Hypocontractile airway smooth muscle phenotype exhibits enhanced β2 laminin chain expression in lung allergy model.

Airway smooth muscle: A modulator of airway remodeling in asthma

Objective To explore the differences in airway inflammation, airway remodeling and airway hyperresponsiveness of ovalbumin (OVA) induced murine asthma model during acute and chronic terms, and clear the pathological changes in the pathogenesis of asthma. Methods 48 BALB/c mice were randomly divided into acute and chronic groups, in which acute groups including normal control group (A1 group) and acute asthma group (A2 group), chronic group including normal control group (B1 group) and chronic asthma group (B2 group). Using OVA sensitized and challenged methods induced acute and chronic asthma model, then assessed the airway resistance, total cell numbers and different cell numbers in the BALF, and detected the levels of IL-4, IL-5, transforming growth factor-β1 (TGF-β1), vascular endothelial growth factor (VEGF) and IFN-γ by enzyme-linked immunosorbent assay (ELISA). Then HE staining was used to observe airway inflammation.AB-PAS and Masson staining were used to measure airway remodeling. Results Compared with normal mice, the airway resistance of the acute asthma group and the chronic asthma group were significantly increased, there was no difference between the two groups, but the basic airway resistance in the chronic phase was significantly changed.Compared to the chronic asthmatic mice, the inflammatory changes, such as numbers of total cells and EOS cells, levels of IL-4, IL-5 and IFN-γ, aggregation of inflammatory cells around the airway and vascular and secretion of airway mucus were more obvious during acute phase.However, changes such as the levels of TGF-β1 and VEGF in the BALF, the thickening of airway smooth muscle, the subepithelial collagen deposition and subepithelial fibrosis were more significant in the chronic phase when compared with the acute asthma group. Conclusions In the early stage of asthma is mainly inflammatory changes, but the mild remodeling changes have been presented during this stage.And in the chronic stage of asthma although the inflammatory changes are continued, but the mainly factors that impact asthma symptoms are the organic changes. Key words: Asthma model; Acute asthma; Chronic asthma; Airway inflammation; Airway remodeling

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The present study compared the effects of repeated antigen exposure on the development of hyperresponsiveness and the expression of RhoA in the main bronchial and lower tracheal smooth muscles of sensitized Actively sensitized rats were repeatedly challenged by antigen inhalation. Twenty-four hours after the final antigen challenge the isometrical contractions of the bronchial and tracheal smooth muscles were measured. Immunoblottings were also performed using bronchial and tracheal homogenates and the density ratios of RhoA/beta-actin were calculated to quantify the levels of RhoA. Acetylcholine-induced contraction of bronchial, but not tracheal, smooth muscle of antigen-treated rats was significantly augmented as compared with that of control rats, indicating that airway hyperresponsiveness appeared by antigen challenge in bronchial smooth muscle. RhoA expression in bronchial, but not tracheal, smooth muscle was significantly increased in the antigen-treated animals. The increased expression of RhoA is suggested to have an important role in developing hyperresponsiveness of bronchial smooth muscle.

Gastroesophageal reflux is a common disorder closely related to chronic airway diseases, such as chronic cough, asthma, chronic bronchitis, and chronic obstructive disease. Indeed, gastroesophageal acid reflux into the respiratory tract causes bronchoconstriction, but the underlying mechanisms have still not been clarified. This study aimed to elucidate functional changes of bronchial smooth muscles (BSMs) isolated from guinea pigs in an animal model of gastroesophageal reflux. The marked airway inflammation, hyperresponsiveness and remodeling were observed after guinea pigs were exposed to intraesophageal HCl infusion for 14 days. In addition, contractile responses to acetylcholine (ACh), KCl, electrical field stimulation, and extracellular Ca(2+) were greater in guinea pigs infused with HCl compared with control groups. The L-type voltage-dependent Ca(2+) channels (L-VDCC) blocker, nicardipine, significantly inhibited ACh- and Ca(2+)-enhanced BSM contractions in guinea pigs infused with HCl. The Rho-kinase inhibitor, Y27632, attenuated ACh-enhanced BSM contractions in guinea pigs infused with HCl. Moreover, mRNA and protein expressions for muscarinic M2 and M3 receptors, RhoA, and L-VDCC in BSM were detected by real-time PCR and Western blot. Expressions of mRNA and protein for muscarinic M3 receptors, RhoA, and L-VDCC were greater than in BSM of HCl-infused guinea pigs, whereas levels of muscarinic M2 receptors were unchanged. We demonstrate that acid infusion to the lower esophagus and, subsequently, microaspiration into the respiratory tract in guinea pigs leads to airway hyperresponsiveness and overactive BSM. Functional and molecular results indicate that overactive BSM is the reason for enhancement of extracellular Ca(2+) influx via L-VDCC and Ca(2+) sensitization through Rho-kinase signaling.

The distal airway is more important in the regulation of airflow resistance than is the proximal airway, and volatile anesthetics have a greater inhibitory effect on distal airway muscle tone. The authors investigated the different reactivities of airway smooth muscles to volatile anesthetics by measuring porcine tracheal or bronchial (third to fifth generation) smooth muscle tension and intracellular concentration of free Ca2+ ([Ca2+]i) and by measuring inward Ca2+ currents (ICa) through voltage-dependent Ca2+ channels (VDCs). Intracellular concentration of free Ca2+ was monitored by the 500-nm light emission ratio of Ca2+ indicator fura-2. Isometric tension was measured simultaneously. Whole-cell patch clamp recording techniques were used to investigate the effects of volatile anesthetics on ICa in dispersed smooth muscle cells. Isoflurane (0-1.5 minimum alveolar concentration) or sevoflurane (0-1.5 minimum alveolar concentration) was introduced into a bath solution. The volatile anesthetics tested had greater inhibitory effects on carbachol-induced bronchial smooth muscle contraction than on tracheal smooth muscle contraction. These inhibitory effects by the anesthetics on muscle tension were parallel to the inhibitory effects on [Ca2+]i. Although tracheal smooth muscle cells had only L-type VDCs, some bronchial smooth muscle cells (approximately 30%) included T-type VDC. Each of the two anesthetics significantly inhibited the activities of both types of VDCs in a dose-dependent manner; however, the anesthetics had greater inhibitory effects on T-type VDC activity in bronchial smooth muscle. The existence of the T-type VDC in bronchial smooth muscle and the high sensitivity of this channel to volatile anesthetics seem to be, at least in part, responsible for the different reactivities to the anesthetics in tracheal and bronchial smooth muscles.

BackgroundIt has recently been suggested that RhoA plays an important role in the enhancement of the Ca2+ sensitization of smooth muscle contraction. In the present study, a participation of RhoA-mediated Ca2+ sensitization in the augmented bronchial smooth muscle (BSM) contraction in a murine model of allergic asthma was examined.MethodsOvalbumin (OA)-sensitized BALB/c mice were repeatedly challenged with aerosolized OA and sacrificed 24 hours after the last antigen challenge. The contractility and RhoA protein expression of BSMs were measured by organ-bath technique and immunoblotting, respectively.ResultsRepeated OA challenge to sensitized mice caused a BSM hyperresponsiveness to acetylcholine (ACh), but not to high K+-depolarization. In α-toxin-permeabilized BSMs, ACh induced a Ca2+ sensitization of contraction, which is sensitive to Clostridium botulinum C3 exoenzyme, indicating that RhoA is implicated in this Ca2+ sensitization. Interestingly, the ACh-induced, RhoA-mediated Ca2+ sensitization was significantly augmented in permeabilized BSMs of OA-challenged mice. Moreover, protein expression of RhoA was significantly increased in the hyperresponsive BSMs.ConclusionThese findings suggest that the augmentation of Ca2+ sensitizing effect, probably via an up-regulation of RhoA protein, might be involved in the enhanced BSM contraction in antigen-induced airway hyperresponsiveness.

1. The purpose of the present experiments was to elucidate the differences in actions of two K+ channel openers, KC 128 and levcromakalim, on the carbachol-induced contraction, membrane potential and 86Rb+ efflux of the dog tracheal and bronchial smooth muscles. Furthermore, we compared the effects of these agents on guinea-pig and human airway smooth muscles. 2. In the dog tracheal and bronchial smooth muscle tissues, levcromakalim induced a concentration-dependent relaxation of the carbachol-induced contraction. The IC50 values were 0.35 microM (pIC50: 6.46 +/- 0.10, n = 9) and 0.55 microM (pIC50: 6.26 +/- 0.07, n = 5), respectively. KC 128 relaxed bronchial smooth muscles precontracted by carbachol with an IC50 value of 0.19 microM (pIC50: 6.73 +/- 0.10, n = 7). However, KC 128 had almost no effect on the contraction evoked by carbachol in the trachea (IC50 > 10 microM). The relaxations induced by levcromakalim and KC 128 were antagonized by glyburide (0.03-1 microM) but not by charybdotoxin (100 nM). 3. Levcromakalim (1 microM) hyperpolarized the membrane of both dog tracheal and bronchial smooth muscle cells, whereas KC 128 (1 microM) hyperpolarized the membrane of bronchial but not of tracheal smooth muscle cells. 4. Levcromakalim (10 microM) increased 86Rb+ efflux rate from both tracheal and bronchial smooth muscle tissues but KC 128 (10 microM) increased 86Rb+ efflux rate only from bronchial and not tracheal smooth muscle tissues. Glyburide (1 microM) prevented the hyperpolarization and the 86Rb+ efflux induced by these agents at the same concentration as observed for mechanical responses. 5. Both KC 128 and levcromakalim relaxed the guinea-pig isolated tracheal smooth muscles precontracted by carbachol (100 nM), histamine (3 micro M) or U46619 (10 nM). KC 128 was approximately 10 times more potent than levcromakalim for each agonist.6. In human bronchial smooth muscles, levcromakalim but not KC 128 induced a concentration dependent relaxation of the carbachol-induced contraction.7. It is concluded that KC 128 has relaxant and hyperpolarizing effects in the dog bronchial and guinea-pig tracheal smooth muscles, but not in the dog tracheal and human bronchial smooth muscles.On the other hand, levcromakalim acts consistently on all the above airway smooth muscle tissues.These results indicate that there are regional and species differences in distribution of K+ channels, and at least two different K+ channel opener- and glyburide-sensitive K+ channels are present in the dog airway smooth muscles.

Furosemide attenuates airway obstruction in asthmatic subjects when administered as an aerosol pretreatment. This protective effect of furosemide could be related to relaxation of bronchial smooth muscle or to increased bronchial blood flow. To determine whether furosemide dilates bronchial smooth muscle, isometric contractile responses in distal bronchi from young pigs were studied. In bronchial smooth muscle rings that were precontracted with 10(-5) M acetylcholine, significant relaxation occurred with 10(-8) to 3 x 10(-6) M isoproterenol but not with 10(-8) to 10(-3) M furosemide. In contrast, bronchial arteries that were precontracted with either 10(-4) M norepinephrine or 10(-8) M vasopressin significantly relaxed in response to 10(-4) to 3 x 10(-3) M and 10(-3) to 3 x 10(-3) M furosemide, respectively. We conclude that furosemide, under the described experimental conditions, relaxes airway vascular smooth muscle but not bronchial smooth muscle. These results are consistent with previous suggestions that inhaled furosemide increases blood flow to airway tissues (Gilbert IA, Lenner KA, Nelson JA, Wolin AD, and Fouke JM. J Appl Physiol 76: 409-415, 1994).

Background: We previously showed that treatment with Mycobacterium bovis BCG killed by extended freeze-drying (EFD BCG) modulates inflammation through regulatory T cells (Tregs) in an acute asthma model. In this study, we investigated the kinetics of Treg induction as well as their long-term homing in spleen and lungs correlating with reduced airway hyperresponsiveness (AHR) in a murine model of acute allergic asthma. We then evaluated the therapeutic implication of EFD BCG in a chronic asthma model. Methods: Tregs expressing Foxp3 were analyzed in various organs shortly and long-term after EFD BCG, live- and Heat Killed-(HK-) BCG treatments in an acute model of asthma. We further studied EFD BCG treatment on airway inflammation using a chronic model of asthma in mice. Results: Foxp3 expression peaked in the inguinal draining lymph-nodes (iDLNs) 2-4 days after EFD BCG treatment whereas it was long-term observed in spleen (days 7 to 90). This increase in Foxp3 expression was also found in lungs upon intranasal ovalbumin (OVA) challenge in OVA-sensitized mice. The loss of protection 4 months after EFD BCG treatment was correlated with the end of this phenomenon. Moreover, major lung inflammation hallmarks of severe asthma after multiple allergen challenges promoting chronic airway inflammation in OVA sensitized mice were reduced by EFD BCG treatment: AHR, eosinophils and neutrophils in bronchoalveolar lavage (BAL), mucus metaplasia, Th2 as well as Th17 cytokine levels in BAL and sera. EFD BCG treatment also enhances PPAR-γ expression and regulates NF-κBp65 translocation in lung extracts in this model of chronic asthma. Conclusions: EFD BCG treatment induced long-term protective effect associated to Foxp3 Tregs in the spleen and lungs in an acute model of asthma and inhibits AHR in a chronic model of asthma. EFD BCG could be a new and promising immuno-modulatory alternative treatment to corticoids in severe human asthma.

Airway hyperresponsiveness, caused by excessive contraction of airway smooth muscle, is a characteristic of asthma involving multiple proteins, including various isoforms of actin and myosin. While α-smooth muscle actin (ACTA2) is linked to hypercontractility, the roles of other isoforms are unclear. Our study investigated the expression of proteins involved in airway smooth muscle contraction and their relation to AHR in an allergic asthma model. Male guinea pigs were sensitized and challenged with ovalbumin, with controls receiving saline. We measured broncho-obstruction and AHR using plethysmography. Protein expression in bronchial and tracheal smooth muscle was analyzed through immunohistochemistry, with proteins identified using electrophoresis and MALDI/TOF-TOF mass spectrometry. In the asthma model, guinea pigs exhibited AHR. The expression of ACTA2, β-cytoplasmic actin (ACTB), and myosin light chains (MYL9) increased, while γ-cytoplasmic actin 1 (ACTG1) was reduced in the bronchial smooth muscle compared to controls. ACTB and ACTA2 expression levels were correlated with AHR, and ACTB was associated with ACTA2, MYL9, and filamin A (FLNA), and inversely with ACTG1. ACTA2 and MYL9 levels showed an inverse association with ACTG1, and the expression levels of FLNA and MYL9 were correlated. Reduced ACTG1 expression was linked to greater AHR. Proteomic analysis confirmed these proteins in guinea pig tracheal smooth muscle, although expression changes differed from the bronchus, except for ACTB, which increased in the asthma model. Our data suggest that increased ACTA2 and ACTB, along with reduced ACTG1, are related to AHR in guinea pigs. MYL9 and FLNA emerge as potential regulators of actin dynamics.

1. Quantitative autoradiographic studies were conducted to determine the distributions and densities of ETA and ETB binding site subtypes in porcine tracheal and bronchial smooth muscle. In addition, the roles of ETA and ETB receptors in endothelin-1-mediated contraction of these tissues were assessed. 2. Quantitative autoradiographic studies revealed that both ETA and ETB binding sites for [125I]-endothelin-1 were present in both bronchial and tracheal airway smooth muscle. However, the proportions of these sites were markedly different at these two levels within the respiratory tract. In tracheal smooth muscle, the proportions of ETA and ETB sites were 30 +/- 1% and 70 +/- 1% respectively, whereas in bronchial smooth muscle, these proportions were virtually reversed, being 73 +/- 2% and 32 +/- 8% respectively. 3. Endothelin-1 induced concentration-dependent contraction of porcine tracheal and bronchial airway smooth muscle. Endothelin-1 had similar potency (concentration producing 30% of the maximum carbachol contraction, Cmax) in trachea (22 nM; 95% confidence limits (c.l.), 9-55 nM; n = 9) and bronchus (22 nM; c.l., 9-55 nM; n = 6). Endothelin-1 also produced comparable maximal contractions in trachea (59 +/- 5% Cmax; n = 9) and bronchus (65 +/- 4% Cmax, n = 6). 4. In trachea, endothelin-1 induced contractions were not significantly inhibited by either the ETA receptor-selective antagonist, BQ-123 (3 microM) or the ETB receptor-selective antagonist, BQ-788 (1 microM). However, in the combined presence of BQ-123 and BQ-788, the concentration-effect curve to endothelin-1 was shifted to the right by 3.7 fold (n = 8; P = 0.01). 5. In bronchus, concentration-effect curves to endothelin-1 were shifted to the right by BQ-123 (3 microM; 4.3 fold; P < 0.05), but not by BQ-788 (1 microM). In the presence of both antagonists, concentration-effect curves to endothelin-1 were shifted by at least 6.7 fold (n = 6; P = 0.01). 6. Sarafotoxin S6c induced contraction in both tissue types, although the maximum contraction was greater in trachea (53 +/- 7% Cmax; n = 6) than in bronchus (21 +/- 5% Cmax; n = 6). BQ-788 (1 microM) markedly reduced sarafotoxin S6c potency in both trachea and bronchus (e.g. by 50 fold in trachea; c.l., 14-180; n = 6; P < 0.05). 7. These data demonstrate that the proportions of functional endothelin receptor subtypes mediating contraction of airway smooth muscle to endothelin-1, vary significantly at different levels in the porcine respiratory tract.

We have been assigned the task of showing that “alterations in airway smooth muscle phenotype DO NOT cause the airways hyperresponsiveness of asthma.” In the following paragraphs we will briefly review the overwhelming lack of evidence that any fundamental change in the intrinsic properties of

Autoradiographic localization of M-cholinergic receptors in the lung tissue of experimental asthmatic guinea-pigs

Bronchomotor tone is regulated by contraction and relaxation of airway smooth muscle (ASM). A weakened ASM relaxation might be a cause of airway hyperresponsiveness (AHR), a characteristic feature of bronchial asthma. Pituitary adenylyl cyclase-activating polypeptide (PACAP) is known as a mediator that causes ASM relaxation. To date, whether or not the PACAP responsiveness is changed in asthmatic ASM is unknown. The current study examined the hypothesis that relaxation induced by PACAP is reduced in bronchial smooth muscle (BSM) of allergic asthma. The ovalbumin (OA)-sensitized mice were repeatedly challenged with aerosolized OA to induce asthmatic reaction. Twenty-four hours after the last antigen challenge, the main bronchial smooth muscle (BSM) tissues were isolated. Tension study showed a BSM hyperresponsiveness to acetylcholine in the OA-challenged mice. Both quantitative RT-PCR and immunoblot analyses revealed a significant decrease in PAC1 receptor expression in BSMs of the diseased mice. Accordingly, in the antigen-challenged group, the PACAP-induced PAC1 receptor-mediated BSM relaxation was significantly attenuated, whereas the relaxation induced by vasoactive intestinal polypeptide was not changed. These findings suggest that the relaxation induced by PACAP is impaired in BSMs of experimental asthma due to a downregulation of its binding partner PAC1 receptor. Impaired BSM responsiveness to PACAP might contribute to the AHR in asthma.