scholarly journals Wet-dry-wet drug screen leads to the synthesis of TS1, a novel compound reversing lung fibrosis through inhibition of myofibroblast differentiation

2021 ◽  
Vol 13 (1) ◽  
Author(s):  
Nadja Anneliese Ruth Ring ◽  
Maria Concetta Volpe ◽  
Tomaž Stepišnik ◽  
Maria Grazia Mamolo ◽  
Panče Panov ◽  
...  
Keyword(s):  
Dopamine Receptor ◽  
High Throughput ◽  
Lung Fibrosis ◽  
Transforming Growth Factor ◽  
Ex Vivo ◽  
Interdisciplinary Approach ◽  
Transforming Growth Factor Β ◽  
Lung Fibroblasts ◽  
Myofibroblast Differentiation ◽  
Primary Fibroblasts

SummaryTherapies halting the progression of fibrosis are ineffective and limited. Activated myofibroblasts are emerging as important targets in the progression of fibrotic diseases. Previously, we performed a high-throughput screen on lung fibroblasts and subsequently demonstrated that the inhibition of myofibroblast activation is able to prevent lung fibrosis in bleomycin-treated mice. High-throughput screens are an ideal method of repurposing drugs, yet they contain an intrinsic limitation, which is the size of the library itself. Here, we exploited the data from our “wet” screen and used “dry” machine learning analysis to virtually screen millions of compounds, identifying novel anti-fibrotic hits which target myofibroblast differentiation, many of which were structurally related to dopamine. We synthesized and validated several compounds ex vivo (“wet”) and confirmed that both dopamine and its derivative TS1 are powerful inhibitors of myofibroblast activation. We further used RNAi-mediated knock-down and demonstrated that both molecules act through the dopamine receptor 3 and exert their anti-fibrotic effect by inhibiting the canonical transforming growth factor β pathway. Furthermore, molecular modelling confirmed the capability of TS1 to bind both human and mouse dopamine receptor 3. The anti-fibrotic effect on human cells was confirmed using primary fibroblasts from idiopathic pulmonary fibrosis patients. Finally, TS1 prevented and reversed disease progression in a murine model of lung fibrosis. Both our interdisciplinary approach and our novel compound TS1 are promising tools for understanding and combating lung fibrosis.

scholarly journals Regulation of myofibroblast differentiation by cardiac glycosides

2016 ◽  
Vol 310 (9) ◽  
pp. L815-L823 ◽  
Author(s):  
Jennifer La ◽  
Eleanor B. Reed ◽  
Svetlana Koltsova ◽  
Olga Akimova ◽  
Robert B. Hamanaka ◽  
...  
Keyword(s):  
Cardiac Glycosides ◽  
Transforming Growth Factor ◽  
Serum Response Factor ◽  
Transforming Growth Factor Β ◽  
Fiber Formation ◽  
Lung Fibroblasts ◽  
Myofibroblast Differentiation ◽  
Fibrotic Diseases ◽  
Cox 2 ◽  
Serum Response

Myofibroblast differentiation is a key process in pathogenesis of fibrotic diseases. Cardiac glycosides (ouabain, digoxin) inhibit Na+-K+-ATPase, resulting in increased intracellular [Na+]-to-[K+] ratio in cells. Microarray analysis suggested that increased intracellular [Na+]/[K+] ratio may promote the expression of cyclooxygenase-2 (COX-2), a critical enzyme in the synthesis of prostaglandins. Given antifibrotic effects of prostaglandins through activation of protein kinase A (PKA), we examined if cardiac glycosides stimulate COX-2 expression in human lung fibroblasts and how they affect myofibroblast differentiation. Ouabain stimulated a profound COX-2 expression and a sustained PKA activation, which was blocked by COX-2 inhibitor or by COX-2 knockdown. Ouabain-induced COX-2 expression and PKA activation were abolished by the inhibitor of the Na+/Ca2+ exchanger, KB-R4943. Ouabain inhibited transforming growth factor-β (TGF-β)-induced Rho activation, stress fiber formation, serum response factor activation, and the expression of smooth muscle α-actin, collagen-1, and fibronectin. These effects were recapitulated by an increase in intracellular [Na+]/[K+] ratio through the treatment of cells with K+-free medium or with digoxin. Although inhibition of COX-2 or of the Na+/Ca2+ exchanger blocked ouabain-induced PKA activation, this failed to reverse the inhibition of TGF-β-induced Rho activation or myofibroblast differentiation by ouabain. Together, these data demonstrate that ouabain, through the increase in intracellular [Na+]/[K+] ratio, drives the induction of COX-2 expression and PKA activation, which is accompanied by a decreased Rho activation and myofibroblast differentiation in response to TGF-β. However, COX-2 expression and PKA activation are not sufficient for inhibition of the fibrotic effects of TGF-β by ouabain, suggesting that additional mechanisms must exist.

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

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.

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 Latent Transforming Growth Factor-β Binding Protein-2 Regulates Lung Fibroblast-to-Myofibroblast Differentiation in Pulmonary Fibrosis via NF-κB Signaling

2021 ◽  
Vol 12 ◽  
Author(s):  
Menglin Zou ◽  
Jingfeng Zou ◽  
Xingxing Hu ◽  
Weishuai Zheng ◽  
Mingyang Zhang ◽  
...  
Keyword(s):  
Growth Factor ◽  
Pulmonary Fibrosis ◽  
Transforming Growth Factor ◽  
Nuclear Translocation ◽  
Binding Protein ◽  
Critical Role ◽  
Transforming Growth Factor Β ◽  
Lung Fibroblasts ◽  
Myofibroblast Differentiation

Despite past extensive studies, the mechanisms underlying pulmonary fibrosis (PF) still remain poorly understood. The aberrantly activated lung myofibroblasts, predominantly emerging through fibroblast-to-myofibroblast differentiation, are considered to be the key cells in PF, resulting in excessive accumulation of extracellular matrix (ECM). Latent transforming growth factor-β (TGFβ) binding protein-2 (LTBP2) has been suggested as playing a critical role in modulating the structural integrity of the ECM. However, its function in PF remains unclear. Here, we demonstrated that lungs originating from different types of patients with PF, including idiopathic PF and rheumatoid arthritis-associated interstitial lung disease, and from mice following bleomycin (BLM)-induced PF were characterized by increased LTBP2 expression in activated lung fibroblasts/myofibroblasts. Moreover, serum LTBP2 was also elevated in patients with COVID-19-related PF. LTBP2 silencing by lentiviral shRNA transfection protected against BLM-induced PF and suppressed fibroblast-to-myofibroblast differentiation in vivo and in vitro. More importantly, LTBP2 overexpression was able to induce differentiation of lung fibroblasts to myofibroblasts in vitro, even in the absence of TGFβ1. By further mechanistic analysis, we demonstrated that LTBP2 silencing prevented fibroblast-to-myofibroblast differentiation and subsequent PF by suppressing the phosphorylation and nuclear translocation of NF-κB signaling. LTBP2 overexpression-induced fibroblast-to-myofibroblast differentiation depended on the activation of NF-κB signaling in vitro. Therefore, our data indicate that intervention to silence LTBP2 may represent a promising therapy for PF.

scholarly journals LncRNA SNHG16 promotes pulmonary fibrosis by targeting miR-455-3p to regulate the Notch2 pathway

2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Panpan Liu ◽  
Lei Zhao ◽  
Yuxia Gu ◽  
Meilan Zhang ◽  
Hongchang Gao ◽  
...  
Keyword(s):  
Pulmonary Fibrosis ◽  
Lung Fibrosis ◽  
Transforming Growth Factor ◽  
Transforming Growth Factor Β ◽  
Interstitial Lung Diseases ◽  
Luciferase Reporter ◽  
Qrt Pcr ◽  
Rna Immunoprecipitation ◽  
And Migration

Abstract Background Idiopathic pulmonary fibrosis (IPF) is the most common interstitial lung diseases with a poor prognosis. Long non-coding RNAs (lncRNAs) have been reported to be involved in IPF in several studies. However, the role of lncRNA SNHG16 in IPF is largely unknown. Methods Firstly, experimental pulmonary fibrosis model was established by using bleomycin (BML). Histology and Western blotting assays were used to determine the different stages of fibrosis and expression of several fibrosis biomarkers. The expression of SNHG16 was detected by quantitative real-time polymerase chain reaction (qRT‐PCR). EdU staining and wound-healing assay were utilized to analyze proliferation and migration of lung fibroblast cells. Molecular mechanism of SNHG16 was explored by bioinformatics, dual-luciferase reporter assay, RNA immunoprecipitation assay (RIP), and qRT-PCR. Results The expression of SNHG16 was significantly up-regulated in bleomycin-(BLM) induced lung fibrosis and transforming growth factor-β (TGF-β)-induced fibroblast. Knockdown of SNHG16 could attenuate fibrogenesis. Mechanistically, SNHG16 was able to bind and regulate the expression of miR-455-3p. Moreover, SNHG16 also regulated the expression of Notch2 by targeting miR-455-3p. Finally, SNHG16 could promote fibrogenesis by regulating the expression of Notch2. Conclusion Taken together, our study demonstrated that SNHG16 promoted pulmonary fibrosis by targeting miR-455-3p to regulate the Notch2 pathway. These findings might provide a novel insight into pathologic process of lung fibrosis and may provide prevention strategies in the future.

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.

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