooth muscle cell actin, and tenascin. Treatment with cytokines TGF-beta and PDGF or with corticosteroid modified the colony-forming capacity of fibrotic and neonatal cell lines, however, none of these treatments induced normal lung cell lines to form colonies. The presence of cells in adult fibrotic tissue with growth characteristics similar to those exhibited by neonatal cells is further evidence of fibroblast heterogeneity and suggests newly differentiated fibroblasts may be prevalent in fibrotic tissue and contribute directly to the matrix disorder seen in this disease.

Topics: Cell Division; Cell Line; Dexamethasone; Fibroblasts; Humans; Immunohistochemistry; Lung; Platelet-Derived Growth Factor; Pulmonary Fibrosis; Transforming Growth Factor beta

To study the role of transforming growth factor-beta 1 (TGF-beta 1) in the pathogenesis of pulmonary fibrosis we have examined lung biopsies from nine patients with systemic sclerosis and interstitial lung disease, eight with 'lone' cryptogenic fibrosing alveolitis, two with cystic fibrosis, two with extrinsic allergic alveolitis, two with Langerhans' cell histiocytosis, one with lymphangioleiomyomatosis, one with giant cell interstitial pneumonia, and one adenocarcinoma of the lung. In cryptogenic fibrosing alveolitis, both 'lone' and associated with systemic sclerosis alveolar macrophages, bronchial epithelium and hyperplastic type II pneumonocytes expressed intracellular TGF-beta 1. Extracellular TGF-beta 1 was found in the fibrous tissue immediately beneath the bronchial and hyperplastic alveolar epithelium. In normal lung, however, the alveolar epithelium and alveolar interstitium were negative for both forms of TGF-beta 1. There was strong expression of TGF-beta 1 in hyperplastic mesothelium and its underlying connective tissue and in Langerhans' cells in the two cases of histiocytosis. In the organizing pneumonia in cystic fibrosis, the intraalveolar buds of granulation tissue reacted strongly for the extracellular form of TGF-beta 1 and the overlying hyperplastic epithelium expressed the intracellular form. In lymphangioleiomyomatosis, the aberrant smooth muscle cells strongly expressed intracellular TGF-beta 1 and the extracellular form was expressed in the adjacent connective tissue. In giant cell interstitial pneumonia, the numerous alveolar macrophage including the multinucleate forms, expressed intracellular TGF-beta 1, as did the hyperplastic alveolar epithelium.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Humans; Immunohistochemistry; Lung Diseases, Interstitial; Pulmonary Fibrosis; Scleroderma, Systemic; Transforming Growth Factor beta

Topics: Cell Line; Collagen; DNA; Fibroblasts; Humans; Interleukin-1; Lung; Proline; Pulmonary Fibrosis; Transforming Growth Factor beta; Tritium

Topics: Ascorbic Acid; Cell Line; Collagen; Fibroblasts; Gene Expression; Glutamate Decarboxylase; Humans; Interferon-gamma; Interleukin-1; Lung; Platelet-Derived Growth Factor; Pulmonary Fibrosis; RNA, Messenger; Transcription, Genetic; Transforming Growth Factor beta

Increased production of transforming growth factor beta (TGF-beta) seems to have an important role in the pathophysiology of bleomycin induced lung fibrosis. This is attributed to the ability of TGF-beta to stimulate infiltration of inflammatory cells and promote synthesis of connective tissue, leading to collagen deposition.. The study was designed to evaluate the antifibrotic potential of TGF-beta antibody in mice treated with bleomycin, which is a model of lung fibrosis. Under methoxyflurane anaesthesia, each mouse received intratracheally either 50 microliters sterile isotonic saline or 0.125 units bleomycin in 50 microliters. Within five minutes after the instillation, mice received into the tail vein 100 microliters non-immune rabbit IgG, TGF-beta 2 antibody, or a combination of TGF-beta 2 and TGF-beta 1 antibodies at various dose regimens. Mice were killed 14 days after the instillation and their lungs processed for morphological and biochemical studies.. Administration of 250 micrograms of TGF-beta 2 antibody after instillation of bleomycin followed by 100 micrograms on day 5 and 100 micrograms on day 9 significantly reduced the bleomycin induced increases in the accumulation of lung collagen from 445.8 (42.3) micrograms/lung to 336.7 (56.6) micrograms/lung at 14 days. Similarly, the combined treatment with 250 micrograms TGF-beta 2 antibody and 250 micrograms TGF-beta 1 antibody after bleomycin instillation followed by 100 micrograms of each antibody on day 5 also caused a significant reduction in bleomycin induced increases in lung collagen accumulation and myeloperoxidase activity at 14 days.. These results suggest that TGF-beta has an important role in the aetiology of bleomycin induced lung fibrosis; the neutralisation of TGF-beta by systemic treatment with its antibodies offers a new mode of pharmacological intervention which may be useful in treating lung fibrosis.

Topics: Animals; Antibodies; Bleomycin; Collagen; Hydroxyproline; Immunoglobulin G; Lung; Male; Mice; Pulmonary Fibrosis; Transforming Growth Factor beta

The development of bleomycin-induced pulmonary fibrosis in rats was studied over a period of 21 d after an intratracheal instillation of bleomycin. The expression of three small proteoglycans (biglycan, decorin, and fibromodulin), collagen III and TGF-beta 1 was studied by RNA-transfer blot analysis. The proteoglycans were also studied by SDS-polyacrylamide gel electrophoresis and Western blots. TGF-beta 1 mRNA increased threefold already on day 3 and remained elevated until day 10. After the increase of TGF-beta 1 mRNA the messages for biglycan and collagen III steadily increased to reach a maximum 10 d after bleomycin instillation. The mRNA for biglycan increased maximally fourfold and that of collagen III 2.5-fold. Decorin mRNA, in contrast to biglycan decreased and reached 20% of control on day 10. The message for fibromodulin remained constant throughout the study period. The amounts of biglycan and decorin in the tissue changed in accordance with the mRNA levels. The results corroborate and extend previous in vitro studies concerning the effect of TGF-beta 1 on the metabolism of small proteoglycans and show that these macromolecules are regulated differently also in vivo. The marked alterations of biglycan and decorin during the development of fibrosis suggests that these proteoglycans have a regulating role in this process.

Topics: Animals; Biglycan; Bleomycin; Blotting, Western; Carrier Proteins; Collagen; Decorin; DNA Probes; Extracellular Matrix Proteins; Fibromodulin; Gene Expression; Lung; Male; Proteoglycans; Pulmonary Fibrosis; Rats; Rats, Sprague-Dawley; RNA, Messenger; Transforming Growth Factor beta

Topics: Antibodies; Humans; Pulmonary Fibrosis; Transforming Growth Factor beta

Hepatic veno-occlusive disease and idiopathic interstitial pneumonitis are major causes of morbidity and mortality after bone marrow transplantation. Fibrosis is a characteristic of both conditions, and transforming growth factor beta (TGF beta) has been implicated in the pathogenesis of fibrosis.. Using acid-ethanol extraction to remove TGF beta from human plasma and a mink-lung epithelial-cell growth-inhibition assay to measure TGF beta activity, we quantified plasma TGF beta in 10 normal subjects and 41 patients before and after they underwent high-dose chemotherapy and autologous bone marrow transplantation for advanced breast cancer.. There was no difference in pretransplantation TGF beta levels between the controls and the patients who did not have hepatic veno-occlusive disease or idiopathic interstitial pneumonitis after transplantation. In contrast, pretransplantation TGF beta levels were significantly higher in patients in whom hepatic veno-occlusive disease or idiopathic interstitial pneumonitis developed than in the controls or the patients without these conditions. The predictive value for the development of either condition was 90 percent or more when pretransplantation plasma TGF beta levels were more than 2 SD above the mean established in the controls.. The plasma TGF beta concentration measured after induction chemotherapy but before high-dose chemotherapy and autologous bone marrow transplantation strongly correlates with the risk of hepatic veno-occlusive disease and idiopathic interstitial pneumonitis after these treatments.

Topics: Adenocarcinoma; Adult; Antineoplastic Combined Chemotherapy Protocols; Bone Marrow Transplantation; Breast Neoplasms; Combined Modality Therapy; Female; Hepatic Veno-Occlusive Disease; Humans; Liver Cirrhosis; Middle Aged; Pulmonary Fibrosis; Transforming Growth Factor beta

Since transforming growth factor beta (TGF beta) has been implicated as an important mediator of pulmonary fibrosis, we measured TGF beta protein and gene expression in alveolar epithelial lining fluid (ELF) of fibrotic scleroderma lungs sampled by bronchoalveolar lavage (BAL). TGF beta protein was qualitatively examined by Western blot analysis, and quantitatively by radioreceptor assays. Gene expression was evaluated in BAL mononuclear cells by Northern blot analysis with quantification of relative gene expression by densitometric analysis of the autoradiograms.. Normal and scleroderma subjects had a 24-kd protein that comigrated with defined human TGF beta 1 and immunoreacted with anti-TGF beta antibody. The normal population had a significantly higher average TGF beta concentration (705 pM) compared with the scleroderma subjects (177 pM). The TGF beta 1 gene was expressed in amounts that did not significantly differ between the scleroderma and normal groups. On an individual subject basis, the TGF beta concentration variability did not correlate with variations in BAL cellularity or TGF beta 1 gene expression within the recovered mononuclear cells.. It is concluded that both normal and fibrotic lungs have TGF beta 1 present at the alveolar epithelial surface. However, in the fibrotic scleroderma lungs, TGF beta protein content and gene expression were not increased at the alveolar epithelial surface. The simultaneous analysis of TGF beta protein content, gene expression, and cellular constituents within individual ELF specimens showed that the cellular components of the ELF do not appear to be major determinants of TGF beta protein concentration at the alveolar epithelial surface.

Topics: Adult; Aged; Alabama; Autoradiography; Blotting, Northern; Blotting, Western; Bronchoalveolar Lavage Fluid; Evaluation Studies as Topic; Female; Gene Expression; Hospitals, University; Humans; Male; Middle Aged; Outpatient Clinics, Hospital; Pulmonary Fibrosis; Radioligand Assay; Scleroderma, Systemic; Sensitivity and Specificity; Transforming Growth Factor beta

Pulmonary fibrosis is a crippling, essentially lethal chronic disease due to an interplay of events following irradiation between the pneumonitic and fibrotic phases. In this series of experiments it is demonstrated that there is no latent period after irradiation, but an immediate intercellular communication system which springs into action to initiate recovery. Latency was only a function of our inability to uncover the molecular events that precede and underlie the clinical pathologic course of organ/tissue irradiation. Current advances in understanding the production of growth factors by different cells provides new insights to autocrine, paracrine and endocrine messages as a basis for understanding radiation pathophysiology as a progressive process that is amplified by other injurious events such as chemotoxicity. This is the first demonstrated release of trophic factors (cytokines) after in vivo irradiation that persists up to a month after exposure, suggesting that the persistence of a small incremental stimulus during a silent "latent" period can be the basis for the clinical pathologic expression of a late radiation effect, that is, pulmonary interstitial fibrosis.

Topics: Animals; Dose-Response Relationship, Radiation; Fibroblasts; Lung; Macrophages, Alveolar; Pneumonia; Pulmonary Fibrosis; Rabbits; Radiation Injuries, Experimental; Transforming Growth Factor beta

Firefighters are potentially at increased risk for cancer and non-malignant respiratory disease due to their toxic exposures on the job. Growth factors and oncogene proteins are thought to play a role in the development of various malignancies and pulmonary fibrotic diseases. Therefore, a cohort of firefighters and matched controls have been screened for the presence of nine different growth factors and oncoproteins using an immunoblotting assay. Fourteen of the firefighters were found to be positive for beta-transforming growth factor (beta-TGF) related proteins compared to no positives in the controls (P = 0.0017). These results suggest that beta-TGF may be a possible biomarker for monitoring firefighters and other exposed workers for the potential development of cancer or non-malignant respiratory disease.

Topics: Amino Acid Sequence; Fires; Humans; Mass Screening; Middle Aged; Molecular Sequence Data; Neoplasms; New York City; Occupational Exposure; Oncogene Proteins; Platelet-Derived Growth Factor; Population Surveillance; Pulmonary Fibrosis; Risk Factors; Transforming Growth Factor beta

Topics: Anemia, Aplastic; Animals; Glomerulonephritis; Humans; Liver Regeneration; Myocardial Infarction; Pulmonary Fibrosis; Receptors, Cell Surface; Receptors, Transforming Growth Factor beta; Signal Transduction; Transforming Growth Factor beta

This study examines the hypothesis that mediators from lung endothelial cells could promote lung collagen synthesis in pulmonary fibrosis. Since bleomycin induces pulmonary fibrosis in humans and animals, the effects of this drug on endothelial cells were examined. Endothelial cell conditioned media were prepared in the presence of various doses of bleomycin, and tested for their ability to stimulate lung fibroblast collagen synthesis. The results show a dose-dependent stimulation of endothelial cell secretion of collagen synthesis stimulatory activity by bleomycin, which peaked at a dose greater than or equal to 100 ng/ml. Stimulation was selective for collagenous protein synthesis. Gel filtration analysis showed most of the activity to reside in fractions with an estimated molecular mass range of 10-27 kD. The activity was inhibited by anti-transforming growth factor-beta (TGF-beta)antibody, but not by nonimmune control IgG. The presence of TGF-beta was confirmed using the mink lung epithelial cell assay. Northern blotting revealed significant increases in TGF-beta mRNA in bleomycin-stimulated endothelial cells. Thus in vitro stimulation of endothelial cells by bleomycin upregulates TGF-beta production, presumably by increased transcription. In view of the chemotactic and matrix synthesis stimulatory properties of this cytokine, such an increase in TGF-beta production may play an important role in bleomycin-induced pulmonary fibrosis.

Topics: Animals; Bleomycin; Cells, Cultured; Collagen; Endothelium, Vascular; Lung; Male; Mink; Pulmonary Fibrosis; Rats; Rats, Inbred F344; RNA, Messenger; Transforming Growth Factor beta

The spontaneous elevation of the transcription of the transforming growth factor-beta (TGF-beta) gene in broncho-alveolar mononuclear cells (BMC) of individuals with autoimmune diseases with lung involvement, by nuclear run-on transcription assay, is shown in this study. In quantification analysis of TGF-beta gene transcription, we found more than 10 times the enhanced transcription of the TGF-beta gene in BMC of individuals with autoimmune diseases with lung involvement, in comparison to normal healthy subjects or patients with bronchial asthma used as controls. Our observation suggests that TGF-beta, a potent mitogen for fibroblasts, may be produced in BMC during an active immune response in individuals with systemic autoimmune diseases with lung involvement, and may be involved in autoimmune-related pathophysiological changes of cytokine networks in lung involvement such as lung fibrosis.

Topics: Autoimmune Diseases; Bronchoalveolar Lavage Fluid; Female; Humans; Leukocytes, Mononuclear; Lupus Erythematosus, Systemic; Male; Pulmonary Fibrosis; Scleroderma, Systemic; Transcription, Genetic; Transforming Growth Factor beta

Idiopathic pulmonary fibrosis is an inexorably fatal disorder characterized by connective tissue deposition within the terminal air spaces resulting in loss of lung function and eventual respiratory failure. Previously, we demonstrated that foci of activated fibroblasts expressing high levels of fibronectin, procollagen, and smooth muscle actin and thus resembling those found in healing wounds are responsible for the connective tissue deposition and scarring in idiopathic pulmonary fibrosis. Using in situ hybridization and immunohistochemistry, we now demonstrate the presence of transforming growth factor beta 1 (TGF-beta 1), a potent profibrotic cytokine, in the foci containing these activated fibroblasts. These results suggest that matrix-associated TGF-beta 1 may serve as a stimulus for the persistent expression of connective tissue genes. One potential source of the TGF-beta 1 is the alveolar macrophage, and we demonstrate the expression of abundant TGF-beta 1 mRNA in alveolar macrophages in lung tissue from patients with idiopathic pulmonary fibrosis.

Topics: Fibronectins; Gene Expression; Humans; Hypertension, Pulmonary; Lung; Nucleic Acid Hybridization; Procollagen; Pulmonary Fibrosis; Reference Values; RNA Probes; RNA, Messenger; Transforming Growth Factor beta

Transforming growth factor-beta (TGF-beta) can regulate cell growth and differentiation as well as production of extracellular matrix proteins. Elevated production of TGF-beta has been associated with human and rodent chronic inflammatory and fibrotic diseases. Using immunohistochemical staining, we have examined lung sections of patients with advanced idiopathic pulmonary fibrosis (IPF), a disease characterized by chronic inflammation and fibrosis and demonstrated a marked and consistent increase in TGF-beta production in epithelial cells and macrophages when compared to patients with nonspecific inflammation and those with no inflammation or fibrosis. In patients with advanced IPF, intracellular staining with anti-LC (1-30) TGF-beta antibody was seen prominently in bronchiolar epithelial cells. In addition, epithelial cells of honeycomb cysts and hyperplastic type II pneumocytes stained intensely. Anti-CC (1-30) TGF-beta antibody, which reacts with extracellular TGF-beta, was localized in the lamina propria of bronchioles and in subepithelial regions of honeycomb cysts in areas of dense fibroconnective tissue deposition. The close association of subepithelial TGF-beta to the intracellular form in advanced IPF suggests that TGF-beta was produced and secreted primarily by epithelial cells. Because of the well-known effects of TGF-beta on extracellular matrix formation and on epithelial cell differentiation, the increased production of TGF-beta in advanced IPF may be pathogenic to the pulmonary fibrotic and regenerative responses seen in this disease.

Topics: Biopsy; Humans; Immunohistochemistry; Pulmonary Fibrosis; Transforming Growth Factor beta

Alveolar macrophages are believed to be central in orchestrating the fibrotic response in interstitial lung disease (ILD). To test the hypothesis that macrophages from patients with ILD were dedicated to growth factor production and that this was independent of other indices of macrophage activation, we measured the mRNA of the B chain of PDGF and TGF-beta, as well as HLA-DR-alpha in alveolar macrophages from patients with ILD and from normal control subjects. When alveolar macrophages were examined immediately after lavage, cells from patients with ILD had increased PDGF(B) but similar TGF-beta and HLA-DR-alpha mRNA when compared with control subjects. Discoordinate regulation of these genes was observed when alveolar macrophage PDGF(B) mRNA increased while TGF-beta and HLA-DR-alpha mRNA decreased after culture for 24 h. This response was not disease-related as these changes were similar in cells from patients with ILD and from control subjects. Because a lymphocytic alveolitis is present in many cases of ILD, we asked whether interferon gamma (IFN-gamma) modulated the activation of these genes. In both the patients and the control subjects, PDGF(B) and HLA-DR-alpha, but not TGF-beta, mRNA were increased after incubation with IFN-gamma. These results indicate that PDGF(B) mRNA may be increased in alveolar macrophages in ILD and that PDGF(B), TGF-beta, and HLA-DR-alpha are independently regulated genes in alveolar macrophages, but that IFN-gamma increases both PDGF(B) and HLA-DR-alpha mRNA. We speculate that IFN-gamma induced PDGF(B) gene activation may be an important mechanism by which lymphocytes promote pulmonary fibrosis.

Topics: Adult; Cells, Cultured; Female; Gene Expression Regulation; HLA-DR Antigens; Humans; Interferon-gamma; Macrophages; Male; Middle Aged; Platelet-Derived Growth Factor; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-sis; Pulmonary Alveoli; Pulmonary Fibrosis; RNA, Messenger; Transcriptional Activation; Transforming Growth Factor beta; Up-Regulation

Topics: Extracellular Matrix Proteins; Fibronectins; Growth Substances; Humans; Nucleic Acid Hybridization; Platelet-Derived Growth Factor; Procollagen; Pulmonary Fibrosis; RNA, Messenger; Transforming Growth Factor beta

Topics: Animals; Humans; Immunohistochemistry; Lung; Pulmonary Fibrosis; Rats; Transforming Growth Factor beta