4-(5-benzo(1-3)dioxol-5-yl-4-pyridin-2-yl-1h-imidazol-2-yl)benzamide and Pulmonary-Fibrosis

Pulmonary fibrosis is a chronic progressive fibrotic interstitial lung disease characterized by excessive extracellular matrix (ECM) deposition caused by activated fibroblasts. Increasing evidence shows that matrix stiffness is essential in promoting fibroblast activation and profibrotic changes. Here, we investigated the expression and function of matrix stiffness-regulated ZNF416 in pulmonary fibrotic lung fibroblasts.. 1 kappa (soft), 60 kappa (stiff) gel-coated coverslips, or transforming growth factor-beta 1 (TGF-β1)-cultured lung fibroblasts and the gain- or loss- of the ZNF416 function assays were performed in vitro. We also established two experimental pulmonary fibrosis mouse models by a single intratracheal instillation with 50 mg/kg silica or 6 mg/kg bleomycin (BLM). ZNF416 siRNA-loaded liposomes and TGF-β1 receptor inhibitor SB431542 were administrated in vivo.. Our study identified that ZNF416 could regulate fibroblast differentiation, proliferation, and contraction by promoting the nuclear accumulation of p-Smad2/3. Besides, ZNF416 siRNA-loaded liposome delivery by tail-vein could passively target the fibrotic area in the lung, and co-administration of ZNF416 siRNA-loaded liposomes and SB431542 significantly protects mice against silica or BLM-induced lung injury and fibrosis.. In this study, our results indicate that mechanosensitive ZNF416 is a potential molecular target for the treatment of pulmonary fibrosis. Strategies aimed at silencing ZNF416 could be a promising approach to fight against pulmonary fibrosis.

Topics: Animals; Bleomycin; Fibroblasts; Liposomes; Lung; Mice; Mice, Inbred C57BL; Pulmonary Fibrosis; RNA, Small Interfering; Silicon Dioxide; Transforming Growth Factor beta1

Idiopathic pulmonary fibrosis (IPF) is a specific form of chronic, progressive and fibrosing interstitial pneumonia of unknown cause. The main feature of IPF is a heterogeneous appearance with areas of sub-pleural fibrosis. However, the mechanism of sub-pleural fibrosis was poorly understood. In this study, our in vivo study revealed that pleural mesothelial cells (PMCs) migrated into lung parenchyma and localized alongside lung fibroblasts in sub-pleural area in mouse pulmonary fibrosis. Our in vitro study displayed that cultured-PMCs-medium induced lung fibroblasts transforming into myofibroblast, cultured-fibroblasts-medium promoted mesothelial-mesenchymal transition of PMCs. Furthermore, these changes in lung fibroblasts and PMCs were prevented by blocking TGF-β1/Smad2/3 signaling with SB431542. TGF-β1 neutralized antibody attenuated bleomycin-induced pulmonary fibrosis. Similar to TGF-β1/Smad2/3 signaling, wnt/β-catenin signaling was also activated in the process of PMCs crosstalk with lung fibroblasts. Moreover, inhibition of CD147 attenuated cultured-PMCs-medium induced collagen-I synthesis in lung fibroblasts. Blocking CD147 signaling also prevented bleomycin-induced pulmonary fibrosis. Our data indicated that crosstalk between PMC and lung fibroblast contributed to sub-pleural pulmonary fibrosis. TGF-β1, Wnt/β-catenin and CD147 signaling was involved in the underling mechanism.

Topics: Animals; Benzamides; Cell Movement; Dioxoles; Disease Models, Animal; Epithelial Cells; Epithelium; Fibroblasts; Gene Expression Regulation; Humans; Lung; Mice; Pleura; Pulmonary Fibrosis; Signal Transduction; Smad2 Protein; Transforming Growth Factor beta1

Hydroxysafflor yellow A (HSYA) is one of the chemical component isolated from Chinese medicine Carthamus tinctorius L. Our preliminary study confirmed that HSYA attenuated bleomycin-induced pulmonary fibrosis in mice. In this study, we evaluated the effect of HSYA on TGF-β1-induced activation of human fetal lung fibroblasts (MRC-5) and explored the underlying mechanisms of its activity.. MRC-5 cells activated by TGF-β1 were incubated with HSYA and/or the TGF-β type I receptor inhibitor, SB431542. TGF-β1-induced cell proliferation, α-smooth muscle actin, collagen I alpha 1 and fibronectin expression, Smad, mitogen-activated protein kinase (MAPK) and phosphatidylinositol-3 kinase/Akt signalling pathway activation were observed.. Hydroxysafflor yellow A significantly inhibited TGF-β1-induced cell proliferation and the expression, both mRNA and protein, of α-smooth muscle actin, collagen I alpha 1 and fibronectin. HSYA also suppressed TGF-β1 activation of Smad signal transduction via inhibition of Smad2 and Smad3 phosphorylation, their nuclear translocation and the binding activity of Smad3 to type I collagen promoter in MRC-5 cells. In addition, HSYA inhibited TGF-β1-induced phosphorylation of extracellular signal-regulated kinase (ERK). The inhibitory effects of HSYA were similar to SB431542.. These findings suggest that HSYA inhibits TGF-β1-induced activation of MRC-5 cells associated with TGF-β1/Smad and ERK/MAPK signalling pathways.

Topics: Actins; Benzamides; Cell Proliferation; Cells, Cultured; Chalcone; Collagen Type I; Dioxoles; Extracellular Signal-Regulated MAP Kinases; Fibroblasts; Fibronectins; Humans; Lung; Protein Serine-Threonine Kinases; Pulmonary Fibrosis; Quinones; Receptor, Transforming Growth Factor-beta Type I; Receptors, Transforming Growth Factor beta; Signal Transduction; Smad2 Protein; Smad3 Protein; Transforming Growth Factor beta1

To investigate the effect of fibroblasts on regulating airway stem cell proliferation in idiopathic pulmonary fibrosis.. Lung cell suspension was prepared from β-actin-GFP mice. Airway stem cells were obtained by fluorescence activated cell sorting and co-cultured with lung fibroblasts. The fibroblasts were treated with TGF-β inhibitor SB43142. The expression of growth factors FGF1/2 and the effect of FGF1/2 on stem cell proliferation were observed.. The cloning efficiency of airway stem cells, when co-cultured with normal lung fibroblast cells for 8 days, was (3.5±1.1)%, while the cloning efficiency was reduced to (0.04±0.04)% when co-cultured with lung fibroblasts from idiopathic pulmonary fibrosis patients. The difference between the 2 groups was statistically significant(P=0.002 5). TGF-β receptor inhibitor SB431542 increased lung fibroblast growth factors FGF1/2 expression.FGF1 mRNA expression was increased to the experimental group 0.005 5 from 0.000 2 in the control group.FGF2 mRNA expression of the amount raised to the experimental group 0.000 15 from 0.000 8 in the control group.FGF1/2 promoted the growth of airway stem cells. After FGF1/2 was co-cultured with normal lung fibroblast cells for 8 days, the cloning efficiency of airway stem cells was (0.3±0.1)%.. During the development of idiopathic pulmonary fibrosis, fibroblast secreted FGF1/2 regulate airway stem cell proliferation.

Topics: Actins; Animals; Benzamides; Cell Movement; Cell Proliferation; Cells, Cultured; Dioxoles; Fibroblast Growth Factor 1; Fibroblast Growth Factor 2; Fibroblasts; Gene Expression Regulation; Humans; Idiopathic Pulmonary Fibrosis; Lung; Mice; MicroRNAs; Protein Serine-Threonine Kinases; Pulmonary Fibrosis; Receptor, Transforming Growth Factor-beta Type II; Receptors, Fibroblast Growth Factor; Receptors, Transforming Growth Factor beta; Transforming Growth Factor beta

Idiopathic pulmonary fibrosis is a chronic pulmonary disease that is characterized by formation of scar tissue in lungs. Transforming growth factor-β (TGF-β) is considered an important cytokine in the pathogenesis of this disease. Hence, the antifibrotic effect of an inhibitor of the TGF-β type I receptor, namely, SB 431542, was investigated in our study. SB 431542 was used to treat TGF-β-treated IMR-90 cells; the expression of α-smooth muscle actin (α-SMA) was detected at the protein level by using an anti-α-SMA antibody, and at the gene level by reverse transcription-quantitative PCR. The effect of the inhibitor on cell proliferation was determined by a cell growth assay. The inhibitor was also administered into bleomycin-treated mice. Histopathological assessment and determination of total collagen levels were carried out to evaluate the severity of lung fibrosis in these mice. Our results demonstrated that treatment with SB 431542 inhibits TGF-β‑induced α-SMA expression in lung fibroblasts, at both the protein and the mRNA levels (P<0.05). However, the inhibitor did not significantly reduce lung fibroblast proliferation. In the bleomycin-induced pulmonary fibrosis mouse model, bleomycin treatment caused important morphological changes, accompanied by an increase in the collagen level of the lungs. Early treatment with SB 431542 prevented the manifestation of histopathological alterations, whereas delayed treatment significantly decreased the collagen level (P<0.05). These results suggest that inhibition of TGF-β signaling, via inhibition of the activin receptor-like kinase-5 (ALK-5) by SB 431542, may attenuate pulmonary fibrosis.

Topics: Actins; Animals; Benzamides; Cell Line; Cell Proliferation; Cell Survival; Dioxoles; Disease Models, Animal; Female; Fibroblasts; Humans; Hydroxyproline; Mice; Protein Serine-Threonine Kinases; Pulmonary Fibrosis; Receptor, Transforming Growth Factor-beta Type I; Receptors, Transforming Growth Factor beta; Transforming Growth Factor beta

Epithelial-mesenchymal transition (EMT) has been considered to be involved in idiopathic pulmonary fibrosis (IPF). However, the EMT process in vivo is much more complex and controversial. Studies regarding the opposite process, mesenchymal-epithelial transition (MET) in fibroblasts, are limited. Therefore, the aim of this study was to verify the involvement of the transforming growth factor (TGF)-β1-dependent EMT network in the process of pulmonary fibrosis and to explore the possibility of MET. Fibrotic lung tissues were obtained from patients with IPF with histological evidence of usual interstitial pneumonia at the time of surgical lung biopsy. For the controls, histologically normal lung tissues were obtained from patients with primary spontaneous pneumothorax at the time of thoracoscopy with stapling of any air leaks. Real-time RT-PCR and western blot analysis revealed that the mRNA and protein levels of TGF-β1, TGF-β1 receptor type I/II/III (TβRI/II/III), Smad2/3/4 and Snail1/2 were significantly upregulated in the fibrotic lung tissue. Inhibitors of various kinases implicated in EMT, including TGF-β1/Smad, Rho kinase (ROCK), p38 mitogen-activated protein kinase (p38 MAPK) and c-Jun NH-terminal kinase (JNK) were used to determine the MET potential in fibroblasts from fibrotic lung tissue. Western blot analysis or indirect immunofluorescence staining revealed that Smad inhibitor, as well as other kinase inhibitors failed to induce the MET process, determined by cellular morphology and protein markers. Our data suggest that the MET process may not be the exact reversal of EMT. In addition to using kinase inhibitors, other intervention measures should be used to explore the possibility of the MET process in fibroblasts from fibrotic lung tissue.

Topics: Adult; Benzamides; Case-Control Studies; Cell Culture Techniques; Dioxoles; Epithelial-Mesenchymal Transition; Fibroblasts; Gene Expression Regulation; Humans; Idiopathic Pulmonary Fibrosis; JNK Mitogen-Activated Protein Kinases; Male; Middle Aged; p38 Mitogen-Activated Protein Kinases; Protein Kinase Inhibitors; Pulmonary Fibrosis; Receptors, Transforming Growth Factor beta; rho-Associated Kinases; RNA, Messenger; Transforming Growth Factor beta1; Young Adult

Evidence suggests epithelial-mesenchymal transition (EMT) as one potential source of fibroblasts in idiopathic pulmonary fibrosis. To assess the contribution of alveolar epithelial cell (AEC) EMT to fibroblast accumulation in vivo following lung injury and the influence of extracellular matrix on AEC phenotype in vitro, Nkx2.1-Cre;mT/mG mice were generated in which AECs permanently express green fluorescent protein (GFP). On days 17-21 following intratracheal bleomycin administration, ~4% of GFP-positive epithelial-derived cells expressed vimentin or α-smooth muscle actin (α-SMA). Primary AECs from Nkx2.1-Cre;mT/mG mice cultured on laminin-5 or fibronectin maintained an epithelial phenotype. In contrast, on type I collagen, cells of epithelial origin displayed nuclear localization of Smad3, acquired spindle-shaped morphology, expressed α-SMA and phospho-Smad3, consistent with activation of the transforming growth factor-β (TGFβ) signalling pathway and EMT. α-SMA induction and Smad3 nuclear localization were blocked by the TGFβ type I receptor (TβRI, otherwise known as Alk5) inhibitor SB431542, while AEC derived from Nkx2.1-Cre;Alk5(flox/KO) mice did not undergo EMT on collagen, consistent with a requirement for signalling via Alk5 in collagen-induced EMT. Inability of a pan-specific TGFβ neutralizing antibody to inhibit effects of collagen together with absence of active TGFβ in culture supernatants is consistent with TGFβ ligand-independent activation of Smad signalling. These results support the notion that AECs can acquire a mesenchymal phenotype following injury in vivo and implicate type I collagen as a key regulator of EMT in AECs through signalling via Alk5, likely in a TGFβ ligand-independent manner.

Topics: Actins; Alveolar Epithelial Cells; Angiotensin II; Animals; Antibiotics, Antineoplastic; Benzamides; Bleomycin; Cells, Cultured; Collagen Type I; Dioxoles; Disease Models, Animal; Epithelial-Mesenchymal Transition; Female; Ligands; Male; Mice; Mice, Knockout; Protein Serine-Threonine Kinases; Pulmonary Fibrosis; Receptor, Transforming Growth Factor-beta Type I; Receptors, Transforming Growth Factor beta; Signal Transduction; Vimentin

Hypoxia-inducible factor-1α (HIF-1α), a transcription factor that functions as a master regulator of oxygen homeostasis, has been implicated in fibrinogenesis. Here, we explore the role of HIF-1α in transforming growth factor-β (TGF-β) signaling by examining the effects of TGF-β(1) on the expression of plasminogen activator inhibitor-1 (PAI-1). Immunohistochemistry of lung tissue from a mouse bleomycin (BLM)-induced pulmonary fibrosis model revealed that expression of HIF-1α and PAI-1 was predominantly induced in alveolar macrophages. Real-time RT-PCR and ELISA analysis showed that PAI-1 mRNA and activated PAI-1 protein level were strongly induced 7 days after BLM instillation. Stimulation of cultured mouse alveolar macrophages (MH-S cells) with TGF-β(1) induced PAI-1 production, which was associated with HIF-1α protein accumulation. This accumulation of HIF-1α protein was inhibited by SB431542 (type I TGF-β receptor/ALK receptor inhibitor) but not by PD98059 (MEK1 inhibitor) and SB203580 (p38 MAP kinase inhibitor). Expression of prolyl-hydroxylase domain (PHD)-2, which is essential for HIF-1α degradation, was inhibited by TGF-β(1), and this decrease was abolished by SB431542. TGF-β(1) induction of PAI-1 mRNA and its protein expression were significantly attenuated by HIF-1α silencing. Transcriptome analysis by cDNA microarray of MH-S cells after HIF-1α silencing uncovered several pro-fibrotic genes whose regulation by TGF-β(1) required HIF-1α, including platelet-derived growth factor-A. Taken together, these findings expand our concept of the role of HIF-1α in pulmonary fibrosis in mediating the effects of TGF-β(1) on the expression of the pro-fibrotic genes in activated alveolar macrophages.

Topics: Animals; Benzamides; Bleomycin; Cell Hypoxia; Dioxoles; Hypoxia-Inducible Factor 1, alpha Subunit; Macrophages, Alveolar; Male; Mice; Mice, Inbred C57BL; Plasminogen Activator Inhibitor 1; Pulmonary Fibrosis; Transforming Growth Factor beta