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Small lungs and suspect smooth muscle: congenital diaphragmatic hernia and the smooth muscle hypothesis
Journal of Pediatric Surgery (2006) 41, 431 – 435
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Small lungs and suspect smooth muscle: congenital diaphragmatic hernia and the smooth muscle hypothesis Edwin C. Jesudason* Department of Paediatric Surgery, Alder Hey Children’s Hospital and Division of Child Health, School of Reproductive and Developmental Medicine, University of Liverpool, Liverpool, UK Index words: Airway smooth muscle; Lung branching morphogenesis; Congenital diaphragmatic hernia
Abstract Lung hypoplasia and congenital diaphragmatic hernia (CDH) represent an unsolved clinical and scientific problem. Early lung morphogenesis is coupled to development and function of pulmonary smooth muscle. Activity of the latter is abnormal from the earliest stages of hypoplastic lung development and before supervening CDH. A bsmooth muscle hypothesisQ is advanced to help explain embryonic lung malformations, fetal failure of lung growth, and postnatal susceptibility to barotrauma, airway hyperreactivity, and pulmonary hypertension in CDH. Exploring the interaction of smooth muscle function and airway pressures may help optimise tracheal occlusion and provide support for both an adequately powered trial of glucocorticoids and also for experimental bpreventilationQ strategies in fetal CDH. D 2006 Elsevier Inc. All rights reserved.
Alongside necrotising enterocolitis, neuroblastoma and short gut syndrome, congenital diaphragmatic hernia (CDH) remains one of the most perplexing problems facing modern paediatric surgery. Together with the persistent therapeutic hurdles, there is an ongoing intellectual challenge to understand the aetiology and pathogenesis of this lethal disease. Moreover, the scientific enquiry is given major impetus by growing appreciation of the size of the health problem: CDH is almost as common as cystic fibrosis and has been calculated to be twice as common as infant cancer
Presented at the 52nd Annual Congress of British Association of Paediatric Surgeons, Dublin, Ireland, July 12-15, 2005. * National Clinician Scientist/Specialist Registrar in Paediatric Surgery, Institute of Child Health, Alder Hey Children’s Hospital, Liverpool, UK. Tel.: +44 151 252 5440; fax: +44 151 228 2024. E-mail address: [email protected]. 0022-3468/$ – see front matter D 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.jpedsurg.2005.11.021
[1]. Given the ubiquitous battle for healthcare resources, the short and long-term financial costs of CDH are also not to be underestimated [2].
1. Pressure for change This article advances a new hypothesis to try and explain several key clinical manifestations of CDH and hopes to show that consideration of the issues raised may help to improve current and future therapy. Finally, this article aims to demonstrate that far from being a troublesome evolutionary remnant (bthe appendix of the lungQ), airway smooth muscle (ASM) may be a crucial regulator of both early and late prenatal lung growth [3]. To do this, one can begin by highlighting cardinal features of CDH that both impair prognosis and also merit explanation by any new hypothesis. At the basic level, CDH comprises prenatal development of a
432 diaphragmatic defect, intrathoracic herniation of abdominal viscera, and lung hypoplasia. It may be associated with other structural anomalies (eg, heart, vertebrae, etc) and chromosomal lesions [4]. Postnatally, refractory respiratory failure represents the greatest threat to survival of the nonsyndromic CDH newborn and is thought to arise because of a varied combination of lung hypoplasia, pulmonary hypertension, and barotrauma [5]. In the longer term, CDH survivors may suffer with chronic problems including airway hyperresponsiveness and feeding difficulties [6-8].
2. The early mesenchymal hit To explain these manifestations, one may start uncontroversially with a mesenchymal lesion: the diaphragm develops from several mesenchymal derivatives [9]. Beyond traditional embryological descriptions, a range of modern data supports this ontogeny and its disruption in CDH. For example, homozygous murine mutants for Wilms Tumour suppressor–1 feature a diaphragmatic defect: Wilms Tumour suppressor–1 is normally expressed in the developing mesenchyme [10]. Moreover, teratogenic induction of CDH in the nitrofen model is associated with apoptotic cell deletion in the cervical myotomes [11]. Such a mesenchymal lesion appears to occur in embryogenesis, given the constellation of recognised associated anomalies in humans and the required timing of nitrofen administration (and of remedial Vitamin A) in the rodent model [12,13]. Hence, an early mesenchymal lesion could neatly explain the diaphragmatic defect, the consequent visceral hernia, and, depending on its extent, certain associated anomalies. However, how does this framework help explain the development of lung hypoplasia and its devastating sequelae?
3. Relating lung hypoplasia and CDH Of 5 possibilities, 4 are usually entertained when trying to explain lung hypoplasia and its temporal relationship with the diaphragmatic hernia (the fifth—that they are entirely unconnected—is readily discarded for obvious statistical reasons): (1) the lung lesion precedes and may even cause the diaphragmatic hernia; (2) the lung lesion succeeds the diaphragmatic hernia and may be caused by it; (3) both lung and diaphragmatic lesions (even if becoming apparent at different times) result from common cause; and (4) a combination of 1 and 2, in which there are 2 lung lesions, one precedes CDH, the other follows it (dual-hit). Iritani [14] first demonstrated that nitrofen reduced embryonic lung length before supervention of CDH in mice. Abnormal lung branching morphogenesis was also reported before CDH in the nitrofen model [15]. This observation was subsequently replicated by 2 groups [16,17]. Near-term, tracheobronchial anomalies persist [18]. Whilst the lung lesion may therefore precede CDH, we now know that the hypoplastic lung itself
E.C. Jesudason does not cause CDH: teratogens remain capable of inducing diaphragmatic defects in a murine model of lung agenesis [19]. This evidence from the nitrofen model has led to gradual revision of the traditional teaching embodied by the second proposition. Nonetheless, extensive experience in the fetal ovine model of CDH indicates that a form of lung hypoplasia can be generated by the surgical creation of CDH [20]. In a fusion of these 2 positions, the bdual-hitQ was advanced. Interestingly, in the eponymous paper, the authors actually failed to show the primary hit: in contrast to 2 previous demonstrations (and one subsequent), there were no significant differences in airway bud number between normal and unselected lungs exposed to nitrofen in vivo [21]. Nevertheless, the bdual-hitQ concept is supportable, given previous reports that the lung lesion may not only precede teratogenic CDH but also succeed surgically created CDH.
4. Pulmonary smooth muscle and hypoplastic lung development Given an early mesenchymal hit to explain the diaphragmatic defect and hernia (F associated anomalies), is it necessary to postulate further insults to arrive at clinical CDH? Examination of rat embryos readily reveals that a distinct mesenchyme surrounds the developing lung buds. Distally, this mesenchyme is in close approximation to key mesenchymal precursors of the future diaphragm. If one accepts a mesenchymal hit to explain subsequent failure of diaphragm formation, it is tenable to suggest that such a lesion encompasses the early pulmonary mesenchyme to varying degree. Given that epithelial-mesenchymal interactions are pivotal to early lung morphogenesis, this would go a long way to explaining early lung lesions in experimental CDH [22]. However, recent studies allow us to generate a more sophisticated hypothesis in this regard: distal pulmonary mesenchyme elaborates essential growth factors for lung morphogenesis. Foremost among these is fibroblast growth factor 10 (FGF10) [22]. Lack of FGF10 leads to lung agenesis in mice [23]. Nitrofen-exposed hypoplastic lungs are deficient in FGF10 and show remedial growth when treated with it in vitro [24,25]. We now know that FGF10-producing mesenchymal cells are specific progenitors of ASM [26]. They differentiate into ASM during embryogenesis and rapidly become synthetically and mechanically active [26,27]. Their coordinated contractility (airway peristalsis) appears to be coupled to lung growth [27]. Modulating either airway peristalsis or growth consistently leads to parallel changes in the other in vitro: for example, FGF10 significantly enhances ASM contraction rates and growth [27]. Abolishing ASM tone impairs overall lung size, whereas supranormal tone appears to halt branching morphogenesis [27-29]. These relationships are disrupted in embryonic lung hypoplasia and accompany fundamental abnormalities of underlying ASM calcium signalling [30]. Notably, the plateau phase of the ASM calcium wave is prolonged in
Small lungs and suspect smooth muscle
Fig. 1 Putative regulation of lung growth by ASM activity via feedback regulation of intraluminal pressure in the expanding lung.
embryonic lung hypoplasia [31]. Furthermore, ASM from near-term hypoplastic lung develops abnormally increased force [32]. Therefore, far from being the appendix of the lung, these findings indicate that ASM has (a) a key role in regulating early lung growth and (b) abnormal function before experimental CDH [27]. Based on these observations, one can postulate a normal feedback loop, for example, to stabilise intrapulmonary pressures that is compromised during fetal development of the CDH lung: FGF10-driven expansion of luminal lung volume should proceed in tandem with ASM elaboration; ASM modulation of airway tone then maintains or bfine-tunesQ intraluminal pressures to facilitate
433 further growth (Fig. 1). Intraluminal pressure is already known to regulate prenatal lung development; for example, drainage of lung liquid via fetal tracheostomy yields a form of lung hypoplasia [33]. Moreover, smooth muscle is recognised to regulate organ compliance in, for example, bladder [34]. In CDH, therefore, a single mesenchymal hit may start both future diaphragmatic and lung defects (Fig. 2). Reduced mesenchymal FGF10 directly impairs growth (via epithelialmesenchymal interactions) and also indirectly via disruption of growth-related ASM peristalsis. Later, in lung development, as epithelial-mesenchymal interactions and embryogenesis give way to fetal growth, ASM dysfunction impairs the hypoplastic lung’s ability to adequately regulate its compliance [35]. This dysregulation further hampers growth and increases vulnerability of the hypoplastic lung to compression by the hernia, fetal lung liquid loss, and postnatal barotrauma [36]. Persistence of ASM dysfunction may also help explain increased rates of airway hyperresponsiveness in CDH survivors [6,7]. Hence, the smooth muscle hypothesis may help explain several of the key manifestations of clinical CDH. Moreover, if mesenchymal progenitors of pulmonary vascular smooth muscle (VSM) were similarly affected, this might help explain vascular hypermuscularisation and pulmonary hypertension in CDH
Fig. 2 An overview of the smooth muscle hypothesis of CDH: note the potential for a single early lesion (top) to generate early and late features of CDH (bottom). PSM indicates pulmonary smooth muscle.
434 [37]. Interestingly, the gene whose mutation has most recently been linked to CDH/eventration and lung hypoplasia in mice and human (Fog-2) is again expressed in relevant early mesenchyme before subsequent restriction to ASM and VSM lineages [38].
E.C. Jesudason stone, Mrs MG Connell, Dr DG Fernig, Ms NP Smith, Dr DG Spiller, Dr MRH White, Dr TV Burdyga, and Professor S Wray. This work was funded by The Academy of Medical Sciences/Health Foundation, The Medical Research Council, The Royal College of Surgeons of England and The Birth Defects Foundation, London, UK.
5. Future research and clinical opportunities The implications of the smooth muscle hypothesis for research and clinical practice merit brief consideration at this point. Firstly, given the careful application and resulting success of postnatal bgentilation,Q it seems logical to devote detailed consideration to prenatal airway pressures and compliance (particularly when applying tracheal occlusion). To do so may help optimise the PLUG technique (Plug the Lung Until it Grows) and assist it to become less quixotic in outcomes [39,40]. Secondly, given the intrinsic rhythmicity of prenatal ASM (and striated muscle breathing movements), it may be worthwhile testing a bdynamicQ PLUG that actually generates fluctuations in intraluminal pressure. One might even conceive of a remotely operated endotracheal bballoon-pumpQ providing a sort of gentle prenatal ventilation or bpreventilationQ [27]. Thirdly, given that prenatal steroids improve lung compliance in experimental CDH, it seems essential to approach an adequately powered multicentre trial once more [35].
6. Conclusion In summary, this article has drawn on clinical and recent experimental evidence to advance the hypothesis that CDH and lung hypoplasia result from an early lesion of diaphragmatic precursors and mesenchymal pulmonary smooth muscle progenitors. The latter then impairs embryonic FGF10-driven lung growth and persists to alter fetal lung compliance (with consequent vulnerability to prenatal compression and postnatal barotrauma). Moreover, vascular and airway hyperreactivity in CDH may thus both be understood in an early smooth muscle lesion. These elements of the smooth muscle hypothesis are eminently testable: doing so may help clinicians to unlock a further slender advantage in the management of this troubling human disease.
Acknowledgments This article is based on the author’s invited lecture Small lungs and suspect smooth muscle at The Congenital Diaphragmatic Hernia Symposium, chaired by Professor PD Losty and Professor P Puri at The British Association of Paediatric Surgeons 52nd Annual International Congress, 2005, Dublin, Ireland. The author thanks the particular contribution of Professor PD Losty and of Mr NC Feather-
References [1] Sydorak RM, Harrison MR. Congenital diaphragmatic hernia: advances in prenatal therapy. World J Surg 2003;27:68 - 76. [2] Metkus AP, Esserman L, Sola A, et al. Cost per anomaly: what does a diaphragmatic hernia cost? J Pediatr Surg 1995;30:226 - 30. [3] Mitzner W. Airway smooth muscle: the appendix of the lung. Am J Respir Crit Care Med 2004;169:787 - 90. [4] Sweed Y, Puri P. Congenital diaphragmatic hernia: influence of associated malformations on survival. Arch Dis Child 1993;69:68 - 70. [5] Smith NP, Jesudason EC, Featherstone NC, et al. Recent advances in congenital diaphragmatic hernia. Arch Dis Child 2005;90:426 - 8. [6] Boas SR, Kurland G, Greally PG, et al. Evolution of airway hyperresponsiveness in infants with severe congenital diaphragmatic hernia. Pediatr Pulmonol 1996;22:295 - 304. [7] Muratore CS, Kharasch V, Lund DP, et al. Pulmonary morbidity in 100 survivors of congenital diaphragmatic hernia monitored in a multidisciplinary clinic. J Pediatr Surg 2001;36:133 - 40. [8] Muratore CS, Utter S, Jaksic T, et al. Nutritional morbidity in survivors of congenital diaphragmatic hernia. J Pediatr Surg 2001;36: 1171 - 6. [9] Kluth D, Losty PD, Schnitzer JJ, et al. Toward understanding the developmental anatomy of congenital diaphragmatic hernia. Clin Perinatol 1996;23:655 - 69. [10] Kreidberg JA, Sariola H, Loring JM, et al. WT-1 is required for early kidney development. Cell 1993;74:679 - 91. [11] Alles AJ, Losty PD, Donahoe PK, et al. Embryonic cell death patterns associated with nitrofen-induced congenital diaphragmatic hernia. J Pediatr Surg 1995;30:353 - 8. [12] Kluth D, Kangah R, Reich P, et al. Nitrofen-induced diaphragmatic hernias in rats: an animal model. J Pediatr Surg 1990;25:850 - 4. [13] Thebaud B, Tibboel D, Rambaud C, et al. Vitamin A decreases the incidence and severity of nitrofen-induced congenital diaphragmatic hernia in rats. Am J Physiol 1999;277:L423 - 9. [14] Iritani I. Experimental study on embryogenesis of congenital diaphragmatic hernia. Anat Embryol (Berl) 1984;169:133 - 9. [15] Jesudason EC, Connell MG, Fernig DG, et al. Early lung malformations in congenital diaphragmatic hernia. J Pediatr Surg 2000;35:124 - 7. [16] Guilbert TW, Gebb SA, Shannon JM. Lung hypoplasia in the nitrofen model of congenital diaphragmatic hernia occurs early in development. Am J Physiol Lung Cell Mol Physiol 2000;279:L1159 - 71. [17] Leinwand MJ, Tefft JD, Zhao J, et al. Nitrofen inhibition of pulmonary growth and development occurs in the early embryonic mouse. J Pediatr Surg 2002;37:1263 - 8. [18] Xia H, Migliazza L, Diez-Pardo JA, et al. The tracheobronchial tree is abnormal in experimental congenital diaphragmatic hernia. Pediatr Surg Int 1999;15:184 - 7. [19] Babiuk RP, Greer JJ. Diaphragm defects occur in a CDH hernia model independently of myogenesis and lung formation. Am J Physiol Lung Cell Mol Physiol 2002;283:L1310 - 4. [20] Starrett RW, de Lorimier AA. Congenital diaphragmatic hernia in lambs: hemodynamic and ventilatory changes with breathing. J Pediatr Surg 1975;10:575 - 82. [21] Keijzer R, Liu J, Deimling J, et al. Dual-hit hypothesis explains pulmonary hypoplasia in the nitrofen model of congenital diaphragmatic hernia. Am J Pathol 2000;156:1299 - 306.
Small lungs and suspect smooth muscle [22] Bellusci S, Grindley J, Emoto H, et al. Fibroblast growth factor 10 (FGF10) and branching morphogenesis in the embryonic mouse lung. Development 1997;124:4867 - 78. [23] Min H, Danilenko DM, Scully SA, et al. Fgf-10 is required for both limb and lung development and exhibits striking functional similarity to Drosophila branchless. Genes Dev 1998;12:3156 - 61. [24] Acosta JM, Thebaud B, Castillo C, et al. Novel mechanisms in murine nitrofen-induced pulmonary hypoplasia: FGF-10 rescue in culture. Am J Physiol Lung Cell Mol Physiol 2001;281:L250 - 7. [25] Teramoto H, Yoneda A, Puri P. Gene expression of fibroblast growth factors 10 and 7 is downregulated in the lung of nitrofen-induced diaphragmatic hernia in rats. J Pediatr Surg 2003;38:1021 - 4. [26] Mailleux AA, Kelly R, Veltmaat JM, et al. Fgf10 expression identifies parabronchial smooth muscle cell progenitors and is required for their entry into the smooth muscle cell lineage. Development 2005;132: 2157 - 66. [27] Jesudason EC, Smith NP, Connell MG, et al. Developing rat lung has a sided pacemaker region for morphogenesis-related airway peristalsis. Am J Respir Cell Mol Biol 2005;32:118 - 27. [28] Roman J. Effects of calcium channel blockade on mammalian lung branching morphogenesis. Exp Lung Res 1995;21:489 - 502. [29] Featherstone NC, Jesudason EC, Connell MG, et al. Sarcoplasmic reticulum regulates calcium transients, airway peristalsis and branching morphogenesis of lung explants in vitro. Proc Am Thorac Soc 2005;2:A503 [abstr]. [30] Featherstone NC, Jesudason EC, Connell MG, et al. Spontaneous propagating calcium waves underpin airway peristalsis in embryonic rat lung. Am J Respir Cell Mol Biol 2005;33:153 - 60. [31] Featherstone NC, Jesudason EC, Connell MG, et al. Carbachol and high K+ depolarisation-induced Ca2+ transients in airway smooth
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muscle in normal and hypoplastic lungs. Proc Am Thorac Soc 2005; 2:A503 [abstr]. Belik J, Davidge ST, Zhang W, et al. Airway smooth muscle changes in the nitrofen-induced congenital diaphragmatic hernia rat model. Pediatr Res 2003;53:737 - 43. Harding R, Hooper SB. Regulation of lung expansion and lung growth before birth. J Appl Physiol 1996;81:209 - 24. Dik P, Tsachouridis GD, Klijn AJ, et al. Detrusorectomy for neuropathic bladder in patients with spinal dysraphism. J Urol 2003; 170(4 Pt 1):1351 - 4. Schnitzer JJ, Hedrick HL, Pacheco BA, et al. Prenatal glucocorticoid therapy reverses pulmonary immaturity in congenital diaphragmatic hernia in fetal sheep. Ann Surg 1996;224:430 - 7. Boloker J, Bateman DA, Wung JT, et al. Congenital diaphragmatic hernia in 120 infants treated consecutively with permissive hypercapnea/spontaneous respiration/elective repair. J Pediatr Surg 2002; 37:357 - 66. Newell MA, Au-Fliegner M, Coppola CP, et al. Hypoxic pulmonary vasoconstriction is impaired in rats with nitrofen-induced congenital diaphragmatic hernia. J Pediatr Surg 1998;33:1358 - 62. Ackerman KG, Herron BJ, Vargas SO, et al. Fog2 is required for normal diaphragm and lung development in mice and humans. PLoS Genet 2005;1:e10. Harrison MR, Keller RL, Hawgood SB, et al. A randomized trial of fetal endoscopic tracheal occlusion for severe fetal congenital diaphragmatic hernia. N Engl J Med 2003;349:1916 - 24. Deprest J, Jani J, Gratacos E, et al. Fetal intervention for congenital diaphragmatic hernia: the European experience. Semin Perinatol 2005;29:94 - 103.
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Small lungs and suspect smooth muscle: congenital diaphragmatic hernia and the smooth muscle hypothesis Edwin C. Jesudason* Department of Paediatric Surgery, Alder Hey Children’s Hospital and Division of Child Health, School of Reproductive and Developmental Medicine, University of Liverpool, Liverpool, UK Index words: Airway smooth muscle; Lung branching morphogenesis; Congenital diaphragmatic hernia
Abstract Lung hypoplasia and congenital diaphragmatic hernia (CDH) represent an unsolved clinical and scientific problem. Early lung morphogenesis is coupled to development and function of pulmonary smooth muscle. Activity of the latter is abnormal from the earliest stages of hypoplastic lung development and before supervening CDH. A bsmooth muscle hypothesisQ is advanced to help explain embryonic lung malformations, fetal failure of lung growth, and postnatal susceptibility to barotrauma, airway hyperreactivity, and pulmonary hypertension in CDH. Exploring the interaction of smooth muscle function and airway pressures may help optimise tracheal occlusion and provide support for both an adequately powered trial of glucocorticoids and also for experimental bpreventilationQ strategies in fetal CDH. D 2006 Elsevier Inc. All rights reserved.
Alongside necrotising enterocolitis, neuroblastoma and short gut syndrome, congenital diaphragmatic hernia (CDH) remains one of the most perplexing problems facing modern paediatric surgery. Together with the persistent therapeutic hurdles, there is an ongoing intellectual challenge to understand the aetiology and pathogenesis of this lethal disease. Moreover, the scientific enquiry is given major impetus by growing appreciation of the size of the health problem: CDH is almost as common as cystic fibrosis and has been calculated to be twice as common as infant cancer
Presented at the 52nd Annual Congress of British Association of Paediatric Surgeons, Dublin, Ireland, July 12-15, 2005. * National Clinician Scientist/Specialist Registrar in Paediatric Surgery, Institute of Child Health, Alder Hey Children’s Hospital, Liverpool, UK. Tel.: +44 151 252 5440; fax: +44 151 228 2024. E-mail address: [email protected]. 0022-3468/$ – see front matter D 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.jpedsurg.2005.11.021
[1]. Given the ubiquitous battle for healthcare resources, the short and long-term financial costs of CDH are also not to be underestimated [2].
1. Pressure for change This article advances a new hypothesis to try and explain several key clinical manifestations of CDH and hopes to show that consideration of the issues raised may help to improve current and future therapy. Finally, this article aims to demonstrate that far from being a troublesome evolutionary remnant (bthe appendix of the lungQ), airway smooth muscle (ASM) may be a crucial regulator of both early and late prenatal lung growth [3]. To do this, one can begin by highlighting cardinal features of CDH that both impair prognosis and also merit explanation by any new hypothesis. At the basic level, CDH comprises prenatal development of a
432 diaphragmatic defect, intrathoracic herniation of abdominal viscera, and lung hypoplasia. It may be associated with other structural anomalies (eg, heart, vertebrae, etc) and chromosomal lesions [4]. Postnatally, refractory respiratory failure represents the greatest threat to survival of the nonsyndromic CDH newborn and is thought to arise because of a varied combination of lung hypoplasia, pulmonary hypertension, and barotrauma [5]. In the longer term, CDH survivors may suffer with chronic problems including airway hyperresponsiveness and feeding difficulties [6-8].
2. The early mesenchymal hit To explain these manifestations, one may start uncontroversially with a mesenchymal lesion: the diaphragm develops from several mesenchymal derivatives [9]. Beyond traditional embryological descriptions, a range of modern data supports this ontogeny and its disruption in CDH. For example, homozygous murine mutants for Wilms Tumour suppressor–1 feature a diaphragmatic defect: Wilms Tumour suppressor–1 is normally expressed in the developing mesenchyme [10]. Moreover, teratogenic induction of CDH in the nitrofen model is associated with apoptotic cell deletion in the cervical myotomes [11]. Such a mesenchymal lesion appears to occur in embryogenesis, given the constellation of recognised associated anomalies in humans and the required timing of nitrofen administration (and of remedial Vitamin A) in the rodent model [12,13]. Hence, an early mesenchymal lesion could neatly explain the diaphragmatic defect, the consequent visceral hernia, and, depending on its extent, certain associated anomalies. However, how does this framework help explain the development of lung hypoplasia and its devastating sequelae?
3. Relating lung hypoplasia and CDH Of 5 possibilities, 4 are usually entertained when trying to explain lung hypoplasia and its temporal relationship with the diaphragmatic hernia (the fifth—that they are entirely unconnected—is readily discarded for obvious statistical reasons): (1) the lung lesion precedes and may even cause the diaphragmatic hernia; (2) the lung lesion succeeds the diaphragmatic hernia and may be caused by it; (3) both lung and diaphragmatic lesions (even if becoming apparent at different times) result from common cause; and (4) a combination of 1 and 2, in which there are 2 lung lesions, one precedes CDH, the other follows it (dual-hit). Iritani [14] first demonstrated that nitrofen reduced embryonic lung length before supervention of CDH in mice. Abnormal lung branching morphogenesis was also reported before CDH in the nitrofen model [15]. This observation was subsequently replicated by 2 groups [16,17]. Near-term, tracheobronchial anomalies persist [18]. Whilst the lung lesion may therefore precede CDH, we now know that the hypoplastic lung itself
E.C. Jesudason does not cause CDH: teratogens remain capable of inducing diaphragmatic defects in a murine model of lung agenesis [19]. This evidence from the nitrofen model has led to gradual revision of the traditional teaching embodied by the second proposition. Nonetheless, extensive experience in the fetal ovine model of CDH indicates that a form of lung hypoplasia can be generated by the surgical creation of CDH [20]. In a fusion of these 2 positions, the bdual-hitQ was advanced. Interestingly, in the eponymous paper, the authors actually failed to show the primary hit: in contrast to 2 previous demonstrations (and one subsequent), there were no significant differences in airway bud number between normal and unselected lungs exposed to nitrofen in vivo [21]. Nevertheless, the bdual-hitQ concept is supportable, given previous reports that the lung lesion may not only precede teratogenic CDH but also succeed surgically created CDH.
4. Pulmonary smooth muscle and hypoplastic lung development Given an early mesenchymal hit to explain the diaphragmatic defect and hernia (F associated anomalies), is it necessary to postulate further insults to arrive at clinical CDH? Examination of rat embryos readily reveals that a distinct mesenchyme surrounds the developing lung buds. Distally, this mesenchyme is in close approximation to key mesenchymal precursors of the future diaphragm. If one accepts a mesenchymal hit to explain subsequent failure of diaphragm formation, it is tenable to suggest that such a lesion encompasses the early pulmonary mesenchyme to varying degree. Given that epithelial-mesenchymal interactions are pivotal to early lung morphogenesis, this would go a long way to explaining early lung lesions in experimental CDH [22]. However, recent studies allow us to generate a more sophisticated hypothesis in this regard: distal pulmonary mesenchyme elaborates essential growth factors for lung morphogenesis. Foremost among these is fibroblast growth factor 10 (FGF10) [22]. Lack of FGF10 leads to lung agenesis in mice [23]. Nitrofen-exposed hypoplastic lungs are deficient in FGF10 and show remedial growth when treated with it in vitro [24,25]. We now know that FGF10-producing mesenchymal cells are specific progenitors of ASM [26]. They differentiate into ASM during embryogenesis and rapidly become synthetically and mechanically active [26,27]. Their coordinated contractility (airway peristalsis) appears to be coupled to lung growth [27]. Modulating either airway peristalsis or growth consistently leads to parallel changes in the other in vitro: for example, FGF10 significantly enhances ASM contraction rates and growth [27]. Abolishing ASM tone impairs overall lung size, whereas supranormal tone appears to halt branching morphogenesis [27-29]. These relationships are disrupted in embryonic lung hypoplasia and accompany fundamental abnormalities of underlying ASM calcium signalling [30]. Notably, the plateau phase of the ASM calcium wave is prolonged in
Small lungs and suspect smooth muscle
Fig. 1 Putative regulation of lung growth by ASM activity via feedback regulation of intraluminal pressure in the expanding lung.
embryonic lung hypoplasia [31]. Furthermore, ASM from near-term hypoplastic lung develops abnormally increased force [32]. Therefore, far from being the appendix of the lung, these findings indicate that ASM has (a) a key role in regulating early lung growth and (b) abnormal function before experimental CDH [27]. Based on these observations, one can postulate a normal feedback loop, for example, to stabilise intrapulmonary pressures that is compromised during fetal development of the CDH lung: FGF10-driven expansion of luminal lung volume should proceed in tandem with ASM elaboration; ASM modulation of airway tone then maintains or bfine-tunesQ intraluminal pressures to facilitate
433 further growth (Fig. 1). Intraluminal pressure is already known to regulate prenatal lung development; for example, drainage of lung liquid via fetal tracheostomy yields a form of lung hypoplasia [33]. Moreover, smooth muscle is recognised to regulate organ compliance in, for example, bladder [34]. In CDH, therefore, a single mesenchymal hit may start both future diaphragmatic and lung defects (Fig. 2). Reduced mesenchymal FGF10 directly impairs growth (via epithelialmesenchymal interactions) and also indirectly via disruption of growth-related ASM peristalsis. Later, in lung development, as epithelial-mesenchymal interactions and embryogenesis give way to fetal growth, ASM dysfunction impairs the hypoplastic lung’s ability to adequately regulate its compliance [35]. This dysregulation further hampers growth and increases vulnerability of the hypoplastic lung to compression by the hernia, fetal lung liquid loss, and postnatal barotrauma [36]. Persistence of ASM dysfunction may also help explain increased rates of airway hyperresponsiveness in CDH survivors [6,7]. Hence, the smooth muscle hypothesis may help explain several of the key manifestations of clinical CDH. Moreover, if mesenchymal progenitors of pulmonary vascular smooth muscle (VSM) were similarly affected, this might help explain vascular hypermuscularisation and pulmonary hypertension in CDH
Fig. 2 An overview of the smooth muscle hypothesis of CDH: note the potential for a single early lesion (top) to generate early and late features of CDH (bottom). PSM indicates pulmonary smooth muscle.
434 [37]. Interestingly, the gene whose mutation has most recently been linked to CDH/eventration and lung hypoplasia in mice and human (Fog-2) is again expressed in relevant early mesenchyme before subsequent restriction to ASM and VSM lineages [38].
E.C. Jesudason stone, Mrs MG Connell, Dr DG Fernig, Ms NP Smith, Dr DG Spiller, Dr MRH White, Dr TV Burdyga, and Professor S Wray. This work was funded by The Academy of Medical Sciences/Health Foundation, The Medical Research Council, The Royal College of Surgeons of England and The Birth Defects Foundation, London, UK.
5. Future research and clinical opportunities The implications of the smooth muscle hypothesis for research and clinical practice merit brief consideration at this point. Firstly, given the careful application and resulting success of postnatal bgentilation,Q it seems logical to devote detailed consideration to prenatal airway pressures and compliance (particularly when applying tracheal occlusion). To do so may help optimise the PLUG technique (Plug the Lung Until it Grows) and assist it to become less quixotic in outcomes [39,40]. Secondly, given the intrinsic rhythmicity of prenatal ASM (and striated muscle breathing movements), it may be worthwhile testing a bdynamicQ PLUG that actually generates fluctuations in intraluminal pressure. One might even conceive of a remotely operated endotracheal bballoon-pumpQ providing a sort of gentle prenatal ventilation or bpreventilationQ [27]. Thirdly, given that prenatal steroids improve lung compliance in experimental CDH, it seems essential to approach an adequately powered multicentre trial once more [35].
6. Conclusion In summary, this article has drawn on clinical and recent experimental evidence to advance the hypothesis that CDH and lung hypoplasia result from an early lesion of diaphragmatic precursors and mesenchymal pulmonary smooth muscle progenitors. The latter then impairs embryonic FGF10-driven lung growth and persists to alter fetal lung compliance (with consequent vulnerability to prenatal compression and postnatal barotrauma). Moreover, vascular and airway hyperreactivity in CDH may thus both be understood in an early smooth muscle lesion. These elements of the smooth muscle hypothesis are eminently testable: doing so may help clinicians to unlock a further slender advantage in the management of this troubling human disease.
Acknowledgments This article is based on the author’s invited lecture Small lungs and suspect smooth muscle at The Congenital Diaphragmatic Hernia Symposium, chaired by Professor PD Losty and Professor P Puri at The British Association of Paediatric Surgeons 52nd Annual International Congress, 2005, Dublin, Ireland. The author thanks the particular contribution of Professor PD Losty and of Mr NC Feather-
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