Airway Smooth Muscle Remodeling Research Articles

Smooth muscle CaMKIIδ promotes allergen-induced airway hyperresponsiveness and inflammation.

Airway smooth muscle (ASM) is a key target cell in allergen-induced asthma known to contribute to airway hyperresponsiveness (AHR) and chronic airway remodeling. Changes in ASM calcium homeostasis have been shown to contribute to AHR although the mechanisms and Ca(2+) signal effectors are incompletely understood. In the present study, we tested the function of ASM multifunctional protein kinase Ca(2+)/calmodulin-dependent kinase II (CaMKII) isoforms CaMKIIδ and CaMKIIγ in allergen-induced AHR and airway remodeling in vivo. Using a murine model of atopic asthma, we demonstrate that CaMKIIδ protein is upregulated in ASM derived from ovalbumin (OVA)-treated animals compared to controls. A genetic approach to conditionally knock out smooth muscle CaMKIIδ and CaMKIIγ in separate Cre-loxp systems was validated, and using this loss-of-function approach, the function of these CaMKII isoforms was tested in ovalbumin (OVA)-induced airway remodeling and AHR. OVA treatment in control mice had no effect on ASM remodeling in this model of AHR, and CaMKIIδ knockouts had no independent effects on ASM content. However, at 1 day post-final OVA challenge, OVA-induced AHR was eliminated in the CaMKIIδ knockouts. OVA-induced peribronchial inflammation and bronchoalveolar lavage fluid (BALF) levels of the Th2 cytokine IL-13 were significantly decreased in the CaMKIIδ knockouts. Unexpectedly, we found increased peribronchial eosinophils in the smooth muscle CaMKIIδ knockouts compared to control animals at 1 day post-final challenge, suggesting that lack of ASM CaMKIIδ delays the progression of AHR rather than inhibiting it. Indeed, when AHR was determined at 7 days post-final OVA challenge, CaMKIIδ knockouts showed robust AHR while AHR was fully resolved in OVA-challenged control mice. These in vivo studies demonstrate a role for smooth muscle CaMKIIδ in promoting airway inflammation and AHR and suggest a complex signaling role for CaMKIIδ in regulating ASM function. These studies confirm the diverse roles of ASM cells as immune effectors that control AHR and call for further studies into CaMKIIδ-mediated signaling in ASM cells during disease.

Contribution of SRF, Elk-1, and myocardin to airway smooth muscle remodeling in heaves, an asthma-like disease of horses.

Myocyte hyperplasia and hypertrophy contribute to the increased mass of airway smooth muscle (ASM) in asthma. Serum-response factor (SRF) is a transcription factor that regulates myocyte differentiation in vitro in vascular and intestinal smooth muscles. When SRF is associated with phosphorylated (p)Elk-1, it promotes ASM proliferation while binding to myocardin (MYOCD) leading to the expression of contractile elements in these tissues. The objective of this study was therefore to characterize the expression of SRF, pElk-1, and MYOCD in ASM cells from central and peripheral airways in heaves, a spontaneously occurring asthma-like disease of horses, and in controls. Six horses with heaves and five aged-matched controls kept in the same environment were studied. Nuclear protein expression of SRF, pElk-1, and MYOCD was evaluated in peripheral airways and endobronchial biopsies obtained during disease remission and after 1 and 30 days of naturally occurring antigenic exposure using immunohistochemistry and immunofluorescence techniques. Nuclear expression of SRF (P = 0.03, remission vs. 30 days) and MYOCD (P = 0.05, controls vs. heaves at 30 days) increased in the peripheral airways of horses with heaves during disease exacerbation, while MYOCD (P = 0.04, remission vs. 30 days) decreased in the central airways of control horses. No changes were observed in the expression of pElk-1 protein in either tissue. In conclusion, SRF and its cofactor MYOCD likely contribute to the hypertrophy of peripheral ASM observed in equine asthmatic airways, while the remodeling of the central airways is more static or involves different transcription factors.

Oxidative stress–induced mitochondrial dysfunction drives inflammation and airway smooth muscle remodeling in patients with chronic obstructive pulmonary disease

BackgroundInflammation and oxidative stress play critical roles in patients with chronic obstructive pulmonary disease (COPD). Mitochondrial oxidative stress might be involved in driving the oxidative stress–induced pathology.ObjectiveWe sought to determine the effects of oxidative stress on mitochondrial function in the pathophysiology of airway inflammation in ozone-exposed mice and human airway smooth muscle (ASM) cells.MethodsMice were exposed to ozone, and lung inflammation, airway hyperresponsiveness (AHR), and mitochondrial function were determined. Human ASM cells were isolated from bronchial biopsy specimens from healthy subjects, smokers, and patients with COPD. Inflammation and mitochondrial function in mice and human ASM cells were measured with and without the presence of the mitochondria-targeted antioxidant MitoQ.ResultsMice exposed to ozone, a source of oxidative stress, had lung inflammation and AHR associated with mitochondrial dysfunction and reflected by decreased mitochondrial membrane potential (ΔΨm), increased mitochondrial oxidative stress, and reduced mitochondrial complex I, III, and V expression. Reversal of mitochondrial dysfunction by the mitochondria-targeted antioxidant MitoQ reduced inflammation and AHR. ASM cells from patients with COPD have reduced ΔΨm, adenosine triphosphate content, complex expression, basal and maximum respiration levels, and respiratory reserve capacity compared with those from healthy control subjects, whereas mitochondrial reactive oxygen species (ROS) levels were increased. Healthy smokers were intermediate between healthy nonsmokers and patients with COPD. Hydrogen peroxide induced mitochondrial dysfunction in ASM cells from healthy subjects. MitoQ and Tiron inhibited TGF-β–induced ASM cell proliferation and CXCL8 release.ConclusionsMitochondrial dysfunction in patients with COPD is associated with excessive mitochondrial ROS levels, which contribute to enhanced inflammation and cell hyperproliferation. Targeting mitochondrial ROS represents a promising therapeutic approach in patients with COPD.

L-thyroxine promotes a proliferative airway smooth muscle phenotype in the presence of TGF-β1.

Hypothyroidism may reduce, whereas hyperthyroidism may aggravate, asthma symptoms. The mechanisms underlying this relationship are largely unknown. Since thyroid hormones have central roles in cell growth and differentiation, we hypothesized that airway remodeling, in particular increased airway smooth muscle (ASM) mass, may be involved. To address this hypothesis, we investigated the effects of triiodothyronine (T3) and l-thyroxine (T4) in the absence and presence of the profibrotic transforming growth factor (TGF)-β1 on human ASM cell phenotype switching. T3 (1-100 nM) and T4 (1-100 nM) did not affect basal ASM proliferation. However, when combined with TGF-β1 (2 ng/ml), T4 synergistically increased the proliferative response, whereas only a minor effect was observed for T3. In line with a switch from a contractile to a proliferative ASM phenotype, T4 reduced the TGF-β1-induced contractile protein expression by ∼50%. Cotreatment with T3 reduced TGF-β1-induced contractile protein expression by ∼25%. The synergistic increase in proliferation was almost fully inhibited by the integrin αvβ3 antagonist tetrac (100 nM), whereas no significant effects of the thyroid receptor antagonist 1-850 (3 μM) were observed. Inhibition of MEK1/2, downstream of the integrin αvβ3, also inhibited the T4- and TGF-β1-induced proliferative responses. Collectively, the results indicate that T4, and to a lesser extent T3, promotes a proliferative ASM phenotype in the presence of TGF-β1, which is predominantly mediated by the membrane-bound T4 receptor αvβ3. These results indicate that thyroid hormones may enhance ASM remodeling in asthma, which could be of relevance for hyperthyroid patients with this disease.

Interaction between endoplasmic/sarcoplasmic reticulum stress (ER/SR stress), mitochondrial signaling and Ca(2+) regulation in airway smooth muscle (ASM).

Airway inflammation is a key aspect of diseases such as asthma. Several inflammatory cytokines (e.g., TNFα and IL-13) increase cytosolic Ca(2+) ([Ca(2+)]cyt) responses to agonist stimulation and Ca(2+) sensitivity of force generation, thereby enhancing airway smooth muscle (ASM) contractility (hyper-reactive state). Inflammation also induces ASM proliferation and remodeling (synthetic state). In normal ASM, the transient elevation of [Ca(2+)]cyt induced by agonists leads to a transient increase in mitochondrial Ca(2+) ([Ca(2+)]mito) that may be important in matching ATP production with ATP consumption. In human ASM (hASM) exposed to TNFα and IL-13, the transient increase in [Ca(2+)]mito is blunted despite enhanced [Ca(2+)]cyt responses. We also found that TNFα and IL-13 induce reactive oxidant species (ROS) formation and endoplasmic/sarcoplasmic reticulum (ER/SR) stress (unfolded protein response) in hASM. ER/SR stress in hASM is associated with disruption of mitochondrial coupling with the ER/SR membrane, which relates to reduced mitofusin 2 (Mfn2) expression. Thus, in hASM it appears that TNFα and IL-13 result in ROS formation leading to ER/SR stress, reduced Mfn2 expression, disruption of mitochondrion-ER/SR coupling, decreased mitochondrial Ca(2+) buffering, mitochondrial fragmentation, and increased cell proliferation.

Distribution of airway smooth muscle remodelling in asthma: Relation to airway inflammation

Pathological phenotypes of asthma have been based predominantly on inflammation, rather than airway wall remodelling. Differences in the distribution of airway smooth muscle (ASM) remodelling between large and small airways may affect clinical outcomes in asthma. The aim of this study was to examine the distribution of ASM remodelling and its relation to airway inflammation. Post-mortem cases of asthma (n = 68) were categorized by the distribution of increased thickness of the ASM layer (relative to nonasthmatic controls, n = 37), into 'large only' (LO, n = 15), 'small only' (SO, n = 4) 'large/small' (LS, n = 24) or no increase (NI, n = 25). Subject characteristics, ASM and airway wall dimensions and inflammatory cell numbers were compared between groups. Apart from reduced clinical severity of asthma in NI cases (P = 0.002), subject characteristics did not distinguish asthma groups. Compared with control subjects, ASM cell number, reticular basement membrane thickness, airway wall thickness, percent muscle shortening and eosinophil number were increased (P < 0.05) in both large and small airways in LS cases and only the large airways in LO cases. Increased numbers of neutrophils were observed only in the small airways of LO cases. Distinct distributions of ASM remodelling are seen in asthma. Pathology limited to the small airways was uncommon. Increased thickness of the ASM layer was associated with airway remodelling and eosinophilia, but not neutrophilia. These data support the presence of distinct pathological phenotypes based on the site of increased ASM.

Low-intensity aerobic exercise training attenuates airway inflammation and remodeling in a rat model of steroid-resistant asthma

Background Aerobic exercise can improve symptoms, reduce airway inflammation, and even ameliorate airway remodeling in asthmatic animals and patients. However, previous studies have focused mainly on the effect of aerobic exercise on steroid-sensitive asthma (SSA). The goals of this study were to determine the effect of low-intensity aerobic exercise training on airway hyperresponsiveness, inflammation, and remodeling in a rat model of steroid-resistant asthma (SRA) and to identify the potential mechanisms underlying these effects. Methods Endotoxin-free ovalbumin with or without lipopolysaccharide were applied to establish rat models of SRA and SSA, respectively. Airway hyperresponsiveness, inflammation, remodeling, expression of interleukin (IL)-25, IL-33, thymic stromal lymphopoietin (TSLP), high mobility group box-1 (HMGB1), and IL-17 in bronchoalveolar lavage fluid (BALF), and the role of dexamethasone (DXM) were compared between these two asthmatic rat models. The effect of low-intensity aerobic exercise training and anti-HMGB1 treatment on airway hyperresponsiveness, inflammation, and remodeling in SRA rats also was evaluated. Results SRA rats developed neutrophil-dominated airway inflammation ((29.5±4.1)% of the total cell numbers in BALF), whereas SSA rats developed eosinophil-dominated airway inflammation ((24.0±6.1)% of the total cell numbers in BALF). Compared with SSA rats, SRA rats had more severe airway hyperresponsiveness, lower levels of IL-25 ((33.6±10.3) vs. (104.8±24.9) pg/ml), IL-33 ((87.5±25.0) vs. (226.6±40.7) pg/ml), and TSLP ((1 933.2±899.5) vs. (7 224.0±992.1) pg/ml), and higher levels of HMGB1 ((21.2±4.5) vs. (5.4±1.6) ng/ml) and IL-17 ((780.5±261.7) vs. (291.4±76.4) pg/ml) in BALF (all P &lt;0.05). However, there was no significant difference in goblet cell hyperplasia, subepithelial collagen thickness, and airway smooth muscle remodeling between the two groups. Compared with control SSA rats, airway hyperresponsiveness, inflammation, and remodeling in SRA rats were less sensitive to DXM treatment. Anti-HMGB1 treatment attenuated airway hyperresponsiveness, inflammation, and remodeling in SRA rats to a certain extent and was accompanied by lower levels of IL-17 ((369.2±126.7) vs. (780.5±261.7) pg/ml in control SRA rats) in BALF (P &lt;0.05). Low-intensity aerobic exercise training decreased the expression of both HMGB1 ((14.1±2.9) vs. (21.2±4.5) ng/ml in control SRA rats) and IL-17 ((545.3±148.6) vs. (780.5±261.7) pg/ml in control SRA rats) in BALF (all P &lt;0.05) and was accompanied by improved airway hyperresponsiveness, inflammation, and remodeling in SRA rats (all P &lt;0.05). Conclusions Low-intensity aerobic exercise training attenuated airway hyperresponsiveness, inflammation, and remodeling in a rat model of SRA. Decreased HMGB1 and IL-17 levels in BALF by aerobic exercise training at least partly contributed to the improvements of SRA.

Nauji astmos patogenezės aspektai

Astma yra lėtinė apatinių kvėpavimo takų uždegimo liga, kuriai būdinga praeinanti bronchų obstrukcija, bronchų hiperreakty-vumas; vyrauja uždegimo ląstelės ir jų išskiriami mediatoriai, struktūriniai bronchų pokyčiai (remodeliavimasis). Bronchų lygiųjų raumenų ląstelės aktyviai dalyvauja bronchų remodeliavimosi procese išskirdamos prouždegimo citokinus, chemokinus ir tarpląstelinės medžiagos baltymus. Eozinofilai, vienos pagrindinių uždegimo ląstelių, yra svarbūs vykstant bronchų remodeliavimuisi. Eozinofilai, išskirdami cisteinil-leukotrienus, skatina bronchų lygiųjų raumenų proliferaciją. Manoma, kad eozinofilai bronchų lygiųjų raumenų ląsteles veikia ir tiesiogiai adhezijos būdu. Duomenys rodo, kad naujas Wingless baltymo/integrazės-1 (WNT) signalų tarp ląstelių perdavimo kelias taip pat gali būti svarbus astmos patogenezėje ir bronchų remodeliavimosi procesuose. Nustatyta, kad, sergant astma, būna padidėjusi WNT-5a ligando raiška. Deja, eozinofilų poveikis WNT sekrecijai bronchų lygiųjų raumenų ląstelėse kol kas nėra gerai ištirtas. Nepaisant plaãai pripažįstamos eozinofilų reikšmės astmos patogenezėje, eozinofilų sukeltas bronchų lygiųjų raumenų remodeliavimosi mechanizmas nėra išsiaiškintas.

The Pro-Proliferative Effects of Nicotine and Its Underlying Mechanism on Rat Airway Smooth Muscle Cells

Recent studies have shown that nicotine, a major component of cigarette smoke, can stimulate the proliferation of non-neuronal cells. Cigarette smoking can promote a variety of pulmonary and cardiovascular diseases, such as chronic obstructive pulmonary disease (COPD), atherosclerosis, and cancer. A predominant feature of COPD is airway remodeling, which includes increased airway smooth muscle (ASM) mass. The mechanisms underlying ASM remodeling in COPD have not yet been fully elucidated. Here, we show that nicotine induces a profound and time-dependent increase in DNA synthesis in rat airway smooth muscle cells (RASMCs) in vitro. Nicotine also significantly increased the number of RASMCs, which was associated with the increased expression of Cyclin D1, phosphorylation of the retinoblastoma protein (RB) and was dependent on the activation of Akt. The activation of Akt by nicotine occurred within minutes and depended upon the nicotinic acetylcholine receptors (nAchRs). Activated Akt increased the phosphorylation of downstream substrates such as GSK3β. Our data suggest that the binding of nicotine to the nAchRs on RASMCs can regulate cellular proliferation by activating the Akt pathway.

Chronic obstructive pulmonary disease and anaesthesia

Chronic obstructive pulmonary disease (COPD) is a commonly encountered respiratory disorder. Patients with COPD pose a challenge to the anaesthetist because intraoperative and post- operative complications occur more commonly than in those without the disease, and can lead to prolonged hospital stay and increased mortality. This article provides an overview of COPD and discusses implications for the anaesthetic management of patients with the disease. Pathophysiology COPD is a chronic and progressive inflammatory condition affecting central and peripheral airways, lung parenchyma, and pulmonary vas- culature. This leads to poorly reversible narrow- ing of the airways, remodelling of airway smooth muscle, increased numbers of goblet cells and mucus-secreting glands, and pulmon- ary vasculature changes resulting in pulmonary hypertension. It is widely accepted that cigarette smoking is the key noxious stimulus leading to the devel- opment of COPD. However, more recently it has been suggested that genetic factors are also implicated, with the finding that a genetic variant (FAM13A) is associated with the devel- opment of COPD in the COPDGene study. 1 COPD is characterized by expiratory airflow limitation because of a combination of small airway inflammation (obstructive bronchiolitis) and parenchymal destruction (emphysema). In the former, inflammation in the small airways causes obstruction and air trapping, leading to dynamic hyperinflation, which adversely affects both ventilation/perfusion (V/Q) matching and the mechanics of the respiratory muscles. In em- physema the end result of inflammation is elastin breakdown and subsequent loss of alveolar structural integrity leading to decreased gas transfer, reduction in the pulmonary capillary bed, and further worsening of V/Q matching. Further airflow limitation results from reduced parenchymal support of small airways. Often it is not possible to make clear distinctions between the two subtypes and the relative contri- bution of each varies from patient to patient. In patients with advanced COPD, the combin- ation of V/Q mismatch, decreased gas transfer, and alveolar hypoventilation ultimately leads to respiratory failure. COPD is often associated with a number of coexisting diseases that may complicate the an- aesthetic management of these patients. A high proportion of patients with COPD are smokers, hence the disease is associated with the develop- ment of lung cancer. Pulmonary hypertension is prevalent in a third of patients with COPD and has been shown to be an indicator of poor long- term survival. Inflammatory processes in the lung not only cause pulmonary effects but also contribute to the extrapulmonary effects of the disease. The origin of this systemic inflamma- tion is unclear and probably multifactorial, but results in weight loss, skeletal muscle dysfunc- tion (with further adverse effects on respiratory muscle function), cardiovascular disease, de- pression, and osteoporosis. Weight loss occurs in 50% of patients with severe COPD and indi- cates a poor prognosis. Clinical features Epidemiology It has been estimated that 3 million people have COPD in the UK, two-thirds of these being undiagnosed. Diagnosis is most common in the sixth decade of life. COPD confers increased risk of hospitaliza- tion in general, and in the critically ill it has been shown to increase mortality both in those with ventilator-associated pneumonia and in those with non-exacerbated disease. The long- term survival of patients with severe COPD undergoing surgery is poor, with postoperative pulmonary complications being common. A recent study identified COPD as an independent Key points Chronic obstructive pulmonary disease (COPD) is a progressive inflammatory condition resulting in expiratory airflow limitation. Treatment involves smoking cessation, inhaled therapy, pulmonary rehabilitation, and appropriate and timely treatment of exacerbations. Patients with COPD are at increased riskof developing perioperative complications and have an increased mortality. Anaesthetic management centres on preoperative optimization and the use of regional techniques wherever possible. If general anaesthesia is used, then artificial ventilation is challenging because of the development of intrinsic positive end-expiratory pressure.

Effects of Zileuton on Airway Smooth Muscle Remodeling after Repeated Allergen Challenge in Brown Norway Rats

Background: Chronic asthma is characterized by airway inflammation and remodeling. Objective: This study aimed to evaluate the effects of zileuton on bronchial hyperresponsiveness, airway inflammation and airway smooth muscle (ASM) remodeling. Methods: Two experimental groups of brown Norway rats sensitized and repeatedly challenged with aerosolized ovalbumin (OA) were given oral zileuton (OA-zileuton group) and oral saline only (OA-saline group). A third, control group was sensitized and challenged by saline. The rats were anesthetized and paralyzed. Pulmonary function tests were performed at baseline and after varying doses of acetylcholine. Bronchoalveolar lavage fluid and lung tissues were examined. Results: Zileuton had beneficial effects on pulmonary function, airway inflammation and ASM remodeling in the OA-zileuton group compared to the OA-saline group. Zileuton inhibited an OA-stimulated increase in ASM by inhibiting hypertrophy, hyperplasia and increased extracellular matrix via the phosphatidylinositol 3-kinase (PI3K)/Akt pathway, thereby reducing cyclin D1 expression and attenuating bronchial hyperresponsiveness. Conclusion: OA increases airway inflammation and ASM mass. Zileuton effectively prevents bronchial hyperresponsiveness, airway inflammation and ASM remodeling in sensitized rats through the PI3K/Akt pathway, which reduces cyclin D1 expression.

Neuronal chemorepellent Semaphorin 3E inhibits human airway smooth muscle cell proliferation and migration

Chronic airway diseases, including asthma, are characterized by increased airway smooth muscle (ASM) mass that is due in part to growth factor-mediated ASM cell proliferation and migration. However, the molecular mechanisms underlying these effects are not completely understood. Semaphorin 3E (Sema3E) has emerged as an essential mediator involved in cell migration, proliferation, and angiogenesis, although its role in ASM cell function is not investigated. We sought to determine the expression of Sema3E receptor, plexinD1, in human ASM cells (HASMCs); effect of Sema3E on basal and platelet-derived growth factor (PDGF)-induced proliferation and migration; and underlying signaling pathways. Expression of plexinD1 in HASMCs was studied with RT-PCR, immunostaining, and flow cytometry. The effect of Sema3E on HASMC proliferation and migration was evaluated by 5-ethynyl-2'-deoxyuridine (EdU) incorporation, cell count, and Boyden chamber assay. Sema3E-mediated intracellular signaling was investigated with fluorescent microscopy, flow cytometry, Rac1 activation, and Western blot analysis. HASMCs from healthy persons expressed plexinD1 more than HASMCs from asthmatic patients. Sema3E increased plexinD1 expression in HASMCs from asthmatic patients. Recombinant Sema3E inhibited PDGF-mediated HASMC proliferation and migration, which was associated with F-actin depolymerization, suppression of PDGF-induced Rac1 guanosine triphosphatase activity, and Akt and extracellular signal-regulated kinase 1 and 2 phosphorylation. Bronchial biopsies from patients with mild asthma displayed immunoreactivity of plexinD1, suggesting the potential in vivo role of Sema3E-PlexinD1 axis in HASMC function. This study provides the first evidence that Sema3E receptor is expressed and plays functional roles in HASMCs. Our data suggest a regulatory role of Sema3E in PDGF-mediated proliferation and migration, leading to downregulation of ASM remodeling.

Thymic Stromal Lymphopoietin Induces Migration in Human Airway Smooth Muscle Cells

Airway remodeling due to increased airway smooth muscle (ASM) mass, likely due to enhanced migration and proliferation, has been shown to be highly associated with decline in lung function in asthma. Thymic stromal lymphopoietin (TSLP) is an IL-7-like, pro-allergic cytokine that has been shown to be necessary and sufficient for the development of allergic asthma. Human ASM (HASM) cells express TSLP receptor (TSLPR), the activation of which leads to enhanced release of proinflammatory mediators such as IL-6, CCL11/eotaxin-1, and CXCL8/IL-8. We show here that TSLP induces HASM cell migration through STAT3 activation since lentiviral-shRNA inhibition of STAT3 abrogated the TSLP-induced cell migration. Moreover, TSLP induced multiple cytoskeleton changes in HASM cells such as actin polymerization, cell polarization, and activation of small GTPase Rac1. Collectively, our data suggest a pro-migratory function of TSLP in ASM remodeling and provides better rationale for targeting TSLP/TSLPR pathway for therapeutic approaches in allergic asthma.

Bronchoconstriction Induces TGF-β Release and Airway Remodelling in Guinea Pig Lung Slices

Airway remodelling, including smooth muscle remodelling, is a primary cause of airflow limitation in asthma. Recent evidence links bronchoconstriction to airway remodelling in asthma. The mechanisms involved are poorly understood. A possible player is the multifunctional cytokine TGF-β, which plays an important role in airway remodelling. Guinea pig lung slices were used as an in vitro model to investigate mechanisms involved in bronchoconstriction-induced airway remodelling. To address this aim, mechanical effects of bronchoconstricting stimuli on contractile protein expression and TGF-β release were investigated. Lung slices were viable for at least 48 h. Both methacholine and TGF-β1 augmented the expression of contractile proteins (sm-α-actin, sm-myosin, calponin) after 48 h. Confocal fluorescence microscopy showed that increased sm-myosin expression was enhanced in the peripheral airways and the central airways. Mechanistic studies demonstrated that methacholine-induced bronchoconstriction mediated the release of biologically active TGF-β, which caused the increased contractile protein expression, as inhibition of actin polymerization (latrunculin A) or TGF-β receptor kinase (SB431542) prevented the methacholine effects, whereas other bronchoconstricting agents (histamine and KCl) mimicked the effects of methacholine. Collectively, bronchoconstriction promotes the release of TGF-β, which induces airway smooth muscle remodelling. This study shows that lung slices are a useful in vitro model to study mechanisms involved in airway remodelling.

The modulation of large airway smooth muscle phenotype and effects of epidermal growth factor receptor inhibition in the repeatedly allergen-challenged rat

Allergen challenges induce airway hyperresponsiveness (AHR) and increased airway smooth muscle (ASM) mass in the sensitized rat. Whether the remodeled ASM changes its phenotype is uncertain. We examined, in sensitized Brown Norway rats, the effects of multiple ovalbumin (Ova) challenges on ASM remodeling and phenotype and the role of the epidermal growth factor receptor (EGFR) in these processes. Rats were sensitized with Ova and challenged three times at 5-day intervals with phosphate-buffered saline or Ova and pretreated with the EGFR inhibitor AG-1478 (5 mg/kg) or its vehicle dimethyl sulfoxide. Ova challenges increased ASM mass in all-sized airways and in large airway mRNA expression of smooth muscle myosin heavy chain (sm-MHC), assessed by laser capture. Myosin light chain kinase and the fast myosin isoform SM-B mRNA expressions were not affected. Ova induced AHR to methacholine, and, based on the constant-phase model, this was largely attributable to the small airways and lung derecruitment at 48 h that recovered by 1 wk. The EGFR ligands amphiregulin and heparin-binding epidermal growth factor (HB-EGF) were increased in bronchoalveolar lavage fluid at 48 h after Ova exposure. AG-1478 inhibited AHR and prevented ASM growth. Epithelial gene expression of EGFR, HB-EGF, matrix metalloproteinase (MMP)-9, Gro-α, and transforming growth factor-β was unaffected by Ova challenges. We conclude that EGFR drives remodeling of ASM, which results from repeated Ova challenge. Furthermore, the latter results in excessive small airway and, to a lesser degree, large airway narrowing to methacholine, and large airway gene expression of contractile protein is conserved.

5-Aza-2′-deoxycytidine inhibited PDGF-induced rat airway smooth muscle cell phenotypic switching

Airway smooth muscle (ASM) cell phenotypic switching played an important role in airway remodeling in asthma. In vitro platelet-derived growth factor (PDGF) induced ASM cell phenotypic switching from a mature to pro-remodeling phenotype, but the mechanism remained incompletely understood. This study was to explore the effect of DNA methyltransferase inhibitor 5-Aza-2'-deoxycytidine (Aza-CdR) on PDGF-induced rat ASM cell phenotypic switching and biological behaviors. Rat airway smooth muscle (RASM) cells were obtained by primary explant techniques. Western blot, 3-dimensional gel contraction, transwell and wound healing assay, and MTT were applied to detect cell phenotypic switching, contractility, migration and proliferation, respectively. Cytoskeleton rearrangement was observed by immunofluorescence. Results showed Aza-CdR inhibited PDGF-induced down-regulation of contractile markers in RASM cells and increased cell contractility. Aza-CdR inhibited PDGF-induced RASM cell migration by abrogating cell morphology change and cytoskeletal reorganization and attenuated the effect of PDGF on proliferating cell nuclear antigen expression and cell cycle progression, ultimately cell proliferation. PDGF-induced DNA methyltransferase 1 (DNMT1) expression was mediated by activation of PI3K/Akt and ERK signaling in RASM cells. Selective depletion of DNMT1 protein by Aza-CdR inhibited PDGF-induced RASM cell phenotypic switching, revealing DNMT1-mediated DNA methylation was implicated in asthmatic ASM remodeling. We proposed for the first time that DNMT1 played a key role in PDGF-induced RASM cell phenotypic switching and Aza-CdR is promising in intervening ASM remodeling in asthma. Although study of abnormal DNA methylation in PDGF-stimulated ASM cells is in its infancy, this work contributes to providing new insights into the mechanism of ASM remodeling and may be helpful for developing effective treatments for airway remodeling in asthma.

Muscarinic receptor stimulation augments TGF-β1-induced contractile protein expression by airway smooth muscle cells

Acetylcholine (ACh) is the primary parasympathetic neurotransmitter in the airways. Recently, it was established that ACh, via muscarinic receptors, regulates airway remodeling in animal models of asthma and chronic obstructive pulmonary disease (COPD). The mechanisms involved are not well understood. Here, we investigated the functional interaction between muscarinic receptor stimulation and transforming growth factor (TGF)-β(1) on the expression of contractile proteins in human airway smooth muscle (ASM) cells. ASM cells expressing functional muscarinic M(2) and M(3) receptors were stimulated with methacholine (MCh), TGF-β(1), or their combination for up to 7 days. Western blot analysis revealed a strong induction of sm-α-actin and calponin by TGF-β(1), which was increased by MCh in ASM cells. Immunocytochemistry confirmed these results and revealed that the presence of MCh augmented the formation of sm-α-actin stress fibers by TGF-β(1). MCh did not augment TGF-β(1)-induced gene transcription of contractile phenotype markers. Rather, translational processes were involved in the augmentation of TGF-β(1)-induced contractile protein expression by muscarinic receptor stimulation, including phosphorylation of glycogen synthase kinase-3β and 4E-binding protein 1, which was enhanced by MCh. In conclusion, muscarinic receptor stimulation augments functional effects of TGF-β(1) in human ASM cells on cellular processes that underpin ASM remodeling in asthma and COPD.

Corticosteroids and Antigen Avoidance Decrease Airway Smooth Muscle Mass in an Equine Asthma Model

Recent studies suggest that airway smooth muscle remodeling is an early event in the course of asthma. Little is known of the effects of long-term antigen avoidance and inhaled corticosteroids on chronically established airway remodeling. We sought to measure the effects of inhaled corticosteroids and antigen avoidance on airway remodeling in the peripheral airways of horses with heaves, a naturally occurring asthma-like disease. Heaves-affected adult horses with ongoing airway inflammation and bronchoconstriction were treated with fluticasone propionate (with and without concurrent antigen avoidance) (n = 6) or with antigen avoidance alone (n = 5). Lung function and bronchoalveolar lavage were performed at multiple time points, and peripheral lung biopsies were collected before and after 6 and 12 months of treatment. Lung function improved more quickly with inhaled corticosteroids, but eventually normalized in both groups. Inflammation was better controlled with antigen avoidance. During the study period, corrected smooth muscle mass decreased from 12.1 ± 2.8 × 10(-3) and 11.3 ± 1.2 × 10(-3) to 8.3 ± 1.4 × 10(-3) and 7.9 ± 1.0 × 10(-3) in the antigen avoidance and fluticasone groups, respectively (P = 0.03). At 6 months, smooth muscle mass was significantly smaller compared with baseline only in the fluticasone-treated animals. The subepithelial collagen area was lower at 12 months than at baseline in both groups. During the study period, airway smooth muscle remodeling decreased by approximately 30% in both groups, although the decrease was faster in horses receiving inhaled corticosteroids. Inhaled corticosteroids may accelerate the reversal of smooth muscle remodeling, even if airway inflammation is better controlled with antigen avoidance.

Regulator of G protein signaling 2 is a key modulator of airway hyperresponsiveness

Drugs targeting individual G protein-coupled receptors are used as asthma therapies, but this strategy is limited because of G protein-coupled receptor signal redundancy. Regulator of G protein signaling 2 (RGS2), an intracellular selective inhibitor of multiple bronchoconstrictor receptors, may play a central role in the pathophysiology and treatment of asthma. We defined functions and mechanisms of RGS2 in regulating airway hyperresponsiveness (AHR), the pathophysiologic hallmark of asthma. Real-time PCR and Western blot were used to determine changes in RGS2 expression in ovalbumin-sensitized/-challenged mice. We also used immunohistochemistry and real-time PCR to compare RGS2 expression between human asthmatic and control subjects. The AHR of RGS2 knockout mice was assessed by using invasive tracheostomy and unrestrained plethysmography. Effects of loss of RGS2 on mouse airway smooth muscle (ASM) remodeling, contraction, intracellular Ca(2+), and mitogenic signaling were determined in vivo and in vitro. RGS2 was highly expressed in human and murine bronchial epithelium and ASM and was markedly downregulated in lungs of ovalbumin-sensitized/-challenged mice. Lung tissues and blood monocytes from asthma patients expressed significantly lower RGS2 protein (lung) and mRNA (monocytes) than from nonasthma subjects. The extent of reduction of RGS2 on human monocytes correlated with increased AHR. RGS2 knockout caused spontaneous AHR in mice. Loss of RGS2 augmented Ca(2+) mobilization and contraction of ASM cells. Loss of RGS2 also increased ASM mass and stimulated ASM cell growth via extracellular signal-regulated kinase and phosphatidylinositol 3-kinase pathways. We identified RGS2 as a potent modulator of AHR and a potential novel therapeutic target for asthma.

The presence of LPS in OVA inhalations affects airway inflammation and AHR but not remodeling in a rodent model of asthma

Ovalbumin (OVA) is the most frequently used allergen in animal models of asthma. Lipopolysaccharide (LPS) contaminating commercial OVA may modulate the evoked airway inflammatory response to OVA. However, the effect of LPS in OVA on airway remodeling, especially airway smooth muscle (ASM) has not been evaluated. We hypothesized that LPS in commercial OVA may enhance allergen-induced airway inflammation and remodeling. Brown Norway rats were sensitized with OVA on day 0. PBS, OVA, or endotoxin-free OVA (Ef-OVA) was instilled intratracheally on days 14, 19, 24. Bronchoalveolar lavage (BAL) fluid, lung, and intrathoracic lymph node tissues were collected 48 h after the last challenge. Immunohistochemistry for α-smooth muscle actin, Periodic-Acid-Schiff staining, and real-time qPCR were performed. Airway hyperresponsiveness (AHR) was also measured. BAL fluid macrophages, eosinophils, neutrophils, and lymphocytes were increased in OVA-challenged animals, and macrophages and neutrophils were significantly lower in Ef-OVA-challenged animals. The ASM area in larger airways was significantly increased in both OVA and Ef-OVA compared with PBS-challenged animals. The mRNA expression of IFN-γ and IL-13 in lung tissues and IL-4 in lymph nodes was significantly increased by both OVA and Ef-OVA compared with PBS and were not significantly different between OVA and Ef-OVA. Monocyte chemoattractant protein (MCP)-1 in BAL fluid and AHR were significantly increased in OVA but not in Ef-OVA. LPS contamination in OVA contributes to the influx of macrophages and MCP-1 increase in the airways and to AHR after OVA challenges but does not affect OVA-induced Th1 and Th2 cytokine expression, goblet cell hyperplasia, and ASM remodeling.