Altered Airway Smooth Muscle Research Articles

Advances in respiratory physiology in mouse models of experimental asthma.

Recent advances in mouse models of experimental asthma coupled with vast improvements in systems that assess respiratory physiology have considerably increased the accuracy and human relevance of the outputs from these studies. In fact, these models have become important pre-clinical testing platforms with proven value and their capacity to be rapidly adapted to interrogate emerging clinical concepts, including the recent discovery of different asthma phenotypes and endotypes, has accelerated the discovery of disease-causing mechanisms and increased our understanding of asthma pathogenesis and the associated effects on lung physiology. In this review, we discuss key distinctions in respiratory physiology between asthma and severe asthma, including the magnitude of airway hyperresponsiveness and recently discovered disease drivers that underpin this phenomenon such as structural changes, airway remodeling, airway smooth muscle hypertrophy, altered airway smooth muscle calcium signaling, and inflammation. We also explore state-of-the-art mouse lung function measurement techniques that accurately recapitulate the human scenario as well as recent advances in precision cut lung slices and cell culture systems. Furthermore, we consider how these techniques have been applied to recently developed mouse models of asthma, severe asthma, and asthma-chronic obstructive pulmonary disease overlap, to examine the effects of clinically relevant exposures (including ovalbumin, house dust mite antigen in the absence or presence of cigarette smoke, cockroach allergen, pollen, and respiratory microbes) and to increase our understanding of lung physiology in these diseases and identify new therapeutic targets. Lastly, we focus on recent studies that examine the effects of diet on asthma outcomes, including high fat diet and asthma, low iron diet during pregnancy and predisposition to asthma development in offspring, and environmental exposures on asthma outcomes. We conclude our review with a discussion of new clinical concepts in asthma and severe asthma that warrant investigation and how we could utilize mouse models and advanced lung physiology measurement systems to identify factors and mechanisms with potential for therapeutic targeting.

Interactions of a high‐fat Western diet and crystalline silica inhalation on airway epithelial ion transport and airway reactivity

Silicosis, an irreversible occupational lung disease caused by crystalline silica inhalation, is a serious health risk for silica‐exposed workers. NIOSH reports that Appalachian coal miners have higher rates of obesity and metabolic dysfunction (MetDys) compared to the general U.S. adult population. MetDys is a risk factor for lung function impairment, pulmonary hypertension, and asthma. Consumption of a high‐fat Western diet (HFWD) is associated with obesity and MetDys. In this study, we investigated the effects of, and determine interactions between, HFWD‐consumption and silica‐exposure on airway epithelial ion transport and smooth muscle reactivity in the F344 rat. Six‐week‐old male F344 rats were fed either a HFWD [40.6% fat (19.5% lard), 40.6% total carbohydrate (20% sucrose), 14.8 % protein] or standard rat chow (STD) [6.2 % fat, 44.2 % carbohydrate (grain sources), 18.6 % protein] for the duration of the study. Following 16 weeks of diet‐consumption, inhalation exposure to respirable crystalline silica (Min‐U‐Sil 5®, 15 mg/m3, 6 h/d, 5 d/wk, for 39 d) or filtered air began, with endpoint experiments conducted at 0, 4, and 8 wk post‐exposure. Airway epithelial ion transport maintains airway surface liquid osmolarity and depth required for effective cilia motility and clearance of xenogens. Changes in ion transport were determined ex vivo by measurement of transepithelial potential difference (Vt), short‐circuit current (ISC) and transepithelial resistance (Rt) in rat tracheal segments mounted in Ussing chambers, and administered the ion transport inhibitors amiloride (Na+ channel blocker; apical), 5‐nitro‐2‐(3‐phenylpropylamino) benzoic acid (NPPB; Clˉ channel blocker; apical), and ouabain (Na+, K+‐pump blocker; basolateral). Airway hyperresponsiveness is associated with obesity and pulmonary diseases such as asthma and COPD; thus, the isolated perfused trachea apparatus was employed to ascertain whether silica or HFWD altered airway smooth muscle reactivity to serosal or mucosal applied methacholine (MCh). HFWD‐consumption had no effect on basal Vt. Silica exposure increased Na+ transport at 0 wk, decreased basal ISC at 4 wk, and reduced Cl‐ channel and Na+, K+‐pump activity at both 4 wk and 8 wk compared to STD+AIR controls. HFWD‐consumption caused a reduction in Cl‐ transport and Na+, K+‐pump activity at 4 wk, while increasing Rt in response to ouabain at 0 wk and NPPB at 8 wk compared to STD+AIR. HFWD+SIL increased basal ISC at 0 and 4 wk, caused reduction in Cl‐ transport and Na+, K+‐pump activity at 4 wk, while reducing Rtin response to ouabain at 4 wk compared to STD+SIL. No significant changes in tracheal reactivity to MCh were observed. In conclusion, HFWD and silica altered epithelial ion transport, but the combined effects of HFWD+SIL were not synergistic.

Design for a multicenter, randomized, sham-controlled study to evaluate safety and efficacy after treatment with the Nuvaira\xae lung denervation system in subjects with chronic obstructive pulmonary disease (AIRFLOW-3)

BackgroundTargeted lung denervation (TLD) is a bronchoscopically delivered ablation therapy that selectively interrupts pulmonary parasympathetic nerve signaling. The procedure has the potential to alter airway smooth muscle tone and reactivity, decrease mucous secretion, and reduce airway inflammation and reflex airway hyperresponsiveness. Secondary outcome analysis of a previous randomized, sham-controlled trial showed a reduction in moderate-to-severe exacerbations in patients with COPD after TLD treatment. A pivotal trial, AIRFLOW-3 has been designed to evaluate the safety and efficacy of TLD combined with optimal medical therapy to reduce moderate or severe exacerbations throughout 1 year, compared with optimal medical therapy alone.MethodsThe study design is a multicenter, randomized, full sham bronchoscopy controlled, double-blind trial that will enroll 400 patients (1:1 randomization). Key inclusion criteria are FEV1/FVC < 0.7, FEV1 30 to 60% of predicted, post-bronchodilator, ≥ 2 moderate or 1 severe COPD exacerbations in the prior year, and COPD assessment test (CAT) ≥ 10. Primary objective will be the comparison of moderate or severe COPD exacerbations through 12 months of TLD therapy with optimal medical therapy versus optimal medical therapy alone. The sham group will be allowed to cross over at 1 year. Patients will be followed for up to 5 years.DiscussionThe multicenter, randomized, full sham bronchoscopy controlled, double-blind AIRFLOW-3 trial will evaluate the efficacy of TLD to reduce moderate or severe COPD exacerbations beyond optimal medical therapy alone. The target population are patients with COPD, who suffer persistent symptoms and exacerbations despite optimal treatment, defining an unmet medical need requiring novel therapeutic solutions. This trial is registered at clinicaltrials.gov: NCT03639051.

Differential estrogen-receptor activation regulates extracellular matrix deposition in human airway smooth muscle remodeling via NF-κB pathway.

Altered airway smooth muscle (ASM) mass and extracellular matrix (ECM) deposition in airways are characteristic features of remodeling in asthma. Increased ECM production modulates ASM cell proliferation and leads to airway remodeling. Our previous studies showed that ASM from patients with asthma exhibited increased expression of estrogen receptor (ER)-β, which upon activation down-regulated ASM proliferation, implicating an important role for estrogen signaling in airway physiology. There is no current information on the effect of differential ER activation on ECM production. In this study, we evaluated the effect of ER-α vs. ER-β activation on ECM production, deposition, and underlying pathways. Primary human ASM cells isolated from asthmatics and nonasthmatics were treated with E2, an ER-α agonist [propylpyrazoletriol (PPT)], and an ER-β agonist [WAY-200070 (WAY)] with TNF-α or platelet-derived growth factor (PDGF) followed by evaluation of ECM production and deposition. Expression of proteins and genes corresponding to ECM were measured using Western blotting and quantitative RT-PCR with subsequent matrix metalloproteinase (MMP) activity. Molecular mechanisms of ER activation in regulating ECM were evaluated by luciferase reporter assays for activator protein 1 (AP-1) and NF-κB. TNF-α or PDGF significantly (P < 0.001) increased ECM deposition and MMP activity in human ASM cells, which was significantly reduced with WAY treatment but not with PPT. Furthermore, TNF-α- or PDGF-induced ECM gene expression in ASM cells was significantly reduced with WAY (P < 0.001). Moreover, WAY significantly down-regulated the activation of NF-κB (P < 0.001) and AP-1 (P < 0.01, P < 0.05) in ASM cells from asthmatics and nonasthmatics. Overall, we demonstrate differential ER signaling in controlling ECM production and deposition. Activation of ER-β diminishes ECM deposition via suppressing the NF-κB pathway activity and might serve as a novel target to blunt airway remodeling.-Ambhore, N. S., Kalidhindi, R. S. R., Pabelick, C. M., Hawse, J. R., Prakash, Y. S., Sathish, V. Differential estrogen-receptor activation regulates extracellular matrix deposition in human airway smooth muscle remodeling via NF-κB pathway.

Contractile Properties of Intrapulmonary Airway Smooth Muscle in Cystic Fibrosis.

Cystic fibrosis (CF) is an autosomal-recessive disease caused by mutations in the CF transmembrane conductance regulator gene. Many patients with CF have asthma-like symptoms and airway hyperresponsiveness, which are potentially associated with altered airway smooth muscle (ASM) contractility. Our goal in this study was to assess the contractility of the CF intrapulmonary ASM. ASM strips were dissected from human control and CF intrapulmonary airways, and assessed for methacholine-induced shortening velocity, maximal force, and stress. We also assessed isoproterenol responses in maximally methacholine-contracted ASM. ASM strips were then incubated for 16 hours with IL-13 and measurements were repeated. Myosin light chain kinase (MLCK) expression was assessed by Western blotting. Airways were immunostained for morphometry. ASM mass was increased in CF airways, which likely contributes to airway hyperresponsiveness. Although ASM contractile properties were not intrinsically different between patients with CF and control subjects, CF ASM responded differently in the presence of the inflammatory mediator IL-13, showing impairment in β-adrenergic-induced relaxation. Indeed, the percentage of relaxation measured at maximal isoproterenol concentrations in the CF ASM was significantly lower after incubation with IL-13 (46.0% ± 6.7% relaxation) than without IL-13 (74.0% ± 7.7% relaxation, P = 0.018). It was also significantly lower than that observed in control ASM incubated with IL-13 (68.8% ± 4.9% relaxation, P = 0.048) and without IL-13 (82.4% ± 9.9%, P = 0.0035). CF ASM incubated with IL-13 also expressed greater levels of MLCK. Thus, our data suggest that the combination of an increase in ASM mass, increased MLCK expression, and inflammation-induced β-adrenergic hyporesponsiveness may contribute to airway dysfunction in CF.

CD38 and airway hyper-responsiveness: studies on human airway smooth muscle cells and mouse models.

Asthma is an inflammatory disease in which altered calcium regulation, contractility, and airway smooth muscle (ASM) proliferation contribute to airway hyper-responsiveness and airway wall remodeling. The enzymatic activity of CD38, a cell-surface protein expressed in human ASM cells, generates calcium mobilizing second messenger molecules such as cyclic ADP-ribose. CD38 expression in human ASM cells is augmented by cytokines (e.g., TNF-α) that requires the activation of MAP kinases and the transcription factors, NF-κB and AP-1, and is post-transcriptionally regulated by miR-140-3p and miR-708 by binding to 3' Untranslated Region of CD38 as well as by modulating the activation of signaling mechanisms involved in its regulation. Mice deficient in Cd38 exhibit reduced airway responsiveness to inhaled methacholine relative to the response in wild-type mice. Intranasal challenge of Cd38-deficient mice with TNF-α or IL-13, or the environmental fungus Alternaria alternata, causes significantly attenuated methacholine responsiveness compared with wild-type mice, with comparable airway inflammation. Reciprocal bone marrow transfer studies revealed partial restoration of airway hyper-responsiveness to inhaled methacholine in the Cd38-deficient mice. These studies provide evidence for CD38 involvement in the development of airway hyper-responsiveness; a hallmark feature of asthma. Future studies aimed at drug discovery and delivery targeting CD38 expression and (or) activity are warranted.

The effect of hyperpolarization-activated cyclic nucleotide-gated ion channel inhibitors on the vagal control of guinea pig airway smooth muscle tone.

Subtypes of the hyperpolarization-activated cyclic nucleotide-gated (HCN) family of cation channels are widely expressed on nerves and smooth muscle cells in many organ systems, where they serve to regulate membrane excitability. Here we have assessed whether HCN channel inhibitors alter the function of airway smooth muscle or the neurons that regulate airway smooth muscle tone. The effects of the HCN channel inhibitors ZD7288, zatebradine and Cs(+) were assessed on agonist and nerve stimulation-evoked changes in guinea pig airway smooth muscle tone using tracheal strips in vitro, an innervated tracheal tube preparation ex vivo or in anaesthetized mechanically ventilated guinea pigs in vivo. HCN channel expression in airway nerves was assessed using immunohistochemistry, PCR and in situ hybridization. HCN channel inhibition did not alter airway smooth muscle reactivity in vitro to exogenously administered smooth muscle spasmogens, but significantly potentiated smooth muscle contraction evoked by the sensory nerve stimulant capsaicin and electrical field stimulation of parasympathetic cholinergic postganglionic neurons. Sensory nerve hyperresponsiveness was also evident in in vivo following HCN channel blockade. Cs(+) , but not ZD7288, potentiated preganglionic nerve-dependent airway contractions and over time induced autorhythmic preganglionic nerve activity, which was not mimicked by inhibitors of potassium channels. HCN channel expression was most evident in vagal sensory ganglia and airway nerve fibres. HCN channel inhibitors had a previously unrecognized effect on the neural regulation of airway smooth muscle tone, which may have implications for some patients receiving HCN channel inhibitors for therapeutic purposes.

Models to understand contractile function in the airways

Although the role of contractile function in the airways is controversial, there is general consensus on the importance of airway smooth muscle (ASM) as a therapeutic target for diseases characterized by airway obstruction, such as asthma or chronic obstructive pulmonary disease. Indeed, the use of bronchodilators to relax ASM is the most common and effective practice to treat airflow obstruction. Excessive pathologic bronchoconstriction may originate from primary alterations of ASM mechanical function and/or from the effects exerted on ASM function by disease processes, such as inflammation and remodeling. An in depth knowledge of the potentially multiple mechanisms that distinctively regulate primary and secondary alterations in ASM contractile function would be essential for the development of new therapeutic approaches aimed at preventing the occurrence or reducing the severity of bronchoconstriction. The present review discusses studies that have addressed the mechanisms of altered ASM contractile function in models of airway hyperresponsiveness. Although not comprehensively, in the present review, animal models of intrinsic airway hyperresponsiveness, normal ontogenesis, and allergic sensitization are analyzed in the attempt to summarize the current knowledge on regulatory mechanisms of ASM contractile function in health and disease. Studies in human ASM and the need for additional models to understand contractile function in the airways are also discussed.

Cyclic GMP-dependent protein kinase regulation of airway smooth muscle in asthma

The mechanisms regulating altered airway smooth muscle (ASM) cell tone and airway reactivity in asthma remain incompletely understood. For vascular smooth muscle (VSM), the most important endogenous relaxation pathway is the nitric oxide (NO)-guanylate cyclase-cyclic GMP-dependent protein kinase type I (PKGI) pathway in which endogenous NO from NO synthases or from exogenous nitrovasodilators activate guanylate cyclase, increasing cGMP levels, and activating PKGI. PKGI in turn targets specific VSM molecules that mediate smooth muscle relaxation. Recent evidence supports that in ASM, like in VSM, activators of guanylyl cyclases have significant cGMP-mediated relaxant and antiproliferative effects. PKGI is therefore a prime but unexplored candidate molecule to mediate ASM relaxation. Recently, we have used mice harbouring a mutation in the leucine zipper N-terminal interaction domain (LZM mice [1]) to test the hypothesis that PKGIα is a critical mediator of ASM relaxation that normally protects against airway hyperresponsivity via interactions with specific molecular targets, including ASM myosin phosphate and Rho/Rho-kinase, and that this regulatory system is perturbed in the setting of asthma. Using this knock-in mouse harboring a discrete mutation in the protein-protein interaction domain of PKGI, which disrupts NO-PKG-mediated smooth muscle relaxation by disrupting the PKGI-myosin phosphatase complex we have begun to explore the mechanism of PKGI-mediated ASM relaxation and the role of PKGI in models of asthma. Several asthma studies in LZM mice and WT littermates have now been performed and analyzed using both the acute and the chronic House Dust Mite (HDM) asthma model in WT and LZM mice. Endpoints examined include airway responsiveness to methacholine; quantification of inflammatory cell populations from Bronchoalveolar lavage (BAL); and studies of airway smooth muscle in the mice. In the LZM mice, there is a significant increase in bronchospasm noted in the airways of mutant mice in both acute and chronic asthma models. No differences between WT and LZM mice in the extent of increase in either total cells or in eosinophilia in BALF samples following HDM was noted in the either the acute or chronic models. The extent and volume of Airway SMC a-actin was also measured in the WT and LZM mice following HDM-induced asthma in the acute model. SMC labeling in the airways was quantified using immunohistochemistry and computerized morphometrics and for each measure, a significant increase in SMC number and total volume was noted in the LZM mice. Studies of PKG expression level and of SMC proliferation in the asthma models are underway. These experiments will provide better understanding of ASM relaxation at the molecular level and have the potential to identify novel gene targets for the development of new therapeutic approaches to bronchoconstriction in asthma.

γ-Aminobutryic Acid

γ-Aminobutryic Acid

Influence of obesity on the prevalence and clinical features of asthma

Obesity has been associated with an increased prevalence of asthma and poorer control of this disease. However, the mechanisms by which obesity can influence airway function and make asthma more difficult to control remain uncertain. The physiological changes associated with obesity can contribute to respiratory symptoms and these should be differentiated from those caused by asthma. Obesity can possibly influence the development of asthma through genetic, developmental, hormonal, neurogenic or mechanical influences. Breathing at low lung volumes and changes in the pattern of breathing in obese subjects may alter airway smooth muscle plasticity and airway function. The release by adipocytes of various cytokines and mediators such as Interleukin-6, TNF-alpha, eotaxin, and leptin, and the reduction of anti-inflammatory adipokines in obese subjects may possibly contribute to the development or increased clinical expression of asthma in promoting airway inflammation. Reduced asthma control and impaired response to asthma therapy have been reported in obese patients. Obesity-related co-morbidities such as Sleep Apnea and Gastro-esophageal reflux may also contribute to this poor control. Weight loss improves asthma control and reduces medication needs. Research is needed to better define the optimal management of obese asthmatic patients.

Mechanisms Altering Airway Smooth Muscle Cell Ca2+ Homeostasis in Two Asthma Models

Background: Asthma is characterized by airway remodeling, altered mucus production and airway smooth muscle cell (ASMC) contraction causing extensive airway narrowing. In particular, alterations of ASMC contractility seem to be of crucial importance. The elevation of the cytoplasmic Ca²⁺ concentration is a key event leading to ASMC contraction and changes in the agonist-induced Ca²⁺ increase in ASMC have been reported in asthma. Objective: The aim of this study was to investigate mechanisms underlying these changes. Methods: Murine tracheal smooth muscle cells (MTSMC) from T-bet KO mice and human bronchial smooth muscle cells (HBSMC) incubated with IL-13 and IL-4 served as asthma models. Acetylcholine-induced changes in the cytoplasmic Ca²⁺ concentration were recorded using fluorescence microscopy and the expression of Ca²⁺ homeostasis regulating proteins was investigated with Western blot analysis. Results: Acetylcholine-induced Ca²⁺ transients were elevated in both asthma models. This correlated with an increased Ca²⁺ content of the sarcoplasmic reticulum (SR). In MTSMC from T-bet KO mice, the expression of the SR Ca²⁺ buffers calreticulin and calsequestrin was higher compared to wild-type mice. In HBSMC incubated with IL-13 or IL-4, the expression of ryanodine receptors, inositol-3-phosphate receptors and sarcoplasmic/endoplasmic reticulum Ca²⁺ ATPases 2 was increased compared to HBSMC without incubation with interleukins. The enlarged acetylcholine-induced Ca²⁺ transients could be reversed by blocking inositol-3-phosphate receptors. Conclusions: We conclude that in the murine asthma model the SR Ca²⁺ buffer capacity is increased, while in the human asthma model the expression of SR Ca²⁺ channels is altered. The investigation of the Ca²⁺ homeostasis of ASMC has the potential to provide new therapeutical options in asthma.

Extracellular Matrix Regulates Enhanced Eotaxin Expression in Asthmatic Airway Smooth Muscle Cells

Altered airway smooth muscle (ASM) function and enrichment of the extracellular matrix (ECM) with fibronectin and collagen are key features of asthma. Previously, we have reported these ECM proteins enhance ASM synthetic function. We compared ASM cultured from endobronchial biopsies from subjects with and without asthma to assess if asthmatic cells were hypersecretory and determined whether the underlying mechanism involved autocrine ECM production. Cells from subjects with and without asthma were cultured on plastic or in plates precoated with ECM proteins. Cytokine production was evaluated by enzyme-linked immunosorbent assay and by reverse transcriptase-polymerase chain reaction. Function-blocking integrin antibodies were used to identify integrin involvement. Baseline eotaxin and its production after stimulation with interleukin (IL)-13, IL-1beta, or tumor necrosis factor-alpha was increased (2.5- to 6.0-fold) in ASM cells cultured from subjects with asthma compared with healthy subjects. When seeded on ECM from asthmatic ASM, IL-13-dependent eotaxin release from healthy or asthmatic ASM was enhanced compared with culture on healthy ECM. The ECM substrates fibronectin and type I collagen each enhanced IL-13-dependent eotaxin release, and Western immunoblot indicated that fibronectin expression was higher in asthmatic ASM cells. Integrin-blocking antibodies revealed that alpha5beta1 was required for more than 50% of the enhanced IL-13-dependent eotaxin release by ASM cells from subjects with asthma, whereas alpha2beta1 or alphavbeta3 neutralization lacked effect. The data indicate that ASM cells cultured from subjects with asthma are hypersecretory compared with cells from healthy donors and that autocrine fibronectin secretion acting via alpha5beta1 in part underlies this effect.

Regulation of Toll-like receptor 4-induced proasthmatic changes in airway smooth muscle function by opposing actions of ERK1/2 and p38 MAPK signaling

Activation of Toll-like receptors (TLRs) on immune surveillance cells in the lung has been implicated in the pathobiology of allergic asthma, a condition associated with altered airway smooth muscle (ASM) contractility. Because ASM is known to directly respond to various proasthmatic stimuli, the potential role of TLR signaling in ASM in regulating airway expression of the proasthmatic phenotype was investigated. Cultured human ASM cells were found to express TLR4 and TLR9 mRNA transcripts and, whereas TLR9 stimulation had little effect, TLR4 activation with LPS elicited significant increases in IL-6 release and evoked proasthmatic-like changes in the constrictor and relaxation responsiveness of isolated rabbit ASM tissues. Complementary studies further demonstrated that the ASM responses to LPS were associated with activation of the ERK1/2 and p38 MAPK signaling pathways, IKK-mediated activation of NF-kappaB, and coupling of phosphorylated ERK1/2 with the p65 subunit of NF-kappaB. Moreover, the induced NF-kappaB activity and changes in ASM responsiveness were prevented in LPS-exposed ASM that were pretreated with inhibitors of ERK1/2 signaling, whereas inhibition of p38 MAPK augmented the proasthmatic responses to LPS. Finally, activation of p38 MAPK with anisomycin prevented both the LPS-induced stimulation of ERK1/2-mediated NF-kappaB activity and associated changes in ASM responsiveness. Collectively, these data support the novel concept that TLR4 activation in ASM elicits changes in ASM function that are regulated by opposing effects of MAPK signaling, wherein LPS-induced ERK1/2 activation mediates NF-kappaB-dependent proasthmatic-like changes in ASM function, whereas coactivation of p38 MAPK serves to homeostatically downregulate the proasthmatic effects of ERK1/2 activation.

TNFα reduces tachykinin, PGE2‐dependent, relaxation of the cultured mouse trachea by increasing the activity of COX‐2

1. Chronic inflammation is a central feature of asthma. The inflammatory cytokine tumour necrosis factor alpha (TNFalpha) has been implicated in this disease, and is known to alter airway smooth muscle functionally. 2. The aim of this study was to investigate the influence of TNFalpha on tachykinin-induced airway relaxation. Mouse tracheae were cultured in the absence and presence of TNFalpha for 1 or 4 days. 3. In the absence of TNFalpha, substance P (SP) and neurokinin A (NKA) induced comparable levels of relaxation in fresh and cultured segments. Functional studies with selective antagonists/inhibitors indicated that the relaxation was mediated by the NK(1) receptor coupled to cyclooxygenase (COX)-2 activation and subsequent release of prostaglandin E(2) (PGE(2)). TNFalpha attenuated SP- and NKA-induced relaxation in a time- and concentration-dependent manner, decreasing the ability of PGE(2) to relax tissues. 4. Further studies indicated that TNFalpha elevated COX-2 activity and that concomitant inhibition of COX-2 reversed TNFalpha-attenuated PGE(2) relaxation. Culture with PGE(2) decreased SP- and PGE(2)-mediated relaxation, further implicating the activity of COX-2 in the attenuation of tachykinin signalling. 5. Gene expression analysis demonstrated that TNFalpha increased the expression of smooth muscle COX-2, PGE(2) synthase and EP(2) receptor mRNA, and decreased the expression of the EP(4) receptor. 6. Overall, these results show that NK(1) receptor-mediated relaxation induced by PGE(2) is attenuated by prolonged TNFalpha stimulation. Increased COX-2 activity induced by TNFalpha appears to be central to this process.

Reduction in airway hyperresponsiveness to methacholine by the application of RF energy in dogs.

We delivered controlled radio frequency energy to the airways of anesthetized, ventilated dogs to examine the effect of this treatment on reducing airway narrowing caused by a known airway constrictor. The airways of 11 dogs were treated with a specially designed bronchial catheter in three of four lung regions. Treatments in each of the three treated lung regions were controlled to a different temperature (55, 65, and 75 degrees C); the untreated lung region served as a control. We measured airway responsiveness to local methacholine chloride (MCh) challenge before and after treatment and examined posttreatment histology to 3 yr. Treatments controlled to 65 degrees C as well as 75 degrees C persistently and significantly reduced airway responsiveness to local MCh challenge (P < or = 0.022). Airway responsiveness (mean percent decrease in airway diameter after MCh challenge) averaged from 6 mo to 3 yr posttreatment was 79 +/- 2.2% in control airways vs. 39 +/- 2.6% (P < or = 0.001) for airways treated at 65 degrees C, and 26 +/- 2.7% (P < or = 0.001) for airways treated at 75 degrees C. Treatment effects were confined to the airway wall and the immediate peribronchial region on histological examination. Airway responsiveness to local MCh challenge was inversely correlated to the extent of altered airway smooth muscle observed in histology (r = -0.54, P < 0.001). We conclude that the temperature-controlled application of radio frequency energy to the airways can reduce airway responsiveness to MCh for at least 3 yr in dogs by reducing airway smooth muscle contractility.

IL-13-dependent autocrine signaling mediates altered responsiveness of IgE-sensitized airway smooth muscle.

In testing the hypothesis that interleukin-4 receptor alpha-subunit (IL-4R alpha)-coupled signaling mediates altered airway smooth muscle (ASM) responsiveness in the atopic sensitized state, isolated rabbit tracheal ASM segments were passively sensitized with immunoglobulin E (IgE) immune complexes, both in the absence and presence of an IL-4R alpha blocking antibody (anti-IL-4R alpha Ab). Relative to control ASM, IgE-sensitized tissues exhibited enhanced isometric constrictor responses to administered ACh and attenuated relaxation responses to isoproterenol. These proasthmatic-like effects were prevented in IgE-sensitized ASM that were pretreated with anti-IL-4R alpha Ab. In complementary experiments, IgE-sensitized cultured human ASM cells exhibited upregulated expression of IL-13 mRNA and protein, whereas IL-4 expression was undetected. Moreover, extended studies demonstrated that 1) exogenous IL-13 administration to naïve ASM elicited augmented contractility to ACh and impaired relaxation to isoproterenol, 2) these effects of IL-13 were prevented by pretreating the tissues with an IL-5 receptor blocking antibody, and 3) IL-13 administration induced upregulated mRNA expression and release of IL-5 protein from cultured ASM cells. Collectively, these findings provide new evidence demonstrating that the altered responsiveness of IgE-sensitized ASM is largely attributed to activation of an intrinsic Th2-type autocrine mechanism involving IL-13/IL-4R alpha-coupled release and action of IL-5 in the sensitized ASM itself.

Peripheral airway smooth muscle mechanics in obstructive airways disease.

The purpose of this study was to determine whether altered airway smooth muscle (ASM) contractility contributes to the pathogenesis of obstructive airways diseases such as chronic obstructive pulmonary disease (COPD) and asthma. The passive and active mechanical properties of isolated human peripheral airways were measured in vitro by myography. The amount of ASM was measured by morphometry. Pulmonary function was assessed before surgery by the FEV(1) (%pred) and the FEV(1)/ FVC (%). Fifteen airways were studied from nonobstructed (NOB) patients, and 15 from obstructed (OB, FEV(1)/FVC < 70%) patients (62 +/- 10 yr, mean +/- SD). The maximal isometric force (Fmax), stress (Fmax/ASM), airway diameter at Lmax (Dmax), maximal isotonic shortening (%Lmax), and normalized airway smooth muscle (ASM/Dmax) were determined in all patients. There was a significant correlation between Fmax and FEV(1) (%pred) (r = -0.579, p < 0.004), between Fmax and FEV(1)/FVC (%) (r = -0.720, p < 0.003), and between stress and FEV(1)/FVC (%) (-0.611, p < 0.002). There was no correlation between isotonic shortening and either measure of pulmonary function. A positive correlation was found between force and shortening (r = 0.442, p < 0.05), and stress and shortening (r = 0.538, p < 0.01). Both force and stress were significantly increased (p < 0.05) in OB (Fmax = 0.87 +/- 0.8 g, stress = 76 +/- 47 mN/mm(2)) versus NOB (Fmax = 0.42 +/- 0.18 g, stress = 51 +/- 21 mN/mm(2)) patients, while isotonic shortening was not different between the two groups. ASM and ASM/Dmax were both significantly increased in the OB patient group (p < 0.05). These results suggest that obstructive airways disease is associated with an increase in the ability of the ASM to generate force. (Values represent means +/- SD.)

Moxonidine acting centrally inhibits airway reflex responses.

We examined the role of I1-imidazoline (I1-IR) receptors in control of airway function, by testing the effects of systemic administration of the I1-IR agonist moxonidine on reflex responses of tracheal smooth muscle (TSM) tone to either lung deflation or mechanical stimulation of intrapulmonary rapidly adapting receptors. Experiments were performed in either alpha-chloralose anesthetized or decorticate, paralyzed, and mechanically ventilated beagle dogs. Moxonidine (10-100 micrograms/kg) administered via three different routes (femoral vein, muscular branch of superior thyroid artery, and vertebral artery) attenuated TSM responses to stimulation of airway sensory nerve fibers by two different ways and caused a decrease in arterial pressure and heart rate. These effects were dose dependent and were significantly reversed by efaroxan (an I1-IR and alpha 2-adrenergic blocker) administered via the vertebral artery. Intravertebral efaroxan abolished the hemodynamic effects of moxonidine. Intravenous moxonidine (10-100 micrograms/kg) did not alter airway smooth muscle responses to electrical stimulation of the peripheral vagus nerve. In addition, in vitro moxonidine (1-100 micrograms/ml) had no effect on contractile responses to increasing doses of acetylcholine. These findings indicate that moxonidine may act at a central site to suppress reflex airway constriction, even when given into the systemic circulation. Given the presence of I1-IR sites and alpha 2-adrenergic receptors in brain regions participating in airway reflexes, these receptor classes may be involved in brainstem control of the cholinergic outflow to the airways.

I 1-imidazoline receptors and cholinergic outflow to the airways

We examined the role of I 1-imidazoline receptors in the control of airway function, by testing the effects of systemic administration of the I 1-imidazoline agonist moxonidine on reflex responses of tracheal smooth muscle (TSM) tone to either lung deflation or mechanical stimulation of intrapulmonary rapidly adapting receptors. Experiments were performed in either α-chloralose anaesthetized or decorticate, paralyzed and mechanically ventilated beagle dogs. Moxonidine (10–100 μg/kg) administered via three different routes (the femoral vein, muscular branch of superior thyroid artery, and vertebral artery) attenuated TSM responses to stimulation of airway sensory nerve fibers by two different ways, and caused a decrease in arterial pressure and heart rate. These effects were dose-dependent, and were significantly reversed by efaroxan (an I 1-imidazoline and α 2-adrenergic blocker) administered via the vertebral artery. Intravertebral efaroxan abolished the hemodynamic effects of moxonidine. Intravenous moxonidine (10–100 μg/kg) did not alter airway smooth muscle responses to electrical stimulation of the peripheral vagus nerve. In addition, in vitro moxonidine (1–100 μg/ml) had no effect on contractile responses to increasing doses of acetylcholine. These findings indicate that moxonidine may act at a central site to suppress reflex airway constriction, even when given into the systemic circulation. Given the presence of I 1-imidazoline sites and α 2-adrenergic receptors in brain regions participating in airway reflexes, these receptor classes may be involved in brainstem control of the cholinergic outflow to the airways.