Citations

Cigarette smoke is a common environmental insult associated with increased risk of developing airway diseases such as wheezing and asthma in neonates and children. In adults, asthma involves airway remodeling characterized by increased airway smooth muscle (ASM) cell proliferation and increased extracellular matrix (ECM) deposition, as well as airway hyperreactivity. The effects of cigarette smoke on remodeling and contractility in the developing airway are not well-elucidated. In this study, we used canalicular-stage (18–20 wk gestational age) human fetal airway smooth muscle (fASM) cells as an in vitro model of the immature airway. fASM cells were exposed to cigarette smoke extract (CSE; 0.5–1.5% for 24–72 h), and cell proliferation, ECM deposition, and intracellular calcium ([Ca 2+ ] i ) responses to agonist (histamine 10 μM) were used to evaluate effects on remodeling and hyperreactivity. CSE significantly increased cell proliferation and deposition of ECM molecules collagen I, collagen III, and fibronectin. In contrast, [Ca 2+ ] i responses were not significantly affected by CSE. Analysis of key signaling pathways demonstrated significant increase in extracellular signal-related kinase (ERK) and p38 activation with CSE. Inhibition of ERK or p38 signaling prevented CSE-mediated changes in proliferation, whereas only ERK inhibition attenuated the CSE-mediated increase in ECM deposition. Overall, these results demonstrate that cigarette smoke may enhance remodeling in developing human ASM through hyperplasia and ECM production, thus contributing to development of neonatal and pediatric airway disease.

Abstract Enhanced airway smooth muscle (ASM) contractility contributes to increased resistance to airflow in diseases such as bronchitis and asthma that occur in passive smokers exposed to secondhand smoke. Little information exists on the cellular mechanisms underlying such airway hyperreactivity. Sputum samples of patients with chronic sinusitis, bronchitis, and asthma show increased concentrations of growth factors called neurotrophins, including brain-derived growth factor (BDNF), but their physiological significance remains unknown. In human ASM, we tested the hypothesis that BDNF contributes to increased contractility with cigarette smoke exposure. The exposure of ASM to 1% or 2% cigarette smoke extract (CSE) for 24 hours increased intracellular calcium ([Ca2+]i) responses to histamine, and further potentiated the enhancing effects of a range of BDNF concentrations on such histamine responses. CSE exposure increased the expression of the both high-affinity and low-affinity neurotrophin receptors tropomyosin-related kinase (Trk)–B and p75 pan-neurotrophin receptor, respectively. Quantitative ELISA showed that CSE increased BDNF secretion by human ASM cells. BDNF small interfering (si)RNA and/or the chelation of extracellular BDNF, using TrkB-fragment crystallizable, blunted the effects of CSE on [Ca2+]i responses as well as the CSE enhancement of cell proliferation, whereas TrkB siRNA blunted the effects of CSE on ASM contractility. These data suggest that cigarette smoke is a potent inducer of BDNF and TrkB expression and signaling in ASM, which then contribute to cigarette smoke–induced airway hyperresponsiveness.

Caveolae are flask-shaped plasma membrane invaginations expressing the scaffolding caveolin proteins. Although caveolins have been found in endothelium and epithelium (where they regulate nitric oxide synthase activity), their role in smooth muscle is still under investigation. We and others have previously shown that caveolae of human airway smooth muscle (ASM), which express caveolin-1, contain Ca 2+ and force regulatory proteins and are involved in mediating the effects of inflammatory cytokines such as TNF-α on intracellular Ca 2+ concentration responses to agonist. Accordingly, we tested the hypothesis that in vivo, absence of caveolin-1 leads to reduced airway hyperresponsiveness, using a knockout (KO) (Cav1 KO) mouse and an ovalbumin-sensitized/challenged (OVA) model of allergic airway hyperresponsiveness. Surprisingly, airway responsiveness to methacholine, tested by use of a FlexiVent system, was increased in Cav1 KO control (CTL) as well as KO OVA mice, which could not be explained by a blunted immune response to OVA. In ASM of wild-type (WT) OVA mice, expression of caveolin-1, the caveolar adapter proteins cavins 1–3, and caveolae-associated Ca 2+ and force regulatory proteins such as Orai1 and RhoA were all increased, effects absent in Cav1 KO CTL and OVA mice. However, as with WT OVA, both CTL and OVA Cav1 KO airways showed signs of enhanced remodeling, with high expression of proliferation markers and increased collagen. Separately, epithelial cells from airways of all three groups displayed lower endothelial but higher inducible nitric oxide synthase and arginase expression. Arginase activity was also increased in these three groups, and the inhibitor nor-NOHA ( N-omega-nor-l-arginine) enhanced sensitivity of isolated tracheal rings to ACh, especially in Cav1 KO mice. On the basis of these data disproving our original hypothesis, we conclude that caveolin-1 has complex effects on ASM vs. epithelium, resulting in airway hyperreactivity in vivo mediated by altered airway remodeling and bronchodilation.

Diseases such as asthma are characterized by airway hyperresponsiveness. Enhanced airway smooth muscle (ASM) intracellular Ca2+ ([Ca2+]i) response to agonist stimulation leading to increased airway constriction has been suggested to contribute to airway hyperresponsiveness. Caveolae are flask-shaped plasma membrane invaginations that express the scaffolding protein caveolin and contain multiple proteins important in [Ca2+]i signaling (e.g., agonist receptors, ion channels). We recently demonstrated that caveolae and caveolin-1 are important in [Ca2+]i regulation in human ASM. Proinflammatory cytokines such as tumor necrosis factor (TNF)-α and interleukin (IL)-13 modulate [Ca2+]i in ASM. We hypothesized that cytokine upregulation of caveolar signaling in ASM contributes to enhanced agonist-induced [Ca2+]i in inflammation. Enzymatically dissociated human ASM cells were exposed to medium (control), 20 ng/ml TNF-α, or 50 ng/ml IL-13 for 24 h. Caveolae-enriched membrane fractions displayed substantial increase in caveolin-1 and -2 expressions by TNF-α and IL-13. Transfection with caveolin-1-mRed DNA substantially accelerated and increased plasma membrane caveolin-1 expression by TNF-α and to a lesser extent by IL-13. Caveolin-1 enhancement was inhibited by nuclear factor-κB and mitogen-activated protein kinase inhibitors. In fura 2-loaded ASM cells, [Ca2+]i responses to 1 μM ACh, 10 μM histamine, or 10 nM bradykinin were all exaggerated by TNF-α as well as IL-13 exposure. However, disruption of caveolae using caveolin-1 suppression via small-interfering RNA resulted in significant blunting of agonist-induced [Ca2+]i responses of vehicle and TNF-α-exposed cells. These functional data were correlated to the presence of TNFR1 receptor (but not the IL-4/IL-13 receptor) within caveolae. Overall, these results indicate that caveolin-1 plays an important role in airway inflammation by modulating the effect of specific cytokines on [Ca2+]i.

Caveolae are specialized membrane microdomains expressing the scaffolding protein caveolin-1. We recently demonstrated the presence of caveolae in human airway smooth muscle (ASM) and the contribution of caveolin-1 to intracellular calcium ([Ca2+]i) regulation. In the present study, we tested the hypothesis that caveolin-1 regulates ASM contractility. We examined the role of caveolins in force regulation of porcine ASM under control conditions as well as TNF-α-induced airway inflammation. In porcine ASM strips, exposure to 10 mM methyl-β-cyclodextrin (CD) or 5 μM of the caveolin-1 specific scaffolding domain inhibitor peptide (CSD) resulted in time-dependent decrease in force responses to 1 μM ACh. Overnight exposure to the cytokine TNF-α (50 ng/ml) accelerated and increased caveolin-1 expression and enhanced force responses to ACh. Suppression of caveolin-1 with small interfering RNA mimicked the effects of CD or CSD. Regarding mechanisms by which caveolae contribute to contractile changes, inhibition of MAP kinase with 10 μM PD98059 did not alter control or TNF-α-induced increases in force responses to ACh. However, inhibiting RhoA with 100 μM fasudil or 10 μM Y27632 resulted in significant decreases in force responses, with lesser effects in TNF-α exposed samples. Furthermore, Ca2+sensitivity for force generation was substantially reduced by fasudil or Y27632, an effect even more enhanced in the absence of caveolin-1 signaling. Overall, these results indicate that caveolin-1 is a critical player in enhanced ASM contractility with airway inflammation.