scholarly journals Improved throughput traction microscopy reveals pivotal role for matrix stiffness in fibroblast contractility and TGF-β responsiveness

2012 ◽  
Vol 303 (3) ◽  
pp. L169-L180 ◽  
Author(s):  
Aleksandar Marinković ◽  
Justin D. Mih ◽  
Jin-Ah Park ◽  
Fei Liu ◽  
Daniel J. Tschumperlin
Keyword(s):  
Extracellular Matrix ◽  
Lung Tissue ◽  
Transforming Growth Factor ◽  
Pivotal Role ◽  
Lung Fibroblasts ◽  
Normal Lung Tissue ◽  
Fluorescent Label ◽  
Normal Lung ◽  
Tgf Β1 ◽  
Traction Microscopy

Lung fibroblast functions such as matrix remodeling and activation of latent transforming growth factor-β1 (TGF-β1) are associated with expression of the myofibroblast phenotype and are directly linked to fibroblast capacity to generate force and deform the extracellular matrix. However, the study of fibroblast force-generating capacities through methods such as traction force microscopy is hindered by low throughput and time-consuming procedures. In this study, we improved at the detail level methods for higher-throughput traction measurements on polyacrylamide hydrogels using gel-surface-bound fluorescent beads to permit autofocusing and automated displacement mapping, and transduction of fibroblasts with a fluorescent label to streamline cell boundary identification. Together these advances substantially improve the throughput of traction microscopy and allow us to efficiently compute the forces exerted by lung fibroblasts on substrates spanning the stiffness range present in normal and fibrotic lung tissue. Our results reveal that lung fibroblasts dramatically alter the forces they transmit to the extracellular matrix as its stiffness changes, with very low forces generated on matrices as compliant as normal lung tissue. Moreover, exogenous TGF-β1 selectively accentuates tractions on stiff matrices, mimicking fibrotic lung, but not on physiological stiffness matrices, despite equivalent changes in Smad2/3 activation. Taken together, these results demonstrate a pivotal role for matrix mechanical properties in regulating baseline and TGF-β1-stimulated contraction of lung fibroblasts and suggest that stiff fibrotic lung tissue may promote myofibroblast activation through contractility-driven events, whereas normal lung tissue compliance may protect against such feedback amplification of fibroblast activation.

Fibrotic extracellular matrix induces release of extracellular vesicles with pro-fibrotic miRNA from fibrocytes

Thorax ◽  
2021 ◽  
pp. thoraxjnl-2020-215962
Author(s):  
Seidai Sato ◽  
Sy Giin Chong ◽  
Chandak Upagupta ◽  
Toyoshi Yanagihara ◽  
Takuya Saito ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Pulmonary Fibrosis ◽  
Extracellular Vesicles ◽  
Mirna Expression ◽  
Magnetic Beads ◽  
Transforming Growth Factor ◽  
Lipid Vesicles ◽  
Normal Lung ◽  
Intratracheal Administration ◽  
Tgf Β1

RationaleExtracellular vesicles (EVs) are small lipid vesicles, and EV-coupled microRNAs (miRNAs) are important modulators of biological processes. Fibrocytes are circulating bone marrow-derived cells that migrate into the injured lungs and contribute to fibrogenesis. The question of whether EV-coupled miRNAs derived from fibrocytes are able to regulate pulmonary fibrosis has not been addressed yet.MethodsPulmonary fibrosis was induced in rats by intratracheal administration of an adenoviral gene vector encoding active transforming growth factor-β1 (TGF-β1) or control vector. Primary fibrocytes and fibroblasts were cultured from rat lungs and were sorted by anti-CD45 magnetic beads. Human circulating fibrocytes and fibrocytes in bronchoalveolar lavage fluid (BALF) were isolated by fibronectin-coated dishes. Fibrocytes were cultured on different stiffness plates or decellularised lung scaffolds. We also determined the effects of extracellular matrix (ECM) and recombinant TGF-β1 on the cellular and EV-coupled miRNA expression of fibrocytes.ResultsThe EVs of fibrocytes derived from fibrotic lungs significantly upregulated the expression of col1a1 of fibroblasts. Culturing on rigid plates or fibrotic decellularised lung scaffolds increased miR-21-5 p expression compared with soft plates or normal lung scaffolds. Dissolved ECM collected from fibrotic lungs and recombinant TGF-β1 increased miR-21-5 p expression on fibrocytes, and these effects were attenuated on soft plates. Fibrocytes from BALF collected from fibrotic interstitial pneumonia patients showed higher miR-21-5 p expression than those from other patients.ConclusionsOur results indicate that ECM contributes to fibrogenesis through biomechanical and biochemical effects on miRNA expression in fibrocytes.

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.

TRIM33 prevents pulmonary fibrosis by impairing TGF-β1 signalling

2020 ◽  
Vol 55 (6) ◽  
pp. 1901346 ◽  
Author(s):  
Pierre-Marie Boutanquoi ◽  
Olivier Burgy ◽  
Guillaume Beltramo ◽  
Pierre-Simon Bellaye ◽  
Lucile Dondaine ◽  
...  
Keyword(s):  
Pulmonary Fibrosis ◽  
Lung Tissue ◽  
Transforming Growth Factor ◽  
Small Heat Shock Protein ◽  
Negative Regulator ◽  
Lung Fibroblasts ◽  
Tissue Slices ◽  
Tgf Β1

BackgroundIdiopathic pulmonary fibrosis (IPF) is a devastating disease characterised by myofibroblast proliferation and abnormal extracellular matrix accumulation in the lungs. Transforming growth factor (TGF)-β1 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-β/SMAD signalling, but its role in fibrogenesis remains unknown. The objective of this study was to elucidate the role of TRIM33 in IPF.MethodsTRIM33 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-β1 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.ResultsTRIM33 was overexpressed in alveolar macrophages and fibroblasts in IPF patients and rodent fibrotic lungs. Trim33 inhibition in BMDM increased TGF-β1 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-β1. 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.ConclusionOur 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.

scholarly journals Extracellular matrix alterations in low-grade lung adenocarcinoma compared with normal lung tissue by imaging mass spectrometry

2020 ◽  
Vol 55 (4) ◽  
pp. e4450 ◽  
Author(s):  
Peggi M. Angel ◽  
Evelyn Bruner ◽  
Jennifer Bethard ◽  
Cassandra L. Clift ◽  
Lauren Ball ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Extracellular Matrix ◽  
Lung Adenocarcinoma ◽  
Lung Tissue ◽  
Imaging Mass Spectrometry ◽  
Normal Lung Tissue ◽  
Normal Lung ◽  
Low Grade

Extracellular matrix deposition by primary human lung fibroblasts in response to TGF-β1 and TGF-β3

1999 ◽  
Vol 276 (5) ◽  
pp. L814-L824 ◽  
Author(s):  
Oliver Eickelberg ◽  
Eleonore Köhler ◽  
Frank Reichenberger ◽  
Sybille Bertschin ◽  
Thomas Woodtli ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Transforming Growth Factor ◽  
Human Lung ◽  
Lung Fibroblasts ◽  
Human Lung Fibroblasts ◽  
Matrix Deposition ◽  
Proline Incorporation ◽  
Primary Fibroblasts ◽  
Extracellular Matrix Deposition ◽  
Tgf Β1

Increased collagen and extracellular matrix (ECM) deposition within the lung is a characteristic feature of lung fibrosis. Transforming growth factor (TGF)-β isoforms play a pivotal role in the production of collagen and ECM. In this study, we investigated the effects of TGF-β1 and TGF-β3 on the main processes controlling ECM deposition using primary human lung fibroblasts. We analyzed 1) collagen metabolism by [3H]proline incorporation, 2) matrix metalloproteinase (MMP) expression by substrate gel zymography, and 3) tissue inhibitor of metalloproteinases (TIMP) expression by Western blot analysis. TGF-β1 and TGF-β3 increased the percentage of secreted collagens in supernatants of primary fibroblasts from 8.0 ± 1.2 (control) to 23.6 ± 4.6 and 22.3 ± 1.3%, respectively. The collagen percentage in deposited ECM was increased from 5.8 ± 0.3 (control) to 9.0 ± 0.5 and 8.8 ± 0.5% by TGF-β1 and TGF-β3, respectively. Secretion of MMP-1 (interstitial collagenase) by fibroblasts was reduced by both TGF-β isoforms, whereas secretion of MMP-2 (gelatinase A) was unaffected by either of the two isoforms. Both TGF-β isoforms increased TIMP-1 protein expression, whereas TIMP-2 protein was decreased. We thus conclude that TGF-β1 and TGF-β3 are equally potent in increasing ECM deposition. Their fibrotic effect in lung fibroblasts results from 1) an increase in the secretion and deposition of total ECM and collagens, 2) a decrease in MMP-1 secretion, and 3) an increase of TIMP-1 expression.

scholarly journals Pirfenidone attenuates lung fibrotic fibroblast-mediated fibrotic responses to transforming growth factor-β1

2018 ◽  
Author(s):  
Jin Jin ◽  
Shinsaku Togo ◽  
Kotaro Kadoya ◽  
Miniwan Tulafu ◽  
Yukiko Namba ◽  
...  
Keyword(s):  
Growth Factor ◽  
Therapeutic Target ◽  
Lung Fibrosis ◽  
Transforming Growth Factor ◽  
Smooth Muscle Actin ◽  
Collagen Gel ◽  
Lung Fibroblasts ◽  
Normal Lung ◽  
Collagen Gel Contraction ◽  
Tgf Β1

AbstractPirfenidone, an antifibrotic agent used for treatment of idiopathic pulmonary fibrosis (IPF), functions by inhibiting myofibroblast differentiation, which is involved in transforming growth factor (TGF)-β1-induced IPF pathogenesis. However, unlike normal lung fibroblasts, the relationship between pirfenidone responses of TGF-β1-induced human fibrotic lung fibroblasts and lung fibrosis is unknown. Here, we investigated the effect of pirfenidone on the functions of two new targets, collagen triple helix repeat containing protein 1 (CTHRC1) and four-and-a-half LIM domain protein 2 (FHL2), which included fibroblast activity, collagen gel contraction, and migration toward fibronectin. Compared to control lung fibroblasts, pirfenidone restored TGF-β1-stimulated fibroblast-mediated collagen gel contraction, migration, and CTHRC1 release in lung fibrotic fibroblasts. Furthermore, pirfenidone attenuated TGF-β1- and CTHRC1-induced fibroblast activity, bone morphogenic protein-4/Gremlin1 upregulation, and α-smooth muscle actin, fibronectin, and FHL2 downregulation, similar to that observed post-CTHRC1 inhibition. In contrast, FHL2 inhibition suppressed migration and fibronectin expression but did not downregulate CTHRC1. Overall, pirfenidone suppressed fibrotic fibroblast-mediated fibrotic processes via inverse regulation of CTHRC1-induced lung fibroblast activity. Thus, CTHRC1 can be used for predicting pirfenidone response and developing new therapeutic target for lung fibrosis.Summary statementPirfenidone suppressed TGF-β1-mediated fibrotic processes in fibrotic lung fibroblasts by attenuating CTHRC1 expression, suggesting that CTHRC1 may be a novel therapeutic target for treating patients with lung fibrosis.

scholarly journals Metformin Protects against Radiation-Induced Acute Effects by Limiting Senescence of Bronchial-Epithelial Cells

2021 ◽  
Vol 22 (13) ◽  
pp. 7064
Author(s):  
Christine Hansel ◽  
Samantha Barr ◽  
Alina V. Schemann ◽  
Kirsten Lauber ◽  
Julia Hess ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Lung Tissue ◽  
Cellular Stress ◽  
Lung Epithelial Cells ◽  
Normal Lung Tissue ◽  
Normal Lung ◽  
Metformin Treatment ◽  
Acute Effects ◽  
Lung Epithelial ◽  
Radiation Induced

Radiation-induced damage to normal lung parenchyma remains a dose-limiting factor in thorax-associated radiotherapy (RT). Severe early and late complications with lungs can increase the risk of morbidity in cancer patients after RT. Herein, senescence of lung epithelial cells following RT-induced cellular stress, or more precisely the respective altered secretory profile, the senescence-associated secretory phenotype (SASP), was suggested as a central process for the initiation and progression of pneumonitis and pulmonary fibrosis. We previously reported that abrogation of certain aspects of the secretome of senescent lung cells, in particular, signaling inhibition of the SASP-factor Ccl2/Mcp1 mediated radioprotection especially by limiting endothelial dysfunction. Here, we investigated the therapeutic potential of a combined metformin treatment to protect normal lung tissue from RT-induced senescence and associated lung injury using a preclinical mouse model of radiation-induced pneumopathy. Metformin treatment efficiently limited RT-induced senescence and SASP expression levels, thereby limiting vascular dysfunctions, namely increased vascular permeability associated with increased extravasation of circulating immune and tumor cells early after irradiation (acute effects). Complementary in vitro studies using normal lung epithelial cell lines confirmed the senescence-limiting effect of metformin following RT finally resulting in radioprotection, while fostering RT-induced cellular stress of cultured malignant epithelial cells accounting for radiosensitization. The radioprotective action of metformin for normal lung tissue without simultaneous protection or preferable radiosensitization of tumor tissue might increase tumor control probabilities and survival because higher radiation doses could be used.

scholarly journals Butyrate Prevents TGF-β1-Induced Alveolar Myofibroblast Differentiation and Modulates Energy Metabolism

Metabolites ◽  
2021 ◽  
Vol 11 (5) ◽  
pp. 258
Author(s):  
Hyo Yeong Lee ◽  
Somi Nam ◽  
Mi Jeong Kim ◽  
Su Jung Kim ◽  
Sung Hoon Back ◽  
...  
Keyword(s):  
Energy Metabolism ◽  
Pulmonary Fibrosis ◽  
Transforming Growth Factor ◽  
Mitochondrial Dynamics ◽  
Tca Cycle ◽  
Lung Fibroblasts ◽  
Myofibroblast Differentiation ◽  
Pulmonary Fibroblasts ◽  
Matrix Deposition ◽  
Tgf Β1

Idiopathic pulmonary fibrosis (IPF) is a serious lung disease characterized by excessive collagen matrix deposition and extracellular remodeling. Signaling pathways mediated by fibrotic cytokine transforming growth factor β1 (TGF-β1) make important contributions to pulmonary fibrosis, but it remains unclear how TGF-β1 alters metabolism and modulates the activation and differentiation of pulmonary fibroblasts. We found that TGF-β1 lowers NADH and NADH/NAD levels, possibly due to changes in the TCA cycle, resulting in reductions in the ATP level and oxidative phosphorylation in pulmonary fibroblasts. In addition, we showed that butyrate (C4), a short chain fatty acid (SCFA), exhibits potent antifibrotic activity by inhibiting expression of fibrosis markers. Butyrate treatment inhibited mitochondrial elongation in TGF-β1-treated lung fibroblasts and increased the mitochondrial membrane potential (MMP). Consistent with the mitochondrial observations, butyrate significantly increased ADP, ATP, NADH, and NADH/NAD levels in TGF-β1-treated pulmonary fibroblasts. Collectively, our findings indicate that TGF-β1 induces changes in mitochondrial dynamics and energy metabolism during myofibroblast differentiation, and that these changes can be modulated by butyrate, which enhances mitochondrial function.

Regulation of extracellular matrix synthesis by TNF-α and TGF-β1 in type II cells exposed to coal dust

1998 ◽  
Vol 275 (4) ◽  
pp. L637-L644 ◽  
Author(s):  
Yu-Chen Lee ◽  
D. Eugene Rannels
Keyword(s):  
Extracellular Matrix ◽  
Cell Cultures ◽  
Transforming Growth Factor ◽  
Coal Dust ◽  
Transforming Growth Factor Β1 ◽  
Type Ii ◽  
Tnf Α ◽  
Type Ii Cell ◽  
Primary Type ◽  
Tgf Β1

Type II pulmonary epithelial cells respond to anthracite coal dust PSOC 867 with increased synthesis of extracellular matrix (ECM) components. Alveolar macrophages modulate this response by pathways that may involve soluble mediators, including tumor necrosis factor-α (TNF-α) or transforming growth factor-β1 (TGF-β1). The effects of TNF-α (10 ng/ml) and/or TGF-β1 (2 ng/ml) were thus investigated in dust-exposed primary type II cell cultures. In control day 1 or day 3 cultures, TNF-α and/or TGF-β1 had little or no effect on the synthesis of type II cellular proteins, independent of whether the cells were exposed to dust. With PSOC 867 exposure, where ECM protein synthesis is elevated, TNF-α and TGF-β1 further increased both the absolute and relative rates of ECM synthesis on day 3 but had little effect on day 1. Each mediator increased expression of fibronectin mRNA, as well as of ECM fibronectin content, in a manner qualitatively similar to their effects on synthesis. Thus TNF-α and TGF-β1 modulate both ECM synthesis and fibronectin content in coal dust-exposed type II cell cultures.

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