cAMP-elevating agents and adenylyl cyclase overexpression promote an antifibrotic phenotype in pulmonary fibroblasts

2004 ◽  
Vol 286 (5) ◽  
pp. C1089-C1099 ◽  
Author(s):  
Xiaoqiu Liu ◽  
Rennolds S. Ostrom ◽  
Paul A. Insel
Keyword(s):  
Cell Proliferation ◽  
Pulmonary Fibrosis ◽  
Adenylyl Cyclase ◽  
Collagen Synthesis ◽  
Lung Fibroblasts ◽  
Prostaglandin E ◽  
Pulmonary Fibroblasts ◽  
Adenosine Receptor Agonist ◽  
Proline Incorporation ◽  
Camp Levels

Pulmonary fibroblasts are recruited to sites of lung injury, where they are activated to produce extracellular matrix proteins and to facilitate repair. However, these cells become dysregulated in pulmonary fibrosis, producing excess collagen at sites of injury and forming fibrotic loci that impair lung function. In this study, we used WI-38 human lung fibroblasts and evaluated the ability of G protein-coupled receptor agonists to increase cAMP production and regulate cell proliferation and collagen synthesis. WI-38 cells increase cAMP in response to the β-adrenergic agonist isoproterenol (Iso), prostaglandin E2 (PGE2), certain prostanoid receptor-selective agonists (beraprost, butaprost), an adenosine receptor agonist, and the direct adenylyl cyclase (AC) activator forskolin (Fsk). Responses to Iso, PGE2, and Fsk were studied in more detail. Each induced a dose-dependent inhibition of serum-stimulated cell proliferation (as measured by [3H]thymidine incorporation) and collagen synthesis (as measured by [3H]proline incorporation, collagenase-sensitive [3H]proline incorporation, or levels of procollagen type 1 C-peptide). Quantitative RT-PCR analyses indicated that elevation in cellular cAMP levels decreases expression of collagen types 1α(II) and 5α(I) and increases expression and activity of matrix metalloproteinase 2 (MMP-2). Overexpression of AC type 6 or inhibition of cyclic nucleotide phosphodiesterases also increased cellular cAMP levels and decreased cell proliferation and collagen synthesis. Thus multiple approaches that increase cAMP signaling reduce proliferation and differentiated function in human pulmonary fibroblasts. These results suggest that therapies that raise cAMP levels may prove useful in the treatment of pulmonary fibrosis.

Impaired synthesis of prostaglandin E2 by lung fibroblasts and alveolar epithelial cells from GM-CSF−/− mice: implications for fibroproliferation

2003 ◽  
Vol 284 (6) ◽  
pp. L1103-L1111 ◽  
Author(s):  
Ryan P. Charbeneau ◽  
Paul J. Christensen ◽  
Cara J. Chrisman ◽  
Robert Paine ◽  
Galen B. Toews ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Pulmonary Fibrosis ◽  
Fluorescein Isothiocyanate ◽  
Surfactant Protein ◽  
Alveolar Epithelial Cells ◽  
Lung Fibroblasts ◽  
Prostaglandin E ◽  
Pulmonary Fibroblasts ◽  
Alveolar Epithelial ◽  
Gm Csf

Prostaglandin E2 (PGE2) is a potent suppressor of fibroblast activity. We previously reported that bleomycin-induced pulmonary fibrosis was exaggerated in granulocyte-macrophage colony-stimulating factor knockout (GM-CSF−/−) mice compared with wild-type (GM-CSF+/+) mice and that increased fibrosis was associated with decreased PGE2 levels in lung homogenates and alveolar macrophage cultures. Pulmonary fibroblasts and alveolar epithelial cells (AECs) represent additional cellular sources of PGE2 within the lung. Therefore, we examined fibroblasts and AECs from GM-CSF−/− mice, and we found that they elaborated significantly less PGE2 than did cells from GM-CSF+/+ mice. This defect was associated with reduced expression of cyclooxygenase-1 and -2 (COX-1 and COX-2), key enzymes in the biosynthesis of PGE2. Additionally, proliferation of GM-CSF−/− fibroblasts was greater than that of GM-CSF+/+ fibroblasts, and GM-CSF−/− AECs were impaired in their ability to inhibit fibroblast proliferation in coculture. The addition of GM-CSF to fibroblasts from GM-CSF−/− mice increased PGE2 production and decreased proliferation. Similarly, AECs isolated from GM-CSF−/− mice with transgenic expression of GM-CSF under the surfactant protein C promoter (SpC-GM mice) produced more PGE2 than did AEC from control mice. Finally, SpC-GM mice were protected from fluorescein isothiocyanate-induced pulmonary fibrosis. In conclusion, these data demonstrate that GM-CSF regulates PGE2 production in pulmonary fibroblasts and AECs and thus plays an important role in limiting fibroproliferation.

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.

scholarly journals Probing into the Mechanism of Alkaline Citrus Extract Promoted Apoptosis in Pulmonary Fibroblasts of Bleomycin-Induced Pulmonary Fibrosis Mice

2018 ◽  
Vol 2018 ◽  
pp. 1-11
Author(s):  
Qi Wu ◽  
Yao Zhou ◽  
Fan-chao Feng ◽  
Yi-han Jin ◽  
Zhi-chao Wang ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Pulmonary Fibrosis ◽  
Fas Ligand ◽  
Prostaglandin E ◽  
Dependent Manner ◽  
Expression Intensity ◽  
Pulmonary Fibroblasts ◽  
Cox 2 ◽  
Pulmonary Fibroblast ◽  
Functioning Mechanism

We extracted the primary pulmonary fibroblasts of the normal and bleomycin-induced pulmonary fibrosis mice and investigated the functioning mechanism of citrus alkaline extract (CAE) in the induction of pulmonary fibroblast apoptosis. The expression intensity of vimentin of the pulmonary fibroblasts in the model mice was higher than that in the normal mice. Meanwhile, the positive expression rate and expression intensity of alpha smooth muscle actin (α-SMA) of the pulmonary fibroblasts in the model mice were higher than those in the normal mice. Results of MTT showed that pulmonary fibroblast activity of the normal and model mice has been significantly inhibited by CAE in a concentration-dependent manner. The results of flow cytometer analysis showed that the proportion of pulmonary fibroblast apoptosis in the model mice has been profoundly increased by CAE treatment in a dosage-dependent manner. Besides we found that the expression of Cleaved-Caspase 3, Cleaved-Caspase 8, Cleaved-poly-ADP-ribose polymerase (Cleaved-PARP), and Fas and Fas Ligand (FasL) was markedly increased after CAE treatment. A further study showed that the expression of Cyclooxygenase-2 (COX-2) and prostaglandin E receptor 2 (EP2) was dependant on the concentration of CAE, indicating that CAE-regulated receptor apoptosis of Fas was probably related to COX-2. The results of fluorescence detection of oxidative stress showed that the level of oxidative stress was significantly increased after CAE treatment. Furthermore, the results of Western Blot showed that the phosphorylation level of p38 (p-p38) was markedly increased, suggesting that CAE probably has regulated COX-2 through increased p-p38 following oxidative stress. Our results therefore suggest that CAE can effectively induce pulmonary fibroblast apoptosis of the normal and model mice, and its functioning mechanism is probably related to the p38/COX-2/Fas signaling pathway regulated by oxidative stress.

scholarly journals Calcitonin gene-related peptide down-regulates bleomycin-induced pulmonary fibrosis

2016 ◽  
Vol 94 (12) ◽  
pp. 1315-1324 ◽  
Author(s):  
Xian-Wei Li ◽  
Xiao-Hui Li ◽  
Jie Du ◽  
Dai Li ◽  
Yuan-Jian Li ◽  
...  
Keyword(s):  
Pulmonary Fibrosis ◽  
Calcitonin Gene Related Peptide ◽  
Collagen I ◽  
Lung Fibroblasts ◽  
Brdu Incorporation ◽  
Pulmonary Fibroblasts ◽  
Related Peptide ◽  
Calcitonin Gene ◽  
Collagen Iii ◽  
Tgf Β1

We have found that eIF3a plays an important role in bleomycin-induced pulmonary fibrosis, and up-regulation of eIF3a induced by TGF-β1 is mediated via the ERK1/2 pathway. Whether ERK1/2 – eIF3a signal pathway is involved in calcitonin gene-related peptide (CGRP)-mediated pathogenesis of bleomycin-induced pulmonary fibrosis remains unknown. Pulmonary fibrosis was induced by intratracheal instillation of bleomycin (5 mg/kg) in rats. Primary pulmonary fibroblasts were cultured to investigate the proliferation by BrdU incorporation method and flow cytometry. Sensory CGRP depletion by capsaicin exacerbated bleomycin-induced pulmonary fibrosis in rats, as shown by a significant disturbed alveolar structure, marked thickening of the interalveolar septa and dense interstitial infiltration by inflammatory cells and fibroblasts, accompanied with increased expression of TGF-β1, eIF3a, phosphorylated ERK1/2, α-SMA, collagen I, and collagen III. Exogenous application of CGRP significantly inhibited TGF-β1-induced proliferation and differentiation of pulmonary fibroblasts concomitantly with decreased expression of eIF3a, phosphorylated ERK1/2, α-SMA, collagen I, and collagen III. These effects of CGRP were abolished in the presence of CGRP8-37. These results suggest that endogenous CGRP is related to the development of pulmonary fibrosis induced by bleomycin, and the inhibitory effect of CGRP on proliferation of lung fibroblasts involves the ERK1/2 – eIF3a signaling pathway.

scholarly journals Dysfunction of Collagen Synthesis and Secretion in Chondrocytes Induced byWisp3Mutation

2013 ◽  
Vol 2013 ◽  
pp. 1-9 ◽  
Author(s):  
Min Wang ◽  
Xiao-Fei Man ◽  
Ya-Qing Liu ◽  
Er-Yuan Liao ◽  
Zhi-Feng Shen ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Collagen Synthesis ◽  
Underlying Mechanism ◽  
Expression Vectors ◽  
Protein Distribution ◽  
Collagen Secretion ◽  
Proline Incorporation ◽  
Spondyloepiphyseal Dysplasia Tarda ◽  
Peak Phase ◽  
Intracellular Collagen

Wisp3gene mutation was shown to cause spondyloepiphyseal dysplasia tarda with progressive arthropathy (SRDT-PA), but the underlying mechanism is not clear. To clarify this mechanism, we constructed the wild and mutatedWisp3expression vectors and transfected into human chondrocytes lines C-20/A4;Wisp3proteins subcellular localization, cell proliferation, cell apoptosis, andWisp3-mediated gene expression were determined, and dynamic secretion of collagen in transfected chondrocytes was analyzed by14C-proline incorporation experiment. MutatedWisp3protein increased proliferation activity, decreased apoptosis of C-20/A4 cells, and aggregated abnormally in cytoplasm. Expression of collagen II was also downregulated in C-20/A4 cells transfected with mutatedWisp3. Wild typeWisp3transfection increased intracellular collagen content and extracellular collagen secretion, but the mutatedWisp3lost this function, and the peak phase of collagen secretion was delayed in mutatedWisp3transfected cells. Thus abnormal protein distribution, cell proliferation, collagen synthesis, and secretion inWisp3mutated chondrocytes might contribute to the pathogenesis of SEDT-PA.

Targeted inhibition of PI3 kinase/mTOR specifically in fibrotic lung fibroblasts suppresses pulmonary fibrosis in experimental models

2020 ◽  
Vol 12 (567) ◽  
pp. eaay3724
Author(s):  
Suraj U. Hettiarachchi ◽  
Yen-Hsing Li ◽  
Jyoti Roy ◽  
Fenghua Zhang ◽  
Estela Puchulu-Campanella ◽  
...  
Keyword(s):  
Pulmonary Fibrosis ◽  
Targeted Delivery ◽  
Collagen Synthesis ◽  
Lung Parenchyma ◽  
Therapeutic Agents ◽  
Experimental Models ◽  
Pi3k Inhibitor ◽  
Lung Fibroblasts ◽  
Average Life Expectancy ◽  
Pi3k Activation

Idiopathic pulmonary fibrosis (IPF) is a lethal disease with an average life expectancy of 3 to 5 years. IPF is characterized by progressive stiffening of the lung parenchyma due to excessive deposition of collagen, leading to gradual failure of gas exchange. Although two therapeutic agents have been approved from the FDA for IPF, they only slow disease progression with little impact on outcome. To develop a more effective therapy, we have exploited the fact that collagen-producing myofibroblasts express a membrane-spanning protein, fibroblast activation protein (FAP), that exhibits limited if any expression on other cell types. Because collagen-producing myofibroblasts are only found in fibrotic tissues, solid tumors, and healing wounds, FAP constitutes an excellent marker for targeted delivery of drugs to tissues undergoing pathologic fibrosis. We demonstrate here that a low–molecular weight FAP ligand can be used to deliver imaging and therapeutic agents selectively to FAP-expressing cells. Because induction of collagen synthesis is associated with phosphatidylinositol 3-kinase (PI3K) activation, we designed a FAP-targeted PI3K inhibitor that selectively targets FAP-expressing human IPF lung fibroblasts and potently inhibited collagen synthesis. Moreover, we showed that administration of the inhibitor in a mouse model of IPF inhibited PI3K activation in fibrotic lungs, suppressed production of hydroxyproline (major building block of collagen), reduced collagen deposition, and increased mouse survival. Collectively, these studies suggest that a FAP-targeted PI3K inhibitor might be promising for treating IPF.

scholarly journals Mir-21 Mediates the Inhibitory Effect of Ang (1–7) on AngII-induced NLRP3 Inflammasome Activation by Targeting Spry1 in lung fibroblasts

2017 ◽  
Vol 7 (1) ◽  
Author(s):  
Na-Na Sun ◽  
Chang-Hui Yu ◽  
Miao-Xia Pan ◽  
Yue Zhang ◽  
Bo-Jun Zheng ◽  
...  
Keyword(s):  
Pulmonary Fibrosis ◽  
Nlrp3 Inflammasome ◽  
Lung Fibrosis ◽  
Collagen Synthesis ◽  
Inhibitory Effect ◽  
Lung Fibroblasts ◽  
Inflammasome Activation ◽  
Apoptosis Resistance ◽  
Nlrp3 Inflammasome Activation

AbstractMicroRNA-21 (mir-21) induced by angiotensin II (AngII) plays a vital role in the development of pulmonary fibrosis, and the NLRP3 inflammasome is known to be involved in fibrogenesis. However, whether there is a link between mir-21 and the NLRP3 inflammasome in pulmonary fibrosis is unknown. Angiotensin-converting enzyme 2/angiotensin(1–7) [ACE2/Ang(1–7)] has been shown to attenuate AngII-induced pulmonary fibrosis, but it is not clear whether ACE2/Ang(1–7) protects against pulmonary fibrosis by inhibiting AngII-induced mir-21 expression. This study’s aim was to investigate whether mir-21 activates the NLRP3 inflammasome and mediates the different effects of AngII and ACE2/Ang(1–7) on lung fibroblast apoptosis and collagen synthesis. In vivo, AngII exacerbated bleomycin (BLM)-induced lung fibrosis in rats, and elevated mir-21 and the NLRP3 inflammasome. In contrast, ACE2/Ang(1–7) attenuated BLM-induced lung fibrosis, and decreased mir-21 and the NLRP3 inflammasome. In vitro, AngII activated the NLRP3 inflammasome by up-regulating mir-21, and ACE2/Ang(1–7) inhibited NLRP3 inflammasome activation by down-regulating AngII-induced mir-21. Over-expression of mir-21 activated the NLRP3 inflammasome via the ERK/NF-κB pathway by targeting Spry1, resulting in apoptosis resistance and collagen synthesis in lung fibroblasts. These results indicate that mir-21 mediates the inhibitory effect of ACE2/Ang(1–7) on AngII-induced activation of the NLRP3 inflammasome by targeting Spry1 in lung fibroblasts.

scholarly journals Downregulation of NOX4 Expression by Roflumilast N-Oxide Reduces Markers of Fibrosis in Lung Fibroblasts

2013 ◽  
Vol 2013 ◽  
pp. 1-8 ◽  
Author(s):  
Daniela Vecchio ◽  
Alessandra Acquaviva ◽  
Beatrice Arezzini ◽  
Hermann Tenor ◽  
Piero A. Martorana ◽  
...  
Keyword(s):  
Collagen Synthesis ◽  
Lung Fibroblasts ◽  
Ros Generation ◽  
Phosphodiesterase 4 ◽  
Nadph Oxidase 4 ◽  
Proline Incorporation ◽  
Per Se ◽  
Oxidative Effects

The phosphodiesterase 4 inhibitor roflumilast prevents bleomycin- (BLM-) induced lung fibrosis in animal models. However, its mechanism of action remains unknown. We investigated whether roflumilast N-oxide (RNO), the active metabolite of roflumilast, can modulatein vitrothe oxidative effects of BLM on human lung fibroblasts (HLF). In addition, since BLM increases the production of F2-isoprostanes that haveper sefibrogenic activity, the effect of RNO on oxidative stress and fibrogenesis induced by the F2-isoprostane 8-epi-PGF2αwas investigated. HLF were preincubated either with the vehicle or with RNO and exposed to either BLM or 8-epi-PGF2α. Proliferation and collagen synthesis were assessed as [3H]-thymidine and [3H]-proline incorporation. Reactive oxygen species (ROS) and F2-isoprostanes were measured. NADPH oxidase 4 (NOX4) protein and mRNA were also evaluated. BLM increased both cell proliferation and collagen synthesis and enhanced ROS and F2-isoprostane production. These effects were significantly prevented by RNO. Also, RNO significantly reduced the increase in both NOX4 mRNA and protein, induced by BLM. Finally, 8-epi-PGF2α  per sestimulated HLF proliferation, collagen synthesis, and NOX4 expression and ROS generation, and RNO prevented these effects. Thus, the antifibrotic effect of RNO observedin vivomay be related to its ability to mitigate ROS generation via downregulation of NOX4.

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