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Published ahead of print on December 2, 2004, doi:10.1165/rcmb.2004-0304OC
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American Journal of Respiratory Cell and Molecular Biology. Vol. 32, pp. 118-127, 2005
© 2005 American Thoracic Society
DOI: 10.1165/rcmb.2004-0304OC

Developing Rat Lung Has a Sided Pacemaker Region for Morphogenesis-Related Airway Peristalsis

Edwin C. Jesudason*, Nicola P. Smith*, Marilyn G. Connell, Dave G. Spiller, Mike R. H. White, Dave G. Fernig and Paul D. Losty

Department of Child Health, The Centre for Cell Imaging, and The Molecular Medicine Group, University of Liverpool, Liverpool, United Kingdom

Correspondence and requests for reprints should be addressed to Edwin C. Jesudason, M.A., M.D., National Clinician Scientist in Paediatric Surgery, Institute of Child Health, Alder Hey Children's Hospital, Eaton Road, Liverpool L12 2AP, UK. E-mail: e.jesudason{at}liv.ac.uk


   Abstract
Top
 Abstract
Introduction
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
Prenatal airways from diverse species are capable of spontaneous peristaltic contractions in each trimester. The function of this smooth muscle activity is unknown. We demonstrate that peristalsis of the embryonic airway originates from a sided pacemaker focus, is stimulated in a calcium-dependent fashion by the pulmonary morphogen fibroblast growth factor-10 (FGF-10), and appears coupled to lung growth. Airway peristalsis may be crucial for lung development (thereby providing a physiologic role for airway smooth muscle) and play a hitherto unanticipated role in reported transgenic mutant lung phenotypes.

Key Words: airway peristalsis • lung branching morphogenesis • pacemaker


   Introduction
Top
 Abstract
 Introduction
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
Airway smooth muscle (ASM) has been described as "the appendix of the lung," implying that it is an evolutionary remnant with no other role in humans than in causation of disease (bronchial hyperresponsiveness) (1). An opposing view is that ASM plays a role in normal mammalian lung development via the phenomenon of airway peristalsis (AP) (2). Resolving these conflicting theories is therefore of potential clinical importance to common diseases associated not only with bronchial hyperreactivity (e.g., asthma, brochiolitis-associated wheeze) but also with inadequate lung development (e.g., congenital diaphragmatic hernia [CDH], lung disease of prematurity) (3).

Spontaneous phasic contractions of isolated prenatal airways and cultured lungs were noted in chick and guinea pig over 75 yr ago (4–6). These contractions produce periodic intraluminal fluid flux in first trimester cultured human lung and distend growing terminal buds of cultured fetal rat lung (2, 7). Using culture and tissue bath, Schittny and coworkers recognized the peristaltic nature of prenatal airway contractility in a variety of mammals (2). Contraction progresses in a smooth wave passing sequentially through contiguous prenatal airway, propelling lung liquid before it and then terminating in, and transiently distending, the fluid-filled buds of the developing lung. This is followed by a similar wave of relaxation before a further peristaltic wave. Nevertheless, what evidence links the physical contractility of AP with regulation of prenatal lung growth?

Mechanical factors are known to regulate diverse aspects of morphogenesis including pre- and postnatal lung development. Fetal breathing movements periodically stretch the lung: their abolition by phrenic nerve ablation or agenesis impairs pulmonary growth (8). Similarly, abolition of diaphragmatic muscularization by deletion of myogenic regulatory factors (MRFs) leads to lung hypoplasia (9). Fetuses with laryngeal atresia or surgical occlusion of the trachea retain lung liquid resulting in stretch-induced pulmonary overgrowth (10). Drainage of this lung liquid by fetal tracheostomy inhibits lung development (11). Postnatally, mechanical ventilation of preterm infants contributes to bronchopulmonary dysplasia in childhood (12). At a cellular level, stretch of fetal lung cells induces signal transduction that may modulate morphogenesis (13). Airway myogenesis in fetal lung explants is also sensitive to stretch (14).

While a range of mechanical factors impact on lung development, is there a specific role for ASM? MRF knockouts abrogate skeletal rather than smooth muscle development (15). Therefore, at present the potential importance of ASM to lung development can only be pieced together from phenomenologic evidence. First, lung hypoplasia in human CDH is associated with increased expression of serum response factor (SRF) isoform, SRF{Delta}5. Overexpression of SRF{Delta}5 impairs normal SRF-mediated stimulation of ASM-specific gene expression (14). Second, lung hypoplasia in teratogen-induced experimental CDH is associated with ASM dysfunction (16). Third, children with asthma with abnormal ASM contractility exhibit airway remodeling comprising not only ASM hypertrophy and hyperplasia but also epithelial and subepithelial changes (17). Fourth, ASM-dependent peristalsis causes airway caliber fluctuations and fluid flux that would be expected to impact on lung growth and cell differentiation via phasic luminal pressure changes (2). Fifth, abolition of AP by L-type calcium channel blockade impairs murine lung growth in vitro (18). Despite this evidence, numerous issues surrounding AP and its relation to lung growth remain unresolved.

First, it remains unclear whether AP resembles normal gut peristalsis in (1) exhibiting a proximo-distal hierarchy of pacemaker areas distributed through the gastrointestinal tract and (2) requiring c-kit–positive interstitial cells of Cajal (ICC) (19). Second, if AP does regulate prenatal lung growth, is it possible to demonstrate this coupling by stimulation/inhibition of one leading to parallel changes in the other? For example, does growth factor stimulation of lung growth increase frequency of AP? Alternatively, does stimulation of AP increase lung growth?

We have used organ culture to show that embryonic rat lung has an autonomous dominant c-kit–negative pacemaker region for AP and that manipulation of either in vitro lung growth or peristalsis frequency is coupled to parallel changes in the other.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 References
 
Manipulation of Lung Growth and Peristalsis in Organ Culture
Lung primordia from Sprague-Dawley rat embryos (Charles River, Margate, UK) were harvested on Day 13.5 of gestation (vaginal plug = Day 0, term = Day 22.5) and cultured on translucent membranes (Millicell; Millipore Corp, Bedford, MA) for 78 h at 37°C in 5% CO2 as previously described (3). Lungs were cultured at the air–medium interface, bathed in serum-free culture medium (DMEM/F12 [Gibco; Invitrogen Life Technologies, Paisley, UK]), with penicillin 100 IU/ml and streptomycin 100 µg/ml (Gibco) ± one of the following (or combination of two as explained in the results): 5% fetal calf serum, 50 ng/ml fibroblast growth factor (FGF)-10, 0.1 µM nicotine (cholinergic smooth muscle stimulant), 5 µM nifedipine (L-type calcium channel blocker) in dimethylsulfoxide (DMSO) or 20 µM UO126 (noncompetitive MEK1/2 inhibitor). Doses were derived from the cited literature (see RESULTS). All culture media were renewed at 48 h. Bioactivity of FGF-10 was also assessed independently of the lung culture by fibroblast thymidine incorporation assay.

Localization of Pacemaker Activity
Lung primordia were either cultured intact or were divided after dissection into three components (right, left, and tracheal segments) before culture. Frequency of airway contractions was recorded over 10-min periods at 30, 54, and 78 h, with the contraction waves classified according to their site of origin as R- (right), L- (left), or T-waves (tracheal). Video recordings allowed determination of the wave origin.

Lung Morphometry
Lung morphometry was performed as previously described by our laboratory (3). Briefly, lungs were photographed at 30, 54, and 78 h in culture using a Zeiss microscope (Zeiss Axiovert S100+V; Jena, Germany) and Hamamatsu digital camera (EB-CCD; Hamamatsu City, Japan). Images were processed using AQMOrcall (Kinetic Imaging, Liverpool, UK) software to trace specimen outlines and calculate area and perimeter measurements. Terminal lung bud counts were taken at 30, 54, and 78 h in culture.

Immunohistochemistry
Cultured lungs were preserved after 30, 54, and 78 h in 4% paraformaldehyde (0.1 M phosphate-buffered saline [PBS], pH 7.4), rinsed in PBS, cryoprotected with 20% sucrose, gelatine-embedded (7.5% gelatine, 15% sucrose in PBS) before being covered in Cryo-M-Bed (Bright, Huntingdon, UK) and snap-frozen at –40°C. Lung sections were taken at 7 µm and mounted on chrome alum gel slides for storage at –40°C.

Slides were washed in 0.5% hydrogen peroxidase in 60% methanol to quench endogenous peroxidase and rinsed in distilled water. They were then incubated with {alpha}-smooth muscle actin antibody (monoclonal anti–{alpha}-SMA Clone No. 1A4, mouse; Sigma, Gillingham, UK) at 1:500 dilution with 0.1% BSA in PBS overnight at 4°C. Slides were rinsed in PBS. Primary antibody was labeled with Strept ABComplex Duet Kit (K0492; Dako, Ely, UK) as per protocol and visualized with diaminobenzidine (D-0426; Sigma) as per kit protocol. Slides were then lightly counterstained with hematoxylin before being dehydrated, cleared, and mounted.

Similarly, c-kit was labeled with polyclonal rabbit IgG (Calbiochem, San Diego, CA) at 1:50 dilution. Adult rat gut was used as positive control. Proliferation was assessed using anti–proliferating cell nuclear antigen, PCNA (clone PC10, anti-mouse; Dako) at 1:500 dilution. Again the antibodies were labeled with Strept ABComplex Duet Kit (Dako K0492) and visualized with FITC (Sigma) for c-kit or diaminobenzidine (D-0426; Sigma) for PCNA. Negative controls were performed in the absence of the relevant primary antibody.

Western Blotting
Cultured lungs were snap-frozen at the end of the period of culture. Specimens from each treatment group were prepared for Western blotting by resuspension in Tris-HCl buffer (40 mM Tris/pH 7.5), before being homogenized. Specimens were then centrifuged (10 min at 400 x g at 4°C) to remove cellular debris. The supernatant was then respun (30 min at 19,000 x g at 4°C). The pellet was resuspended in equal volumes of sample buffer. Sample loading was normalized to protein content, determined by the BCA assay (Sigma) to ensure equal quantities of protein in each lane. Western blotting was performed with 15% SDS-gel, anti–{alpha}-SMA antibody was used (monoclonal anti {alpha}-SMA Clone No. 1A4, mouse; Sigma), bands were visualized with chemiluminescence (RPN2105; Amersham Biosciences, Amersham, UK).

Statistical Analysis
Proportions of lungs contracting, proportions of R, L, and T waves, and proportions of proliferating cells were analyzed using the chi-squared test. Morphometry data (area, perimeter, and terminal bud-counts) were non-normally distributed and were analyzed by the Mann-Whitney U test for nonparametric data. Intercontraction intervals approximated to the normal distribution and Student's t test was used to compare means. Significance was taken as P < 0.01 throughout. We used SPSS statistics package version 11.0 for Windows (SPSS Inc., Chicago, IL)

Analysis of variability in the intercontraction intervals (as is used for heart rate variability) was deemed inappropriate as the total observation period was of not dissimilar order of magnitude to the intercontraction intervals.


   RESULTS
Top
 Abstract
 Introduction
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 References
 
Airway Peristalsis Emerges in Embryogenesis in this Culture System
At 30 h in vitro 37% (15 of 41) of embryonic lungs harvested at Day 13.5 of gestation and cultured in serum-free medium were contracting. However, at 54 and 78 h in vitro, 100% of cultured lungs were contracting (41 of 41). The contractility exhibited by the cultured lungs in this system was true peristalsis (see video in the online supplement) comprising phasic contractions (frequency ~ 1/min) that propagated in a smooth wave through contiguous airway with consequent propulsion of lung liquid (Figure 1) and followed by a wave of generalized relaxation.



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  		Figure 1. Airway peristalsis causes periodic fluid flux in cultured embryonic lung. Fluid flux within the epithelial lumen (E) of cultured rudiments can be visualized due to the periodic movement of luminal cellular debris (D). Sequential photomicrographs show terminal buds of a single unsectioned lung rudiment after 78 h in culture. These pictures were taken less than 90 s apart at an original x32 magnification. The wavefront of cellular debris (shown between the dark arrows) advances proximally along the epithelial lumen. These pictures depict four instants from a cyclic process featuring rhythmic ebb and flow of luminal debris within the living culture specimen.

 
The Origin of Peristaltic Waves Is Asymmetric and c-kit–Independent
A discrete area at the second bronchial division of the right lung was the site of origin of > 80% of peristaltic waves throughout the 78 h culture of whole lung primordia (n = 41) (Figure 2). Moreover, every harvested lung primordium displayed this right-sided preponderance. Suspecting a true sidedness to the origin of peristaltic waves, we cultured lung primordia after division into right lung, left lung, and trachea (n = 14). Frequency of airway peristalsis waves was again recorded over 10 min at 30, 54, and 78 h. Each wave was classified according to its origin as right (R-wave), left (L-wave), or tracheal (T-wave). Whole and divided specimens were compared for frequency and origin of peristaltic waves. The frequency of R-waves remained similar in whole and divided lung cultures, indicating a right-sided pacemaker focus with autonomy from the remainder of the lung primordia. In contrast, L-wave frequency increased 4-fold in isolated left lung culture (but remained lower than R-wave frequency). This suggests that the right lung pacemaker normally dominates the left lung. Low basal T-wave frequency declined over the culture period in both whole and divided explants. Thus, in contrast to the left lung, the trachea exhibits no "escape" rhythm (Figure 3). The right lung pacemaker focus coincided with the densest initial staining for {alpha}-SMA (Figure 4). However, it was clear that the right-sided pacemaker continued to dominate the rest of the developing lung despite later elaboration of SMA throughout the major airways. ICC are intestinal pacemaker cells and c-kit–positive (19). However, no such pacemaker cells were demonstrated by c-kit immunoreactivity in embryonic lung (data not shown). Mature rat gut was used as a positive control. Having examined the origins of AP, we then studied its putative purpose and relationship to in vitro lung growth. To do this we systematically augmented or inhibited either lung growth or peristalsis and examined the effect upon the other. In particular, we looked for evidence that the two phenomena can be dissociated.



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  		Figure 2. Airway peristalsis originates from a pacemaker region in the embryonic right lung. Series of photomicrographs from a cycle of airway peristalsis. View images clockwise from top left (panels 1–4). Epithelium (E) in the dashed box is constricted in panels 2 and 3 before relaxation in panel 4. t, trachea; b, example of end-bud; R, right side; L, left side.

 


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  		Figure 3. Subdividing the lung primordia increases L-wave frequency without changing R-wave activity. Graphs show the mean rate of peristaltic waves per minute originating in the right lung (R-waves), left lung (L-waves), and trachea (T-waves) for intact (blue) or divided (red) lung primordia at 30 h (top), 54 h (middle), and 78 h (bottom) in culture. Error bars represent the standard deviation.

 


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  		Figure 4. Early airway smooth muscle expression coincides with the pacemaker area. Photomicrographs of a E13.5 lung rudiment sectioned at 7 µm progressing from posterior (panel i) to anterior (panel iv) through the specimen. Every third section is shown to give an overview of the specimen. Sections have been stained for {alpha}-SMA (solid black staining). Smooth muscle staining is focused on the right side (R) around the first major airway bifurcation in the developing right lung (n = 6).

 
Serum Stimulation of Lung Growth Is Associated with Increased Peristalsis Frequency
Serum stimulation acts via wide variety of cellular targets. Foremost among these is serum response factor. SRF plays a key role in smooth muscle gene expression and is notably defective in human lung hypoplasia (14). We reasoned that serum stimulation would be a way to promote in vitro lung growth with particular emphasis on developing ASM within the mesenchyme. Addition of 5% fetal calf serum to lung primordia (n = 16) significantly increased luminal lung area and perimeter at 78 h (measured by image analysis software from photomicrographs) and terminal bud counts at 54 and 78 h (Figures 5 and 6) compared with serum-free controls (n = 41). These morphologic changes were accompanied by increased peristalsis frequency at 30, 54, and 78 h in vitro (Figure 7). SMA expression in serum-stimulated lung cultures did not differ significantly from untreated controls (Figure 8).



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  		Figure 5. Morphometric data for cultured lung primordia. Specimen areas (mm2, top row), perimeter (mm, middle row), and total bud counts (bottom row) at 30 h (left column), 54 h (middle column), and 78 h (right column) in vitro. Bars represent median and inter-quartile range. Additions to the culture medium are represented by the color coding of the bars (given at the right of the figure). *Significant increase compared with serum-free controls; **significant decrease compared with serum-free controls (P < 0.01). #No significant difference compared with nifedipine alone.

 


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  		Figure 6. Morphologic development of cultured lung primordia. Photomicrographs of lung primordia taken at 30 h (left column), 54 h (center column), and 78 h (right column) in vitro. Top row are cultured in standard serum-free medium, subsequent rows had the following additions: second row, serum; third row, FGF-10; fourth row, nicotine; fifth row, nifedipine; sixth row, nifedipine + FGF-10; seventh row, U0126. Scale bar: 0.5 mm.

 


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  		Figure 7. Serum, FGF-10, and nicotine all increase peristalsis frequency. Intercontraction intervals at 30, 54, and 78 h in vitro; bars represent mean intercontraction interval and 99% confidence intervals. *Significant decrease compared with serum-free controls, **Significant increase (P < 0.01).

 


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  		Figure 8. {alpha}-SMA expression is not changed by manipulating growth or peristalsis. Western blot for {alpha}-SMA from embryonic lungs (n = 6 per group) cultured in: 1, serum-free medium; 2, FGF-10; 3, nifedipine; 4, nicotine; 5, U0126. Molecular weights of protein standards (kD) are given on the left.

 
FGF-10 Stimulation of Lung Growth Is Associated with Increased Peristalsis Frequency
We then targeted the developing lung epithelium with the classical morphogen FGF-10 to stimulate lung growth and assess the impact on AP frequency. The interaction of the classical morphogen FGF-10 and its epithelial receptor FGFR2IIIb is essential to mammalian lung development (20). FGF-10 is known to enhance normal lung growth in vitro (21). Culture of lung primordia with 50 ng/ml FGF-10 (n = 11) produced significant increases in luminal lung area at 54 and 78 h, whereas perimeter and bud count were not significantly changed (Figures 5 and 6). Similar early dilatation (at the expense of branching) has been described by others and may depend on culture system in use (21, 22). FGF-10 significantly increased cell proliferation (PCNA immunopositivity) in both epithelium and mesenchyme (Figure 9). In addition, peristaltic frequency was significantly augmented throughout the 78-h culture period (Figure 7). Nevertheless, there was no increase in SMA expression on Western blots from FGF-10–treated lungs compared with untreated lung (Figure 8).



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  		Figure 9. Lung cell proliferation is increased by FGF-10 and nicotine. The graph shows mean (± SE) proportions of PCNA positive cells in epithelium, mesenchyme, and overall. Table shows mean ± SE by tissue compartment and culture medium used. Cell counts were performed on >= 18 fields from >= 6 lung primordia for each group. Both FGF-10 and nicotine significantly increase % PCNA positive cells in each tissue area compared with serum-free specimens (* P < 0.05 by Chi-squared test).

 
Nicotine Stimulation of Peristalsis Is Associated with Increased Lung Growth
We then studied whether increasing AP would impact on lung growth. Cholinergic agonists increase the peristalsis frequency of porcine airway in tissue bath (23). Nicotine (0.1 µM) (n = 11) significantly increased peristalsis frequency in lung primordia after 30 and 54 h in culture (Figure 7). Enhancement of AP was accompanied by significantly increased luminal lung area at 54 and 78 h but no alteration of perimeter or bud count (Figures 5 and 6). Cell proliferation (PCNA immunopositivity) was increased in epithelium and mesenchyme (Figure 9). Western blotting again revealed no increase in SMA expression when lungs were nicotine treated (Figure 8).

Blockade of Airway Peristalsis Impairs Lung Growth and Abolishes FGF-10 Growth Stimulation
Roman demonstrated that addition of nifedipine to cultured murine lung primordia abolishes airway peristalsis and impairs in vitro lung growth (18). Addition of 5 µM nifedipine in DMSO to our cultures (n = 11) completely abolished AP during the 78-h culture and significantly reduced lung luminal area at 30 and 54 h and perimeter at all time points. Total bud count was not significantly altered (Figures 5 and 6). Lungs cultured in DMSO alone (n = 10) showed no morphologic or peristaltic differences to control lungs with serum-free medium only (n = 41) (data not shown).

We tried then to dissociate lung growth and AP by stimulating the former with 50 ng/ml FGF-10 while simultaneously inhibiting the latter with 5 µM nifedipine in DMSO (n = 14). Compared with untreated lungs (n = 41), luminal area was significantly reduced at 30 and 54 h, lung perimeter was significantly decreased at all time points, and bud counts were not significantly different (Figures 5 and 6). Luminal area and perimeter were significantly reduced compared with lungs cultured with FGF-10 alone (Figures 5 and 6). There were no significant morphologic differences between nifedipine treated lungs with and without FGF-10 (Figures 5 and 6).

Inhibition of Lung Growth Reduces the Prevalence and Frequency of Airway Peristalsis
Kling and coworkers showed that UO126 (a MEK1/2 inhibitor) impairs in vitro lung growth, increases mesenchymal apoptosis, and decreases epithelial proliferation (24). We hypothesized that this growth inhibition is accompanied by reductions in AP. We showed that 20 µM UO126 significantly reduced luminal area, perimeter, and bud count of cultured lung at all time points (n = 16) (Figures 5 and 6). This growth inhibition was accompanied by significant reductions in the proportion of lungs contracting at 30, 54, and 78 h (19% versus 37%, 56% versus 100%, and 69% versus 100%, respectively; all P < 0.05 Chi-squared test). In the reduced number of lungs peristalsing there was a significant lengthening of intercontraction interval at 54 h (with similar trends at 30 and 78 h) compared with lung cultured in the absence of UO126 (Figure 7).

Addition of 0.1 µM nicotine to lungs cultured with UO126 (n = 12) did not alter the proportion of peristalsing lungs, but did increase AP frequency at 54 h (with similar trends at 30 and 78 h) compared with lungs cultured with UO126 alone (data not shown). Luminal lung area was only significantly increased at 30 h in lungs cultured with UO126 and nicotine compared with lungs with UO126 alone (data not shown). Overall, addition of nicotine to UO126-treated lungs failed to restore normal levels of in vitro lung growth.


   DISCUSSION
Top
 Abstract
 Introduction
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
References
 
Postnatal function of ASM is subject to intensive investigation and therapeutic modulation due to its role in the worldwide morbidity and mortality of reactive airways disease (e.g., asthma). By comparison, prenatal ASM function has been little explored. We have shown that prenatal lung has a sided, dominant, autonomous, c-kit–independent pacemaker region for airway peristalsis. The focal pacemaker emerges in spatiotemporal parallel with SMA expression and persists as SMA is elaborated throughout the main airways. Manipulation of either AP or lung growth could have shown that (1) changing one does not affect the other, (2) changing one yields opposing alterations in the other, or (3) modulating one produces parallel changes in the other. We have demonstrated the latter. Furthermore, we have shown that attempts to uncouple the two phenomena have been thus far unsuccessful. Together, this supports the concept that myogenic pacemaker-driven AP has a function in lung development.

The Origin of Airway Peristalsis and the Pulmonary Pacemaker
We demonstrated that AP commences in embryonic lung development in tandem with the earliest elaboration of SMA. In their study of cultured murine lungs Schittny and colleagues made the qualitative comment that peristaltic waves could originate from the proximal part of isolated lung lobes and from the trachea in cultured whole lung primordia (2). We have specifically measured the site of origin of embryonic AP waves in a substantially larger quantitative study. Peristaltic activity originates predominately from a specific region of the proximal right lung. Several contractions originating in the trachea do occur, but their incidence decreases with time in vitro and they are far less common than AP waves starting in right and left lung, respectively. Moreover, isolation of the trachea from the right lung pacemaker does not increase T-wave frequency (indicating that the trachea is quiescent relative to right and left lungs). Ablation of the right-sided pacemaker area permits faster "escape" rhythms to emerge from the developing left lung. This accelerated left lung activity not only persists, but appears to increase with time in isolated culture. In contrast, isolation of the right lung leaves the rate of R-waves unaffected. Together this indicates that the right lung pacemaker dominates other areas of the developing lung and is autonomous of them. This arrangement resembles the hierarchy of pacemakers in that other endodermal derivative, the gut (19). However, airway and gastrointestinal tract differ in that AP is independent of c-kit–positive ICC. Our demonstration of absent c-kit expression in peristalsing airway is consistent with the absence of an overt lung phenotype in c-kit null murine mutants (19). Instead, AP appears to be myogenic in origin and most likely to depend upon gap-junction–mediated transmission (2). The site of the pacemaker focus in the right lung coincides with the earliest and strongest expression of SMA. Nevertheless, the dominance of the right-sided pacemaker persists even when SMA expression has spread throughout the primitive airway at later stages in vitro. This implies that although SMA may play a time-specific role in initiation of pacemaker activity, the continued dominance of the right-sided pacemaker is likely to depend on other yet to be determined biochemical and/or physiologic factors. For example, anatomical left-right asymmetry of the developing lung is already established by the time AP commences. Therefore, sidedness of the physiologic pacemaker is likely to be downstream of the genetic determinants of anatomical left-right asymmetry.

Airway Peristalsis Appears Linked to Embryonic Lung Growth In Vitro
Having demonstrated a sided c-kit–independent pacemaker for AP, we examined links between peristalsis and lung growth. Lung morphogenesis in vitro is critically dependent on the specifics of the culture and is recognized to vary between early luminal dilatation and early branching responses (22). This variation is apparent, for example, in the published reports of FGF-10 stimulation of lung growth in culture (21). To assay in vitro lung growth, we were therefore careful to measure not only terminal bud counts but also luminal lung area and perimeter (from photomicrograph tracings) and, where relevant, pulmonary cell proliferation. Consistent with this, we have avoided linking peristalsis with any one specific aspect of morphologic lung development (e.g., overall size or branching). Given these caveats, we explored the relationship between airway peristalsis and lung growth by systematically manipulating one to examine the effects on the other.

Serum successfully stimulated lung branching morphogenesis in association with significant rise in AP frequency. Given the particular importance of SRF to smooth muscle development, the combined and concordant impact of serum on lung growth and peristalsis appears biologically significant.

Targeting the lung epithelium instead (with FGF-10), we again stimulated lung growth to assess the impact on AP frequency. We demonstrated that specific FGF-10 growth enhancement is accompanied by a significant increase in peristaltic activity in lung primordia. Enhanced lung growth was observed as increased epithelial area and increased pulmonary cell proliferation rather than branching morphogenesis. This is consistent with previous studies in which FGF-10 induced airway dilatation followed only later by branching (21). Moreover, as mentioned, whether the lung responds with dilatation or branching appears to be exquisitely sensitive to culture conditions (22).

FGF-10 is elaborated by lung mesenchyme and acts upon epithelial FGFR2IIIb (21). However, AP is generated by mesenchyme-derived ASM. Therefore, in enhancing embryonic airway peristalsis, exogenous FGF-10 appears to act either in a direct unreported manner on developing ASM or indirectly via an FGFR2IIIb-mediated epithelial response. A direct FGF-10 action on developing ASM is not supported by our observation that SMA expression was unaltered in FGF-10–treated lungs. Supporting an indirect action, FGF-10 increased cell proliferation in both lung epithelium and mesenchyme. Like lung growth itself, airway peristalsis may therefore be regulated by cardinal epithelial–mesenchymal interactions. However, endogenous FGF-10 is elaborated predominantly in the distal mesenchyme and acts to induce distal epithelial branching. With time, proximal epithelium becomes unresponsive to this branching morphogen. How then might distally expressed FGF-10 interact with a proximal mesodermally derived pacemaker? Once again, such an interaction would appear to be necessarily indirect. One possibility is that distal FGF-10 alters the distal propagation speed of the calcium transients and hence peristaltic wave velocity in the distal portions of the developing lung. This then might allow the proximal pacemaker to "fire" again sooner than if distal conduction (and hence refractory period) had been prolonged.

We used nicotine to increase AP frequency. Nicotine also increased lung luminal area and epithelial cell proliferation, but not branching morphogenesis, in our culture system. Previously it has been shown that in a defined culture system nicotine increases murine lung branching morphogenesis (25). However a recent study has indicated that nicotine impairs lung growth in vivo while not altering lung branching morphogenesis in vitro (26). The variation in these findings may once more be dependent on the specific culture system in use. Furthermore, in vivo studies indicate that nicotine's effects are highly dose dependent (26). Finally, it is possible that nicotine has different effects on early and late gestation lung. Wuenschell and coworkers have now confirmed that nicotine not only modulates in vitro lung growth but also alters expression of specific genes (27). Together with our findings, this indicates that nicotine does indeed alter in vitro lung morphogenesis.

Serum, FGF-10, and nicotine all raised the frequency of AP waves. Increased smooth muscle activity often results in smooth muscle hypertrophy and/or hyperplasia (as is apparent in reactive airways disease). Therefore, it is reassuring that augmented peristalsis (due to serum, FGF-10, or nicotine) was not associated with increased SMA expression. Increased peristalsis (and lung growth) may therefore be achievable without pathologic airway hypermuscularization.

We completely inhibited AP using L-type calcium channel blockade and, like Roman, we showed this impaired lung growth (18). We then tried to dissociate peristalsis and lung growth by inhibiting the former with nifedipine while augmenting the latter with FGF-10. However, nifedipine abolished both AP and FGF-10 stimulation of lung growth. Nifedipine's action on FGF-10–stimulated lung growth is at least in part a direct effect on FGFR2IIIb-expressing lung endoderm. Nifedipine blocks L-type calcium channels and influx of extracellular calcium. Extracellular calcium regulates proliferation of human bronchial epithelial cells in mesenchyme-free static culture (28). Moreover, extracellular calcium modulates the cardinal FGF-10–FGFR2IIIb signaling pathway in isolated epithelial cells (29, 30). However, proliferation and biochemical signaling in lung epithelial cells is also modulated by stretch (13). These stretch-induced responses are regulated by mesenchyme and FGF-10, and not blocked by nifedipine acting directly on isolated lung epithelium (31, 32). Therefore, it remains plausible that nifedipine also indirectly antagonizes FGF-10 growth stimulation via abolition of peristalsis-mediated epithelial stretch.

Kling and colleagues demonstrated that MEK1/2 inhibitor, UO126, impairs lung growth and epithelial proliferation and increases mesenchymal apoptosis in murine lung culture (24). We showed that UO126 inhibition of lung growth was associated with decreased prevalence and frequency of airway peristalsis. We again tried to dissociate lung growth and peristalsis by inhibiting the former with UO126 and augmenting the latter with nicotine. Nicotine treatment of UO126 cultured lungs modestly improved peristalsis frequency and (again in parallel) lung growth.

Each manipulation of either AP or lung growth therefore yields a similar response in the other. We were unable to stimulate one while inhibiting the other. Persistent congruence between lung growth and airway peristalsis supports their coupling during normal lung development.

Scientific and Clinical Implications of AP Regulating Lung Growth
Coupling of AP and lung growth has consequences for developmental lung biology and respiratory disease. First, lung dysmorphogenesis observed in several transgenic knockouts may result not simply from absent morphogen gradients but also disrupted AP (33). Second, maternal smoking increases the risk of sudden infant death syndrome (SIDS) (34). Nicotine-induced impairment of central responses to hypoxia and apnea have been proposed as a potential cause (35). An alternative explanation is that altered central nervous system control of respiratory function is secondary to nicotine-induced modulation of airway peristalsis and hence lung compliance and development. Indeed, prenatal nicotine exposure in monkeys has been shown to cause abnormalities of lung development (36). Third, surgical occlusion of the fetal trachea (PLUG technique) was developed to augment lung growth in high-risk cases of CDH. However, a recent randomized trial demonstrated no survival benefit (37). In fact, the PLUG has been shown to impair important aspects of fetal lung development (38). The PLUG could be impeding normal lung development and/or maturation by interfering with phasic AP. A dynamic PLUG-unPLUG technique may therefore represent a superior technique to stimulate lung growth in CDH. Fourth, although increasingly premature babies are surviving to maturity, many develop bronchopulmonary dysplasia (BPD) in childhood. This lung dysmorphology is associated with chronically impaired lung function and has been attributed to barotrauma associated with artificial ventilation (39). Could artificial ventilation contribute to BPD by prematurely disrupting fetal airway peristalsis? Finally, if AP is important to lung growth and originates from a focal pacemaker in vivo, exogenous endobronchial pacing of immature ASM may be a future strategy to enhance lung development and/or maturation in utero or perhaps in prematurity. If these possibilities are to be realized, further exploration of airway peristalsis is required.

AP has been demonstrated in each trimester in both cultured lung and freshly explanted fetal airway in tissue bath (2). Documenting AP in vivo is therefore an important next step. Second, in separate studies, we have shown that AP is underpinned by periodic calcium oscillations that propagate along the long axis of the developing airway. Showing that morphogens such as FGF-10 modulate the characteristics of these calcium waves would be further support for the idea that AP and lung growth are coupled.

In conclusion, we have demonstrated for the first time that developing lung has a right-sided, c-kit–independent pacemaker area for airway peristalsis that dominates and is autonomous of the rest of the lung. The rhythmicity of this pacemaker emerges during embryogenesis in parallel with early SMA expression. This focal origin of AP waves then persists despite the proximodistal spread of SMA expression with time. Manipulation of either AP or lung growth consistently yields parallel changes in the other. Attempts to uncouple the two phenomena have been thus far unsuccessful. This supports the concept that pacemaker-driven AP is linked to lung development and that ASM is not merely a troublesome evolutionary remnant. There is now an opportunity to determine whether this distinctive physiology of AP can be harnessed to ameliorate the morbidity and mortality associated with disordered human lung development.


   Footnotes
 
This work was supported by The Academy of Medical Sciences/The Health Foundation, The Royal College of Surgeons of England and The Birth Defects Foundation.

This article has an online supplement, which is accessible from this issue's table of contents at www.atsjournals.org

Conflict of Interest Statement: E.C.J. has no declared conflicts of interest; N.P.S. has no declared conflicts of interest; M.G.C. has no declared conflicts of interest; D.G.S. has no declared conflicts of interest; M.R.H.W. has no declared conflicts of interest; D.G.F. has no declared conflicts of interest; and P.D.L. has no declared conflicts of interest.

* Joint first authors. Back

Received in original form September 27, 2004

Received in final form November 29, 2004


   References
Top
 Abstract
 Introduction
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 

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