Role of airway smooth muscle cell phenotypes in airway tone and obstruction in guinea pig asthma model

Background Airway obstruction (AO) in asthma is driven by airway smooth muscle (ASM) contraction. AO can be induced extrinsically by direct stimulation of ASM with contractile agonists as histamine, or by indirect provocation with antigens as ovalbumin, while the airway tone is dependent on intrinsic mechanisms. The association of the ASM phenotypes involved in different types of AO and airway tone in guinea pigs was evaluated. Methods Guinea pigs were sensitized to ovalbumin and challenged with antigen. In each challenge, the maximum OA response to ovalbumin was determined, and before the challenges, the tone of the airways. At third challenge, airway responsiveness (AR) to histamine was evaluated and ASM cells from trachea were disaggregated to determinate: (a) by flow cytometry, the percentage of cells that express transforming growth factor-β1 (TGF-β1), interleukin-13 (IL-13) and sarco-endoplasmic Ca2+ ATPase-2b (SERCA2b), (b) by RT-PCR, the SERCA2B gene expression, (c) by ELISA, reduced glutathione (GSH) and, (d) Ca2+ sarcoplasmic reticulum refilling rate by microfluorometry. Control guinea pig group received saline instead ovalbumin. Results Antigenic challenges in sensitized guinea pigs induced indirect AO, AR to histamine and increment in airway tone at third challenge. No relationship was observed between AO induced by antigen and AR to histamine with changes in airway tone. The extent of antigen-induced AO was associated with both, TGF-β1 expression in ASM and AR degree. The magnitude of AR and antigen-induced AO showed an inverse correlation with GSH levels in ASM. The airway tone showed an inverse association with SERCA2b expression. Conclusions Our data suggest that each type of AO and airway tone depends on different ASM phenotypes: direct and indirect AO seems to be sensitive to the level of oxidative stress; indirect obstruction induced by antigen appears to be influenced by the expression of TGF-β1 and the SERCA2b expression level plays a role in the airway tone.

Scopoletin inhibits PDGF-BB-induced proliferation and migration of airway smooth muscle cells by regulating NF-κκB signaling pathway

Background: Asthma is a common chronic inflammatory disease of the airway, and airway remodeling and the proliferation mechanism of airway smooth muscle cells (ASMCs) is of great significance to combat this disease.Objective: To assess possible effects of scopoletin on asthma and the potential signaling pathway.Materials and methods: ASMCs were treated PDGF-BB and scopoletin and subjected to cell viability detection by CCK-8 assay. Cell migration of ASMCs was determined by a wound closure assay and transwell assay. The protein level of MMP2, MMP9, calponin and α-SMA were measured using western blot. The levels of NF-κB signaling pathway were detected by Western blotting.Results: Scopoletin inhibited proliferation of PDGF-BB - induced ASMCs. Also it suppressed the migration and invasion of PDGF-BB - induced ASMCs. We further showed that Scopoletin regulated phenotypic transition of ASMCs. Mechanically, Scopoletin inhibited proliferation and invasion of ASMCs by regulating NF-κB signaling pathway.Conclusions: We therefore thought Scopoletin could serve as a promising drug for the treatment of asthma.

Curcumol inhibits PDGF-BB-induced proliferation and migration of airway smooth muscle cells by suppressing ERK/CREB pathway

Background: Curcumol, possessing antiviral, antifungal, antimicrobial, anticancer, and anti-inflammatory properties, has been widely used in treating cancers and liver fibrosis. The aim of this study was to determine the effect of curcumol on the progression of asthma.Materials and methods: Curcumol was administrated to platelet-derived growth factor (PDGF)- BB-stimulated airway smooth muscle cells (ASMCs). The proliferation of ASMCs was assessed by MTT and EdU incorporation assays. The apoptosis of ASMCs was measured by flow cytometry and Western blotting. The migration of ASMCs was evaluated by Transwell migration assay and Western blotting. The regulatory effects of curcumol on extracellular signal-regulated protein kinase (ERK)/cAMP response element-binding protein (CREB) pathway was evaluated by Western blotting.Results: The proliferation and migration of ASMCs induced by PDGF-BB was suppressed, and the apoptosis of ASMCs was elevated by curcumol in a dose-dependent manner. The activation of ERK/CREB pathway induced by PDGF-BB was suppressed by curcumol.Conclusion: Curcumol could suppress ERK/CREB pathway to inhibit proliferation and migration and promote apoptosis of PDGF-BB-stimulated ASMCs. These findings suggest that curcumol may act as a potential drug for asthma treatment.

STIM1 is a core trigger of airway smooth muscle remodeling and hyperresponsiveness in asthma

Airway remodeling and airway hyperresponsiveness are central drivers of asthma severity. Airway remodeling is a structural change involving the dedifferentiation of airway smooth muscle (ASM) cells from a quiescent to a proliferative and secretory phenotype. Here, we show up-regulation of the endoplasmic reticulum Ca2+ sensor stromal-interacting molecule 1 (STIM1) in ASM of asthmatic mice. STIM1 is required for metabolic and transcriptional reprogramming that supports airway remodeling, including ASM proliferation, migration, secretion of cytokines and extracellular matrix, enhanced mitochondrial mass, and increased oxidative phosphorylation and glycolytic flux. Mechanistically, STIM1-mediated Ca2+ influx is critical for the activation of nuclear factor of activated T cells 4 and subsequent interleukin-6 secretion and transcription of pro-remodeling transcription factors, growth factors, surface receptors, and asthma-associated proteins. STIM1 drives airway hyperresponsiveness in asthmatic mice through enhanced frequency and amplitude of ASM cytosolic Ca2+ oscillations. Our data advocates for ASM STIM1 as a target for asthma therapy.

Effects of airway smooth muscle contraction and inflammation on lung tissue compliance

There are renewed interests in using the parameter K of Salazar-Knowles' equation to assess lung tissue compliance. K either decreases or increases when the lung's parenchyma stiffens or loosens, respectively. However, whether K is affected by other common features of respiratory diseases, such as inflammation and airway smooth muscle (ASM) contraction, is unknown. Herein, male C57BL/6 mice were treated intranasally with either saline or lipopolysaccharide (LPS) at 1 mg/Kg to induce pulmonary inflammation. They were then subjected to either a multiple or a single-dose challenge with methacholine to activate ASM to different degrees. A quasi-static pressure-driven partial pressure-volume maneuver was performed before and after methacholine. The Salazar-Knowles' equation was then fitted to the deflation limb of the P-V loop to obtain K, as well as the parameter A, an estimate of lung volume (inspiratory capacity). The fitted curve was also used to derive the quasi-static elastance (Est) at 5 cmH2O. The results demonstrate that LPS and both methacholine challenges increased Est. LPS also decreased A, but did not affect K. In contradistinction, methacholine decreased both A and K in the multiple-dose challenge, while it decreased K but not A in the single-dose challenge. These results suggest that LPS increases Est by reducing the open lung volume (A) and without affecting tissue compliance (K), while methacholine increases Est by decreasing tissue compliance with or without affecting lung volume. We conclude that lung tissue compliance, assessed using the parameter K of Salazar-Knowles' equation, is insensitive to inflammation but sensitive to ASM contraction.

MiR-18a Inhibits PI3K/AKT Signaling Pathway to Regulate PDGF BB-Induced Airway Smooth Muscle Cell Proliferation and Phenotypic Transformation

The increased proliferation and migration of airway smooth muscle cells (ASMCs) is a key process in the formation of airway remodeling in asthma. In this study, we focused on the expression of mircoRNA-18a (miR-18a) in airway remodeling in bronchial asthma and its related mechanisms. ASMCs are induced by platelet-derived growth factor BB (PDGF-BB) for in vitro airway remodeling. The expression of miR-18a in sputum of asthmatic patients and healthy volunteers was detected by qRT-PCR. The expression of miR-18a was over-expressed or interfered with in PDGF-BB-treated ASMCs. Cell proliferation, apoptosis and migration were detected by MTT, flow cytometry and Transwell, respectively; the expression of contractile phenotype marker proteins (SM-22α, α-SM-actin, calponin) and key molecules of the phosphatidylinositol 3-kinase (PI3K)/AKT pathway (PI3K, p-PI3K, AKT and p-AKT) in ASMCs were detected by Western blot. The expression of miR-18a was down-regulated in the sputum and PDGF-BB-treated ASMCs of asthma patients. PDGF-BB could promote the proliferation and migration of ASMCs and inhibit their apoptosis; it could also promote the phenotypic transformation of ASMCs and activate the PI3K/AKT pathway. MiR-18a could inhibit the proliferation, migration ability and phenotypic transformation of ASMCs induced by PDGF-BB to a certain extent and alleviate the effect of PDGF-BB in supressing apoptosis, while miR-18a could inhibit the activation of the PI3K/AKT pathway. MiR-18a inhibits PDGF-BB-induced proliferation, migration and phenotypic conversion of ASMCs by inhibiting the PI3K/AKT pathway, thus attenuating airway remodeling in asthma.