Effects of thymosin β4 and its N-terminal fragment Ac-SDKP on TGF-β-treated human lung fibroblasts and in the mouse model of bleomycin-induced lung fibrosis

Carbon nanotubes (CNTs) induce rapid interstitial lung fibrosis, but the underlying mechanisms are unclear. Previous studies indicated that the ability of CNTs to penetrate lung epithelium, enter interstitial tissue, and stimulate fibroblasts to produce collagen matrix is important to lung fibrosis. In this study, we investigated the activation of transforming growth factor-β receptor-1 [TGF-β R1; i.e., activin receptor-like kinase 5 (ALK5) receptor] and TGF-β/Smad signaling pathway in CNT-induced collagen production in human lung fibroblasts. Human lung fibroblasts and epithelial cells were exposed to low, physiologically relevant concentrations (0.02–0.6 μg/cm2) of single-walled CNTs (SWCNT) and multiwalled CNTs (MWCNT) in culture and analyzed for collagen, TGF-β1, TGF-β R1, and SMAD proteins by Western blotting and immunofluorescence. Chemical inhibition of ALK5 and short-hairpin (sh) RNA targeting of TGF-β R1 and Smad2 were used to probe the fibrogenic mechanism of CNTs. Both SWCNT and MWCNT induced an overexpression of TGF-β1, TGF-β R1 and Smad2/3 proteins in lung fibroblasts compared with vehicle or ultrafine carbon black-exposed controls. SWCNT- and MWCNT-induced collagen production was blocked by ALK5 inhibitor or shRNA knockdown of TGF-β R1 and Smad2. Our results indicate the critical role of TGF-β R1/Smad2/3 signaling in CNT-induced fibrogenesis by upregulating collagen production in lung fibroblasts. This novel finding may aid in the design of mechanism-based risk assessment and development of rapid screening tests for nanomaterial fibrogenicity.

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Relaxin induces an extracellular matrix-degrading phenotype in human lung fibroblasts in vitro and inhibits lung fibrosis in a murine model in vivo.

Journal of Clinical Investigation ◽

10.1172/jci119099 ◽

1996 ◽

Vol 98 (12) ◽

pp. 2739-2745 ◽

Cited By ~ 226

Author(s):

E N Unemori ◽

L B Pickford ◽

A L Salles ◽

C E Piercy ◽

B H Grove ◽

...

Keyword(s):

Extracellular Matrix ◽

Murine Model ◽

Lung Fibrosis ◽

Human Lung ◽

Lung Fibroblasts ◽

Human Lung Fibroblasts

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Phosphatidylethanolamine Induces an Antifibrotic Phenotype in Normal Human Lung Fibroblasts and Ameliorates Bleomycin-Induced Lung Fibrosis in Mice

International Journal of Molecular Sciences ◽

10.3390/ijms19092758 ◽

2018 ◽

Vol 19 (9) ◽

pp. 2758 ◽

Cited By ~ 2

Author(s):

Luis Vazquez-de-Lara ◽

Beatriz Tlatelpa-Romero ◽

Yair Romero ◽

Nora Fernández-Tamayo ◽

Fernando Vazquez-de-Lara ◽

...

Keyword(s):

Phospholipase C ◽

Lung Fibrosis ◽

Human Lung ◽

Complex Mixture ◽

Lung Fibroblasts ◽

Collagen Concentration ◽

Human Lung Fibroblasts ◽

Collagen Expression ◽

Normal Human

Lung surfactant is a complex mixture of phospholipids and specific proteins but its role in the pathogenesis of interstitial lung diseases is not established. Herein, we analyzed the effects of three representative phospholipid components, that is, dipalmitoilphosphatidylcoline (DPPC), phosphatidylglycerol (PG) and phosphatidylethanolamine (PE), on collagen expression, apoptosis and Ca2+ signaling in normal human lung fibroblasts (NHLF) and probed their effect in an experimental model of lung fibrosis. Collagen expression was measured with RT-PCR, apoptosis was measured by using either the APOPercentage assay kit (Biocolor Ltd., Northern Ireland, UK) or the Caspase-Glo 3/7 assay (Promega, Madison, WI, USA) and Ca2+ signaling by conventional epifluorescence imaging. The effect in vivo was tested in bleomycin-induced lung fibrosis in mice. DPPC and PG did not affect collagen expression, which was downregulated by PE. Furthermore, PE promoted apoptosis and induced a dose-dependent Ca2+ signal. PE-induced Ca2+ signal and apoptosis were both blocked by phospholipase C, endoplasmic reticulum pump and store-operated Ca2+ entry inhibition. PE-induced decrease in collagen expression was attenuated by blocking phospholipase C. Finally, surfactant enriched with PE and PE itself attenuated bleomycin-induced lung fibrosis and decreased the soluble collagen concentration in mice lungs. This study demonstrates that PE strongly contributes to the surfactant-induced inhibition of collagen expression in NHLF through a Ca2+ signal and that early administration of Beractant enriched with PE diminishes lung fibrosis in vivo.

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Application of a High-Content Screening Assay Utilizing Primary Human Lung Fibroblasts to Identify Antifibrotic Drugs for Rapid Repurposing in COVID-19 Patients

SLAS DISCOVERY Advancing Life Sciences ◽

10.1177/24725552211019405 ◽

2021 ◽

pp. 247255522110194

Author(s):

John A. Marwick ◽

Richard J. R. Elliott ◽

James Longden ◽

Ashraff Makda ◽

Nik Hirani ◽

...

Keyword(s):

Lung Fibrosis ◽

Transforming Growth Factor ◽

Human Lung ◽

Rapid Development ◽

Transforming Growth Factor Β ◽

Lung Fibroblasts ◽

High Signal ◽

Screening Assay ◽

Human Lung Fibroblasts ◽

Screening Assays

Lung imaging and autopsy reports among COVID-19 patients show elevated lung scarring (fibrosis). Early data from COVID-19 patients as well as previous studies from severe acute respiratory syndrome, Middle East respiratory syndrome, and other respiratory disorders show that the extent of lung fibrosis is associated with a higher mortality, prolonged ventilator dependence, and poorer long-term health prognosis. Current treatments to halt or reverse lung fibrosis are limited; thus, the rapid development of effective antifibrotic therapies is a major global medical need that will continue far beyond the current COVID-19 pandemic. Reproducible fibrosis screening assays with high signal-to-noise ratios and disease-relevant readouts such as extracellular matrix (ECM) deposition (the hallmark of fibrosis) are integral to any antifibrotic therapeutic development. Therefore, we have established an automated high-throughput and high-content primary screening assay measuring transforming growth factor-β (TGFβ)-induced ECM deposition from primary human lung fibroblasts in a 384-well format. This assay combines longitudinal live cell imaging with multiparametric high-content analysis of ECM deposition. Using this assay, we have screened a library of 2743 small molecules representing approved drugs and late-stage clinical candidates. Confirmed hits were subsequently profiled through a suite of secondary lung fibroblast phenotypic screening assays quantifying cell differentiation, proliferation, migration, and apoptosis. In silico target prediction and pathway network analysis were applied to the confirmed hits. We anticipate this suite of assays and data analysis tools will aid the identification of new treatments to mitigate against lung fibrosis associated with COVID-19 and other fibrotic diseases.

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Tropomyosin 2.1 collaborates with fibronectin to promote TGF-β1-induced contraction of human lung fibroblasts

Respiratory Research ◽

10.1186/s12931-021-01730-y ◽

2021 ◽

Vol 22 (1) ◽

Author(s):

Peta Bradbury ◽

Cassandra P. Nader ◽

Aylin Cidem ◽

Sandra Rutting ◽

Dianne Sylvester ◽

...

Keyword(s):

Lung Fibrosis ◽

Human Lung ◽

Collagen Gel ◽

Protein Profiling ◽

Lung Fibroblasts ◽

Human Lung Fibroblasts ◽

Collagen Gel Contraction ◽

The Matrix ◽

Cytokine Stimulation ◽

Tgf Β1

AbstractMany lung diseases are characterized by fibrosis, leading to impaired tissue patency and reduced lung function. Development of fibrotic tissue depends on two-way interaction between the cells and the extra-cellular matrix (ECM). Concentration-dependent increased stiffening of the ECM is sensed by the cells, which in turn increases intracellular contraction and pulling on the matrix causing matrix reorganization and further stiffening. It is generally accepted that the inflammatory cytokine growth factor β1 (TGF-β1) is a major driver of lung fibrosis through the stimulation of ECM production. However, TGF-β1 also regulates the expression of members of the tropomyosin (Tm) family of actin associating proteins that mediate ECM reorganization through intracellular-generated forces. Thus, TGF-β1 may mediate the bi-directional signaling between cells and the ECM that promotes tissue fibrosis. Using combinations of cytokine stimulation, mRNA, protein profiling and cellular contractility assays with human lung fibroblasts, we show that concomitant induction of key Tm isoforms and ECM by TGF-β1, significantly accelerates fibrotic phenotypes. Knocking down Tpm2.1 reduces fibroblast-mediated collagen gel contraction. Collectively, the data suggest combined ECM secretion and actin cytoskeleton contractility primes the tissue for enhanced fibrosis. Our study suggests that Tms are at the nexus of inflammation and tissue stiffening. Small molecules targeting specific Tm isoforms have recently been designed; thus targeting Tpm2.1 may represent a novel therapeutic target in lung fibrosis.

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Inhibition of Raf1 ameliorates bleomycin-induced pulmonary fibrosis through attenuation of TGF-β1 signaling

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.00093.2018 ◽

2018 ◽

Vol 315 (2) ◽

pp. L241-L247 ◽

Cited By ~ 3

Author(s):

Shuang Li ◽

Jia Liu ◽

Jiangning Tan ◽

Lian Li ◽

Mary J. Kaltreider ◽

...

Keyword(s):

Pulmonary Fibrosis ◽

Lung Fibrosis ◽

Molecular Mechanisms ◽

Human Lung ◽

Smooth Muscle Actin ◽

Lung Fibroblasts ◽

Human Lung Fibroblasts ◽

And Migration ◽

Fibrotic Lung Disease ◽

Tgf Β1

Idiopathic pulmonary fibrosis (IPF) is a fatal fibrotic lung disease associated with aberrant activation and differentiation of fibroblasts, leading to abnormal extracellular matrix production. Currently, it is still an untreatable disease (except for lung transplantation). Here, we demonstrate that the Raf1 inhibitor GW5074 ameliorates lung fibrosis in bleomycin-induced pulmonary fibrosis. Posttreatment with GW5074 reduced fibronectin (FN) expression, collagen deposition, and inflammatory cell infiltration in bleomycin-challenged mice, suggesting an antifibrotic property of GW5074. To determine the molecular mechanisms by which inhibition of Raf1 ameliorates lung fibrosis, we investigated the role of Raf1 in TGF-β1 signaling in human lung fibroblasts. GW5074 or downregulation of Raf1 by siRNAs significantly attenuated TGF-β1-induced smooth muscle actin, FN, and collagen I expression, whereas overexpression of Raf1 promoted the effects of TGF-β1 in lung fibroblasts. Furthermore, we found that Raf1-promoted TGF-β1 signaling was through the Raf1/ERK/Smad pathway and contributed to the cell proliferation and migration in human lung fibroblasts. This study provides preclinical and mechanistic evidence for development of Raf1 inhibitors as potential antifibrotic drugs for the treatment of IPF.

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Cell-surface phenotyping identifies CD36 and CD97 as novel markers of fibroblast quiescence in lung fibrosis

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.00439.2017 ◽

2018 ◽

Vol 315 (5) ◽

pp. L682-L696 ◽

Cited By ~ 3

Author(s):

Katharina Heinzelmann ◽

Mareike Lehmann ◽

Michael Gerckens ◽

Nina Noskovičová ◽

Marion Frankenberger ◽

...

Keyword(s):

Growth Factor ◽

Cell Surface ◽

Lung Tissue ◽

Lung Fibrosis ◽

Replicative Senescence ◽

Human Lung ◽

Lung Fibroblasts ◽

Surface Markers ◽

Human Lung Fibroblasts ◽

Extracellular Matrix Components

Fibroblasts play an important role in lung homeostasis and disease. In lung fibrosis, fibroblasts adopt a proliferative and migratory phenotype, with increased expression of α-smooth muscle actin (αSMA) and enhanced secretion of extracellular matrix components. Comprehensive profiling of fibroblast heterogeneity is limited because of a lack of specific cell-surface markers. We have previously profiled the surface proteome of primary human lung fibroblasts. Here, we sought to define and quantify a panel of cluster of differentiation (CD) markers in primary human lung fibroblasts and idiopathic pulmonary fibrosis (IPF) lung tissue, using immunofluorescence and FACS analysis. Fibroblast function was assessed by analysis of replicative senescence. We observed the presence of distinct fibroblast phenotypes in vivo, characterized by various combinations of Desmin, αSMA, CD36, or CD97 expression. Most markers demonstrated stable expression over passages in vitro, but significant changes were observed for CD36, CD54, CD82, CD106, and CD140a. Replicative senescence of fibroblasts was observed from passage 10 onward. CD36- and CD97-positive but αSMA-negative cells were present in remodeled areas of IPF lungs. Transforming growth factor (TGF)-β treatment induced αSMA and collagen I expression but repressed CD36 and CD97 expression. We identified a panel of stable surface markers in human lung fibroblasts, applicable for positive-cell isolation directly from lung tissue. TGF-β exposure represses CD36 and CD97 expression, despite increasing αSMA expression; we therefore identified complex surface protein changes during fibroblast-myofibroblast activation. Coexistence of quiescence and activated fibroblast subtypes in the IPF lung suggests dynamic remodeling of fibroblast activation upon subtle changes to growth factor exposure in local microenvironmental niches.

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