Gene expression in asthmatic airway smooth muscle

Airway smooth muscle abnormalities are central to the pathophysiology of asthma. These airway smooth muscle cell abnormalities may include changes in cell number, size, phenotype, or function. Gene expression studies performed using asthmatic airway smooth muscle cells represent one approach to identifying the abnormalities of airway smooth muscle that occur in asthma in vivo. However, due to the technical challenges involved, only two studies have been performed to date using freshly obtained tissue from subjects with asthma. The first of these studies suggested increased expression of myosin light-chain kinase in airway smooth muscle from patients with asthma, whereas the second study found no difference in myosin light-chain kinase expression, nor any difference in other markers of smooth muscle phenotype in asthma. Studies performed in cell culture through the application of gene expression microarrays to profile airway smooth muscle cells exposed to potential mediators of asthma yield more consistent results, including induction by IL-13 of tenascin, the H1 histamine receptor, and IL-13 receptor subunits. However, the significance of these microarray findings for smooth muscle function is uncertain. Furthermore, gene expression studies have a fundamental limitation in that many functional properties of airway smooth muscle are regulated at other levels (e.g., protein phosphorylation). Thus, gene expression studies ultimately must be integrated with other methodological approaches to adequately study airway smooth muscle in asthma in vivo.     

Mechanical properties of asthmatic airway smooth muscle

Airway smooth muscle (ASM) is the major effector of excessive airway narrowing in asthma. Changes in some of the mechanical properties of ASM could contribute to excessive narrowing and have not been systematically studied in human ASM from nonasthmatic and asthmatic subjects. Human ASM strips (eight asthmatic and six nonasthmatic) were studied at in situ length and force was normalised to maximal force induced by electric field stimulation (EFS). Measurements included: passive and active force versus length before and after length adaptation, the force-velocity relationship, maximal shortening and force recovery after length oscillation. Force was converted to stress by dividing by cross-sectional area of muscle. The only functional differences were that the asthmatic tissue was stiffer at longer lengths (p<0.05) and oscillatory strain reduced isometric force in response to EFS by 19% as opposed to 36% in nonasthmatics (p<0.01). The mechanical properties of human ASM from asthmatic and nonasthmatic subjects are comparable except for increased passive stiffness and attenuated decline in force generation after an oscillatory perturbation. These data may relate to reduced bronchodilation induced by a deep inspiration in asthmatic subjects.     

Depressed cyclic AMP levels in airway smooth muscle from asthmatic dogs

We tested mongrel dogs by intradermal injection and tracheobronchial aerosol challenge with Ascaris suum antigen extract. All dogs were skin-test positive but could be segregated, on the basis of airways resistance measurements, into "asthmatic" (bronchoreactive) and "nonasthmatic" (nonbronchoreactive) groups. By using tracheal rings from these dogs, we measured the abilities of the beta-adrenergic agonist, isoproterenol, to relax tracheal smooth muscle contracted by methacholine and to cause cyclic AMP (cAMP) accumulation in the presence and absence of methacholine. The magnitude of relaxation induced by any concentration of isoproterenol was always less in the smooth muscle from "asthmatic" dogs. In the same tissues the concentrations of cAMP after in vitro equilibration, but prior to isoproterenol addition, were significantly less in the "asthmatic" than "nonasthmatic" samples. The accumulation of cAMP due to isoproterenol was similar in both groups for every dose of isoproterenol so that the initial difference between groups in cAMP concentration was maintained in an additive fashion over the entire dose-response curve. Total protein content of trachealis muscles from both groups of dogs was the same. We conclude that beta-adrenergically sensitive adenylate cyclase is not impaired in tracheal smooth muscle from "asthmatic" dogs; rather, the basal concentration of cAMP is depressed in "asthmatic" airway smooth muscle, and this difference is maintained throughout the isoproterenol dose-response curve. The depressed intracellular cAMP concentrations may be related to the decreased relaxation induced by isoproterenol in the "asthmatic" tracheal smooth muscle.     

Histamine bronchoconstriction reduces airway responsiveness in asthmatic subjects

Tachyphylaxis occurs to repeated challenges with inhaled histamine but not with inhaled acetylcholine in asthmatic subjects. This study was undertaken to determine whether prior histamine bronchoconstriction reduces airway responsiveness to inhaled acetylcholine in mild asthmatic subjects demonstrating histamine tachyphylaxis. All subjects developed histamine tachyphylaxis with repeated histamine challenge. The mean histamine PC20 increased from 3.74 to 5.92 mg/ml (p less than 0.005) when the histamine challenges were separated by 1 h. Prior acetylcholine bronchoconstriction did not reduce airway responsiveness to subsequent inhalation of either acetylcholine or histamine in these subjects. However, histamine inhalation did reduce airway responsiveness to acetylcholine in all subjects. The mean acetylcholine PC20 following acetylcholine inhalation was 3.37 mg/ml (%SD 2.17) and this increased to 7.76 mg/ml (%SD 1.80) after histamine inhalation (p less than 0.0005). Thus, this study demonstrates that prior histamine, but not acetylcholine, bronchoconstriction can partially protect against bronchoconstriction caused by both histamine and acetylcholine. Therefore, reduced airway responsiveness caused by histamine bronchoconstriction is specific for histamine and is not due to bronchoconstriction per se. However, the reduced airway responsiveness following histamine bronchoconstriction, is nonspecific.     

Expression of connective tissue growth factor in asthmatic airway smooth muscle cells

There is strong evidence to implicate transforming growth factor-beta in the remodeling that occurs in asthma, as levels are increased in bronchial lavage fluid and gene expression is increased in bronchial tissue. Transforming growth factor-beta is also known to increase the release of collagen from airway smooth muscle. Here we identify for the first time a possible mechanism for the effects of transforming growth factor-beta. Transforming growth factor-beta specifically induces mRNA and protein for connective tissue growth factor in airway smooth muscle, and moreover, we report that the connective tissue growth factor response is greater in airway smooth muscle cultured from patients with asthma compared with patients without asthma. This occurs at both the level of mRNA (37.53 +/- 11.62- and 13.59 +/- 3.12-fold increase at 24 hours compared with time 0, respectively, p < 0.02) and protein production (67.57 +/- 27.80- and 3.58 +/- 0.6-fold increase at 24 hours compared with time 0, respectively, p < 0.03). The differential connective tissue growth factor response to transforming growth factor-beta in asthmatic airway smooth muscle identifies a potential role for connective tissue growth factor in the remodeling that is characteristic of severe persistent asthma.     

Connective tissue growth factor induces extracellular matrix in asthmatic airway smooth muscle

Transforming growth factor (TGF)-beta and connective tissue growth factor may be implicated in extracellular matrix protein deposition in asthma. We have recently reported that TGF-beta increased connective tissue growth factor expression in airway smooth muscle cells isolated from patients with asthma. In this study, we examined fibronectin and collagen production and signal transduction pathways after stimulation with TGF-beta and connective tissue growth factor. In both asthmatic and nonasthmatic airway smooth muscle cells, TGF-beta and connective tissue growth factor led to the production of fibronectin and collagen I. Fibronectin and collagen expression was extracellular regulated kinase-dependent in both cell types but phosphoinositide-3 kinase-dependent only in asthmatic airway smooth muscle cells. p38 was implicated in fibronectin but not collagen expression in both cell types. TGF-beta induction of fibronectin and collagen was in part mediated by an autocrine action of connective tissue growth factor. Phosphorylation of SMAD-2 may represent an additional pathway because this was increased in asthmatic cells. Our results suggest that these two cytokines may be important in the deposition of extracellular matrix proteins and that the signal transduction pathways may be different in asthmatic and nonasthmatic cells.     

Inhalant corticosteroids inhibit mechanical strain-induced RANTES and eotaxin production by human airway smooth muscle cells

BackgroundRANTES and eotaxin play an important role in the production of allergic inflammation of the airway through a chemotactic activity for eosinophils. Airway smooth muscle (ASM) cells are known to produce these cytokines in response to pro-inflammatory stimuli, such as tumor necrosis factor (TNF)-α; however, it has not been determined whether mechanical strain could induce cytokine production by ASM cells. In addition, in the present study we also examined the effect of inhalant corticosteroids, namely fluticasone propionate (FP) and budesonide (BUD), on RANTES and eotaxin production.MethodsTo clarify these issues, human ASM cells cultured on silicone culture rubber dishes that had been incubated with or without inhalant corticosteroids were stretched or compressed. The ASM cells were simultaneously stimulated with TNF-α. The concentration of RANTES and eotaxin in the culture supernatants was determined.ResultsThe results showed that: (i) mechanical strain, including stretch and compression, stimulated ASM cells to produce RANTES and eotaxin; and (ii) FP and BUD inhibited mechanical strain-induced and TNF-α-stimulated RANTES and eotaxin production by ASM cells. These results indicate that mechanical strain is capable of inducing RANTES and eotaxin production by ASM cells and that inhalant corticosteroids effectively inhibit RANTES and eotaxin production by mechanical strain-loaded ASM cells, as well as by TNF-α-stimulated cells.ConclusionsMechanical strain and inflammatory stimuli are capable of inducing chemokine production; therefore, suppressing the contraction of, as well as chemokine production by, ASM cells is important for controlling the production and the progression of allergic inflammation.     

L-精氨酸对哮喘大鼠气道平滑肌细胞增殖的影响

目的 观察L-精氨酸(L-Arg)对哮喘气道重构大鼠气道平滑肌细胞(ASMC)的细胞周期分布及细胞周期调节蛋白(CCRP)表达水平的影响,探讨L-Arg体内干预哮喘大鼠ASMC增殖的可能作用机制.方法 实验分成对照组、哮喘组、L-Arg组,建立大鼠哮喘气道重构模型,检测血清NO-2/NO-3含量、肺内支气管内管壁和平滑肌层厚度及ASMC核数、ASMC的细胞周期分布以及ASMC内细胞周期素E(cyclin E)、cyclin A、cyclin B、蛋白27kip1(P27kip1)的表达.结果 哮喘组大鼠支气管内管壁、平滑肌层的厚度和ASMC数目显著大于对照组(P<0.05);L-Arg组大鼠支气管内管壁的厚度、平滑肌层的厚度和ASMC数目显著小于哮喘组(P<0.05).哮喘组血清一氧化氮(nitricoxide,NO)水平显著低于对照组(P<0.05);L-Arg组血清NO水平显著高于哮喘组(P<0.01).哮喘组G0/G1期ASMC比例及P27kip1表达水平显著低于对照组(P<0.01),G2/M+S期ASMC比例及cyclin E、cyclin A和cyclin B表达水平显著高于对照组(P<0.01);L-Arg组G0/G1期ASMC比例及P27kip1表达水平显著高于哮喘组(P<0.05),G2/M+S期ASMC比例及cyclin E和cyclin A表达水平显著低于哮喘组(P<0.05).结论 L-Arg通过调控CCRP的表达水平阻滞细胞从G1期进入S期而抑制哮喘ASMC的增殖.     

哮喘豚鼠气管平滑肌钾离子通道特性的改变

目的为探讨哮喘时电压依赖性钾离子通道（ⅠＫ）特性的改变。方法在支气管平滑肌细胞膜上，用膜片钳技术进行了研究。结果哮喘平滑肌细胞有如下改变：（１）静息跨膜电位差值降低。（２）通道电导值与正常组相比下降了约５１％。（３）开放时间常数的快、慢成分明显缩短，分别下降到正常对照组的２５％和７％。关闭时间常数明显延长。（４）开放概率有电压依赖性，但在相同的钳制电压范围内明显降低。结论哮喘气道高反应性时钾离子通道动力学活性明显衰减，提示将导致细胞兴奋阈降低。     

Bronchoalveolar lavage fluid from asthmatic subjects is mitogenic for human airway smooth muscle

Airway smooth muscle proliferation may contribute to the airway wall remodeling seen in asthma. In this study we tested for the presence of airway smooth muscle mitogenic activity in bronchoalveolar lavage (BAL) fluid obtained from 12 atopic asthmatics before and serially after segmental allergen challenge, and from four normal subjects who did not undergo allergen challenge. Mitogenic effect was assessed by coincubating BAL fluid with human airway smooth muscle cells, and measuring its effect on (3)[H]thymidine incorporation and cell number. Induction of ERK phosphorylation and cyclin D(1) protein abundance were also assessed. Compared with serum-free medium alone, BAL fluid obtained from normal subjects increased thymidine incorporation, cell number, ERK phosphorylation, and cyclin D(1) abundance. BAL fluid from asthmatic subjects prior to allergen challenge induced even greater increases in all measures, except for cell number, which was similar to that observed with normal subjects' BAL fluid. Incubation with lavage fluid obtained 48 h after segmental allergen challenge in atopic asthmatics caused yet further increases in thymidine incorporation, cell number, and cyclin D(1) protein abundance. Molecular sieving of prechallenge BAL fluid from three asthmatic subjects demonstrated that mitogenic activity was present exclusively in the > 10 kD fraction. These results provide the first direct demonstration that fluid lining the airways of asthmatics contains excess mitogenic activity for human airway smooth muscle, and that this activity increases further after allergen challenge.     

L-精氨酸对哮喘大鼠气道平滑肌细胞增殖的影响

目的 观察L-精氨酸（L-Arg）对哮喘气道重构大鼠气道平滑肌细胞（ASMC）的细胞周期分布及细胞周期调节蛋白（CCRP）表达水平的影响,探讨L-Arg体内干预哮喘大鼠ASMC增殖的可能作用机制。方法实验分成对照组、哮喘组、L-Arg组,建立大鼠哮喘气道重构模型,检测血清NO2^-／NO3^-含量、肺内支气管内管壁和平滑肌层厚度及ASMC核数、ASMC的细胞周期分布以及ASMC内细胞周期素E（cyclin E）、cyclinA、cyclinB、蛋白27^kip1（P27^kip1）的表达。结果 哮喘组大鼠支气管内管壁、平滑肌层的厚度和ASMC数目显著大于对照组（P〈0．05）;L-Arg组大鼠支气管内管壁的厚度、平滑肌层的厚度和ASMC数目显著小于哮喘组（P〈0．05）。哮喘组血清一氧化氮（nitricoxide,NO）水平显著低于对照组（P〈0．05）;L-Arg组血清NO水平显著高于哮喘组（P〈0．01）。哮喘组G0／G1期ASMC比例及P27^kip1表达水平显著低于对照组（P〈0．01）,G2／M＋S期ASMC比例及cyclinE、cyclinA和cyclinB表达水平显著高于对照组（P〈0．01）;L-Arg组G0／G1期ASMC比例及P27^kip1表达水平显著高于哮喘组（P〈0．05）,G2／M＋S期ASMC比例及cyclinE和cyclinA表达水平显著低于哮喘组（P〈0．05）。结论 L-Arg通过调控CCRP的表达水平阻滞细胞从G1期进入S期而抑制哮喘ASMC的增殖。     

Histamine tachyphylaxis in human airway smooth muscle. The role of H2-receptors and the bronchial epithelium.

The role of airway epithelium and H2-receptors in the development of histamine tachyphylaxis was studied using human isolated bronchial smooth muscle strips obtained from 18 patients undergoing thoracotomy. In epithelium-intact strips, a 38% reduction in the maximal contractile response (Emax) (p less than 0.002) and a 2.14-fold increase in the EC50 (p less than 0.02; n = 18) was observed after three separate histamine cumulative concentration effect curves (CCEC). In contrast, significant differences were not seen for either Emax (p greater than 0.4; n = 10) or EC50 (p greater than 0.26; n = 10) in epithelium-denuded strips. In separate experiments, both intact and denuded muscle strips were treated with the H2-receptor antagonist ranitidine (60 microM), either 30 min prior to the first or 30 min prior to the second histamine CCEC. In epithelium-intact strips, pretreatment with ranitidine caused a 1.8-fold increase in Emax in the initial CCEC (p less than 0.02), and both ranitidine schedules prevented tachyphylaxis (n = 8). In epithelium-denuded preparations, ranitidine did not enhance the responsiveness to histamine beyond that seen in untreated epithelium-denuded strips (n = 6). These data suggest that histamine-induced tachyphylaxis occurs in human airway smooth muscle and is mediated, at least in part, via H2-receptors resident on airway epithelium. In vivo, this may function as a protective mechanism, but damage to the epithelium and loss of H2-receptors may be significant in the development of histamine bronchial hyperreactivity as seen in asthma.     

支气管哮喘大鼠气道重塑中气道平滑肌细胞凋亡及地塞米松的干预作用

目的 观察支气管哮喘(简称哮喘)大鼠气道重塑中气道平滑肌细胞(airway smoothmuscle cells,ASMC)凋亡及地塞米松对ASMC凋亡的影响.方法 将清洁级雄性SD大鼠按随机数字表法分为正常对照组、哮喘组和地塞米松干预组,每组12只.以卵蛋白致敏和激发的方法制备大鼠慢性哮喘模型.用脱氧核糖核苷酸末端转移酶(TdT酶)介导的dUTP切口末端标记法(TUNEL法)检测ASMC凋亡并计算凋亡指数.用免疫组织化学和原位杂交法分别检测气道平滑肌Bcl-2、Bax蛋白及其mRNA的表达情况.经SPSS 11.5软件进行统计学分析,实验数据用x±s表示.多组间比较采用方差分析,多组样本均数两两比较,两变量的相关程度采用直线相关分析.结果 (1)对照组、哮喘组和干预组大鼠ASMC的凋亡指数分别为:0.201±0.022、0.030±0.016和0.118±0.043;(2)对照组、哮喘组和干预组大鼠气道平滑肌层Bcl-2蛋白表达的吸光度值分别为0.060±0.012、0.112±0.028和0.080±0.010,Bcl-2 mRNA表达的吸光度值分别为0.065±0.019、0.157±0.019和0.099±0.029;(3)对照组、哮喘组和干预组大鼠气道平滑肌层Bax蛋白表达的吸光度值为0.120±0.020、0.062±0.012和0.093±0.010,Bax mRNA表达的吸光度值分别为0.155±0.025、0.074±0.019和0.118±0.031;(4)相关分析结果显示,ASMC的凋亡指数与气道平滑肌厚度以及Bcl-2蛋白的相对含量呈显著负相关(r值分别为-0.860、-0.783,P<0.01);ASMC的凋亡指数与气道平滑肌Bax蛋白相对含量呈正相关(r=0.837,P<0.01).结论 ASMC凋亡的减少可能参与了哮喘气道重塑过程;地塞米松可以增加促凋亡蛋白Box的表达,同时减少抑凋亡蛋白Bcl-2的表达,从而增加ASMC的凋亡.     

Histamine receptors in esophageal smooth muscle of the opossum.

Esophageal smooth muscle was examined for histamine receptors. The effects of histamine, the histamine analogs 4-methylhistamine (4-MH) and 2-(2-pyridyl) ethylamine (PEA), the histamine receptor antagonists mepyramine and metiamide, and the histamine-releasing substance compound 48/80, on lower esophageal sphincter (LES) and esophageal body (EB) smooth muscle of the opossum were studied in a superfused tissue bath. Histamine, PEA, an H1 receptor agonist, and compound 48/80 caused a dose-related increase in LES basal tension and in EB off response amplitude, the threshold for histamine being 6.7 X 10(-8) M and that for PEA being 6.7 X 10(-7) M. In the presence of mepyramine, and H1 receptor antagonist, the effects of histamine and compound 48/80 were reversed to inhibition of both LES basal tension and EB off response amplitude, while the effect of PEA was abolished. Metiamide, an H2 receptor antagonist, did not alter responses to histamine, PEA, or compound 48/80. The H2 receptor agonist 4-methylhistamine caused a reduction of LES tension and EB off response amplitude, but caused an increase in those parameters in the presence of metiamide. A combination of mepyramine and metiamide abolished responses to all agonist drugs. The results indicate that LES and EB smooth muscle contain both excitatory H1 and inhibitory H2 receptors for histamine. Endogenous histamine released from storage sites in LES and EB and exogenous histamine both preferentially activate H1 receptors.     

线粒体膜电位和细胞色素C在哮喘大鼠气道平滑肌细胞中的表达

目的 观察线粒体膜电位和细胞色素C在哮喘大鼠气道平滑肌细胞中的表达,探讨其在哮喘大鼠气道平滑肌细胞发病机制中的作用.方法 将SD大鼠随机分为对照组和哮喘组,以卵清白蛋白致敏和激发的方法 制备大鼠慢性哮喘模型,用组织贴壁法体外培养气道平滑肌细胞,流式细胞术测细胞线粒体膜电位,免疫蛋白印迹检测细胞色素C.结果 哮喘组线粒体...     

The contribution of airway smooth muscle to airway narrowing and airway hyperresponsiveness in disease.

Airway hyperresponsiveness (AHR), the exaggerated response to constrictor agonists in asthmatic subjects, is incompletely understood. Changes in either the quantity or properties of airway smooth muscle (ASM) are possible explanations for AHR. Morphometric analyses demonstrate structural changes in asthmatic airways, including subepithelial fibrosis, gland hyperplasia/hypertrophy, neovascularization and an increase in ASM mass. Mathematical modelling of airway narrowing suggests that, of all the changes in structure, the increase in ASM mass is the most probable cause of AHR. An increase in ASM mass in the large airways is more closely associated with a greater likelihood of dying from asthma than increases in ASM mass in other locations within the airway tree. ASM contraction is opposed by the elastic recoil of the lungs and airways, which appears to limit the degree of bronchoconstriction in vivo. The cyclical nature of tidal breathing applies stresses to the airway wall that enhance the bronchodilating influence of the lung tissues on the contracting ASM, in all probability by disrupting cross-bridges. However, the increase in ASM mass in asthma may overcome the limitation resulting from the impedances to ASM shortening imposed by the lung parenchyma and airway wall tissues. Additionally, ASM with the capacity to shorten rapidly may achieve shorter lengths and cause a greater degree of bronchoconstriction when stimulated to contract than slower ASM. Changes in ASM properties are induced by the process of sensitization and allergen-exposure such as enhancement of phospholipase C activity and inositol phosphate turnover, and increases in myosin light chain kinase activity. Whether changes in ASM mass or biochemical/biomechanical properties form the basis for asthma remains to be determined.     

Migration of airway smooth muscle cells

Migration of smooth muscle cells is a process fundamental to development of hollow organs, including blood vessels and the airways. Migration is also thought to be part of the response to tissue injury. It has also been suggested to contribute to airways remodeling triggered by chronic inflammation. In both nonmuscle and smooth muscle cells numerous external signaling molecules and internal signal transduction pathways contribute to cell migration. The review includes evidence for the functional significance of airway smooth muscle migration, a summary of promigratory and antimigratory agents, and summaries of important signaling pathways mediating migration. Important signaling pathways and effector proteins described include small G proteins, phosphatidylinositol 3-kinases (PI3-K), Rho activated protein kinase (ROCK), p21-activated protein kinases (PAK), Src family tyrosine kinases, and mitogen-activated protein kinases (MAPK). These signaling modules control multiple critical effector proteins including actin nucleating, capping and severing proteins, myosin motors, and proteins that remodel microtubules. Actin filament remodeling, focal contact remodeling and propulsive force of molecular motors are all coordinated to move cells along gradients of chemical cues, matrix adhesiveness, or matrix stiffness. Airway smooth muscle cell migration can be modulated in vitro by drugs commonly used in pulmonary medicine including beta-adrenergic agonists and corticosteroids. Future studies of airway smooth muscle cell migration may uncover novel targets for drugs aimed at modifying airway remodeling.