Human Bronchial Smooth Muscle Research Articles

Artesunate ameliorates cigarette smoke-induced airway remodelling via PPAR-\u03b3/TGF-\u03b21/Smad2/3 signalling pathway

BackgroundAirway remodelling is the major pathological feature of chronic obstructive pulmonary disease (COPD), and leads to poorly reversible airway obstruction. Current pharmacological interventions are ineffective in controlling airway remodelling. In the present study, we investigated the potential role of artesunate in preventing and treating airway remodelling and the underlying molecular mechanisms in vitro and in vivo.MethodsA COPD rat model was established by cigarette smoke (CS) exposure. After 12 weeks of artesunate treatment, pathological changes in the lung tissues of COPD rats were examined by ELISA and histochemical and immunohistochemical staining. A lung functional experiment was also carried out to elucidate the effects of artesunate. Human bronchial smooth muscle (HBSM) cells were used to clarify the underlying molecular mechanisms.ResultsArtesunate treatment inhibited CS-induced airway inflammation and oxidative stress in a dose-dependent manner and significantly reduced airway remodelling by inhibiting α-smooth muscle actin (α-SMA) and cyclin D1 expression. PPAR-γ was upregulated and TGF-β1/Smad2/3 signalling was inactivated by artesunate treatment in vivo and in vitro. Furthermore, PPAR-γ knockdown by siRNA transfection abolished artesunate-mediated inhibition of HBSM cell proliferation by activiting the TGF-β1/Smad2/3 signalling pathway and downregulating the expression of α-SMA and cyclin D1 in HBSM cells.ConclusionsThese findings suggest that artesunate could be used to treat airway remodelling by regulating PPAR-γ/TGF-β1/Smad signalling in the context of COPD.

MicroRNA-21 inhibition attenuates airway inflammation and remodelling by modulating the transforming growth factor β-Smad7 pathway.

Background/AimsCurrent asthma therapies remain unsatisfactory for controlling airway remodelling in asthma. MicroRNA-21 is a key player in asthma pathogenesis, but the molecular mechanisms underlying its effects on airway remodelling are not completely understood. We investigated the effects of inhibition of microRNA-21 on allergic airway inflammation and remodelling.MethodsFemale BALB/c mice were divided into four groups: control, ovalbumin-sensitized and -challenged for 3 months, microRNA-negative control-treated ovalbumin-treated, and microRNA-21 inhibitor-treated ovalbumin-treated groups. Parameters related to airway remodelling, cytokine production, airway inflammation, and airway hyperresponsiveness were compared between groups. Human bronchial smooth muscle cells were used in a mechanism study.ResultsIn this asthma model, ovalbumin-sensitized and -challenged mice exhibited allergic airway inflammation and airway remodelling. MicroRNA-21 inhibitor-treated mice had fewer inflammatory cells, lower TH2 cytokine production, and suppressed parameters related to remodelling such as goblet cell hyperplasia, collagen deposition, hydroxyproline content, and expression of smooth muscle actin. Inhibition of microRNA-21 decreased transforming growth factor β1 expression and induced Smad7 expression in lung tissue. In human bronchial smooth muscle cells stimulated with transforming growth factor β1, microRNA-21 inhibition upregulated Smad7 expression and decreased markers of airway remodelling.ConclusionsInhibition of microRNA-21 had both anti-inflammatory and anti-remodelling effects in this model of ovalbumin-induced chronic asthma. Our data suggest that the microRNA-21–transforming growth factor β1–Smad7 axis modulates the pathogenesis of ovalbumin-induced chronic asthma and in human bronchial smooth muscle cells. MicroRNA-21 inhibitors may be a novel therapeutic target in patients with allergic asthma, especially those with airway remodelling.

NOX4-Derived ROS Promotes Collagen I Deposition in Bronchial Smooth Muscle Cells by Activating Noncanonical p38MAPK/Akt-Mediated TGF-β Signaling.

Background Airway smooth muscle (ASM) remodeling is a hallmark in chronic obstructive pulmonary disease (COPD). NADPH oxidase 4- (NOX4-) mediated reactive oxygen species (ROS) production plays a crucial role in cell differentiation and extracellular matrix (ECM) synthesis in ASM remodeling. However, the precise mechanisms underpinning its pathogenic roles remain elusive. Methods The expression of NOX4 and TGF-β1 in the airway of the lung was measured in COPD patients and the control group. Cigarette smoke- (CS-) induced emphysema mice were generated, and the alteration of α-SMA, NOX4, TGF-β1, and collagen I was accessed. The changes of the expression of ECM markers, NOX4, components of TGF-β/Smad, and MAPK/Akt signaling in human bronchial smooth muscle cells (HBSMCs) were ascertained for delineating mechanisms of NOX4-mediated ROS production on cell differentiation and remodeling in human ASM cells. Results An increased abundance of NOX4 and TGF-β1 proteins in the epithelial cells and ASM of lung was observed in COPD patients compared with the control group. Additionally, an increased abundance expression of NOX4 and α-SMA was observed in the lungs of the CS-induced emphysema mouse model. TGF-β1 displayed abilities to increase the oxidative burden and collagen I production, along with enhanced phosphorylation of ERK, p38MAPK, and p-Akt473 in HBSMCs. These effects of TGF-β1 could be inhibited by the ROS scavenger N-acetylcysteine (NAC), siRNA-mediated knockdown of Smad3 and NOX4, and pharmacological inhibitors SB203580 (p38MAPK inhibitor) and LY294002 (Akt inhibitor). Conclusions NOX4-mediated ROS production alters TGF-β1-induced cell differentiation and collagen I protein synthesis in HBSMCs in part through the p38MAPK/Akt signaling pathway in a Smad-dependent manner.

Visualized heterooligomeric subunit structures of 817 human cellular proteins by correlating native protein 2D maps with protein interaction databases

Previously, we have reported on the reconstruction of 4323 native protein 2D maps of human bronchial smooth muscle cells (HBSMC) by combining nondenaturing 2DE, grid gel-cutting, and quantitative LC-MS/MS. In this work, we report on the visualization of the heterooligomeric subunit structures of HBSMC proteins by correlating the native protein 2D maps with the information in protein interaction databases. Image analysis of the 2D maps was employed as the first approach. Each of the native protein 2D maps of 2327 proteins, which had three or more detected squares in the native protein 2D maps, was compared with the 2D maps of the remaining 2326 proteins scoring the degree of overlap in an area around the quantity peak and the protein partner which showed the best score was decided. The protein pairs were examined on their reported interactions referring to protein interaction databases. In order to consider the cases where a protein has multiple functions and the heterooligomeric subunit structures might not be detected from the image analysis, prior database search was employed as the second approach. Each of the 1689 HBSMC proteins, which had five or more detected squares in the native protein 2D maps, was examined on its interactor proteins described in the databases, then the native 2D map was compared with the maps of the interactor proteins to find the overlap which reasonably supported the interaction. Summarizing these examinations, 215 heterooligomeric subunit structures of 817 human cellular proteins could be visualized on the native protein 2D maps.

Arsenic trioxide-eluting electrospun nanofiber-covered self-expandable metallic stent reduces granulation tissue hyperplasia in rabbit trachea

Stent-related granulation tissue hyperplasia is a major complication that limits the application of stents in airways. In this study, an arsenic trioxide-eluting electrospun nanofiber-covered self-expandable metallic stent (ATO-NFCS) was developed. Poly-L-lactide-caprolactone (PLCL) was selected as the drug-carrying polymer. Stents with two different ATO contents (0.4% ATO/PLCL and 1.2% ATO/PLCL) were fabricated. The in vitro release in simulated airway fluid suggested that the total ATO release time was 1 d. The growth of human embryonic pulmonary fibroblasts (CCC-HPF-1), normal human bronchial epithelial cells and airway smooth muscle cells was inhibited by ATO. When embedded in paravertebral muscle, the nanofiber membrane showed good short-term and long-term biological effects. In an animal study, placement of the ATO-NFCS in the trachea through a delivery system under fluoroscopy was feasible. The changes in liver and kidney function 1 and 7 d after ATO-NFCS placement were within the normal range. On pathological examination, the heart, liver, spleen, lungs and kidneys were normal. The effectiveness of the ATO-NFCS in reducing granulation tissue hyperplasia and collagen deposition was demonstrated in the rabbit airway (n = 18) at 4 weeks. The present study preliminarily investigated the efficacy of the ATO-NFCS in reducing granulation tissue formation in the trachea of rabbits. The results suggest that the ATO-NFCS is safe in vivo, easy to place, and effective for the suppression of granulation tissue formation.

P62-dependent autophagy in airway smooth muscle cells regulates metabolic reprogramming and promotes airway remodeling

AimsGrowing evidence indicates insufficient autophagy is crucial to airway remodeling in asthma. However, it is uncertain whether p62, an autophagy major regulator, mediates the airway remodeling process. This study aimed to evaluate the role and underlying mechanism of p62 in airway remodeling in asthma. Materials and methodsAirway remodeling was confirmed via histopathology. Western blotting and RT-PCR were used to detect the expression of autophagic and glycolytic proteins, as well as glycolytic genes. Glycolysis was measured by glucose consumption and lactate production. Cell proliferation was analyzed by CCK8 assays while and the scratch test and transwell method were used for cell migration. Key findingsWe found that insufficient autophagic flux and increased p62 expression existed in chronic asthma mice. Additionally, knockdown of p62 inhibited asthmatic human bronchial smooth muscle cells (BSMCs) proliferation and migration in vitro. To elucidate the underlying mechanism of p62-mediated autophagy flux in directing BSMCs function, we demonstrated that knockdown of p62 decreased the glucose consumption and lactate production in BSMCs, whereas p62 overexpression had the opposite effect. Furthermore, we showed that p62 regulated glycolysis in BSMCs by the mTOR/c-Myc/hexokinase 2 (HK2) pathway. SignificanceOur findings suggest that p62 is involved in BSMCs proliferation and migration via the mTOR/c-Myc/HK2-mediated glycolysis, thereby providing a new target for airway remodeling treatment.

Downregulation of miR-140-3p Contributes to Upregulation of CD38 Protein in Bronchial Smooth Muscle Cells.

In allergic bronchial asthma, an increased smooth muscle contractility of the airways is one of the causes of the airway hyperresponsiveness (AHR). Increasing evidence also suggests a possible involvement of microRNAs (miRNAs) in airway diseases, including asthma, although their roles in function and pathology largely unknown. The current study aimed to determine the role of a miRNA, miR-140-3p, in the control of protein expression of CD38, which is believed to regulate the contraction of smooth muscles, including the airways. In bronchial smooth muscles (BSMs) of the mice that were actively sensitized and repeatedly challenged with ovalbumin antigen, an upregulation of CD38 protein concurrently with a significant reduction of miR-140-3p was observed. In cultured human BSM cells (hBSMCs), transfection with a synthetic miR-140-3p inhibitor caused an increase in CD38 protein, indicating that its basal protein expression is regulated by endogenous miR-140-3p. Treatment of the hBSMCs with interleukin-13 (IL-13), an asthma-related cytokine, caused both an upregulation of CD38 protein and a downregulation of miR-140-3p. Transfection of the hBSMCs with miR-140-3p mimic inhibited the CD38 protein upregulation induced by IL-13. On the other hand, neither a CD38 product cyclic ADP-ribose (cADPR) nor its antagonist 8-bromo-cADPR had an effect on the BSM contraction even in the antigen-challenged mice. Taken together, the current findings suggest that the downregulation of miR-140-3p induced by IL-13 might cause an upregulation of CD38 protein in BSM cells of the disease, although functional and pathological roles of the upregulated CD38 are still unclear.

Targeting of protease activator receptor-2 (PAR-2) antagonist FSLLRY-NH2 as an asthma adjuvant therapy.

Asthma is a chronic inflammatory and multifactorial respiratory tract disease. It affects over 18 million adults and 6 million children in the USA with Puerto Ricans showing the highest prevalence (12%–19%). This airways illness can be triggered by an environmental stimulus such as grass pollen, fungi spores, cockroaches allergens, dust mites metabolic compounds, and importantly, by environmental proteases such as trypsin and tryptase. Because of the pivotal role of proteases in the onset of asthma pathophysiology, we focused this study on the serine Protease Activated Receptor-2 (PAR-2), a G-protein-coupled receptor widely expressed in cells across the respiratory tract. Herein, we measured the activation of PAR-2 on primary pulmonary bronchial/tracheal epithelial cells, human small airway epithelial cells, lung bronchial smooth muscle cells (with and without asthma). We tested human-derived eosinophils from 61 Puerto Rican participants (33 asthmatic and 28 non-asthmatic). As surrogate of PAR-2 activation or inhibition we used intracellular calcium mobilization assay. We hypothesized that following exposure of the PAR-2 agonist (AC264613), the studied human primary cell types will increase the mobilization of intracellular calcium levels. In contrast, we expected a decrease of the intracellular calcium levels upon exposure to a PAR-2 antagonist (FSLLRY-NH2). The Puerto Rican-derived eosinophils were analyzed for the proinflammatory markers MAPK/PI3K using flow cytometry (n = 8). As expected, the PAR-2 agonist significantly increased the activation of PAR-2 on the bronchial/tracheal epithelial cells, bronchial smooth muscle cells and human small airway epithelial cells (P = .01). The PAR-2 antagonist significantly decreased the intracellular calcium levels of these lung primary down to undetectable levels (P = .01). Remarkably, the asthmatic-derived eosinophils showed a striking 300% increase of intracellular calcium mobilization suggesting a severe response to the PAR-2 agonist stimuli in asthmatics. In contrast, there were no significant changes between groups after adding the PAR-2 antagonist. Our outcomes revealed that PAR-2 antagonist effectively inhibited the studied primary cells, expecting to decrease the immune response of eosinophils. Most importantly, our results reveal a promising role for the PAR-2 antagonist in targeting bronchial/tracheal epithelial cells, human small airway epithelial cells and bronchial smooth muscle cells with the potential to oblige an asthma adjuvant therapy.

Long noncoding RNA MCM3AP antisense RNA 1 is downregulated in chronic obstructive pulmonary disease and regulates human bronchial smooth muscle cell proliferation.

ObjectivesThis study aimed to investigate the involvement of MCM3AP antisense RNA 1 (MCM3AP-AS1) in chronic obstructive pulmonary disease (COPD).MethodsThe expression levels of plasma MCM3AP-AS1 in COPD patients and healthy controls were measured by quantitative PCR before treatment and at 3 months after the initiation of treatment (post-treatment) from COPD patients. The role of MCM3AP-AS1 in regulating the proliferation of human bronchial smooth muscle cells (HBSMCs) was explored by a cell proliferation assay.ResultsWe found that MCM3AP-AS1 expression was downregulated in the plasma of COPD patients compared with controls. Among controls, MCM3AP-AS1 expression was lower in smokers than never-smokers. A 3-year follow-up study showed that, among smokers, patients with low MCM3AP-AS1 expression showed a higher incidence of COPD. After treatment for COPD, MCM3AP-AS1 expression significantly increased. The cell proliferation assay showed that MCM3AP-AS1 overexpression decreased the proliferation rate of HBSMCs. MCM3AP-AS1 silencing had the opposite effect.ConclusionsMCM3AP-AS1 appears to be downregulated in COPD and to predict its occurrence. MCM3AP-AS1 regulates the proliferation of HBSMCs to participate in airway remodeling.

Asthmatic Bronchial Smooth Muscle Increases CCL5-Dependent Monocyte Migration in Response to Rhinovirus-Infected Epithelium.

Asthma exacerbations, a major concern in therapeutic strategies, are most commonly triggered by viral respiratory infections, particularly with human rhinovirus (HRV). Infection of bronchial epithelial (BE) cells by HRV triggers inflammation, notably monocyte recruitment. The increase of bronchial smooth muscle (BSM) mass in asthma, a hallmark of bronchial remodeling, is associated with the annual rate of exacerbations. The aim of the present study was to assess whether or not BSM could increase monocyte migration induced by HRV-infected BE. We used an advanced in vitro model of co-culture of human BE cells in air-liquid interface with human BSM cells from control and asthmatic patients. Inflammation triggered by HRV infection (HRV-16, MOI 0.1, 1 h) was assessed at 24 h with transcriptomic analysis and multiplex ELISA. In vitro CD14+ monocyte migration was evaluated with modified Boyden chamber. Results showed that HRV-induced monocyte migration was substantially increased in the co-culture model with asthmatic BSM, compared with control BSM. Furthermore, the well-known monocyte migration chemokine, CCL2, was not involved in this increased migration. However, we demonstrated that CCL5 was further increased in the asthmatic BSM co-culture and that anti-CCL5 blocking antibody significantly decreased monocyte migration induced by HRV-infected BE. Taken together, our findings highlight a new role of BSM cells in HRV-induced inflammation and provide new insights in mucosal immunology which may open new opportunities for prevention and/or treatment of asthma exacerbation.

Non-coding RNAs and bronchial smooth muscle hyperresponsiveness in allergic bronchial asthma

Non-coding RNAs, such as microRNAs (miRNAs) and long non-coding RNAs (lncRNAs), play important roles in normal and diseased cell functions. A small GTPase RhoA is a key protein of bronchial smooth muscle (BSM) contraction, and an up-regulation of RhoA has been demonstrated in BSMs of experimental asthma. Our previous study also demonstrated that RhoA translation was controlled by a miRNA, miR-133a, in BSMs. In human BSM cells (hBSMCs), an up-regulation of RhoA was observed when the function of endogenous miR-133a was inhibited by its antagomir. Treatment of hBSMCs with interleukin-13 (IL-13) caused an up-regulation of RhoA and a down-regulation of miR-133a. In a murine experimental asthma, increased expression of IL-13 and RhoA and the BSM hyperresponsiveness were observed. Interestingly, the level of miR-133a was significantly decreased in BSMs of the diseased animals. These findings suggest that RhoA expression is negatively regulated by miR-133a in BSMs, and that the miR-133a down-regulation causes an up-regulation of RhoA, resulting in an augmentation of the contraction. Recent studies also revealed an inhibitory effect of lncRNA Malat1 on the miR-133a function. Thus, lncRNAs/miRNAs might be key regulators of BSM hyperresponsiveness, and provide us a new insight into the treatment of airway hyperresponsiveness in asthmatics.

Non-coding RNAs and bronchial smooth muscle hyperresponsiveness in allergic bronchial asthma

Non-coding RNAs, such as microRNAs (miRNAs) and long non-coding RNAs (lncRNAs), play important roles in normal and diseased cell functions. A small GTPase RhoA is a key protein of bronchial smooth muscle (BSM) contraction, and an up-regulation of RhoA has been demonstrated in BSMs of experimental asthma. Our previous study also demonstrated that RhoA translation was controlled by a miRNA, miR-133a, in BSMs. In human BSM cells (hBSMCs), an up-regulation of RhoA was observed when the function of endogenous miR-133a was inhibited by its antagomir. Treatment of hBSMCs with interleukin-13 (IL-13) caused an up-regulation of RhoA and a down-regulation of miR-133a. In a murine experimental asthma, increased expression of IL-13 and RhoA and the BSM hyperresponsiveness were observed. Interestingly, the level of miR-133a was significantly decreased in BSMs of the diseased animals. These findings suggest that RhoA expression is negatively regulated by miR-133a in BSMs, and that the miR-133a down-regulation causes an up-regulation of RhoA, resulting in an augmentation of the contraction. Recent studies also revealed an inhibitory effect of lncRNA Malat1 on the miR-133a function. Thus, lncRNAs/miRNAs might be key regulators of BSM hyperresponsiveness, and provide us a new insight into the treatment of airway hyperresponsiveness in asthmatics.

Effects of 1,8-cineole (eucalyptol) on the activity of human bronchial tissue

1,8-cineole (eucalyptol) is used for the treatment of bronchial complaints, sinusitis and colds. Experiments have previously shown that 1,8-cineole, a monoterpene (C-10), has a very pronounced spasmolytic effect on smooth muscle fibre. In nearly all clinical applications with 1,8-cineole, especially when used in conjunction with allergic symptoms, histamine receptors prove to be crucial for treatment (use of histamine inhibitors). Results: The desired 1,8-cineole effects are attained through: specifically blocking H1 histamine receptors, without influencing ACh receptors Inhibiting the contractile activity of human bronchial smooth muscle by activating H2 histamine receptors. The ultimate goal of this study was to address the bronchodilatory effect of this compound, using human airway smooth muscle in order to demonstrate a possible role for 1,8-cineole in airway diseases. Keywords: Smooth muscle fibre; 1,8-cineole (eucalyptol); Human bronchial tissue; Bronchitis; Monoterpene

Long Noncoding RNA COPDA1 Promotes Airway Smooth Muscle Cell Proliferation in Chronic Obstructive Pulmonary Disease.

Abnormal expression of long noncoding RNAs (lncRNAs) has been confirmed to be associated with many diseases, including chronic obstructive pulmonary disease (COPD). To gain better understanding of the mechanism of COPD, we investigated the lncRNA and mRNA profiles in the lung tissue of patients with COPD. According to the analysis, one of the significantly different lncRNAs, COPDA1, might participate in the occurrence and development of COPD. Lung tissues were collected from nonsmokers, smokers, or smokers with COPD for RNA sequencing. Bioinformatic analysis and cell experiments were used to define the function of COPDA1, and the effects of COPDA1 on intracellular Ca2+ concentration and cell proliferation were examined after knockdown or overexpression of COPDA1. A number of variations of lncRNAs were found in the comparison of nonsmokers, smokers, and smokers with COPD. GO (Gene Ontology) and KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway analyses indicated that smoking was involved in the activation of cytokines and the cell cycle, which is associated with COPD. According to the lncRNA-mRNA-coexpressing network and enrichment analysis, COPDAz1 and one of its target genes, MS4A1 (membrane-spanning 4-domains family, subfamily A) were investigated, and we discovered that the expression of MS4A1 was closely associated with lncRNA COPDA1 expression in human bronchial smooth muscle cells (HBSMCs). Further study showed that lncRNA COPDA1 upregulated the expression of MS4A1 to increase store-operated calcium entry in the HBSMCs, resulting in the promotion of the proliferation of smooth muscle cells as well as of airway remodeling. COPDA1 might be involved in the regulation of certain signaling pathways in COPD, might promote the proliferation of HBSMCs, and might also be involved in facilitating airway remodeling.

Prevention and relaxation effects of Liriope platyphylla on bronchial asthma in vitro model by suppressing the activities of MAPK/NF-κB pathway

Backgrounds: Bronchial asthma (BA) is a common type of asthma, defined as a chronic inflammation, hyperplasia, and hypercontraction of the airway. In this study, we investigated whether Liriope platyphylla extract (LPP) can help prevent inflammation, suppress hyperplasia, and support relaxation via regulation of MAPK/NF-κB activity. Methods: To investigate the inflammation prevention effects, A549 cells, a human airway epithelial cell line, were pretreated with various concentration of LPP and then treated with EGF recombinant (10 ng/mL). Anti-hyperplasia and muscle relaxation experiments were performed with asthma condition human bronchial smooth muscle (hBSM) cells. The effects of LPP on MAPK/NF-κB activity and MAPK/NF-κB-related signaling, such as COX-2 and iNOS expression, was analyzed through western blot analysis. The mRNA expression levels of Th-2-cell-related cytokines, including IL-6 and TNF-α, and contraction-related factors, such as PLCβ, IP3R, and MLCK, were determined by real-time PCR. Results: LPP pretreatment significantly reduced MAPK/NF-κB activity in EGF-induced asthma condition A549 cells. Therefore, there was significant suppression of both COX-2 and iNOS expression. In addition, the mRNA expression of IL-6 and TNF-α was dose depen-dently reduced. Although there was no statistical significance, factors related to contraction showed a tendency to decrease. Conclusion: Based on our results, we believe that LPP shows good potential for application as a therapeutic agent for asthma through inflammation prevention and bronchial smooth muscle relaxation.

Niclosamide ethanolamine induces trachea relaxation and inhibits proliferation and migration of trachea smooth muscle cells

Our previous study found that the anthelmintic drug niclosamide relaxed the constricted arteries and inhibited proliferation and migration of vascular smooth muscle cells. Here, we investigated the effect of niclosamide ethanolamine (NEN) on trachea function and the proliferation and migration of trachea smooth muscle cells. Isometric tension of trachea was recorded by multi-channel myograph system. The cell proliferation was detected by using BrdU cell proliferation assay. The cell migration ability was evaluated by using scratch assay. The protein level was measured by using western blot technique. Acute treatment with NEN dose-dependently relaxed acetylcholine chloride (Ach)- and High K+ physiological salt solution (KPSS)-induced constriction of mice trachea. Pre-treatment with NEN inhibited Ach- and KPSS-induced constriction of mice trachea. NEN treatment inhibited proliferation of human bronchial smooth muscle cells (HBSMCs), inhibited migration of HBSMCs and rat primary trachea smooth muscle cells. NEN treatment activated adenosine monophosphate activated protein kinase (AMPK) activity and inhibited signal transducer and activator of transcription 3 (STAT3) activity in HBSMCs. In conclusion, niclosamide ethanolamine induces trachea relaxation and inhibits proliferation and migration of trachea smooth muscle cells, indicating that niclosamide might be a potential drug for chronic asthma treatment.

Formoterol counteracts the inhibitory effect of cigarette smoke on glucocorticoid-induced leucine zipper (GILZ) transactivation in human bronchial smooth muscle cells

Cigarette smokers with asthma and chronic obstructive pulmonary disease (COPD) are less responsive to glucocorticoids (GCs). The anti-inflammatory action of GCs depends also on their ability to transactivate genes such as GC-induced leucine zipper (GILZ). We investigated the effects of aqueous cigarette smoke extract (CSE) on GILZ transactivation evoked by 17-beclomethasone monopropionate (BMP) or fluticasone propionate (FP) in the presence or absence of the long acting β2-adrenoceptor agonist (LABA) bronchodilator formoterol or salmeterol in human primary cultures of human bronchial smooth muscle cells (HBSMC). We monitored GC receptor Ser211 phosphorylation by western blot analysis and GC receptor nuclear translocation by immunostaining followed high-content imaging analysis. BMP, as well as FP, induced GILZ expression in a concentration-dependent manner (EC50 of 0.87 and 0.16 nM respectively). Pre-incubation with CSE inhibited GC-evoked GILZ transactivation (>50%), GC receptor Ser211 phosphorylation and nuclear translocation. Both formoterol and salmeterol counteracted the effect of CSE on GC-induced GILZ expression but not on nuclear translocation or phosphorylation. The effect of formoterol was mimicked by the cAMP-elevating agent forskolin and blocked by ICI 118,551, a selective β2-adrenoceptor antagonist. Pre-incubation with TNF-α also reduced GC-evoked GILZ transactivation but was not counteracted by formoterol undercovering a different responsiveness to LABAs of TNF-α in comparison to CSE.In sum, CSE inhibits GC-evoked transactivation of GILZ and such effect is counteracted by LABAs, through β2-adrenoceptors and a cAMP-dependent mechanism. This study sheds light on a mechanism underlying complementary interactions between LABAs and inhaled GCs that could be relevant in smokers with asthma and COPD.

Hydroxysafflor yellow A (HSYA) targets the platelet-activating factor (PAF) receptor and inhibits human bronchial smooth muscle activation induced by PAF.

Hydroxysafflor yellow A (HSYA) is the main active ingredient of edible plant safflower. HSYA has demonstrated anti-inflammatory effects. The inflammatory response is the key mechanism responsible for asthma, and the pro-inflammatory platelet-activating factor (PAF) is known to play a role in the pathology of bronchial asthma. In this study, we stimulated human bronchial smooth muscle cells (HBSMCs) with PAF and examined the effects of HSYA on the resulting asthma-related process. PAF stimulation induced HBSMC activation, induced proliferation, increased expression of the pro-inflammatory cytokines interleukin (IL)-6, IL-1β, and tumor necrosis factor-α, and activated asthma-related signaling pathways. All these effects were significantly inhibited by treatment with HSYA (9, 27, 81 μmol L-1). The effects of HSYA were prevented by the addition of a PAF receptor (PAFR) antagonist or by PAFR gene silencing with small interfering RNA. These results suggest that HSYA may inhibit PAF-induced activation of HBSMCs by targeting the PAFR. Overall, these findings provide evidence that HSYA can be applied as a potential therapeutic agent in the treatment of bronchial asthma.

Comparison of the performance of 1D SDS-PAGE with nondenaturing 2DE on the analysis of proteins from human bronchial smooth muscle cells using quantitative LC-MS/MS

The supernatant and precipitate protein fractions from human bronchial smooth muscle cells (HBSMC) were analyzed using 1D SDS-PAGE-LC-MS/MS and the performance of the method was compared with that of nondenaturing 2DE-LC-MS/MS applied to the supernatant fraction, the methods and the results have been reported previously. The 1D gel lanes were cut into pieces of the same size, in order to enable the reproduction of digital 1D images of the individual MS-assigned proteins. When the obtained information on individual HBSMC proteins was compared between the two methods, SDS-PAGE is advantageous in visualizing the quantity differences between differently treated samples, whereas nondenatuing 2DE is advantageous in visualizing protein interactions. SDS-PAGE-MS of the supernatant fraction provided the assignment of 2552 proteins and their percent abundance ranged from 3.5% to 2 × 10−4%. 2DE-MS of the supernatant fraction provided 4323 proteins with percent abundance ranged from 3.6% to 1 × 10−5%, suggesting that the step of isoelectric focusing served to raise the sensitivity of the method. The proteins in the precipitate fraction, which could not be analyzed by 2DE-MS, were characterized by the abundance of proteins allocated to “membrane” within the category “Cellular component” of UniProtKB database and especially those allocated to “transmembrane” within the subcategory “membrane.” On the other hand, there were about 600 “membrane” proteins which showed more than two-fold higher percent abundance in 2DE-MS than in SDS-PAGE-MS. These results showed that 1D SDS-PAGE and nondenaturing 2DE would provide complementary information on the analysis of proteins and protein interactions in cells.

Na+/Ca2+ Exchanger 1 in Airway Smooth Muscle of Allergic Inflammation Mouse Model.

Cytosolic free Ca2+ ([Ca2+]cyt) is essential for airway contraction, secretion and remodeling. [Ca2+]cyt homeostasis is controlled by several critical molecules, one of which is the Na+/Ca2+ exchanger 1 (NCX1) in the plasma membrane. Since little is currently known about NCX1 in the airway smooth muscle and its involvement in airway diseases, the present study was designed to investigate the expression and function of NCX1 in normal airway smooth muscle and its relevance to airway inflammation. Western blot analysis, tracheal smooth muscle contraction, and [Ca2+]cyt measurements were performed in mouse tracheal smooth muscle tissues and primary airway smooth muscle cell cultures. Additional studies were performed in a mouse model of allergic airway inflammation. Our data showed that NCX1 proteins were expressed in the human bronchial smooth muscle cells (HBSMCs), murine airway and whole lung. Carbachol raised [Ca2+]cyt in mouse tracheal smooth muscle cells and induced murine tracheal contraction, all of which were significantly attenuated by KB-R7943, a selective NCX inhibitor. Removal of extracellular Na+ increased [Ca2+]cyt in HBSMCs and mouse tracheal SMCs, which was dependent on extracellular Ca2+ and sensitive to KB-R7943. TNF-α treatment of HBSMCs significantly upregulated mRNA and protein expression of NCX1 and enhanced NCX activity. Finally, KB-R7943 abolished the airway hyperresponsiveness to methacholine in an ovalbumin-induced mouse model of allergic airway inflammation. Together, these findings indicate that NCX1 in airway smooth muscle may play an important role in the development of airway hyperresponsiveness, and downregulation or inhibition of NCX1 may serve as a potential therapeutic approach for asthma.