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Lung development involves lung bud initiation, branching morphogenesis, saccular formation, alveolar septation, and accompanying vascular development that begins in the embryonic period and continues through fetal and postnatal periods.
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These developmental processes are regulated by diverse crosstalk between the airway epithelium and surrounding mesenchyme, which are highly coordinated by localized expression of transcriptional factors, growth factors, and extracellular matrix.
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Temporospatially regulated specific cell differentiation, proliferation, and survival and extracellular matrix deposition give rise to the final complex lung structure.
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Bronchopulmonary dysplasia is a chronic lung disease of premature infants characterized by a developmental arrest of the immature lung caused by injurious stimuli such as mechanical ventilation, oxygen exposure, and intrauterine or postnatal infections.
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Animal studies suggest that dysregulation of key signaling pathways plays an important role in neonatal lung injury, repair and subsequent development of bronchopulmonary dysplasia.
The first breath taken by newborns after birth initiates the transition from fetal to neonatal life. Successful transition is dependent on the lung to transport oxygen from the atmosphere into the bloodstream and to release carbon dioxide from the bloodstream into the atmosphere. This exchange of gases takes place in the alveoli, the terminal units of the lung that consist of an epithelial layer surrounded by capillaries, and supported by extracellular matrix (ECM). The alveolocapillary barrier is thin and covers a large surface area to maximize gas exchange. The human lung achieves a final gas diffusion surface of 70 m 2 with 0.2 mm in thickness by young adulthood and is capable of supporting systemic oxygen consumption ranging between 250 mL/min at rest and 5500 mL/min during maximal exercise. To facilitate the development of such a large, diffusible interface of the epithelial layer with the circulation, the embryonic lung undergoes branching morphogenesis to form a vast network of branched airways and subsequent formation and multiplication of alveoli by septation during the late stage of fetal lung development. By the time the full-term infant is born there are about 50 million alveoli in the lungs that provide sufficient gas exchange to sustain extrauterine life. Postnatally, alveoli continue to grow in size and number by septation to form approximately 300 million units in the adult lung. A matching capillary network develops in close apposition to the alveolar surface beginning in the middle to late stage of fetal development and continuing through postnatal development, which in the adult can accommodate pulmonary blood flow rising from 4 L/min at rest to 40 L/min during maximal exercise.
Our understanding of basic lung developmental processes has been improved through extensive studies in mouse molecular genetics and genomics. It is well recognized that these developmental processes are regulated by diverse crosstalk between the airway epithelium and surrounding mesenchyme, which are highly coordinated by transcriptional factors, growth factors and ECM residing in the lung microenvironment. Specific temporospatial cell proliferation, differentiation, migration, and apoptosis orchestrated by this interplay give rise to the complex lung structure that prepares for the first breath. Genetic mutations, physical forces, intrauterine infection, and particularly premature birth, can disrupt these developmental processes, thus resulting in defective lung development that can lead to respiratory failure and death.
Bronchopulmonary dysplasia (BPD) is a chronic lung disease of premature infants characterized by a developmental arrest of the immature lung caused by injurious stimuli such as mechanical ventilation, oxygen exposure, and intrauterine or postnatal infections. Data from animal studies suggest that dysregulation of key signaling pathways plays an important role in neonatal lung injury, repair, and subsequent development of BPD. Therefore basic knowledge about lung developmental processes and their cellular and molecular regulatory mechanisms is essential to understand lung injury and repair. This may lead to novel strategies to prevent and manage neonatal lung diseases, particularly BPD.
This chapter provides a brief overview of normal lung developmental processes, the key signaling pathways and proposed models of regulation of lung budding, branching morphogenesis, alveolarization, and vascular development. It also describes how injury from mechanical ventilation and oxygen exposure modulates key pathways, thus affecting neonatal lung development in the context of prematurity.
Overview of Lung Developmental Stages
Human lung development begins with the formation of airway primordia from the embryonic foregut that subsequently undergoes branching morphogenesis to form the conducting airways with expansion of the terminal airways, in combination with epithelial cell differentiation and vascular development to form the alveoli. Based on histologic appearance, lung development is classically divided into five overlapping stages: embryonic, pseudoglandular, canalicular, saccular, and alveolar ( Fig. 1.1 ). Distinctive histologic and structural changes at each stage of lung development have been well described, although the regulatory mechanisms responsible for these changes are not fully understood. There are striking similarities between human and mouse lung development. In fact, most of the current knowledge of lung developmental biology is acquired from mouse molecular genetics and genomic studies. This section reviews the key events during each of the lung developmental stages in mice and humans with a goal to better understand the regulatory mechanisms controlling this intricate process.
Embryonic Stage
The embryonic stage of human lung development occurs from 4 to 7 weeks of gestation. The lung bud originates as the laryngotracheal groove from the ventral surface of the primitive foregut. The proximal portion of the laryngotracheal groove separates dorsoventrally from the primitive esophagus to form the tracheal rudiment, which gives rise to the left and right main stem bronchi by branching into the ventrolateral mesenchyme derived from the splanchnic mesoderm. Subsequently, the right main bronchus branches to form three lobar bronchi, and the left main bronchus branches to form two lobar bronchi. The embryonic stage of mouse lung development occurs from embryonic day (E) 9 to E14, which begins as the formation of two endodermal buds from the ventral side of the primitive foregut. The single foregut tube then separates into the trachea containing the two primary lung buds and esophagus by inward movement of lateral mesodermal ridges, which proceed in a posterior to anterior direction. The two primary lung buds subsequently grow and branch into the splanchnic mesenchyme, with the right bud giving rise to four lobar bronchi and the left bud giving rise to a single lobar bronchus. During this stage, the trachea, primary bronchi, and major airways are lined with undifferentiated columnar epithelium.
Molecular Regulation of Lung Bud Initiation and Tracheoesophageal Separation
The processes and molecular regulators for lung bud initiation and tracheoesophageal separation are not fully established. However, mouse models have demonstrated that localized expression of key transcription factors and growth factors is essential during these processes ( Fig. 1.2 ). Nkx2.1 (also known as thyroid transcription factor 1) is the earliest known transcriptional factor expressed in endodermal cells in the prospective lung/tracheal region of the anterior foregut. Deletion of the Nkx2.1 gene in mice results in abnormal lung formation with two main bronchi that give rise to cystic structures. Additional studies have also demonstrated that Nkx2.1 is essential for distal lung epithelial cell differentiation and expression of surfactant protein C (SP-C). Expression of Nkx2.1 in the foregut endoderm is regulated by wingless/int (Wnt)-β-catenin signaling. Combined loss of Wnt2 and Wnt2b , which are expressed in the mesoderm surrounding the anterior foregut, or of β-catenin in the endoderm, leads to loss of Nkx2.1 expression and failure of foregut separation. Nkx2.1 expression is inhibited by transforming growth factor β (TGF-β) signaling for lung epithelial progenitor cell fate determination.
Sonic hedgehog ( Shh ) is expressed in the ventral foregut endoderm as early as E9.5 and appears to mediate early signaling between the endoderm and mesoderm. Shh mediates its effects via GLI–Kruppel family member ( Gli ) 2/3 transcriptional factors present in the mesoderm. Mice with a targeted deletion of Shh gene have foregut defects with tracheoesophageal atresia/stenosis, tracheoesophageal fistula, and tracheal and lung anomalies. Gli2 –/– mice have unilobar left and right lungs and GLi3 –/– mice present with reductions in shape and size of pulmonary segmental branches. Recent studies found that Gli2 –/– ;GLi3 –/– double-knockout mice have a hypoplastic foregut that completely lacked Nkx2.1-positive respiratory progenitors. In addition, Wnt2/2b transcripts were also lost or dramatically downregulated in these embryos.
Signaling mediated by fibroblast growth factor 10 (FGF10) and its receptor 2b (FGFR2b) is crucial for lung bud initiation. FGF10 belongs to an increasingly large and complex family of growth factors that signal through four cognate tyrosine kinases FGFRs. FGF10 is a chemotactic and proliferation factor for lung endoderm that is expressed in the mesenchyme at the prospective sites of lung bud formation. T-box transcription factors Tbx2 , Tbx3 , and Tbx4 are coexpressed with Fgf10 in the mesenchyme, which may positively regulate Fgf10 expression. The essential role of FGF10 in lung bud initiation is highlighted by the findings that deletion of Fgf10 gene results in lung agenesis in mice.
Retinoic acid (RA) also plays a role in lung bud formation. RA is produced in the mesoderm and can act on mesoderm and adjacent endoderm. RA, acting via the RA receptor β (RARβ) in the mesoderm, regulates Fgf10 expression by integrating the Wnt and TGF-β pathways, to promote lung bud formation. Dysregulation of bioactive RA synthesis through genetic mutation of retinaldehyde dehydrogenase 2 ( Raldh2 –/– ), and pharmacologic inhibition of RA generation results in upregulation of TGF-β in the foregut and lung agenesis. Thus RA–TGF-β–FGF10 interact with each other to control the initiation of the lung bud.
Our understanding of the critical signals required for initial lung budding and tracheoesophageal separation is incomplete and many other factors are likely to be involved. Knowledge gained from mouse studies will likely provide new insights into human congenital anomalies such as lung or tracheal agenesis, esophageal atresia, and tracheoesophageal fistula.
Pseudoglandular Stage
During the pseudoglandular stage (5–17 weeks in humans, E14–E16.5 in mice), the airway epithelial tubules undergo reproducible, bilaterally asymmetric, and stereotypical branching to form a treelike structure, which gives rise to 16 generations of conducting airways up to the level of terminal bronchioles. There is also proximal airway epithelial differentiation with the appearance of basal cells, goblet cells, pulmonary neuroendocrine cells, ciliated cells, and nonciliated columnar (Clara) cells. The surrounding mesenchymal cells differentiate into fibroblasts, myofibroblasts, smooth muscle cells, and chondrocytes to form muscle and cartilage around the proximal airways. Vascular growth is in close proximity to airway branching during this stage. By the end of the pseudoglandular stage, the conducting airways and their accompanying pulmonary and bronchial arteries develop in a pattern corresponding to that found in the adult lung.
Airway branching morphogenesis in the pseudoglandular stage is controlled not only by intrinsic factors but also by physical space in the pleural cavity. Formation of the diaphragm begins in the embryonic stage, continues through the pseudoglandular stage, and separates the pleural from the peritoneal cavities. Defects in the formation of the diaphragm, as in congenital diaphragmatic hernia, result in a decrease in the space within the pleural cavity, which in turn limits branching morphogenesis and vascularization, leading to variable degrees of pulmonary and vascular hypoplasia.
Epithelial-Mesenchymal Interactions Control Branching Morphogenesis
Following the formation of primary lung buds, the airway epithelial tubules undergo branching morphogenesis to form the respiratory tree. Although the process of airway branching morphogenesis is still far from being fully understood, the interactions between epithelium and mesenchyme orchestrated by compartmental transcriptional factors, growth factors, as well as ECM play critical roles. These interactions involve FGF10, heparan sulfate proteoglycan, SHH, bone morphogenetic protein (BMP), TGF-β, Wnts, Hox, and RA. These molecules are expressed in specific temporal, spatial, and cellular fashion, and together these signaling pathways coordinate reciprocal interactions between the epithelium and mesenchyme that control cell proliferation, differentiation, survival, and ultimately the number and size of airway branches ( Fig. 1.3 ).
FGF10-FGFR2b Signaling: Driving Force for Branching Morphogenesis
At the early stages of branching morphogenesis, Fgf10 is expressed in the mesenchyme surrounding the distal lung bud tip, whereas Fgfr2b is expressed at high levels along the entire proximal-distal axis of the airway endoderm. Extensive in vitro studies have demonstrated the critical role of FGF10 in stimulating budding in mouse embryonic lung explants. In mesenchyme-free embryonic lung bud cultures, addition of recombinant FGF10 to culture medium induces budding. Furthermore, placing an FGF10-soaked heparin bead, either in mesenchyme-free or whole lung bud cultures, induces bud elongation toward the FGF10 bead. These in vitro data combined with in vivo data showing lung agenesis in Fgf10 mutant mice indicate a critical role of FGF10 in driving branching morphogenesis. Interestingly, heparan sulfate side chains bind to growth factors, in particular, FGF10, to provide location-specific patterning that drives airway branching. Thus spatiotemporal expression and the signaling activity of FGF10 need to be precisely regulated during branching morphogenesis to control the specific sites of budding, bud elongation, and branching. Further, recent studies of lung explant cultures have found that Rac1, a small Rho guanosine triphosphatase, regulates FGF10 expression and branching morphogenesis.
Control of FGF10-FGFR2 Signaling by SHH and Sprouty
The exchange of signals between the growing bud and the surrounding mesenchyme establishes feedback responses that control the size and shape of the bud during branching. SHH, which is highly expressed in the distal lung epithelium, plays a role in controlling localized Fgf10 expression in the mesenchyme surrounding the distal lung bud tip (see Fig. 1.3 ). In lung explant cultures, expression of Fgf10 is inhibited by SHH. Similarly, in Shh transgenic mice, Fgf10 expression is downregulated in the lungs. Furthermore, in Shh − / − mice, Fgf10 expression is no longer restricted to the focal mesenchyme surrounding the distal bud, but becomes widespread throughout the distal mesenchyme. Recent studies have identified the PEA3 group ETS domain transcription factors ETV4 and ETV5 as mediators of the FGF-SHH negative feedback loop during branching morphogenesis.
Another antagonistic mechanism that interacts with FGF10 signaling is through Sprouty (Spry). In the developing mouse lung, Spry2 is present at the tips of the growing epithelial buds. FGF10 induces Spry2 expression in lung epithelium. Overexpression of Spry2 in the distal lung epithelium of transgenic mice severely impairs branching. However, Spry2- deficient mice have reduced branching and cystic formation and these structural anomalies are accompanied by reduced Fgf10 expression and increased Shh and Bmp4 expression. Thus the interplay of FGF10-SHH-Spry2 regulates early lung branching morphogenesis.
BMP Signaling Regulates Branching Morphogenesis
The BMP family contains more than 20 members that regulate many developmental processes, including lung development, and BMP4 is the best studied in lung branching morphogenesis. Bmp4 , Alk3 (the type I receptor of Bmp4 ), and the BMP signaling transducer, Smad1 , are present both in the epithelium and mesenchyme of the embryonic lung during early branching morphogenesis. Transgenic overexpression of Bmp4 in the distal epithelium causes abnormal lung morphogenesis with cystic terminal sacs. Interestingly, blockade of endogenous Bmp4 in embryonic mouse lung epithelium results in abnormal lung development with dilated terminal sacs, similar to those observed in Bmp4 transgenic mice. Conditional deletion of Alk3 in embryonic lung epithelium causes retardation of branching morphogenesis. Furthermore, abrogation of lung epithelial-specific Smad1 also resulted in retardation of lung branching morphogenesis. These findings suggest that balanced BMP4 signaling is important for in vivo lung branching morphogenesis, although the precise mechanisms remain unclear.
TGF-β Signaling Inhibits Branching
Members of the TGF-β family—TGF-β1, TGF-β2, and TGF-β3—have been implicated in lung airway branching. During lung branching morphogenesis, Tgf β messenger ribonucleic acid (mRNA) is expressed in the mesenchyme adjacent to the epithelium. However, TGF-β1 protein accumulates in stalks and in regions between buds, where ECM components collagen I, collagen III, and fibronectin are also present. Tgfβ1 −/− mice develop severe pulmonary inflammation postnatally, whereas Tgfβ2 gene deletion results in embryonic lethality at E14.5 with abnormal branching morphogenesis. Tgfβ3 −/− mice present with cleft palate, retarded lung development, and neonatal lethality. In contrast, overexpression of Tgfβ1 in embryonic lung epithelium decreases airway and vascular development as well as epithelial cell differentiation. Many in vitro studies have demonstrated that exogenous TGF-β1 severely inhibits embryonic lung branching and epithelial differentiation, but stimulates mesenchymal differentiation by inducing ectopic expression of α-smooth muscle actin (α-SMA) and collagen. TGF-β1 also markedly inhibits Fgf10 expression in lung explant culture. Abrogation of TGF-β signaling transducers Smad2 , Smad3 , and Smad4 significantly affects branching. Cumulatively, TGFβ signaling may be part of a mechanism that prevents FGF10 from being expressed in the mesenchyme of bud stalks or in more proximal regions of the lung. At these sites, TGF-β can also induce synthesis of ECM and prevent budding locally (see Fig. 1.3 ).
Wnt Signaling: Autocrine and Paracrine Effects on Branching Morphogenesis
The Wnt family constitutes a large family of secreted glycoproteins with highly conserved cysteine residues. Wnt ligands bind to the membrane receptors, frizzled (Fzd) and low-density lipoprotein receptor-related protein (LRP) 5 or 6, thus activating a diverse array of intracellular signaling, targeted gene transcription, and cellular responses. Canonical Wnt signaling, one of the best studied systems in lung development, involves nuclear translocation of β-catenin with subsequent interaction with members of T-cell–specific transcription factor (Tcf)/lymphoid enhancer-binding factor (Lef) family to induce target gene transcription. Several Wnt ligands, receptors, and components of the canonical pathway, such as β-catenin and Tcf/Lef transcription factors, are expressed in a highly cell-specific fashion in the developing lung. The role of Wnt/β-catenin signaling in branching morphogenesis has been examined by mouse mutagenesis as well as in embryonic lung explant culture. Epithelial-specific overexpression of Wnt5a results in decreased branching morphogenesis and increased enlargement of distal air spaces. These lungs showed increased FGF signaling in the mesenchyme and decreased SHH signaling in the epithelium. Conversely, targeted deletion of Wnt5a leads to overexpansion of distal airways and expanded interstitium, accompanied by increased Shh expression. Deletion of the Wnt4 gene results in severe lung hypoplasia, tracheal abnormalities, and decreased expression of Fgf10 and Wnt2. Loss of Fzd2 specifically in the developing lung epithelium results in defects in domain branch-point formation that alter the primary branching program of the lung. Epithelial-specific deletion of β-catenin or overexpression of Wnt inhibitor, dickkopf1 (Dkk1) results in disruption of distal airway development and expansion of proximal airways. Furthermore, inhibition of Wnt signaling by Dkk1 in vitro also results in disruption of branching morphogenesis and defective formation of pulmonary vascular network in embryonic lung explants. Clearly, the mechanisms by which Wnt signaling regulates lung branching morphogenesis are very complex. These may be related to the fact that multiple Wnt ligands exist in the embryonic lung and Wnt signaling is known to regulate epithelial and mesenchymal cell biology in an autocrine and paracrine fashion. In addition, both canonical/β-catenin and noncanonical Wnt signaling pathways probably play a role in lung branching morphogenesis. Furthermore, how Wnt signaling interacts with other key signaling pathways such as FGF, SHH, and BMP remains unclear.
Hox5 Genes Control Lung Patterning by Regulating Wnt/BMP4 Signaling
Hox genes are a highly conserved group of transcription factors controlling complex developmental processes in various organs. In human and mouse, 39 Hox genes are categorized into 13 paralog groups. Recent studies demonstrated that Hoxa5;Hoxb5;Hoxc5 triple mutation results in severe embryonic lung hypoplasia with reduced branching and proximal-distal patterning defects. Interestingly, loss of function of Hox5 leads to loss of expression of Wnt2/2b in the distal lung mesenchyme and downregulation of downstream targets, including Left1 , Axin2 , and Bmp4 . Thus Hox5 genes act as key upstream mesenchymal regulators of Wnt2/Wnt2b-BMP4 signaling that is critical for lung branching morphogenesis.
In summary, lung branching morphogenesis is controlled by epithelial-mesenchymal interactions that are orchestrated by a network of groups of transcriptional factors, growth factors, and ECM. Additionally, other molecules, such as integrins, fibronectin, and matrix metalloproteinases, that are dynamically expressed during lung development also play a role in lung branching morphogenesis. Along with airway tubule budding, elongation, and branching, specific cell differentiation occurs in the endoderm and mesenchymal compartments. Furthermore, the regulatory mechanisms for proximal-distal patterning, establishing cell fate, and maintaining progenitor cells are likely even more complex. Physical forces such as intraluminal fluid pressure also play an important role in branching morphogenesis. Understanding the mechanisms of how the fluid is produced, and how fluid pressure is sensed and maintained has clinical implications in understanding congenital pulmonary hypoplasia. More importantly, defining these mechanisms may help developing fetal therapies to enhance lung growth in the face of lung hypoplasia.
Canalicular Stage
During the canalicular stage (16–26 weeks in humans, E16.5–E17.5 in mice), the terminal bronchioles continue to branch to form the final seven generations of the respiratory tree. The respiratory bronchioles branch out from the terminal bronchioles to form the prospective acini, which are accompanied by development of the capillary bed, the beginning of alveolar type II epithelial (ATII) cell differentiation to synthesize surfactant phospholipids and proteins, and the thinning of the surrounding mesenchymal tissues. The lung appears “canalized” as capillaries begin to arrange themselves around the air space and come into close apposition with the overlying epithelium. At sites of apposition, thinning of the epithelium occurs to form the first sites of the air-blood barrier. Thus if a fetus is born at around 24 weeks, the end of the canalicular stage, these primitive acini have the capacity to perform some gas exchange.
Saccular Stage
The saccular stage in humans occurs from 24 to 36 weeks. During this stage, primary septation results in clusters of thin-walled saccules in the distal lung to form the alveolar ducts, the last generation of airways before the development of alveoli. Small mesenchymal ridges develop on the saccular walls to initiate secondary septation. The capillaries form a “double capillary network” within the relatively broad and cellular intersaccular septae. The ATII cells are further differentiated and become functionally mature with the ability to produce surfactant. The alveolar type I epithelial (ATI) cells differentiate from the ATII cells in close apposition to the thinning capillaries to produce the final gas-exchange unit. The interstitium between the air spaces becomes thinner as the result of decreased collagen fiber deposition. Furthermore, elastic fibers are deposited in the interstitium, which lays the foundation for subsequent secondary septation and the formation of alveoli. The process of saccular formation in mice is quite similar to that in humans; however, the timing of the saccular stage in mice begins at E17.5 and continues up to postnatal day (P) 5.
Alveolar Stage
During the alveolar stage (36 weeks to childhood in humans, P5 to P30 in mice), the saccules are subdivided by the ingrowth of ridges or crests known as secondary septae. The ATII and ATI cells continue to differentiate. Postnatally, the alveoli continue to multiply by increasing secondary septation. Between birth and adulthood, the alveolar surface area expands nearly 20-fold. In this stage, the double capillary network undergoes maturation, fusing into a single layer to allow for efficient gas exchange. Thus capillary volume increases 35-fold from birth to adulthood. In mouse lung development, alveolarization is completely a postnatal event. At birth, the mouse lung is in the saccular stage, equivalent to the human lung at 26 to 32 weeks’ gestation. Mouse alveologenesis begins around P5 and continues up to P30. This postnatal pattern of mouse alveolar development provides an excellent model system for mechanistic studies to understand neonatal lung injury and repair in preterm infants.
Regulatory Mechanisms of Alveologenesis
During the saccular stage, the primary septae are tightly associated with the vascular plexus, with ECM rich in elastin, and with still poorly defined mesenchymal cell types, including precursors of myofibroblasts. The endoderm begins to differentiate into two main specialized cell types of the future ATII and ATI cells. During alveolarization the sacs are subdivided by the ingrowth of secondary septae. Both myofibroblast progenitors and endothelial cells migrate into these crests, and a scaffold of matrix proteins is deposited, enriched in elastin at the tip. This development of secondary septae and formation of alveoli involves highly coordinated interactions of myofibroblasts, epithelial cells and microvascular endothelial cells, and proper deposition of ECM, particularly elastin ( Fig. 1.4 ). In contrast to the extensive knowledge of the regulatory mechanisms of branching morphogenesis, it has been challenging to identify the molecular mechanisms that regulate cell proliferation, differentiation, migration, and ECM deposition in alveologenesis. This is in part because mouse mutagenesis often profoundly affects the earliest stages of lung development, thus resulting in cessation of lung development and/or death before the initiation of sacculation and alveolarization. Nevertheless, several signaling pathways have been proposed to play a role in regulating alveolar development.
Myofibroblast Differentiation and Elastin Deposition: Key Events for Alveolar Septation
The alveolar myofibroblasts have long been recognized to play an essential role in alveolar septation, and platelet-derived growth factor (PDGF) is probably one of the most important factors that regulates myofibroblast differentiation. It has been proposed that myofibroblasts are differentiated from alveolar interstitial lipofibroblasts, which “traffic” lipids and store retinoids. Confocal microscopy revealed that lipofibroblasts with high lipid content are located at the base of alveolar septa and express low levels of PDGF receptor α (PDGFRα). The same study showed that cells expressing high levels of PDGFRα have the characteristic of myofibroblasts located at the alveolar entry ring. These cells express α-SMA, contain contractile elements, and also produce tropoelastin, the soluble precursor of elastin. Elastin is assembled by cross-linking of tropoelastin under the action of lysyl oxidase in the ECM environment. PDGFα, a strong chemoattractant for fibroblasts, is produced by alveolar epithelial cells. The importance of PDGFα, myofibroblasts, and elastin in alveolar septation was demonstrated by early studies in Pdgf α − / − mice that have a profound deficiency in alveolar myofibroblasts and associated bundles of elastin fibers, resulting in absence of secondary septa and definitive alveoli. Inhibition of PDGF signaling by imatinib, a PDGF receptor antagonist, impaired alveolarization in the postnatal lung. It has been suggested that in the absence of PDGFα, alveolar myofibroblasts or their precursors fail to migrate to the sites where elastin deposition and septation should occur. Furthermore, this migration to the sites of septal budding is not a random phenomenon, and a morphogen gradient is likely established to tightly regulate PDGFα production, myofibroblast differentiation and migration, and elastin deposition, thus providing instruction for the precise and specific localization of secondary septae ( Fig. 1.5 ).
In addition, other molecules have been implicated in alveolarization by directly or indirectly affecting PDGF signaling, myofibroblast differentiation and migration, and elastin assembly. Members of the FGF family play an important role not only in branching morphogenesis but also in alveolarization. Multiple FGFs and FGFRs are expressed in the lung during late-stage development. The critical role for the FGF pathway in alveolar development was demonstrated by the phenotype of lungs of Fgfr3/Fgfr4 double-mutant mice that failed to undergo secondary septation. RA is known to be involved in not just early lung morphogenesis but also alveolar development. Synthesizing enzymes, receptors, and signaling transducers of RA are abundant during alveolar septation. Mice with deletions of RA receptors fail to form normal alveoli. Precisely how RA signaling regulates alveolarization is not well understood. There is evidence for RA crosstalk with PDGF and FGF signaling. There is also evidence that epithelial Notch signaling regulates alveolar formation through induction of PDGFα in ATII cells and activation of PDGFRα signaling in alveolar myofibroblast progenitors.
External stressors also affect alveolarization (see Fig. 1.4 ). Thus exposure to starvation results in inhibition of alveolarization, which is reversed with refeeding. Further, both hypoxia and hyperoxia during the neonatal period in mice are associated with increased TGF-β and disruption of alveolarization. Interestingly, exposure to even low doses of corticosteroids completely inhibits alveolarization, which is irreversible and extends to adulthood. These are relevant studies because preterm infants are often nutritionally deprived, exposed to repeated hyperoxemia and hypoxemia, and are given corticosteroids for blood pressure support and as antiinflammatory therapy to allow weaning from respiratory support.
Development of Airway Epithelial Lineages
Following branching morphogenesis, epithelial cell differentiation along distinct lineages into mature conducting airway and distal alveolar epithelial cells is necessary for appropriate lung function. Many transcription factors regulate proximal epithelial cell fate, including Sox2, FoxJ1, and Notch. Thus Sox2 regulates differentiation of proximal endotherm progenitors into mature lineages. FoxJ1 is required for the formation of the multiciliated cells from Sox2-positive progenitors, and Notch signaling is important in establishing and maintaining a proper balance of the various differentiated proximal airway cell types as well as directing the differentiation of basal cells in the adult lung.
As lung development goes through canalicular, saccular, and alveolar stages, distal airway multipotent progenitors begin to generate differentiated distal alveolar epithelial cells, ATI and ATII. Although classical studies suggested that ATI cells arise from ATII cells, recent studies propose that distal tip progenitors contain bipotent progenitor cells that express both ATI and ATII markers late in gestation. These data suggest that these pluripotent progenitors can differentiate into ATI and ATII cells as development prodeeds.
The Surfactant System
Surfactant proteins (SPs) and lipids are synthesized primarily by ATII cells. There are four SPs—SP-A, SP-B, SP-C, and SP-D—that have been characterized to date. SP-A and SP-D are members of the collectin family and confer innate immunity as they have carbohydrate recognition domains that allow them to bind bacteria and viruses, promoting phagocytosis by macrophages. SP-B and SP-C are highly hydrophobic, segregate into the lipid fraction of surfactant, and are assembled with surfactant phospholipids into lamellar bodies that are the storage organelles for surfactant before secretion. Nkx2.1 regulates the surfactant-associated genes and is required for normal surfactant synthesis at birth. Phospholipids, the principal component of pulmonary surfactant, consist primarily of phosphatidylcholine (PC) and phosphatidylglycerol. PC and phosphatidylglycerol are produced by ATII cells, dramatically increasing in late gestation. Adenosine triphosphate–binding cassette transporter 3 (ABCA3), located on the limiting membrane of the lamellar body, is required for PC to be transferred to the lamellar body. In the lamellar body, SP-B and SP-C are assembled with PC into bilayer membranes. The surfactant contents in the lamellar body are secreted into the airway upon stimulation by catecholamines, purinoreceptor agonists, and cell stretch. GPR116, an orphan G-protein–coupled receptor located on respiratory epithelial cells, regulates surfactant secretion because Gpr116 mutation in mouse results in excess secretion of surfactant that accumulates in the air spaces after birth.
Regulation of Pulmonary Vascular Development
The lung vasculature is constituted by the pulmonary and bronchial vascular systems. The pulmonary system consists of pulmonary arteries that carry blood to the alveolar capillary network to be oxygenated; oxygenated blood returns through pulmonary veins back to the heart. The bronchial system supplies oxygen and nutrients to the nonrespiratory portion of the lung, including the bronchial walls and perihilar region. The molecular basis of pulmonary vascular development is not well understood but is increasingly recognized as being controlled by epithelial-endothelial as well as endothelial-mesenchymal crosstalk.
Vascular Morphogenesis
It is generally believed that early pulmonary vascular development involves three processes to establish a circulatory network: angiogenesis, vasculogenesis, and fusion. Angiogenesis is defined as formation of new blood vessels from preexisting vasculature. New vessels sprout via a well-defined program: degradation of the basement membrane, endothelial cell differentiation and migration, formation of solid sprouts of endothelial cells, and restructuring of the sprout into a luminal line by endothelial cells that is finally integrated into the vascular network. Vasculogenesis is defined as de novo formation of blood vessels from angioblasts or endothelial precursor cells arising in the mesodermal mesenchyme. Earlier studies by DeMello et al. indicated that the proximal vessels are generated by angiogenesis, whereas the distal vessels are formed by vasculogenesis during lung morphogenesis. The proximal and distal vessels fuse to establish the luminal connection via a lytic process. Using analysis of serial sections of human embryos, this group suggested that the same processes also occur during human lung formation. However, this concept has been challenged in later studies. Work from Schachtner et al. suggested that vasculogenesis is primarily responsible for both proximal and distal vascular formation during lung development. Studies by Hall et al. in human embryos have also indicated that intrapulmonary arteries originate from a continuous expansion and coalescence of a primary capillary plexus that would form by vasculogenesis during the pseudoglandular stage. They have also indicated that the pulmonary veins are formed by the same mechanism. Parera et al. have proposed distal angiogenesis as a new concept for early pulmonary vascular morphogenesis. Schwarz et al. have also proposed that initial pulmonary vessel formation within the mesenchyme is predominantly angiogenic.
VEGF-Mediated Epithelial-Endothelial Interaction in Vascular and Alveolar Development
There is increasing evidence that epithelial-endothelial interactions play important roles in vascularization and alveolarization. Vascular endothelial growth factor (VEGF) is a key angiogenic factor known to play important roles in these processes. VEGF stimulates proliferation, migration, differentiation, and tube formation in endothelial cells. These effects are largely elicited by VEGF binding to the high-affinity VEGF receptor 2 (VEGFR2) or Flk-1 on endothelial cells. During normal mouse lung development, various VEGF isoforms (VEGF122, VEGF164, VEGF188) are present in epithelial cells, and their expression increases during later canalicular and saccular stages, when most of the vessel growth occurs in the lung. In contrast, VEGFR2 and VEGFR1 (Flt-1) are expressed in the adjacent endothelial cells. Individual knockouts for Vegf, Vegfr2 , and Vegfr1 result in embryonic lethality before the development of the lung capillary plexus. Targeted deletion of the Vegf gene in respiratory epithelium results in an almost complete absence of pulmonary capillaries and this defective vascular formation is associated with a defect in primary septal formation. Interestingly, these structural defects are coupled with suppression of epithelial proliferation and decreased hepatocyte growth factor (HGF) expression in endothelial cells. Furthermore, targeted deletion of the Hgf receptor gene in the epithelium results in a similar septation defect as seen in VEGF-deleted lungs. These data highlight the mechanisms by which VEGF and HGF signaling pathways orchestrate the reciprocal interactions between airway epithelium and the surrounding endothelium during septation. Recent studies showed that conditional inactivation of Vegf during alveologenesis not only decreased pulmonary capillary and alveolar development but also altered RA expression. Treatment with RA partially improved vascular and alveolar development induced by VEGF inhibition. Thus VEGF and RA signaling interact to regulate vascularization and alveologenesis. VEGF signaling is also differentially regulated by FGF9 and SHH signaling during mouse lung development. Mesenchymal expression of VEGF is regulated by gain and loss of function of Fgf9 and Vegf is required for Fgf9 -induced pulmonary blood vessel formation. Shh , on the other hand, regulates the pattern of Vegf expression rather than the content because loss of Shh signaling did not affect Vegf expression in subepithelial mesenchyme, but decreased Vegf expression in the submesothelial mesenchyme. Nitric oxide (NO) is known to mediate VEGF angiogenic activity. In a neonatal rat model, treatment with SU5416, a VEGFR inhibitor, results in both disrupted angiogenesis and alveolarization and this is associated with a decreased content of endothelial NO synthase (eNOS) and NO production. In contrast, inhaled NO improves alveolar development and pulmonary hypertension in VEGFR inhibitor-treated rats. Further evidence of the importance of NO in alveolarization and vascularization was demonstrated by the combined disruption of alveolarization and paucity of distal arteries observed in Nos- deficient fetal and neonatal mice.
Additional Angiogenic Factors in Vascular Development
Angiopoietins (Ang) and Tie signaling are also known to play important roles in vascular morphogenesis and homeostasis. Ang/Tie signaling is known to play a primary role in the later stages of vascular development and in adult vasculature, where they control remodeling and stabilization of vessels. Ang1 appears to work in complementary fashion with VEGF during early vascular development. VEGF appears to initiate vascular formation, and Ang1 promotes subsequent vascular remodeling, maturation, and stabilization, perhaps, in part, by supporting interactions between endothelial cells and surrounding support cells and ECM. The role of Ang/Tie in developmental angiogenesis is highlighted by the early embryonic lethality and significant abnormal vascular development observed in offspring of Ang1 − / − , Tie1 − / − , Tie2 − − as well as Tie1 − / − /Tie2 − − mice. The specific role of Ang/Tie signaling in pulmonary vascular development is poorly understood. Studies have shown that Ang1 is expressed in lungs of newborn mice and its expression is increased from P1 to P14, whereas Ang2 is abundantly expressed at birth and decreases inversely with Ang1. Transgenic overexpression of a potent form of Ang1 protein, COMP- Ang1 in lung epithelium resulted in abnormal alveolar and vascular structure and 50% lethality at birth owing to respiratory failure. During postnatal lung development, Ang/Tie2 signaling is regulated by the Wnt ligand-receptor, LRP5. Thus precise regulation of Tie2 signaling through an Ang1 and Ang2 expression switch is important to construct a mature lung vascular network required for normal lung development. Ang1 also plays a role in pulmonary hypertension because lung overexpression of Ang1 causes severe pulmonary hypertension and Ang1 is increased in lungs from pulmonary hypertensive patients. However, cell-based Ang1 gene transfer protects against monocrotaline-induced experimental pulmonary hypertension.
Other angiogenic signaling pathways, such as BMP, Notch, TGF-β, Wnts, and the Eph family of receptor tyrosine kinases and their membrane tethered ligands Ephrins, are likely involved in pulmonary vascular development. More studies are needed to define the regulatory mechanisms of these important pathways and the interactions among these pathways during normal and abnormal lung vascular morphogenesis.
Lung Injury and Repair: Disruption of Normal Lung Development
With its vast airway and alveolar epithelium open to the atmosphere, the newborn lung is at great risk for harmful environmental insults, such as oxidative stress, physical forces, and infective agents. These environmental challenges place the lung under constant threat of injury, requiring coordinated defense repair and remodeling processes. The lungs of full-term neonates have a great ability to overcome various injuries, to generate needed repair and remodeling appropriately, and ultimately to maintain and/or restore normal lung architecture and function. When premature delivery occurs, particularly between 24 and 28 weeks, the lungs of these preterm infants are in the late canalicular to early saccular stage. Alveolarization has not yet begun and surfactant production is minimal. Lungs in the canalicular to early saccular stage are poorly compliant, whereas the chest wall is extremely compliant. A large proportion of infants born at these early gestational ages have significant respiratory failure and often require respiratory support including mechanical ventilation and oxygen therapy. These lungs are therefore at great risk for injury, altered development, and BPD.
Over the past four decades, with advances in neonatal intensive care—such as the introduction of antenatal corticosteroids, exogenous surfactant therapy, and gentler ventilator strategies—the survival of extremely premature infants has been significantly improved. However, during the same period, the incidence of BPD has not changed appreciably. This is likely due to the fact that infants of lower gestational ages are now surviving. BPD is now recognized as a developmental arrest of lung development at the saccular stage caused by injurious stimuli, such as mechanical ventilation, oxygen exposure, and intrauterine or postnatal infections. Larger and simplified distal air spaces and decreased vascular growth are the key pathologic features observed in the lungs of infants dying of BPD. The combination of decreased vascular growth and excessive pulmonary vascular remodeling leads to pulmonary hypertension, which significantly contributes to the morbidity and mortality of these infants. The largely unchanged incidence of BPD has not only provided tremendous challenges in the management of these patients, but has also spurned the need for new knowledge of the molecular basis of neonatal lung injury and repair. Experimental models of BPD have used both larger animals such as preterm baboons and sheep, and smaller animals such as rats and mice. These studies attempt to create the BPD phenotype by exposing immature baboons and sheep, or neonatal rats and mice to noxious stimuli such as mechanical ventilation, hyperoxia, and/or infection. Extensive data generated from these studies indicate that the key signaling pathways regulating normal lung development can be disrupted by injurious stimuli in the immature lung, thereby playing important roles in the pathogenesis of BPD. Although many factors are involved in neonatal lung injury, this section emphasizes the role of inflammasomes, VEGF, TGF-β, and connective tissue growth factor (CTGF) ( Fig. 1.6 ).
