Morphogenetic implications of peristaltic fluid-tissue dynamics in the embryonic lung.

Peristalsis begins in the lung as soon as the smooth muscle forms, and persists until birth. Since the prenatal lung is liquid-filled, smooth muscle action can deform tissues and transport fluid far from the immediately adjacent tissues. Stretching of embryonic tissues and sensation of internal fluid flows have been shown to have potent morphogenetic effects. We hypothesize that these effects are at work in lung morphogenesis. To place that hypothesis in a quantitative framework, we analyze a model of the fluid-structure interactions between embryonic tissues and lumen fluid resulting from peristaltic waves that partially occlude the airway. We find that if the airway is closed, deformations are synchronized; by contrast, if the trachea is open, maximal occlusion precedes maximal pressure. We perform a parametric analysis of how occlusion, stretch, and flow depend on tissue stiffnesses, smooth muscle force, tissue shape and size, and fluid viscosity. We find that most of these relationships are governed by simple ratios.

Airway branching has conserved needs for local parasympathetic innervation but not neurotransmission.

BACKGROUND: Parasympathetic signaling has been inferred to regulate epithelial branching as well as organ regeneration and tumor development. However, the relative contribution of local nerve contact versus secreted signals remains unclear. Here, we show a conserved (vertebrates to invertebrates) requirement for intact local nerves in airway branching, persisting even when cholinergic neurotransmission is blocked. RESULTS: In the vertebrate lung, deleting enhanced green fluorescent protein (eGFP)-labeled intrinsic neurons using a two-photon laser leaves adjacent cells intact, but abolishes branching. Branching is unaffected by similar laser power delivered to the immediately adjacent non-neural mesodermal tissue, by blocking cholinergic receptors or by blocking synaptic transmission with botulinum toxin A. Because adjacent vasculature and epithelial proliferation also contribute to branching in the vertebrate lung, the direct dependence on nerves for airway branching was tested by deleting neurons in Drosophila embryos. A specific deletion of neurons in the Drosophila embryo by driving cell-autonomous RicinA under the pan-neuronal elav enhancer perturbed Drosophila airway development. This system confirmed that even in the absence of a vasculature or epithelial proliferation, airway branching is still disrupted by neural lesioning. CONCLUSIONS: Together, this shows that airway morphogenesis requires local innervation in vertebrates and invertebrates, yet neurotransmission is dispensable. The need for innervation persists in the fly, wherein adjacent vasculature and epithelial proliferation are absent. Our novel, targeted laser ablation technique permitted the local function of parasympathetic innervation to be distinguished from neurotransmission.

Structure and epitope distribution of heparan sulfate is disrupted in experimental lung hypoplasia: a glycobiological epigenetic cause for malformation?

BACKGROUND: Heparan sulfate (HS) is present on the surface of virtually all mammalian cells and is a major component of the extracellular matrix (ECM), where it plays a pivotal role in cell-cell and cell-matrix cross-talk through its large interactome. Disruption of HS biosynthesis in mice results in neonatal death as a consequence of malformed lungs, indicating that HS is crucial for airway morphogenesis. Neonatal mortality (~50%) in newborns with congenital diaphragmatic hernia (CDH) is principally associated with lung hypoplasia and pulmonary hypertension. Given the importance of HS for lung morphogenesis, we investigated developmental changes in HS structure in normal and hypoplastic lungs using the nitrofen rat model of CDH and semi-synthetic bacteriophage ('phage) display antibodies, which identify distinct HS structures. RESULTS: The pulmonary pattern of elaborated HS structures is developmentally regulated. For example, the HS4E4V epitope is highly expressed in sub-epithelial mesenchyme of E15.5 - E17.5 lungs and at a lower level in more distal mesenchyme. However, by E19.5, this epitope is expressed similarly throughout the lung mesenchyme.We also reveal abnormalities in HS fine structure and spatiotemporal distribution of HS epitopes in hypoplastic CDH lungs. These changes involve structures recognised by key growth factors, FGF2 and FGF9. For example, the EV3C3V epitope, which was abnormally distributed in the mesenchyme of hypoplastic lungs, is recognised by FGF2. CONCLUSIONS: The observed spatiotemporal changes in HS structure during normal lung development will likely reflect altered activities of many HS-binding proteins regulating lung morphogenesis. Abnormalities in HS structure and distribution in hypoplastic lungs can be expected to perturb HS:protein interactions, ECM microenvironments and crucial epithelial-mesenchyme communication, which may contribute to lung dysmorphogenesis. Indeed, a number of epitopes correlate with structures recognised by FGFs, suggesting a functional consequence of the observed changes in HS in these lungs. These results identify a novel, significant molecular defect in hypoplastic lungs and reveals HS as a potential contributor to hypoplastic lung development in CDH. Finally, these results afford the prospect that HS-mimetic therapeutics could repair defective signalling in hypoplastic lungs, improve lung growth, and reduce CDH mortality.

Long Covid in adults discharged from UK hospitals after Covid-19: A prospective, multicentre cohort study using the ISARIC WHO Clinical Characterisation Protocol.

BACKGROUND: This study sought to establish the long-term effects of Covid-19 following hospitalisation. METHODS: 327 hospitalised participants, with SARS-CoV-2 infection were recruited into a prospective multicentre cohort study at least 3 months post-discharge. The primary outcome was self-reported recovery at least ninety days after initial Covid-19 symptom onset. Secondary outcomes included new symptoms, disability (Washington group short scale), breathlessness (MRC Dyspnoea scale) and quality of life (EQ5D-5L). FINDINGS: 55% of participants reported not feeling fully recovered. 93% reported persistent symptoms, with fatigue the most common (83%), followed by breathlessness (54%). 47% reported an increase in MRC dyspnoea scale of at least one grade. New or worse disability was reported by 24% of participants. The EQ5D-5L summary index was significantly worse following acute illness (median difference 0.1 points on a scale of 0 to 1, IQR: -0.2 to 0.0). Females under the age of 50 years were five times less likely to report feeling recovered (adjusted OR 5.09, 95% CI 1.64 to 15.74), were more likely to have greater disability (adjusted OR 4.22, 95% CI 1.12 to 15.94), twice as likely to report worse fatigue (adjusted OR 2.06, 95% CI 0.81 to 3.31) and seven times more likely to become more breathless (adjusted OR 7.15, 95% CI 2.24 to 22.83) than men of the same age. INTERPRETATION: Survivors of Covid-19 experienced long-term symptoms, new disability, increased breathlessness, and reduced quality of life. These findings were present in young, previously healthy working age adults, and were most common in younger females. FUNDING: National Institute for Health Research, UK Medical Research Council, Wellcome Trust, Department for International Development and the Bill and Melinda Gates Foundation.

Heparan sulfate in lung morphogenesis: The elephant in the room.

Heparan sulfate (HS) is a structurally complex polysaccharide located on the cell surface and in the extracellular matrix, where it participates in numerous biological processes through interactions with a vast number of regulatory proteins such as growth factors and morphogens. HS is crucial for lung development; disruption of HS synthesis in flies and mice results in a major aberration of airway branching, and in mice, it results in neonatal death as a consequence of malformed lungs and respiratory distress. Epithelial-mesenchymal interactions governing lung morphogenesis are directed by various diffusible proteins, many of which bind to, and are regulated by HS, including fibroblast growth factors, sonic hedgehog, and bone morphogenetic proteins. The majority of research into the molecular mechanisms underlying defective lung morphogenesis and pulmonary pathologies, such as bronchopulmonary dysplasia and pulmonary hypoplasia associated with congenital diaphragmatic hernia (CDH), has focused on abnormal protein expression. The potential contribution of HS to abnormalities of lung development has yet to be explored to any significant extent, which is somewhat surprising given the abnormal lung phenotype exhibited by mutant mice synthesizing abnormal HS. This review summarizes our current understanding of the role of HS and HS-binding proteins in lung morphogenesis and will present in vitro and in vivo evidence for the fundamental importance of HS in airway development. Finally, we will discuss the future possibility of HS-based therapeutics for ameliorating insufficient lung growth associated with lung diseases such as CDH.

Heparan sulfate phage display antibodies identify distinct epitopes with complex binding characteristics: insights into protein binding specificities.

Heparan sulfate (HS) binds and modulates the transport and activity of a large repertoire of regulatory proteins. The HS phage display antibodies are powerful tools for the analysis of native HS structure in situ; however, their epitopes are not well defined. Analysis of the binding specificities of a set of HS antibodies by competitive binding assays with well defined chemically modified heparins demonstrates that O-sulfates are essential for binding; however, increasing sulfation does not necessarily correlate with increased antibody reactivity. IC50 values for competition with double modified heparins were not predictable from IC50 values with corresponding singly modified heparins. Binding assays and immunohistochemistry revealed that individual antibodies recognize distinct epitopes and that these are not single linear sequences but families of structurally similar motifs in which subtle variations in sulfation and conformation modify the affinity of interaction. Modeling of the antibodies demonstrates that they possess highly basic CDR3 and surrounding surfaces, presenting a number of possible orientations for HS binding. Unexpectedly, there are significant differences between the existence of epitopes in tissue sections and observed in vitro in dot blotted tissue extracts, demonstrating that in vitro specificity does not necessarily correlate with specificity in situ/vivo. The epitopes are therefore more complex than previously considered. Overall, these data have significance for structure-activity relationships of HS, because the model of one antibody recognizing multiple HS structures and the influence of other in situ HS-binding proteins on epitope availability are likely to reflect the selectivity of many HS-protein interactions in vivo.

Neonatal endosurgical congenital diaphragmatic hernia repair: a systematic review and meta-analysis.

OBJECTIVE: To compare outcomes of open and endosurgical neonatal congenital diaphragmatic hernia (CDH) repairs. BACKGROUND: Historically a surgical emergency, neonatal CDH repair is now deferred pending stabilization of characteristically labile cardiopulmonary physiology. Usually accomplished via laparotomy, surgical repair may acutely worsen lung function; conversely, by reducing the visceral hernia, surgery might improve it. Theoretically, endosurgical repair could minimize deleterious effects of surgery while garnering benefits from decompressing the CDH lung. As endosurgical repair gains popularity, it is important to investigate whether or not minimally-invasive neonatal CDH repair has benefits. METHODS: We searched Medline, Embase, and Cochrane Trials databases for studies comparing open with endosurgical CDH repair. Non-neonatal series and reports without comparison groups were excluded. References from papers and conference proceedings were also hand searched. Meta-analysis used a fixed effects model and was reported in accordance with PRISMA. RESULTS: We included 3 studies (1 unpublished; none randomized); all compared thoracoscopic and open CDH repair and together described 143 patients. All studies had limitations, including use of historical controls. Demographics, CDH sidedness, APGAR and associated anomaly prevalence were similar between groups. For endosurgical repair, recurrence was higher (RR: 3.2 [1.1, 9.3], P = 0.03) and operative time longer (WMD 50 minutes [32, 69], P < 0.00001). Survival and patch usage were not different between open and endosurgical groups. CONCLUSIONS: Neonatal thoracoscopic CDH repair has greater recurrence rates and operative times but similar survival and patch usage compared with open surgery. A prospective registry for all such cases would guide development of trials (Stage 2b; IDEAL recommendations).

Embryonic lung growth is normal in a cftr-knockout mouse model.

The role of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel in embryonic lung growth remains uncertain. The authors used an established embryonic lung culture model to investigate the impact of cftr knockout on lung growth, airway peristalsis, and airway smooth muscle (ASM) distribution. Lung area, perimeter, lung bud count, and frequency of contraction were similar in wild-type (cftr +/+) and cftr knockout mice (cftr -/-). The percentage of mitotic cells was also consistent between genotypes in mesenchyme and epithelium. Smooth muscle distribution surrounding the airway appeared normally distributed in all genotypes. These data suggest that normal embryonic lung growth, ASM differentiation and airway peristalsis are CFTR independent.

SERCA directs cell migration and branching across species and germ layers.

Branching morphogenesis underlies organogenesis in vertebrates and invertebrates, yet is incompletely understood. Here, we show that the sarco-endoplasmic reticulum Ca2+ reuptake pump (SERCA) directs budding across germ layers and species. Clonal knockdown demonstrated a cell-autonomous role for SERCA in Drosophila air sac budding. Live imaging of Drosophila tracheogenesis revealed elevated Ca2+ levels in migratory tip cells as they form branches. SERCA blockade abolished this Ca2+ differential, aborting both cell migration and new branching. Activating protein kinase C (PKC) rescued Ca2+ in tip cells and restored cell migration and branching. Likewise, inhibiting SERCA abolished mammalian epithelial budding, PKC activation rescued budding, while morphogens did not. Mesoderm (zebrafish angiogenesis) and ectoderm (Drosophila nervous system) behaved similarly, suggesting a conserved requirement for cell-autonomous Ca2+ signaling, established by SERCA, in iterative budding.

Quantifying cellular and subcellular stretches in embryonic lung epithelia under peristalsis: where to look for mechanosensing.

Peristalsis begins in the lung as soon as the smooth muscle (SM) forms, and persists until birth. As the prenatal lung is filled with liquid, SM action can, through lumen pressure, deform tissues far from the immediately adjacent tissues. Stretching of embryonic tissues has been shown to have potent morphogenetic effects. We hypothesize that these effects are at work in lung morphogenesis. In order to refine that broad hypothesis in a quantitative framework, we geometrically analyse cell shapes in an epithelial tissue, and individual cell deformations resulting from peristaltic waves that completely occlude the airway. Typical distortions can be very large, with opposite orientations in the stalk and tip regions. Apical distortions are always greater than basal distortions. We give a quantitative estimate of the relationship between length of occluded airway and the resulting tissue stretch in the distal tip. We refine our analysis of cell stresses and strains from peristalsis with a simple mechanical model of deformation of cells within an epithelium, which accounts for basic subcellular geometry and material properties. The model identifies likely stress concentrations near the nucleus and at the apical cell-cell junction. The surprisingly large strains of airway peristalsis may serve to rearrange cells and stimulate other mechanosensitive processes by repeatedly aligning cytoskeletal components and/or breaking and reforming lateral cell-cell adhesions. Stress concentrations between nuclei of adjacent cells may serve as a mechanical control mechanism guiding the alignment of nuclei as an epithelium matures.

Morphogenetic Implications of Peristalsis-Driven Fluid Flow in the Embryonic Lung.

Epithelial organs are almost universally secretory. The lung secretes mucus of extremely variable consistency. In the early prenatal period, the secretions are of largely unknown composition, consistency, and flow rates. In addition to net outflow from secretion, the embryonic lung exhibits transient reversing flows from peristalsis. Airway peristalsis (AP) begins as soon as the smooth muscle forms, and persists until birth. Since the prenatal lung is liquid-filled, smooth muscle action can transport fluid far from the immediately adjacent tissues. The sensation of internal fluid flows has been shown to have potent morphogenetic effects, as has the transport of morphogens. We hypothesize that these effects play an important role in lung morphogenesis. To test these hypotheses in a quantitative framework, we analyzed the fluid-structure interactions between embryonic tissues and lumen fluid resulting from peristaltic waves that partially occlude the airway. We found that if the airway is closed, fluid transport is minimal; by contrast, if the trachea is open, shear rates can be very high, particularly at the stenosis. We performed a parametric analysis of flow characteristics' dependence on tissue stiffnesses, smooth muscle force, geometry, and fluid viscosity, and found that most of these relationships are governed by simple ratios. We measured the viscosity of prenatal lung fluid with passive bead microrheology. This paper reports the first measurements of the viscosity of embryonic lung lumen fluid. In the range tested, lumen fluid can be considered Newtonian, with a viscosity of 0.016 ± 0.008 Pa-s. We analyzed the interaction between the internal flows and diffusion and conclude that AP has a strong effect on flow sensing away from the tip and on transport of morphogens. These effects may be the intermediate mechanisms for the enhancement of branching seen in occluded embryonic lungs.

Cortical Tension Allocates the First Inner Cells of the Mammalian Embryo.

Every cell in our body originates from the pluripotent inner mass of the embryo, yet it is unknown how biomechanical forces allocate inner cells in vivo. Here we discover subcellular heterogeneities in tensile forces, generated by actomyosin cortical networks, which drive apical constriction to position the first inner cells of living mouse embryos. Myosin II accumulates specifically around constricting cells, and its disruption dysregulates constriction and cell fate. Laser ablations of actomyosin networks reveal that constricting cells have higher cortical tension, generate tension anisotropies and morphological changes in adjacent regions of neighboring cells, and require their neighbors to coordinate their own changes in shape. Thus, tensile forces determine the first spatial segregation of cells during mammalian development. We propose that, unlike more cohesive tissues, the early embryo dissipates tensile forces required by constricting cells via their neighbors, thereby allowing confined cell repositioning without jeopardizing global architecture.

Subspecialization may improve an esophageal atresia service but has not addressed declining trainee experience.

BACKGROUND: Subspecialization defined pediatric surgery using Alder Hey innovations in neonatal surgical units (Rickham) and anesthesia (Jackson-Rees). In neonatal surgery, United Kingdom subspecialization for cloacal extrophy and biliary atresia acknowledges their dependence on multidisciplinary management and the desirability of caseload for training. We phased in regional subspecialization for esophageal atresia (EA) repair and replacement surgery while trainee numbers increased nationally to reduce hours. We examined EA outcomes and training during subspecialization. METHODS: We analyzed EA cases (1999-2009) treated at Alder Hey Children's Hospital in two 5-year cohorts, the first early phase of incomplete subspecialization and the later near-total or "comprehensive" subspecialization phase. These periods approximated those before and after trainee numbers rose sharply to reduce working hours. RESULTS: Of 119 cases, 60 in the early cohort shared similar characteristics with the 59 in the later cohort. Near-complete subspecialization was achieved in the second 5 years with 97% of cases performed under a surgeon with an EA specialty interest; in the earlier cohort, 25% of surgeries were undertaken by surgeons without EA subspecialty interest. With near-complete subspecialization, pediatric intensive care unit stay fell from 5 (4-11) to 4 (2-7) days (median (IQR); P = .005), and approaches such as the Bianchi axillary repair and Bax single-stage jejunal interposition were introduced; hospital stay went from 25 (12-63) to 17 (13-28) days (P = .27), and deaths, from 6 to 3 children (P = .49). Mortality was 7.6% (9/119) compared with 14% (19/134) in our previous institutional series (χ(2) = 2.8, P = .09), and neonatal mortality fell from 6% to 0 (P = .008). Near doubling of trainee numbers accompanied an approximately 3-fold fall in repairs per trainee over the study. CONCLUSION: Near-complete subspecialization for EA coincided with reduced pediatric intensive care unit stay, successful introduction of cosmetic axillary approaches, and extension of our replacement service to offer all interposition types. It has not reversed the steep decline in trainee experience of EA that has been associated with the greater numbers of trainees that have been employed to reduce working hours.

Enhancing safety of laparoscopic vascular control for neonatal sacrococcygeal teratoma.

Life-threatening bleeding is a hazard of major tumor excision in children. However, fatalities from inadvertent arterial ligation should not be overlooked. Sacrococcygeal teratoma is the commonest neonatal tumor. Laparotomy to ligate the median sacral artery has been used to preempt potentially fatal resectional bleeding. Use of laparoscopy to achieve the same is an evolving technique, with only 7 neonatal cases described. As such, the Idea, Development, Exploration, Assessment, Long-term study (IDEAL) guidelines on surgical innovation recommend case reports addressing proof of concept, technical factors and safety tips. Fortunately, mistaken arterial division is so far unreported during laparoscopic median sacral artery ligation. However, as uptake widens, anatomical distortion by tumor and surgeon disorientation at endosurgery are risk factors for even such inconceivable complications. We report a successful case of laparoscopic vascular control for neonatal sacrococcygeal teratoma and demonstrate an observation that serves as a useful safety check for this procedure (as well as the open alternative).

Laparoscopic excision of a retroperitoneal lymphatic malformation in a newborn.

Abdominal lymphatic malformations may be challenging to eradicate. Retroperitoneal lesions may more difficult to resect than mesenteric ones; however, the latter may predispose to intestinal volvulus, leading to calls for their prompt excision. Such lesions identified perinatally may pose particular challenges: in one case, respiratory failure caused by abdominal distension required emergency drainage followed by later laparoscopic excision; laparoscopy has also been used promptly to diagnose and resect neonatal mesenteric lymphatic malformations with their inherent volvulus risk. We illustrate that even if neonatal laparoscopy identifies a retroperitoneal rather than mesenteric lymphatic malformation, curative endosurgical excision remains feasible.

Contrasting expression of canonical Wnt signaling reporters TOPGAL, BATGAL and Axin2(LacZ) during murine lung development and repair.

Canonical WNT signaling plays multiple roles in lung organogenesis and repair by regulating early progenitor cell fates: investigation has been enhanced by canonical Wnt reporter mice, TOPGAL, BATGAL and Axin2(LacZ). Although widely used, it remains unclear whether these reporters convey the same information about canonical Wnt signaling. We therefore compared beta-galactosidase expression patterns in canonical Wnt signaling of these reporter mice in whole embryo versus isolated prenatal lungs. To determine if expression varied further during repair, we analyzed comparative pulmonary expression of beta-galactosidase after naphthalene injury. Our data show important differences between reporter mice. While TOPGAL and BATGAL lines demonstrate Wnt signaling well in early lung epithelium, BATGAL expression is markedly reduced in late embryonic and adult lungs. By contrast, Axin2(LacZ) expression is sustained in embryonic lung mesenchyme as well as epithelium. Three days into repair after naphthalene, BATGAL expression is induced in bronchial epithelium as well as TOPGAL expression (already strongly expressed without injury). Axin2(LacZ) expression is increased in bronchial epithelium of injured lungs. Interestingly, both TOPGAL and Axin2(LacZ) are up regulated in parabronchial smooth muscle cells during repair. Therefore the optimal choice of Wnt reporter line depends on whether up- or down-regulation of canonical Wnt signal reporting in either lung epithelium or mesenchyme is being compared.

Excision of ganglioneuroma from skull base to aortic arch.

High retropharyngeal neuroblastic tumors in children have been excised and debulked transorally or cervically, often with a covering tracheostomy. Although we and others have approached high thoracic lesions thoracoscopically, the trapdoor incision (or modification thereof) is generally reserved for cervicothoracic tumors with significant vessel encasement around the thoracic inlet. We report a case of symptomatic ganglioneuroma extending from the nasopharynx, at the level of the skull base, down to the aortic arch: macroscopic clearance was achieved via an extended trapdoor incision and without recourse to tracheostomy, transoral surgery, or transfusion.

Surgical practice for infantile hypertrophic pyloric stenosis in the United Kingdom and Ireland--a survey of members of the British Association of Paediatric Surgeons.

PURPOSE: Operative strategy and antibiotic policy in treating infantile hypertrophic pyloric stenosis (IHPS) may vary widely. This study surveys current practice in the United Kingdom and Ireland among members of the British Association of Paediatric Surgeons. METHODS: The study used postal and email survey of consultant pediatric surgeons. RESULTS: One hundred five questionnaires were distributed, and 94 replies (90% response) were received. Umbilical pyloromyotomy is performed exclusively by 57 surgeons. Fourteen surgeons (15%) use laparoscopy, whereas 5 (randomized trial in progress) use the umbilical or laparoscopic route. Eight reported that the umbilical or classical right upper quadrant (RUQ) incision is undertaken at their institution according to surgeon's preference. Ten surgeons only deploy an RUQ incision. Antibiotic practice showed that 40 (70%) using the umbilical incision prescribe prophylactic therapy, whereas only 6 adopting other operative techniques (RUQ incision or laparoscopy) found this policy beneficial. More than 50% surveyed do not routinely recommend antibiotics. CONCLUSION: Umbilical pyloromyotomy is used by most pediatric surgeons in the United Kingdom and Ireland. Laparoscopy is increasingly popular in minimally invasive centers. The RUQ incision is used by a minority of surgeons. Antibiotic prophylaxis was common with the umbilical incision only. The superior cosmetic results offered by umbilical pyloromyotomy and laparoscopy are a benchmark for surgeons currently providing contemporary care for babies with IHPS.

Exploiting mechanical stimuli to rescue growth of the hypoplastic lung.

Impaired lung development afflicts a range of newborns cared for by paediatric surgeons. As a result the speciality has led in the development of surgical models that illustrate the biomechanical regulation of lung growth. Using transgenic mutants, biologists have similarly discovered much about the biochemical regulation of prenatal lung growth. Airway smooth muscle (ASM) and its prenatal contractility airway peristalsis (AP) represent a novel link between these areas: ASM progenitors produce an essential biochemical factor for lung morphogenesis, whilst calcium-driven biomechanical ASM activity appears to regulate the same. In this invited paper, I take the opportunity both to review our recent findings on lung growth and prenatal ASM, and also to discuss mechanisms by which ASM contractility can regulate growth. Finally, I will introduce some novel ideas for exploration: ASM contractility could help to schedule parturition (pulmonary parturition clock) and could even be a generic model for smooth muscle regulation of morphogenesis in similar organs.