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

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.

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Regorafenib-Attenuated, Bleomycin-Induced Pulmonary Fibrosis by Inhibiting the TGF-β1 Signaling Pathway

International Journal of Molecular Sciences ◽

10.3390/ijms22041985 ◽

2021 ◽

Vol 22 (4) ◽

pp. 1985

Author(s):

Xiaohe Li ◽

Ling Ma ◽

Kai Huang ◽

Yuli Wei ◽

Shida Long ◽

...

Keyword(s):

Pulmonary Fibrosis ◽

Signaling Pathway ◽

Transforming Growth Factor ◽

Kinase Inhibitor ◽

In Vivo Experiments ◽

Age Related ◽

And Migration ◽

Tgf Β1

Idiopathic pulmonary fibrosis (IPF) is a fatal and age-related pulmonary disease. Nintedanib is a receptor tyrosine kinase inhibitor, and one of the only two listed drugs against IPF. Regorafenib is a novel, orally active, multi-kinase inhibitor that has similar targets to nintedanib and is applied to treat colorectal cancer and gastrointestinal stromal tumors in patients. In this study, we first identified that regorafenib could alleviate bleomycin-induced pulmonary fibrosis in mice. The in vivo experiments indicated that regorafenib suppresses collagen accumulation and myofibroblast activation. Further in vitro mechanism studies showed that regorafenib inhibits the activation and migration of myofibroblasts and extracellular matrix production, mainly through suppressing the transforming growth factor (TGF)-β1/Smad and non-Smad signaling pathways. In vitro studies have also indicated that regorafenib could augment autophagy in myofibroblasts by suppressing TGF-β1/mTOR (mechanistic target of rapamycin) signaling, and could promote apoptosis in myofibroblasts. In conclusion, regorafenib attenuates bleomycin-induced pulmonary fibrosis by suppressing the TGF-β1 signaling pathway.

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Alcohol induces TGFβ1 via downregulation of miR-1946a in murine lung fibroblast

Scientific Reports ◽

10.1038/s41598-020-76148-5 ◽

2020 ◽

Vol 10 (1) ◽

Author(s):

Xian Fan ◽

Stephen T. Mills ◽

Mevelyn J. Kaalla ◽

Viranuj Sueblinvong

Keyword(s):

Protein Expression ◽

Transforming Growth Factor Beta ◽

Transforming Growth Factor ◽

Smooth Muscle Actin ◽

Direct Interaction ◽

Lung Fibroblasts ◽

Murine Lung ◽

Gene And Protein Expression

Abstract Exaggerated transforming growth factor-beta 1 (TGFβ1) expression worsens fibroproliferation following bleomycin-induced lung injury in alcohol-fed mice. MicroRNA (miR)-1946a is predicted to bind to the TGFβ1 3′ untranslated region (UTR), thereby inhibiting its transcription. We hypothesize that alcohol suppresses miR-1946a and induces TGFβ1. Primary murine lung fibroblasts (PLFs) were cultured ± alcohol, miR-1946a mimic or inhibitor, and TGFβ1 signaling inhibitors. miR-1946a was analyzed after alcohol treatment in vitro and in vivo. TGFβ1 expression and TGFβ1 3′UTR-luciferase activity was quantified. We showed that alcohol suppressed miR-1946a in the alcohol-fed mouse lungs and PLFs. MiR-1946a inhibitor increased TGFβ1 expression in the fibroblast. MiR-1946a mimic treatment suppressed TGFβ1 gene expression and TGFβ1 3′UTR activity. Overexpression of miR1946a inhibited alcohol-induced TGFβ1 gene and protein expression as well as alcohol-induced TGFβ1 and α-smooth muscle actin (SMA) protein expression in PLFs. In conclusion, miR-1946a modulates TGFβ1 expression through direct interaction with TGFβ1 3′UTR. These findings identify a novel mechanism by which alcohol induces TGFβ1 in the lung.

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Elevated expression of NEU1 sialidase in idiopathic pulmonary fibrosis provokes pulmonary collagen deposition, lymphocytosis, and fibrosis

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.00346.2015 ◽

2016 ◽

Vol 310 (10) ◽

pp. L940-L954 ◽

Cited By ~ 15

Author(s):

Irina G. Luzina ◽

Virginia Lockatell ◽

Sang W. Hyun ◽

Pavel Kopach ◽

Phillip H. Kang ◽

...

Keyword(s):

Gene Expression ◽

Endothelial Cells ◽

Idiopathic Pulmonary Fibrosis ◽

Pulmonary Fibrosis ◽

Molecular Mechanisms ◽

Transforming Growth Factor ◽

Recombinant Adenovirus ◽

Global Gene Expression

Idiopathic pulmonary fibrosis (IPF) poses challenges to understanding its underlying cellular and molecular mechanisms and the development of better therapies. Previous studies suggest a pathophysiological role for neuraminidase 1 (NEU1), an enzyme that removes terminal sialic acid from glycoproteins. We observed increased NEU1 expression in epithelial and endothelial cells, as well as fibroblasts, in the lungs of patients with IPF compared with healthy control lungs. Recombinant adenovirus-mediated gene delivery of NEU1 to cultured primary human cells elicited profound changes in cellular phenotypes. Small airway epithelial cell migration was impaired in wounding assays, whereas, in pulmonary microvascular endothelial cells, NEU1 overexpression strongly impacted global gene expression, increased T cell adhesion to endothelial monolayers, and disrupted endothelial capillary-like tube formation. NEU1 overexpression in fibroblasts provoked increased levels of collagen types I and III, substantial changes in global gene expression, and accelerated degradation of matrix metalloproteinase-14. Intratracheal instillation of NEU1 encoding, but not control adenovirus, induced lymphocyte accumulation in bronchoalveolar lavage samples and lung tissues and elevations of pulmonary transforming growth factor-β and collagen. The lymphocytes were predominantly T cells, with CD8+ cells exceeding CD4+ cells by nearly twofold. These combined data indicate that elevated NEU1 expression alters functional activities of distinct lung cell types in vitro and recapitulates lymphocytic infiltration and collagen accumulation in vivo, consistent with mechanisms implicated in lung fibrosis.

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The Protective Effect of Naringenin on Airway Remodeling after Mycoplasma Pneumoniae Infection by Inhibiting Autophagy-Mediated Lung Inflammation and Fibrosis

Mediators of Inflammation ◽

10.1155/2018/8753894 ◽

2018 ◽

Vol 2018 ◽

pp. 1-10 ◽

Cited By ~ 12

Author(s):

Yan Lin ◽

Dan Tan ◽

Qianna Kan ◽

Zhen Xiao ◽

Zhiyan Jiang

Keyword(s):

Pulmonary Fibrosis ◽

Therapeutic Effect ◽

Mycoplasma Pneumoniae ◽

Transforming Growth Factor Beta ◽

Transforming Growth Factor ◽

Airway Remodeling ◽

Enzyme Linked Immunosorbent Assay ◽

The Relationship

Our previous study has shown that Chinese medicine, Qingfei Tongluo formula (QTF), has a significantly therapeutic effect to Mycoplasma pneumoniae (MP) pneumonia (MPP). The aim of this study was to investigate the therapeutic effect and mechanism of naringenin (NRG) on MPP which was an important component of QTF. Here, we studied 124 children with or without MPP and compared inflammatory cytokines and fibrinogen-related protein expression with enzyme-linked immunosorbent assay. We also employed a BALB/c mouse model of MPP and divided the mice into three groups: ctrl (normal control mice), MPP (MP-infected mice), and MPP + NRG (MP-infected mice treated with NRG). BEAS-2B cells were used to confirm the relationship between autophagy, inflammation, and fibrosis. The results show proinflammatory cytokines (interleukin- [IL-] 6, IL-1β, and tumor necrosis factor-α), and transforming growth factor beta (TGF-β) expression was significantly increased after MP infection from both clinical and animal experiment. In vivo experimental confirmation showed that NRG treatment decreased MPP-induced lung injury in mice by inhibiting autophagy-mediated inflammatory cytokine expression and pulmonary fibrosis. In vitro experiments confirmed it. These results indicate that NRG treatment suppressed the inflammatory response and pulmonary fibrosis by inhibition of autophagy after MP infection.

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Kasugamycin is a novel chitinase 1 inhibitor with strong antifibrotic effects on pulmonary fibrosis

10.1101/2021.02.25.432796 ◽

2021 ◽

Author(s):

Jae-Hyun Lee ◽

Chang-Min Lee ◽

Mun-Ock Kim ◽

Jin Wook Park ◽

Suchitra Kamle ◽

...

Keyword(s):

Growth Factor ◽

Pulmonary Fibrosis ◽

Transforming Growth Factor Beta ◽

Transforming Growth Factor ◽

Glycosyl Hydrolase ◽

Aminoglycoside Antibiotic ◽

Therapeutic Drug ◽

Antifibrotic Effect

Rationale: Pulmonary fibrosis is a devastating lung disease with few therapeutic options. Chitinase 1 (CHIT1), an 18 glycosyl hydrolase family member, contributes to the pathogenesis of pulmonary fibrosis through regulation of Transforming Growth Factor (TGF)-β signaling and effector function. Therefore, CHIT1 is a potential therapeutic target of pulmonary fibrosis. Objectives: This study aimed to identify and characterize a druggable CHIT1 inhibitor with strong antifibrotic activity and minimal toxicity for therapeutic application to pulmonary fibrosis. Methods: Extensive screening of small molecule libraries identified the aminoglycoside antibiotic Kasugamycin as a potent CHIT1 inhibitor. Measurements and Main Results: Elevated levels of CHIT1 were detected in the lungs of patients with pulmonary fibrosis. In vivo bleomycin- and TGF-β-stimulated murine models of pulmonary fibrosis, Kasugamycin showed impressive anti-fibrotic effects in both preventive and therapeutic conditions. In vitro studies also demonstrated that Kasugamycin inhibits fibrotic macrophage activation, fibroblast proliferation and myofibroblast transformation. Null mutation of transforming growth factor beta associated protein 1 (TGFBRAP1), a recently identified CHIT1 interacting signaling molecule, phenocopied antifibrotic effects of Kasugamycin in in vivo lungs and in vitro fibroblasts responses. Kasugamycin inhibits physical association between CHIT1 and TGFBRAP1 with decreased levels of SMAD4 association, suggesting that antifibrotic effect of Kasugamycin is mediated through regulation of TGFBRAP1, at least in part. Conclusions: These studies demonstrate that Kasugamycin is a novel CHIT1 inhibitor with strong antifibrotic effect that can be further developed as an effective and safe therapeutic drug for pulmonary fibrosis.

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Nifuroxazide Ameliorates Pulmonary Fibrosis By Blocking Myofibroblast Genesis And Stat3 Activation: A Drug Repurposing Study

10.21203/rs.3.rs-557091/v1 ◽

2021 ◽

Author(s):

Cailing Gan ◽

Qianyu Zhang ◽

Hongyao Liu ◽

Guan Wang ◽

Liqun Wang ◽

...

Keyword(s):

Epithelial Cells ◽

Pulmonary Fibrosis ◽

Western Blot ◽

Immune Cells ◽

Transforming Growth Factor ◽

Lung Fibroblasts ◽

Mesenchymal Transition ◽

Tgf Β1

Abstract Background: Idiopathic pulmonary fibrosis (IPF) is a lung disease with complex pathogenesis, high mortality. The development of new drugs is time-consuming and laborious, and the research on new use of old drugs can save time and clinical costs and even avoid serious side effects. Nifuroxazide (NIF) was originally used to treat diarrhoea, but in recent years it has been found to have other pharmacological effects such as anti-tumor and inhibiting inflammatory diseases related to diabetic nephropathy. However, there are no reports about its role in pulmonary fibrosis.Methods: The therapeutic effect of NIF on bleomycin (BLM)-induced pulmonary fibrosis in vivo was measured by ELISA, hydroxyproline content, H&E and Masson staining, IHC and Western blot. The content of immune cells in lung tissue was analyzed by flow cytometry. NIF cytotoxicity were evaluated in NIH/3T3, Human pulmonary fibroblasts (HPF), A549 and Rat primary lung fibroblasts (RPLF) using MTT assay. Finally, a cell model induced by transforming growth factor-β1 (TGF-β1) stimulation and different in vitro experiments (Immunofluorescence, Western blot, Wound migration assay) were conducted to determine the effect of NIF on the activation of fibroblasts and the epithelial-mesenchymal transition (EMT) and migration of epithelial cells.Results: In vivo, intraperitoneal injection of NIF relieved and reversed pulmonary fibrosis caused by BLM bronchial instillation. In addition, nifuroxazide inhibited the expression of a variety of cellular inflammatory factors and immune cells. Furthermore, nifuroxazide suppressed the activation of fibroblasts and the EMT of epithelial cells induced by TGF-β1. Most importantly, we used an analytical docking experiment and thermal shift assay to further verify that nifuroxazide worked in conjunction with Stat3. Moreover, nifuroxazide decreased the expression of p-Stat3 in vitro and in vivo.Conclusion: These results suggest that NIF inhibits and reverses pulmonary fibrosis and support NIF as a viable treatment option that may bring benefits to patients with IPF.

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Caveolin-1–derived peptide limits development of pulmonary fibrosis

Science Translational Medicine ◽

10.1126/scitranslmed.aat2848 ◽

2019 ◽

Vol 11 (522) ◽

pp. eaat2848 ◽

Cited By ~ 10

Author(s):

Amarnath Satheesh Marudamuthu ◽

Yashodhar Prabhakar Bhandary ◽

Liang Fan ◽

Vijay Radhakrishnan ◽

BreAnne MacKenzie ◽

...

Keyword(s):

Pulmonary Fibrosis ◽

Transforming Growth Factor ◽

Human Peripheral Blood ◽

Transforming Growth Factor Β1 ◽

Interstitial Lung Diseases ◽

Full Length ◽

Lung Fibroblasts ◽

Caveolin 1

Idiopathic pulmonary fibrosis (IPF) is a fatal fibrotic lung disease with a median 5-year survival of ~20%. Current U.S. Food and Drug Administration–approved pharmacotherapies slow progression of IPF, providing hope that even more effective treatments can be developed. Alveolar epithelial progenitor type II cell (AEC) apoptosis and proliferation, and accumulation of activated myofibroblasts or fibrotic lung fibroblasts (fLfs) contribute to the progression of IPF. Full-length caveolin-1 scaffolding domain peptide (CSP; amino acids 82 to 101 of Cav1: DGIWKASFTTFTVTKYWFYR) inhibits AEC apoptosis and fLf activation and expansion and attenuates PF in bleomycin (BLM)–induced lung injury in mice. Like full-length CSP, a seven–amino acid deletion fragment of CSP, CSP7 (FTTFTVT), demonstrated antifibrotic effects in murine models of lung fibrosis. When CSP7 was administered during the fibrotic phase in three preclinical models [single-dose BLM, repeated-dose BLM, and adenovirus expressing constitutively active transforming growth factor–β1 (Ad-TGF-β1)–induced established PF], CSP7 reduced extracellular matrix (ECM) markers characteristic of PF, increased AEC survival, and improved lung function. CSP7 is amenable to both systemic (intraperitoneal) or direct lung delivery in a nebulized or dry powder form. Furthermore, CSP7 treatment of end-stage human IPF lung tissue explants attenuated ECM production and promoted AEC survival. Ames testing for mutagenicity and in vitro human peripheral blood lymphocyte and in vivo mouse micronucleus transformation assays indicated that CSP7 is not carcinogenic. Together, these findings support the further development of CSP7 as an antifibrotic treatment for patients with IPF or other interstitial lung diseases.

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

10.1101/2021.01.15.425870 ◽

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.

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Transforming growth factor beta 1 (TGF-beta 1) induced neutrophil recruitment to synovial tissues: implications for TGF-beta-driven synovial inflammation and hyperplasia.

Journal of Experimental Medicine ◽

10.1084/jem.173.5.1121 ◽

1991 ◽

Vol 173 (5) ◽

pp. 1121-1132 ◽

Cited By ~ 129

Author(s):

R A Fava ◽

N J Olsen ◽

A E Postlethwaite ◽

K N Broadley ◽

J M Davidson ◽

...

Keyword(s):

Growth Factor ◽

Synovial Tissue ◽

Transforming Growth Factor Beta ◽

Transforming Growth Factor ◽

Biologically Active ◽

Tgf Beta ◽

Histological Techniques ◽

Growth Factor Beta

We have studied the consequences of introducing human recombinant transforming growth factor beta 1 (hrTGF-beta 1) into synovial tissue of the rat, to begin to better understand the significance of the fact that biologically active TGF-beta is found in human arthritic synovial effusions. Within 4-6 h after the intra-articular injection of 1 microgram of hrTGF-beta 1 into rat knee joints, extensive recruitment of polymorphonuclear leukocytes (PMNs) was observed. Cytochemistry and high resolution histological techniques were used to quantitate the influx of PMNs, which peaked 6 h post-injection. In a Boyden chamber assay, hrTGF-beta 1 at 1-10 fg/ml elicited a chemotactic response from PMNs greater in magnitude than that evoked by FMLP, establishing that TGF-beta 1 is an effective chemotactic agent for PMNs in vitro as well as in vivo. That PMNs may represent an important source of TGF-beta in inflammatory infiltrates was strongly suggested by a demonstration that stored TGF-beta 1 was secreted during phorbol myristate acetate-stimulated degranulation in vitro. Acid/ethanol extracts of human PMNs assayed by ELISA contained an average of 355 ng of TGF/beta 1 per 10(9) cells potentially available for secretion during degranulation of PMNs. [3H]Thymidine incorporation in vivo and autoradiography of tissue sections revealed that widespread cell proliferation was triggered by TGF-beta 1 injection. Synovial lining cells and cells located deep within the subsynovial connective tissue were identified as sources of at least some of the new cells that contribute to TGF-beta 1-induced hyperplasia. Our results demonstrate that TGF-beta is capable of exerting pathogenic effects on synovial tissue and that PMNs may represent a significant source of the TGF-beta present in synovial effusions.

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An In Vitro Model for Assessing Corneal Keratocyte Spreading and Migration on Aligned Fibrillar Collagen

Journal of Functional Biomaterials ◽

10.3390/jfb9040054 ◽

2018 ◽

Vol 9 (4) ◽

pp. 54 ◽

Cited By ~ 7

Author(s):

Pouriska Kivanany ◽

Kyle Grose ◽

Nihan Yonet-Tanyeri ◽

Sujal Manohar ◽

Yukta Sunkara ◽

...

Keyword(s):

Growth Factor ◽

Transforming Growth Factor Beta ◽

Transforming Growth Factor ◽

Cell Movement ◽

Time Lapse ◽

Collagen Fibrils ◽

Fibrillar Collagen ◽

Freeze Injury

Background: Corneal stromal cells (keratocytes) are responsible for developing and maintaining normal corneal structure and transparency, and for repairing the tissue after injury. Corneal keratocytes reside between highly aligned collagen lamellae in vivo. In addition to growth factors and other soluble biochemical factors, feedback from the extracellular matrix (ECM) itself has been shown to modulate corneal keratocyte behavior. Methods: In this study, we fabricate aligned collagen substrates using a microfluidics approach and assess their impact on corneal keratocyte morphology, cytoskeletal organization, and patterning after stimulation with platelet derived growth factor (PDGF) or transforming growth factor beta 1 (TGFβ). We also use time-lapse imaging to visualize the dynamic interactions between cells and fibrillar collagen during wound repopulation following an in vitro freeze injury. Results: Significant co-alignment between keratocytes and aligned collagen fibrils was detected, and the degree of cell/ECM co-alignment further increased in the presence of PDGF or TGFβ. Freeze injury produced an area of cell death without disrupting the collagen. High magnification, time-lapse differential interference contrast (DIC) imaging allowed cell movement and subcellular interactions with the underlying collagen fibrils to be directly visualized. Conclusions: With continued development, this experimental model could be an important tool for accessing how the integration of multiple biophysical and biochemical signals regulate corneal keratocyte differentiation.

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