Bronchial Smooth Muscle Cells Research Articles

Mechanism of Periostin Production in Human Bronchial Smooth Muscle Cells

Background: Asthma is a chronic airway inflammatory disease characterized by airway remodeling, in which the bronchial smooth muscle (BSM) cells play an important role. Periostin, a biomarker that reflects Th2-driven inflammatory diseases such as asthma, may play an important role in the asthmatic airway. Although periostin is mainly produced in airway epithelial cells and fibroblasts after interleukin (IL)-13 stimulation, whether BSM cells produce periostin remains unclear. Therefore, we investigated periostin production in BSM cells and the mechanisms involved. Methods: Human BSM cells were cultured, and the effect of IL-13 stimulation on periostin production was evaluated using quantitative polymerase chain reaction and enzyme-linked immunosorbent assay (ELISA). We evaluated the phosphorylation of signal transducer and activator of transcription factor 6 (STAT6), extracellular signal-regulated kinase (ERK)1/2, and Akt after IL-13 stimulation. Furthermore, using ELISA, we evaluated the influence of several phosphorylation inhibitors on periostin production. Results: Periostin mRNA expression increased in a dose- and time-dependent manner after IL-13 stimulation; periostin production was induced 24 and 48 h after stimulation. IL-13 stimulation induced the phosphorylation of STAT6, ERK1/2, and Akt. IL-13-induced periostin production was attenuated by inhibiting STAT6 phosphorylation and strongly suppressed by inhibiting mitogen-activated protein kinase kinase 1/2 phosphorylation or phosphatidylinositol 3-kinase (PI3K) phosphorylation. Conclusions: BSM cells produced periostin after IL-13 stimulation, via the JAK/STAT6, ERK1/2, and PI3K/Akt pathways. Understanding the mechanism of periostin production in BSM cells may help to clarify asthma pathogenesis.

Overexpression of miR-155-5p Inhibits the Proliferation and Migration of IL-13-Induced Human Bronchial Smooth Muscle Cells by Suppressing TGF-β-Activated Kinase 1/MAP3K7-Binding Protein 2.

PurposeMolecular mechanisms leading to asthma is still ill-defined. Though the function of microRNAs (miRNAs) in asthma was previously reported, the involvement of miR-155 in important features of this disease remains unknown. The present study was designed to uncover the probable involvement of miR-155-5p in the proliferation and migration of IL-13-induced human bronchial smooth muscle cells (BSMCs) and the intrinsic regulatory mechanism.MethodsThe effects of different concentrations of IL-13 on the proliferation and migration of BSMCs as well as the expression of miR-155-5p and its predicted target transforming growth factor (TGF)-β-activated kinase 1/MAP3K7-binding protein 2 (TAB2) were investigated. The effects of miR-155-5p on the proliferation and migration of interleukin (IL)-13-induced BSMCs was determined in vitro using BSMCs transfected with miR-155 mimic/inhibitor and induced by a high concentration of IL-13. The quantitative real-time polymerase chain reaction (qRTPCR) was employed for determining the expression of miR-155-5p and TAB2. Western blotting was applied to analyze the expression of TAB2 at the protein level. Cell proliferation and migration were assessed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and Transwell assays, respectively.ResultsThe proliferation and migration of BSMCs were dose-dependently increased with IL-13 treatment. Contrariwise, IL-13 dose-dependently inhibited the expression of miR-155-5p in BSMCs. Mechanistic studies showed that inhibition of miR-155-5p further promoted the stimulatory effects of IL-13, whereas overexpression of miR-155 significantly inhibited these effects. In silico studies and luciferase reporter assays indicated that TAB2 was a negatively regulated miR-155-5p target.ConclusionsThese results suggested that miR-155-5p-inhibit the IL-13-induced proliferation and migration of BSMCs by targeting TAB2 and that the IL-13/miR-155/TAB2 pathway could serve as a therapeutic target for pulmonary diseases, especially asthma.

Synthetic cardamonin inhibits migration of serum-induced human bronchial smooth muscle cells (HBSMCs)

Event Abstract Back to Event Synthetic cardamonin inhibits migration of serum-induced human bronchial smooth muscle cells (HBSMCs) Nazmi F. Musa1* 1 Faculty of Medicine and Health Sciences, Universiti Putra Malaysia, Malaysia Background Increased smooth muscle mass is a prominent features of airways remodelling. This leads to persistent airflow limitation and impaired lung function which contribute to asthma. Inhaled corticosteroid, a primary treatment in asthma management, is unable to inhibit airway remodelling. Hence, effective treatments targeting airway remodelling in asthma are urgently needed. Increased smooth muscle mass may be attributed to both the proliferation and migration of smooth muscle cells. Cardamonin is a chalcone analog that has been reported to inhibit migration of vascular smooth muscle cells. However, its effects upon the migratory capability of bronchial smooth muscle cells have yet to be determined. The ability of synthetic cardamonin to suppress serum-induced Human Bronchial Smooth Muscle Cells (HBSMCs) migration and the mechanisms involved were therefore be evaluated. Methods Briefly, HBSMCs were treated with the cardamonin (0.75-12.5uM) in a complete media for 48 hours. The effects of cardamonin on HBSMCs migration were assessed through scratch and transwell assays. Meanwhile the effects of cardamonin (12.5uM) on the expression of proteins associated with cell migration (RhoA and phospho-cofillin) were determined by western blot. Results The scratch and transwell assays demonstrated that synthetic cardamonin (0.78-12.5uM) reduced the HBSMCs migration in a dose-dependent manner. The inhibitory effect of cardamonin on migration of serum-induced HBSMCs is associated with a reduced expression of RhoA and increased phosphorylation of cofillin. Conclusion Synthetic cardamonin is able to reduce the migration of serum-induced HBSMCs via downregulation of RhoA and upregulation of phosphorylation of cofillin. Acknowledgements Daud Ahmad Israf Ali, Tham Chau Ling, Hanis Hazeera Harith, Manraj Singh Cheema Keywords: Airway remodeling in asthma, Cardamonin (PubChem CID: 10424762), airways smooth muscle migration, serum-induced Human Bronchial Smooth Muscle Cells, Actin cytoskeletal remodeling Conference: International Conference on Drug Discovery and Translational Medicine 2018 (ICDDTM '18) “Seizing Opportunities and Addressing Challenges of Precision Medicine”, Putrajaya, Malaysia, 3 Dec - 5 Feb, 2019. Presentation Type: Oral Presentation Topic: Inflammatory diseases Citation: Musa NF (2019). Synthetic cardamonin inhibits migration of serum-induced human bronchial smooth muscle cells (HBSMCs). Front. Pharmacol. Conference Abstract: International Conference on Drug Discovery and Translational Medicine 2018 (ICDDTM '18) “Seizing Opportunities and Addressing Challenges of Precision Medicine”. doi: 10.3389/conf.fphar.2018.63.00049 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 30 Oct 2018; Published Online: 17 Jan 2019. * Correspondence: Mr. Nazmi F Musa, Faculty of Medicine and Health Sciences, Universiti Putra Malaysia, Serdang, Malaysia, nazmi908991@gmail.com Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Nazmi F Musa Google Nazmi F Musa Google Scholar Nazmi F Musa PubMed Nazmi F Musa Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Omalizumab, the first available antibody for biological treatment of severe asthma: more than a decade of real-life effectiveness.

Omalizumab was the first, and for a long time the only available monoclonal antibody for the add-on treatment of severe allergic asthma. In particular, omalizumab selectively targets human immunoglobulin (Ig)E, forming small-size immune complexes that inhibit IgE binding to its high- and low-affinity receptors. Therefore, omalizumab effectively blunts the immune response in atopic asthmatic patients, thus significantly improving the control of asthma symptoms and successfully preventing disease exacerbations. These very positive effects of omalizumab make it possible to drastically decrease both referrals to the emergency room and hospitalizations for asthma exacerbations. Such important therapeutic actions of omalizumab have been documented by several randomized clinical trials, and especially by more than 10 years of real-life experience in daily clinical practice. Omalizumab can also interfere with airway remodelling by inhibiting the activation of IgE receptors located on structural cells such as bronchial epithelial cells and airway smooth muscle cells. Moreover, omalizumab is characterized by a very good safety and tolerability profile. Hence, omalizumab represents a valuable therapeutic option for the add-on biological treatment of severe allergic asthma.

Selective inhibition of histamine-evoked Ca2+ signals by compartmentalized cAMP in human bronchial airway smooth muscle cells

Intracellular Ca2+ and cAMP typically cause opposing effects on airway smooth muscle contraction. Receptors that stimulate these pathways are therapeutic targets in asthma and chronic obstructive pulmonary disease. However, the interactions between different G protein-coupled receptors (GPCRs) that evoke cAMP and Ca2+ signals in human bronchial airway smooth muscle cells (hBASMCs) are poorly understood. We measured Ca2+ signals in cultures of fluo-4-loaded hBASMCs alongside measurements of intracellular cAMP using mass spectrometry or [3H]-adenine labeling. Interactions between the signaling pathways were examined using selective ligands of GPCRs, and inhibitors of Ca2+ and cAMP signaling pathways. Histamine stimulated Ca2+ release through inositol 1,4,5-trisphosphate (IP3) receptors in hBASMCs. β2-adrenoceptors, through cAMP and protein kinase A (PKA), substantially inhibited histamine-evoked Ca2+ signals. Responses to other Ca2+-mobilizing stimuli were unaffected by cAMP (carbachol and bradykinin) or minimally affected (lysophosphatidic acid). Prostaglandin E2 (PGE2), through EP2 and EP4 receptors, stimulated formation of cAMP and inhibited histamine-evoked Ca2+ signals. There was no consistent relationship between the inhibition of Ca2+ signals and the amounts of intracellular cAMP produced by different stimuli. We conclude that β-adrenoceptors, EP2 and EP4 receptors, through cAMP and PKA, selectively inhibit Ca2+ signals evoked by histamine in hBASMCs, suggesting that PKA inhibits an early step in H1 receptor signaling. Local delivery of cAMP within hyperactive signaling junctions mediates the inhibition.

Regulating effect of glycyrrhetinic acid on bronchial asthma smooth muscle proliferation and apoptosis as well as inflammatory factor expression through ERK1/2 signaling pathway.

To study the influence of glycyrrhetinic acid (GA) on bronchial asthma (BA) smooth muscle proliferation and apoptosis as well as inflammatory factor expression and its molecular mechanism. Male SD guinea pigs were selected and made into asthma models, bronchial asthma smooth muscle cells were cultured and divided into BA group, GA group and GA+LM group that were treated with serum-free RPMI1640 culture medium, serum-free RPMI1640 culture medium containing 50ng/mL glycyrrhetinic acid, serum-free RPMI1640 culture medium containing 50ng/mL glycyrrhetinic acid and 100ng/mL LM22B-10 respectively; normal guinea pigs were collected and bronchial smooth muscle cells were cultured as control group. The cell proliferation activity as well as the expression of proliferation and apoptosis genes, inflammatory factors and p-ERK1/2 was determined. Proliferation activity value and mRNA expression of Bcl-2, TNF-α, IL-4, IL-6, YKL-40, protein expression of p-ERK1/2 of airway smooth muscle cell in BA group were significantly higher than those of control group while mRNA expression levels of Bax, caspase-9 as well as caspase-3 were significantly lower than that of control group (P<0.05); proliferation activity value and mRNA expression of Bcl-2, TNF-α, IL-4, IL-6, YKL-40, protein expression of p-ERK1/2 of airway smooth muscle cell in GA group were significantly lower than those of BA group (P<0.05) while the mRNA expression levels of Bax, caspase-9 as well as caspase-3 were significantly higher than those of BA group (P<0.05); proliferation activity value and mRNA expression of Bcl-2, TNF-α, IL-4, IL-6, YKL-40 of airway smooth muscle cell in GA+LM group were significantly higher than those of GA group (P<0.05) while mRNA expression levels of Bax, caspase-9 as well as caspase-3 were significantly lower that of GA group (P<0.05). GA can inhibit the proliferation of bronchial smooth muscle cells and reduce the expression of inflammatory factors by inhibiting the phosphorylation of ERK1/2.

An impedance-based cell contraction assay using human primary smooth muscle cells and fibroblasts

IntroductionMany cell types (including muscle cells and fibroblasts) can contract at physiological conditions and their contractility may change during tissue injury and repair or other diseases such as allergy and asthma. The conventional gel contraction assay is commonly used to monitor the cellular contractility. It is a manual assay and the experiment usually takes hours even days to complete. As its readout is not always accurate and reliable, the gel contraction assay is often used to qualitatively (but not quantitatively) characterize cellular contractility under various conditions. MethodTo overcome the limits of the gel contraction assay, we developed an impedance-based contraction assay using the xCELLigence RTCA MP system. This technology utilizes special 96-well E-plates with gold microelectrode arrays printed in individual wells to monitor cellular adhesion by recording the electrical impedance in real time. The impedance change (percentage vs. control) can be used as the readout for cellular contraction. ResultsWe demonstrated that the impedance-based contraction assay can be performed within 2h. Using this new method, we quantitatively characterized the effects of several contractile stimulators and inhibitors on human primary bronchial smooth muscle cells and primary lung fibroblasts. DiscussionThe impedance-based contraction assay can be applied to both basic research and drug discovery for characterizing cellular contraction quantitatively. Because it has high throughput capacity and high reproducibility, the impedance-based contraction assay is useful for high throughput functional screening in drug industry.

COPD affected lung tissue remodelling due to the local distribution of MMP-2, TIMP-2, TGF-β1 and HSP-70

Chronic obstructive pulmonary disease (COPD) is strongly associated with progressive airway limitation where the key mechanism is abnormal airway remodelling of bronchial mucosa maintained by complex crosstalk of tissue remodelling and regulatory factors. The aim of this study was to determine the appearance and local distribution of tissue remodelling factors in COPD-affected lung tissue and to compare the findings with the control group. In this study, lung tissue specimens were obtained from 27 patients with COPD and 49 control patients. Tissue samples were examined by routine hematoxylin and eosin staining. Remodelling and regulatory factors matrix metalloproteinase-2 (MMP-2), tissue inhibitor of metalloproteinase-2 (TIMP-2), transforming growth factor-β1 (TGF-β1), as well as heat shock protein-70 (Hsp-70) were detected by immunohistochemistry in airway mucosa. The numbers of positive structures were evaluated semiquantitatively. Non-parametrical statistical analysis was performed. Overall COPD-affected lung tissue presented chronic inflammation and tissue remodelling in routine histological analysis. Compared to the control group, statistically significant (P&lt;0.05) difference was calculated between COPD-affected lung tissue and control group with overall more immunoreactive cells containing MMP-2, TIMP-2 and TGF-β1 and less Hsp-70 immunoreactive bronchial epithelial cells, subepithelial connective tissue fibroblasts, bronchial smooth muscle cells and secretory cells of bronchial glands with more pronounced findings of TGF-β1, however, less Hsp-70. Study findings suggest pronounced tissue damage and remodelling with the local regulatory environment of up-regulated TGF-β1. Increased numbers of MMP-2, TIMP-2 and TGF-β1 immunoreactive cells suggest the key role of these remodelling factors in COPD pathogenesis. Decrease of Hsp-70 proves increased cell damage in COPD-affected airway mucosa.

Functional Cooperation between KCa3.1 and TRPV4 Channels in Bronchial Smooth Muscle Cell Proliferation Associated with Chronic Asthma.

Airway smooth muscle cells (SMC) proliferation contributes to the airways remodeling and irreversible airway obstruction during severe asthma, but the mechanisms of airway SMC proliferation are poorly understood. Intracellular Ca2+ levels play an important role in regulating cell proliferation. We have previously reported KCa3.1 channels regulated human bronchial smooth muscle (HBSM) cells proliferation via the Ca2+ influx as a consequence of membrane hyperpolarization. However, the role of potassium channels KCa3.1 in airway remodeling as well as the mechanism for extracellular Ca2+ influx induced by the activation of KCa3.1 remains unknown. Here we demonstrated that KCa3.1 channels deficiency attenuated airway remodeling, airway inflammation, and airway hyperresponsiveness (AHR) in a mouse model of chronic asthma. The gene expressions of repressor element 1-silencing transcription factor (REST) and c-Jun, two transcriptional regulators of KCa3.1 channels, were correlated negatively or positively with KCa3.1 channels expressions both in vivo and in vitro using real-time PCR and Western blot analyses. RNAi-mediated knockdown or pharmacological blockade of KCa3.1 and TRPV4 significantly attenuated HBSM cells proliferation. Using confocal imaging and custom data analysis software, blockade of TRPV4 decreased the Ca2+ influx induced by 1-EBIO-mediated KCa3.1 activation. Double-labeled staining showed that KCa3.1 and TRPV4 channels colocalized in HBSM cells. These results demonstrate that KCa3.1 channels regulate the proliferation phenotype of HBSM cells via TRPV4 channels in the process of chronic asthma, making it a potential therapeutic target to treat chronic asthma.

Increased neutrophil gelatinase-associated lipocalin (NGAL) promotes airway remodelling in chronic obstructive pulmonary disease

Airway remodelling is an important component of chronic obstructive pulmonary disease (COPD). Neutrophil gelatinase-associated lipocalin (NGAL) from neutrophils may drive COPD epithelial-mesenchymal transition (EMT). NGAL expression was quantified in the lungs of COPD patients and bronchoalveolar lavage fluid (BALF) of ozone-treated mice. Reticular basement membrane (RBM) thickness and E-cadherin and α-smooth muscle actin (α-SMA) expression were determined in mice airways. Effects of cigarette smoke extract (CSE) and inflammatory factors on NGAL expression in human neutrophils as well as the effects of NGAL on airway structural cells was assessed. NGAL was mainly distributed in neutrophils and enhanced in lung tissues of both COPD patients and BALF of ozone-treated mice. We showed decreased E-cadherin and increased α-SMA expression in bronchial epithelium and increased RBM thickness in ozone-treated animals. In vitro, CSE, IL-1β and IL-17 enhanced NGAL mRNA expression in human neutrophils. NGAL, in turn, down-regulated the expression of E-cadherin and up-regulated α-SMA expression in 16HBE cells via the WNT/glycogensynthase kinase-3β (GSK-3β) pathway. Furthermore, NGAL promoted the proliferation and migration of human bronchial smooth muscle cells (HASMCs). The present study suggests that elevated NGAL promotes COPD airway remodelling possibly through altered EMT. NGAL may be a potential target for reversing airway obstruction and remodelling in COPD.

Transforming Growth Factor β1 Function in Airway Remodeling and Hyperresponsiveness. The Missing Link?

The pathogenesis of asthma includes a complex interplay among airway inflammation, hyperresponsiveness, and remodeling. Current evidence suggests that airway structural cells, including bronchial smooth muscle cells, myofibroblasts, fibroblasts, and epithelial cells, mediate all three aspects of asthma pathogenesis. Although studies show a connection between airway remodeling and changes in bronchomotor tone, the relationship between the two remains unclear. Transforming growth factor β1 (TGF-β1), a growth factor elevated in the airway of patients with asthma, plays a role in airway remodeling and in the shortening of various airway structural cells. However, the role of TGF-β1 in mediating airway hyperresponsiveness remains unclear. In this review, we summarize the literature addressing the role of TGF-β1 in airway remodeling and shortening. Through our review, we aim to further elucidate the role of TGF-β1 in asthma pathogenesis and the link between airway remodeling and airway hyperresponsiveness in asthma and to define TGF-β1 as a potential therapeutic target for reducing asthma morbidity and mortality.

Mechanisms underlying the pathogenesis of hyper-contractility of bronchial smooth muscle in allergic asthma.

Airway hyperresponsiveness (AHR) and inflammation are key pathophysiological features of asthma. Enhanced contraction of bronchial smooth muscle (BSM) is one of the causes of the AHR. It is thus important for development of asthma therapy to understand the change in the contractile signaling of airway smooth muscle cells associated with the AHR. In addition to the Ca2+-mediated phosphorylation of myosin light chain (MLC), contractile agonists also enhance MLC phosphorylation level, Ca2+-independently, by inactivating MLC phosphatase (MLCP), called Ca2+ sensitization of contraction, in smooth muscle cells including airways. To date, involvements of RhoA/ROCKs and PKC/Ppp1r14a (also called as CPI-17) pathways in the Ca2+ sensitization have been identified. Our previous studies revealed that the agonist-induced Ca2+ sensitization of contraction is markedly augmented in BSMs of animal models of allergen-induced AHR. In BSMs of these animal models, the expression of RhoA and CPI-17 proteins were significantly increased, indicating that both the Ca2+ sensitizing pathways are augmented. Interestingly, incubation of BSM cells with asthma-associated cytokines, such as interleukin-13 (IL-13), IL-17, and tumor necrosis factor-α (TNF-α), caused up-regulations of RhoA and CPI-17 in BSM cells of naive animals and cultured human BSM cells. In addition to the transcription factors such as STAT6 and NF-κB activated by these inflammatory cytokines, an involvement of down-regulation of miR-133a, a microRNA that negatively regulates RhoA translation, has also been suggested in the IL-13- and IL-17-induced up-regulation of RhoA. Thus, the Ca2+ sensitizing pathways and the cytokine-mediated signaling including microRNAs in BSMs might be potential targets for treatment of allergic asthma, especially the AHR.

Role of mast cells in bronchial contraction in nonallergic obstructive lung pathology.

The role of mast cells in contractile bronchial smooth muscle activity has been evaluated in a model of chronic obstructive pulmonary disease induced in rats that were intermittently exposed to nitrogen dioxide (NO2) for 60 days. Starting from the 31st day, one group of rats inhaled sodium cromoglycate before exposure to NO2 to stabilize mast cell membranes. The second group (control) was not treated. Isometric smooth muscle contraction was analysed in isolated bronchial samples in response to nerve and smooth muscle stimulation. Histological analysis revealed large numbers of mast cells in lung tissue of COPD model rats. The inhibition of mast cell degranulation by sodium cromoglycate prevented the development of nerve-stimulated bronchial smooth muscle hyperactivity in COPD model rats. Histamine or adenosine-induced hyperactivity on nerve stimulation was also inhibited by sodium cromoglycate in bronchial smooth muscle in both control and COPD model rats. This suggests that the mechanism of contractile activity enhancement of bronchial wall smooth muscle cells may be mediated through the activation of resident mast cells transmembrane adenosine receptors resulting in their partial degranulation, with the released histamine acting upon histamine H1-receptors which trigger reflex pathways via intramural ganglion neurons.

Interleukin-17A directly acts on bronchial smooth muscle cells and augments the contractility.

Although interleukin-17 (IL-17) contributes to the induction of airway hyperresponsiveness in asthma, its effect on bronchial smooth muscle (BSM) remains largely unknown. Evidence support an involvement of RhoA/Rho-kinase in BSM contraction, and the pathway has now been proposed as a novel target for asthma therapy. To clarify the role of IL-17 on the development of BSM hyperresponsiveness, effects of IL-17A on BSM contractility and RhoA expression were investigated. Male BALB/c mice and cultured human BSM cells (hBSMCs) were used. In the murine model of allergic asthma, BSM hyperresponsiveness with an IL-17A up-regulation in bronchoalveolar lavage fluids were observed. RT-PCR analyses revealed the expression of receptors for IL-17A in mouse BSMs and hBSMCs. In the hBSMCs, incubation with IL-17A caused an up-regulation of RhoA protein. Western blot analyses also revealed phosphorylations of JNKs/ERKs and a down-regulation of IκB-α in the IL-17A-treated hBSMCs, indicating that IL-17A could act on BSM cells directly. However, IL-17A did not activate STAT6, which is also known as a signaling molecule that causes an up-regulation of RhoA when activated by IL-13. On the other hand, IL-17A caused a down-regulation of miR-133a-3p, a microRNA that negatively regulates RhoA translation. In the naive mice, in vivo IL-17A treatment to the airways by intranasal instillation induced a BSM hyperresponsiveness with RhoA protein up-regulation. These findings indicate that IL-17 directly acts on BSM cells and up-regulates RhoA protein probably via a down-regulation of miR-133a-3p, resulting in an induction of the BSM hyperresponsiveness.

Simvastatin alleviates airway inflammation and remodelling through up-regulation of autophagy in mouse models of asthma.

Statins have been widely used in inflammatory diseases including asthma, because of their anti-inflammatory and immunomodulatory properties. It has been shown that simvastatin induces autophagy and cell death in some circumstances. However, the possible cross-talk between simvastatin and autophagic processes in lung disease is largely unknown. Thus, we investigated the impact of simvastatin on airway inflammation and airway remodelling and the possible relationship of these processes to a simvastatin-induced autophagic pathway in mouse models of asthma. Ovalbumin (OVA)-sensitized and challenged mice were treated with simvastatin and sacrificed. The autophagy-related proteins Atg5, LC3B and Beclin1 were quantified, as well as the autophagy flux in bronchial smooth muscle cells (BSMCs). The relationship between airway inflammation and the autophagic process was investigated. We show that simvastatin treatment mediates activation of autophagy in BSMCs, which is correlated with airway inflammation and airway remodelling in mouse models of asthma. Simvastatin increases autophagy-related protein Atg5, LC3B and Beclin1 expression and autophagosome formation in lung tissue. Simvastatin-induced autophagy is associated with increased interferon-gamma (IFN-γ) and decreased IL-4, IL-5 and IL-13 cytokines production in BSMCs, as well as reversed extracellular matrix (ECM) deposition. In contrast, autophagy inhibitor 3-methyladenine (3-MA) eliminates the therapeutic effect of simvastatin. These findings demonstrate that simvastatin inhibits airway inflammation and airway remodelling through an activated autophagic process in BSMCs. We propose a crucial function of autophagy in statin-based therapeutic approaches in asthma.

Leukotriene B4 receptor type 2 protects against pneumolysin-dependent acute lung injury

Although pneumococcal infection is a serious problem worldwide and has a high mortality rate, the molecular mechanisms underlying the lethality caused by pneumococcus remain elusive. Here, we show that BLT2, a G protein-coupled receptor for leukotriene B4 and 12(S)-hydroxyheptadecatrienoic acid (12-HHT), protects mice from lung injury caused by a pneumococcal toxin, pneumolysin (PLY). Intratracheal injection of PLY caused lethal acute lung injury (ALI) in BLT2-deficient mice, with evident vascular leakage and bronchoconstriction. Large amounts of cysteinyl leukotrienes (cysLTs), classically known as a slow reactive substance of anaphylaxis, were detected in PLY-treated lungs. PLY-dependent vascular leakage, bronchoconstriction, and death were markedly ameliorated by treatment with a CysLT1 receptor antagonist. Upon stimulation by PLY, mast cells produced cysLTs that activated CysLT1 expressed in vascular endothelial cells and bronchial smooth muscle cells, leading to lethal vascular leakage and bronchoconstriction. Treatment of mice with aspirin or loxoprofen inhibited the production of 12-HHT and increased the sensitivity toward PLY, which was also ameliorated by the CysLT1 antagonist. Thus, the present study identifies the molecular mechanism underlying PLY-dependent ALI and suggests the possible use of CysLT1 antagonists as a therapeutic tool to protect against ALI caused by pneumococcal infection.

Caveolin-1 and Caveolin-3: Structural and Functional Insights into Their Role in Murine Airway Smooth Muscle Constriction

Serotonin and acetylcholine are bronchoconstrictors clinically relevant for airway diseases associated with airway hyperreactivity. Caveolins (cav-1, -2 and -3), which are binding partners for receptors and enzymes, are structural components of caveolae. The process of cav assembling into caveolae requires cytoplasmic adapter proteins (cavin-1 to -4). Here, we addressed the role of cav-1, cav-3, cavin-1 and -4 in airway smooth muscle (SM) constriction utilizing immunofluorescence, RT-PCR, electron microscopy, western blotting, co-immunoprecipitation and functional analysis (organ bath and videomicroscopy) in cav-1 and cav-3 deficient (KO) and wild-type mice. In mouse airways, cavin-1 and -4 interact with cav-1 and cav-3, respectively. Cav-1 expression was decreased in tracheal SM in cav-3-KO mice. Caveolar numbers were diminished in cav-1-KO but not in cav-3-KO tracheal SM. Muscarine-induced tracheal constriction was not altered in either KO-strain but was increased in intrapulmonary bronchi of cav-3-KO mice. However, serotonin-induced constriction was lost in the trachea of both KO-strains. Protein and mRNA for cavin-1, -4 and cav-1 and -3 were also detected in human intrapulmonary bronchi and primary SM cells. In conclusion, cav-1 and cav-3 are essential for serotonin-induced tracheal SM constriction pointing to an involvement of different serotonin receptors in the trachea and bronchi that differ in their coupling to caveolae. Cav-3 was previously found to interact with muscarinic receptor type-2 (m2AChR) in bronchial SM cells. Thus, depletion of cav-3 might cause a disturbance of m2AChR coupling to caveolae resulting in increased muscarine-induced bronchoconstriction. Funding: DFG (SFB-TR84)

MicroRNA-146a expression inhibits the proliferation and promotes the apoptosis of bronchial smooth muscle cells in asthma by directly targeting the epidermal growth factor receptor.

The present study aimed to determine the expression of microRNA-146a (miR-146a) in the plasma of children with asthma, and to investigate the effect of miR-146a on the proliferation and apoptosis of bronchial smooth muscle cells (BSMCs). Reverse transcription-quantitative polymerase chain reaction was used to determine the expression levels of miR-146a mimics and its inhibitor. A Cell Counting kit-8 assay was performed to examine the proliferation of BSMCs. Caspase-3/7 activity was determined using a Caspase-Glo 3/7 kit. To measure the expression levels of proteins associated with apoptosis, western blotting was performed. The target gene of miR-146a was identified using a dual-luciferase reporter assay. The plasma levels of miR-146a in children with asthma were significantly higher compared with those in healthy children. Enhanced miR-146a expression inhibited the proliferation of BSMCs. BSMC apoptosis was promoted by miR-146a. The mechanism underlying the miR-146a-induced promotion of BSMC apoptosis may be its direct targeting of epidermal growth factor receptor (EGFR), which affects downstream signaling pathways. In conclusion, miR-146a expression in asthma inhibits the proliferation and promotes the apoptosis of BSMCs by direct targeting of EGFR.

Bronchial Smooth Muscle Remodeling in Nonsevere Asthma.

Increased bronchial smooth muscle (BSM) mass is a key feature of airway remodeling that classically distinguishes severe from nonsevere asthma. Proliferation of BSM cells involves a specific mitochondria-dependent pathway in individuals with severe asthma. However, BSM remodeling and mitochondrial biogenesis have not been examined in nonsevere asthma. We aimed to assess whether an increase in BSM mass was also implicated in nonsevere asthma and its relationship with mitochondria and clinical outcomes. We enrolled 34 never-smoker subjects with nonsevere asthma. In addition, we recruited 56 subjects with nonsevere asthma and 19 subjects with severe asthma as comparative groups (COBRA cohort [Cohorte Obstruction Bronchique et Asthme; Bronchial Obstruction and Asthma Cohort; sponsored by the French National Institute of Health and Medical Research, INSERM]). A phenotypic characterization was performed using questionnaires, atopy and pulmonary function testing, exhaled nitric oxide measurement, and blood collection. Bronchial biopsy specimens were processed for immunohistochemistry and electron microscopy analysis. After BSM remodeling assessment, subjects were monitored over a 12-month period. We identified characteristic features of remodeling (BSM area >26.6%) and increased mitochondrial number within BSM in a subgroup of subjects with nonsevere asthma. The number of BSM mitochondria was positively correlated with BSM area (r = 0.78; P < 0.001). Follow-up analysis showed that subjects with asthma with high BSM had worse asthma control and a higher rate of exacerbations per year compared with subjects with low BSM. This study reveals that BSM remodeling and mitochondrial biogenesis may play a critical role in the natural history of nonsevere asthma (Mitasthme study). Clinical trial registered with www.clinicaltrials.gov (NCT00808730).

Dipeptidyl peptidase-4 is highly expressed in bronchial epithelial cells of untreated asthma and it increases cell proliferation along with fibronectin production in airway constitutive cells.

BackgroundType 2 helper T-cell cytokines including IL-13 play a central role in the pathogenesis of bronchial asthma (BA). During the course of our research, our attention was drawn to dipeptidyl peptidase-4 (DPP4) as one of the molecules that were induced from bronchial epithelial cells (BECs) by IL-13 stimulation. DPP4 could become a new biomarker or therapeutic target. The aim of this study was to investigate the expression of DPP4 in the asthmatic airway, and its role in the pathophysiology of asthma.MethodsBECs were isolated from patients with inhaled corticosteroid-treated asthma (stBA) and inhaled corticosteroid-naïve asthma (snBA) using bronchoscopy.DPP4 mRNA expression in freshly isolated BECs and primary cultured BECs with or without IL-13 stimulation was investigated by microarray analysis and quantitative real-time PCR (qPCR). The distribution of DPP4 protein was determined by immunostaining of transbronchial lung biopsy specimens from asthma patients. The effect of recombinant human (rh) DPP4 on the proliferation of lung fibroblasts (HFL-1) and bronchial smooth muscle cells (BSMCs) was examined, as well as its effect on the production of fibronectin (FN).ResultsDPP4 mRNA was strongly expressed in freshly isolated BECs in snBA, and its expression was significantly enhanced by IL-13 stimulation. DPP4 mRNA expression in BECs of snBA significantly correlated with exhaled nitric oxide. Biopsied tissues of the asthmatic airway revealed strong expression of DPP4 protein in BECs from snBA subjects. rhDPP4 stimulated the proliferation of HFL-1 and BSMCs, and it also enhanced production of FN from these airway cells.ConclusionDPP4 may be involved in the pathologic features of asthmatic airway inflammation and cell proliferation and FN production.Electronic supplementary materialThe online version of this article (doi:10.1186/s12931-016-0342-7) contains supplementary material, which is available to authorized users.