Nitric Oxide-Releasing Drug Glyceryl Trinitrate Targets JAK2/STAT3 Signaling, Migration and Invasion of Triple-Negative Breast Cancer Cells.

Triple-negative breast cancer (TNBC) is a highly aggressive disease with invasive and metastasizing properties associated with a poor prognosis. The STAT3 signaling pathway has shown a pivotal role in cancer cell migration, invasion, metastasis and drug resistance of TNBC cells. IL-6 is a main upstream activator of the JAK2/STAT3 pathway. In the present study we examined the impact of the NO-donor glyceryl trinitrate (GTN) on the activation of the JAK2/STAT3 signaling pathway and subsequent migration, invasion and metastasis ability of TNBC cells through in vitro and in vivo experiments. We used a subtoxic dose of carboplatin and/or recombinant IL-6 to activate the JAK2/STAT3 signaling pathway and its functional outcomes. We found an inhibitory effect of GTN on the activation of the JAK2/STAT3 signaling, migration and invasion of TNBC cells. We discovered that GTN inhibits the activation of JAK2, the upstream activator of STAT3, and mediates the S-nitrosylation of JAK2. Finally, the effect of GTN (Nitronal) on lung metastasis was investigated to assess its antitumor activity in vivo.

TRIM33 prevents pulmonary fibrosis by impairing TGF-ß1 signalling.

BACKGROUND: Idiopathic pulmonary fibrosis (IPF) is a devastating disease characterised by myofibroblast proliferation and abnormal extracellular matrix accumulation in the lungs. Transforming growth factor (TGF)-ß1 initiates key profibrotic signalling involving the SMAD pathway and the small heat shock protein B5 (HSPB5). Tripartite motif-containing 33 (TRIM33) has been reported to negatively regulate TGF-ß/SMAD signalling, but its role in fibrogenesis remains unknown. The objective of this study was to elucidate the role of TRIM33 in IPF. METHODS: TRIM33 expression was assessed in the lungs of IPF patients and rodent fibrosis models. Bone marrow-derived macrophages (BMDM), primary lung fibroblasts and 3D lung tissue slices were isolated from Trim33-floxed mice and cultured with TGF-ß1 or bleomycin (BLM). Trim33 expression was then suppressed by adenovirus Cre recombinase (AdCre). Pulmonary fibrosis was evaluated in haematopoietic-specific Trim33 knockout mice and in Trim33-floxed mice that received AdCre and BLM intratracheally. RESULTS: TRIM33 was overexpressed in alveolar macrophages and fibroblasts in IPF patients and rodent fibrotic lungs. Trim33 inhibition in BMDM increased TGF-ß1 secretion upon BLM treatment. Haematopoietic-specific Trim33 knockout sensitised mice to BLM-induced fibrosis. In primary lung fibroblasts and 3D lung tissue slices, Trim33 deficiency increased expression of genes downstream of TGF-ß1. In mice, AdCre-Trim33 inhibition worsened BLM-induced fibrosis. In vitro, HSPB5 was able to bind directly to TRIM33, thereby diminishing its protein level and TRIM33/SMAD4 interaction. CONCLUSION: Our results demonstrate a key role of TRIM33 as a negative regulator of lung fibrosis. Since TRIM33 directly associates with HSPB5, which impairs its activity, inhibitors of TRIM33/HSPB5 interaction may be of interest in the treatment of IPF.

Pathogenesis of fibrosis in interstitial lung disease.

PURPOSE OF REVIEW: Pulmonary fibrosis is a chronic and progressive lung disease involving unclear pathological mechanisms. The present review presents and discusses the major and recent advances in our knowledge of the pathogenesis of lung fibrosis. RECENT FINDINGS: The past months have deepened our understanding on the cellular actors of fibrosis with a better characterization of the abnormal lung epithelial cells observed during lung fibrosis. Better insight has been gained into fibroblast biology and the role of immune cells during fibrosis. Mechanistically, senescence appears as a key driver of the fibrotic process. Extracellular vesicles have been discovered as participating in the impaired cellular cross-talk during fibrosis and deeper understanding has been made on developmental signaling in lung fibrosis. SUMMARY: This review emphasizes the contribution of different cell types and mechanisms during pulmonary fibrosis, highlights new insights for identification of potential therapeutic strategies, and underlines where future research is needed to answer remaining open questions.

Cell-specific expression of runt-related transcription factor 2 contributes to pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a fatal lung disease with limited therapeutic options and unknown etiology. IPF is characterized by epithelial cell injury, impaired cellular crosstalk between epithelial cells and fibroblasts, and the formation of fibroblast foci with increased extracellular matrix deposition (ECM). We investigated the role of runt-related transcription factor 2 (RUNX2), a master regulator of bone development that has been linked to profibrotic signaling. RUNX2 expression was up-regulated in lung homogenates from patients with IPF and in experimental bleomycin-induced lung fibrosis. The RUNX2 level correlated with disease severity as measured by decreased diffusing capacity and increased levels of the IPF biomarker, matrix metalloproteinase 7. Nuclear RUNX2 was observed in prosurfactant protein C-positive hyperplastic epithelial cells and was rarely found in myofibroblasts. We discovered an up-regulation of RUNX2 in fibrotic alveolar epithelial type II (ATII) cells as well as an increase of RUNX2-negative fibroblasts in experimental and human pulmonary fibrosis. Functionally, small interfering RNA-mediated RUNX2 knockdown decreased profibrotic ATII cell function, such as proliferation and migration, whereas fibroblasts displayed activation markers and increased ECM expression after RUNX2 knockdown. This study reveals that RUNX2 is differentially expressed in ATII cells vs. fibroblasts in lung fibrosis, which contributes to profibrotic cell function. Cell-specific targeting of RUNX2 pathways may represent a therapeutic approach for IPF.-Mümmler, C., Burgy, O., Hermann, S., Mutze, K., Günther, A., Königshoff, M. Cell-specific expression of runt-related transcription factor 2 contributes to pulmonary fibrosis.

Increased Extracellular Vesicles Mediate WNT5A Signaling in Idiopathic Pulmonary Fibrosis.

Rationale: Idiopathic pulmonary fibrosis (IPF) is a lethal lung disease characterized by lung epithelial cell injury, increased (myo)fibroblast activation, and extracellular matrix deposition. Extracellular vesicles (EVs) regulate intercellular communication by carrying a variety of signaling mediators, including WNT (wingless/integrated) proteins. The relevance of EVs in pulmonary fibrosis and their potential contribution to disease pathogenesis, however, remain unexplored.Objectives: To characterize EVs and study the role of EV-bound WNT signaling in IPF.Methods: We isolated EVs from BAL fluid (BALF) from experimental lung fibrosis as well as samples from IPF, non-IPF interstitial lung disease (ILD), non-ILD, and healthy volunteers from two independent cohorts. EVs were characterized by transmission electron microscopy, nanoparticle tracking analysis, and Western blotting. Primary human lung fibroblasts (phLFs) were used for EV isolation and analyzed by metabolic activity assays, cell counting, quantitative PCR, and Western blotting upon WNT gain- and loss-of-function studies.Measurements and Main Results: We found increased EVs, particularly exosomes, in BALF from experimental lung fibrosis as well as from patients with IPF. WNT5A was secreted on EVs in lung fibrosis and induced by transforming growth factor-ß in primary human lung fibroblasts. The phLF-derived EVs induced phLF proliferation, which was attenuated by WNT5A silencing and antibody-mediated inhibition and required intact EV structure. Similarly, EVs from IPF BALF induced phLF proliferation, which was mediated by WNT5A.Conclusions: Increased EVs function as carriers for signaling mediators, such as WNT5A, in IPF and thus contribute to disease pathogenesis. Characterization of EV secretion and composition may lead to novel approaches to diagnose and develop treatments for pulmonary fibrosis.

Pleural inhibition of the caspase-1/IL-1ß pathway diminishes profibrotic lung toxicity of bleomycin.

BACKGROUND: Idiopathic and toxic pulmonary fibrosis are severe diseases starting classically in the subpleural area of the lung. It has recently been suggested that pleural mesothelial cells acquire a myofibroblast phenotype under fibrotic conditions induced by TGF-ß1 or bleomycin. The importance and role of inflammation in fibrogenesis are still controversial. In this work, we explored the role of IL-1ß/caspase-1 signaling in bleomycin lung toxicity and in pleural mesothelial cell transformation. METHODS: C57BL/6 mice were intravenously injected with either bleomycin or nigericin or NaCl as control. In vitro, the Met5A cell line was used as a model of human pleural mesothelial cells. RESULTS: Intravenous injections of bleomycin induced lung fibrosis with histologically-proven peripheral distribution, collagen accumulation in the pleural and subpleural area, and overexpression of markers of myofibroblast transformation of pleural cells which migrated into the lung. These events were associated with an inflammatory process with an increase in neutrophil recruitment in pleural lavage fluid and increased caspase-1 activity. TGF-ß1 was also overexpressed in pleural lavage fluid and was produced by pleural cells following intravenous bleomycin. In this model, local pleural inhibition of IL-1ß with the IL-1ß inhibitor anakinra diminished TGF-ß1 and collagen accumulation. In vitro, caspase-1 inhibition interfered with Met5A cell transformation into the myofibroblast-like phenotype induced by bleomycin or TGF-ß1. Moreover, nigericin, a caspase-1 activator, triggered transformation of Met5A cells and its intra-pleural delivery induced fibrogenesis in mice. CONCLUSIONS: We demonstrated, after intravenous bleomycin injection in mice, the role of the pleura and highlighted the key role of IL-1ß/caspase-1 axis in this fibrogenesis process.

Antifibrotic role of αB-crystallin inhibition in pleural and subpleural fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a devastating disease characterized by myofibroblast proliferation and extracellular-matrix accumulation. IPF typically starts in subpleural lung regions, and recent studies suggest that pleural mesothelial cells play a role in the onset of the disease. The transition of mesothelial cells into myofibroblasts (mesothelio-mesenchymal transition) is induced by the profibrotic cytokine, transforming growth factor (TGF)-ß1, and is thought to play a role in the development and progression of IPF. The Mothers Against Decapentaplegic homolog (Smad)-dependent pathway is the main TGF-ß1 pathway involved in fibrosis. αB-crystallin is constitutively expressed in the lungs, and is inducible by stress, acts as a chaperon, and is known to play a role in cell cytoskeleton architecture. We recently showed that the lack of αB-crystallin hampered TGF-ß1 signaling by favoring Smad4 monoubiquitination and nuclear export. We demonstrate here, for the first time, that αB-crystallin is strongly overexpressed in the pleura of fibrotic lungs from patients with IPF and in rodent models of pleural/subpleural fibrosis. αB-crystallin-deficient mice are protected from pleural/subpleural fibrosis induced by the transient adenoviral-mediated overexpression of TGF-ß1 or the intrapleural injection of bleomycin combined with carbon particles. We show that αB-crystallin inhibition hampers Smad4 nuclear localization in pleural mesothelial cells and the consequent characteristics of mesothelio-mesenchymal transition. αB-crystallin-deficient mesothelial cells fail to acquire the properties of myofibroblasts, thus limiting their migration in vivo and the progression of fibrosis in the lung parenchyma. In conclusion, our work demonstrates that αB-crystallin may be a key target for the development of specific drugs in the treatment of IPF.

The small heat-shock protein αB-crystallin is essential for the nuclear localization of Smad4: impact on pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a devastating disease characterized by the proliferation of myofibroblasts and the accumulation of extracellular matrix (ECM) in the lungs. TGF-ß1 is the major profibrotic cytokine involved in IPF and is responsible for myofibroblast proliferation and differentiation and ECM synthesis. αB-crystallin is constitutively expressed in the lungs and is inducible by stress, acts as a chaperone and is known to play a role in cell cytoskeleton architecture homeostasis. The role of αB-crystallin in fibrogenesis remains unknown. The principal signalling pathway involved in this process is the Smad-dependent pathway. We demonstrate here that αB-crystallin is strongly expressed in fibrotic lung tissue from IPF patients and in vivo rodent models of pulmonary fibrosis. We also show that αB-crystallin-deficient mice are protected from bleomycin-induced fibrosis. Similar protection from fibrosis was observed in αB-crystallin KO mice after transient adenoviral-mediated over-expression of IL-1ß or TGF-ß1. We show in vitro in primary epithelial cells and fibroblasts that αB-crystallin increases the nuclear localization of Smad4, thereby enhancing the TGF-ß1-Smad pathway and the consequent activation of TGF-ß1 downstream genes. αB-crystallin over-expression disrupts Smad4 mono-ubiquitination by interacting with its E3-ubiquitin ligase, TIF1γ, thus limiting its nuclear export. Conversely, in the absence of αB-crystallin, TIF1γ can freely interact with Smad4. Consequently, Smad4 mono-ubiquitination and nuclear export are favoured and thus TGF-ß1-Smad4 pro-fibrotic activity is inhibited. This study demonstrates that αB-crystallin may be a key target for the development of specific drugs in the treatment of IPF or other fibrotic diseases.

HSPB5 Inhibition by NCI-41356 Reduces Experimental Lung Fibrosis by Blocking TGF-ß1 Signaling.

Idiopathic pulmonary fibrosis is a chronic, progressive and lethal disease of unknown etiology that ranks among the most frequent interstitial lung diseases. Idiopathic pulmonary fibrosis is characterized by dysregulated healing mechanisms that lead to the accumulation of large amounts of collagen in the lung tissue that disrupts the alveolar architecture. The two currently available treatments, nintedanib and pirfenidone, are only able to slow down the disease without being curative. We demonstrated in the past that HSPB5, a low molecular weight heat shock protein, was involved in the development of fibrosis and therefore was a potential therapeutic target. Here, we have explored whether NCI-41356, a chemical inhibitor of HSPB5, can limit the development of pulmonary fibrosis. In vivo, we used a mouse model in which fibrosis was induced by intratracheal injection of bleomycin. Mice were treated with NaCl or NCI-41356 (six times intravenously or three times intratracheally). Fibrosis was evaluated by collagen quantification, immunofluorescence and TGF-ß gene expression. In vitro, we studied the specific role of NCI-41356 on the chaperone function of HSPB5 and the inhibitory properties of NCI-41356 on HSPB5 interaction with its partner SMAD4 during fibrosis. TGF-ß1 signaling was evaluated by immunofluorescence and Western Blot in epithelial cells treated with TGF-ß1 with or without NCI-41356. In vivo, NCI-41356 reduced the accumulation of collagen, the expression of TGF-ß1 and pro-fibrotic markers (PAI-1, α-SMA). In vitro, NCI-41356 decreased the interaction between HSPB5 and SMAD4 and thus modulated the SMAD4 canonical nuclear translocation involved in TGF-ß1 signaling, which may explain NCI-41356 anti-fibrotic properties. In this study, we determined that inhibition of HSPB5 by NCI-41356 could limit pulmonary fibrosis in mice by limiting the synthesis of collagen and pro-fibrotic markers. At the molecular level, this outcome may be explained by the effect of NCI-41356 inhibiting HSPB5/SMAD4 interaction, thus modulating SMAD4 and TGF-ß1 signaling. Further investigations are needed to determine whether these results can be transposed to humans.

Extracellular Lipids in the Lung and Their Role in Pulmonary Fibrosis.

Lipids are major actors and regulators of physiological processes within the lung. Initial research has described their critical role in tissue homeostasis and in orchestrating cellular communication to allow respiration. Over the past decades, a growing body of research has also emphasized how lipids and their metabolism may be altered, contributing to the development and progression of chronic lung diseases such as pulmonary fibrosis. In this review, we first describe the current working model of the mechanisms of lung fibrogenesis before introducing lipids and their cellular metabolism. We then summarize the evidence of altered lipid homeostasis during pulmonary fibrosis, focusing on their extracellular forms. Finally, we highlight how lipid targeting may open avenues to develop therapeutic options for patients with lung fibrosis.

HSP47: a potential target for fibrotic diseases and implications for therapy.

Introduction: Chronic fibrotic disorders are challenging clinical problems. The major challenge is the identification of specific targets expressed selectively in fibrotic tissues. Collagen accumulation is the hallmark fibrosis. HSP47 is a collagen-specific chaperon with critical role in collagen folding. This review discusses the anti-fibrotic potential of HSP47. Areas covered: This review compiles data retrieved from the PubMed database with keywords 'HSP47+fibrosis' from 01/2005 to 06/2020. We examined 1) collagen biology and its role in fibrotic diseases, 2) HSP47 role in fibrosis, 3) HSP47 inhibition strategies and 4) clinical investigations. The identification of the HSP47-collagen binding site led to the development of methods to screen HSP47 inhibitors with anti-fibrotic potential. Specific in vivo  delivery systems of HSP47 siRNA to fibrotic tissue reduced collagen production/secretion associated with fibrosis inhibition in preclinical models. This strategy is about to be tested in clinical trials. Expert opinion: As a collagen-specific chaperon, HSP47 is a promising therapeutic target in fibrosis. Preclinical models have shown encouraging anti-fibrotic results. Anti-HSP47 strategies need to be further evaluated in clinical trials. The increase in circulating-HSP47 in lung fibrosis patients highlights the potential of HSP47 as a noninvasive biomarker and may represent an important step toward personalized medicine in fibrotic disorders.

Exosome Isolation by Ultracentrifugation and Precipitation and Techniques for Downstream Analyses.

Exosomes are 50- to 150-nm-diameter extracellular vesicles secreted by all mammalian cells except mature red blood cells and contribute to diverse physiological and pathological functions within the body. Many methods have been used to isolate and analyze exosomes, resulting in inconsistencies across experiments and raising questions about how to compare results obtained using different approaches. Questions have also been raised regarding the purity of the various preparations with regard to the sizes and types of vesicles and to the presence of lipoproteins. Thus, investigators often find it challenging to identify the optimal exosome isolation protocol for their experimental needs. Our laboratories have compared ultracentrifugation and commercial precipitation- and column-based exosome isolation kits for exosome preparation. Here, we present protocols for exosome isolation using two of the most commonly used methods, ultracentrifugation and precipitation, followed by downstream analyses. We use NanoSight nanoparticle tracking analysis and flow cytometry (Cytek® ) to determine exosome concentrations and sizes. Imaging flow cytometry can be utilized to both size exosomes and immunophenotype surface markers on exosomes (ImageStream® ). High-performance liquid chromatography followed by nano-flow liquid chromatography-mass spectrometry (LCMS) of the exosome fractions can be used to determine the presence of lipoproteins, with LCMS able to provide a proteomic profile of the exosome preparations. We found that the precipitation method was six times faster and resulted in a ∼2.5-fold higher concentration of exosomes per milliliter compared to ultracentrifugation. Both methods yielded extracellular vesicles in the size range of exosomes, and both preparations included apoproteins. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Pre-analytic fluid collection and processing Basic Protocol 2: Exosome isolation by ultracentrifugation Alternate Protocol 1: Exosome isolation by precipitation Basic Protocol 3: Analysis of exosomes by NanoSight nanoparticle tracking analysis Alternate Protocol 2: Analysis of exosomes by flow cytometry and imaging flow cytometry Basic Protocol 4: Downstream analysis of exosomes using high-performance liquid chromatography Basic Protocol 5: Downstream analysis of the exosome proteome using nano-flow liquid chromatography-mass spectrometry.

In the spotlight: first ERS Early Career Member Award winner, the ERS Lung Science Conference 2020 and new ECMC members.

All you need to know about @LiesLahousse, first ECM to be granted the Early Career Member Award; #LSC2020; the newly elected assembly representatives for @EarlyCareerERS; and new @EuroRespSoc fellowship opportunities http://bit.ly/2OTgQlo.

Syndecan-1 promotes lung fibrosis by regulating epithelial reprogramming through extracellular vesicles.

Idiopathic pulmonary fibrosis (IPF) is a chronic and fatal lung disease. A maladaptive epithelium due to chronic injury is a prominent feature and contributor to pathogenic cellular communication in IPF. Recent data highlight the concept of a "reprogrammed" lung epithelium as critical in the development of lung fibrosis. Extracellular vesicles (EVs) are potent mediator of cellular crosstalk, and recent evidence supports their role in lung pathologies such as IPF. Here, we demonstrate that syndecan-1 is overexpressed by the epithelium in the lungs of IPF patients and in murine models after bleomycin injury. Moreover, we find that syndecan-1 is a pro-fibrotic signal that alters alveolar type II (ATII) cell phenotypes by augmenting TGFß and Wnt signaling among other pro-fibrotic pathways. Importantly, we demonstrate that syndecan-1 controls the packaging of several anti-fibrotic microRNAs into EVs that have broad effects over several fibrogenic signaling networks as a mechanism of regulating epithelial plasticity and pulmonary fibrosis. Collectively, our work reveals new insight into how EVs orchestrate cellular signals that promote lung fibrosis and demonstrate the importance of syndecan-1 in coordinating these programs.

The WNT signaling pathways in wound healing and fibrosis.

The WNT signaling pathways are major regulators of organ development. Ample research over the past few decades revealed that these pathways are critically involved in adult tissue homeostasis and stem cell function as well as the development of chronic diseases, such as cancer and fibrosis. In this review, we will describe the different WNT signal pathways, summarize the current evidence of WNT signal involvement in wound healing and fibrosis, and highlight potential novel therapeutic options for fibrotic disorders targeting WNT signaling pathways.