Intratracheal Bleomycin Research Articles

Changes in pulmonary endothelial cell properties during bleomycin-induced pulmonary fibrosis

BackgroundPulmonary fibrosis is a progressive and lethal disease characterized by damage to the lung parenchyma with excess extracellular matrix deposition. The involvement of endothelial cells in fibrosis development is unclear.MethodsWe isolated pulmonary endothelial cells, using a magnetic-activated cell sorting system, from mice with pulmonary fibrosis induced by intratracheal bleomycin. We characterized endothelial cells isolated at various times in the course of pulmonary fibrosis development.ResultsInflammatory cell infiltration was observed at 7 days after bleomycin administration, and fibrotic changes with increased collagen content were observed on day 21. Endothelial cells were isolated at these two timepoints. Levels of von Willebrand factor, plasminogen activator inhibitor-1 and matrix metalloproteinase-12 were elevated in lung endothelial cells isolated from bleomycin-treated mice at days 7 and 21. This indicated that intratracheal bleomycin administration induced endothelium injury. Expression of fibrogenic mediators, transforming growth factor (TGF)-β, connective tissue growth factor and platelet-derived growth factor-C was elevated in the cells from bleomycin-treated, compared with untreated, lungs. When endothelial cells were treated with TGF-β, α-smooth muscle actin (SMA) expression and collagen production were increased only in those cells from bleomycin-treated mouse lungs. Thapsigargin-induced prostaglandin I2 and nitric oxide production, decreased in endothelial cells from bleomycin-treated mouse lungs, compared with controls, was further suppressed by TGF-β.ConclusionBleomycin administration induced functional changes in lung endothelial cells, indicating potential involvement of endothelium in pulmonary fibrogenesis.

Endothelial-derived endothelin-1 promotes pulmonary vascular remodeling in bleomycin-induced pulmonary fibrosis.

Endothelin-1 (ET-1) induces pulmonary vascular remodeling and pulmonary hypertension secondary to pulmonary fibrosis. Given that endothelial cells are the main source of ET-1 and ET-1 from other cells may encounter difficulty penetrating vascular compartments, we hypothesize that endothelial-derived ET-1 promotes vascular remodeling secondary to pulmonary fibrosis. We used vascular endothelial ET-1 knock-out (VEETKO) and Wild type mice for this research. They were given intratracheal bleomycin and euthanized at day 28. We quantified pulmonary fibrosis, measured lung ET-1 and its receptors' expression, and assessed pulmonary vascular remodeling by calculating medial wall index, muscularization index, adventitial collagen and adventitial fibroblast and macrophage accumulation. Right ventricle remodeling was also assessed. Both VEETKO and Wild type mice developed comparable pulmonary fibrosis and similar fibrosis-related gene expression. Compared to Wild type mice, bleomycin-induced VEETKO mice had lower ET-1 peptide levels (15.4 pg/mg vs. 31.2 pg/mg, p<0.01). Expression of both ET-1 receptors mRNAs were increased in fibrosis models. Bleomycin-induced fibrosis VEETKO mice had significantly less muscularized arterioles, lower muscularization index and attenuated adventitial collagen, fibroblast and macrophage accumulation as compared to that of Wild type mice. Right ventricular pressure, hypertrophy and fibrosis did not increase both in VEETKO and Wild type mice despite the more enhanced vascular remodeling in Wild type. In conclusion, endothelial-derived endothelin-1 promotes pulmonary vascular remodeling secondary to bleomycin-induced pulmonary fibrosis.

A Multimodal Imaging Approach Based on Micro-CT and Fluorescence Molecular Tomography for Longitudinal Assessment of Bleomycin-Induced Lung Fibrosis in Mice.

Idiopathic pulmonary fibrosis (IPF) is a fatal lung disease characterized by the progressive and irreversible destruction of lung architecture, which causes significant deterioration in lung function and subsequent death from respiratory failure. The pathogenesis of IPF in experimental animal models has been induced by bleomycin administration. In this study, we investigate an IPF-like mouse model induced by a double intratracheal bleomycin instillation. Standard histological assessments used for studying lung fibrosis are invasive terminal procedures. The goal of this work is to monitor lung fibrosis through noninvasive imaging techniques such as Fluorescent Molecular Tomography (FMT) and Micro-CT. These two technologies validated with histology findings could represent a revolutionary functional approach for real time non-invasive monitoring of IPF disease severity and progression. The fusion of different approaches represents a step further for understanding the IPF disease, where the molecular events occurring in a pathological condition can be observed with FMT and the subsequent anatomical changes can be monitored by Micro-CT.

Submicron emulsion of cinnamaldehyde ameliorates bleomycin-induced idiopathic pulmonary fibrosis via inhibition of inflammation, oxidative stress and epithelial-mesenchymal transition

AimsIdiopathic pulmonary fibrosis (IPF) is the most frequent and severe form of idiopathic interstitial pneumonias. The pathogenesis is associated with inflammation and oxidative stress and epithelial-mesenchymal transition (EMT). Cinnamaldehyde exhibits antiinflammatory and antioxidant properties, but its effect on IPF is unknown. The present study is to investigate the anti-fibrotic effect and action mechanism of cinnamaldehyde on IPF. Materials and methodsIPF was induced by intratracheal bleomycin in mice. Submicron emulsion of cinnamaldehyde was given by intraperitoneal injection once everyday for 7 or 21 continuous days after bleomycin administration. Lung histological and injury indexes were analyzed. The protein expressions of inflammation and oxidative stress as well as EMT markers alpha-smooth muscle actin (α-SMA) and E-cadherin in mice and cultured A549 cells were measured. ResultsCinnamaldehyde attenuated the bleomycin-induced histological injury, reduced hydroxyproline level and improved pulmonary function by the inhibiting inflammatory cytokines and reactive oxygen species production as well as enhancing total superoxide dismutase activity in bleomycin-induced mice. Cinnamaldehyde also inhibited EMT in both bleomycin-induced mice and TGF-β1-stimulated A549 cells. ConclusionsCinnamaldehyde ameliorated bleomycin-induced IPF via inhibition of inflammation and oxidative stress and EMT.

Thyroid hormone inhibits lung fibrosis in mice by improving epithelial mitochondrial function.

Thyroid hormone (TH) is critical for the maintenance of cellular homeostasis during stress responses, but its role in lung fibrosis is unknown. Here, we found that the activity and expression of iodothyronine deiodinase 2 (DIO2), an enzyme that activates TH, was higher in lungs of patients with idiopathic pulmonary fibrosis compared to control individuals and correlated with disease severity. We also found that Dio2 knockout mice exhibited enhanced bleomycin-induced lung fibrosis. Aerosolized TH delivery increased survival and resolved fibrosis in two models of pulmonary fibrosis in mice (intratracheal bleomycin and inducible TGF-β1). Sobetirome, a TH mimetic, also blunted bleomycin-induced lung fibrosis. Given after bleomycin-induced injury, TH promoted mitochondrial biogenesis, improved mitochondrial bioenergetics and attenuated mitochondria-regulated apoptosis in alveolar epithelial cells both in vivo and in vitro. TH did not blunt fibrosis in Ppargc1a or Pink1 knockout mice suggesting dependence on these pathways. We conclude that the TH anti-fibrotic properties are associated with protection of alveolar epithelial cells and restoration of mitochondrial function and thus may represent an effective therapy for pulmonary fibrosis.

Essential involvement of the CX3CL1-CX3CR1 axis in bleomycin-induced pulmonary fibrosis via regulation of fibrocyte and M2 macrophage migration

The potential role of macrophages in pulmonary fibrosis (PF) prompted us to evaluate the roles of CX3CR1, a chemokine receptor abundantly expressed in macrophages during bleomycin (BLM)-induced PF. Intratracheal BLM injection induced infiltration of leukocytes such as macrophages into the lungs, which eventually resulted in fibrosis. CX3CR1 expression was mainly detected in the majority of macrophages and in a small portion of α-smooth muscle actin-positive cells in the lungs, while CX3CL1 was expressed in macrophages. BLM-induced fibrotic changes in the lungs were reduced without any changes in the number of leukocytes in Cx3cr1−/− mice, as compared with those in the wild-type (WT) mice. However, intrapulmonary CX3CR1+ macrophages displayed pro-fibrotic M2 phenotypes; lack of CX3CR1 skewed their phenotypes toward M1 in BLM-challenged lungs. Moreover, fibrocytes expressed CX3CR1, and were increased in BLM-challenged WT lungs. The number of intrapulmonary fibrocytes was decreased in Cx3cr1−/− mice. Thus, locally-produced CX3CL1 can promote PF development primarily by attracting CX3CR1-expressing M2 macrophages and fibrocytes into the lungs.

Depletion of club cells attenuates bleomycin-induced lung injury and fibrosis in mice

BackgroundThe role of bronchiolar epithelial cells in the pathogenesis of pulmonary fibrosis has not been clarified. We previously demonstrated DNA damage in murine bronchioles in the early stages of bleomycin-induced pulmonary fibrosis that subsequently extended to alveolar cells at the advanced stages of the disease. Club cells are progenitor cells for bronchioles and are known to play protective roles against lung inflammation and damage. The aim of the present study was to elucidate the role of club cells in the development of pulmonary fibrosis.MethodsC57BL/6 J mice received naphthalene intraperitoneally on day −2 to deplete club cells and were given intratracheal bleomycin or a vehicle on day 0. Lung tissues were obtained on days 1, 7, and 14, and bronchoalveolar lavage was performed on day 14. Bronchiolar epithelial cells sampled by laser capture microdissection were analyzed by gene expression microarray analysis on day 14.ResultsClub cell depletion induced by naphthalene protected mice from bleomycin-induced lung injury and fibrosis. Bleomycin-triggered bronchiolar TGF-β1 expression was reduced. Gene expression microarray analysis revealed that genes associated with inflammatory response and chemokine activity were downregulated in the bleomycin-injured bronchiolar epithelium with club cell injury compared to that in bronchiolar epithelium without cell injury.ConclusionsClub cells are involved in the development of lung injury and fibrosis.

Antifibrotic role of vascular endothelial growth factor in pulmonary fibrosis.

The chronic progressive decline in lung function observed in idiopathic pulmonary fibrosis (IPF) appears to result from persistent nonresolving injury to the epithelium, impaired restitution of the epithelial barrier in the lung, and enhanced fibroblast activation. Thus, understanding these key mechanisms and pathways modulating both is essential to greater understanding of IPF pathogenesis. We examined the association of VEGF with the IPF disease state and preclinical models in vivo and in vitro. Tissue and circulating levels of VEGF were significantly reduced in patients with IPF, particularly in those with a rapidly progressive phenotype, compared with healthy controls. Lung-specific overexpression of VEGF significantly protected mice following intratracheal bleomycin challenge, with a decrease in fibrosis and bleomycin-induced cell death observed in the VEGF transgenic mice. In vitro, apoptotic endothelial cell–derived mediators enhanced epithelial cell injury and reduced epithelial wound closure. This process was rescued by VEGF pretreatment of the endothelial cells via a mechanism involving thrombospondin-1 (TSP1). Taken together, these data indicate beneficial roles for VEGF during lung fibrosis via modulating epithelial homeostasis through a previously unrecognized mechanism involving the endothelium.

5-HT7 receptor antagonism (SB-269970) attenuates bleomycin-induced pulmonary fibrosis in rats via downregulating oxidative burden and inflammatory cascades and ameliorating collagen deposition: Comparison to terguride

The neurotransmitter 5-hydroxytryptamine (5-HT) is involved in regulation of local tissue inflammation and repair through a set of receptors (5-HT1-7 receptors), which are expressed in the lung. Considering the protective importance of 5-HT receptor antagonists against development of pulmonary fibrosis, we evaluated whether 5-HT7 receptor antagonist (SB-269970) modulates lung inflammatory and fibrogenic processes in comparison with 5-HT2A/B receptor antagonist (terguride), in bleomycin (BLM)-induced idiopathic pulmonary fibrosis (IPF) model.IPF model induced by a single dose of intra-tracheal BLM instillation (5mg/kg), and rats were treated with intraperitoneal injection of SB-269970 (1mg/kg day) or terguride (1.2mg/kg/d). The experiment was carried out on two separate sets of rats that were killed at day 7th and day 21st to evaluate the endpoint of the IPF inflammatory and fibrogenic phases, respectively.During the inflammatory phase 5-HT2A/B and 5-HT7 receptor antagonists attenuated the BLM-induced increase in the lung fluid content, the inflammatory cytokines levels and oxidative stress burden. In the fibrogenic phase, both SB-269970 and terguride reduced the serotonin concentrations in lung homogenates and significantly protected against IPF fibrogenic phase by attenuating collagen deposition and mRNA expression of both transforming growth factor-β1 (TGF- β1), and procollagen type Ӏ (PINP). 5-hydroxytryptamine 5-HT7 receptor antagonist showed more benefits than 5-HT2A/B receptor antagonist on the deleterious effects accompanied BLM instillation. The present study showed involvement of 5-HT7 receptor in the pathophysiology of BLM-induced IPF in rats and identified it as a potential therapeutic target in lung fibrotic disorders.

Involvement of midkine in the development of pulmonary fibrosis.

Midkine is a low‐molecular‐weight heparin‐binding protein that is strongly expressed mainly in the midgestation period and has various physiological activities such as in development and cell migration. Midkine has been reported to be strongly expressed in cancer cells and in inflammation and repair processes, and to be involved in the pathogenesis of various diseases. However, its role in the lung is poorly understood. In this study, we analyzed the clinical characteristics of idiopathic pulmonary fibrosis patients in relation to midkine expression and used a mouse bleomycin‐induced pulmonary fibrosis model to investigate the role of midkine in pulmonary fibrosis. In the idiopathic pulmonary fibrosis patients, the serum midkine level was significantly higher than in healthy subjects, and midkine levels in the serum and bronchoalveolar lavage (BAL) fluid correlated positively with the percentage of inflammatory cells in the BAL fluid. In wild‐type mice, intratracheal bleomycin administration increased midkine expression in lung tissue. Additionally, compared with wild‐type mice, midkine‐deficient mice showed low expression of both collagen and α‐smooth muscle actin, as well as a low value for the pathological lung fibrosis score after bleomycin administration. Furthermore, the total cell count and lymphocyte percentage in the BAL fluid, as well as TNF‐α and transforming growth factor‐β expression in lung tissue, were significantly lower in the midkine‐deficient mice compared with wild‐type mice. These results suggest that midkine is involved in the development of pulmonary fibrosis by regulating inflammatory cell migration into the lung, and TNF‐α and transforming growth factor‐β expression.

Pulmonary fibrosis requires cell-autonomous mesenchymal fibroblast growth factor (FGF) signaling

Idiopathic pulmonary fibrosis (IPF) is characterized by progressive pulmonary scarring, decline in lung function, and often results in death within 3-5 five years after diagnosis. Fibroblast growth factor (FGF) signaling has been implicated in the pathogenesis of IPF; however, the mechanism through which FGF signaling contributes to pulmonary fibrosis remains unclear. We hypothesized that FGF receptor (FGFR) signaling in fibroblasts is required for the fibrotic response to bleomycin. To test this, mice with mesenchyme-specific tamoxifen-inducible inactivation of FGF receptors 1, 2, and 3 (Col1α2-CreER; TCKO mice) were lineage labeled and administered intratracheal bleomycin. Lungs were collected for histologic analysis, whole lung RNA and protein, and dissociated for flow cytometry and FACS. Bleomycin-treated Col1α2-CreER; TCKO mice have decreased pulmonary fibrosis, collagen production, and fewer α-smooth muscle actin-positive (αSMA+) myofibroblasts compared with controls. Freshly isolated Col1α2-CreER; TCKO mesenchymal cells from bleomycin-treated mice have decreased collagen expression compared with wild type mesenchymal cells. Furthermore, lineage labeled FGFR-deficient fibroblasts have decreased enrichment in fibrotic areas and decreased proliferation. These data identify a cell autonomous requirement for mesenchymal FGFR signaling in the development of pulmonary fibrosis, and for the enrichment of the Col1α2-CreER-positive (Col1α2+) mesenchymal lineage in fibrotic tissue following bleomycin exposure. We conclude that mesenchymal FGF signaling is required for the development of pulmonary fibrosis, and that therapeutic strategies aimed directly at mesenchymal FGF signaling could be beneficial in the treatment of IPF.

An Official American Thoracic Society Workshop Report: Use of Animal Models for the Preclinical Assessment of Potential Therapies for Pulmonary Fibrosis.

Numerous compounds have shown efficacy in limiting development of pulmonary fibrosis using animal models, yet few of these compounds have replicated these beneficial effects in clinical trials. Given the challenges associated with performing clinical trials in patients with idiopathic pulmonary fibrosis (IPF), it is imperative that preclinical data packages be robust in their analyses and interpretations to have the best chance of selecting promising drug candidates to advance to clinical trials. The American Thoracic Society has convened a group of experts in lung fibrosis to discuss and formalize recommendations for preclinical assessment of antifibrotic compounds. The panel considered three major themes (choice of animal, practical considerations of fibrosis modeling, and fibrotic endpoints for evaluation). Recognizing the need for practical considerations, we have taken a pragmatic approach. The consensus view is that use of the murine intratracheal bleomycin model in animals of both genders, using hydroxyproline measurements for collagen accumulation along with histologic assessments, is the best-characterized animal model available for preclinical testing. Testing of antifibrotic compounds in this model is recommended to occur after the acute inflammatory phase has subsided (generally after Day 7). Robust analyses may also include confirmatory studies in human IPF specimens and validation of results in a second system using in vivo or in vitro approaches. The panel also strongly encourages the publication of negative results to inform the lung fibrosis community. These recommendations are for preclinical therapeutic evaluation only and are not intended to dissuade development of emerging technologies to better understand IPF pathogenesis.

Disease Specific Signatures Identified by RNA‐seq of Sorted Lung Cellular Populations

RationaleFibrotic interstitial lung disease (ILD) is among the top indications for patients undergoing lung transplantation worldwide. While our group and others have demonstrated a role for monocyte‐derived alveolar macrophages in the development of lung fibrosis in murine models, it is unknown if human alveolar macrophages play a similar role in the development of fibrotic ILD. We hypothesized that transcriptional profiling of AMs isolated from lung transplant donors and recipients as well as from murine alveolar macrophages in a bleomycin‐induced lung fibrosis model would demonstrate homology of involved genes.MethodsFluorescence‐activated cell sorting (FACS) was used to isolate AMs (CD45+ CD169+ HLA‐DR+) from samples of 18 donor lungs and 22 explanted lungs from patients at the time of lung transplantation and to isolate separately tissue‐resident and monocyte‐derived AMs from mice at fourteen and nineteen days following the administration of intratracheal bleomycin. RNA extraction from sorted cells was performed with poly(A) enrichment, followed by single‐end RNA‐seq. Exploratory data analysis using principle component analysis (PCA) and K‐mean clustering were performed. Differentially expressed genes were estimated between donors and patients with interstitial lung disease, and between murine tissue‐resident and monocyte‐derived AMs during bleomycin‐induced fibrosis.ResultsThe median age of the 22 lung transplant recipients was 56 (IQR 13–64). Among recipients, there were eleven patients with the diagnosis of fibrotic ILD (five with scleroderma‐associated ILD, three with myositis‐ILD, two with idiopathic pulmonary fibrosis, and one with mixed connective tissue disease ILD). PCA of sorted populations showed grouping by diagnosis. 61 genes were identified that were differentially expressed (adjusted p &lt; 0.05) in AMs between donors and patients with fibrotic ILD, and that were also homologs of genes highly expressed in murine monocyte‐derived AMs relative to tissue‐resident AMs during bleomycin‐induced lung fibrosis. Hierarchical clustering of samples according to expression of these homologous genes showed just one ILD sample clustering among donors, and of 61 total differentially expressed homologous genes, 51 were upregulated in AMs from patients with fibrotic ILD and only 10 were downregulated.ConclusionsTranscriptional profiling of AMs collected at the time of lung transplantation identifies homology between human fibrotic interstitial lung disease and a murine model of bleomycin‐induced lung fibrosis. By focusing on key lung cellular populations such as alveolar macrophages, future transcriptional studies of lung disease in humans may identify new pathways and new potential targets for therapy for patients with lung disease.Support or Funding Information5T32HL076139‐13, AG049665, HL071643, ES013995, The Veterans Administration, DOD W81XWH‐15‐1‐0215

Prevention of Bleomycin-Induced Pulmonary Inflammation and Fibrosis in Mice by Paeonol.

Pulmonary fibrosis is a severe and progressive disease that is characterized by an abnormal deposition of extracellular matrix, such as collagens. The pathogenesis of this disease may be initiated by oxidative damage of lung epithelial cells by fibrogenic stimuli, leading to lung inflammation, which in turn promotes various lung fibrotic responses. The profibrogenic effect of transforming growth factor-β1 (TGF-β1) on lung fibroblasts is crucial for the pathogenesis of this disease. Paeonol, the main phenolic compound present in the Chinese herb Paeonia suffruticosa, has antioxidant and anti-inflammatory properties. However, whether paeonol has therapeutic effects against pulmonary fibrosis remains unclear. Using a murine model, we showed that 21 days after the insult, intratracheal bleomycin caused pulmonary inflammation and fibrosis, as evidenced by lung histopathological manifestations and increase in various indices. The inflammatory indices included an increase in total cell count, differential cell count, and total protein concentration in bronchoalveolar lavage fluid. The fibrotic indices included an increase in lung levels of TGF-β1, total collagen, type 1α1 collagen (COL1A1), and α-smooth muscle actin (α-SMA; a marker of myofibroblasts). Bleomycin also was found to cause an increase in oxidative stress as reflected by increased levels of malondialdehyde and 4-hydroxynonenal in the lungs. Importantly, all these pathophysiological events were suppressed by daily treatment with paeonol. Using human lung fibroblasts, we further demonstrated that exposure of human lung fibroblasts to TGF-β1 increased productions of α-SMA and COL1A1, both of which were inhibited by inhibitors of Jun N-terminal kinase (JNK), p38, and Smad3. JNK and p38 are two subfamily members of mitogen-activated protein kinases (MAPKs), whereas Smad3 is a transcription factor. TGF-β1 exposure also increased the phosphorylation of JNK, p38, and Smad3 prior to the induction of α-SMA and COL1A1. Notably, all these TGF-β1-induced cellular events were suppressed by paeonol treatment. Our findings suggest that paeonol has antioxidant, anti-inflammatory, and anti-fibrotic functions against bleomycin-induced pulmonary fibrosis in mice. The beneficial effect of paeonol may be, at least in part, mediated through the inhibition of the MAPKs/Smad3 signaling.

Effects of nintedanib on the microvascular architecture in a lung fibrosis model.

Nintedanib, a tyrosine kinase inhibitor approved for the treatment of idiopathic pulmonary fibrosis, has anti-fibrotic, anti-inflammatory, and anti-angiogenic activity. We explored the impact of nintedanib on microvascular architecture in a pulmonary fibrosis model. Lung fibrosis was induced in C57Bl/6 mice by intratracheal bleomycin (0.5mg/kg). Nintedanib was started after the onset of lung pathology (50mg/kg twice daily, orally). Micro-computed tomography was performed via volumetric assessment. Static lung compliance and forced vital capacity were determined by invasive measurements. Mice were subjected to bronchoalveolar lavage and histologic analyses, or perfused with a casting resin. Microvascular corrosion casts were imaged by scanning electron microscopy and synchrotron radiation tomographic microscopy, and quantified morphometrically. Bleomycin administration resulted in a significant increase in higher-density areas in the lungs detected by micro-computed tomography, which was significantly attenuated by nintedanib. Nintedanib significantly reduced lung fibrosis and vascular proliferation, normalized the distorted microvascular architecture, and was associated with a trend toward improvement in lung function and inflammation. Nintedanib resulted in a prominent improvement in pulmonary microvascular architecture, which outperformed the effect of nintedanib on lung function and inflammation. These findings uncover a potential new mode of action of nintedanib that may contribute to its efficacy in idiopathic pulmonary fibrosis.

Glycosides Based Standardized Fenugreek Seed Extract Ameliorates Bleomycin-induced Liver Fibrosis in Rats Via Modulation of Endogenous Enzymes.

Background:Liver fibrosis a complex process of excess collagen deposition resulted in disturbance of hepatic cellar function. Glycosides based standardized fenugreek seed extract (SFSE-G) has potent anti-inflammatory, antioxidant, and anti-fibrotic properties.Objective:The aim of this study is to evaluate the hepatoprotective potential of SFSE-G against bleomycin (BLM)-induced liver fibrosis in laboratory animals.Materials and Methods:Sprague-Dawley rats (180–220 g) were assigned to various groups, namely, normal, sham, BLM control, SFSE-G (5, 10, 20, and 40 mg/kg, p.o.), methylprednisolone (10 mg/kg, p.o.), and sildenafil (25 mg/kg, p.o.). Liver fibrosis was induced in various groups (except normal and sham) by single intratracheal BLM (6 IU/kg) injection. Various biochemical, molecular (reverse transcription polymerase chain reaction) and histological parameters were evaluated.Results:Intratracheal BLM administration caused significant induction (P < 0.001) of hepatotoxicity and liver fibrosis reflected by elevated levels of serum aspartate transaminase (AST), alanine transaminase (ALT), total as well as direct bilirubin, and gamma-glutamyl transferase (GGT). Administration of SFSE-G (20 and 40 mg/kg, p.o.) significantly reduced (P < 0.001) levels of AST, ALT, and GGT and significantly increased (P < 0.001) the level of serum albumin. BLM-induced elevated liver oxidative stress and decreased total antioxidant capacity was significantly restored (P < 0.001) by SFSE-G (20 and 40 mg/kg) treatment. It also significantly inhibited BLM-induced alteration in liver Farnesoid X receptor (FXR) mRNA expression. SFSE-G treatment reduced histopathological alteration induced by BLM in liver.Conclusion:SFSE-G exerts its hepatoprotective potential via inhibition of oxido-nitrosative stress and modulation of FXR mRNA expression thus ameliorates BLM-induced liver fibrosis.

Aberrant lung remodeling in a mouse model of surfactant dysregulation induced by modulation of the Abca3 gene

The lipid transporter, ATP binding cassette class A3 (ABCA3), plays a critical role in the biogenesis of alveolar type 2 (AT2) cell lamellar bodies (LBs). A relatively large number of mutations in the ABCA3 gene have been identified in association with diffuse parenchymal lung disease (DPLD), the most common of which is a missense mutation (valine substitution for lysine at residue 292 (ABCA3E292V)) that leads to functional impairment of the transporter in vitro. The consequences of ABCA3E292V gene expression in vivo are unknown. To address this question, we developed mouse models expressing ABCA3E292V knocked-in to the endogenous mouse locus. The parental (F1) mouse line (mAbca3E292V) that retained an intronic pgk-Neo selection cassette (inserted in reverse orientation) (mAbca3E292V–rNeo) demonstrated an allele dependent extracellular surfactant phospholipid (PL) deficiency. We hypothesize that this PL deficiency leads to aberrant parenchymal remodeling contributing to the pathophysiology of the DPLD phenotype. Compared to wild type littermates, baseline studies of mice homozygous for the pgk-Neo insert (mAbca3E292V–rNeo+/+) revealed nearly 50% reduction in bronchoalveolar lavage (BAL) PL content that was accompanied by quantitative reduction in AT2 LB size with a compensatory increase in LB number. The phenotypic alteration in surfactant lipid homeostasis resulted in an early macrophage predominant alveolitis which peaked at 8 weeks of age. This was followed by age-dependent development of histological DPLD characterized initially by peribronchial inflammatory cell infiltration and culminating in both an emphysema-like phenotype (which included stereologically quantifiable reductions in both alveolar septal surface area and volume of septal wall tissue) plus foci of trichrome-positive collagen deposition together with substantial proliferation of hyperplastic AT2 cells. In addition to spontaneous lung remodeling, mABCA3E292V–rNeo mice were rendered more vulnerable to exogenous injury. Three weeks following intratracheal bleomycin challenge, mAbca3–rNeo mice demonstrated allele-dependent susceptibility to bleomycin including enhanced weight loss, augmented airspace destruction, and increased fibrosis. Removal of the rNeo cassette from mAbca3 alleles resulted in restoration of BAL PL content to wild-type levels and an absence of changes in lung histology up to 32 weeks of age. These results support the importance of surfactant PL homeostasis as a susceptibility factor for both intrinsic and exogenously induced lung injury/remodeling.

The antifibrotic effects of alveolar macrophages 5-HT2C receptors blockade on bleomycin-induced pulmonary fibrosis in rats.

The most widespread chronic fibrosing lung disease is idiopathic pulmonary fibrosis. Lung serotonin (5-HT) content is increased during pulmonary fibrosis with the implication of 5-HT2 receptors in the pathogenesis. Serotonin plays important roles in alveolar macrophages function through 5-HT2C receptors activation. Numerous studies described the important role of 5-HT2A/B receptor blockers in suppressing different types of fibrosis as idiopathic pulmonary fibrosis. The current study pointed to examine the antifibrotic effects of RS-102221 and/or terguride through in vivo model of pulmonary fibrosis. Induction of pulmonary fibrosis was through a single dose of intra-tracheal bleomycin instillation (5mg/kg dissolved in phosphate buffer saline) in adult male albino Wistar rats. Next day of bleomycin instillation, intraperitoneal injection of RS-102221 (0.5mg/kg/d) and/or terguride (1.2mg/kg/d) were administered and continued for 14 days. Noticeable increase in 5-HT2C receptors expression was observed in fibrotic lung tissues with marked allocation belonging to alveolar macrophages. Either RS-102221 or terguride reduced the increments in lung water contents, grading of lung fibrosis, lung tumor necrosis factor-alpha (TNF-α), transforming growth factor-beta1 (TGF-β1) and vascular endothelial growth factor (VEGF) levels in lung injury and fibrosis-induced by bleomycin. Moreover, collagen content and myofibroblasts-alpha smooth muscle actin (α-SМA) were significantly decreased. Additionally, the simultaneous administration of RS-102221 with terguride had a synergistic outcome compared to that obtained by individual monotherapy. These findings suggested the potential use of 5-HT2A/B/C antagonists as anti-fibrotic agents in lung fibrosis.

Nuclear Thioredoxin-1 Overexpression Attenuates Alcohol-Mediated Nrf2 Signaling and Lung Fibrosis.

BackgroundAlcohol abuse, which impairs antioxidant defenses and promotes acute lung injury, increases Nrf2 nuclear translocation but nevertheless inhibits its activation of the antioxidant response element (ARE). Thioredoxin‐1 (Trx1) is required for optimal Nrf2 binding and activation of the ARE, and we hypothesized that its inhibition contributes to impaired Nrf2‐ARE signaling in the alcoholic lung.MethodsLung tissue and primary lung fibroblasts (PLFs) were isolated from C57/BL6 wild‐type (WT) and transgenic mice overexpressing the human Trx1 gene with a nuclear localizing sequence (NLS‐Tg); some mice consumed alcohol in water prior to lung tissue and PLF isolation; in some mice, acute lung injury was induced with intratracheal bleomycin. In other experiments, PLFs were isolated from WT and NLS‐Tg mice and then exposed to alcohol. Finally, PLF isolated from WT mice were transfected with Trx1 expression vector containing either a cytosolic localized sequence (NES) or a nuclear localized sequence (NLS) prior to alcohol exposure.ResultsAlcohol treatment in vivo or in vitro decreased Trx1 expression, and bleomycin‐treated alcohol‐fed mice had fibrotic disrepair in their lungs. In parallel, whereas alcohol exposure in vitro increased TGF β1 expression and decreased Nrf2‐ARE activity in PLF from WT mice, these effects were not observed in PLF from NLS‐Tg mice. Finally, selective overexpression of Trx1 in the nucleus but not in the cytosol preserved Nrf2‐ARE activity during alcohol exposure.ConclusionsAlthough alcohol‐induced redox stress actually promotes Nrf2 nuclear translocation, the coincident suppression of Trx1 impairs Nrf2‐ARE activity within the nuclear compartment. Nuclear overexpression of Trx1 restored Nrf2‐ARE activity and attenuated alcohol‐induced TGF β1 expression and alcohol‐induced exaggerate response to bleomycin‐induced acute lung injury.

CD4+CD25hiFoxp3+ Cells Exacerbate Bleomycin-Induced Pulmonary Fibrosis

Idiopathic pulmonary fibrosis is a fatal lung disease with a median survival of 2 to 5 years. A decade of studies has downplayed inflammation contributing to its pathogenesis. However, these studies preceded the discovery of regulatory T cells (Tregs) and all of their functions. On the basis of human studies demonstrating Tregs can decrease graft-versus-host disease and vasculitides, there is consideration of their use to treat idiopathic pulmonary fibrosis. We hypothesized that Treg therapy would attenuate the fibroplasia involved in a preclinical murine model of pulmonary fibrosis. IL-2 complex was used invivo to expand CD4(+)CD25(hi)Foxp3(+) cells in the lung during intratracheal bleomycin challenge; however, this unexpectedly led to an increase in lung fibrosis. More important, this increase in fibrosis was a lymphocyte-dependent process. We corroborated these results using a CD4(+)CD25(hi)Foxp3(+) cellular-based therapy. Mechanistically, we demonstrated that CD4(+)CD25(hi)Foxp3(+) cells undergo alterations during bleomycin challenge and the IL-2 complex had no effect on profibrotic (eg, transforming growth factor-β) or type 17 immune response cytokines; however, there was a marked down-regulation of the type 1 and augmentation of the type 2 immune response cytokines from the lungs. Collectively, our animal studies show that a specific lung injury can induce Treg alterations, which can augment pulmonary fibrosis.