scholarly journals Lysyl oxidase directly contributes to extracellular matrix production and fibrosis in systemic sclerosis

2021 ◽  
Vol 320 (1) ◽  
pp. L29-L40
Author(s):  
Xinh-Xinh Nguyen ◽  
Tetsuya Nishimoto ◽  
Takahisa Takihara ◽  
Logan Mlakar ◽  
Amy D. Bradshaw ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Systemic Sclerosis ◽  
Pulmonary Fibrosis ◽  
Nuclear Localization ◽  
Amine Oxidase ◽  
Ex Vivo ◽  
Lysyl Oxidase ◽  
Lung Fibroblasts ◽  
Transcriptional Level

Pulmonary fibrosis is one of the important causes of morbidity and mortality in fibroproliferative disorders such as systemic sclerosis (SSc) and idiopathic pulmonary fibrosis (IPF). Lysyl oxidase (LOX) is a copper-dependent amine oxidase whose primary function is the covalent crosslinking of collagens in the extracellular matrix (ECM). We investigated the role of LOX in the pathophysiology of SSc. LOX mRNA and protein levels were increased in lung fibroblasts of SSc patients compared with healthy controls and IPF patients. In vivo, bleomycin induced LOX mRNA expression in lung tissues, and LOX activity increased in the circulation of mice with pulmonary fibrosis, suggesting that circulating LOX parallels levels in lung tissues. Circulating levels of LOX were reduced upon amelioration of fibrosis with an antifibrotic peptide. LOX induced ECM production at the transcriptional level in lung fibroblasts, human lungs, and human skin maintained in organ culture. In vivo, LOX synergistically exacerbated fibrosis in bleomycin-treated mice. Further, LOX increased the production of interleukin (IL)-6, and the increase was mediated by LOX-induced c-Fos expression, the nuclear localization of c-Fos, and its engagement with the IL-6 promoter region. Our findings demonstrate that LOX expression and activity correlate with fibrosis in vitro, ex vivo, and in vivo. LOX induced ECM production via upregulation of IL-6 and nuclear localization of c-Fos. Thus, LOX has a direct pathogenic role in SSc-associated fibrosis that is independent of its crosslinking function. Our findings also suggest that measuring circulating LOX levels and activity can be used for monitoring response to antifibrotic therapy.

scholarly journals Saracatinib, a Selective Src Kinase Inhibitor, Blocks Fibrotic Responses in In Vitro, In Vivo and Ex Vivo Models of Pulmonary Fibrosis

2022 ◽  
Author(s):  
Farida Ahangari ◽  
Christine Becker ◽  
Daniel G Foster ◽  
Maurizio Chioccioli ◽  
Meghan Nelson ◽  
...  
Keyword(s):  
Pulmonary Fibrosis ◽  
Transforming Growth Factor Beta ◽  
Transforming Growth Factor ◽  
Kinase Inhibitor ◽  
Recombinant Adenovirus ◽  
Ex Vivo ◽  
Src Kinase ◽  
Lung Fibroblasts

Idiopathic Pulmonary Fibrosis (IPF) is a chronic, progressive, and often fatal disorder. Two FDA approved anti-fibrotic drugs, nintedanib and pirfenidone, slow the rate of decline in lung function, but responses are variable and side effects are common. Using an in-silico data-driven approach, we identified a robust connection between the transcriptomic perturbations in IPF disease and those induced by saracatinib, a selective Src kinase inhibitor, originally developed for oncological indications. Based on these observations, we hypothesized that saracatinib would be effective at attenuating pulmonary fibrosis. We investigated the anti-fibrotic efficacy of saracatinib relative to nintedanib and pirfenidone in three preclinical models: (i) in vitro in normal human lung fibroblasts (NHLFs); (ii) in vivo in bleomycin and recombinant adenovirus transforming growth factor-beta (Ad-TGF-β) murine models of pulmonary fibrosis; and (iii) ex vivo in precision cut lung slices from these mouse models. In each model, the effectiveness of saracatinib in blocking fibrogenic responses was equal or superior to nintedanib and pirfenidone.

scholarly journals NADPH oxidase DUOX1 sustains TGF-β1 signalling and promotes lung fibrosis

2020 ◽  
pp. 1901949
Author(s):  
Ruy Andrade Louzada ◽  
Raphaël Corre ◽  
Rabii Ameziane El Hassani ◽  
Lydia Meziani ◽  
Madeleine Jaillet ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Pulmonary Fibrosis ◽  
Nadph Oxidase ◽  
Lung Fibrosis ◽  
Transforming Growth Factor ◽  
Lung Fibroblasts ◽  
Mouse Lung ◽  
Epithelial Surface ◽  
Tgf Β1

Interstitial lung fibroblast activation coupled with extracellular matrix production is a pathological signature of pulmonary fibrosis, and is governed by transforming growth factor (TGF-β1)/Smad signalling. TGF-β1 and oxidative stress cooperate to drive fibrosis. Cells can produce reactive oxygen species (ROS) through activation and/or induction of NADPH oxidases, such as dual oxidase (DUOX1/2). Since DUOX enzymes, as extracellular H2O2-generating systems, are involved in extracellular matrix formation and in wound healing in different experimental models, we hypothesised that DUOX-based NADPH oxidase plays a role in the pathophysiology of pulmonary fibrosis.Our in vivo data (IPF patients and mouse models of lung fibrosis) showed that the NADPH oxidase DUOX1 is induced in response to lung injury. DUOX1-deficient mice (DUOX1+/- and DUOX1-/-) had an attenuated fibrotic phenotype. In addition to being highly expressed at the epithelial surface of airways, DUOX1 appears to be also well expressed in the fibroblastic foci of remodelled lungs. By using primary human and mouse lung fibroblasts, we showed that TGF-β1 upregulates DUOX1 and its maturation factor DUOXA1 and that DUOX1-derived H2O2 promoted the duration of TGF-β1-activated Smad3 phosphorylation by preventing phospho-Smad3 degradation. Analysis of the mechanism revealed that DUOX1 inhibited the interaction between phospho-Smad3 and the ubiquitin ligase NEDD4L, preventing NEDD4L-mediated ubiquitination of phospho-Smad3 and its targeting for degradation.These findings highlight a role for DUOX1-derived H2O2 in a positive feedback that amplifies the signalling output of the TGF-β1 pathway and identify DUOX1 as a new therapeutic target in pulmonary fibrosis.

scholarly journals 2027 The role of lysyl oxidase in systemic sclerosis-associated lung fibrosis

2018 ◽  
Vol 2 (S1) ◽  
pp. 32-33
Author(s):  
Xinh-Xinh Nguyen ◽  
Tetsuya Nishimoto ◽  
Takahisa Takihara ◽  
Logan Mlakar ◽  
Ellen Riemer ◽  
...  
Keyword(s):  
Lung Fibrosis ◽  
Human Lung ◽  
Ex Vivo ◽  
Lysyl Oxidase ◽  
Lung Fibroblasts ◽  
Primary Fibroblasts ◽  
Expression And Activity

OBJECTIVES/SPECIFIC AIMS: Systemic sclerosis (SSc) is a connective tissue disease of unknown etiology characterized by progressive fibrosis of the skin and multiple visceral organs. Effective therapies for SSc are needed. Lysyl oxidase (LOX) is a copper-dependent amide oxidase that plays a critical role in the crosslinking of the extracellular matrix (ECM). In this study, we investigated the role of LOX in the pathophysiology of SSc. METHODS/STUDY POPULATION: LOX expression and protein levels were measured in lung tissues and primary fibroblasts from patients with SSc and healthy controls. The effects of recombinant LOX (rLOX) were measured in vitro in primary fibroblasts, ex vivo in human lung tissues and in vivo in mice given bleomycin in combination with rLOX. LOX levels and activity were evaluated in lung fibroblasts treated with an endostatin-derived peptide that ameliorates fibrosis and in mice treated with bleomycin in combination with the peptide. Further, to differentiate the crosslinking activity of LOX from other potential effects, primary human fibroblasts were cultured with rLOX in the presence of the inhibitor, beta-aminopropionitrile. The expression levels of ECM (collagen and fibronectin), pro-fibrotic factors (IL-6 and TGF-beta), and transcription factor (c-Fos) were examined by real-time PCR, ELISA, immunoblotting, or hydroxyproline assay. RESULTS/ANTICIPATED RESULTS: LOX mRNA was increased in lung tissues and matching fibroblasts of SSc patients. rLOX-induced ECM production in vitro and ex vivo in lung fibroblasts and in human lung tissues maintained in organ culture, respectively. Additionally, TGF-beta and bleomycin induced ECM production, LOX mRNA expression and activity. Endostatin peptide abrogated these effects. In vivo, rLOX synergistically exacerbated pulmonary fibrosis in bleomycin-treated mice. The inhibition of LOX catalytic activity by beta-aminopropionitrile failed to abrogate LOX-induced ECM production. LOX increased the production of IL-6. IL-6 neutralization blocked the effects of LOX. Further, LOX induced c-Fos expression and its nuclear localization. DISCUSSION/SIGNIFICANCE OF IMPACT: LOX expression and activity were increased with fibrosis in vitro, ex vivo, and in vivo. LOX induced fibrosis via increasing ECM, IL-6 and c-Fos translocation to the nucleus. These effects were independent of the crosslinking activity of LOX and mediated by IL-6. Our findings suggest that inhibition of LOX may be a viable option for the treatment of lung fibrosis. Further, the use of human lung in organ culture establishes the relevance of our findings to human disease.

scholarly journals Nanoscale dysregulation of collagen structure-function disrupts mechano-homeostasis and mediates pulmonary fibrosis

eLife ◽  
2018 ◽  
Vol 7 ◽  
Author(s):  
Mark G Jones ◽  
Orestis G Andriotis ◽  
James JW Roberts ◽  
Kerry Lunn ◽  
Victoria J Tear ◽  
...  
Keyword(s):  
Pulmonary Fibrosis ◽  
Structure Function ◽  
Ex Vivo ◽  
Lysyl Oxidase ◽  
Cross Linking ◽  
Collagen Structure ◽  
Tissue Stiffness ◽  
Collagen Concentration ◽  
Dual Inhibition

Matrix stiffening with downstream activation of mechanosensitive pathways is strongly implicated in progressive fibrosis; however, pathologic changes in extracellular matrix (ECM) that initiate mechano-homeostasis dysregulation are not defined in human disease. By integrated multiscale biomechanical and biological analyses of idiopathic pulmonary fibrosis lung tissue, we identify that increased tissue stiffness is a function of dysregulated post-translational collagen cross-linking rather than any collagen concentration increase whilst at the nanometre-scale collagen fibrils are structurally and functionally abnormal with increased stiffness, reduced swelling ratio, and reduced diameter. In ex vivo and animal models of lung fibrosis, dual inhibition of lysyl oxidase-like (LOXL) 2 and LOXL3 was sufficient to normalise collagen fibrillogenesis, reduce tissue stiffness, and improve lung function in vivo. Thus, in human fibrosis, altered collagen architecture is a key determinant of abnormal ECM structure-function, and inhibition of pyridinoline cross-linking can maintain mechano-homeostasis to limit the self-sustaining effects of ECM on progressive fibrosis.

scholarly journals Glycogen Synthase Kinase-3β Inhibition with 9-ING-41 Attenuates the Progression of Pulmonary Fibrosis

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Ann Jeffers ◽  
Wenyi Qin ◽  
Shuzi Owens ◽  
Kathleen B. Koenig ◽  
Satoshi Komatsu ◽  
...  
Keyword(s):  
Pulmonary Fibrosis ◽  
Signaling Pathway ◽  
Glycogen Synthase ◽  
Ex Vivo ◽  
Morphological Changes ◽  
Alveolar Epithelial Cell ◽  
Lung Fibroblasts ◽  
Myofibroblast Differentiation ◽  
Gsk 3Β

AbstractIdiopathic pulmonary fibrosis (IPF) is a progressive interstitial lung disease with a median survival of 3 years after diagnosis. Although the etiology of IPF is unknown, it is characterized by extensive alveolar epithelial cell apoptosis and proliferation of myofibroblasts in the lungs. While the origins of these myofibroblast appear to be diverse, fibroblast differentiation contributes to expansion of myofibroblasts and to disease progression. We found that agents that contribute to neomatrix formation and remodeling in pulmonary fibrosis (PF); TGF-β, Factor Xa, thrombin, plasmin and uPA all induced fibroblast/myofibroblast differentiation. These same mediators enhanced GSK-3β activation via phosphorylation of tyrosine-216 (p-Y216). Inhibition of GSK-3β signaling with the novel inhibitor 9-ING-41 blocked the induction of myofibroblast markers; α-SMA and Col-1 and reduced morphological changes of myofibroblast differentiation. In in vivo studies, the progression of TGF-β and bleomycin mediated PF was significantly attenuated by 9-ING-41 administered at 7 and 14 days respectively after the establishment of injury. Specifically, 9-ING-41 treatment significantly improved lung function (compliance and lung volumes; p < 0.05) of TGF-β adenovirus treated mice compared to controls. Similar results were found in mice with bleomycin-induced PF. These studies clearly show that activation of the GSK-3β signaling pathway is critical for the induction of myofibroblast differentiation in lung fibroblasts ex vivo and pulmonary fibrosis in vivo. The results offer a strong premise supporting the continued investigation of the GSK-3β signaling pathway in the control of fibroblast-myofibroblast differentiation and fibrosing lung injury. These data provide a strong rationale for extension of clinical trials of 9-ING-41 to patients with IPF.

Lysyl oxidase regulation and protein aldehydes in the injured newborn lung

2021 ◽  
Author(s):  
Ying Zhong ◽  
Rose C Mahoney ◽  
Zehedina Khatun ◽  
Howard H Chen ◽  
Christopher T. Nguyen ◽  
...  
Keyword(s):  
Lung Injury ◽  
Protein Expression ◽  
Lysyl Oxidase ◽  
Critical Role ◽  
Molecular Probes ◽  
Lung Fibroblasts ◽  
Transcriptional Level ◽  
Newborn Mouse ◽  
Mouse Pup

During newborn lung injury, excessive activity of lysyl oxidases (LOXs) disrupts extracellular matrix (ECM) formation. Previous studies indicate that TGFβ activation in the O2-injured mouse pup lung increases lysyl oxidase (LOX) expression. But how TGFβ regulates this, and whether the LOXs generate excess pulmonary aldehydes are unknown. First, we determined that O2-mediated lung injury increases LOX protein expression in TGFβ-stimulated pup lung interstitial fibroblasts. This regulation appeared to be direct; this is because TGFβ treatment also increased LOX protein expression in isolated pup lung fibroblasts. Then using a fibroblast cell line, we determined that TGFβ stimulates LOX expression at a transcriptional level via Smad2/3-dependent signaling. LOX is translated as a pro-protein that requires secretion and extracellular cleavage before assuming amine oxidase activity and, in some cells, reuptake with nuclear localization. We found that pro-LOX is processed in the newborn mouse pup lung. Also, O2-mediated injury was determined to increase pro-LOX secretion and nuclear LOX immunoreactivity particularly in areas populated with interstitial fibroblasts and exhibiting malformed ECM. Then, using molecular probes, we detected increased aldehyde levels in vivo in O2-injured pup lungs, which mapped to areas of increased pro-LOX secretion in lung sections. Increased activity of LOXs plays a critical role in the aldehyde generation; an inhibitor of LOXs prevented the elevation of aldehydes in the O2-injured pup lung. These results reveal new mechanisms of TGFβ and LOX in newborn lung disease and suggest that aldehyde-reactive probes might have utility in sensing the activation of LOXs in vivo during lung.

scholarly journals CYP2E1 regulates the development of radiation-induced pulmonary fibrosis via ER stress- and ROS-dependent mechanisms

2017 ◽  
Vol 313 (5) ◽  
pp. L916-L929 ◽  
Author(s):  
Beomseok Son ◽  
TaeWoo Kwon ◽  
Sungmin Lee ◽  
IkJoon Han ◽  
Wanyeon Kim ◽  
...  
Keyword(s):  
Pulmonary Fibrosis ◽  
Er Stress ◽  
Epithelial To Mesenchymal Transition ◽  
Profile Analysis ◽  
Lung Fibroblasts ◽  
In Vivo Studies ◽  
Transcriptional Level ◽  
Mesenchymal Transition ◽  
Radiation Induced

Radiation-induced pulmonary fibrosis (RIPF) is one of the most common side effects of lung cancer radiotherapy. This study was conducted to identify the molecular mechanism responsible for RIPF. We revealed that the transcriptional level of cytochrome P450 2E1 (CYP2E1) was elevated by examining expression profile analysis of RIPF mouse models. We also confirmed that CYP2E1 regulated levels of endoplasmic reticulum (ER) stress and reactive oxygen species (ROS) in alveolar epithelial type II (AE2) cells and lung fibroblasts. Inhibition of CYP2E1 via its siRNA or inhibitor significantly attenuated epithelial-to-mesenchymal transition and apoptosis of AE2 cells, as well as myofibroblast formation induced by radiation. Finally, the effects of a CYP2E1 inhibitor on development of RIPF were evaluated by in vivo studies. Taken together, the results of the present study suggest that CYP2E1 is an important mediator of RIPF development that functions by increasing cellular ER stress and ROS levels.

scholarly journals Identification of Paired-related Homeobox Protein 1 as a key mesenchymal transcription factor in Idiopathic Pulmonary Fibrosis

2021 ◽  
Author(s):  
E. Marchal-Duval ◽  
M. Homps-Legrand ◽  
A. Froidure ◽  
M. Jaillet ◽  
M. Ghanem ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Idiopathic Pulmonary Fibrosis ◽  
Pulmonary Fibrosis ◽  
Ex Vivo ◽  
Lung Fibroblasts ◽  
Matrix Remodeling ◽  
Dependent Manner ◽  
Homeobox Protein

ABSTRACTMatrix remodeling is a salient feature of idiopathic pulmonary fibrosis (IPF). Targeting cells driving matrix remodeling could be a promising avenue for IPF treatment. Analysis of transcriptomic database identified the mesenchymal transcription factor PRRX1 as upregulated in IPF.PRRX1, strongly expressed by lung fibroblasts, was regulated by a TGF-β/PGE2 balance in vitro in control and IPF fibroblasts, while IPF fibroblast-derived matrix increased PRRX1 expression in a PDGFR dependent manner in control ones.PRRX1 inhibition decreased fibroblast proliferation by downregulating the expression of S phase cyclins. PRRX1 inhibition also impacted TGF-β driven myofibroblastic differentiation by inhibiting SMAD2/3 phosphorylation through phosphatase PPM1A upregulation and TGFBR2 downregulation, leading to TGF-β response global decrease.Finally, targeted inhibition of Prrx1 attenuated fibrotic remodeling in vivo with intra-tracheal antisense oligonucleotides in bleomycin mouse model of lung fibrosis and ex vivo using precision-cut lung slices.Our results identified PRRX1 as a mesenchymal transcription factor driving lung fibrogenesis.Brief SummaryInhibition of a single fibroblast-associated transcription factor, namely paired-related homeobox protein 1, is sufficient to dampen lung fibrogenesis.

scholarly journals Targeting Lysyl oxidase-like 2 in Idiopathic Pulmonary Fibrosis

2019 ◽  
Author(s):  
Milena S. Espindola ◽  
David M Habiel ◽  
Ana Lucia Coelho ◽  
Amanda Mikels-Vigdal ◽  
Cory M. Hogaboam
Keyword(s):  
Pulmonary Fibrosis ◽  
Mouse Model ◽  
Lysyl Oxidase ◽  
Lung Fibroblasts ◽  
Normal Lung ◽  
Dependent Manner ◽  
Humanized Mouse ◽  
Humanized Mouse Model

AbstractThe composition of extracellular matrix (ECM) is altered during pathologic scarring in damaged organs including the lung. One major change in the ECM involves the cross-linking of collagen, which promotes fibroblast to myofibroblast differentiation.ObjectiveWe examined the role of lysyl oxidase (LOX)-like 2 in lung fibroblasts cultured from normal or IPF lung samples and in a humanized mouse model of IPF using a monoclonal antibody (Simtuzumab).Research Design and MethodsPrimary lung fibroblasts from normal donor lungs and IPF lung explants were examined for expression of LOXL2. Targeting LOXL2 with Simtuzumab on normal and IPF fibroblasts was examined both in vitro and in vivo for synthetic, functional, and profibrotic properties.ResultsLOXL2 was increased at transcript and protein level in IPF compared with normal lung samples. In a dose-dependent manner, Simtuzumab enhanced differentiation of fibroblasts into myofibroblasts. Inhibition of LOXL2 also enhanced fibroblast invasion and accelerated the outgrowth of fibroblasts from dissociated human lung cell preparations. Finally, preventative or delayed delivery of Simtuzumab enhanced lung fibrosis in a humanized mouse model of pulmonary fibrosis.ConclusionConsistent with its failure in a Phase 2 clinical trial, Simtuzumab exhibited no therapeutic efficacy in translational in vitro and in vivo assays.

scholarly journals Amplified canonical transforming growth factor-β signalling via heat shock protein 90 in pulmonary fibrosis

2016 ◽  
Vol 49 (2) ◽  
pp. 1501941 ◽  
Author(s):  
Zaneta Sibinska ◽  
Xia Tian ◽  
Martina Korfei ◽  
Baktybek Kojonazarov ◽  
Janina Susanne Kolb ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Growth Factor ◽  
Pulmonary Fibrosis ◽  
Transforming Growth Factor ◽  
A549 Cells ◽  
Lung Fibroblasts ◽  
Hsp90 Inhibition ◽  
Extracellular Matrix Production ◽  
Matrix Production ◽  
Β Receptor

Interstitial lung fibroblast activation coupled with extracellular matrix production is a pathological signature of idiopathic pulmonary fibrosis (IPF), and is governed by transforming growth factor (TGF)-β/Smad signalling. We sought to define the role of heat shock protein (HSP)90 in profibrotic responses in IPF and to determine the therapeutic effects of HSP90 inhibition in a murine model of pulmonary fibrosis.We investigated the effects of HSP90 inhibition in vitro by applying 17-AAG (17-allylamino-17-demethoxygeldanamycin) to lung fibroblasts and A549 cells and in vivo by administering 17-DMAG (17-dimethylaminoethylamino-17-demethoxygeldanamycin) to mice with bleomycin-induced pulmonary fibrosis.HSP90 expression was increased in (myo)fibroblasts from fibrotic human and mouse lungs compared with controls. 17-AAG inhibited TGF-β1-induced extracellular matrix production and transdifferentiation of lung fibroblasts and epithelial–mesenchymal transition of A549 cells. The antifibrotic effects were associated with TGF-β receptor disruption and inhibition of Smad2/3 activation. Co-immunoprecipitation revealed that HSP90β interacted with TGF-β receptor II and stabilised TGF-β receptors. Furthermore, 17-DMAG improved lung function and decreased fibrosis and matrix metalloproteinase activity in the lungs of bleomycin-challenged mice.In conclusion, this is the first study to demonstrate that HSP90 inhibition blocks pulmonary fibroblast activation and ameliorates bleomycin-induced pulmonary fibrosis in mice.

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