Airway Smooth Muscle Function Research Articles

Rho-Kinase Inhibition of Active Force and Passive Tension in Airway Smooth Muscle: A Strategy for Treating Airway Hyperresponsiveness in Asthma.

Rho-kinase inhibitors have been identified as a class of potential drugs for treating asthma because of their ability to reduce airway inflammation and active force in airway smooth muscle (ASM). Past research has revealed that, besides the effect on the ASM's force generation, rho-kinase (ROCK) also regulates actin filament formation and filament network architecture and integrity, thus affecting ASM's cytoskeletal stiffness. The present review is not a comprehensive examination of the roles played by ROCK in regulating ASM function but is specifically focused on passive tension, which is partially determined by the cytoskeletal stiffness of ASM. Understanding the molecular basis for maintaining active force and passive tension in ASM by ROCK will allow us to determine the suitability of ROCK inhibitors and its downstream enzymes as a class of drugs in treating airway hyperresponsiveness seen in asthma. Because clinical trials using ROCK inhibitors in the treatment of asthma have yet to be conducted, the present review focuses on the in vitro effects of ROCK inhibitors on ASM's mechanical properties which include active force generation, relaxation, and passive stiffness. The review provides justification for future clinical trials in the treatment of asthma using ROCK inhibitors alone and in combination with other pharmacological and mechanical interventions.

CFTR dysfunction in smooth muscle drives TGFβ dependent airway hyperreactivity

BackgroundThe primary underlying defect in cystic fibrosis (CF) is disrupted ion transport in epithelia throughout the body. It is unclear if symptoms such as airway hyperreactivity (AHR) and increased airway smooth muscle (ASM) volume in people with CF are due to inherent abnormalities in smooth muscle or are secondary to epithelial dysfunction. Transforming Growth Factor beta 1 (TGFβ) is an established genetic modifier of CF lung disease and a known driver of abnormal ASM function. Prior studies have demonstrated that CF mice develop greater AHR, goblet cell hyperplasia, and ASM hypertrophy after pulmonary TGFβ exposure. However, the mechanism driving these abnormalities in CF lung disease, specifically the contribution of CFTR loss in ASM, was unknown.MethodsIn this study, mice with smooth muscle-specific loss of CFTR function (Cftrfl/fl; SM-Cre mice) were exposed to pulmonary TGFβ. The impact on lung pathology and physiology was investigated through examination of lung mechanics, Western blot analysis, and pulmonary histology.ResultsCftrfl/fl; SM-Cre mice treated with TGFβ demonstrated greater methacholine-induced AHR than control mice. However, Cftrfl/fl; SM-Cre mice did not develop increased inflammation, ASM area, or goblet cell hyperplasia relative to controls following TGFβ exposure.ConclusionsThese results demonstrate a direct smooth muscle contribution to CF airway obstruction mediated by TGFβ. Dysfunction in non-epithelial tissues should be considered in the development of CF therapeutics, including potential genetic therapies.

Membrane adhesion junctions regulate airway smooth muscle phenotype and function.

The local environment surrounding airway smooth muscle (ASM) cells has profound effects on the physiological and phenotypic properties of ASM tissues. ASM is continually subjected to the mechanical forces generated during breathing and to the constituents of its surrounding extracellular milieu. The smooth muscle cells within the airways continually modulate their properties to adapt to these changing environmental influences. Smooth muscle cells connect to the extracellular cell matrix (ECM) at membrane adhesion junctions that provide mechanical coupling between smooth muscle cells within the tissue. Membrane adhesion junctions also sense local environmental signals and transduce them to cytoplasmic and nuclear signaling pathways in the ASM cell. Adhesion junctions are composed of clusters of transmembrane integrin proteins that bind to ECM proteins outside the cell and to large multiprotein complexes in the submembranous cytoplasm. Physiological conditions and stimuli from the surrounding ECM are sensed by integrin proteins and transduced by submembranous adhesion complexes to signaling pathways to the cytoskeleton and nucleus. The transmission of information between the local environment of the cells and intracellular processes enables ASM cells to rapidly adapt their physiological properties to modulating influences in their extracellular environment: mechanical and physical forces that impinge on the cell, ECM constituents, local mediators, and metabolites. The structure and molecular organization of adhesion junction complexes and the actin cytoskeleton are dynamic and constantly changing in response to environmental influences. The ability of ASM to rapidly accommodate to the ever-changing conditions and fluctuating physical forces within its local environment is essential for its normal physiological function.

Mechanistic Links Between Obesity and Airway Pathobiology Inform Therapies for Obesity-Related Asthma.

Obesity-related asthma is associated with a high disease burden and a poor response to existent asthma therapies, suggesting that it is a distinct asthma phenotype. The proposed mechanisms that contribute to obesity-related asthma include the effects of the mechanical load of obesity, adipokine perturbations, and immune dysregulation. Each of these influences airway smooth muscle function. Mechanical fat load alters airway smooth muscle stretch affecting airway wall geometry, airway smooth muscle contractility, and agonist delivery; weight loss strategies, including medically induced weight loss, counter these effects. Among the metabolic disturbances, insulin resistance and free fatty acid receptor activation influence distinct signaling pathways in the airway smooth muscle downstream of both the M2 muscarinic receptor and the β2 adrenergic receptor, such as phospholipase C and the extracellular signal-regulated kinase signaling cascade. Medications that decrease insulin resistance and dyslipidemia are associated with a lower asthma disease burden. Leptin resistance is best understood to modulate muscarinic receptors via the neural pathways but there are no specific therapies for leptin resistance. From the immune perspective, monocytes and T helper cells are involved in systemic pro-inflammatory profiles driven by obesity, notably associated with elevated levels of interleukin-6. Clinical trials on tocilizumab, an anti-interleukin antibody, are ongoing for obesity-related asthma. This armamentarium of therapies is distinct from standard asthma medications, and once investigated for its efficacy and safety among children, will serve as a novel therapeutic intervention for pediatric obesity-related asthma. Irrespective of the directionality of the association between asthma and obesity, airway-specific mechanistic studies are needed to identify additional novel therapeutic targets for obesity-related asthma.

Airway Smooth Muscle Regulated by Oxidative Stress in COPD.

Since COPD is a heterogeneous disease, a specific anti-inflammatory therapy for this disease has not been established yet. Oxidative stress is recognized as a major predisposing factor to COPD related inflammatory responses, resulting in pathological features of small airway fibrosis and emphysema. However, little is known about effects of oxidative stress on airway smooth muscle. Cigarette smoke increases intracellular Ca2+ concentration and enhances response to muscarinic agonists in human airway smooth muscle. Cigarette smoke also enhances proliferation of these cells with altered mitochondrial protein. Hydrogen peroxide and 8-isoprostans are increased in the exhaled breath condensate in COPD. These endogenous oxidants cause contraction of tracheal smooth muscle with Ca2+ dynamics through Ca2+ channels and with Ca2+ sensitization through Rho-kinase. TNF-α and growth factors potentiate proliferation of these cells by synthesis of ROS. Oxidative stress can alter the function of airway smooth muscle through Ca2+ signaling. These phenotype changes are associated with manifestations (dyspnea, wheezing) and pathophysiology (airflow limitation, airway remodeling, airway hyperresponsiveness). Therefore, airway smooth muscle is a therapeutic target against COPD; oxidative stress should be included in treatable traits for COPD to advance precision medicine. Research into Ca2+ signaling related to ROS may contribute to the development of a novel agent for COPD.

Transgenic overexpression of α7 integrin in smooth muscle attenuates allergen-induced airway inflammation in a murine model of asthma.

Asthma is a chronic inflammatory disorder of the lower airways characterized by modulation of airway smooth muscle (ASM) function. Infiltration of smooth muscle by inflammatory mediators is partially regulated by transmembrane integrins and the major smooth muscle laminin receptor α7β1 integrin plays a critical role in the maintenance of ASM phenotype. The goal of the current study was to investigate the role of α7 integrin in asthma using smooth muscle-specific α7 integrin transgenic mice (TgSM-Itgα7) using both acute and chronic OVA sensitization and challenge protocols that mimic mild to severe asthmatic phenotypes. Transgenic over-expression of the α7 integrin in smooth muscle resulted in a significant decrease in airway resistance relative to controls, reduced the total number of inflammatory cells and substantially inhibited the production of crucial Th2 and Th17 cytokines in airways. This was accompanied by decreased secretion of various inflammatory chemokines such as eotaxin/CCL11, KC/CXCL3, MCP-1/CCL2, and MIP-1β/CCL4. Additionally, α7 integrin overexpression significantly decreased ERK1/2 phosphorylation in the lungs of TgSM-Itgα7 mice and affected proliferative, contractile, and inflammatory downstream effectors of ERK1/2 that drive smooth muscle phenotype in the lung. Taken together, these results support the hypothesis that enhanced expression of α7 integrin in vivo inhibits allergic inflammation and airway resistance. Moreover, we identify ERK1/2 as a potential target by which α7 integrin signals to regulate airway inflammation. We conclude that identification of therapeutics targeting an increase in smooth muscle α7 integrin expression could serve as a potential novel treatment for asthma.

Extracellular cAMP-Adenosine Pathway Signaling: A Potential Therapeutic Target in Chronic Inflammatory Airway Diseases.

Adenosine is a purine nucleoside that, via activation of distinct G protein-coupled receptors, modulates inflammation and immune responses. Under pathological conditions and in response to inflammatory stimuli, extracellular ATP is released from damaged cells and is metabolized to extracellular adenosine. However, studies over the past 30 years provide strong evidence for another source of extracellular adenosine, namely the “cAMP-adenosine pathway.” The cAMP-adenosine pathway is a biochemical mechanism mediated by ATP-binding cassette transporters that facilitate cAMP efflux and by specific ectoenzymes that convert cAMP to AMP (ecto-PDEs) and AMP to adenosine (ecto-nucleotidases such as CD73). Importantly, the cAMP-adenosine pathway is operative in many cell types, including those of the airways. In airways, β2-adrenoceptor agonists, which are used as bronchodilators for treatment of asthma and chronic respiratory diseases, stimulate cAMP efflux and thus trigger the extracellular cAMP-adenosine pathway leading to increased concentrations of extracellular adenosine in airways. In the airways, extracellular adenosine exerts pro-inflammatory effects and induces bronchoconstriction in patients with asthma and chronic obstructive pulmonary diseases. These considerations lead to the hypothesis that the cAMP-adenosine pathway attenuates the efficacy of β2-adrenoceptor agonists. Indeed, our recent findings support this view. In this mini-review, we will highlight the potential role of the extracellular cAMP-adenosine pathway in chronic respiratory inflammatory disorders, and we will explore how extracellular cAMP could interfere with the regulatory effects of intracellular cAMP on airway smooth muscle and innate immune cell function. Finally, we will discuss therapeutic possibilities targeting the extracellular cAMP-adenosine pathway for treatment of these respiratory diseases.

NMDA receptor modulation and severe acute respiratory syndrome treatment.

N-Methyl-D-aspartate (NMDA) subtype of glutamate receptors is expressed in the human lungs and central nervous system. NMDA receptor potentiation could increase calcium ion influx and promote downstream signaling mechanisms associated with cellular contractions that are disrupted in severe acute respiratory syndrome. Pharmacological effects generated by triggering glutamate receptor function in the brain, coupled with concurrent stimulation of the respiratory tract, may produce a synergetic effect, improving the airway smooth muscle function. A novel multipronged intervention to simultaneously potentiate NMDA receptors expressed both in the central nervous system and airway muscles would be helpful for the treatment of severe acute respiratory syndrome that deteriorates peripheral and central nervous system function before causing death in humans.

Retraction Note: The anti-proliferative and anti-inflammatory response of COPD airway smooth muscle cells to hydrogen sulfide

COPD is a common, highly debilitating disease of the airways, primarily caused by smoking. Chronic inflammation and structural remodelling are key pathological features of this disease caused, in part, by the aberrant function of airway smooth muscle (ASM). We have previously demonstrated that hydrogen sulfide (H2S) can inhibit ASM cell proliferation and CXCL8 release, from cells isolated from non-smokers. We examined the effect of H2S upon ASM cells from COPD patients. ASM cells were isolated from non-smokers, smokers and patients with COPD (n = 9). Proliferation and cytokine release (IL-6 and CXCL8) of ASM was induced by FCS, and measured by bromodeoxyuridine incorporation and ELISA, respectively. Exposure of ASM to H2S donors inhibited FCS-induced proliferation and cytokine release, but was less effective upon COPD ASM cells compared to the non-smokers and smokers. The mRNA and protein expression of the enzymes responsible for endogenous H2S production (cystathionine-β-synthase [CBS] and 3-mercaptopyruvate sulphur transferase [MPST]) were inhibited by H2S donors. Finally, we report that exogenous H2S inhibited FCS-stimulated phosphorylation of ERK–1/2 and p38 mitogen activated protein kinases (MAPKs), in the non-smoker and smoker ASM cells, with little effect in COPD cells. H2S production provides a novel mechanism for the repression of ASM proliferation and cytokine release. The ability of COPD ASM cells to respond to H2S is attenuated in COPD ASM cells despite the presence of the enzymes responsible for H2S production.

The Evaluation of Acute Lung Injury Induced by Nitrogen Mustard in Mouse Precision Cut Lung Slices

Nitrogen Mustard (NM; mechlorethamine hydrochloride) is a cytotoxic alkylating agent known to cause progressive pulmonary injury. Its toxic properties elicit extensive tissue remodeling within the respiratory tract, contributing to the development of chronic lung disease. Precision-Cut Lung Slices (PCLS) represent an ex vivo alternative to in vivo studies, minimizing animal numbers while increasing experimental replicates, which is advantageous for mechanistic toxicology studies and evaluating potential therapeutics. PCLS are three-dimensional excerpts of the lung that include all resident cell types; they remain viable in culture for up to 14 days, allowing for evaluation of functional activity, structural alterations and cytotoxicity. In the present studies, PCLS were used to assess NM-induced lung injury and alterations in pulmonary mechanics. C57BL/6 mice were euthanized, the lungs filled with agarose, isolated after congealment, and sliced (300 mM in thickness); slices were cultured in Dulbecco's Modified Eagle's Medium (DMEM) and Ham's F-12 Nutrient Mixture (DMEM/F-12). Twenty-four hours later, PCLS were treated with NM (1, 10, and 100 mM) for one hour, washed, refed with medium and cultured for 3 days. NM caused a dose-related increase in cytotoxicity and a decrease in mitochondrial activity as measured by lactate dehydrogenase (LDH) and WST-1 assays, respectively. Analysis of histologic sections revealed that NM caused damage to the airway epithelium; this was associated with decreased mRNA expression of collagen1A1 and collagen3A1, markers of tissue repair. To determine if these changes were linked to altered pulmonary function, we measured airway responsiveness using video microscopy. In these studies, changes in airway area after stimulation with increasing concentrations of methacholine were assessed in cross-sectional airways. Consistent with structural alterations, NM caused a ~30% decrease in maximal airway contraction after methacholine, relative to control, indicating a loss in airway smooth muscle function. These data demonstrate that PCLS can be used effectively to mechanistically assess the progression of injury induced by NM. Further understanding of the progression of damage and repair processes following exposure to mustards is essential in order to develop therapeutics for treating pulmonary toxicity.

ORMDL3 expression in ASM regulates hypertrophy, hyperplasia via TPM1 and TPM4, and contractility.

ORM1-like 3 (ORMDL3) has strong genetic linkage to childhood onset asthma. To determine whether ORMDL3 selective expression in airway smooth muscle (ASM) influences ASM function, we used Cre-loxP techniques to generate transgenic mice (hORMDL3Myh11eGFP-cre), which express human ORMDL3 selectively in smooth muscle cells. In vitro studies of ASM cells isolated from the bronchi of hORMDL3Myh11eGFP-cre mice demonstrated that they developed hypertrophy (quantitated by FACS and image analysis), developed hyperplasia (assessed by BrdU incorporation), and expressed increased levels of tropomysin proteins TPM1 and TPM4. siRNA knockdown of TPM1 or TPM4 demonstrated their importance to ORMDL3-mediated ASM proliferation but not hypertrophy. In addition, ASM derived from hORMDL3Myh11eGFP-cre mice had increased contractility to histamine in vitro, which was associated with increased levels of intracellular Ca2+; increased cell surface membrane Orai1 Ca2+ channels, which mediate influx of Ca2+ into the cytoplasm; and increased expression of ASM contractile genes sarco/endoplasmic reticulum Ca2+ ATPase 2b and smooth muscle 22. In vivo studies of hORMDL3Myh11eGFP-cre mice demonstrated that they had a spontaneous increase in ASM and airway hyperreactivity (AHR). ORMDL3 expression in ASM thus induces changes in ASM (hypertrophy, hyperplasia, increased contractility), which may explain the contribution of ORMDL3 to the development of AHR in childhood onset asthma, which is highly linked to ORMDL3 on chromosome 17q12-21.

Inhaled triple therapy and airway hyperresponsiveness in persistent asthma

Adding a long-acting muscarinic antagonist (LAMA) is recommended in the guidelines for a patient with asthma not controlled on inhaled corticosteroid (ICS)/long-acting β-agonist (LABA). The change in forced expiratory volume in 1 second (FEV1) resulting from the addition of LAMA to ICS/LABA may be relatively small or less than the minimal clinically important difference, and as such, improved airway caliber is not the primary mechanism for a reduction in exacerbations with LAMA therapy. 1 Lipworth B.J. Emerging role of long acting muscarinic antagonists for asthma. Br J Clin Pharmacol. 2014; 77: 55-62 Crossref PubMed Scopus (33) Google Scholar Exacerbations are, in part, determined by the effects on airway hyperresponsiveness (AHR) in which a LAMA might be expected to confer additional airway smooth muscle–stabilizing effects when added to ICS/LABA. 2 Currie G.P. Jackson C.M. Ogston S.A. Lipworth B.J. Airway-stabilizing effect of long-acting beta2-agonists as add-on therapy to inhaled corticosteroids. Qjm. 2003; 96: 435-440 Crossref PubMed Scopus (23) Google Scholar Aside from effects on airway smooth muscle, there may be neuronal and paracrine actions of LAMAs that may reduce exacerbations. 1 Lipworth B.J. Emerging role of long acting muscarinic antagonists for asthma. Br J Clin Pharmacol. 2014; 77: 55-62 Crossref PubMed Scopus (33) Google Scholar Indirect challenge tests with agents such as mannitol are thought to be more physiological than direct agents such as histamine or methacholine in that they better reflect the underlying inflammation status as opposed to the function of airway smooth muscle alone. 3 Nair P. Martin J.G. Cockcroft D.C. et al. Airway hyperresponsiveness in asthma: measurement and clinical relevance. J Allergy Clin Immunol Pract. 2017; 5: 649-659.e2 Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar

Subacute TGFβ Exposure Drives Airway Hyperresponsiveness in Cystic Fibrosis Mice through the PI3K Pathway.

Cystic fibrosis (CF) is a lethal genetic disease characterized by progressive lung damage and airway obstruction. The majority of patients demonstrate airway hyperresponsiveness (AHR), which is associated with more rapid lung function decline. Recent studies in the neonatal CF pig demonstrated airway smooth muscle (ASM) dysfunction. These findings, combined with observed CF transmembrane conductance regulator (CFTR) expression in ASM, suggest that a fundamental defect in ASM function contributes to lung function decline in CF. One established driver of AHR and ASM dysfunction is transforming growth factor (TGF) β1, a genetic modifier of CF lung disease. Prior studies demonstrated that TGFβ exposure in CF mice drives features of CF lung disease, including goblet cell hyperplasia and abnormal lung mechanics. CF mice displayed aberrant responses to pulmonary TGFβ, with elevated PI3K signaling and greater increases in lung resistance compared with controls. Here, we show that TGFβ drives abnormalities in CF ASM structure and function through PI3K signaling that is enhanced in CFTR-deficient lungs. CF and non-CF mice were exposed intratracheally to an adenoviral vector containing the TGFβ1 cDNA, empty vector, or PBS only. We assessed methacholine-induced AHR, bronchodilator response, and ASM area in control and CF mice. Notably, CF mice demonstrated enhanced AHR and bronchodilator response with greater ASM area increases compared with non-CF mice. Furthermore, therapeutic inhibition of PI3K signaling mitigated the TGFβ-induced AHR and goblet cell hyperplasia in CF mice. These results highlight a latent AHR phenotype in CFTR deficiency that is enhanced through TGFβ-induced PI3K signaling.

Estrogen Receptors Differentially Regulate the Overall Contractility of Human Airway Smooth Muscle

PurposeEpidemiological evidences suggest higher incidence and severity of asthma in pre‐menopausal women and aging men, indicating the role of sex steroids, especially estrogen. Our recent studies demonstrated that airway smooth muscle (ASM) cells from both male and female expresses estrogen receptors (ERα and β) and these receptors differentially regulate intracellular calcium ([Ca2+]i) which is a key role player in contraction. The degree of force for contraction can also be altered in a Ca2+‐independent manner via RhoA. Both Ca2+‐dependent and Ca2+‐independent pathways in ASM thus together contribute towards the airway contractility. In this study, we hypothesized that the ERs differentially regulate cyclic adenosine monophosphate (cAMP) and RhoA mechanisms, thereby affecting the overall contractility of human ASM cells.MethodsAsthmatic and non‐asthmatic primary human ASM cells (isolated from lung tissue, Mayo Clinic, IRB‐approved) were seeded and grown to 70% confluence. After serum‐deprivation, cells were exposed to 1nM 17β‐estradiol (E2), 10nM PPT (ERα agonist) or WAY (ERβ agonist) for 24hr. IBMX (10μM) was used as phosphodiesterase inhibitor and Isoproterol (100nM for 10min) served as a positive control group for estimation of cAMP. In parallel studies, phosphorylation of myosin light chain (MLC20) and myosin phosphatase targeting subunit‐1 (MYPT1) was assessed through Western analyses. Activated RhoA and cAMP measurements were done through ELISA kits. Cell contraction was accessed through traction force microscopy (TFM) using softgel plates coated with collagen and fluorescent beads. Upon treatments, phase‐contrast and fluorescent images were captured before, during and after 10μM histamine exposure and net traction force was calculated using advanced software.ResultsProlonged (24h) activation of ERβ significantly increased the baseline cAMP levels whereas ERα activation decreased it. This effect was more profound in asthmatic ASM cells compared to non‐asthmatics. Furthermore, ERβ activation lowered the active RhoA levels in non‐asthmatic human ASM cells. The phosphorylation of MLC20 and MYPT1 was significantly attenuated with ERβ activation compared to vehicle. TFM studies demonstrated reduced traction force in non‐asthmatic ASM cells treated with ERβ agonist compared to the vehicle, whereas ERα activation did not show any significant changes.ConclusionOur findings highlight the crucial role of divergent ER signaling in regulating Ca2+‐dependent and Ca2+‐independent pathways, which together govern the contractile function of ASM. Particularly, ERβ activation (and not ERα) mediates ASM relaxation by increasing the cAMP, reducing active RhoA and decreasing phosphorylation of MLC20/MYPT1. Thus, ERβ activation might serve as a novel target to manage the increased ASM contractility in asthma.Support or Funding InformationAcknowledgementSupported by NIH grants R01‐HL0123494 (Venkatachalem), R01‐HL 088029 (Prakash) and R01‐HL142061 (Pabelick, Prakash).

Laminin α4 contributes to airway remodeling and inflammation in asthma.

Airway inflammation and remodeling are characteristic features of asthma, with both contributing to airway hyperresponsiveness (AHR) and lung function limitation. Airway smooth muscle (ASM) accumulation and extracellular matrix deposition are characteristic features of airway remodeling, which may contribute to persistent AHR. Laminins containing the α2-chain contribute to characteristics of ASM remodeling in vitro and AHR in animal models of asthma. The role of other laminin chains, including the laminin α4 and α5 chains, which contribute to leukocyte migration in other diseases, is currently unknown. The aim of the current study was to investigate the role of these laminin chains in ASM function and in AHR, remodeling, and inflammation in asthma. Expression of both laminin α4 and α5 was observed in the human and mouse ASM bundle. In vitro, laminin α4 was found to promote a pro-proliferative, pro-contractile, and pro-fibrotic ASM cell phenotype. In line with this, treatment with laminin α4 and α5 function-blocking antibodies reduced allergen-induced increases in ASM mass in a mouse model of allergen-induced asthma. Moreover, eosinophilic inflammation was reduced by the laminin α4 function-blocking antibody as well. Using airway biopsies from healthy subjects and asthmatic patients, we found inverse correlations between ASM α4-chain expression and lung function and AHR, whereas eosinophil numbers correlated positively with expression of laminin α4 in the ASM bundle. This study, for the first time, indicates a prominent role for laminin α4 in ASM function and in inflammation, AHR, and remodeling in asthma, whereas the role of laminin α5 is more subtle.

TRPPing up bronchiectasis.

TRPPing up bronchiectasis.

Bnip3 regulates airway smooth muscle cell focal adhesion and proliferation.

Increased airway smooth muscle (ASM) mass is a key contributor to airway narrowing and airway hyperresponsiveness in asthma. Besides conventional pathways and regulators of ASM proliferation, recent studies suggest that changes in mitochondrial morphology and function play a role in airway remodeling in asthma. In this study, we aimed at determining the role of mitochondrial Bcl-2 adenovirus E1B 19 kDa-interacting protein, Bnip3, in the regulation of ASM proliferation. Bnip3 is a member of the Bcl-2 family of proteins critical for mitochondrial health, mitophagy, and cell survival/death. We found that Bnip3 expression is upregulated in ASM cells from asthmatic donors compared with that in ASM cells from healthy donors and transient downregulation of Bnip3 expression in primary human ASM cells using an siRNA approach decreased cell adhesion, migration, and proliferation. Furthermore, Bnip3 downregulation altered the structure (electron density) and function (cellular ATP levels, membrane potential, and reacitve oxygen species generation) of mitochondria and decreased expression of cytoskeleton proteins vinculin, paxillin, and actinin. These findings suggest that Bnip3 via regulation of mitochondria functions and expression of adhesion proteins regulates ASM adhesion, migration, and proliferation. This study reveals a novel role for Bnip3 in ASM functions and establishes Bnip3 as a potential target in mitigating ASM remodeling in asthma.

Estrogen Signaling on Mitochondrial Dynamics in Human Airway Smooth Muscle Cells

BackgroundThe pathophysiology of asthma involves inflammation and altered structure and function of airway smooth muscle (ASM). There is emerging evidence that asthma affects adult women disproportionately with more severity than men. Also, our recent findings suggest that differential estrogen receptors (ER), ERα and ERβ expression and function are present in asthmatic ASM cells. Due to high oxygen environment in the lungs and the presence of inflammation, asthmatic ASM cells undergo oxidative stress. Mitochondrial dysfunction due to this oxidative stress plays a crucial role in the bioenergetics pathways involved in asthma. In this study, we hypothesize that differential ER signaling affects mitochondrial dynamics (fission/fusion) in ASM, which subsequently affects reactive oxygen species (ROS) generation and intracellular Ca2+ ([Ca2+]i) buffering capacity of mitochondria.MethodsHuman ASM cells were enzymatically dissociated from lung samples of non‐asthmatics undergoing thoracic surgery at Mayo Clinic. Cells were grown onto 8‐well labtek plates and treated with pro‐inflammatory cytokine, TNFα (20 mg/mL) in the presence or absence of 1 nM 17β‐estradiol (E2) which is a nonselective ER agonist, 10 nM PPT (ERα agonist) or 10 nM WAY (ERβ agonist) for 24h. For morphology studies, the cells were loaded with Mitotracker Green and then imaged on the Bio‐Tek Lionheart FX Live Cell Imager. Form factor and aspect ratio are used as parameters for determining fission vs fusion in mitochondria and these were calculated using MATLAB. Western blot and qPCR techniques were performed to access the protein and gene expression of the fission and fusion proteins Dynamin Related Protein1 (Drp1) and Mitofusion2 (Mfn2). ROS measurement was performed by loading cells with MitoSOX Red and their fluorescence intensity was recorded on the Live Cell Imager.ResultsIn mitotracker loaded ASM cells, ERβ agonist WAY was effective in increasing the form factor and aspect ratio as compared to vehicle. In addition, ERβ was effective in reducing TNFα induced reduction in form factor and aspect ratio. However, both E2 and ERα agonist, PPT, did not show any changes in form factor and aspect ratio of TNFα exposed ASM cells. Western analysis and gene expression studies showed higher expression of Drp1 and lower expression of Mfn2 in TNFα treated cells. Pre‐treatment with ERβ‐agonist reversed the altered expression of Drp1 and Mfn2 in TNFα treated cells. Using MitoSOX Red, it was observed that ERβ agonist WAY treated cells showed lower ROS generation as compared to TNFα.ConclusionOur data suggests a novel estrogen signaling in ASM cells for maintaining the structure of mitochondria. ERβ is effective in ameliorating the inflammation due to TNFα and hence preserves the morphology of mitochondria during inflammation. Further studies will confirm the subsequent alterations in mitochondrial ROS generation and [Ca2+]i buffering capacity due to this altered mitochondrial morphology.Support or Funding InformationSupported by the UGSRF fellowship by APS (Jesme) and NIH grants R01 HL123494, (Venkatachalem), and R01 HL 088029 (Prakash)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Cigarette Smoke Disrupts Calcium Homeostasis in Airway Smooth Muscle Cells

Tobacco smoking is the largest risk factor for developing chronic obstructive pulmonary disease (COPD), and is associated with airway hyperresponsiveness. Hyperresponsiveness is linked to abnormal calcium metabolism, which regulates the contractility and hence the tone of airway smooth muscle (ASM). However, whether cigarette smoke (CS) directly alters or regulates calcium signaling in ASM cells (ASMC) is not fully known. In airway epithelial cells, acute CS exposure increases intracellular Ca2+ concentrations ([Ca2+]i). We hypothesise that CS impedes ASM function by disrupting calcium homeostasis in ASMC, and the present study aims to unravel the effects of CS on calcium handling in ASMC. Primary rat and human ASMC were isolated via enzymatic digestion, and maintained in culture up to 8 passages. ASMC were seeded onto glass coverslips, and deprived of serum for 24–48 hours prior to calcium imaging studies using fura‐2‐AM. ASMC were exposed to either whole CS or CS extract (CSE) from 3R4F reference cigarettes (University of Kentucky) with the particulate phase filtered out. We found that both CS and CSE elevated [Ca2+]i in rat and human ASMC, and this increase was not abolished following depletion of sarcoplasmic‐reticulum calcium stores with thapsigargin or cyclopiazonic acid. The increase in [Ca2+]i in response to CSE was concentration‐dependent. Removal of extracellular calcium attenuated the CSE‐induced elevation of [Ca2+]i in hASMC, suggesting that CSE may activate calcium influx pathways in cultured hASMC. We speculate that this elevation of [Ca2+]i would lead to exacerbated ASM contraction, especially under inflamed conditions in COPD, and hence narrowing of the airway lumen. Further investigations into the source of [Ca2+]i increase, identity of potential calcium influx channels activated, and the direct effects of CS/CSE on ASM contraction are on‐going.Support or Funding InformationThis work is supported by Newcastle University's JJ Hunter Bequest scholarship.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Molecular Mechanisms for the Mechanical Modulation of Airway Responsiveness.

The smooth muscle of the airways is exposed to continuously changing mechanical forces during normal breathing. The mechanical oscillations that occur during breathing have profound effects on airway tone and airway responsiveness both in experimental animals and humans in vivo and in isolated airway tissues in vitro. Experimental evidence suggests that alterations in the contractile and mechanical properties of airway smooth muscle tissues caused by mechanical perturbations result from adaptive changes in the organization of the cytoskeletal architecture of the smooth muscle cell. The cytoskeleton is a dynamic structure that undergoes rapid reorganization in response to external mechanical and pharmacologic stimuli. Contractile stimulation initiates the assembly of cytoskeletal/extracellular matrix adhesion complex proteins into large macromolecular signaling complexes (adhesomes) that undergo activation to mediate the polymerization and reorganization of a submembranous network of actin filaments at the cortex of the cell. Cortical actin polymerization is catalyzed by Neuronal-Wiskott–Aldrich syndrome protein (N-WASP) and the Arp2/3 complex, which are activated by pathways regulated by paxillin and the small GTPase, cdc42. These processes create a strong and rigid cytoskeletal framework that may serve to strengthen the membrane for the transmission of force generated by the contractile apparatus to the extracellular matrix, and to enable the adaptation of smooth muscle cells to mechanical stresses. This model for the regulation of airway smooth muscle function can provide novel perspectives to explain the normal physiologic behavior of the airways and pathophysiologic properties of the airways in asthma.