Oxygen dose responsiveness of human fetal airway smooth muscle cells

Am J Physiol Lung Cell Mol Physiol 303: L711–L719, 2012. First published August 24, 2012; doi:10.1152/ajplung.00037.2012.

Oxygen dose responsiveness of human fetal airway smooth muscle cells William R. Hartman,1 Dan F. Smelter,1 Venkatachalem Sathish,1,2 Michael Karass,1 Sunchin Kim,1 Bharathi Aravamudan,1 Michael A. Thompson,1 Yassine Amrani,3 Hitesh C. Pandya,4 Richard J. Martin,5 Y. S. Prakash,1,2 and Christina M. Pabelick1,2 Departments of 1Anesthesiology, and 2Physiology and Biomedical Engineering, Mayo Clinic, Rochester Minnesota; Departments of 3Infection, Immunity, and Inflammation, and 4Pediatrics, University of Leicester, Leicester, United Kingdom; 5 Department of Pediatrics, Division of Neonatology, Rainbow Babies Children’s Hospital, Case Western Reserve University, Cleveland, Ohio Submitted 25 January 2012; accepted in final form 21 August 2012

Hartman WR, Smelter DF, Sathish V, Karass M, Kim S, Aravamudan B, Thompson MA, Amrani Y, Pandya HC, Martin RJ, Prakash YS, Pabelick CM. Oxygen dose responsiveness of human fetal airway smooth muscle cells. Am J Physiol Lung Cell Mol Physiol 303: L711–L719, 2012. First published August 24, 2012; doi:10.1152/ajplung.00037.2012.—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 Ca2⫹ ([Ca2⫹]i) and cellular proliferation. fASM cells expressed substantial smooth muscle actin and myosin and several Ca2⫹ regulatory proteins but not fibroblast or epithelial markers, profiles qualitatively comparable to adult human ASM. Fluorescence Ca2⫹ imaging showed robust [Ca2⫹]i responses to 1 ␮M acetylcholine (ACh) and 10 ␮M histamine (albeit smaller and slower than adult ASM), partly sensitive to zero extracellular Ca2⫹. 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. [Ca2⫹]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; asthma; neonate; proliferation; apoptosis; mitochondria

during lung development involves multiple cell types and complex processes that are only partly understood. Airway smooth muscle (ASM) is undoubtedly important to the regulation of airway tone throughout life and contributes to the pathophysiology of several diseases including asthma (3, 7, 8, 16, 22, 51, 54). Furthermore, in lung diseases of the neonate, such as neonatal respiratory distress syndrome (6, 47) as well as bronchopulmonary dysplasia resulting from exposure to high inspired oxygen levels (hyperoxia) and/or mechanical ventilation (23, 33, 54), detrimental

FORMATION OF AIRWAY COMPONENTS

Address for reprint requests and other correspondence: C. M. Pabelick, Dept. of Anesthesiology, 4-184 W Jos SMH, Mayo Clinic, Rochester, MN 55905 (e-mail: [email protected]). http://www.ajplung.org

changes in airway structure and function can contribute to both immediate and long-term enhancement of airway reactivity with predisposition to asthma. Indeed, preterm infants continue to be at very high risk for airway hyperreactivity in childhood, with greater need for asthma medication at 2 yr of age in former very-low-birth-weight infants. Although several factors including prematurity itself are likely contributory, hyperoxia clearly affects airway structure and function. There is a clinical trend toward reducing the extent and duration of oxygen exposure to avoid hyperoxia while minimizing detrimental effects attributable to insufficient oxygen saturations. Regardless, to maintain oxygen saturations, exposure to some level of hyperoxia does occur, placing the developing airway at risk. Thus it is important to determine the extent and form of hyperoxia effects on developing airway. As in adults, neonatal asthma also involves enhanced airway contractility (13, 15, 23, 34) as well as airway remodeling (21, 30), both of which involve ASM to a large extent. The role of oxygen in modulating either aspect of ASM is not known. Although in vitro and in vivo studies in animal models such as rat (19, 29, 31), mouse (9, 13), and sheep (7, 25) have provided much insight into potential mechanisms underlying neonatal lung disease, species differences in lung development (60) as well as mechanisms that regulate ASM structure and function (4, 28) necessitate a need for an age-appropriate human model. Some studies have used adult human ASM cells or tissues with interventions such as hyperoxia (48) or inflammation (14). However, it is unknown whether developing ASM can be considered comparable to adult ASM at baseline (considering the substantial need for rapid growth during development compared with the adult) or in its response to detrimental interventions. Regardless of whether it is neonatal or adult airway, intracellular Ca2⫹ ([Ca2⫹]i) regulation is a key determinant of ASM contractility and thus airway tone (20, 45). However, compared with adult ASM, the mechanisms by which [Ca2⫹]i is regulated in neonatal ASM, especially in humans, is still under investigation. Furthermore, ASM mass, i.e., size and number of ASM cells, is also an important factor in determining airway contractility. In adults, dysregulated ASM cellular proliferation contributes to airway remodeling and hyperresponsiveness in asthma (12, 27). Whether factors such as hypoxia, hyperoxia, and/or inflammation also result in altered cell proliferation of the developing airway, especially in humans, is not known. A limited number of studies have used isolated human fetal or neonatal lung tissues to obtain mechanistic information (14, 34, 35). However, the specific properties of developing human fetal ASM (fASM) in terms of [Ca2⫹]i and/or cell proliferation

1040-0605/12 Copyright © 2012 the American Physiological Society

L711

L712

OXYGEN TOXICITY IN FETAL AIRWAY

or their responsiveness to oxygen have not been previously examined. In the present study, we characterized isolated human fASM cells in the context of their suitability for studying regulation of [Ca2⫹]i and cellular proliferation. Furthermore, we determined the effects of different oxygen concentrations on fASM properties, with the hypothesis that developing ASM can tolerate hyperoxia to only a certain point beyond which detrimental effects occur. In this regard, we examined the effect of hypoxia as well as different levels of hyperoxia on ASM proliferation vs. apoptosis. Furthermore, a major aspect of cellular proliferation and apoptosis is the role of mitochondrial proteins. In addition to being the powerhouse of the cell, mitochondria buffer cytoplasmic Ca2⫹ (17, 32, 44, 49, 55) and modulate cell proliferation/death (57, 59). Given the relationship between oxygen and mitochondria, we examined the effect of different oxygen concentrations on mitochondrial fission vs. fusion. MATERIALS AND METHODS

Materials. Chemicals and supplies were obtained from Sigma (St. Louis, MO) unless mentioned otherwise. Tissue culture reagents, including DMEM F/12 Hanks Balanced Salt Solution (HBSS) and fetal bovine serum (FBS), were obtained from Invitrogen (Carlsbad, CA). Human fASM cells. fASM cells were obtained from canalicular stage (18 –20 wk gestation) fetal trachea (deidentified samples, in vitro processing only; courtesy of MRC-Wellcome Trust HDBR Group, Newcastle University & University College London; approved by Ethics Committees in the UK, and considered exempt by Mayo Institutional Review Board). Cells were enzymatically dissociated using previously described techniques (34, 35). Tracheobronchial tissue from adults were obtained from lung samples incidental to patients undergoing thoracic surgery at Mayo Clinic (2nd to 6th generation, deidentified, typically lobectomies or pneumonectomies, considered surgical waste; approved and considered exempt by Mayo IRB) and adult ASM cells isolated as described previously (39, 40). Both fASM and adult ASM cells were plated and grown in a 95% air-5% CO2 humidified incubator using DMEM F/12 (phenol red free) supplemented with 10% FBS. Cells were placed in 0.5% serum for 24 h before experiments to arrest growth. Smooth muscle markers were verified as described previously (18, 56). Experiments were performed in 2.5 mM Ca2⫹ HBSS. Western blot analysis. Proteins from prepared cellular extracts were separated using SDS-PAGE (10% gradient gels, although lower percentage gels were required for Western blot of the ryanodine receptor; Criterion Gel System; Bio-Rad, Hercules, CA) at 200 V for 1 h and transferred to polyvinylidene difluoride membranes (Bio-Rad) for 60 min. Membranes were then blocked for 1 h with 5% milk at room temperature in Tris-buffered saline with 0.1% Tween (TBST) and incubated overnight with gentle rocking at 4°C with 1 ␮g/ml of appropriate antibody. The following antibodies were used (from Santa Cruz Biotechnology, Santa Cruz, CA) unless otherwise noted: Cyclin E, proliferating cell nuclear antigen (PCNA), BCL-2, caspase-9, cytochrome c, p27Kip1, Cavolin-1, Drp1, Mfn2 (Abcam, Cambridge, MA), GAPDH (Cell Signaling Technology, Danvers, MA), smooth muscle actin and myosin heavy chain (MHC), M3 muscarinic receptor (acetylcholine receptor, AChR), H1 histaminergic receptor, neurokinin receptors NK1R and NK2R, sarcoendoplasmic reticulum (SR) Ca2⫹ ATPase (SERCA, Abcam), IP3 receptor (IP3R) and ryanodine receptors (RyR), transient receptor potential channel TRPC3, storeoperated Ca2⫹ entry regulator STIM1 (Abcam), E-cadherin (Abcam), and fibroblast surface protein (FSP). Following three washes with TBST, secondary antibodies were added. For this step, either horse-

radish peroxidase-conjugated antibodies with chemiluminescence substrate or far-red fluorescent dye conjugated antibodies were used. Blots were imaged on a Kodak Image Station 4000MM (Carestream Health, New Haven, CT) or a LiCor OdysseyXL system (Lincoln, NE) and quantified using densitometry. Normalization of protein blots was performed using GAPDH. Cellular proliferation assay. Proliferation was assessed in 96-well plates using the CyQuant NF cellular proliferation fluorescence kit (Invitrogen) using a recently published protocol (2). Briefly, cells were plated at ⬃5,000 cells/well. To analyze proliferation under different conditions, cells were grown in completely serum-free media or different amounts of serum or in the presence of the commonly used mitogen platelet-derived growth factor (10 ng/ml, PDGF-BB; Sigma Aldrich, St. Louis, MO). Interventions of hypoxia or hyperoxia were superimposed throughout the cell proliferative period of 48 h, following which media was aspirated and cells incubated at 37°C for 1 h with CyQuant dye in HBSS. The resultant fluorescence was determined on a Flexstation 3 microplate reader (Molecular Devices, Sunnyvale, CA; excitation 480 nm/emission 530 nm). Oxygen exposure. Serum-starved fASM cells were exposed for 48 h to a single oxygen concentration between 10% (hypoxia) and 90%, in 10% increments (21% was used for normoxia) in a 5% CO2 humidified incubator. Cells were then evaluated for [Ca2⫹]i responses (see below), cellular proliferation (using CyQuant Assay as described above), or Western analysis of lysates. [Ca2⫹]i imaging. Techniques for imaging of [Ca2⫹]i in ASM cells has been previously published (39, 40). Briefly, cells were incubated in 5 ␮M fluo-4-AM (Invitrogen) at room temperature for 45 min. Fluorescence measurements were made using the FlexStation3 microplate reader along with its micropipetting capabilities to add agents of interest. Fluorescence was calibrated in situ empirically by comparing fluorescence intensity for known Ca2⫹ levels in ASM cells exposed to the ionophore A-23187. Mitochondrial analysis. fASM cells were stained for 15 min with 50 nM of the mitochondrial marker MitoTracker Green. Cells were then imaged using a Nikon TE2000 inverted microscope equipped with a ⫻40/1.3 NA oil immersion lens, a 12-bit Roper Scientific Cascade high-sensitivity camera (1024 ⫻ 1024 pixels), and a Metamorph imaging system (Molecular Devices). Mitochondrial patterns within populations of cells were imaged before and after exposure to different oxygen concentrations. In a second set of studies, the expression of mitochondrial fission vs. fusion proteins was evaluated using Western blots. Statistical analysis. Experiments were performed using cells from five fetal airway samples. Comparisons between groups were made using Sigma Plot (SYSTAT, San Jose, CA) software statistical package, with independent Student’s t-test or 2-way ANOVA with repeated measures and Bonferroni correction. Statistical significance was established at P ⬍ 0.05. Values are means ⫾ SE. RESULTS

Characteristics of fASM cells. Brightfield microscopy of gross morphological features showed that serum-deprived fASM cells (Fig. 1) have a spindle-shaped morphology typical of smooth muscle cells in general and are comparable to the well-known shape of adult ASM cells. In culture, confluent sheets of such spindle-shaped cells were observed and were found to persist even after six passages of subculture. Western blot analysis of whole cell lysates of fASM cells showed substantial expression of smooth muscle actin as well as myosin heavy chain. Indeed expression of these contractile proteins was higher than that in adult ASM cells (Fig. 2; P ⬍ 0.05). In contrast, both FSP and the epithelial cell marker, E-cadherin, were absent (Fig. 2), whereas vimentin was expressed at low levels (data not shown), confirming that the

AJP-Lung Cell Mol Physiol • doi:10.1152/ajplung.00037.2012 • www.ajplung.org


Fig. 1. Serum-deprived human fetal airway smooth muscle (fASM) cells display mononucleated spindle-shaped morphology typical of smooth muscle cells. Scale bar ⫽ 50 ␮m for left and 10 ␮m for right.

population of fASM cells was relatively pure smooth muscle phenotype. To demonstrate fASM cells as a viable model for cell signaling mechanisms during early development, it was necessary to determine the presence of proteins known to be important for smooth muscle [Ca2⫹]i and force regulation. Western analysis of whole cell fASM extracts showed substantial expression of M3 AChR (comparable to adult ASM, Fig. 2), suggesting the ability for this well-known bronchoconstric-

Fig. 2. Expression of intracellular Ca2⫹ ([Ca2⫹]i) regulatory proteins and smooth muscle markers in fASM cells. Western analysis of whole cell lysates showed substantial expression of contractile proteins (smooth muscle actin and myosin heavy chain, MHC), receptors for bronchoconstrictor agonists (M3 muscarinic receptor; acetylcholine receptor, AChR); H1 histaminergic receptor; neurokinin receptors (NK1R and NK2R) as well a number of [Ca2⫹]i regulatory mechanisms including sarcoendoplasmic reticulum Ca2⫹ ATPase (SERCA) and IP3 receptor (IP3R), and to lesser extents ryanodine receptors (RyR), transient receptor potential channel TRPC3, and store-operated Ca2⫹ entry regulator STIM1. In contrast, markers of epithelial cells (E-cadherin) or fibroblasts (fibroblast surface protein; FSP) were absent. Values (means ⫾ SE) from n of 5 samples normalized to GAPDH in bar graph. *Significant difference between fASM and adult ASM cells (P ⬍ 0.05).

L713

tor to function in these cells. However, H1R expression was substantially lower in fASM (Fig. 2; P ⬍ 0.05). In addition to ACh and histamine, adult airways are also known to be sensitive to tachykinins such as substance P (11) that act via NK receptors. Western blot analysis showed that NK1 receptors are expressed in fASM cells at levels somewhat higher than in adult ASM cells (Fig. 2 P ⬍ 0.05). Compared with NK1 receptor expression, lower band intensities were detected for NK2 receptor expression in both fetal and adult ASM cells. In adult ASM, [Ca2⫹]i regulation involves both SR Ca2⫹ release/reuptake as well as Ca2⫹ influx/efflux across the plasma membrane (20, 45). Western blot analysis of fASM cells showed the expression of both IP3R and RyR SR Ca2⫹ release channels. Expression of RyR was substantially lower in fASM cells, whereas IP3R expression was slightly but significantly higher compared with adult ASM (Fig. 2; P ⬍ 0.05). Substantial expression of the SR Ca2⫹ reuptake protein SERCA2 at levels comparable to adult ASM was detected (Fig. 2). Compared with adult ASM, the expression of Ca2⫹ influx proteins such as TRPC3, as well as the influx regulatory protein STIM1, was much lower in fASM cells (Fig. 2, P ⬍ 0.05). We have recently demonstrated that [Ca2⫹]i regulation in adult ASM cells is highly regulated by caveolae (plasma membrane invaginations rich in cholesterol and sphingolipids) and the constitutive caveolin-1 protein (41, 46). Preparation of plasma membrane fractions enriched in caveolae (41) showed substantial expression of caveolin-1 in fASM cells (Fig. 3). Furthermore, the cholesterol-chelating agent methyl-␤-cyclodextrin (CD; 10 mM) was used to verify that the caveolin-1

Fig. 3. Expression of caveolin-1 in fASM cells. Compared with adult ASM cells, fASM cells expressed significantly greater amounts of the constituent caveolar protein caveolin-1. Exposure to the cholesterol-chelating agent methyl␤-cyclodextrin (CD) significantly reduced caveolin-1 expression, verifying membrane expression of this protein. Values (means ⫾ SE) from n of 5 samples normalized to GAPDH in bar graph. *Significant CD effect; #significant difference between fASM and adult ASM cells (P ⬍ 0.05).


L714


was truly within caveolar fractions. Exposure of fASM cells to 10 mM CD for 2 h resulted in substantial reduction in caveolin-1 expression (Fig. 3, P ⬍ 0.05). [Ca2⫹]i regulation in fASM cells. In serum-deprived, fluo4-loaded fASM cells, exposure to 1 ␮M ACh resulted in robust [Ca2⫹]i responses that showed a typical higher peak followed by a lower plateau level still above baseline (Fig. 4A). Compared with adult ASM cells that also showed a robust [Ca2⫹]i response, the peak [Ca2⫹]i response of fASM cells was significantly smaller (Fig. 4C), as was the plateau level (P ⬍ 0.05 for both peak and plateau). In a separate set of fASM cells, [Ca2⫹]i responses to 10 ␮M histamine were also typical, with a transient higher peak, and lower plateau level (Fig. 4A). As with ACh, the amplitude was smaller in fASM cells, compared with adult cells (Fig. 4C, P ⬍ 0.05). However, if anything, the [Ca2⫹]i responses of fASM cells to histamine were generally more robust than for ACh. In adult ASM, the tachykinin Substance P is known to enhance [Ca2⫹]i (11, 31). Although there are currently no data on this aspect in developing ASM, airway irritability and a role of innervation have been suggested (58). We found that overnight exposure to 1 ␮M Substance P enhanced the subsequent [Ca2⫹]i response to ACh, compared with vehicle alone (Fig. 4B; P ⬍ 0.05). Here, the effect of Substance P on fASM cells compared with vehicle control was proportionately greater (Fig. 4C). All of the above experiments confirmed the existence of functional agonist receptors. A number of protocols was then used to determine mechanisms underlying [Ca2⫹]i responses in fASM cells. Removal of extracellular Ca2⫹ significantly blunted the [Ca2⫹]i responses to ACh (Fig. 5, A and D, P ⬍ 0.05), confirming a component of Ca2⫹ influx. Conversely, fASM cells showed a transient [Ca2⫹]i response to 5 mM caffeine (albeit slower than adult ASM), indicating the presence of functional RyR channels (Fig. 5, B and D). Exposure to 10 mM CD for 1 h decreased baseline [Ca2⫹]i and furthermore blunted [Ca2⫹]i responses to ACh (Fig. 5, C and D), indicating a role for caveolae in [Ca2⫹]i regulation of fASM cells. Proliferation of fASM cells. Cellular proliferation of fASM cells was determined using the Invitrogen CyQuant NF proliferation assay. As shown in Fig. 6, fASM proliferation in serum-free medium was minimal, compared with a small level of baseline proliferation in adult ASM cells. However, in the presence of even 0.5% serum, or the mitogen PDGF alone, fASM cell proliferation was substantial (Fig. 6; P ⬍ 0.05 for serum or PDGF effect, and difference between fASM and adult). Oxygen effects on human fASM cells. Compared with cell proliferation in normoxia and 0.5% serum, fASM cells grown in 10% hypoxia demonstrated robust proliferation (Fig. 7, P ⬍ 0.05). Between oxygen concentrations of 20% and 50% oxygen, there was a progressive and significant increase in proliferation (Fig. 7; P ⬍ 0.05). However, beyond 50% hyperoxia, cell counts progressively declined (P ⬍ 0.05 compared with values at 50%), suggesting ongoing apoptosis. To evaluate the changing levels of proliferation at different oxygen concentrations, we performed a series of Western blot analyses examining markers of proliferative vs. apoptotic pathways. At 10% hypoxia, fASM cells demonstrated increased expression of the proproliferative markers PCNA and Cyclin E (Fig. 8; P ⬍ 0.05). In addition, there was a trend toward

Fig. 4. [Ca2⫹]i responses of fASM cells to agonists. Exposure of fASM cells to 1 ␮M ACh or 10 ␮M histamine induced a large peak [Ca2⫹]i response followed by a lower plateau level above baseline (A). HBSS, Hanks’s Balanced Salt Solution. Overnight exposure to the neurokinin receptor agonist Substance P (SP; 1 ␮M) resulted in enhanced [Ca2⫹]i responses to subsequent 1 ␮M ACh exposure (B). In general, compared with adult ASM cells that also produced qualitatively similar responses, the peak [Ca2⫹]i responses to agonist were significantly smaller in fASM cells for ACh (with or without SP) and histamine. Values (means ⫾ SE) from n of 5 samples in bar graph (C). *Significant difference between fASM and adult ASM cells; #significant SP effect (P ⬍ 0.05).


L715


Fig. 5. Mechanisms of [Ca2⫹]i responses in fASM cells. Removal of extracellular Ca2⫹ significantly blunted the [Ca2⫹]i responses to ACh (shown) (A), confirming a component of Ca2⫹ influx. The influx component, relative to overall [Ca2⫹]i response, was larger in fASM cells compared with adult ASM cells. Conversely, fASM cells showed a transient [Ca2⫹]i response to 5 mM caffeine (albeit slower and smaller than adult ASM), indicating the presence of SR Ca2⫹ release and functional RyR channels (B). Separately, exposure to 10 mM of the plasma membrane cholesterol-chelating agent methyl-␤-cyclodextrin (CD) for 1 h decreased baseline [Ca2⫹]i and furthermore blunted [Ca2⫹]i responses to ACh, indicating a role for plasma membrane caveolae in [Ca2⫹]i regulation of fASM cells (C). Values (means ⫾ SE) from n of 5 samples in bar graph (D). *Significant difference between fASM and adult ASM cells (P ⬍ 0.05). #Significant effect of 0 Ca2⫹ HBSS or CD.

enhanced expression of the antiapoptosis marker BCL-2 (Fig. 8), as well as a slight decrease in p27Kip1 expression. These changes in expression were not statistically significant. With increased oxygen levels above normoxia, the expression of proproliferative markers rapidly declined (especially above 40%), whereas the expression of proapoptotic markers such as caspase 9, cytochrome c, and p27Kip1 all increased, albeit at different rates (Fig. 8; P ⬍ 0.05 for each marker at different oxygen concentrations above normoxia). At the highest oxygen concentration tested (70%), the expression of apoptosis markers was still significantly higher than the normoxia control. However, the expression observed was slightly less compared with 50% and 60% hyperoxia. This may reflect a diminished cell number attributed to cellular death.

Hyperoxia effects on mitochondria. Fluorescence imaging of MitoTracker-labeled fASM cells in normoxia showed reticular mitochondrial patterns (Fig. 9). With increasing oxygen concentrations (beyond 50%), mitochondria took on a more punctate and aggregated appearance with short branching patterns such that, at 80% oxygen, mostly aggregated mitochondria were observed (Fig. 9). Furthermore, Western blot analysis showed that, compared with normoxia, expression of the mitochondrial fusion protein Mfn2 was significantly decreased at 50%, 60%, and 70% oxygen, whereas expression of Drp1 was significantly decreased only at 60% oxygen (Fig. 9; P ⬍ 0.05). However, at higher oxygen concentrations, Drp1 expression was substantially increased.

Fig. 6. Proliferation of fASM cells. Cellular proliferation was determined using a CyQuant assay. In serum-depleted media, fASM cells showed minimal proliferation at 48 h. The presence of 0.5% serum substantially enhanced proliferation, especially in fASM cells, as did the standard mitogen plateletderived growth factor (PDGF). Values (means ⫾ SE) from n of 5 samples. Percent increase from initial cell count of 5,000. *Significant difference between fASM and adult ASM cells (P ⬍ 0.05). #Significant effect of serum or PDGF.

Fig. 7. Proliferation responses of fASM cells to oxygen. Cellular proliferation was determined using a fluorescent CyQuant assay. Compared with normoxic controls, fASM cells showed enhanced proliferation in 10% hypoxia at 48 h. However, with increasing oxygen concentration beyond normoxia, cell proliferation progressively increased until 50%, beyond which proliferation quickly dropped off to below normoxic levels, suggesting increasing cell death. Values (means ⫾ SE) from n of 5 samples. Percent increase from initial cell count of 5,000. *Significant difference in cell number from normoxia (P ⬍ 0.05).


L716


with normoxia, hypoxia as well as moderate levels of hyperoxia enhance cell proliferation, but higher levels of oxygen enhance apoptosis. Thus it appears that even moderate levels of hyperoxia that are used clinically to maintain oxygen satura-

Fig. 8. Effect of oxygen on expression of proliferation and apoptosis proteins in fASM cells. A: Western analysis of whole cell lysates showed increased expression for markers of cellular proliferation (proliferating cell nuclear antigen, PCNA, Cyclin E) when fASM cells were exposed for 48 h to 10% hypoxia or to moderate levels of hyperoxia until 40%. Correspondingly, antiapoptotic proteins such as BCL-2 were higher. However, beyond 40%, expression of proapoptotic proteins such as p27Kip1, caspase 9, and cytochrome c progressively increased with increasing oxygen concentration. Values (means ⫾ SE) from n of 5 samples normalized to GAPDH in bar graph (B). *Significant difference from normoxia (P ⬍ 0.05).

DISCUSSION

In the present study, we demonstrate that human fASM cells are an appropriate model for examining signaling mechanisms relevant to ASM structure and function within the developing airway. Human fASM cells express a portfolio of agonist receptors and other signaling mechanisms likely to be involved in [Ca2⫹]i regulation and airway contractility during development. Although fASM cells show robust [Ca2⫹]i responses to bronchoconstrictor agonists, these responses are generally smaller and slower than in adult ASM cells, suggesting relative immaturity of the regulatory apparatus. Furthermore, consistent with their role in rapid growth of the airway, fASM cells display robust cell proliferation. These properties of [Ca2⫹]i responsiveness and cell proliferation appear to be substantially modulated by oxygen in a bimodal fashion where, compared

Fig. 9. Mitochondrial fragmentation in fASM cells. A: fluorescence imaging of MitoTracker-labeled fASM cells in normoxia showed reticular mitochondrial patterns. With increasing oxygen concentrations, mitochondria took on a more punctate and aggregated appearance with short branching patterns such that at 80% oxygen, mostly aggregated mitochondria were observed. B: mitochondrial fusion protein (Mfn2) expression was significantly decreased at higher oxygen tensions, whereas the mitochondrial fission protein Drp1 was decreased. Values (means ⫾ SE) from n of 5 samples normalized to GAPDH in bar graph. *Significant difference from normoxia (P ⬍ 0.05).



tions in premature infants can lead to changes in developing ASM, which may have long-term consequences on the structure and function of the airway. In adults, diseases such as asthma are well known to be characterized by airway hyperresponsiveness and airway wall thickening, which involve increased ASM contractility and proliferation (12, 20, 27). Infants with chronic lung disease of prematurity also exhibit airway hyperresponsiveness, and postmortem examination shows airway wall thickening (26). Furthermore, airway development may be influenced by the altering levels of oxygen within the setting of the intensive care unit and environmental factors such as household cigarette smoke in survivors of the premature period. Finally, adult disease such as asthma may have roots in detrimental processes that occur in the newborn lung (38). Thus ASM structure and function in the developing lung have important implications for both neonatal and adult lung disease. However, studies to date have relied on animal models for examining changes in ASM of developing lung due to relative lack of availability of age-appropriate human tissues. Although these studies in small or large animal models (7, 9, 13, 19, 25, 29, 31) have provided much insight into potential mechanisms underlying neonatal lung disease, it is also recognized that species differences in lung development do exist (60) as do differences in mechanisms that regulate ASM structure and function (4, 28). Adult human ASM has been used as a surrogate (14, 48), but, considering the substantial need for rapid growth during development compared with the adult, it is unclear whether developing ASM behave differently in response to interventions such as altered oxygen concentration or inflammation. In the present study, we characterized a human fASM cell model system as a relatively age-appropriate tool toward understanding mechanisms that control ASM structure and function, at least in vitro. With the understanding that substantial structural and functional changes occur in the airway in the perinatal period, to examine mechanisms that affect the airway of premature infants and neonates, it would obviously be more appropriate to examine tissues from these specific age groups. However, such an avenue is not feasible due to the understandable lack of availability of tissues from early postnatal age groups. Nonetheless, based on the present study, human fASM cells may serve at least as a species-appropriate model, and perhaps age-appropriate as well for the airways in premature birth. Similar to adult ASM cells in vitro (18, 24, 36), fASM cells retain a fusiform appearance. We found that fASM cells express receptors for the most common bronchoconstricting agents ACh and histamine, as well as contractile proteins such as actin and myosin (but not markers of fibroblasts or epithelial cells; Ref. 50). Furthermore, fASM cells produce robust [Ca2⫹]i responses to these agonist receptors, albeit smaller and slower than adult ASM cells. In this regard, the relative maturity of the histaminergic vs. muscarinic signaling systems appear to be different. Furthermore, fASM cells show robust ability to proliferate and maintain their ASM characteristics despite multiple cell passages. In adult ASM, [Ca2⫹]i regulation in response to agonist stimulation involves several mechanisms including both IP3R and RyR SR Ca2⫹ release channels, SR Ca2⫹ reuptake via SERCA, and plasma membrane Ca2⫹ influx that occurs through voltage-gated, receptor-operated, and store-operated

L717

channels (20, 45). In the present study, we found substantial expression of proteins involved in both SR Ca2⫹ regulation (IP3R and SERCA, but not RyR) and lower expression of proteins that regulate store-operated Ca2⫹ entry (TRPC3, STIM1). An important caveat here would be that expression of proteins in whole cell lysates does not necessarily reflect function of these proteins, especially because many of the plasma membrane proteins may be dependent on caveolae or other mechanisms for membrane expression and function. However, we also found that both ACh and histamine produce robust [Ca2⫹]i responses that involve both plasma membrane Ca2⫹ influx (responses reduced by removal of extracellular Ca2⫹) as well as SR Ca2⫹ release/reuptake (persistent, albeit smaller, response even in the presence of zero extracellular Ca2⫹; response to caffeine). These data are among the first regarding [Ca2⫹]i regulation in developing human ASM. A single study in fASM cells of even earlier gestation than in our study reported spontaneous electrical and force oscillations that were enhanced by ACh but suppressed by removal of extracellular Ca2⫹ or inhibition of voltage-gated Ca2⫹ influx, suggesting a prominent role for influx (34). Whether SR Ca2⫹ regulation was also involved is not known. However, we propose that influx mechanisms likely play a more important role early in ASM development, whereas the SR is immature. With advancing gestation, SR regulation also makes a contribution that is even further developed through postnatal growth. Here, the relative expressions and function of proteins such as IP3R vs. RyR may be important, as would the second messenger cascades. These aspects of [Ca2⫹]i regulation in fASM remain to be examined. Nonetheless, the relevance of these developmental changes lies in how extrinsic factors such as hypoxia or hyperoxia may influence ASM function in terms of [Ca2⫹]i and non-Ca2⫹ effects of these regulatory proteins, topics for future studies. In adult ASM cells, we and others have recently demonstrated the importance of caveolae and caveolin-1 in [Ca2⫹]i regulation (41, 46). Caveolae are plasma membrane invaginations containing the constituent caveolin proteins as well as a number of [Ca2⫹]i and force regulatory proteins including agonist receptors and influx channels including TRPCs (41). There is currently no information on the expression of role of caveolae or caveolins in developing airway. Although a caveolin-1 knockout mouse exists (43) and develops normally to adulthood, it is currently unknown whether ASM structure or function is altered early in development. In the present study, we found substantial expression of caveolin-1 in fASM cells. Furthermore, chelation of membrane lipid rafts using CD substantially reduced baseline [Ca2⫹]i and blunted [Ca2⫹]i responses to agonist, suggesting a functional role for caveolae within fASM cells. Overall, these data demonstrate a relatively well-developed [Ca2⫹]i regulatory mechanism in fASM cells. Neural regulation of airway tone is an important aspect of airway reactivity throughout life (11). Neurally mediated bronchoconstriction involves nonadrenergic/noncholinergic pathways (10, 42) that are mediated in part via tachykinins (neurokinin A and B, and substance P). Substance P can elicit a direct bronchoconstrictive effect on ASM via NK receptors. Neurokinin signaling in the developing airway has been examined only to a limited extent. Substance P responsiveness has been shown in fetal pigs (53), whereas neurokinin A produces bronchoconstriction in human fetal lung tissue (14). The pres-


L718


ent study is the first to demonstrate the existence of NK receptors in fASM and [Ca2⫹]i responsiveness to SP, suggesting that SP can potentially enhance airway reactivity in the neonate. In neonatal rat, hyperoxia is associated with increased expression of the preprotachykinin gene, which encodes SP, as well SP itself (1), and functionally with increased cholinergically mediated airway contractile responses in vitro and in vivo (5, 19, 58). Whether neurokinin signaling is important in the setting of airway hyperreactivity with hyperoxia has not been examined in the human and is the focus of ongoing studies in our group. Overall, the results of our study demonstrate that robust [Ca2⫹]i regulatory mechanisms exist in human fASM that are comparable in some ways to adult ASM, but not in others. These Ca2⫹ data are generally consistent with the findings of Sparrow and Mitchell (52) showing lower contractility of the fetal pig trachea compared with adults. However, it is also important to note that no differences in bronchial responsiveness was observed. Because our fASM cells were derived from trachea, it remains to be determined whether human bronchial ASM differ in [Ca2⫹]i or contractility in early development. The present study found that fASM cells respond robustly to serum by proliferating. In this regard, a high potential for proliferation is to be expected from these cells, considering their importance in the growing lung. The relevance of fASM cells further lies in the effect of hyperoxia or other insults on fASM proliferation and thus airway thickening and remodeling. There is currently no information on this topic. Our data, however, by investigating a dose responsiveness of these cells to increasing levels of oxygen tension have shown that hyperoxia levels lower than 50 – 60% O2 favored increased fASM cell proliferation/apoptosis ratio phenotype characterized by limited cellular apoptosis, a predominance of proliferation/ antiapoptosis proteins compared with apoptosis proteins, and diminished mitochondrial fractionation compared with fASM cells grown in normoxia. As cells were exposed to higher oxygen levels, there was a phenotypic shift where cells favored an apoptosis phenotype demonstrated by significantly enhanced apoptosis, a predominance of proapoptosis proteins, and increased mitochondrial fission. In this regard, the proproliferative effect of hypoxia, comparable to moderate hyperoxia, is also interesting. The relevance of these novel findings lies in recognizing the potential detrimental effects of oxygen on developing airway (here ASM), beyond the commonly quoted issues of retinopathy of prematurity and bronchopulmonary dysplasia. Here, enhanced ASM proliferation may lead to airway thickening and increased airway stiffness (37) and contribute to increased work of breathing, especially in the setting of a compliant chest wall in the infant. On the one hand, hypoxia is clearly detrimental as a whole, and the limited data in our study suggest that hypoxia would only enhance airway remodeling. On the other hand, even moderate hyperoxia could be detrimental in enhancing proliferation, whereas hyperoxia ⬎60% with proapoptotic effects is unlikely to be beneficial in any way. Although not a focus of this study, such detrimental effects likely occur in other cell types including bronchial and alveolar epithelium. Overall, even though the extent of hyperoxia will be eventually determined by the need to maintain oxygen saturations, it may be important to consider effects on the developing airway in evaluating the risks vs. benefits, especially in the long term.

GRANTS This study was supported by the Flight Attendants Medical Research Institute (FAMRI, to C. Pabelick, V. Sathish), NIH R01 grants HL090595 (C. Pabelick) and HL056470 (Y. Prakash, R. Martin), and the Foundation of Anesthesia Education and Research (FAER, to W. Hartman). DISCLOSURES No conflicts of interest, financial or otherwise, are declared by the authors. AUTHOR CONTRIBUTIONS Author contributions: W.R.H., D.F.S., V.S., B.A., M.A.T., Y.A., H.C.P., R.J.M., Y.S.P., and C.M.P. conception and design of research; W.R.H., D.F.S., V.S., M.K., S.K., B.A., M.A.T., and C.M.P. performed experiments; W.R.H., D.F.S., M.K., S.K., B.A., M.A.T., H.C.P., Y.S.P., and C.M.P. analyzed data; W.R.H., D.F.S., V.S., B.A., M.A.T., Y.A., H.C.P., R.J.M., Y.S.P., and C.M.P. interpreted results of experiments; W.R.H., D.F.S., V.S., M.K., S.K., B.A., M.A.T., Y.S.P., and C.M.P. prepared figures; W.R.H., D.F.S., B.A., M.A.T., Y.A., H.C.P., R.J.M., Y.S.P., and C.M.P. drafted manuscript; W.R.H., D.F.S., V.S., B.A., M.A.T., Y.A., H.C.P., R.J.M., Y.S.P., and C.M.P. edited and revised manuscript; W.R.H., D.F.S., V.S., M.K., S.K., B.A., M.A.T., Y.A., H.C.P., R.J.M., Y.S.P., and C.M.P. approved final version of manuscript. REFERENCES 1. Agani FH, Kuo NT, Chang CH, Dreshaj IA, Farver CF, Krause JE, Ernsberger P, Haxhiu MA, Martin RJ. Effect of hyperoxia on substance P expression and airway reactivity in the developing lung. Am J Physiol Lung Cell Mol Physiol 273: L40 –L45, 1997. 2. Aravamudan B, Thompson M, Pabelick C, Prakash YS. Brain-derived neurotropic factor induces proliferation of human airway smooth muscle cells. J Cell Mol Med 16: 812–823, 2012. 3. Badri KR, Zhou Y, Schuger L. Embryological origin of airway smooth muscle. Proc Am Thorac Soc 5: 4 –10, 2008. 4. Bates JH, Rincon M, Irvin CG. Animal models of asthma. Am J Physiol Lung Cell Mol Physiol 297: L401–L410, 2009. 5. Belik J, Jankov RP, Pan J, Yi M, Chaudhry I, Tanswell AK. Chronic O2 exposure in the newborn rat results in decreased pulmonary arterial nitric oxide release and altered smooth muscle response to isoprostane. J Appl Physiol 96: 725–730, 2004. 6. Bertrand JM, Riley SP, Popkin J, Coates AL. The long-term pulmonary sequelae of prematurity: the role of familial airway hyperreactivity and the respiratory distress syndrome. N Engl J Med 312: 742–745, 1985. 7. Bland RD. Neonatal chronic lung disease in the post-surfactant era. Biol Neonate 88: 181–191, 2005. 8. Brightling CE, Bradding P, Symon FA, Holgate ST, Wardlaw AJ, Pavord ID. Mast-cell infiltration of airway smooth muscle in asthma. N Engl J Med 346: 1699 –1705, 2002. 9. Bry K, Hogmalm A, Backstrom E. Mechanisms of inflammatory lung injury in the neonate: lessons from a transgenic mouse model of bronchopulmonary dysplasia. Semin Perinatol 34: 211–221, 2010. 10. Canning BJ. Reflex regulation of airway smooth muscle tone. J Appl Physiol 101: 971–985, 2006. 11. Canning BJ, Fischer A. Neural regulation of airway smooth muscle tone. Respir Physiol 125: 113–127, 2001. 12. Dekkers BG, Maarsingh H, Meurs H, Gosens R. Airway structural components drive airway smooth muscle remodeling in asthma. Proc Am Thorac Soc 6: 683–692, 2009. 13. deLemos RA, Coalson JJ. The contribution of experimental models to our understanding of the pathogenesis and treatment of bronchopulmonary dysplasia. Clin Perinatol 19: 521–539, 1992. 14. Fayon M, Ben-Jebria A, Elleau C, Carles D, Demarquez JL, Savineau JP, Marthan R. Human airway smooth muscle responsiveness in neonatal lung specimens. Am J Physiol Lung Cell Mol Physiol 267: L180 –L186, 1994. 15. Fiascone JM, Rhodes TT, Grandgeorge SR, Knapp MA. Bronchopulmonary dysplasia: a review for the pediatrician. Curr Probl Pediatr 19: 169 –227, 1989. 16. Ford LE, Gilbert SH. The importance of maturational studies in airway smooth muscle. Am J Physiol Lung Cell Mol Physiol 289: L898 –L901, 2005. 17. Hajnoczky G, Csordas G, Yi M. Old players in a new role: mitochondria-associated membranes, VDAC, and ryanodine receptors as contribu-



18.

19.

20. 21. 22. 23. 24.

25. 26. 27.

28.

29. 30. 31.

32.

33.

34.

35.

36.

37.

38.

tors to calcium signal propagation from endoplasmic reticulum to the mitochondria. Cell Calcium 32: 363–377, 2002. Halayko AJ, Salari H, Ma X, Stephens NL. Markers of airway smooth muscle cell phenotype. Am J Physiol Lung Cell Mol Physiol 270: L1040 – L1051, 1996. Hershenson MB, Garland A, Kelleher MD, Zimmermann A, Hernandez C, Solway J. Hyperoxia-induced airway remodeling in immature rats. Correlation with airway responsiveness. Am Rev Respir Dis 146: 1294 – 1300, 1992. Hirota S, Helli P, Janssen LJ. Ionic mechanisms and Ca2⫹ handling in airway smooth muscle. Eur Respir J 30: 114 –133, 2007. Homer RJ, Elias JA. Airway Remodeling in Asthma: Therapeutic Implications of Mechanisms. Physiology (Bethesda) 20: 28 –35, 2005. Jesudason EC. Airway smooth muscle: an architect of the lung? Thorax 64: 541–545, 2009. Jobe AH, Bancalari E. Bronchopulmonary dysplasia. Am J Respir Crit Care Med 163: 1723–1729, 2001. Johnson PR, Roth M, Tamm M, Hughes M, Ge Q, King G, Burgess JK, Black JL. Airway smooth muscle cell proliferation is increased in asthma. Am J Respir Crit Care Med 164: 474 –477, 2001. Kallapur SG, Jobe AH. Contribution of inflammation to lung injury and development. Arch Dis Child Fetal Neonatal Ed 91: F132–F135, 2006. Kwinta P, Pietrzyk JJ. Preterm birth and respiratory disease in later life. Expert Rev Respir Med 4: 593–604, 2010. Mahn K, Ojo OO, Chadwick G, Aaronson PI, Ward JPT, Lee TH. Ca2⫹ homeostasis and structural and functional remodelling of airway smooth muscle in asthma. Thorax 65: 547–552, 2010. Martin JG, Ramos-Barbon D. Airway smooth muscle growth from the perspective of animal models. Respir Physiol Neurobiol 137: 251–261, 2003. Martin RJ, Walsh MC. Inhaled nitric oxide for preterm infants—who benefits? N Engl J Med 353: 82–84, 2005. Mauad T, Bel EH, Sterk PJ. Asthma therapy and airway remodeling. J Allergy Clin Immunol 120: 997–1009; quiz 1010 –1001, 2007. Meuchel LW, Stewart A, Smelter DF, Abcejo AJ, Thompson MA, Zaidi SIA, Martin RJ, Prakash YS. Neurokinin-neurotrophin interactions in airway smooth muscle. Am J Physiol Lung Cell Mol Physiol 301: L91–L98, 2011. Nassar A, Simpson AW. Elevation of mitochondrial calcium by ryanodine-sensitive calcium-induced calcium release. J Biol Chem 275: 23661– 23665, 2000. Northway WH Jr, Moss RB, Carlisle KB, Parker BR, Popp RL, Pitlick PT, Eichler I, Lamm RL, Brown BW Jr. Late pulmonary sequelae of bronchopulmonary dysplasia. N Engl J Med 323: 1793–1799, 1990. Pandya HC, Innes J, Hodge R, Bustani P, Silverman M, Kotecha S. Spontaneous contraction of pseudoglandular-stage human airspaces is associated with the presence of smooth muscle-alpha-actin and smooth muscle-specific myosin heavy chain in recently differentiated fetal human airway smooth muscle. Biol Neonate 89: 211–219, 2006. Pandya HC, Snetkov VA, Twort CHC, Ward JPT, Hirst SJ. Oxygen regulates mitogen-stimulated proliferation of fetal human airway smooth muscle cells. Am J Physiol Lung Cell Mol Physiol 283: L1220 –L1230, 2002. Panettieri RA, Murray RK, DePalo LR, Yadvish PA, Kotlikoff MI. A human airway smooth muscle cell line that retains physiological responsiveness. Am J Physiol Cell Physiol 256: C329 –C335, 1989. Panitch HB, Allen JL, Ryan JP, Wolfson MR, Shaffer TH. A comparison of preterm and adult airway smooth muscle mechanics. J Appl Physiol 66: 1760 –1765, 1989. Piippo-Savolainen E, Korppi M. Long-term outcomes of early childhood wheezing. Curr Opin Allergy Clin Immunol 9: 190 –196, 2009.

L719

39. Prakash YS, Sathish V, Thompson MA, Pabelick CM, Sieck GC. Asthma and sarcoplasmic reticulum Ca2⫹ reuptake in airway smooth muscle. Am J Physiol Lung Cell Mol Physiol 297: L794, 2009. 40. Prakash YS, Thompson MA, Pabelick CM. Brain-derived neurotrophic factor in TNF␣ modulation of Ca2⫹ in human airway smooth muscle. Am J Respir Cell Mol Biol 41: 603–611, 2009. 41. Prakash YS, Thompson MA, Vaa B, Matabdin I, Peterson TE, He T, Pabelick CM. Caveolins and intracellular calcium regulation in human airway smooth muscle. Am J Physiol Lung Cell Mol Physiol 293: L1118 – L1126, 2007. 42. Racke K, Matthiesen S. The airway cholinergic system: physiology and pharmacology. Pulm Pharmacol Ther 17: 181–198, 2004. 43. Razani B, Lisanti MP. Caveolin-deficient mice: insights into caveolar function human disease. J Clin Invest 108: 1553–1561, 2001. 44. Rizzuto R, Pozzan T. Microdomains of intracellular Ca2⫹: molecular determinants and functional consequences. Physiol Rev 86: 369 –408, 2006. 45. Sanderson MJ, Delmotte P, Bai Y, Perez-Zogbhi JF. Regulation of airway smooth muscle cell contractility by Ca2⫹ signaling and sensitivity. Proc Am Thorac Soc 5: 23–31, 2008. 46. Sathish V, Yang B, Meuchel LW, VanOosten SK, Ryu AJ, Thompson MA, Prakash YS, Pabelick CM. Caveolin-1 and force regulation in porcine airway smooth muscle. Am J Physiol Lung Cell Mol Physiol 300: L920 –L929, 2011. 47. Schreiber MD, Gin-Mestan K, Marks JD, Huo D, Lee G, Srisuparp P. Inhaled nitric oxide in premature infants with the respiratory distress syndrome. N Engl J Med 349: 2099 –2107, 2003. 48. Shenberger JS, Dixon PS. Oxygen induces S-phase growth arrest and increases p53 and p21WAF1/CIP1 expression in human bronchial smoothmuscle cells. Am J Respir Cell Mol Biol 21: 395–402, 1999. 49. Shkryl VM, Shirokova N. Transfer and tunneling of Ca2⫹ from sarcoplasmic reticulum to mitochondria in skeletal muscle. J Biol Chem 281: 1547–1554, 2006. 50. Singh SR, Billington CK, Sayers I, Hall IP. Can lineage-specific markers be identified to characterize mesenchyme-derived cell populations in the human airways? Am J Physiol Lung Cell Mol Physiol 299: L169 – L183, 2010. 51. Smith PG, Moreno R, Ikebe M. Strain increases airway smooth muscle contractile and cytoskeletal proteins in vitro. Am J Physiol Lung Cell Mol Physiol 272: L20 –L27, 1997. 52. Sparrow MP, Mitchell HW. Contraction of smooth muscle of pig airway tissues from before birth to maturity. J Appl Physiol 68: 468 –477, 1990. 53. Sparrow MP, Warwick SP, Mitchell HW. Foetal airway motor tone in prenatal lung development of the pig. Eur Respir J 7: 1416 –1424, 1994. 54. Sward-Comunelli SL, Mabry SM, Truog WE, Thibeault DW. Airway muscle in preterm infants: Changes during development. J Pediatr 130: 570 –576, 1997. 55. Szalai G, Csordas G, Hantash BM, Thomas AP, Hajnoczky G. Calcium signal transmission between ryanodine receptors and mitochondria. J Biol Chem 275: 15305–15313, 2000. 56. Townsend EA, Thompson MA, Pabelick CM, Prakash YS. Rapid effects of estrogen on intracellular Ca2⫹ regulation in human airway smooth muscle. Am J Physiol Lung Cell Mol Physiol 298: L521–L530, 2010. 57. Wang X, Su B, Lee HG, Li X, Perry G, Smith MA, Zhu X. Impaired balance of mitochondrial fission and fusion in Alzheimer’s disease. J Neurosci 29: 9090 –9103, 2009. 58. Yao Q, Zaidi SI, Haxhiu MA, Martin RJ. Neonatal lung and airway injury: a role for neurotrophins. Semin Perinatol 30: 156 –162, 2006. 59. Youle RJ, Karbowski M. Mitochondrial fission in apoptosis. Nat Rev Mol Cell Biol 6: 657–663, 2005. 60. Zoetis T, Hurtt ME. Species comparison of lung development. Birth Defects Res B Dev Reprod Toxicol 68: 121–124, 2003.
