Bronchial Smooth Muscle Hyperresponsiveness Research Articles

Investigation of the relaxing effect of a camphor nanoemulsion on rat isolated trachea

Asthma is a chronic inflammatory disease that targeting lower airways, being characterized by bronchial smooth muscle hyper responsiveness and mucus hypersecretion. Asthma is considered the most common respiratory disease in the world, affecting approximately 235 million individuals. The main therapy sometimes fails to establish clinical improvement in patients, which leads to a constant search for new alternatives. Camphor is a transparent solid monoterpene with a strong aroma, which due to its high lipophilicity is insoluble in water. Nanostructured carrier systems have shown promise as a delivery system for lipophilic compounds such as monoterpenes. Therefore, the objective of this work was to evaluate the relaxant effect of nanoemulsified camphor (NEC), as well as the mechanism of action of that monoterpene, in isolated rat trachea. The results obtained demonstrated that NEC promote relaxation of the isolated rat trachea when smooth muscle contraction was induced by both carbachol (CCh) and KCl, presenting a pCE50 of 2.25 ± 0.27 and 3.30 ± 0.07, respectively. In the presence of dexamethasone (DEXA), tetraethylammonium (TEA), glibenclamide (GLIB), 1H-[1,2,4]-oxadiazole-[4,3,-a]-quinoxaline-1-one (ODQ) and ruthenium red (RR) there was a significant difference in at least one of the evaluated pharmacological parameters, such as concentration-response curves shape, Emax or pCE50.As conclusion, NEC may be involved with β-adrenergic receptors, channels for K+ sensitive to ATP (KATP) or Channels for K+ opened by Ca2+ (KCa), increase in prostanoids and with receptor channel with transient potential (TRPv). In conclusion, β-adrenergic receptors, prostanoids, nitric oxide (NO), ATP-sensitive K+ channels (KATP), Ca2+-opened K+ channels (KCa), and transient receptor potential cation channel subfamily V (TRPV) are involved in the relaxing effect of NEC.In addition, the mechanism of action of NEC may be involved with the signal transduction pathway Nitric Oxide/soluble guanylyl cyclase/cGMP/cGMP-activated protein kinase. NEC, therefore, demonstrates spasmolytic activity when presenting tracheal relaxation compared to CCh and KCl contracturants.

Attenuation of relaxing response induced by pituitary adenylate cyclase-activating polypeptide in bronchial smooth muscle of experimental asthma.

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.

Non-coding RNAs and bronchial smooth muscle hyperresponsiveness in allergic bronchial asthma

Non-coding RNAs, such as microRNAs (miRNAs) and long non-coding RNAs (lncRNAs), play important roles in normal and diseased cell functions. A small GTPase RhoA is a key protein of bronchial smooth muscle (BSM) contraction, and an up-regulation of RhoA has been demonstrated in BSMs of experimental asthma. Our previous study also demonstrated that RhoA translation was controlled by a miRNA, miR-133a, in BSMs. In human BSM cells (hBSMCs), an up-regulation of RhoA was observed when the function of endogenous miR-133a was inhibited by its antagomir. Treatment of hBSMCs with interleukin-13 (IL-13) caused an up-regulation of RhoA and a down-regulation of miR-133a. In a murine experimental asthma, increased expression of IL-13 and RhoA and the BSM hyperresponsiveness were observed. Interestingly, the level of miR-133a was significantly decreased in BSMs of the diseased animals. These findings suggest that RhoA expression is negatively regulated by miR-133a in BSMs, and that the miR-133a down-regulation causes an up-regulation of RhoA, resulting in an augmentation of the contraction. Recent studies also revealed an inhibitory effect of lncRNA Malat1 on the miR-133a function. Thus, lncRNAs/miRNAs might be key regulators of BSM hyperresponsiveness, and provide us a new insight into the treatment of airway hyperresponsiveness in asthmatics.

Non-coding RNAs and bronchial smooth muscle hyperresponsiveness in allergic bronchial asthma

Non-coding RNAs, such as microRNAs (miRNAs) and long non-coding RNAs (lncRNAs), play important roles in normal and diseased cell functions. A small GTPase RhoA is a key protein of bronchial smooth muscle (BSM) contraction, and an up-regulation of RhoA has been demonstrated in BSMs of experimental asthma. Our previous study also demonstrated that RhoA translation was controlled by a miRNA, miR-133a, in BSMs. In human BSM cells (hBSMCs), an up-regulation of RhoA was observed when the function of endogenous miR-133a was inhibited by its antagomir. Treatment of hBSMCs with interleukin-13 (IL-13) caused an up-regulation of RhoA and a down-regulation of miR-133a. In a murine experimental asthma, increased expression of IL-13 and RhoA and the BSM hyperresponsiveness were observed. Interestingly, the level of miR-133a was significantly decreased in BSMs of the diseased animals. These findings suggest that RhoA expression is negatively regulated by miR-133a in BSMs, and that the miR-133a down-regulation causes an up-regulation of RhoA, resulting in an augmentation of the contraction. Recent studies also revealed an inhibitory effect of lncRNA Malat1 on the miR-133a function. Thus, lncRNAs/miRNAs might be key regulators of BSM hyperresponsiveness, and provide us a new insight into the treatment of airway hyperresponsiveness in asthmatics.

Augmented Pla2g4c/Ptgs2/Hpgds axis in bronchial smooth muscle tissues of experimental asthma.

RationaleAugmented smooth muscle contractility of the airways is one of the causes of airway hyperresponsiveness in asthmatics. However, the mechanism of the altered properties of airway smooth muscle cells is not well understood.ObjectivesTo identify differentially expressed genes (DEGs) related to the bronchial smooth muscle (BSM) hyper-contractility in a murine asthma model.MethodsThe ovalbumin (OA)-sensitized mice were repeatedly challenged with aerosolized OA to induce asthmatic reaction. Transcriptomic profiles were generated by microarray analysis of BSM tissues from the OA-challenged and control animals, and KEGG (Kyoto Encyclopedia of Genes and Genomes) Pathway Analysis was applied.Measurements and main resultsTension study showed a BSM hyperresponsiveness to acetylcholine (ACh) in the OA-challenged mice. A total of 770 genes were differentially expressed between the OA-challenged and control animals. Pathway analysis showed a significant change in arachidonic acid (AA) metabolism pathway in BSM tissues of the OA-challenged mice. Validation of DEGs by quantitative RT-PCR showed a significant increase in PLA2 group 4c (Pla2g4c)/COX-2 (Ptgs2)/PGD2 synthase 2 (Hpgds) axis. PGD2 level in bronchoalveolar fluids of the OA-challenged mice was significantly increased. A 24-h incubation of BSM tissues with PGD2 caused a hyperresponsiveness to ACh in naive control mice.ConclusionsAA metabolism is shifted towards PGD2 production in BSM tissues of asthma. Increased PGD2 level in the airways might be a cause of the BSM hyperresponsiveness in asthma.

Intranasal administration of recombinant progranulin inhibits bronchial smooth muscle hyperresponsiveness in mouse allergic asthma.

Progranulin (PGRN) is a growth factor with multiple biological functions and has been suggested as an endogenous inhibitor of Tumor necrosis factor-α (TNF-α)-mediated signaling. TNF-α is believed to be one of the important mediators of the pathogenesis of asthma, including airway hyperresponsiveness (AHR). In the present study, effects of recombinant PGRN on TNF-α-mediated signaling and antigen-induced hypercontractility were examined in bronchial smooth muscles (BSMs) both in vitro and in vivo. Cultured human BSM cells (hBSMCs) and male BALB/c mice were used. The mice were sensitized and repeatedly challenged with ovalbumin antigen. Animals also received intranasal administrations of recombinant PGRN into the airways 1 h before each antigen inhalation. In hBSMCs, PGRN inhibited both the degradation of IκB-α (an index of NF-κB activation) and the upregulation of RhoA (a contractile machinery-associated protein that contributes to the BSM hyperresponsiveness) induced by TNF-α, indicating that PGRN has an ability to inhibit TNF-α-mediated signaling also in the BSM cells. In BSMs of the repeatedly antigen-challenged mice, an augmented contractile responsiveness to acetylcholine with an upregulation of RhoA was observed: both the events were ameliorated by pretreatments with PGRN intranasally. Interestingly, a significant decrease in PGRN expression was found in the airways of the repeatedly antigen-challenged mice rather than those of control animals. In conclusion, exogenously applied PGRN into the airways ameliorated the antigen-induced BSM hyperresponsiveness, probably by blocking TNF-α-mediated response. Increasing PGRN levels might be a promising therapeutic for AHR in allergic asthma.

Interleukin-17A directly acts on bronchial smooth muscle cells and augments the contractility.

Although interleukin-17 (IL-17) contributes to the induction of airway hyperresponsiveness in asthma, its effect on bronchial smooth muscle (BSM) remains largely unknown. Evidence support an involvement of RhoA/Rho-kinase in BSM contraction, and the pathway has now been proposed as a novel target for asthma therapy. To clarify the role of IL-17 on the development of BSM hyperresponsiveness, effects of IL-17A on BSM contractility and RhoA expression were investigated. Male BALB/c mice and cultured human BSM cells (hBSMCs) were used. In the murine model of allergic asthma, BSM hyperresponsiveness with an IL-17A up-regulation in bronchoalveolar lavage fluids were observed. RT-PCR analyses revealed the expression of receptors for IL-17A in mouse BSMs and hBSMCs. In the hBSMCs, incubation with IL-17A caused an up-regulation of RhoA protein. Western blot analyses also revealed phosphorylations of JNKs/ERKs and a down-regulation of IκB-α in the IL-17A-treated hBSMCs, indicating that IL-17A could act on BSM cells directly. However, IL-17A did not activate STAT6, which is also known as a signaling molecule that causes an up-regulation of RhoA when activated by IL-13. On the other hand, IL-17A caused a down-regulation of miR-133a-3p, a microRNA that negatively regulates RhoA translation. In the naive mice, in vivo IL-17A treatment to the airways by intranasal instillation induced a BSM hyperresponsiveness with RhoA protein up-regulation. These findings indicate that IL-17 directly acts on BSM cells and up-regulates RhoA protein probably via a down-regulation of miR-133a-3p, resulting in an induction of the BSM hyperresponsiveness.

Augmented bronchial smooth muscle contractility induced by aqueous cigarette smoke extract in rats

Cigarette smoking is the main risk factor for the development of chronic obstructive pulmonary disease (COPD). However, little is known about the mechanisms of cigarette smoke-induced bronchial smooth muscle (BSM) hyperresponsiveness. In the present study, we investigated the effects of aqueous cigarette smoke extract (ACSE) on the BSM contraction in rats. The bronchial strips of rats were incubated with ACSE or control-extract for 24 h. The acetylcholine (ACh), high K+ depolarization and sodium fluoride (NaF)-induced BSM contraction of the ACSE-treated group was significantly augmented as compared to that of the control one. The expression levels of both myosin light-chain kinase (MLCK) and RhoA were significantly increased in the ACSE-treated BSM. These findings suggest that the water-soluble components of cigarette smoke may cause BSM hyperresponsiveness via an increase in MLCK and RhoA.

MicroRNAs in asthma

Asthma is a global disease affecting millions of people. Current treatments are largely symptomatic and, although often effective, can be associated with various side effects. microRNAs (miRNAs/miRs) are regulatory RNAs that affect protein synthesis. They represent new therapeutic targets, and medicines that target specific miRNAs may have potential in the treatment of asthma. There have been a number of studies in the field of miRNA that implicate specific miRNAs in the pathophysiology of asthma. For example, studies using mouse models have identified miRNAs that are altered in response to allergen challenge. Certain miRNAs that are involved in the regulation of interleukin-13 and the TH2 response, key components of the asthmatic response, have been shown to be amenable to modulation by premiRs and antimiRs. Other studies have identified miRNAs that are implicated in bronchial smooth muscle hyperresponsiveness and proliferation. Single-nucleotide polymorphisms in miRNA responsive elements within asthma susceptibility genes, and also in miRNAs themselves, can also contribute to the asthma phenotype. Developing miRNA-based medicines to treat the pulmonary manifestations of asthma could yield therapeutics with new properties that have the potential to treat both the inflammation and hyperresponsivesness associated with this disease.

Different effects of smoke from heavy and light cigarettes on the induction of bronchial smooth muscle hyperresponsiveness in rats

Cigarette smoking is one of the main risk factors in the development of chronic obstructive pulmonary disease (COPD). It has been suggested that an augmented agonist-induced, RhoA mediated Ca²⁺ sensitization is responsible for the enhanced bronchial smooth muscle contraction induced by cigarette smoking. In the present study, to determine whether or not these phenomena are dependent on the degree of exposure to the components of cigarette smoke, we examined the effects of exposure to mainstream smoke derived from either light or heavy cigarettes on both the contractile responsiveness and the expression of RhoA in bronchial smooth muscle. Male Wistar rats were exposed to mainstream cigarette smoke for 2 hr/day for 2 weeks. Twenty-four hr after the last cigarette smoke exposure, we measured isometrical contractions of the bronchial smooth muscle. The concentration-response curve to ACh was significantly shifted upward after heavy cigarette smoke (HCS) exposure, whereas no significant difference was observed in the case of light cigarette smoke (LCS) exposure compared with control rats. No significant difference in K⁺ responsiveness was observed between the groups. The expression of RhoA protein in bronchial preparations from rats repeatedly exposed to HCS, but not to LCS, was significantly increased as compared with that of the control animals. On the other hand, inhalation of nicotine had no effect on either the ACh- and high K⁺ depolarization-induced contractions or the expression of RhoA protein. The increased expression of RhoA seems to have an important role in the augmented contractile responses of the airways in rats, a characteristic feature of early COPD.

Involvement of Src family kinase activation in angiotensin II-induced hyperresponsiveness of rat bronchial smooth muscle

Angiotensin II (Ang II) might be an important mediator in pathogenesis of airway hyperresponsiveness (AHR) that is the asthmatic characteristic feature of asthma, although the mechanisms of AHR caused by Ang II are not yet clear. Presently, the RT-PCR analyses revealed that all the Src family kinases (SFKs), such as Fyn, Lck, Lyn, Hck, Src, Yes, Blk, Fgr and Frk, were expressed in the lungs and main bronchi of rats. The phosphorylation (activation) of SFK (Tyr416) was increased in bronchial smooth muscle (BSM) by Ang II. The Ang II-induced SFK phosphorylation was inhibited by pretreatment with SU6656, an SFK inhibitor. The concentration–contraction curves to carbachol (CCh) were shifted to the left in the presence of Ang II. The maximal contraction of CCh was also significantly increased by pretreatment with Ang II. These results indicate that Ang II causes BSM hyperresponsiveness. The Ang II-induced BSM hyperresponsiveness was significantly inhibited by SU6656, although the carbachol (CCh)-induced contraction itself was not changed by SU6656. In conclusion, Ang II induced a BSM hyperresponsiveness through activation of SFK, and might play an important role in pathophysiology of bronchial asthma.

Angiotensin II induces hyperresponsiveness of bronchial smooth muscle via an activation of p42/44 ERK in rats

Angiotensin II (Ang II) might be an important mediator in the pathogenesis of bronchial asthma, although the mechanisms of airway hyperresponsiveness caused by Ang II are not yet clear. Whether p42/44 ERK contributes to the Ang II-elicited bronchial smooth muscle (BSM) hyperresponsiveness in rats was presently examined. The RT-PCR analyses revealed that Ang II AT(1A), AT(1B), and AT(2) receptors, angiotensinogen, angiotensin-converting enzyme, but not renin, were expressed in the lungs, trachea, and main bronchi of rats. Only a small and transient contraction was induced by the application of Ang II in the main bronchial smooth muscle; the contraction was inhibited by losartan, an AT(1) receptor antagonist. The contractions induced by carbachol (CCh), high K(+) depolarization, and sodium fluoride (NaF; a G protein activator) were augmented by pretreatment with Ang II. The BSM hyperresponsiveness induced by Ang II was abolished by losartan. Furthermore, the Ang II-induced BSM hyperresponsiveness to CCh was attenuated by pretreatment with U-0126, a p42/44 ERK kinase (MEK-1/2) inhibitor. In conclusion, Ang II-induced BSM hyperresponsiveness through the activation of p42/44 ERK may play an important role in the pathophysiology of bronchial asthma, although Ang II itself caused a small force development in the bronchial smooth muscle.

SKI-II, an Inhibitor of Sphingosine Kinase, Ameliorates Antigen-Induced Bronchial Smooth Muscle Hyperresponsiveness, but Not Airway Inflammation, in Mice

To determine if endogenously generated sphingosine-1-phosphate (S1P) is involved in the development of allergic bronchial asthma, the effects of systemic treatments with SKI-II, a specific inhibitor of sphingosine kinase, on antigen-induced bronchial smooth muscle (BSM) hyperresponsiveness and airway inflammation were examined in mice. Male BALB/c mice were actively sensitized with ovalbumin (OA) antigen and were repeatedly challenged with aerosolized antigen. Animals also received intraperitoneal injections with SKI-II (50 mg/kg) 1 h prior to each antigen challenge. The acetylcholine (ACh)-induced contraction of BSM isolated from the repeatedly antigen-challenged mice was significantly augmented, that is, BSM hyperresponsiveness, as compared with that from the control animals (P < 0.05). The BSM hyperresponsiveness induced by antigen exposure was ameliorated by the systemic treatment with SKI-II, whereas the treatments had no effect on BSM responsiveness to ACh in control animals. On the other hand, the systemic treatments with SKI-II had no effect on antigen-induced inflammatory signs, such as increase in cell counts in bronchoalveolar lavage fluids (BALFs) and change in airway histology; upregulation of BALF cytokines, such as interleukin-4 (IL-4) and IL-13; and elevation of total and OA-specific immunoglobulin E (IgE) in sera. These findings suggest that sphingosine kinase inhibitors such as SKI-II have an ability to prevent the development of BSM hyperresponsiveness, but not of allergic airway inflammation. The endogenously generated S1P might be one of the exacerbating factors for the airway hyperresponsiveness, one of the characteristic features of allergic bronchial asthma.

MicroRNAs and Their Therapeutic Potential for Human Diseases: MiR-133a and Bronchial Smooth Muscle Hyperresponsiveness in Asthma

MicroRNAs (miRNAs) play important roles in normal and diseased cell functions. The small-GTPase RhoA is one of the key proteins of bronchial smooth muscle (BSM) contraction, and an upregulation of RhoA has been demonstrated in BSMs of experimental asthma. Although the mechanism of RhoA upregulation in the diseased BSMs is not fully understood, recent observations suggest that RhoA translation is controlled by a miRNA, miR-133a, in cardiomyocytes. Similarly, in human BSM cells (hBSMCs), our recent studies revealed that an upregulation of RhoA was induced when the function of endogenous miR-133a was inhibited by its antagomir. Treatment of hBSMCs with interleukin-13 (IL-13) caused an upregulation of RhoA and a downregulation of miR-133a. In a mouse model of allergic bronchial asthma, increased expression of IL-13 and RhoA and the BSM hyperresponsiveness were observed. The level of miR-133a was significantly decreased in BSMs of the diseased animals. These findings suggest that RhoA expression is negatively regulated by miR-133a in BSMs and that the miR-133a downregulation causes an upregulation of RhoA, resulting in an augmentation of the contraction. MiR-133a might be a key regulator of BSM hyperresponsiveness and provide us with new insight into the treatment of airway hyperresponsiveness in asthmatics.

RhoA, a Possible Target for Treatment of Airway Hyperresponsiveness in Bronchial Asthma

Airway hyperresponsiveness to nonspecific stimuli is one of the characteristic features of allergic bronchial asthma. An elevated contractility of bronchial smooth muscle has been considered as one of the causes of the airway hyperresponsiveness. The contraction of smooth muscles including airway smooth muscles is mediated by both Ca²+-dependent and Ca²+-independent pathways. The latter Ca²+-independent pathway, termed Ca²+ sensitization, is mainly regulated by a monomeric GTP-binding protein, RhoA, and its downstream target Rho-kinase. In animal models of allergic bronchial asthma, an augmented agonist-induced, RhoA-mediated contraction of bronchial smooth muscle has been suggested. The RhoA/Rho-kinase signaling is now proposed as a novel target for the treatment of airway hyperresponsiveness in asthma. Herein, we will discuss the mechanism of development of bronchial smooth muscle hyperresponsiveness, one of the causes of the airway hyperresponsiveness, based on the recent studies using animal models of allergic bronchial asthma and/or cultured airway smooth muscle cells. The possibility of RhoA as a therapeutic target in asthma, especially airway hyperresponsiveness, will also be described.

Inhibition of geranylgeranyltransferase inhibits bronchial smooth muscle hyperresponsiveness in mice

Recent studies revealed an involvement of RhoA/Rho-kinase in the contraction of bronchial smooth muscle (BSM), and this pathway has now been proposed as a new target for asthma therapy. A posttranslational geranylgeranylation of RhoA is required for its activation. Thus selective inhibition of geranylgeranyltransferase may be a novel strategy for treatment of the BSM hyperresponsiveness in asthmatics. To test this hypothesis, we investigated the effect of a geranylgeranyltransferase inhibitor, GGTI-2133, on antigen-induced BSM hyperresponsiveness by using mice with experimental asthma. Mice were sensitized and repeatedly challenged with ovalbumin antigen. Animals also were treated with GGTI-2133 (5 mg/kg ip) once a day before and during the antigen inhalation period. Repeated antigen inhalation caused a BSM hyperresponsiveness to acetylcholine with the increased expressions of RhoA and the anti-farnesyl-positive 21-kDa proteins, probably geranylgeranylated RhoA. The in vivo GGTI-2133 treatments significantly inhibited BSM hyperresponsiveness induced by antigen exposure. In another series of experiments, BSM tissues isolated from the repeatedly antigen-challenged mice were cultured for 48 h in the absence or presence of GGTI-2133. Under these conditions, the putative geranylgeranylated RhoA was decreased in a GGTI-2133 concentration-dependent manner. The in vitro incubation with GGTI-2133 also inhibited BSM hyperresponsiveness induced by antigen exposure. These findings suggest that GGTI-2133 inhibits antigen-induced BSM hyperresponsiveness, probably by reducing downstream signal transduction of RhoA. Selective geranylgeranyltransferase inhibitors may be beneficial for the treatment of airway hyperresponsiveness, one of the characteristic features of allergic bronchial asthma.

A Novel STAT6 Inhibitor AS1517499 Ameliorates Antigen-Induced Bronchial Hypercontractility in Mice

Interleukin-13 (IL-13) is one of the central mediators for development of airway hyperresponsiveness in asthma. The signal transducer and activation of transcription 6 (STAT6) is one of the major signal transducers activated by IL-13, and a possible involvement of IL-13/STAT6 pathway in the augmented bronchial smooth muscle (BSM) contraction has been suggested. In the present study, the effect of a novel STAT6 inhibitor, AS1517499, on the development of antigen-induced BSM hyperresponsiveness was investigated. In cultured human BSM cells, IL-13 (100 ng/ml) caused a phosphorylation of STAT6 and an up-regulation of RhoA, a monomeric GTPase responsible for Ca2+ sensitization of smooth muscle contraction: both events were inhibited by co-incubation with AS1517499 (100 nM). In BALB/c mice that were actively sensitized and repeatedly challenged with ovalbumin antigen, an increased IL-13 level in bronchoalveolar lavage fluids and a phosphorylation of STAT6 in bronchial tissues were observed after the last antigen challenge. These mice had an augmented BSM contractility to acetylcholine together with an up-regulation of RhoA in bronchial tissues. Intraperitoneal injections of AS1517499 (10 mg/kg) 1 hour before each ovalbumin exposure inhibited both the antigen-induced up-regulation of RhoA and BSM hyperresponsiveness, almost completely. A partial but significant inhibition of antigen-induced production of IL-13 was also found. These findings suggest that the inhibitory effects of STAT6 inhibitory agents, such as AS1517499, both on RhoA and IL-13 up-regulations might be useful for asthma treatment.

Activation of signal transducer and activator of transcription factor 1 by interleukins-13 and -4 in cultured human bronchial smooth muscle cells

The family of signal transducer and activator of transcription (STAT) factors play a critical role in the signaling of many cytokines. In addition to the involvement of STAT6 in allergic bronchial asthma, both STAT1 and STAT3 have also been implicated. However, there is little information whether or not the T helper 2 cytokines, which cause several key features of allergic asthma, really induce the activation of STAT1 and/or STAT3 in bronchial smooth muscle (BSM) cells. In the present study, the effects of interleukin-13 (IL-13) and IL-4 on activation of these STAT molecules were examined in cultured human bronchial smooth muscle cells (hBSMCs). After a starvation period, the hBSMCs were treated with 100 ng/ml of IL-13 or IL-4. Total protein samples were prepared at intervals of 1, 3, 6, 12 and 24 hours after the cytokine treatment, and Western blot analyses for total and tyrosine-phosphorylated STATs molecules were conducted. As a result, ut was found that both IL-13 and IL-4 caused a significant increase in the levels of phosphorylated STAT1. Examination of the time-course revealed a peak of STAT1 phosphorylation at 1 hr after cytokine application. In contrast, neither IL-13 nor IL-4 induced phosphorylation of STAT3. Neither of these cytokines changed the protein expression of the STATs themselves. These findings suggest that STAT1, but not STAT3, might also be one of the crucial signal transducers in the development of BSM hyper-responsiveness, which is one of the causes of AHR in asthmatics.

Interleukin-13 Augments Bronchial Smooth Muscle Contractility with an Up-Regulation of RhoA Protein

Interleukin-13 (IL-13) is one of the central mediators for development of airway hyperresponsiveness in asthma. However, its effect on bronchial smooth muscle (BSM) is not well known. Recent studies revealed an involvement of RhoA/Rho-kinase in BSM contraction, and this pathway has now been proposed as a new target for asthma therapy. To elucidate the role of IL-13 on the induction of BSM hyperresponsiveness, effects of IL-13 on contractility and RhoA expression in BSMs were investigated. Male BALB/c mice were sensitized and repeatedly challenged with ovalbumin antigen. In the repeatedly antigen-challenged mice, marked airway inflammation and BSM hyperresponsiveness with an up-regulation of IL-13 in bronchoalveolar lavage fluids were observed. In cultured human BSM cells, IL-13 caused an up-regulation of RhoA. The IL-13-induced up-regulation of RhoA was inhibited by leflunomide, an inhibitor of signal transducer and activator of transcription 6 (STAT6). In isolated BSM tissues of naive mice, the contractility was significantly enhanced by organ culture in the presence of IL-13. Moreover, in vivo treatment of airways with IL-13 by intranasal instillation caused a BSM hyperresponsiveness with an up-regulation of RhoA in naive mice. These findings suggest that IL-13/STAT6 signaling is critical for development of antigen-induced BSM hyperresponsiveness and that agents that specifically inhibit this pathway in BSM may provide a novel strategy for the treatment of asthma.

Glucocorticoids inhibited airway hyperresponsiveness through downregulation of CPI-17 in bronchial smooth muscle

Glucocorticoids are the most effective anti-inflammatory treatment for asthma, and inhaled corticosteroids are the most effective long-term control therapy for persistent asthma. In the present study, to determine the mechanism of the inhibitory effect of glucocorticoids on airway hyperresponsiveness, the effects of glucocorticoids on the expression and activation of PKC-potentiated protein phosphatase 1 inhibitory protein of 17 kDa (CPI-17) were examined in bronchial smooth muscles of antigen-induced airway hyperresponsive rats. Repeated antigen inhalation to animals sensitized with DNP- Ascaris antigen caused a marked bronchial smooth muscle hyperresponsiveness to acetylcholine, accompanied by upregulation and acetylcholine-induced activation of CPI-17 to result in an increase in myosin light chain (MLC) phosphorylation. Treatment with glucocorticoids (prednisolone or beclomethasone, 10 mg/kg, i.p., respectively) significantly inhibited the airway hyperresponsiveness, and markedly reduced both the protein and mRNA levels of CPI-17 in bronchial smooth muscle. The acetylcholine-induced activation of CPI-17, i.e., phosphorylation of CPI-17, was also significantly inhibited by glucocorticoids. Glucocorticoids also prevented the augmented acetylcholine-induced MLC phosphorylation observed in the airway hyperresponsive rats. Therefore, glucocorticoids might inhibit the airway hyperresponsiveness through the inhibition of overexpression and activation of CPI-17.