scholarly journals Targeting MAP3K19 prevents human lung myofibroblast activation both in vitro and in a humanized SCID model of idiopathic pulmonary fibrosis

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Isabelle C. Jones ◽  
Milena S. Espindola ◽  
Rohan Narayanan ◽  
Ana L. Coelho ◽  
David M. Habiel ◽  
...  
Keyword(s):  
Idiopathic Pulmonary Fibrosis ◽  
Pulmonary Fibrosis ◽  
Smooth Muscle Actin ◽  
Mitogen Activated Protein Kinase ◽  
Lung Fibroblasts ◽  
Alpha Smooth Muscle Actin ◽  
Biochemical Assays ◽  
Mitogen Activated Protein ◽  
Si Rna

AbstractIdiopathic Pulmonary Fibrosis (IPF) is a disease with a devastating prognosis characterized by unrelenting lung scarring. Aberrant activation of lung fibroblasts is a key feature of this disease, yet the key pathways responsible for this are poorly understood. Mitogen-activated protein kinase, kinase, kinase- 19 (MAP3K19) was recently shown to be upregulated in IPF and this MAPK has a key role in target gene transcription in the TGF-β pathway. Herein, we further investigate the role of MAP3K19 in cultured normal and IPF fibroblasts and in a humanized SCID mouse model of IPF employing both short interfering (si) RNA and novel small-molecule inhibitors directed at this kinase. Targeting MAP3K19 had significant inhibitory effects on the expression of both alpha smooth muscle actin and extracellular matrix in cultured human IPF fibroblasts. Quantitative protein and biochemical assays, as well as histological analysis, showed that MAP3K19 was required for the development of lung fibrosis in SCID mice humanized with IPF lung fibroblasts. MAP3K19 was required for IPF myofibroblast differentiation, and targeting its activity attenuated the profibrotic activity of these cells both in vitro and in an adoptive transfer SCID model of pulmonary fibrosis.

scholarly journals RXFP1 expression is regulated by miR-144-3p in Fibroblasts from Patients with Idiopathic Pulmonary Fibrosis

2017 ◽  
Author(s):  
Harinath Bahudhanapati ◽  
Jiangning Tan ◽  
Justin A Dutta ◽  
Stephen B Strock ◽  
Yingze Zhang ◽  
...  
Keyword(s):  
Idiopathic Pulmonary Fibrosis ◽  
Pulmonary Fibrosis ◽  
Smooth Muscle Actin ◽  
Negative Regulator ◽  
Lung Fibroblasts ◽  
Luciferase Reporter ◽  
Alpha Smooth Muscle Actin ◽  
Protein Levels ◽  
Donor Lung ◽  
Luciferase Reporter Vector

ABSTRACTRelaxin has been considered as a potential therapy for patients with pulmonary fibrosis. We have previously shown, however, that a potential limitation of relaxin-based therapy for Idiopathic Pulmonary Fibrosis (IPF) is the loss of expression of the relaxin receptor Relaxin/Insulin Like Receptor 1 (RXFP1) expression in fibroblasts. The molecular mechanism for RXFP1 down-regulation in IPF patients remains unclear. To determine whether microRNAs play a role in RXFP1 gene expression, we employed a bioinformatics approach to identify microRNAs (miRs) that are predicted to target RXFP1. By in silico analysis, we identified a putative target site in the RXFP1 mRNA for the miR-144 family. We found that miR-144-3p was upregulated in IPF fibroblasts compared to donor lung fibroblast controls. Forced miR-144-3p mimic expression reduced RXFP1 mRNA and protein levels and increased expression of the myofibroblast marker alpha-smooth muscle actin (α-SMA) in donor lung fibroblasts. IPF lung fibroblasts transfected with a miR-144-3p inhibitor increased RXFP1 expression and reduced α-SMA expression. A lentiviral luciferase reporter vector carrying the WT 3’UTR of RXFP1 was repressed more in lung fibroblasts whereas vector carrying a mutated miR-144-3p binding site exhibited less sensitivity to endogenous miR-144-3p expression, suggesting that RXFP1 is a direct target of miR-144-3p. Thus, miR-144-3p is highly expressed in IPF fibroblasts and acts as a negative regulator of RXFP1 protein expression.

scholarly journals LTBP2 is secreted from lung myofibroblasts and is a potential biomarker for idiopathic pulmonary fibrosis

2018 ◽  
Vol 132 (14) ◽  
pp. 1565-1580 ◽  
Author(s):  
Yasunori Enomoto ◽  
Sayomi Matsushima ◽  
Kiyoshi Shibata ◽  
Yoichiro Aoshima ◽  
Haruna Yagi ◽  
...  
Keyword(s):  
Idiopathic Pulmonary Fibrosis ◽  
Pulmonary Fibrosis ◽  
Transforming Growth Factor ◽  
Smooth Muscle Actin ◽  
Bootstrap Method ◽  
Transforming Growth Factor Β ◽  
Lung Fibroblasts ◽  
Potential Biomarker ◽  
Fibrotic Process ◽  
Mouse Lungs

Although differentiation of lung fibroblasts into α-smooth muscle actin (αSMA)-positive myofibroblasts is important in the progression of idiopathic pulmonary fibrosis (IPF), few biomarkers reflecting the fibrotic process have been discovered. We performed microarray analyses between FACS-sorted steady-state fibroblasts (lineage (CD45, TER-119, CD324, CD31, LYVE-1, and CD146)-negative and PDGFRα-positive cells) from untreated mouse lungs and myofibroblasts (lineage-negative, Sca-1-negative, and CD49e-positive cells) from bleomycin-treated mouse lungs. Amongst several genes up-regulated in the FACS-sorted myofibroblasts, we focussed on Ltbp2, the gene encoding latent transforming growth factor-β (TGF-β) binding protein-2 (LTBP2), because of the signal similarity to Acta2, which encodes αSMA, in the clustering analysis. The up-regulation was reproduced at the mRNA and protein levels in human lung myofibroblasts induced by TGF-β1. LTBP2 staining in IPF lungs was broadly positive in the fibrotic interstitium, mainly as an extracellular matrix (ECM) protein; however, some of the αSMA-positive myofibroblasts were also stained. Serum LTBP2 concentrations, evaluated using ELISA, in IPF patients were significantly higher than those in healthy volunteers (mean: 21.4 compared with 12.4 ng/ml) and showed a negative correlation with % predicted forced vital capacity (r = −0.369). The Cox hazard model demonstrated that serum LTBP2 could predict the prognosis of IPF patients (hazard ratio for death by respiratory events: 1.040, 95% confidence interval: 1.026–1.054), which was validated using the bootstrap method with 1000-fold replication. LTBP2 is a potential prognostic blood biomarker that may reflect the level of differentiation of lung fibroblasts into myofibroblasts in IPF.

scholarly journals Mode of action of nintedanib in the treatment of idiopathic pulmonary fibrosis

2015 ◽  
Vol 45 (5) ◽  
pp. 1434-1445 ◽  
Author(s):  
Lutz Wollin ◽  
Eva Wex ◽  
Alexander Pautsch ◽  
Gisela Schnapp ◽  
Katrin E. Hostettler ◽  
...  
Keyword(s):  
Growth Factor ◽  
Idiopathic Pulmonary Fibrosis ◽  
Pulmonary Fibrosis ◽  
Tyrosine Kinases ◽  
Cell Injury ◽  
Transforming Growth Factor ◽  
Alveolar Epithelial Cell ◽  
Phase Iii ◽  
Lung Fibroblasts

Idiopathic pulmonary fibrosis (IPF) is a progressive and ultimately fatal disease characterised by fibrosis of the lung parenchyma and loss of lung function. Although the pathogenic pathways involved in IPF have not been fully elucidated, IPF is believed to be caused by repetitive alveolar epithelial cell injury and dysregulated repair, in which there is uncontrolled proliferation of lung fibroblasts and differentiation of fibroblasts into myofibroblasts, which excessively deposit extracellular matrix (ECM) proteins in the interstitial space. A number of profibrotic mediators including platelet-derived growth factor (PDGF), fibroblast growth factor (FGF) and transforming growth factor-β are believed to play important roles in the pathogenesis of IPF. Nintedanib is a potent small molecule inhibitor of the receptor tyrosine kinases PDGF receptor, FGF receptor and vascular endothelial growth factor receptor. Data from in vitro studies have shown that nintedanib interferes with processes active in fibrosis such as fibroblast proliferation, migration and differentiation, and the secretion of ECM. In addition, nintedanib has shown consistent anti-fibrotic and anti-inflammatory activity in animal models of lung fibrosis. These data provide a strong rationale for the clinical efficacy of nintedanib in patients with IPF, which has recently been demonstrated in phase III clinical trials.

ID: 112: ROLE OF PHOSPHOLIPASE D IN IDIOPATHIC PULMONARY FIBROSIS

2016 ◽  
Vol 64 (4) ◽  
pp. 964.1-964
Author(s):  
V Suryadevara ◽  
T Royston ◽  
E Berdyshev ◽  
L Huang ◽  
V Natarajan ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Idiopathic Pulmonary Fibrosis ◽  
Pulmonary Fibrosis ◽  
Phospholipase D ◽  
Human Lung ◽  
Lung Fibroblasts ◽  
Human Lung Fibroblasts

Idiopathic pulmonary fibrosis (IPF) is a deadly interstitial disease that leads to scarring and fibrosis of the lung tissue. In pulmonary fibrosis, there is injury and denudation of the alveolar epithelium, which further leads to activation of fibroblasts which differentiate into myofibroblasts. This includes several mechanisms including epithelial to mesenchymal transition (EMT). In this study, we investigated the role of phospholipase D (PLD) in IPF and also its underlying mechanism like EMT and fibroblast proliferation and differentiation. An in vivo murine model of bleomycin-induced pulmonary fibrosis (PF) and in vitro models of murine alveolar type-II epithelial cells (MLE-12) and human lung fibroblasts were used. C57BL/6 and genetically engineered PLD2−/− mice were intratracheally challenged with bleomycin (1.5 U/kg animal) for 14 days and markers of inflammation, EMT and fibrosis were determined. MLE-12 cells were treated with specific PLD1 or PLD2 inhibitors prior to bleomycin (10 mU/ml) challenge, and the role of PLD in EMT and apoptosis of alveolar epithelial cells was studied. Human lung fibroblasts were serum-starved (3h), pretreated with PLD1 or PLD2 inhibitors, and the effect of TGF-β (5 ng/ml) on differentiation of lung fibroblast to myofibroblast was determined. Intra-tracheal instillation of bleomycin in the mice for 14 days leads to the progression of fibrosis in the lung. The lung tissues of the bleomycin treated mice were found to have increased PLD2 protein expression, myofibroblast markers like α-SMA, fibronectin, mesenchymal markers like vimentin, inflammatory cytokines and collagen. Genetic deletion of PLD2 in mice attenuated bleomycin-induced lung inflammation and pulmonary fibrosis. In vitro, MLE-12 cells pretreated with either PLD1 or PLD2 inhibitor did not show a profound reduction either in apoptosis or the expression of transcription factors such as SNAIL, and other markers of EMT. However, MLE-12 cells pretreated with both PLD1 (250 nM) and PLD2 (500 nM) inhibitors were resistant to bleomycin-induced apoptosis, and exhibited reduced expression of SNAIL and mesenchymal markers. On the contrary, human lung fibroblasts pretreated with PLD1 and PLD2 inhibitors showed increased fibroblast to myofibroblast differentiation mediated by TGF-β. The present study suggests a role for PLD2 in bleomycin-induced PF. In vitro, inhibition of both PLD1 and PLD2 was necessary to attenuate bleomycin-induced EMT in epithelial cells and TGF-β mediated differentiation of fibroblasts to myofibroblasts. The in vivo and in vitro results identify the mechanism by which PLD regualtes PF and suggest PLD as a potential therapeutic target in pulmonary fibrosis. This work was supported by National Institutes of Health grant P01 HL98050 to VN.

FGF9 and FGF18 in idiopathic pulmonary fibrosis promote survival and migration and inhibit myofibroblast differentiation of human lung fibroblasts in vitro

2016 ◽  
Vol 310 (7) ◽  
pp. L615-L629 ◽  
Author(s):  
Audrey Joannes ◽  
Stéphanie Brayer ◽  
Valérie Besnard ◽  
Joëlle Marchal-Sommé ◽  
Madeleine Jaillet ◽  
...  
Keyword(s):  
Growth Factor ◽  
Idiopathic Pulmonary Fibrosis ◽  
Pulmonary Fibrosis ◽  
Human Lung ◽  
Lung Fibroblasts ◽  
Myofibroblast Differentiation ◽  
Human Lung Fibroblasts ◽  
And Migration ◽  
Fibroblast Foci

Idiopathic pulmonary fibrosis (IPF) is characterized by an accumulation of extracellular matrix proteins and fibroblasts in the distal airways. Key developmental lung signaling pathways are reactivated in IPF. For instance, fibroblast growth factor 9 (FGF9) and FGF18, involved in epithelial-mesenchymal interactions, are critical for lung development. We evaluated the expression of FGF9, FGF18, and FGF receptors (FGFRs) in lung tissue from controls and IPF patients and assessed their effect on proliferation, survival, migration, and differentiation of control and IPF human lung fibroblasts (HLFs). FGF9, FGF18, and all FGFRs were present in the remodeled alveolar epithelium close to the fibroblast foci in IPF lungs. FGFR3 was generally detected in fibroblast foci by immunohistochemistry. In vitro, HLFs mainly expressed mesenchyme-associated FGFR isoforms (FGFR1c and FGFR3c) and FGFR4. FGF9 did not affect fibroblast proliferation, whereas FGF18 inhibited cell growth in control fibroblasts. FGF9 and FGF18 decreased Fas-ligand-induced apoptosis in control but not in IPF fibroblasts. FGF9 prevented transforming growth factor β1-induced myofibroblast differentiation. FGF9 and FGF18 increased the migratory capacities of HLF, and FGF9 actively modulated matrix metalloproteinase activity. In addition, FGFR3 inhibition by small interfering RNA impacted p-ERK activation by FGF9 and FGF18 and their effects on differentiation and migration. These results identify FGF9 as an antiapoptotic and promigratory growth factor on HLF, maintaining fibroblasts in an undifferentiated state. The biological effects of FGF9 and FGF18 were partially driven by FGFR3. FGF18 was a less potent molecule. Both growth factors likely contribute to the fibrotic process in vivo.

The Strain Response of Lung Myofibroblasts Cultured on Electrospun Polycaprolactone Nanofibers

2013 ◽  
Author(s):  
Timothy J. Fee ◽  
Yong Zhou ◽  
Lauren E. Marshall ◽  
Joel L. Berry
Keyword(s):  
Smooth Muscle ◽  
Idiopathic Pulmonary Fibrosis ◽  
Pulmonary Fibrosis ◽  
Smooth Muscle Actin ◽  
Strain Response ◽  
Muscle Actin ◽  
Alpha Smooth Muscle Actin

Idiopathic Pulmonary Fibrosis is a devastating condition characterized by excessive localized production of collagen in the lungs. Over 131,000 people are living with IPF in America (1, 2). There is currently no known treatment or cure for the disease. It has recently been shown that IPF myofibroblasts are sensitive to the stiffness of their substrate. Specifically, alpha Smooth Muscle Actin (alpha-SMA), a known indicator of IPF activity, was differentially produced on soft vs. stiff substrates (3). This suggests a mechanotransduction pathway within the IPF myofibroblasts.

scholarly journals Akt1 regulates pulmonary fibrosis via modulating IL-13 expression in macrophages

2019 ◽  
Vol 25 (7) ◽  
pp. 451-461 ◽  
Author(s):  
Yunjuan Nie ◽  
Yudong Hu ◽  
Kaikai Yu ◽  
Dan Zhang ◽  
Yinze Shi ◽  
...  
Keyword(s):  
Idiopathic Pulmonary Fibrosis ◽  
Pulmonary Fibrosis ◽  
Smooth Muscle Actin ◽  
In Vivo Studies ◽  
Muscle Actin ◽  
Alpha Smooth Muscle Actin ◽  
Fibrosis Progression ◽  
Matrix Components ◽  
Deficient Mice

Idiopathic pulmonary fibrosis is a progressive interstitial pneumonia characterised by fibroblast accumulation, collagen deposition and extracellular matrix (ECM) remodelling. It was reported that Akt1 mediated idiopathic pulmonary fibrosis progression through regulating the apoptosis of alveolar macrophage, while its effect on macrophage-produced cytokines remains largely unknown. In the present study, we first examined the phosphorylation of Akt1 in lung sections from idiopathic pulmonary fibrosis patients by immunohistochemistry before applying a bleomycin-induced idiopathic pulmonary fibrosis model using Akt1−/− mice and Akt1+/+ littermates. The results showed that Akt1 was remarkably up-regulated in idiopathic pulmonary fibrosis patients, while in vivo studies revealed that Akt1-deficient mice had well-preserved alveolar structure and fewer collagens, secreted fewer matrix components, including alpha smooth-muscle actin and fibronectin and survived significantly longer than Akt1+/+ littermates. Additionally, the pro-fibrogenic cytokine IL-13 was down-regulated at least twofold in Akt1−/−mice compared to the Akt1+/+group on d 3 and 7 after bleomycin treatment. Furthermore, it was found that Akt1–/– macrophages displayed down-regulation of IL-13 compared to Akt1+/+ macrophages in which Akt1 was phosphorylated in response to IL-33 stimulation. These findings indicate that Akt1 modulates pulmonary fibrosis through inducing IL-13 production by macrophages, suggesting that targeting Akt1 may simultaneously block the fibrogenic processes of idiopathic pulmonary fibrosis.

scholarly journals Interaction of long noncoding RNAs and microRNAs in the pathogenesis of idiopathic pulmonary fibrosis

2015 ◽  
Vol 47 (10) ◽  
pp. 463-469 ◽  
Author(s):  
Chaoqun Huang ◽  
Ye Yang ◽  
Lin Liu
Keyword(s):  
Idiopathic Pulmonary Fibrosis ◽  
Pulmonary Fibrosis ◽  
Smooth Muscle Actin ◽  
Noncoding Rnas ◽  
Long Noncoding Rnas ◽  
Lung Fibroblasts ◽  
Competitive Endogenous Rnas ◽  
New Research ◽  
Actin Expression ◽  
Regulatory Functions

Long noncoding RNAs (lncRNAs) are transcribed RNAs with more than 200 nucleotides in length. A growing body of evidence supports the notion that lncRNAs act as competitive endogenous RNAs for microRNAs and play roles in physiological and pathological processes. Several studies have demonstrated the roles of microRNAs in the pathogenesis of idiopathic pulmonary fibrosis (IPF). However, it is unknown whether lncRNAs are involved in IPF. To investigate the roles of lncRNAs in IPF, we determined the interaction of lncRNAs and microRNAs by motif search and manual comparison. The sequences of the dysregulated microRNAs in IPF including miR-21, miR-31, miR-101, miR-29, miR-199, and let-7d were used to search NONCODE database containing 33,829 human lncRNAs. A total of 34 lncRNAs with potential binding sites to these microRNAs were identified. We then examined the expression levels of the identified lncRNAs in the lungs of IPF patients by real-time PCR. Of 34 lncRNAs, nine lncRNAs were dysregulated in the IPF lungs. Four of them were inversely correlated to the microRNA expression in IPF. Further studies revealed that silencing the lncRNA CD99 molecule pseudogene 1 (CD99P1) inhibited proliferation and α-smooth muscle actin expression of lung fibroblasts, while knockdown of the lncRNA n341773 increased collagen expression in lung fibroblasts. These results suggest that CD99P1 and n341773 may be involved in the regulation of lung fibroblast proliferation and differentiation. The identification of regulatory functions of lncRNAs in lung fibroblasts may provide new research directions for the therapy of IPF.

scholarly journals Transglutaminase 2: a novel therapeutic target for idiopathic pulmonary fibrosis using selective small molecule inhibitors

Amino Acids ◽  
2021 ◽  
Author(s):  
Shaun Fell ◽  
Zhuo Wang ◽  
Andy Blanchard ◽  
Carmel Nanthakumar ◽  
Martin Griffin
Keyword(s):  
Idiopathic Pulmonary Fibrosis ◽  
Pulmonary Fibrosis ◽  
Therapeutic Target ◽  
Transforming Growth Factor ◽  
Smooth Muscle Actin ◽  
Transglutaminase 2 ◽  
Transforming Growth Factor Β1 ◽  
Lung Fibroblasts ◽  
Myofibroblast Phenotype ◽  
Novel Therapeutic

AbstractThis study investigates the effects of a site-directed TG2-selective inhibitor on the lung myofibroblast phenotype and ECM deposition to elucidate TG2 as a novel therapeutic target in idiopathic pulmonary fibrosis (IPF)—an incurable progressive fibrotic disease. IPF fibroblasts showed increased expression of TG2, α smooth muscle actin (αSMA) and fibronectin (FN) with increased extracellular TG2 and transforming growth factor β1 (TGFβ1) compared to normal human lung fibroblasts (NHLFs) which do not express αSMA and express lower levels of FN. The myofibroblast phenotype shown by IPF fibroblasts could be reversed by selective TG2 inhibition with a reduction in matrix FN and TGFβ1 deposition. TG2 transduction or TGFβ1 treatment of NHLFs led to a comparable phenotype to that of IPF fibroblasts which was reversible following selective TG2 inhibition. Addition of exogenous TG2 to NHLFs also induced the myofibroblast phenotype by a mechanism involving TGFβ1 activation which could be ameliorated by selective TG2 inhibition. SMAD3-deleted IPF fibroblasts via CRISPR-cas9 genome editing, showed reduced TG2 protein levels following TGFβ1 stimulation. This study demonstrates a key role for TG2 in the induction of the myofibroblast phenotype and shows the potential for TG2-selective inhibitors as therapeutic agents for the treatment of fibrotic lung diseases like IPF.

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