Bleomycin Model Of Pulmonary Fibrosis Research Articles

The mRNA-binding protein DDX3 mediates TGF-β1 upregulation of translation and promotes pulmonary fibrosis.

Pulmonary fibrosis is potentiated by a positive feedback loop involving the extracellular sialidase enzyme neuraminidase 3 (NEU3) causing release of active TGF-β1 and TGF-β1 upregulating NEU3 by increasing translation without affecting mRNA levels. In this report, we elucidate the TGF-β1 upregulation of the translation mechanism. In human lung fibroblasts, TGF-β1 increased levels of proteins, including NEU3, by increasing translation of the encoding mRNAs without significantly affecting levels of these mRNAs. A total of 180 of these mRNAs shared a common 20-nucleotide motif. Deletion of this motif from NEU3 mRNA eliminated the TGF-β1 upregulation of NEU3 translation, while insertion of this motif in 2 mRNAs insensitive to TGF-β1 caused TGF-β1 to upregulate their translation. RNA-binding proteins including DEAD box helicase 3, X-linked (DDX3), bind the RNA motif, and TGF-β1 regulates their protein levels and/or binding to the motif. We found that DDX3 was upregulated in the fibrotic lesions in patients with pulmonary fibrosis, and inhibiting DDX3 in fibroblasts reduced TGF-β1 upregulation of NEU3 levels. In the mouse bleomycin model of pulmonary fibrosis, injections of the DDX3 inhibitor RK-33 potentiated survival and reduced lung inflammation, fibrosis, and tissue levels of DDX3, TGF-β1, and NEU3. These results suggest that inhibiting an mRNA-binding protein that mediates TGF-β1 upregulation of translation can reduce pulmonary fibrosis.

Changes in lung sialidases in male and female mice after bleomycin aspiration

ABSTRACT Aim of the study: Sialidases, also called neuraminidases, are enzymes that cleave terminal sialic acids from glycoconjugates. In humans and mice, lung fibrosis is associated with desialylation of glycoconjugates and upregulation of sialidases. There are four mammalian sialidases, and it is unclear when the four mammalian sialidases are elevated over the course of inflammatory and fibrotic responses, whether tissue resident and inflammatory cells express different sialidases, and if sialidases are differentially expressed in male and females. Materials and Methods: To determine the time course of sialidase expression and the identity of sialidase expressing cells, we used the bleomycin model of pulmonary fibrosis in mice to examine levels of sialidases during inflammation (days 3 − 10) and fibrosis (days 10 − 21). Results: Bleomycin aspiration increased sialidase NEU1 at days 14 and 21 in male mice and day 10 in female mice. NEU2 levels increased at day 7 in male and day 10 in female mice. NEU3 appears to have a biphasic response in male mice with increased levels at day 7 and then at days 14 and 21, whereas in female mice NEU3 levels increased over 21 days. In control mice, the sialidases were mainly expressed by EpCAM positive epithelial cells, but after bleomycin, epithelial cells, CD45 positive immune cells, and alveolar cells expressed NEU1, NEU2, and NEU3. Sialidase expression was higher in male compared to female mice. There was little expression of NEU4 in murine lung tissue. Conclusions: These results suggest that sialidases are dynamically expressed following bleomycin, that sialidases are differentially expressed in male and females, and that of the four sialidases only NEU3 upregulation is associated with fibrosis in both male and female mice.

Disturbance of serum lipid metabolites and potential biomarkers in the Bleomycin model of pulmonary fibrosis in young mice

BackgroundAltered metabolic pathways have recently been considered as potential drivers of idiopathic pulmonary fibrosis (IPF) for the study of drug therapeutic targets. However, our understanding of the metabolite profile during IPF formation is lacking.MethodsTo comprehensively characterize the metabolic disorders of IPF, a mouse IPF model was constructed by intratracheal injection of bleomycin into C57BL/6J male mice, and lung tissues from IPF mice at 7 days, 14 days, and controls were analyzed by pathology, immunohistochemistry, and Western Blots. Meanwhile, serum metabolite detections were conducted in IPF mice using LC–ESI–MS/MS, KEGG metabolic pathway analysis was applied to the differential metabolites, and biomarkers were screened using machine learning algorithms.ResultsWe analyzed the levels of 1465 metabolites and found that more than one-third of the metabolites were altered during IPF formation. There were 504 and 565 metabolites that differed between M7 and M14 and controls, respectively, while 201 differential metabolites were found between M7 and M14. In IPF mouse sera, about 80% of differential metabolite expression was downregulated. Lipids accounted for more than 80% of the differential metabolite species with down-regulated expression. The KEGG pathway enrichment analysis of differential metabolites was mainly enriched to pathways such as the metabolism of glycerolipids and glycerophospholipids. Eight metabolites were screened by a machine learning random forest model, and receiver operating characteristic curves (ROC) assessed them as ideal diagnostic tools.ConclusionsIn conclusion, we have identified disturbances in serum lipid metabolism associated with the formation of pulmonary fibrosis, contributing to the understanding of the pathogenesis of pulmonary fibrosis.

Mapping the metabolomic and lipidomic changes in the bleomycin model of pulmonary fibrosis in young and aged mice

ABSTRACTAlterations in metabolic pathways were recently recognized as potential underlying drivers of idiopathic pulmonary fibrosis (IPF), translating into novel therapeutic targets. However, knowledge of metabolic and lipid regulation in fibrotic lungs is limited. To comprehensively characterize metabolic perturbations in the bleomycin mouse model of IPF, we analyzed the metabolome and lipidome by mass spectrometry. We identified increased tissue turnover and repair, evident by enhanced breakdown of proteins, nucleic acids and lipids and extracellular matrix turnover. Energy production was upregulated, including glycolysis, the tricarboxylic acid cycle, glutaminolysis, lactate production and fatty acid oxidation. Higher eicosanoid synthesis indicated inflammatory processes. Because the risk of IPF increases with age, we investigated how age influences metabolomic and lipidomic changes in the bleomycin-induced pulmonary fibrosis model. Surprisingly, except for cytidine, we did not detect any significantly differential metabolites or lipids between old and young bleomycin-treated lungs. Together, we identified metabolomic and lipidomic changes in fibrosis that reflect higher energy demand, proliferation, tissue remodeling, collagen deposition and inflammation, which might serve to improve diagnostic and therapeutic options for fibrotic lung diseases in the future.

The sialidase NEU3 promotes pulmonary fibrosis in mice

BackgroundSialic acid is often the distal sugar on glycoconjugates, and sialidases are enzymes that remove this sugar. In fibrotic lesions in human and mouse lungs, there is extensive desialylation of glycoconjugates, and upregulation of sialidases including the extracellular sialidase NEU3. In the bleomycin model of pulmonary fibrosis, mice lacking NEU3 (Neu3−/−) showed strongly attenuated bleomycin-induced weight loss, lung damage, inflammation, and fibrosis. This indicates that NEU3 is necessary for the full spectrum of bleomycin-induced pulmonary fibrosis.MethodsTo determine if NEU3 is sufficient to induce pulmonary fibrosis, recombinant murine NEU3 and a mutated inactive recombinant murine NEU3 protein were produced. Mice were given recombinant NEU3 proteins by oropharyngeal aspiration, either alone or 10 days after bleomycin challenge. Over the course of 21 days, mice were assessed for weight change, and after euthanasia, bronchoalveolar lavage fluid cells and lung tissue were assessed for inflammation and fibrosis.ResultsAspiration of recombinant murine NEU3 caused inflammation and fibrosis in the lungs, while inactive NEU3 caused inflammation but not fibrosis. Mice were also treated with recombinant murine NEU3 starting 10 days after bleomycin. In male but not female mice, recombinant murine NEU3 increased inflammation and fibrosis. Inactive NEU3 did not enhance bleomycin-induced lung fibrosis.ConclusionThese results suggest that NEU3 is sufficient to induce fibrosis in the lungs, that aspiration of NEU3 has a greater effect on male mice, and that this effect is mediated by NEU3’s enzymic activity.

Modulation of Oxidative Phosphorylation with IM156 Attenuates Mitochondrial Metabolic Reprogramming and Inhibits Pulmonary Fibrosis.

Metabolic reprogramming of the myofibroblast plays a fundamental role in the pathogenesis of fibrosing interstitial lung diseases. Here, we characterized the in vitro and in vivo metabolic and antifibrotic effects of IM156, an oxidative phosphorylation (OXPHOS) modulator that acts by inhibiting protein complex 1. In vitro, IM156 inhibited transforming growth factor β (TGFβ)-dependent increases in mitochondrial oxygen consumption rate and expression of myofibroblast markers in human pulmonary fibroblasts without altering cell viability or adding to TGFβ-induced increases in the extracellular acidification rate. IM156 significantly increased cellular AMP-activated protein kinase (AMPK) phosphorylation and was 60-fold more potent than metformin. In vivo, chronic oral administration of IM156 was highly distributed to major peripheral organs (i.e., lung, liver, kidney, heart) and had significant dose-related effects on the plasma metabolome consistent with OXPHOS modulation and AMPK activation. IM156 increased glycolysis, lipolysis, β-oxidation, and amino acids and decreased free fatty acids, tricarboxylic acid cycle activity, and protein synthesis. In the murine bleomycin model of pulmonary fibrosis, daily oral administration of IM156, administered 7 days after lung injury, attenuated body/lung weight changes and reduced lung fibrosis and inflammatory cell infiltration. The plasma exposures of IM156 were comparable to well tolerated doses in human studies. In conclusion, the metabolic and antifibrotic effects of IM156 suggest that OXPHOS modulation can attenuate myofibroblast metabolic reprogramming and support testing IM156 as a therapy for idiopathic pulmonary fibrosis and other fibrotic diseases. SIGNIFICANCE STATEMENT: Fibrosing interstitial lung diseases have a poor prognosis, and current antifibrotic treatments have significant limitations. This study demonstrates that attenuation of fibrogenic metabolic remodeling, by modulation of oxidative phosphorylation with IM156, prevents myofibroblast phenotype/collagen deposition and is a potentially effective and translational antifibrotic strategy.

Abstract 3167: Stromal TIMP-1 drives tumor progression in lung adenocarcinoma through CD63 interaction

Abstract The two most common lung cancer subtypes are adenocarcinoma (ADC) and squamous cell carcinoma (SCC). Even though both subtypes are epithelial in origin, it is now clear that tumor associated fibroblasts (TAFs) are key regulators of tumor progression and response to therapies. Nintedanib is an antifibrotic drug that targets the tumor stroma and has been clinically approved to treat lung ADC patients owing to the therapeutic benefits exhibited by this drug selectively in ADC (but not in SCC) in the LUME-1 cinical trial. We have implicated recently both ADC-TAFs and ADC cancer cells in the selective effects of nintedanib in ADC, since this drug reduced the growth and invasion induction elicited by the secretome of TGF-β-activated ADC-TAFs on a panel of ADC cells, whereas such reduction was not observed in SCC-TAFs. However, the key molecules involved in the aberrant TAF-carcinoma crosstalk in ADC remain unknown. TIMP-1 is a multifunctional protein that has been associated with poor prognosis in lung cancer and is downregulated by nintedanib in a bleomycin model of pulmonary fibrosis. Our preliminary results revealed that the TIMP-1 receptor CD63 is overexpressed in ADC patients compared to SCC. Therefore, our working hypothesis was that nintedanib reduces ADC cells growth and invasion by abrogating the TAF-carcinoma crosstalk driven by TIMP-1 and CD63.To test this hypothesis, we used primary TAFs obtained with the patient informed consent, and using protocols approved by the Ethics Committee of the Hospital Clinic. TGF-β1-activated ADC-TAFs and SCC-TAFs were treated with nintedanib, and their secreted TIMP-1 was determined by ELISA. Two high-CD63 cell lines (H1437 and H23) were used in some experiments, and siRNA was used to knock-down either TIMP-1 in TAFs or CD63 in cancer cellsOur in vitro results showed that the secretion of TIMP-1 was significantly larger in ADC-TAFs compared to SCC-TAFs. Likewise, nintedanib elicited a higher TIMP-1 downregulation in ADC-TAFs compared SCC-TAFs. Of note, TIMP-1 and CD63 were both implicated in the pro-tumorigenic crosstalk, since knocking-down TIMP-1 in ADC-TAFs or CD63 in ADC cells was sufficient to abrogate the growth and invasion enhancement elicited by the secretome of TAFs. Moreover, CD63 was necessary to enhance the invasion of ADC cells upon stimulation with recombinant TIMP-1. In addition, we found that knocking-down TIMP-1 in ADC-TAFs was sufficient to compromise the inhibitory effects of nintedanib on the growth and invasion enhancement elicited by the secretome of TAFs on ADC cells. Collectively, our results support a novel TAF-carcinoma crosstalk driven by TIMP-1 and CD63 in lung ADC, and support that such heterotypic crosstalk may underlie the aberrant tumor-promoting effects of ADC-TAFs that are selectively downregulated by nintedanib. Citation Format: Paula Duch, Natalia Díaz-Valdivia, Marta Gabasa, Rafael Ikemori, Marselina Arshakyan, Frank Hillberg, Noemí Reguart, Derek Radisky, Jordi Alcaraz. Stromal TIMP-1 drives tumor progression in lung adenocarcinoma through CD63 interaction [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 3167.

FK506-Binding Protein 13 Expression Is Upregulated in Interstitial Lung Disease and Correlated with Clinical Severity. A Potentially Protective Role.

Pulmonary fibrosis is a progressive lung disease characterized by myofibroblast accumulation and excessive extracellular matrix deposition. We sought to investigate the role of FKBP13 (13-kD FK506-binding protein), an endoplasmic reticulum-resident molecular chaperone, in various forms of pulmonary fibrosis. We first characterized the gene and protein expression of FKBP13 in lung biopsy specimens from 24 patients with idiopathic pulmonary fibrosis and 17 control subjects. FKBP13 expression was found to be elevated in the fibrotic regions of idiopathic pulmonary fibrosis lung tissues and correlated with declining forced vital capacity and dyspnea severity. FKBP13 expression was also increased in lung biopsy specimens of patients with hypersensitivity pneumonitis, rheumatoid arthritis, and sarcoidosis-associated interstitial lung disease. We next evaluated the role of this protein using FKBP13-/- mice in a bleomycin model of pulmonary fibrosis. Animals were assessed for lung function and histopathology at different stages of lung injury including the inflammatory (Day 7), fibrotic (Day 21), and resolution (Day 50) phases. FKBP13-/- mice showed increased infiltration of inflammatory cells and cytokines at Day 7, increased lung elastance and fibrosis at Day 21, and impaired resolution of fibrosis at Day 50. These changes were associated with an increased number of cells that stained positive for TUNEL and cleaved caspase 3 in the FKBP13-/- lungs, indicating a heightened cellular sensitivity to bleomycin. Our findings suggest that FKBP13 is a potential biomarker for severity of interstitial lung diseases and that it has a biologically relevant role in protecting mice against bleomycin-induced injury, inflammation, and fibrosis.

Inhibiting Sialidase-Induced TGF-β1 Activation Attenuates Pulmonary Fibrosis in Mice.

The active form of transforming growth factor-β1 (TGF-β1) plays a key role in potentiating fibrosis. TGF-β1 is sequestered in an inactive state by a latency-associated glycopeptide (LAP). Sialidases (also called neuraminidases (NEU)) cleave terminal sialic acids from glycoconjugates. The sialidase NEU3 is upregulated in fibrosis, and mice lacking Neu3 show attenuated bleomycin-induced increases in active TGF-β1 in the lungs and attenuated pulmonary fibrosis. Here we observe that recombinant human NEU3 upregulates active human TGF-β1 by releasing active TGF-β1 from its latent inactive form by desialylating LAP. Based on the proposed mechanism of action of NEU3, we hypothesized that compounds with a ring structure resembling picolinic acid might be transition state analogs and thus possible NEU3 inhibitors. Some compounds in this class showed nanomolar IC50 for recombinant human NEU3 releasing active human TGF-β1 from the latent inactive form. The compounds given as daily 0.1-1-mg/kg injections starting at day 10 strongly attenuated lung inflammation, lung TGF-β1 upregulation, and pulmonary fibrosis at day 21 in a mouse bleomycin model of pulmonary fibrosis. These results suggest that NEU3 participates in fibrosis by desialylating LAP and releasing TGF-β1 and that the new class of NEU3 inhibitors are potential therapeutics for fibrosis. SIGNIFICANCE STATEMENT: The extracellular sialidase NEU3 appears to be a key driver of pulmonary fibrosis. The significance of this report is that 1) we show the mechanism (NEU3 desialylates the latency-associated glycopeptide protein that keeps the profibrotic cytokine transforming growth factor-β1 (TGF-β1) in an inactive state, causing active TGF-β1 release), 2) we then use the predicted NEU3 mechanism to identify nM IC50 NEU3 inhibitors, and 3) these new NEU3 inhibitors are potent therapeutics in a mouse model of pulmonary fibrosis.

Mucosa-Associated Lymphoid Tissue Lymphoma Translocation 1 Inhibitor as a Novel Therapeutic Tool for Lung Injury.

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.

Inhibition of Aurora Kinase B attenuates fibroblast activation and pulmonary fibrosis.

Fibroblast activation including proliferation, survival, and ECM production is central to initiation and maintenance of fibrotic lesions in idiopathic pulmonary fibrosis (IPF). However, druggable molecules that target fibroblast activation remain limited. In this study, we show that multiple pro‐fibrotic growth factors, including TGFα, CTGF, and IGF1, increase aurora kinase B (AURKB) expression and activity in fibroblasts. Mechanistically, we demonstrate that Wilms tumor 1 (WT1) is a key transcription factor that mediates TGFα‐driven AURKB upregulation in fibroblasts. Importantly, we found that inhibition of AURKB expression or activity is sufficient to attenuate fibroblast activation. We show that fibrosis induced by TGFα is highly dependent on AURKB expression and treating TGFα mice with barasertib, an AURKB inhibitor, reverses fibroblast activation, and pulmonary fibrosis. Barasertib similarly attenuated fibrosis in the bleomycin model of pulmonary fibrosis. Together, our preclinical studies provide important proof‐of‐concept that demonstrate barasertib as a possible intervention therapy for IPF.

The importance of interventional timing in the bleomycin model of pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a complex disease of unknown aetiology, which makes drug development challenging. Single administration of bleomycin directly to the lungs of mice is a widely used experimental model for studying pulmonary fibrogenesis and evaluating the effect of therapeutic antifibrotic strategies. The model works by inducing an early inflammatory phase, which transitions into fibrosis after 5-7 days. This initial inflammation makes therapeutic timing crucial. To accurately assess antifibrotic efficacy, the intervention should inhibit fibrosis without impacting early inflammation.Studies published between 2008 and 2019 using the bleomycin model to investigate pulmonary fibrosis were retrieved from PubMed, and study characteristics were analysed. Intervention-based studies were classified as either preventative (starting <7 days after bleomycin installation) or therapeutic (>7 days). In addition, studies were cross-referenced with current major clinical trials to assess the availability of preclinical rationale.A total of 976 publications were evaluated. 726 investigated potential therapies, of which 443 (61.0%) were solely preventative, 166 (22.9%) were solely therapeutic and 105 (14.5%) were both. Of the 443 preventative studies, only 70 (15.8%) characterised inflammation during the model's early inflammatory phase. In the reported 145 IPF clinical trials investigating 93 compounds/combinations, only 25 (26.9%) interventions had any preclinical data on bleomycin available on PubMed.Since 2008, we observed a shift (from <5% to 37.4%) in the number of studies evaluating drugs in the therapeutic setting in the bleomycin model. While this shift is encouraging, further characterisation of early inflammation and appropriate preclinical therapeutic testing are still needed. This will facilitate fruitful drug development in IPF, and more therapeutic strategies for patients with this devastating disease.

Intracellular Heat Shock Protein 70 Deficiency in Pulmonary Fibrosis.

Idiopathic pulmonary fibrosis (IPF) pathogenesis has been postulated to involve a variety of mechanisms associated with the aging process, including loss of protein homeostasis (proteostasis). Heat shock proteins are cellular chaperones that serve a number of vital maintenance and repair functions, including the regulation of proteostasis. Previously published data have implicated heat shock protein 70 (Hsp70) in the development of pulmonary fibrosis in animal models. We sought to identify alterations in Hsp70 expression in IPF lung. Hsp70 mRNA and protein were decreased in primary fibroblasts cultured from IPF versus normal donor lung tissue. In addition to cultured fibroblasts, Hsp70 expression was decreased in intact IPF lung, a stressed environment in which upregulation of protective heat shock proteins would be anticipated. In support of a mechanistic association between decreased Hsp70 and fibrosis, cultured primary lung fibroblasts deficient in Hsp70 secreted increased extracellular matrix proteins. Treatment of primary normal human lung fibroblasts in vitro with either of the profibrotic molecules IGFBP5 (insulin-like growth factor-binding protein 5) or transforming growth factor-β1 downregulated Hsp70, suggesting Hsp70 is a downstream target in the fibrotic cascade. Hsp70-knockout mice subjected to an inhalational bleomycin model of pulmonary fibrosis demonstrated accelerated fibrosis versus wild-type control animals. We therefore conclude that reduced Hsp70 protein contributes to fibrosis and that interventions aimed at restoring normal expression of Hsp70 represent a novel therapeutic strategy for pulmonary fibrosis.

Anti-fibrotic Effects of CXCR4-Targeting i-body AD-114 in Preclinical Models of Pulmonary Fibrosis

Idiopathic pulmonary fibrosis (IPF) is a chronic fibrotic lung disease that is prevalent in individuals >50 years of age, with a median survival of 3–5 years and limited therapeutic options. The disease is characterized by collagen deposition and remodeling of the lung parenchyma in a process that is thought to be driven by collagen-expressing immune and structural cells. The G-protein coupled C-X-C chemokine receptor 4, CXCR4, is a candidate therapeutic target for IPF owing to its role in the recruitment of CXCR4+ fibrocytes from the bone marrow to fibrotic lung tissue and its increased expression levels by structural cells in fibrotic lung tissue. We have engineered a novel fully human single domain antibody “i-body” called AD-114 that binds with high affinity to human CXCR4. We demonstrate here that AD-114 inhibits invasive wound healing and collagen 1 secretion by human IPF fibroblasts but not non-diseased control lung fibroblasts. Furthermore, in a murine bleomycin model of pulmonary fibrosis, AD-114 reduced the accumulation of fibrocytes (CXCR4+/Col1+/CD45+) in fibrotic murine lungs and ameliorated the degree of lung injury. Collectively, these studies demonstrate that AD-114 holds promise as a new biological therapeutic for the treatment of IPF.

The purinergic receptor subtype P2Y2 mediates chemotaxis of neutrophils and fibroblasts in fibrotic lung disease.

Idiopathic pulmonary fibrosis (IPF) is a devastating disease with few available treatment options. Recently, the involvement of purinergic receptor subtypes in the pathogenesis of different lung diseases has been demonstrated. Here we investigated the role of the purinergic receptor subtype P2Y2 in the context of fibrotic lung diseases.The concentration of different nucleotides was measured in the broncho-alveolar lavage (BAL) fluid derived from IPF patients and animals with bleomycin-induced pulmonary fibrosis. In addition expression of P2Y2 receptors by different cell types was determined. To investigate the functional relevance of P2Y2 receptors for the pathogenesis of the disease the bleomycin model of pulmonary fibrosis was used. Finally, experiments were performed in pursuit of the involved mechanisms.Compared to healthy individuals or vehicle treated animals, extracellular nucleotide levels in the BAL fluid were increased in patients with IPF and in mice after bleomycin administration, paralleled by a functional up-regulation of P2Y2R expression. Both bleomycin-induced inflammation and fibrosis were reduced in P2Y2R-deficient compared to wild type animals. Mechanistic studies demonstrated that recruitment of neutrophils into the lungs, proliferation and migration of lung fibroblasts as well as IL6 production are key P2Y2R mediated processes.Our results clearly demonstrate the involvement of P2Y2R subtypes in the pathogenesis of fibrotic lung diseases in humans and mice and hence support the development of selective P2Y2R antagonists for the treatment of IPF.

Prophylactic and curative effect of rosemary leaves extract in a bleomycin model of pulmonary fibrosis

Context: Pulmonary fibrosis is a devastating disease without effective treatment. Rosemary is appreciated since ancient times for its medicinal properties, while biomolecules originated from the plant have an antioxidant and antifibrotic effect.Objective: The effects of Rosmarinus officinalis L. (Lamiaceae) leaves extract (RO) on bleomycin-induced lung fibrosis were investigated.Materials and methods: Male Wistar rats were given a single dose of bleomycin (BLM, 4 mg/kg, intratracheal), while RO (75 mg/kg, intraperitoneal) was administered 3 days later and continued for 4 weeks (BLM/RO1-curative group). Alternatively, RO was administered 2 weeks before BLM and continued 15 days thereafter (BLM/RO2-prophylactic group). Antioxidant activities of RO and lung tissues were studied by standard methods. Histological staining revealed lung architecture and collagen deposition. RO was characterized for its polyphenol content and by high-performance liquid chromatography.Results: RO polyphenol content was 60.52 mg/g of dry weight, carnosic and rosmarinic acids being major components (6.886 and 2.351 mg/g). Antioxidant effect of RO (DPPH and FRAP assay) expressed as IC50 values were 2.23 μg/mL and 0.074 μg/mL, respectively. In BLM/RO1 and BLM/RO2 lung architecture was less compromised compared to BLM, which was reflected in lower fibrosis score (2.33 ± 0.33 and 1.8 ± 0.32 vs 3.7 ± 0.3). Malondialdehyde levels were attenuated (141% and 108% vs 258% of normal value). Catalase and glutathione-S-transferase activities were normalized (103% and 117% vs 59%, 85% and 69% vs 23%, respectively).Discussion and conclusion: RO has a protective effect against BLM-induced oxidative stress and lung fibrosis due to its phenolic compounds.

STAT3 as a Multi‐organ Target for Tissue Fibrosis

Despite recent therapeutic developments, pulmonary fibrosis remains a debilitating disease with poor prognosis. In order to create effective treatments, promising molecular targets need to be identified and validated. Across multiple organs, mesenchymal derived myofibroblasts play an essential role in matrix deposition and tissue contraction. We previously carried out a high‐content, RNAi screen to characterize myofibroblast cytoskeletal phenotypes (α‐SMA and organized F‐actin) in cultured fibroblasts. Hits from this screen, including STAT3 (Signal transducer and activator of transcription 3), were validated in primary lung fibroblasts from patients with IPF (idiopathic pulmonary fibrosis) on compliant hydrogels. To further evaluate STAT3 as a therapeutic target, we have focused on LLL12, a small molecule inhibitor of STAT3. LLL12 dose‐dependently (0.3, 1.0μM) reduced RNA expression of profibrotic genes (ACTA2, CTGF, COL1A1, and FN1) as well as protein deposition of collagen III and fibronectin, in TGFβ stimulated cultured fibroblasts. LLL12 also reduced contractile force, as measured by traction force microscopy, to levels comparable to that seen with inhibition of Rho kinase (Y27632, 20μM). In an effort to support the role of STAT3 as a multi‐organ regulator of tissue fibrosis we confirmed similar effects of LLL12 in primary human hepatic stellate cells and cardiac fibroblasts. Using the bleomycin model of pulmonary fibrosis we assessed the in vivo efficacy of STAT3 inhibition. The lungs of the mice treated therapeutically (day 14–28) with LLL12 (5 mg/kg i.p. daily) showed reduced collagen and fibrotic matrix content measured by hydroxyproline and trichrome histology. RNA expression of COL1A1, FN1, CTGF, and ACTA2 also decreased (~50%) in the mice treated with LLL12. Importantly, in the mice which did not receive bleomycin, LLL12 had no effect on RNA expression of these genes. These data not only validate the RNAi screening approach, but also point towards STAT3 as an important molecular target for regulation of myofibroblast activation in pulmonary fibrosis and other fibrotic diseases.Support or Funding InformationSupported by NIH RO1 HL092961 and HL113796, and Boehringer Ingelheim.

Preventive and therapeutic effects of thymosin β4 N-terminal fragment Ac-SDKP in the bleomycin model of pulmonary fibrosis.

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.

Mesenchymal Deficiency of Notch1 Attenuates Bleomycin-Induced Pulmonary Fibrosis

Notch signaling pathway is involved in the regulation of cell fate, differentiation, proliferation, and apoptosis in development and disease. Previous studies suggest the importance of Notch1 in myofibroblast differentiation in lung alveogenesis and fibrosis. However, direct invivo evidence of Notch1-mediated myofibroblast differentiation is lacking. In this study, we examined the effects of conditional mesenchymal-specific deletion of Notch1 on pulmonary fibrosis. Crossing of mice bearing the floxed Notch1 gene with α2(I) collagen enhancer-Cre-ER(T)-bearing mice successfully generated progeny with a conditional knockout (CKO) of Notch1 in collagen I-expressing (mesenchymal) cells on treatment with tamoxifen (Notch1 CKO). Because Notch signaling is known to be activated in the bleomycin model of pulmonary fibrosis, control and Notch1 CKO mice were analyzed for their responses to bleomycin treatment. The results showed significant attenuation of pulmonary fibrosis in CKO relative to control mice, as examined by collagen deposition, myofibroblast differentiation, and histopathology. However, there were no significant differences in inflammatory or immune cell influx between bleomycin-treated CKO and control mouse lungs. Analysis of isolated lung fibroblasts confirmed absence of Notch1 expression in cells from CKO mice, which contained fewer myofibroblasts and significantly diminished collagen I expression relative to those from control mice. These findings revealed an essential role for Notch1-mediated myofibroblast differentiation in the pathogenesis of pulmonary fibrosis.

Disruption of Calcium Signaling in Fibroblasts and Attenuation of Bleomycin-Induced Fibrosis by Nifedipine.

Fibrotic lung disease afflicts millions of people; the central problem is progressive lung destruction and remodeling. We have shown that external growth factors regulate fibroblast function not only through canonical signaling pathways but also through propagation of periodic oscillations in Ca(2+). In this study, we characterized the pharmacological sensitivity of the Ca(2+)oscillations and determined whether a blocker of those oscillations can prevent the progression of fibrosis in vivo. We found Ca(2+) oscillations evoked by exogenously applied transforming growth factor β in normal human fibroblasts were substantially reduced by 1 μM nifedipine or 1 μM verapamil (both L-type blockers), by 2.7 μM mibefradil (a mixed L-/T-type blocker), by 40 μM NiCl2 (selective at this concentration against T-type current), by 30 mM KCl (which partially depolarizes the membrane and thereby fully inactivates T-type current but leaves L-type current intact), or by 1 mM NiCl2 (blocks both L- and T-type currents). In our in vivo study in mice, nifedipine prevented bleomycin-induced fibrotic changes (increased lung stiffness, overexpression of smooth muscle actin, increased extracellular matrix deposition, and increased soluble collagen and hydroxyproline content). Nifedipine had little or no effect on lung inflammation, suggesting its protective effect on lung fibrosis was not due to an antiinflammatory effect but rather was due to altering the profibrotic response to bleomycin. Collectively, these data show that nifedipine disrupts Ca(2+) oscillations in fibroblasts and prevents the impairment of lung function in the bleomycin model of pulmonary fibrosis. Our results provide compelling proof-of-principle that interfering with Ca(2+) signaling may be beneficial against pulmonary fibrosis.