Human Fetal Airway Smooth Muscle Research Articles

Intermittent Hypoxia-Hyperoxia and Oxidative Stress in Developing Human Airway Smooth Muscle.

Premature infants are frequently and intermittently administered supplemental oxygen during hypoxic episodes, resulting in cycles of intermittent hypoxia and hyperoxia. The relatively hypoxic in utero environment is important for lung development while hyperoxia during the neonatal period is recognized as detrimental towards the development of diseases such as bronchopulmonary dysplasia and bronchial asthma. Understanding early mechanisms that link hypoxic, hyperoxic, and intermittent hypoxic-hyperoxic exposures to altered airway structure and function are key to developing advanced therapeutic approaches in the clinic. Changes in oxygen availability can be detrimental to cellular function and contribute to oxidative damage. Here, we sought to determine the effect of oxygen on mitochondria in human fetal airway smooth muscle cells exposed to either 5% O2, 21% O2, 40% O2, or cycles of 5% and 40% O2 (intermittent hypoxia-hyperoxia). Reactive oxygen species production, altered mitochondrial morphology, and changes in mitochondrial respiration were assessed in the context of the antioxidant N-acetylcysteine. Our findings show developing airway smooth muscle is differentially responsive to hypoxic, hyperoxic, or intermittent hypoxic-hyperoxic exposure in terms of mitochondrial structure and function. Cycling O2 decreased mitochondrial branching and branch length similar to hypoxia and hyperoxia in the presence of antioxidants. Additionally, hypoxia decreased overall mitochondrial respiration while the addition of antioxidants increased respiration in normoxic and O2-cycling conditions. These studies show the necessity of balancing oxidative damage and antioxidant defense systems in the developing airway.

Calcium-sensing receptor and CPAP-induced neonatal airway hyperreactivity in mice.

Background:Continuous positive airway pressure (CPAP) in preterm infants is initially beneficial, but animal models suggest longer term detrimental airway effects towards asthma. We used a neonatal CPAP mouse model and human fetal airway smooth muscle (ASM) to investigate the role of extracellular calcium sensing receptor (CaSR) in these effects.Methods:Newborn wild type and smooth muscle-specific CaSR−/− mice were given CPAP for 7 days via a custom device (mimicking CPAP in premature infants), and recovered in normoxia for another 14 days (representing infants at 3–4 years). Airway reactivity was tested using lung slices, and airway CaSR quantified. Role of CaSR was tested using NPS2143 (inhibitor) or siRNA in WT mice. Fetal ASM cells stretched cyclically with/without static stretch mimicking breathing and CPAP were analyzed for intracellular Ca2+ ([Ca2+]i) responses, role of CaSR and signaling cascades.Results:CPAP increased airway reactivity in WT but not CaSR−/− mice, increasing ASM CaSR. NPS2143 or CaSR siRNA reversed CPAP effects in WT mice. CPAP increased fetal ASM [Ca2+]I, blocked by NPS2143, and increased ERK1/2 and RhoA suggesting two mechanisms by which stretch increases CaSR.Conclusions:These data implicate CaSR in CPAP effects on airway function with implications for wheezing in former preterm infants.

Calcium-Sensing Receptor Contributes to Hyperoxia Effects on Human Fetal Airway Smooth Muscle.

Supplemental O2 (hyperoxia), necessary for maintenance of oxygenation in premature infants, contributes to neonatal and pediatric airway diseases including asthma. Airway smooth muscle (ASM) is a key resident cell type, responding to hyperoxia with increased contractility and remodeling [proliferation, extracellular matrix (ECM) production], making the mechanisms underlying hyperoxia effects on ASM significant. Recognizing that fetal lungs experience a higher extracellular Ca2+ ([Ca2+]o) environment, we previously reported that the calcium sensing receptor (CaSR) is expressed and functional in human fetal ASM (fASM). In this study, using fASM cells from 18 to 22 week human fetal lungs, we tested the hypothesis that CaSR contributes to hyperoxia effects on developing ASM. Moderate hyperoxia (50% O2) increased fASM CaSR expression. Fluorescence [Ca2+]i imaging showed hyperoxia increased [Ca2+]i responses to histamine that was more sensitive to altered [Ca2+]o, and promoted IP3 induced intracellular Ca2+ release and store-operated Ca2+ entry: effects blunted by the calcilytic NPS2143. Hyperoxia did not significantly increase mitochondrial calcium which was regulated by CaSR irrespective of oxygen levels. Separately, fASM cell proliferation and ECM deposition (collagens but not fibronectin) showed sensitivity to [Ca2+]o that was enhanced by hyperoxia, but blunted by NPS2143. Effects of hyperoxia involved p42/44 ERK via CaSR and HIF1α. These results demonstrate functional CaSR in developing ASM that contributes to hyperoxia-induced contractility and remodeling that may be relevant to perinatal airway disease.

Cellular clocks in hyperoxia effects on [Ca2+]i regulation in developing human airway smooth muscle.

Supplemental O2 (hyperoxia) is necessary for preterm infant survival but is associated with development of bronchial airway hyperreactivity and childhood asthma. Understanding early mechanisms that link hyperoxia to altered airway structure and function are key to developing advanced therapies. We previously showed that even moderate hyperoxia (50% O2) enhances intracellular calcium ([Ca2+]i) and proliferation of human fetal airway smooth muscle (fASM), thereby facilitating bronchoconstriction and remodeling. Here, we introduce cellular clock biology as a novel mechanism linking early oxygen exposure to airway biology. Peripheral, intracellular clocks are a network of transcription-translation feedback loops that produce circadian oscillations with downstream targets highly relevant to airway function and asthma. Premature infants suffer circadian disruption whereas entrainment strategies improve outcomes, highlighting the need to understand relationships between clocks and developing airways. We hypothesized that hyperoxia impacts clock function in fASM and that the clock can be leveraged to attenuate deleterious effects of O2 on the developing airway. We report that human fASM express core clock machinery (PER1, PER2, CRY1, ARNTL/BMAL1, CLOCK) that is responsive to dexamethasone (Dex) and altered by O2. Disruption of the clock via siRNA-mediated PER1 or ARNTL knockdown alters store-operated calcium entry (SOCE) and [Ca2+]i response to histamine in hyperoxia. Effects of O2 on [Ca2+]i are rescued by driving expression of clock proteins, via effects on the Ca2+ channels IP3R and Orai1. These data reveal a functional fASM clock that modulates [Ca2+]i regulation, particularly in hyperoxia. Harnessing clock biology may be a novel therapeutic consideration for neonatal airway diseases following prematurity.

Hydrogen sulfide, oxygen, and calcium regulation in developing human airway smooth muscle.

Preterm infants can develop airway hyperreactivity and impaired bronchodilation following supplemental O2 (hyperoxia) in early life, making it important to understand mechanisms of hyperoxia effects. Endogenous hydrogen sulfide (H2 S) has anti-inflammatory and vasodilatory effects with oxidative stress. There is little understanding of H2 S signaling in developing airways. We hypothesized that the endogenous H2 S system is detrimentally influenced by O2 and conversely H2 S signaling pathways can be leveraged to attenuate deleterious effects of O2 . Using human fetal airway smooth muscle (fASM) cells, we investigated baseline expression of endogenous H2 S machinery, and effects of exogenous H2 S donors NaHS and GYY4137 in the context of moderate hyperoxia, with intracellular calcium regulation as a readout of contractility. Biochemical pathways for endogenous H2 S generation and catabolism are present in fASM, and are differentially sensitive to O2 toward overall reduction in H2 S levels. H2 S donors have downstream effects of reducing [Ca2+ ]i responses to bronchoconstrictor agonist via blunted plasma membrane Ca2+ influx: effects blocked by O2 . However, such detrimental O2 effects are targetable by exogenous H2 S donors such as NaHS and GYY4137. These data provide novel information regarding the potential for H2 S to act as a bronchodilator in developing airways in the context of oxygen exposure.

Long non-coding RNA00882 contributes to platelet-derived growth factor-induced proliferation of human fetal airway smooth muscle cells by enhancing Wnt/β-catenin signaling via sponging miR-3619–5p

Long non-coding RNAs (lncRNAs) are emerging as novel and critical regulators in the pathogenesis of asthma. However, the precise role of lncRNAs in pediatric asthma remains largely unknown. In this study, we aimed to investigate the biological function of lncRNA00882 (LINC00882) in regulating the proliferation of fetal airway smooth muscle (ASM) cells, which play an important role in airway remodeling during asthma development. Herein, we found that LINC00882 expression was significantly up-regulated in ASM cells stimulated with platelet-derived growth factor (PDGF). Functional experiments showed that the knockdown of LINC00882 markedly reduced the proliferation of fetal ASM cells induced by PDGF, while the overexpression of LINC00882 exhibited the opposite effect. Bioinformatics analysis, the luciferase reporter assay and the RNA pull-down assay revealed that LINC00882 directly interacted with microRNA-3619–5p (miR-3619–5p). LINC00882 negatively regulated miR-3619–5p expression in fetal ASM cells. Notably, β-catenin was identified as a target gene of miR-3619–5p. miR-3619–5p overexpression restricted PDGF-induced cell proliferation through inhibiting Wnt/β-catenin signaling. Moreover, miR-3619–5p overexpression significantly attenuated the LINC00882-induced promotion effect on PDGF-induced cell proliferation and Wnt/β-catenin signaling in fetal ASM cells. In contrast, miR-3619–5p inhibition significantly reversed the LINC00882 knockdown-mediated inhibitory effect on PDGF-induced cell proliferation and Wnt/β-catenin signaling. Taken together, our results demonstrate that LINC00882 promotes PDGF-induced cell proliferation of ASM cells by enhancing Wnt/β-catenin signaling via sponging miR-3619–5p, suggesting a potential role for LINC00882 in airway remodeling in pediatric asthma.

MicroRNA-20b-5p inhibits platelet-derived growth factor-induced proliferation of human fetal airway smooth muscle cells by targeting signal transducer and activator of transcription 3

Pediatric asthma is still a health threat to the pediatric population in recent years. The airway remodeling induced by abnormal airway smooth muscle (ASM) cell proliferation is an important cause of asthma. MicroRNAs (miRNAs) are important regulators of ASM cell proliferation. Numerous studies have reported that miR-20b-5p is a critical regulator for cell proliferation. However, whether miR-20b-5p is involved in regulating ASM cell proliferation remains unknown. In this study, we aimed to investigate the potential role of miR-20b-5p in regulating the proliferation of fetal ASM cell induced by platelet-derived growth factor (PDGF). Here, we showed that miR-20b-5p was significantly decreased in fetal ASM cells treated with PDGF. Biological experiments showed that the overexpression of miR-20b-5p inhibited the proliferation while miR-20b-5p inhibition markedly promoted the proliferation of fetal ASM cells. Bioinformatics analysis and luciferase reporter assay showed that miR-20b-5p directly targeted the 3′-UTR of signal transducer and activator of transcription 3 (STAT3). Further data showed that miR-20b-5p negatively regulated the expression of STAT3 in fetal ASM cells. Moreover, miR-20b-5p regulates the transcriptional activity of STAT3 in fetal ASM cells. Overexpression of STAT3 reversed the inhibitory effect of miR-20b-5p overexpression on fetal ASM cell proliferation while the knockdown of STAT3 abrogated the promoted effect of miR-20b-5p inhibition on fetal ASM cell proliferation. Overall, our results show that miR-20b-5p impedes PDGF-induced proliferation of fetal ASM cells through targeting STAT3. Our study suggests that miR-20b-5p may play an important role in airway remodeling during asthma and suggests that miR-20b-5p may serve as a potential therapeutic target for pediatric asthma.

MicroRNA-874 inhibits TNF-α-induced remodeling in human fetal airway smooth muscle cells by targeting STAT3

Pro-inflammatory cytokines-induced airway remodeling was a significant feature of asthma disease. The aim of the present study was to explore the functional significance of miR-874 in tumor necrosis factor (TNF)-α-treated human fetal airway smooth muscle (fASM) cells. Here, we found that TNF-α treatment significantly down-regulated the expression of miR-874 in fASM cells. MiR-874 overexpression markedly inhibited cell viability and migration, suppressed the expression of PCNA and Ki67, reduced the expression of collagen I and collagen III, decreased the expression and activity of matrix metalloproteinase (MMP)-9 and MMP-2, and induced an obvious elevation of tissue inhibitors of metalloproteinases (TIMPs). In addition, the increased production of interleukin (IL)-6, IL-8 and eotaxin induced by TNF-α were significantly inhibited by miR-874 overexpression. Signal transducers and activators of transcription (STAT) 3 was identified as a direct target of miR-874, and STAT3 overexpression partly reversed the protective effects of miR-874 against TNF-α-induced airway remodeling. Overall, these findings demonstrate that miR-874 inhibits TNF-α-induced remodeling in human fASM cells at least in part by targeting STAT3.

MicroRNA-590-5p represses proliferation of human fetal airway smooth muscle cells by targeting signal transducer and activator of transcription 3.

IntroductionPediatric asthma has remained a health threat to children in recent years. The abnormal proliferation of airway smooth muscle (ASM) cells contributes to airway remodeling during development of asthma. MicroRNAs (miRNAs) are critical regulators of ASM cell proliferation during airway remodeling. miR-590-5p has been reported to regulate cell proliferation in various cell types. However, it remains unclear whether miR-590-5p regulates ASM cell proliferation. In this study, we aimed to investigate the potential role of miR-590-5p in regulating fetal ASM cell proliferation in vitro stimulated by platelet-derived growth factor (PDGF).Material and methodsmiRNA, mRNA, and protein expression was detected by real-time quantitative polymerase chain reaction and western blot. Cell proliferation was detected by CCK-8 and BrdU assays. The target of miR-590-5p was confirmed by dual-luciferase reporter assay.ResultsMiR-590-5p expression was significantly down-regulated in fetal ASM cells stimulated with PDGF (p < 0.05). Overexpression of miR-590-5p inhibited cell proliferation (p < 0.05), whereas the suppression of miR-590-5p promoted cell proliferation of fetal ASM cells stimulated with PDGF (p < 0.05). Signal transducer and activator of transcription 3 (STAT3) was identified as a target gene of miR-590-5p. In addition, miR-590-5p negatively regulated STAT3 expression (p < 0.05). Moreover, miR-590-5p also modulated downstream genes of STAT3 including cyclin D3 and p27 (p < 0.05). The restoration of STAT3 significantly reversed the inhibitory effect of miR-590-5p on fetal ASM cell proliferation.ConclusionsMiR-590-5p inhibits proliferation of fetal ASM cells by down-regulating STAT3, thereby suggesting a novel therapeutic target for the treatment of pediatric asthma.

Overexpression of microRNA-375 impedes platelet-derived growth factor-induced proliferation and migration of human fetal airway smooth muscle cells by targeting Janus kinase 2

The abnormal proliferation and migration of airway smooth muscle (ASM) cells play a critical role in airway remodeling during the development of asthma. MicroRNAs (miRNAs) have emerged as critical regulators of ASM cell proliferation and migration in airway remodeling. In this study, we aimed to investigate the potential role of miR-375 in the regulation of platelet-derived growth factor (PDGF)-induced fetal ASM cell proliferation and migration. Our results showed that miR-375 expression was significantly decreased in fetal ASM cells that were treated with PDGF. Functional data showed that overexpression of miR-375 inhibited the proliferation and migration of fetal ASM cells, whereas inhibition of miR-375 enhanced the proliferation and migration of fetal ASM cells. The results of bioinformatics analysis and a dual-luciferase reporter assay showed that miR-375 binds directly to the 3′-untranslated region of Janus kinase 2 (JAK2). Further data confirmed that miR-375 negatively regulates the expression of JAK2 in fetal ASM cells. Moreover, miR-375 also impeded the PDGF-induced activation of signal transducer and activator of transcription 3 (STAT3) in fetal ASM cells. However, restoration of JAK2 expression partially reversed the inhibitory effect of miR-375 on fetal ASM cell proliferation and migration. Overall, our results demonstrate that miR-375 inhibits fetal ASM cell proliferation and migration by targeting JAK2/STAT3 signaling. Our study provides a potential therapeutic target for the development of novel treatment strategies for pediatric asthma.

Moderate hyperoxia induces extracellular matrix remodeling by human fetal airway smooth muscle cells.

BackgroundPremature infants are at increased risk for airway diseases, such as wheezing and asthma, because of early exposure to risk factors including hyperoxia. As in adult asthma, airway remodeling and increased extracellular matrix (ECM) deposition is involved.MethodsWe assessed the impact of 24-72 h of moderate hyperoxia (50%) on human fetal airway smooth muscle (fASM) ECM deposition through western blot, modified in-cell western, and zymography techniques.ResultsHyperoxia exposure significantly increased collagen I and collagen III deposition, increased pro- and cleaved matrix metalloproteinase 9 (MMP9) activity, and decreased endogenous MMP inhibitor, TIMP1, expression. Hyperoxia-induced change in caveolin-1 (CAV1) expression was assessed as a potential mechanism for the changes in ECM deposition. CAV1 expression was decreased following hyperoxia. Supplementation of CAV1 activity with caveolar scaffolding domain (CSD) peptide abrogated the hyperoxia-mediated ECM changes.ConclusionsThese results demonstrate that moderate hyperoxia enhances ECM deposition in developing airways by altering the balance between MMPs and their inhibitors (TIMPs), and by increasing collagen deposition. These effects are partly mediated by a hyperoxia-induced decrease in CAV1 expression. In conjunction with prior data demonstrating increased fASM proliferation with hyperoxia, these data further demonstrate that hyperoxia is an important instigator of remodeling in developing airways.

TLR3 activation increases chemokine expression in human fetal airway smooth muscle cells.

Viral infections, such as respiratory syncytial virus and rhinovirus, adversely affect neonatal and pediatric populations, resulting in significant lung morbidity, including acute asthma exacerbation. Studies in adults have demonstrated that human airway smooth muscle (ASM) cells modulate inflammation through their ability to secrete inflammatory cytokines and chemokines. The role of ASM in the developing airway during infection remains undefined. In our study, we used human fetal ASM cells as an in vitro model to examine the effect of Toll-like receptor (TLR) agonists on chemokine secretion. We found that fetal ASM express multiple TLRs, including TLR3 and TLR4, which are implicated in the pathogenesis of respiratory syncytial virus and rhinovirus infection. Cells were treated with TLR agonists, polyinosinic-polycytidylic acid [poly(I:C)] (TLR3 agonist), lipopolysaccharide (TLR4 agonist), or R848 (TLR7/8 agonist), and IL-8 and chemokine (C-C motif) ligand 5 (CCL5) secretion were evaluated. Interestingly, poly(I:C), but neither lipopolysaccharide nor R848, increased IL-8 and chemokine (C-C motif) ligand 5 secretion. Examination of signaling pathways suggested that the poly(I:C) effects in fetal ASM involve TLR and ERK signaling, in addition to another major inflammatory pathway, NF-κB. Moreover, there are variations between fetal and adult ASM with respect to poly(I:C) effects on signaling pathways. Pharmacological inhibition suggested that ERK pathways mediate poly(I:C) effects. Overall, our data show that poly(I:C) initiates activation of proinflammatory pathways in developing ASM, which may contribute to immune responses to infection and exacerbation of asthma.

Vitamin D attenuates cytokine-induced remodeling in human fetal airway smooth muscle cells.

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)2 D3 ), 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.

288: Synthetic double stranded RNA increases chemokine expression in human fetal airway smooth muscle cells

288: Synthetic double stranded RNA increases chemokine expression in human fetal airway smooth muscle cells

Hyperoxia-induced changes in estradiol metabolism in postnatal airway smooth muscle.

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.

691: Vitamin D reduces TNF-α induced MMP-9 activity in human fetal airway smooth muscle cells

Premature infants are at risk of developing lung diseases including asthma. There is a link between antenatal inflammation and airway remodeling that predisposes infants and children to development of asthma and/or wheezing. Clinically, these airway diseases appear to be modified by maternal nutritional status, with studies demonstrating inverse relationships between Vitamin D levels and prematurity, chronic lung disease, and childhood asthma. Using human fetal airway smooth muscle cells (fASMC) as an in vitro model of the developing airway, we explored the hypothesis that Vitamin D blunts the effects of inflammation in regard to extracellular matrix remodeling, thus alleviating structural airway changes that would contribute to asthma.

Oxygen dose responsiveness of human fetal airway smooth muscle cells

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.

Hyperoxia enhances intracellular calcium in human fetal airway smooth muscle via cyclic nucleotide regulation of brain‐derived neurotrophic factor release

Supplemental oxygen places the immature airway at risk for bronchopulmonary dysplasia and asthma. Hyperoxia enhances intracellular Ca2+ ([Ca2+]i) and contractility of airway smooth muscle (ASM), and impairs relaxation (potentially by altering cyclic nucleotide levels). The neurotrophin brain‐derived neurotrophic factor (BDNF) enhances [Ca2+]i in adult ASM. We tested the hypothesis that hyperoxia‐induced airway hyperreactivity involves cyclic nucleotide‐mediated enhancement of BDNF release in human fetal (18–22 weeks) ASM cells exposed to 21% (normoxia) or 80% oxygen in the presence/absence of 1mM dibutyryl cAMP (DBcAMP) or 8‐Br‐cGMP. BDNF release (ELISA) was increased by hyperoxia (143% of normoxia). DBcAMP enhanced BDNF release with normoxia (242% of baseline) and even more with hyperoxia (280%), while 8‐Br‐cGMP blunted BDNF release in normoxia (57% of baseline) but to a lesser extent with hyperoxia (68% of baseline). Measurements of [Ca2+]i in fetal ASM cells showed DBcAMP increasing [Ca2+]i responses to histamine or ACh (effects enhanced with hyperoxia). Further, chelation of released BDNF using chimeric TrkB‐Fc (1?g/ml) reversed DBcAMP effects and blunted [Ca2+]i responses to below controls. We conclude that an imbalance between cAMP vs. cGMP enhances BDNF release in the developing airway, resulting in greater airway reactivity with hyperoxia. Supported by NIH grants HL056470 and HL088029

Spontaneous Contractility of Human Fetal Airway Smooth Muscle

Physical forces such as fetal breathing and fluid secretion may influence lung development, perhaps by their ability to distend the fetal lung. We found that airway smooth muscle cells in first-trimester human lung tissue and cultured lung tissue explants spontaneously contract. The contractions caused visible movement of intraluminal fluid and distended the distal ends of the epithelial tubules, suggesting that they produced significant changes in intraluminal pressure. Smooth muscle contractility and responses to pharmacologic manipulations were recorded with video microscopy. The interval between contractions ranged from 8 to 135 s (mean +/- SEM, 54 +/- 5 s; n = 20). Smooth muscle contractility was not inhibited by tetrodotoxin or atropine, implying a myogenic rather than neurogenic origin. Because cholinergic nerves modulate adult airway smooth muscle contractility, we asked whether fetal airway smooth muscle cells were regulated by cholinergic agents. The cholinergic agonists acetylcholine and carbachol both increased fetal airway smooth muscle contractility. Contractions were inhibited by the calcium channel blockers CdCl2 and nifedipine, suggesting that an influx of extracellular Ca2+ accompanied airway smooth muscle contractions. Isoproterenol dilated the contractile regions of the epithelial tubules and stopped contractions. Lemakalim, an activator of smooth muscle ATP-sensitive K+ channels, also arrested contractions and relaxed fetal airway smooth muscle. In conclusion, human fetal airway smooth muscle contacts spontaneously and exhibits pharmacologic responsiveness similar to adult airway smooth muscle. We speculate that fetal airway smooth muscle contractions, possibly exerting effects through phasic changes in intraluminal pressure, may be an important physical force contributing to lung development.