Thompson, Michael A.

Supplemental oxygen, used to treat hypoxia in preterm and term neonates, increases the risk of neonatal lung diseases, such as bronchopulmonary dysplasia (BPD) and asthma. There is a known sex predilection for BPD, but the underlying mechanisms are not clear. We tested the hypothesis that altered, local estradiol following hyperoxia contributes to pathophysiological changes observed in immature lung. In human fetal airway smooth muscle (fASM) cells exposed to normoxia or hyperoxia, we measured the expression of proteins involved in estrogen metabolism and cell proliferation responses to estradiol. In fASM cells, CYP1a1 expression was increased by hyperoxia, whereas hyperoxia-induced enhancement of cell proliferation was blunted by estradiol. Pharmacological studies indicated that these effects were attributable to upregulation of CYP1a1 and subsequent increased metabolism of estradiol to a downstream intermediate 2-methoxyestradiol. Microarray analysis of mouse lung exposed to 14 days of hyperoxia showed the most significant alteration in CYP1a1 expression, with minimal changes in expression of five other genes related to estrogen receptors, synthesis, and metabolism. Our novel results on estradiol metabolism in fetal and early postnatal lung in the context of hyperoxia indicate CYP1a1 as a potential mechanism for the protective effect of estradiol in hyperoxia-exposed immature lung, which may help explain the sex difference in neonatal lung diseases.

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.

Asthma in the pediatric population remains a significant contributor to morbidity and increasing healthcare costs. Vitamin D3 insufficiency and deficiency have been associated with development of asthma. Recent studies in models of adult airway diseases suggest that the bioactive Vitamin D3 metabolite, calcitriol (1,25‐dihydroxyvitamin D3; 1,25(OH)2D3), modulates responses to inflammation; however, this concept has not been explored in developing airways in the context of pediatric asthma. We used human fetal airway smooth muscle (ASM) cells as a model of the early postnatal airway to explore how calcitriol modulates remodeling induced by pro‐inflammatory cytokines. Cells were pre‐treated with calcitriol and then exposed to TNFα or TGFβ for up to 72 h. Matrix metalloproteinase (MMP) activity, production of extracellular matrix (ECM), and cell proliferation were assessed. Calcitriol attenuated TNFα enhancement of MMP‐9 expression and activity. Additionally, calcitriol attenuated TNFα and TGFβ‐induced collagen III expression and deposition, and separately, inhibited proliferation of fetal ASM cells induced by either inflammatory mediator. Analysis of signaling pathways suggested that calcitriol effects in fetal ASM involve ERK signaling, but not other major inflammatory pathways. Overall, our data demonstrate that calcitriol can blunt multiple effects of TNFα and TGFβ in developing airway, and point to a potentially novel approach to alleviating structural changes in inflammatory airway diseases of childhood. J. Cell. Physiol. 230: 1189–1198, 2015. © 2014 Wiley Periodicals, Inc., A Wiley Company

Abstract Plasma membrane Ca2+ influx, especially store-operated Ca2+ entry triggered by sarcoplasmic reticulum (SR) Ca2+ release, is a key component of intracellular calcium concentration ([Ca2+]i) regulation in airway smooth muscle (ASM). Agonist-induced Ca2+ oscillations in ASM that involve both influx and SR mechanisms have been previously demonstrated. In nonexcitable cells, [Ca2+]i oscillations involve Ca2+ influx via arachidonic acid (AA) –stimulated channels, which show similarities to store-operated Ca2+ entry, although their molecular identity remains undetermined. Little is known about AA-regulated Ca2+ channels or their regulation in ASM. In enzymatically dissociated human ASM cells loaded with the Ca2+ indicator, fura-2, AA (1–10 μM) triggered [Ca2+]i oscillations that were inhibited by removal of extracellular Ca2+. Other fatty acids, such as the diacylglycerol analog, 1-oleoyl-2-acetyl-SN-glycerol, oleic acid, and palmitic acid (10 μM each), failed to elicit similar [Ca2+]i responses. Preincubation with LaCl3 (1 μM or 1 mM) inhibited AA-induced oscillations. Inhibition of receptor-operated channels (SKF96,365 [10 μM]), lipoxygenase (zileuton [10 μM]), or cyclooxygenase (indomethacin [10 μM]) did not affect oscillation parameters. Inhibition of SR Ca2+ release (ryanodine [10 μM] or inositol 1,4,5-trisphosphate receptor inhibitor, xestospongin C [1 μM]) decreased [Ca2+]i oscillation frequency and amplitude. Small interfering RNA against caveolin-1, stromal interaction molecule 1, or Orai3 (20 nM each) reduced the frequency and amplitude of AA-induced [Ca2+]i oscillations. In ASM cells derived from individuals with asthma, AA increased oscillation amplitude, but not frequency. These results are highly suggestive of a novel AA-mediated Ca2+–regulatory mechanism in human ASM, reminiscent of agonist-induced oscillations. Given the role of AA in ASM intracellular signaling, especially with inflammation, AA-regulated Ca2+ channels could potentially contribute to increased [Ca2+]i in diseases such asthma.

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.

Clinically observed differences in airway reactivity and asthma exacerbations in women at different life stages suggest a role for sex steroids in modulating airway function although their targets and mechanisms of action are still being explored. We have previously shown that clinically relevant concentrations of exogenous estrogen acutely decrease intracellular calcium ([Ca2+]i) in human airway smooth muscle (ASM), thereby facilitating bronchodilation. In this study, we hypothesized that estrogens modulate cyclic nucleotide regulation, resulting in decreased [Ca2+]iin human ASM. In Fura-2-loaded human ASM cells, 1 nM 17β-estradiol (E2) potentiated the inhibitory effect of the β-adrenoceptor (β-AR) agonist isoproterenol (ISO; 100 nM) on histamine-mediated Ca2+entry. Inhibition of protein kinase A (PKA) activity (KT5720; 100 nM) attenuated E2effects on [Ca2+]i. Acute treatment with E2increased cAMP levels in ASM cells comparable to that of ISO (100 pM). In acetylcholine-contracted airways from female guinea pigs or female humans, E2potentiated ISO-induced relaxation. These novel data suggest that, in human ASM, physiologically relevant concentrations of estrogens act via estrogen receptors (ERs) and the cAMP pathway to nongenomically reduce [Ca2+]i, thus promoting bronchodilation. Activation of ERs may be a novel adjunct therapeutic avenue in reactive airway diseases in combination with established cAMP-activating therapies such as β2-agonists.

Maintenance of blood oxygen saturation dictates supplemental oxygen administration to premature infants, but hyperoxia predisposes survivors to respiratory diseases such as asthma. Although much research has focused on oxygen effects on alveoli in the setting of bronchopulmonary dysplasia, the mechanisms by which oxygen affects airway structure or function relevant to asthma are still under investigation. We used isolated human fetal airway smooth muscle (fASM) cells from 18–20 postconceptual age lungs (canalicular stage) to examine oxygen effects on intracellular Ca 2+ ([Ca 2+ ] i ) and cellular proliferation. fASM cells expressed substantial smooth muscle actin and myosin and several Ca 2+ regulatory proteins but not fibroblast or epithelial markers, profiles qualitatively comparable to adult human ASM. Fluorescence Ca 2+ imaging showed robust [Ca 2+ ] i responses to 1 μM acetylcholine (ACh) and 10 μM histamine (albeit smaller and slower than adult ASM), partly sensitive to zero extracellular Ca 2+ . Compared with adult, fASM showed greater baseline proliferation. Based on this validation, we assessed fASM responses to 10% hypoxia through 90% hyperoxia and found enhanced proliferation at <60% oxygen but increased apoptosis at >60%, effects accompanied by appropriate changes in proliferative vs. apoptotic markers and enhanced mitochondrial fission at >60% oxygen. [Ca 2+ ] i responses to ACh were enhanced for <60% but blunted at >60% oxygen. These results suggest that hyperoxia has dose-dependent effects on structure and function of developing ASM, which could have consequences for airway diseases of childhood. Thus detrimental effects on ASM should be an additional consideration in assessing risks of supplemental oxygen in prematurity.

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.

Regulation of cytosolic Ca2+ concentration ([Ca2+]cyt) in airway smooth muscle (ASM) is a key aspect of airway contractility and can be modulated by inflammation. Mitochondria have tremendous potential for buffering [Ca2+]cyt, helping prevent Ca2+ overload, and modulating other intracellular events. Here, compartmentalization of mitochondria to different cellular regions may subserve different roles. In the present study, we examined the role of Ca2+ buffering by mitochondria and mitochondrial Ca2+ transport mechanisms in the regulation of [Ca2+]cyt in enzymatically dissociated human ASM cells upon exposure to the proinflammatory cytokines TNF-α and IL-13. Cells were loaded simultaneously with fluo-3 AM and rhod-2 AM, and [Ca2+]cyt and mitochondrial Ca2+ concentration ([Ca2+]mito) were measured, respectively, using real-time two-color fluorescence microscopy in both the perinuclear and distal, perimembranous regions of cells. Histamine induced a rapid increase in both [Ca2+]cyt and [Ca2+]mito, with a significant delay in the mitochondrial response. Inhibition of the mitochondrial Na+/Ca2+ exchanger (1 μM CGP-37157) increased [Ca2+]mito responses in perinuclear mitochondria but not distal mitochondria. Inhibition of the mitochondrial uniporter (1 μM Ru360) decreased [Ca2+]mito responses in perinuclear and distal mitochondria. CGP-37157 and Ru360 significantly enhanced histamine-induced [Ca2+]cyt. TNF-α and IL-13 both increased [Ca2+]cyt, which was associated with decreased [Ca2+]mito in the case of TNF-α but not IL-13. The effects of TNF-α on both [Ca2+]cyt and [Ca2+]mito were affected by CGP-37157 but not by Ru360. Overall, these data demonstrate that in human ASM cells, mitochondria buffer [Ca2+]cyt after agonist stimulation and its enhancement by inflammation. The differential regulation of [Ca2+]mito in different parts of ASM cells may serve to locally regulate Ca2+ fluxes from intracellular sources versus the plasma membrane as well as respond to differential energy demands at these sites. We propose that such differential mitochondrial regulation, and its disruption, may play a role in airway hyperreactivity in diseases such as asthma, where [Ca2+]cyt is increased.