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.

Identification and characterization of an atypical Gαs-biased β2AR agonist that fails to evoke airway smooth muscle cell tachyphylaxis

G protein–coupled receptors display multifunctional signaling, offering the potential for agonist structures to promote conformational selectivity for biased outputs. For β2-adrenergic receptors (β2AR), unbiased agonists stabilize conformation(s) that evoke coupling to Gαs (cyclic adenosine monophosphate [cAMP] production/human airway smooth muscle [HASM] cell relaxation) and β-arrestin engagement, the latter acting to quench Gαs signaling, contributing to receptor desensitization/tachyphylaxis. We screened a 40-million-compound scaffold ranking library, revealing unanticipated agonists with dihydroimidazolyl-butyl-cyclic urea scaffolds. The S-stereoisomer of compound C1 shows no detectable β-arrestin engagement/signaling by four methods. However, C1-S retained Gαs signaling—a divergence of the outputs favorable for treating asthma. Functional studies with two models confirmed the biasing: β2AR-mediated cAMP signaling underwent desensitization to the unbiased agonist albuterol but not to C1-S, and desensitization of HASM cell relaxation was observed with albuterol but not with C1-S. These HASM results indicate biologically pertinent biasing of C1-S, in the context of the relevant physiologic response, in the human cell type of interest. Thus, C1-S was apparently strongly biased away from β-arrestin, in contrast to albuterol and C5-S. C1-S structural modeling and simulations revealed binding differences compared with unbiased epinephrine at transmembrane (TM) segments 3,5,6,7 and ECL2. C1-S (R2 = cyclohexane) was repositioned in the pocket such that it lost a TM6 interaction and gained a TM7 interaction compared with the analogous unbiased C5-S (R2 = benzene group), which appears to contribute to C1-S biasing away from β-arrestin. Thus, an agnostic large chemical-space library identified agonists with receptor interactions that resulted in relevant signal splitting of β2AR actions favorable for treating obstructive lung disease.

Network and co-expression analysis of airway smooth muscle cell transcriptome delineates potential gene signatures in asthma

Airway smooth muscle (ASM) is known for its role in asthma exacerbations characterized by acute bronchoconstriction and remodeling. The molecular mechanisms underlying multiple gene interactions regulating gene expression in asthma remain elusive. Herein, we explored the regulatory relationship between ASM genes to uncover the putative mechanism underlying asthma in humans. To this end, the gene expression from human ASM was measured with RNA-Seq in non-asthmatic and asthmatic groups. The gene network for the asthmatic and non-asthmatic group was constructed by prioritizing differentially expressed genes (DEGs) (121) and transcription factors (TFs) (116). Furthermore, we identified differentially connected or co-expressed genes in each group. The asthmatic group showed a loss of gene connectivity due to the rewiring of major regulators. Notably, TFs such as ZNF792, SMAD1, and SMAD7 were differentially correlated in the asthmatic ASM. Additionally, the DEGs, TFs, and differentially connected genes over-represented in the pathways involved with herpes simplex virus infection, Hippo and TGF-β signaling, adherens junctions, gap junctions, and ferroptosis. The rewiring of major regulators unveiled in this study likely modulates the expression of gene-targets as an adaptive response to asthma. These multiple gene interactions pointed out novel targets and pathways for asthma exacerbations.

Essential role of smooth muscle Rac1 in severe asthma-associated airway remodelling

BackgroundSevere asthma is a chronic lung disease characterised by inflammation, airway hyperresponsiveness (AHR) and airway remodelling. The molecular mechanisms underlying uncontrolled airway smooth muscle cell (aSMC) proliferation involved in pulmonary remodelling are still largely unknown. Small G proteins of the Rho family (RhoA, Rac1 and Cdc42) are key regulators of smooth muscle functions and we recently demonstrated that Rac1 is activated in aSMC from allergic mice. The objective of this study was to assess the role of Rac1 in severe asthma-associated airway remodelling.Methods and resultsImmunofluorescence analysis in human bronchial biopsies revealed an increased Rac1 activity in aSMC from patients with severe asthma compared with control subjects. Inhibition of Rac1 by EHT1864 showed that Rac1 signalling controlled human aSMC proliferation induced by mitogenic stimuli through the signal transducer and activator of transcription 3 (STAT3) signalling pathway. In vivo, specific deletion of Rac1 in SMC or pharmacological inhibition of Rac1 by nebulisation of NSC23766 prevented AHR and aSMC hyperplasia in a mouse model of severe asthma. Moreover, the Rac1 inhibitor prevented goblet cell hyperplasia and epithelial cell hypertrophy whereas treatment with corticosteroids had less effect. Nebulisation of NSC23766 also decreased eosinophil accumulation in the bronchoalveolar lavage of asthmatic mice.ConclusionThis study demonstrates that Rac1 is overactive in the airways of patients with severe asthma and is essential for aSMC proliferation. It also provides evidence that Rac1 is causally involved in AHR and airway remodelling. Rac1 may represent as an interesting target for treating both AHR and airway remodelling of patients with severe asthma.

LOXL2 Mediates Airway Smooth Muscle Cell Matrix Stiffness and Drives Asthmatic Airway Remodelling

Airway smooth muscle cells (ASM) are fundamental to asthma pathogenesis, influencing bronchoconstriction, airway hyper-responsiveness, and airway remodelling. Extracellular matrix (ECM) can influence tissue remodelling pathways, however, to date no study has investigated the effect of ASM ECM stiffness and crosslinking on the development of asthmatic airway remodelling. We hypothesised that TGFβ activation by ASM is influenced by ECM in asthma and sought to investigate the mechanisms involved.This study combines in vitro and in vivo approaches: human ASM cells were used in vitro to investigate basal TGFβ activation and expression of ECM crosslinking enzymes. Human bronchial biopsies from asthmatic and non-asthmatic donors were used to confirm LOXL2 expression ASM. A chronic ovalbumin model of asthma was used to study the effect of LOXL2 inhibition on airway remodelling.We found that ASM cells from asthmatics activated more TGFβ basally than non-asthmatic controls and that diseased cell-derived ECM influences levels of TGFβ activated. Our data demonstrate that the ECM crosslinking enzyme LOXL2 is increased in asthmatic ASM cells and in bronchial biopsies. Crucially, we show that LOXL2 inhibition reduces ECM stiffness and TGFβ activation in vitro, and can reduce subepithelial collagen deposition and ASM thickness, two features of airway remodelling, in an ovalbumin mouse model of asthma.These data are the first to highlight a role for LOXL2 in the development of asthmatic airway remodelling and suggest that LOXL2 inhibition warrants further investigation as a potential therapy to reduce remodelling of the airways in severe asthma.