Bleomycin Mouse Model Research Articles

P311 Promotes Lung Fibrosis via Stimulation of Transforming Growth Factor-β1, -β2, and -β3 Translation.

Interstitial lung fibrosis, a frequently idiopathic and fatal disease, has been linked to the increased expression of profibrotic transforming growth factor (TGF)-βs. P311 is an RNA-binding protein that stimulates TGF-β1, -β2, and -β3 translation in several cell types through its interaction with the eukaryotic translation initiation factor 3b. We report that P311 is switched on in the lungs of patients with idiopathic pulmonary fibrosis (IPF) and in the mouse model of bleomycin (BLM)-induced pulmonary fibrosis. To assess the in vivo role of P311 in lung fibrosis, BLM was instilled into the lungs of P311-knockout mice, in which fibrotic changes were significantly decreased in tandem with a reduction in TGF-β1, -β2, and -β3 concentration/activity compared with BLM-treated wild-type mice. Complementing these findings, forced P311 expression increased TGF-β concentration/activity in mouse and human lung fibroblasts, thereby leading to an activated phenotype with increased collagen production, as seen in IPF. Consistent with a specific effect of P311 on TGF-β translation, TGF-β1-, -β2-, and -β3-neutralizing antibodies downregulated P311-induced collagen production by lung fibroblasts. Furthermore, treatment of BLM-exposed P311 knockouts with recombinant TGF-β1, -β2, and -β3 induced pulmonary fibrosis to a degree similar to that found in BLM-treated wild-type mice. These studies demonstrate the essential function of P311 in TGF-β-mediated lung fibrosis. Targeting P311 could prove efficacious in ameliorating the severity of IPF while circumventing the development of autoimmune complications and toxicities associated with the use of global TGF-β inhibitors.

Mitogen-activated Protein Kinase-activated Protein Kinase 2 Inhibition Attenuates Fibroblast Invasion and Severe Lung Fibrosis.

Severe pulmonary fibrosis such as idiopathic pulmonary fibrosis (IPF) is characterized by the accumulation of extracellular matrix and fibroblast activation. Targeting fibroblast activation has contributed to the development of antifibrotic therapeutics for patients with IPF. Mitogen-activated protein kinase-activated protein kinase 2 (MK2), downstream in the transforming growth factor-β/p38 mitogen-activated protein kinase pathway, has been implicated in inflammatory and fibrosing diseases. Increased concentrations of activated MK2 were expressed in IPF lung and in the mouse bleomycin model of lung fibrosis. The aim of the present study was to determine the role and the mechanisms of MK2 in fibroblast invasion and lung fibrosis. Our results showed that an MK2 inhibitor (MMI-0100) was able to inhibit the invasive capacity of lung fibroblasts isolated from patients with IPF, as well as fibroblasts isolated from both wild-type mice and mice with overexpressing hyaluronan synthase 2 (HAS2) in the myofibroblast compartment. We previously showed that hyaluronan and HAS2 regulate fibroblast invasion and lung fibrosis in vivo. The results of the present study showed that MMI-0100 reduced transforming growth factor-β-induced hyaluronan production in human and mouse fibroblasts in vitro and that HAS2 mediated MK2 activation, suggesting a feed-forward loop in fibroblast activation. More importantly, MK2 inhibition attenuated hyaluronan accumulation and reduced collagen content in bleomycin-injured mouse lungs in vivo. Conditional deletion of MK2 in fibroblasts attenuated bleomycin-induced lung fibrosis. These data provide evidence that MK2 has a role in fibroblast invasion and fibrosis and may be a novel therapeutic target in pulmonary fibrosis.

A Lipidomics Approach to Identifying Key Lipid Species Involved in VEGF-Inhibitor Mediated Attenuation of Bleomycin-Induced Pulmonary Fibrosis.

Poor molecular characterization of idiopathic pulmonary fibrosis (IPF) has led to insufficient understanding of the pathogenesis of the disease, resulting in lack of effective therapies and poor prognosis. Particularly, the role of lipid imbalance due to impaired lipid metabolism in the pathogenesis of IPF has been poorly studied. The authors have used shotgun lipidomics in a bleomycin (BLM) mouse model of pulmonary fibrosis with vascular endothelial growth factor (VEGF)-inhibitor CBO-P11 as a therapeutic measure, to identify a comprehensive set of lipids that contribute to the pathogenesis of pulmonary fibrosis. The authors report that attenuation of BLM-induced fibrotic response with CBO-P11 cotreatment is accompanied by a decrease in total lipid content and specific downregulation of lipids, which are upregulated in response to BLM treatment. Dysregulated lipids identified in this study hold the potential of being future biomarkers for IPF.

Sialidase inhibitors attenuate pulmonary fibrosis in a mouse model

Fibrosis involves increasing amounts of scar tissue appearing in a tissue, but what drives this is unclear. In fibrotic lesions in human and mouse lungs, we found extensive desialylation of glycoconjugates, and upregulation of sialidases. The fibrosis-associated cytokine TGF-β1 upregulates sialidases in human airway epithelium cells, lung fibroblasts, and immune system cells. Conversely, addition of sialidases to human peripheral blood mononuclear cells induces accumulation of extracellular TGF-β1, forming what appears to be a sialidase - TGF-β1 - sialidase positive feedback loop. Monocyte-derived cells called fibrocytes also activate fibroblasts, and we found that sialidases potentiate fibrocyte differentiation. A sialylated glycoprotein called serum amyloid P (SAP) inhibits fibrocyte differentiation, and sialidases attenuate SAP function. Injections of the sialidase inhibitors DANA and oseltamivir (Tamiflu) starting either 1 day or 10 days after bleomycin strongly attenuate pulmonary fibrosis in the mouse bleomycin model, and by breaking the feedback loop, cause a downregulation of sialidase and TGF-β1 accumulation. Together, these results suggest that a positive feedback loop involving sialidases potentiates fibrosis, and suggest that sialidase inhibitors could be useful for the treatment of fibrosis.

α1A-Subtype adrenergic agonist therapy for the failing right ventricle.

Failure of the right ventricle (RV) is a serious disease with a poor prognosis and limited treatment options. Signaling by α1-adrenergic receptors (α1-ARs), in particular the α1A-subtype, mediate cardioprotective effects in multiple heart failure models. Recent studies have shown that chronic treatment with the α1A-subtype agonist A61603 improves function and survival in a model of left ventricular failure. The goal of the present study was to determine if chronic A61603 treatment is beneficial in a RV failure model. We used tracheal instillation of the fibrogenic antibiotic bleomycin in mice to induce pulmonary fibrosis, pulmonary hypertension, and RV failure within 2 wk. Some mice were chronically treated with a low dose of A61603 (10 ng·kg-1·day-1). In the bleomycin model of RV failure, chronic A61603 treatment was associated with improved RV fractional shortening and greater in vitro force development by RV muscle preparations. Cell injury markers were reduced with A61603 treatment (serum cardiac troponin I, RV fibrosis, and expression of matrix metalloproteinase-2). RV oxidative stress was reduced (using the probes dihydroethidium and 4-hydroxynonenal). Consistent with lowered RV oxidative stress, A61603 was associated with an increased level of the cellular antioxidant superoxide dismutase 1 and a lower level of the prooxidant NAD(P)H oxidase isoform NOX4. In summary, in the bleomycin model of RV failure, chronic A61603 treatment reduced RV oxidative stress, RV myocyte necrosis, and RV fibrosis and increased both RV function and in vitro force development. These findings suggest that in the context of pulmonary fibrosis, the α1A-subtype is a potential therapeutic target to treat the failing RV.NEW & NOTEWORTHY Right ventricular (RV) failure is a serious disease with a poor prognosis and no effective treatments. In the mouse bleomycin model of RV failure, we tested the efficacy of a treatment using the α1A-adrenergic receptor subtype agonist A61603. Chronic A61603 treatment improved RV contraction and reduced multiple indexes of RV injury, suggesting that the α1A-subtype is a therapeutic target to treat RV failure.

KIF5A transports collagen vesicles of myofibroblasts during pleural fibrosis

Fibrosis involves the production of extracellular matrix proteins in tissues and is often preceded by injury or trauma. In pleural fibrosis excess collagen deposition results in pleural thickening, increased stiffness and impaired lung function. Myofibroblasts are responsible for increased collagen deposition, however the molecular mechanism of transportation of procollagen containing vesicles for secretion is unknown. Here, we studied the role of kinesin on collagen-1 (Col-1) containing vesicle transportation in human pleural mesothelial cells (HPMCs). Among a number of cargo transporting kinesins, KIF5A was notably upregulated during TGF-β induced mesothelial-mesenchymal transition (MesoMT). Using superresolution structured illumination microscopy and the DUO-Link technique, we found that KIF5A colocalized with Col-1 containing vesicles. KIF5A knock-down significantly reduced Col-1 secretion and attenuated TGF-β induced increment in Col-1 localization at cell peripheries. Live cell imaging revealed that GFP-KIF5A and mCherry-Col-1 containing vesicles moved together. Kymography showed that these molecules continuously move with a mean velocity of 0.56 μm/sec, suggesting that the movement is directional but not diffusion limited process. Moreover, KIF5A was notably upregulated along with Col-1 and α-smooth muscle actin in pleural thickening in the carbon-black bleomycin mouse model. These results support our hypothesis that KIF5A is responsible for collagen transportation and secretion from HPMCs.

Longitudinal assessment of bleomycin-induced lung fibrosis by Micro-CT correlates with histological evaluation in mice

BackgroundThe intratracheal instillation of bleomycin in mice induces early damage to alveolar epithelial cells and development of inflammation followed by fibrotic tissue changes and represents the most widely used model of pulmonary fibrosis to investigate human IPF.Histopathology is the gold standard for assessing lung fibrosis in rodents, however it precludes repeated and longitudinal measurements of disease progression and does not provide information on spatial and temporal distribution of tissue damage.Here we investigated the use of the Micro-CT technique to allow the evaluation of disease onset and progression at different time-points in the mouse bleomycin model of lung fibrosis. Micro-CT was throughout coupled with histological analysis for the validation of the imaging results.MethodsIn bleomycin-instilled and control mice, airways and lung morphology changes were assessed and reconstructed at baseline, 7, 14 and 21 days post-treatment based on Micro-CT images. Ashcroft score, percentage of collagen content and percentage of alveolar air area were detected on lung slides processed by histology and subsequently compared with Micro-CT parameters.ResultsExtent (%) of fibrosis measured by Micro-CT correlated with Ashcroft score, the percentage of collagen content and the percentage of alveolar air area (r2 = 0.91; 0.77; 0.94, respectively). Distal airway radius also correlated with the Ashcroft score, the collagen content and alveolar air area percentage (r2 = 0.89; 0.78; 0.98, respectively).ConclusionsMicro-CT data were in good agreement with histological read-outs as micro-CT was able to quantify effectively and non-invasively disease progression longitudinally and to reduce the variability and number of animals used to assess the damage. This suggests that this technique is a powerful tool for understanding experimental pulmonary fibrosis and that its use could translate into a more efficient drug discovery process, also helping to fill the gap between preclinical setting and clinical practice.

Longitudinal assessment of bleomycin-induced lung fibrosis by Micro-CT correlates with histological evaluation in mice

Background: The intratracheal instillation of bleomycin in mice induces early damage to alveolar epithelial cells and development of inflammation followed by fibrotic tissue changes and represents the most widely used model of pulmonary fibrosis to investigate human IPF. Histopathology is the gold standard for assessing lung fibrosis in rodents, however it precludes repeated and longitudinal measurements of disease progression and does not provide information on spatial and temporal distribution of tissue damage. Here we investigated the use of the Micro-CT technique to allow the evaluation of disease onset and progression at different time-points in the mouse bleomycin model of lung fibrosis. Micro-CT was throughout coupled with histological analysis for the validation of the imaging results.
 Methods: In bleomycin-instilled and control mice, airways and lung morphology changes were assessed and reconstructed at baseline, 7, 14 and 21 days post-treatment based on Micro-CT images. Ashcroft score, percentage of collagen content and percentage of alveolar air area were detected on lung slides processed by histology and subsequently compared with Micro-CT parameters.
 Results: Extent (%) of fibrosis measured by Micro-CT correlated with Ashcroft score, the percentage of collagen content and the percentage of alveolar air area (r2 = 0.91; 0.77; 0.94, respectively). Distal airway radius also correlated with the Ashcroft score, the collagen content and alveolar air area percentage (r2 = 0.89; 0.78; 0.98, respectively).
 Conclusions: Micro-CT data were in good agreement with histological read-outs as micro-CT was able to quantify effectively and non-invasively disease progression longitudinally and to reduce the variability and number of animals used to assess the damage. This suggests that this technique is a powerful tool for understanding experimental pulmonary fibrosis and that its use could translate into a more efficient drug discovery process, also helping to fill the gap between preclinical setting and clinical practice.

Heparanase contributes to idiopathic pulmonary fibrosis via regulation of A20/TNFAIP3 expression

We have previously demonstrated that heparanase, a mammalian endoglycosidase that cleaves the extracellular glycosaminoglycan heparan sulfate (HS), is activated during acute lung inflammation, contributing to the acute respiratory distress syndrome. The impact of heparanase on chronic lung injury (such as idiopathic pulmonary fibrosis, IPF) is uncertain. We found increased heparanase expression within the lungs of patients with severe IPF, co‐localized with clusters of pathologically‐activated myofibroblasts. Similar heparanase induction was noted in a bleomycin mouse model of pulmonary fibrosis. Proliferation of myofibroblasts during Idiopathic Pulmonary Fibrosis (IPF) has been attributed to increased resistance to apoptosis. To determine effect of heparanase on apoptosis, normal human lung fibroblasts (HLF) were treated with heparanase, TGFβ1 (a pro‐fibrotic mediator), CXCL10 (anti‐fibrotic chemokine) or combinations thereof for 48 hours. We measured gene expression of A20/TNFAIP3, an anti‐apoptotic protein which protects cells from TNF‐induced cell death. A20/TNFAIP3 expression was increased in heparanase‐treated human lung fibroblasts. Heparanase‐induced A20/TNFAIP3 expression was augmented by CXCL10, and abolished by TGFb1. Pharmacological inhibition of heparanase activity did not change A20/TNFAIP3 expression, suggesting that heparanase may regulate A20/TNFAIP3 expression via non‐enzymatic effects. Taken together, these findings suggest that heparanase induction may promote fibroblast survival and pathological collagen deposition in IPF.Support or Funding InformationR01 HL125371

Activation and overexpression of Sirt1 attenuates lung fibrosis via P300

Persistent fibroblast activation is a predominant feature of idiopathic pulmonary fibrosis (IPF), but the transcriptional and epigenetic mechanisms controlling this process are not well understood. Silent information regulator type-1 (Sirt1) is a member of class Ⅲ histone deacetylase with important regulatory roles in a variety of pathophysiologic processes, but its role in fibrotic lung diseases is not clearly elucidated. Sirt1 expression in lung tissues of IPF patients and in a mouse model of bleomycin (BLM)-induced lung fibrosis were evaluated by immunofluorescence. The function of Sirt1 in BLM-induced lung fibrosis in the mouse model or transforming growth factor β1 (TGF-β1)-mediated lung fibroblast cellular model was investigated by Sirt1 activation, overexpression and knockdown of Sirt1. Finally, the involvement of p300 signaling pathways was assessed. In this study, we found up-regulation of Sirt1 in BLM-induced lung fibrosis, as well as in the lungs of IPF patients, including in the aggregated pulmonary fibroblasts of fibrotic foci. Activation or overexpression of Sirt1 attenuated TGF-β1-mediated lung fibroblast differentiation and activation and diminished the severity of experimental lung fibrosis in mice. Whereas knockdown of Sirt1 promoted the pro-fibrogenic activity of TGF-β1 in lung fibroblasts. A potential mechanism for the role of Sirt1 in lung fibrosis was through regulating the expression of p300. Thus, we characterized Sirt1 as an important regulator of lung fibrosis and provides a proof of principle for activation or overexpression of Sirt1 as a potential novel therapeutic strategy for IPF.

Effects of IL-17A on fibrosis of skin and lung in a mouse model of systemic sclerosis

Objective To analyze the expression of interleukin (IL)-17A in a mouse model of bleomycin (BLM)-induced systemic sclerosis (SSc) and to evaluate its effects on inflammation and fibrosis in skin and lung tissues. Methods Twenty-four female BALB/c mice were randomly divided into four groups: normal control group (mice were subcutaneously injected with phosphate buffer), model group (subcutaneously injected with BLM), antibody group (injected with BLM + IL-17A monoclonal antibody), homotypic control group (injected with BLM + isotype control). Pathological changes in skin and lung tissues of those mice were observed; inflammatory and fibrotic scores were assessed. Immunohistochemistry and real-time fluorescent quantitative PCR (RT-PCR) were used to detect the expression of IL-17A, TGF-β1 and typeⅠ collagen in skin and lung tissues of those mice at mRNA level. Mouse lung fibroblasts (FB) derived from the mice of model group were cultured in vitro and then were cultured with IL-17A cytokines with or without the interference of monoclonal antibodies. Expression of typeⅠ collagen and TGF-β1 at mRNA level and levels of IL-6 and TGF-β1 in the culture supernatants were detected by RT-PCR and enzyme-linked immunosorbent assay (ELISA), respectively. Results Compared with the mice of model and homotypic control groups, those of the antibody group showed mild skin thickening, skin inflammation and lung inflammation as well as lower fibrosis scores (P<0.05). The expression of IL-17A at both protein and mRNA levels and the expression of TGF-β1 and collagen typeⅠ at mRNA level in skin and lung tissues of mice of the antibody group were significantly lower than those of the model and homotypic control group (P<0.05). Results of the in vitro cell culture of SSc mice-derived lung FB with IL-17A showed that the expression of TGF-β1 and typeⅠ collagen at mRNA level and the levels of IL-6 and TGF-β1 in the culture supernatants were decreased with the interference of anti-IL-17A monoclonal antibody (P<0.05), but were still higher than those of the control group (P<0.05). Conclusion IL-17A promotes the development of inflammation and fibrosis in skin and lung tissues in the mouse model of SSc. Blocking IL-17A might inhibit fibrosis in SSc by inhibiting the production of TGF-β1, IL-6 and typeⅠ collagen. Key words: Systemic sclerosis; Anti-IL-17A monoclonal antibody; IL-17A; TGF-β1; TypeⅠ collagen

KIF5A is Responsible for Collagen Transport of Myofibroblasts during Pleural Fibrosis

Fibrosis is the production of extracellular matrix proteins in tissues and often proceeded by injuries. In pleural fibrosis excess collagen deposition occurs, which results in increased stiffness and thickening of pleura during tissue rearrangements. Myofibroblasts are responsible for oversecretion of collagen, however the molecular mechanism of transportation of procollagen containing vesicles for secretion is unknown. Here, we studied the role of kinesin on collagen-1 containing vesicle transportation in human pleural mesothelial cells (HPMCs). Among a number of cargo transporting kinesins, KIF5A was notably upregulated during TGF-β induced mesothelial-mesenchymal transition (MesoMT). Using superresolution structured illumination microscopy (SIM) and DUO-Link technique, we found KIF5A notably colocalizes with collagen-1 containing vesicles. KIF5A knock-down (KD) by specific siRNA significantly reduced collagen-1 secretion but not plasminogen activator inhibitor 1 (PAI-1). KIF5A KD notably attenuated TGF-β induced increase in collagen-1 localization at cell peripheries. Live cell imaging with GFP-KIF5A and mCherry-collagen-1 showed that KIF5A and collagen-1 containing vesicles moved together. Kymograph showed that these molecules continuously move with mean velocity of 0.56 μm/sec, suggesting that the movement is directional but not diffusion process. Moreover, KIF5A was notably upregulated along with collagen-1 and α-smooth muscle actin (α-SMA) (MesoMT marker) in thickening pleura of carbon-black bleomycin mouse model. All the results support that KIF5A is responsible for collagen transportation and secretion from HPMCs.

Quantification of Pulmonary Fibrosis in a Bleomycin Mouse Model Using Automated Histological Image Analysis.

Current literature on pulmonary fibrosis induced in animal models highlights the need of an accurate, reliable and reproducible histological quantitative analysis. One of the major limits of histological scoring concerns the fact that it is observer-dependent and consequently subject to variability, which may preclude comparative studies between different laboratories. To achieve a reliable and observer-independent quantification of lung fibrosis we developed an automated software histological image analysis performed from digital image of entire lung sections. This automated analysis was compared to standard evaluation methods with regard to its validation as an end-point measure of fibrosis. Lung fibrosis was induced in mice by intratracheal administration of bleomycin (BLM) at 0.25, 0.5, 0.75 and 1 mg/kg. A detailed characterization of BLM-induced fibrosis was performed 14 days after BLM administration using lung function testing, micro-computed tomography and Ashcroft scoring analysis. Quantification of fibrosis by automated analysis was assessed based on pulmonary tissue density measured from thousands of micro-tiles processed from digital images of entire lung sections. Prior to analysis, large bronchi and vessels were manually excluded from the original images. Measurement of fibrosis has been expressed by two indexes: the mean pulmonary tissue density and the high pulmonary tissue density frequency. We showed that tissue density indexes gave access to a very accurate and reliable quantification of morphological changes induced by BLM even for the lowest concentration used (0.25 mg/kg). A reconstructed 2D-image of the entire lung section at high resolution (3.6 μm/pixel) has been performed from tissue density values allowing the visualization of their distribution throughout fibrotic and non-fibrotic regions. A significant correlation (p<0.0001) was found between automated analysis and the above standard evaluation methods. This correlation establishes automated analysis as a novel end-point measure of BLM-induced lung fibrosis in mice, which will be very valuable for future preclinical drug explorations.

Lrp5/β-Catenin Signaling Controls Lung Macrophage Differentiation and Inhibits Resolution of Fibrosis.

Previous studies established that attenuating Wnt/β-catenin signaling limits lung fibrosis in the bleomycin mouse model of this disease, but the contribution of this pathway to distinct lung cell phenotypes relevant to tissue repair and fibrosis remains incompletely understood. Using microarray analysis, we found that bleomycin-injured lungs from mice that lack the Wnt coreceptor low density lipoprotein receptor-related protein 5 (Lrp5) and exhibit reduced fibrosis showed enrichment for pathways related to extracellular matrix processing, immunity, and lymphocyte proliferation, suggesting the contribution of an immune-matrix remodeling axis relevant to fibrosis. Activation of β-catenin signaling was seen in lung macrophages using the β-catenin reporter mouse, Axin2+/LacZ. Analysis of lung immune cells by flow cytometry after bleomycin administration revealed that Lrp5-/- lungs contained significantly fewer Siglec Flow alveolar macrophages, a cell type previously implicated as positive effectors of fibrosis. Macrophage-specific deletion of β-catenin in CD11ccre;β-cateninflox mice did not prevent development of bleomycin-induced fibrosis but facilitated its resolution by 8 weeks. In a nonresolving model of fibrosis, intratracheal administration of asbestos in Lrp5-/- mice also did not prevent the development of fibrosis but hindered the progression of fibrosis in asbestos-treated Lrp5-/- lungs, phenocopying the findings in bleomycin-treated CD11ccre;β-cateninflox mice. Activation of β-catenin signaling using lithium chloride resulted in worsened fibrosis in wild-type mice, further supporting that the effects of loss of Lrp5 are directly mediated by Wnt/β-catenin signaling. Together, these data suggest that lung myeloid cells are responsive to Lrp5/β-catenin signaling, leading to differentiation of an alveolar macrophage subtype that antagonizes the resolution of lung fibrosis.

Serum extracellular vesicular miR-21-5p is a predictor of the prognosis in idiopathic pulmonary fibrosis.

BackgroundIdiopathic pulmonary fibrosis (IPF) is a disease with a poor prognosis. Although the median survival is 3 years, the clinical course varies to a large extent among IPF patients. To date, there has been no definitive prognostic marker. Extracellular vesicles (EVs) are known to hold nucleic acid, including microRNAs, and to regulate gene expression in the recipient cells. Moreover, EVs have been shown to express distinct surface proteins or enveloped microRNAs depending on the parent cell or pathological condition. We aimed to identify serum EV microRNAs that would be prognostic for IPF.MethodsTo determine target microRNAs in IPF, we measured serum EV microRNA expression profiles using microRNA PCR arrays in a bleomycin mouse model and validated the microRNAs in additional mice using RT-PCR. Secondly, we enrolled 41 IPF patients and conducted a 30-month prospective cohort study. Expression of serum EV miR-21-5p was normalized by dividing by the EV amount. The relative amount of EVs was measured using the ExoScreen method. We calculated the correlations between baseline serum EV miR-21-5p expression and other clinical variables. Furthermore, we determined if serum EV miR-21-5p can predict mortality during 30 months using the Cox hazard model. According to the median level, we divided the IPF patients into two groups. Then we compared the survival rate during 30 months between the two groups using the Kaplan-Meier method.ResultsSerum EV miR-21-5p was elevated in both the acute inflammatory phase (day 7) and the chronic fibrotic phase (day 28) in the mouse model. In the clinical setting, serum EV miR-21-5p was significantly higher in IPF patients than in healthy control subjects. The baseline serum EV miR-21-5p was correlated with the rate of decline in vital capacity over 6 months. Furthermore, serum EV miR-21-5p was independently associated with mortality during the following 30 months, even after adjustment for other variables. In the survival analysis, IPF patients whose baseline serum EV miR-21-5p was high had a significantly poorer prognosis over 30 months.ConclusionsOur results suggest that serum EV miR-21-5p has potential as a prognostic biomarker for IPF.Electronic supplementary materialThe online version of this article (doi:10.1186/s12931-016-0427-3) contains supplementary material, which is available to authorized users.

MiR‑221 targets HMGA2 to inhibit bleomycin‑induced pulmonary fibrosis by regulating TGF‑β1/Smad3-induced EMT.

MicroRNA(miR)-221 plays an essential role in the epithelial-mesenchymal transition (EMT). High mobility group AT-hook 2(HMGA2), is a key regulator of EMT. However, the role of miR‑221 in pulmonary fibrosis, and the association between miR‑221 and HMGA2 remain largely unknown. For this purpose, we examined the expression of miR‑221 and HMGA2 in human idiopathic pulmonary fibrosis(IPF) tissues and pulmonary cells, namely the adenocarcinomaA549 and human bronchial epithelium(HBE) cell lines, and found that the expression of miR‑221 was inhibited in both tissues and cells whereas high mRNA and protein expression of HMGA2 was observed. Additionally, transforming growth factor‑β1(TGF‑β1) induced the EMT, characterized by the upregulated expression of the mesenchymal markers, namely N‑cadherin, vimentin, α‑smooth muscle actin, collagenI and collagenIII, and the downregulated expression of the epithelial marker E-cadherin in A549 and HBE cells. We then performed transfection with miR‑221 mimics, and found that the expression of phosphorylated-Smad3 in miR‑221‑overexpressing cells was significantly downregulated, compared with that in the TGF‑β1-treated cells without transfection. Furthermore, the overexpression of miR‑221 decreased the expression of HMGA2, suppressed the EMT, and inhibited the proliferation of A549 and HBE cells. HMGA2 was directly targeted by miR‑221 which was confirmed by the dual-luciferase reporter gene assay. Finally, a mouse model of bleomycin(BLM)‑induced pulmonary fibrosis was used to confirm the effect of miR‑221 on EMT. Hematoxylin and eosin staining showed that BLM induced thicker alveolar walls and more collagen deposition, whereas miR‑221 treatment reduced lung fibrosis and the tissues exhibited thinner alveolar walls and normal lung alveoli. Furthermore, the EMT process was suppressed following miR‑221 injection. Taken together, these findings sugest that miR‑221 targets HMGA2 to inhibit BLM‑induced pulmonary fibrosis through the TGF‑β1/Smad3 signaling pathway.

Autotaxin activity increases locally following lung injury, but is not required for pulmonary lysophosphatidic acid production or fibrosis.

Lysophosphatidic acid (LPA) is an important mediator of pulmonary fibrosis. In blood and multiple tumor types, autotaxin produces LPA from lysophosphatidylcholine (LPC) via lysophospholipase D activity, but alternative enzymatic pathways also exist for LPA production. We examined the role of autotaxin (ATX) in pulmonary LPA production during fibrogenesis in a bleomycin mouse model. We found that bleomycin injury increases the bronchoalveolar lavage (BAL) fluid levels of ATX protein 17-fold. However, the LPA and LPC species that increase in BAL of bleomycin-injured mice were discordant, inconsistent with a substrate-product relationship between LPC and LPA in pulmonary fibrosis. LPA species with longer chain polyunsaturated acyl groups predominated in BAL fluid after bleomycin injury, with 22:5 and 22:6 species accounting for 55 and 16% of the total, whereas the predominant BAL LPC species contained shorter chain, saturated acyl groups, with 16:0 and 18:0 species accounting for 56 and 14% of the total. Further, administration of the potent ATX inhibitor PAT-048 to bleomycin-challenged mice markedly decreased ATX activity systemically and in the lung, without effect on pulmonary LPA or fibrosis. Therefore, alternative ATX-independent pathways are likely responsible for local generation of LPA in the injured lung. These pathways will require identification to therapeutically target LPA production in pulmonary fibrosis.-Black, K. E., Berdyshev, E., Bain, G., Castelino, F. V., Shea, B. S., Probst, C. K., Fontaine, B. A., Bronova, I., Goulet, L., Lagares, D., Ahluwalia, N., Knipe, R. S., Natarajan, V., Tager, A. M. Autotaxin activity increases locally following lung injury, but is not required for pulmonary lysophosphatidic acid production or fibrosis.

Altered Expression of Bone Morphogenetic Protein Accessory Proteins in Murine and Human Pulmonary Fibrosis

Idiopathic pulmonary fibrosis is a chronic, progressive fibrotic disease with a poor prognosis. The balance between transforming growth factor β1 and bone morphogenetic protein (BMP) signaling plays an important role in tissue homeostasis, and alterations can result in pulmonary fibrosis. We hypothesized that multiple BMP accessory proteins may be responsible for maintaining this balance in the lung. Using the bleomycin mouse model for fibrosis, we examined an array of BMP accessory proteins for changes in mRNA expression. We report significant increases in mRNA expression of gremlin 1, noggin, follistatin, and follistatin-like 1 (Fstl1), and significant decreases in mRNA expression of chordin, kielin/chordin-like protein, nephroblastoma overexpressed gene, and BMP and activin membrane-bound inhibitor (BAMBI). Protein expression studies demonstrated increased levels of noggin, BAMBI, and FSTL1 in the lungs of bleomycin-treated mice and in the lungs of idiopathic pulmonary fibrosis patients. Furthermore, we demonstrated that transforming growth factor β stimulation resulted in increased expression of noggin, BAMBI, and FSTL1 in human small airway epithelial cells. These results provide the first evidence that multiple BMP accessory proteins are altered in fibrosis and may play a role in promoting fibrotic injury.

Effects of thymosin β4 and its N-terminal fragment Ac-SDKP on TGF-β-treated human lung fibroblasts and in the mouse model of bleomycin-induced lung fibrosis

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.Objectives: 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.Methods: 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.Results: 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.Conclusion: 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.

IL-27 alleviates the bleomycin-induced pulmonary fibrosis by regulating the Th17 cell differentiation.

BackgroundInterleukin-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.MethodsIn 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.ResultsIncreased 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.ConclusionsThis study demonstrates that IL-27 potentially attenuates BLM-induced pulmonary fibrosis by regulating Th17 differentiation and cytokine secretion.