Recent advances in our understanding of the mechanisms of late lung development and bronchopulmonary dysplasia
Abstract
The objective of lung development is to generate an organ of gas exchange that provides both a thin gas diffusion barrier and a large gas diffusion surface area, which concomitantly generates a steep gas diffusion concentration gradient. As such, the lung is perfectly structured to undertake the function of gas exchange: a large number of small alveoli provide extensive surface area within the limited volume of the lung, and a delicate alveolo-capillary barrier brings circulating blood into close proximity to the inspired air. Efficient movement of inspired air and circulating blood through the conducting airways and conducting vessels, respectively, generates steep oxygen and carbon dioxide concentration gradients across the alveolo-capillary barrier, providing ideal conditions for effective diffusion of both gases during breathing. The development of the gas exchange apparatus of the lung occurs during the second phase of lung development—namely, late lung development—which includes the canalicular, saccular, and alveolar stages of lung development. It is during these stages of lung development that preterm-born infants are delivered, when the lung is not yet competent for effective gas exchange. These infants may develop bronchopulmonary dysplasia (BPD), a syndrome complicated by disturbances to the development of the alveoli and the pulmonary vasculature. It is the objective of this review to update the reader about recent developments that further our understanding of the mechanisms of lung alveolarization and vascularization and the pathogenesis of BPD and other neonatal lung diseases that feature lung hypoplasia.
Introduction
“No organ in the body has been the object of a greater number of erroneous descriptions and interpretation of normal and abnormal processes than has the lung.” (August 24th, 1948)—Edith L. Potter, M.D. (1901–1993)
Edith Louise Potter founded the modern subspecialty of perinatal pathology in the early 1930s. Although Dr. Potter will be best remembered for her extraordinary work on the facial characteristics of infants with bilateral renal agenesis (“Potter’s facies”), with which her name is eponymously linked, her words from 1948 (429), quoted above, remain very true today. Mother Nature has yet to give up most of her secrets about how healthy and diseased lungs develop, and to date there remains little agreement about how an alveolus—the core gas exchange unit of the lung—actually forms and how alveolar development might be disordered in preterm and term newborns in whom disorders of lung development are sometimes present. These disorders include bronchopulmonary dysplasia (BPD) (549), congenital diaphragmatic hernia (CDH) (272), congenital lung malformations that occur in the lung below the carina, including alveolar capillary dysplasia (ACD) (65), airway malformations (412), and a multitude of other clinically challenging neonatal lung pathologies.
The lung undertakes many physiological functions; most notably, the lung facilitates gas exchange: the directional diffusion of oxygen in the inspired air, from the alveolar air spaces into the bloodstream, and, concurrently, the directional movement of carbon dioxide present in the bloodstream into the alveolar air spaces, which is subsequently exhaled. To effectively facilitate gas exchange by diffusion, the structure of the lung must adequately satisfy the requirements of effective diffusion as set out by the German physician and physiologist Adolf Eugen Fick in what has become known as Fick’s laws (156). According to Fick’s laws, the passive diffusion of gases is governed by 1) the available surface area, 2) the diffusion distance, and 3) the concentration gradient. To this end, a lung optimized for gas exchange should have a large surface area and a thin diffusion barrier and should facilitate the establishment of a steep concentration gradient for gas diffusion in the correct direction. The lung structure beautifully fulfils these criteria, and it is the objective of lung development to generate an organ that—insofar as possible—satisfies these criteria. This review largely ignores the early phases of lung development, including the embryonic, pseudoglandular, and canalicular stages (Table 1), and focuses on the saccular and alveolar stages of lung development, which largely comprise so-called late lung development, which is when the development of the gas exchange regions of the lung takes place.
| Period, days | ||||||
|---|---|---|---|---|---|---|
| Animal | Term | Embryonic | Pseudoglandular | Canalicular | Saccular | Alveolar |
| Mouse | 20 | E9.5–E12 | E12–E16.5 | E16.5–E17.5 | E17.5–P4 | P4–P21 |
| Rat | 21–24 | E11–E13 | E13–E18.5 | E18.5–E20 | E21–P4 | P4–P21 |
| Rabbit | 32 | Up to E18 | E21–E24 | E24–E27 | From E27 | From E30 |
| Sheep | 147 | E17–E40 | E40–E80 | E80–E110 | E110–E130 | From E130 |
| Pig | 115 | Up to E25 | E22–E56 | E56–E98 | From E99 | From E104 |
| Baboon | 168–185 | Up to E42 | Up to E80 | E80–E120 | E120–E140 | From E140 |
| Human | 280 | Up to E42 | E52–E112 | E112–E168 | From E168 | From E252 |
The gas exchange unit of the lung is the alveolus, where the “business” of gas exchange is undertaken over the alveolo-capillary barrier. In the alveolus, inspired air is brought into very close proximity to the circulating blood (217), since the blood-air barrier is very thin (200 nm–2 µm in humans) and is permeable to many gases including oxygen and carbon dioxide. These properties of the blood-air barrier facilitate gas exchange and thereby satisfy the demand of Fick’s laws for as narrow as possible a diffusion distance. To this end, as lung development proceeds through the saccular and alveolar stages (Table 1), there is a marked thinning of the walls of the primitive air spaces, which become saccules and, ultimately, alveoli. A reduction in the thickness of the blood-air barrier is achieved largely by thinning of the septal walls of the sacculi and alveoli. In mice, the bulk of septal wall thinning takes place postnatally, when the septal wall thickness decreases from ≈20 μm at postnatal day (P)5 to ≈5 μm at P28 (430). Thus the process of lung development effectively generates a very thin diffusion barrier. Lung development also strives to generate as large a surface area as possible over which gas diffusion may occur. This is ingeniously achieved through progressive subdivisions of the primitive air spaces, where the first acini are born in the pseudoglandular stage, where the acinar airways are formed (472), followed by epithelial cell differentiation in the canalicular stage that allows distinction between the conducting and respiratory airways (472). During the canalicular stage, bronchoalveolar duct junctions are formed. Respiratory bronchioles are absent in the lungs of rodents, where these junctions are thus at the entrance to the acinus. Johannes Schittny and coworkers have recently demonstrated that the number of acini in rodents is determined in the canalicular stage and remains constant throughout adolescence and into adulthood (49). The pulmonary acini are effectively clusters of gas-exchanging airways that are ventilated by terminal bronchioles. In contrast to rodents, in higher mammals such as humans, the acinus is located four generations (of respiratory bronchioles) proximal to the bronchoalveolar duct junctions (472). The expansion of the gas exchange surface begins in earnest in the saccular stage, when the widening ends of the acinar airways form saccules and the widening of these saccules occurs concomitantly with condensation of the mesenchyme. Primary septa are formed by the region between two saccules. Because the capillary network remains closely associated with the saccule, primary septa contain two capillary layers, each capillary layer associated with “its” saccule. Mice and rats are born in the saccular stage of lung development, while in humans normal lung sacculation occurs in utero (472). A massive expansion of the gas exchange surface area of the lung takes place during the alveolar stage, when existing saccules are subdivided by new (secondary) septa that are generated from the primary septa, forming alveoli. How this occurs continues to be a matter of debate, although the remodeling of the extracellular matrix (ECM), and elastin in particular (75), has been ascribed a key mechanistic role. A phase of microvascular maturation accompanies later alveolarization, where the double capillary layer originally present in the saccule walls condenses to form a single capillary layer. It is generally accepted that (at least in rodents) alveolarization is biphasic, with an accelerated rate of generation of alveoli in the immediate postnatal period followed by a comparatively slower rate of generation of alveoli in later life (430, 472, 537). Several exciting new theories have been forwarded in the past 30 months to explain how saccules are subdivided to form alveoli, including the “net or crest” idea of Kelsey Branchfield and Xin Sun (71). Irrespective of how alveoli are generated, the massive burst of alveolarization that occurs during the alveolar stage of lung development generates the very large gas exchange surface area required by the lung for efficient gas diffusion. In mice, the alveolus density increases by 900% over the period P5–P14 (430). The third requirement of Fick’s laws, the need for a steep concentration gradient, to which gas diffusion is proportional, is satisfied by efficient breathing, where the conducting airways allow the delivery of inspired air containing high O2 and low CO2 (relative to the deoxygenated blood) concentrations to the ventilatory unit (the region of the alveolar ducts), which forms a concentration gradient with the correspondingly comparatively low O2 and high CO2 in the blood delivered to the gas exchange regions by the precapillary pulmonary arterioles. As such, the heart and the neuromuscular control of breathing work in concert to generate steep gas concentration gradients that facilitate O2 and CO2 exchange across the alveolo-capillary barrier.
When lung development is disordered, serious life-threatening disease can develop. This is particularly noteworthy in preterm-born or term-born infants, in whom disturbances to late lung development cause dangerous respiratory failure. The most common disorders of late lung development encountered in a neonatal intensive care setting are BPD and CDH, although many others also exist. Given our poor understanding of normal and aberrant late lung development, there is much interest in developing our knowledge about how alveoli develop, as well as understanding how key pathologies that generate disordered lung development, including BPD (109) and CDH (136), evolve. Uncovering new developmental pathways may reveal new targets amenable to pharmacological manipulation for the medical management of clinical disorders of lung development, either to protect the developing lung from damage or to promote lung repair and regeneration (225, 499). To this end, the lung development and clinical neonatology communities continue to explore the molecular mechanisms of normal lung development (364) and the factors that influence the postnatal maturation of the mammalian lung (562). It is the objective of this review to update the reader about advances in our understanding of normal and aberrant lung alveolarization reported in the literature since the beginning of 2015. It is intended that this update will pick up where the last biannual review (485) ended. This article reviews the identification of new players and processes that direct normal and aberrant late lung development and reports the use of pharmacological and other interventions, as well as transgenic animal studies, to study late lung development. As evident in this review, growth factors, inflammation, the lung vasculature, and the ECM continue to be explored as mediators of lung alveolarization. However, recent new developments reported here include very welcome and increasing attention paid to the role of maternal, enteral, and parenteral nutrition in lung alveolarization; as well as attention to extrapulmonary organ systems and the placenta. Increasing consideration of changes in lung function alongside changes in lung structure that are highlighted in this review also represents welcome movement in an important and often neglected direction. Several new technologies as well as extensions of existing technologies to study lung alveolarization are also reviewed, together with the availability of new resources to the lung development community.
Mediators of Lung Development
Growth factors and signal transduction.
A key mechanism by which injurious insults such as hyperoxia deregulate lung development is through disturbances to signal transduction pathways that direct lung organogenesis (428). The transforming growth factor (TGF)-β superfamily of polypeptide growth factors, which has already received much attention as a mediator of both normal (17, 18, 331, 544) and aberrant (16, 366, 380, 575) lung alveolarization in experimental animals and in clinical subjects (533), continues to draw interest. Further validation of a role for deregulated TGF-β signaling in aberrant lung alveolarization includes observations by Vineet Bhandari and coworkers (508) that TGF-β1, ostensibly via type II TGF-β receptor (TGFBR2) signaling, stunts alveolarization in mice. TGF-β signaling may proceed through many pathways, including two canonical pathways, where the activin-like kinase type of type I TGF-β receptor (TGFBR1) may drive TGF-β signaling through the second messengers SMAD family member 2 (SMAD2) and SMAD3 (for classical TGF-β signaling), while the alternative type I TGF-β receptor, activin A receptor-like type 1 (ACVRL1), may drive TGF-β signaling through the second messengers SMAD1, SMAD5, and SMAD8 [typical of bone morphogenetic protein (BMP) signaling]. Both pathways have received recent attention, where, for example, an imbalance in the TGFBR1/SMAD2/SMAD3 and ACVRL1/SMAD1/SMAD5/SMAD8 axes of the canonical TGF-β signaling pathways has been proposed (240). These data are interesting, given the ability of glucocorticoids—which are used in the medical management of preterm birth and BPD—to shift the balance between these two signaling pathways (475).
Notably, other recent developments include the genetic ablation of the unfortunately named “TGF-β induced” (Tgfbi) expression, using a Tgfbi::lacZ gene trap (8) that resulted in blunted alveolarization, with concomitant perturbations to gas exchange efficiency, assessed by the diffusion capacity for carbon monoxide (DlCO), as well as reduced elastic recoil. Interestingly, the lung volumes of wild-type and knockout animals were similar. It is noteworthy that the respiratory function and compliance measurements were made in adult animals, not neonates. This report adds to the list of TGF-β superfamily members that are causal players in lung alveolarization. Hyperoxia exposure of newborn mice caused temporal changes in Tgfbi steady-state levels in the lung, which were reduced early but increased later. However, no lung injury studies using Tgfbi::lacZ mice were undertaken; thus the functional contribution of Tgfbi to arrested alveolarization has yet to be demonstrated.
Peroxynitrite has also been implicated as an upstream regulator of not only TGF-β but also insulin-like growth factor I (IGF-I, product of the Igf1 gene) activity during aberrant lung development (53). In an in vitro system, nitration of the IGF-I receptor (IGF1R) prevented binding of IGF-I and reduced IGF1R activation. Furthermore, these investigators documented a causal role for TGF-β signaling in aberrant alveolarization caused by exposure of newborn rat pups to hyperoxia, using the TGFBR1 inhibitor SB431542, where alveolarization was improved under hyperoxia conditions. In parallel, daily administration of the peroxynitrite decomposition catalyst 5,10,15,20-tetrakis(4-sulfonatophenyl)porphyrinato Fe(III) chloride (FeTPPS) to newborn mouse pups reduced steady-state mRNA levels of Tgfb1 (encoding the TGF-β1 ligand) that were increased by hyperoxia exposure. The investigators thus proposed a multifunctional role for peroxynitrite in oxygen-arrested alveolarization, where peroxynitrite may block IGF-I signaling and may promote TGF-β signaling. A clear limitation of this study was the lack of morphometric studies with the FeTPPs intervention, which are eagerly awaited. However, insight into the regulation of IGF-I in the lung is noteworthy, given the proposed roles for IGF-I in disorders both of fetal development and during early postnatal life (201, 202).
Studies on TGF-β in lung development are not restricted to preclinical BPD models, where recent work has also highlighted deregulated TGF-β signaling after tracheal occlusion of the fetus, a widely employed surgical management strategy for CDH. Both in clinical subjects with CDH and in a preclinical surgical model of CDH in rabbits, tracheal occlusion led to increased steady-state levels of Tgfb1 transcripts, although it is not yet clear whether this phenomenon had any functional pathological consequences (550).
The regulation of TGF-β signaling in the developing lung by endogenous mediators has also received attention, where a role for latent TGF-β-binding protein 4 (LTBP4) in regulation of TGF-β levels was further explored (120). Terminal air space development was defective in Ltbp4−/− mice (500), where, in combination with various TGF-β ligand-knockout mice, TGF-β2 was identified as the ligand that impacts secondary septation in Ltbp4−/− mice. Furthermore, by expressing in vivo an LTBP4 variant that is defective for TGF-β binding, it was demonstrated that the formation of LTBP4-latent TGF-β complexes was not required for normal lung development. Rather than an effect on TGF-β function, the impact of LTBP4 on regulating fibulin-5-dependent elastic fiber assembly was highlighted as the pathogenic pathway underlying defective secondary septation in Ltbp4−/− mice.
The spate of reports on stem cell therapy in preclinical models of BPD has also touched on the growth factor area. Intravenous application of bone marrow mesenchymal stem cell (MSC) cultures together with intraperitoneal erythropoietin to newborn mice exposed to hyperoxia resulted in improved alveolarization (312). This improvement was accompanied by blunted TGF-β signaling, which was evident by reduced TGF-β1 levels, and reduced phosphorylation of SMAD2 and SMAD3. It is noteworthy that vascular endothelial growth factor (VEGF) protein levels and increased capillary density in lung sections also accompanied improved alveolarization.
The alternative branch of the TGF-β superfamily, namely, the BMP family of peptide growth factors, has also been implicated in postnatal lung maturation, where BMP signaling was documented to be essential for surfactant production in neonates, facilitating respiratory adaptation to the extrauterine environment (315). Apart from TGF-β and BMP signaling, several other growth factor signaling systems have been demonstrated to have protective effects in the hyperoxia-based preclinical models of BPD, including administration of substance P (218) and upregulation of epidermal growth factor-like domain 7 (EGFL7) expression in newborn rats by erythropoietin administration (114), with other pathways such as the Hippo signaling pathway awaiting attention (462).
Oxidants and oxidative stress.
The use of oxygen supplementation has highlighted oxygen toxicity and associated oxidative stress as a key pathomechanism underlying defective alveolarization in both a clinical and a preclinical context. In the case of the former, reports continue to emerge documenting reduced levels of antioxidants in preterm infants who go on to develop BPD (3). Interestingly, a recent clinical study suggested that a combination of early oxygen supplementation together with parenteral nutrition was additive, being associated with prolonged oxidative stress and thus increased risk of BPD (362). This report highlighted the need to carefully consider the level of oxygen supplementation, particularly in combination with parenteral nutrition, and further suggested the need to develop safer formulations for parenteral nutrition (362). The supplementation of parenteral nutrition to prevent loss of alveoli in a preclinical model in guinea pigs has also been examined, where guinea pig neonates receiving parenteral nutrition supplemented with oxidized glutathione (glutathione disulfide, GSSG) appeared to have better alveolar development (151). The somewhat unconventional experimental model of loss of alveoli and the unconventional analysis of alveoli number employed in this study highlight a pressing need for the validation of this interesting approach in alternative BPD models using well-established lung structure analyses. Taken together, these studies suggest that parenteral nutrition, in combination either with an additional oxidative stress (in this case, oxygen) or with concomitant redox mediator (in this case, GSSG) application, has the capacity to influence lung alveolarization. This area certainly warrants further exploration.
In a hyperoxia-based BPD model in rats, hyperoxia induced DNA strand breaks, concomitant with upregulated expression of the DNA repair enzyme 8-oxoguanine DNA glycosylase 1 in alveolar type (AT)II cells, suggesting that hyperoxia exposure not only damages DNA in the lung epithelium but also engages the DNA repair machinery (239).
Sequence variants of antioxidant response genes have also been considered in terms of BPD susceptibility, where the hypomorphic NQO1 [encoding NAD(P)H quinone dehydrogenase 1] single-nucleotide polymorphism (SNP; in this case, rs1800566)—in a homozygous state—was associated with increased incidence of BPD (467), while the NFE2L2 (encoding nuclear factor, erythroid derived 2, like 2; also called NRF2) SNP rs6721961 was associated with decreased incidence of severe BPD (467). After adjustment for epidemiological confounders, the NQO1 and NFE2L2 SNPs were associated with BPD and severe BPD, respectively. These findings, which require validation in other cohorts, are most interesting, given other reports in preclinical models. For example, Hye-Youn Cho and colleagues (102) documented that an arrest of alveolarization, as well as lung edema and inflammation provoked by hyperoxia, was more severe in Nfe2l2−/− mouse pups. In stark contrast, Sharon McGrath-Morrow and colleagues (348) documented that while NFE2L2 activation induced the expression of antioxidant genes NFE2L2 did not attenuate arrested alveolarization in neonates. This was documented after exposure of Nfe2l2−/− mice to hyperoxia and by activation of Nfe2l2 expression with sulforaphane in utero. Both groups used the same mouse strain background and approximately the same duration and intensity of oxygen injury; therefore, the explanation for this paradox is not clear, and the physiological role of the NFE2L2 SNP rs6721961 in arrested alveolarization and in BPD remains to be clarified.
The association of the hypomorphic NQO1 SNP rs1800566 with BPD is also interesting, given that this polymorphism has been independently associated with BPD by another group (175). The NQO1 gene product detoxifies genotoxic products of oxidative stress. A role for NQO1 in protecting alveolarization from oxidative stress has been documented in preclinical studies. For example, arrested alveolarization caused by hyperoxia is exaggerated in Cyp1a1−/− mice (which are deficient in cytochrome P-450, family 1, subfamily a, polypeptide 1), and this can be prevented by β-naphthoflavone administration, which, among other effects, increases steady-state levels of Nqo1 mRNA (337). Thus NQO1 appears to be a most interesting candidate pathogenic mediator and candidate intervention target in arrested alveolarization.
A role for the lung antioxidant machinery in affording protection against oxidative insult has been validated for superoxide dismutase 3 (SOD3; also called extracellular superoxide dismutase, EC-SOD), where Sod3−/− mice exhibited arrested alveolarization (129). This arrested alveolarization was exacerbated after intraperitoneal bleomycin treatment and was accompanied by the development of pulmonary hypertension (PH) and right ventricular remodeling (129), in a model thought to involve the angiotensinogen/angiotensin II system (4). These data suggest a role for SOD3 in protecting the lung from oxygen toxicity. In contrast, Sod2+/− mice (which express reduced levels of superoxide dismutase 2) appeared to be affected similarly to wild-type mice after exposure to hyperoxia (homozygous null mutants were not viable). However, the statistical analysis of the alveolar region did not include a comparison of hyperoxia-treated wild-type vs. Sod2+/− mice (188). These data suggested that a heterozygous state of Sod2 does not alter the hyperoxia-induced arrest of lung alveolarization in experimental BPD.
Inflammation.
Several interesting recent reports have expanded our knowledge about how inflammatory processes modulate lung organogenesis, which has also been the subject of recent reviews (44, 367, 479). In the context of epithelial-mesenchymal interactions, the inducible overexpression of an activated inhibitor of κB (IκB) kinase β (IKBKB; also called IKKβ) in the developing lung epithelium, with a reverse tetracycline transactivator (rtTA) induction system together with the secretoglobin family 1A member 1 (Scgb1a1, also called Cc10) promoter, impaired saccular development in embryonic and newborn mouse pups (54), as assessed by mean distal air space area in the immediate postnatal period. The “epithelial-derived inflammation” disrupted elastic fiber organization and reduced steady-state levels of several components of the elastin fiber assembly machinery, including fibulins, lysyl oxidases, and fibrillin 1 (FBN1). Together, these data demonstrate that activation of innate immune pathways in the airway epithelium of developing lungs disrupts elastic fiber assembly and lung saccular development.
Related to the study described above, the pharmacological modulation of nuclear factor (NF)-κB has also been undertaken, where Cristina Alvira and coworkers inhibited NF-κB signaling with BAY 11-7082 (215) in mice injected with Escherichia coli O127:B8 lipopolysaccharide (LPS) to induce systemic (including lung) inflammation and examined the effects of the intervention on distal lung structure. These investigators demonstrated the utility of promoting some NF-κB inflammatory signaling and, using a neutralizing antibody, the utility of suppressing damaging chemokine (C-X-C motif) ligand 2 [CXCL2; also called macrophage inflammatory protein (MIP)-2] production, to promote lung alveolarization. Clearly much remains to be learned about the contribution of NF-κB signaling to postnatal lung maturation.
A key role for forkhead box M1 (FOXM1) in directing the response of the lung to hyperoxia has also been demonstrated (569), where genetic abrogation of Foxm1 gene expression in myeloid-derived inflammatory cells was achieved with LysM-Cre+/−/Foxm1fl/fl mice. Loss of FoxM1 from myeloid-lineage inflammatory cells led to an exaggerated response to hyperoxia on lung structure, which was associated with reduced macrophage abundance in the lungs. These data add to a growing body of evidence of the involvement of forkhead box (FOX) proteins in lung developmental pathology (511).
Chemoattractants that modulate inflammatory cell dynamics have also been studied in animal models of BPD, including aminoacyl tRNA synthetase complex-interacting multifunctional protein 1 (AIMP1, also called endothelial monocyte-activating polypeptide II, EMAPII), which mediates myeloid cell trafficking (279). Recombinant AIMP1 administration to healthy mice worsened alveolar architecture and promoted cardiac remodeling. Administration of an anti-AIMP1 antibody to mice with concomitant exposure to hyperoxia limited macrophage recruitment and attenuated the impact of hyperoxia on alveolar architecture and cardiac remodeling. A second study on inflammatory mediators identified leukotriene B4 as a mediator of both macrophage influx and PH, in an experimental BPD model based on intraperitoneal administration of bleomycin to rat pups (146). However, targeting leukotriene pathways with zileuton (a 5-lipoxygenase inhibitor), montelukast (a cysteinyl leukotriene receptor antagonist), or SC57461A (a leukotriene A4 hydrolase inhibitor) did not prevent abnormal lung morphology caused by bleomycin, although zileuton improved pulmonary hemodynamics and SC57461 improved cardiac remodeling.
A connection between inflammation and TGF-β signaling, elastogenesis, and epithelial-to-mesenchymal transition (EMT) has also been proposed, where two in vivo interventions in mice in the hyperoxia-based BPD model have been undertaken. Using the interleukin (IL)-1 receptor antagonist (IL1RN; also called IL-1Ra) to block IL1A (also called IL-1α) and IL1B (also called IL-1β) signaling led the investigators to suggest that chronic inflammation-derived IL1B, upstream of integrin αvβ6 and TGF-β, misdirected elastogenesis during aberrant lung alveolarization (557). This study built on continued work by Claudia Nold-Petry and coworkers that explored the utility of IL1RN to promote alveolarization in experimental animal models of BPD, where the anti-inflammatory and alveolarization-promoting effects of IL1RN were demonstrated be superior to the effects of protein C in a combined intrauterine E. coli LPS-hyperoxia study (456) and where ILRN exhibited demonstrable utility in limiting hyperoxia-provoked airway fibrosis but not airway hyperreactivity (455). Along these lines, alternative IL-1 receptor (IL1RL1) antagonists, including the competitive inhibitor anakinra (Kineret) and the potent noncompetitive inhibitor 101.10, have also found utility in inflammation-based models of preterm birth (375). In a related study (297), the utility of IL1RN was validated as being protective against hyperoxia-induced perturbations to alveolarization. Subsequently, the role of the NACHT, LRR (NLR), and PYD domains-containing protein (NLRP) 3 inflammasome in experimental BPD was examined, since inflammasomes have already been linked to abnormal fetal airway development (502). Modulation of the NLRP1, NLRP3, and NLR family CARD domain-containing protein (NLRC) 4 inflammasome subsets continues to draw interest in lung pathology, with several exciting approaches now described to modulate inflammasome function in the lung, including through the modulation of expression of the histone deacetylase sirtuin 1 (SIRT1) (171), the purinergic receptor P2X7 receptor (PR2X7) (164), the inflammasome adaptor protein called PYD and CARD domain containing (PYCARD, also called ASC) (88), and the LYN protooncogene, Src family tyrosine kinase (LYN) (172), as well as exposure to carbon monoxide (249). In the context of experimental BPD, the NLRP3 inflammasome was inactivated or inhibited, either with Nlrp3−/− mice or with the inhibitor glyburide, where inactivation or inhibition of the NLRP3 inflammasome protected lung alveolarization from hyperoxia. The inflammasome has also been indirectly targeted with NSC23766, a specific Rac family small GTPase 1 (RAC1) inhibitor, which demonstrated a similar beneficial impact on lung alveolarization, lung vascular development, and cardiac remodeling in experimental BPD (224).
Inflammatory cells per se are also emerging as key mediators of both normal and aberrant lung development. Mark Krasnow and coworkers (519) recently beautifully documented that macrophages populate the lung in three “developmental waves,” with each wave giving rise to a distinct lineage, that in addition to expressing different markers also reside in different locations and, surprisingly, demonstrate little interconversion. This report lays important groundwork for further studies on how development contributes to lung macrophage diversity. In a pathological context, the role of inflammatory cells in arrested lung alveolarization is often speculated at (459, 494). Two recent reports suggest abnormal CD8+ (technically, CD8a+) T cell effector function in cord blood of preterm neonates (470), as well as a proinflammatory cord blood CD4+ T cell status in chorioamnionitis and BPD, with decreased abundance of regulatory T cells in BPD (353). Taking inflammatory cell function into preclinical animal models of BPD, targeting integrin alpha M (ITGAM, also called CD11b)-marked inflammatory cells, with a diphtheria toxin-based CD11b cell-depletion strategy and exposure of mouse pups to hyperoxia, a lung-protective population of anti-inflammatory CD11b+ mononuclear cells was identified (150). This study exclusively addressed lung injury, and follow-up studies on lung architecture are eagerly awaited, particularly considering the emergence of methodologies to target CD11b cells in vivo (1, 343).
Modulation of leukocyte recruitment has been demonstrated to have some utility in preclinical BPD models using leukadherin-1, an agonist of the leukocyte surface integrin CD11b/CD18, which enhances leukocyte adhesion to the endothelium and thus reduces transendothelial migration and influx of leukocytes to sites of recruitment (231). Administration of leukadherin-1 to rat pups exposed to hyperoxia decreased macrophage infiltration and partially restored normal lung alveolarization and vascular development. The modulation of leukocyte recruitment has also been undertaken, targeting C-X-C motif chemokine receptor 4 (CXCR4) (139), where the CXCR4 antagonist AMD3100 was applied to rat pups exposed to hyperoxia. Antagonism of CXCR4 under hyperoxic conditions resulted in improved alveolarization, improved lung vascularization, improved pulmonary hemodynamics and cardiac remodeling, and reduced inflammatory cell abundance in bronchoalveolar lavage (BAL) fluids. All of these studies highlight the utility of modulating inflammatory events to rescue aberrant lung alveolarization.
IL-10 also continues to attract attention in the context of BPD, where steady-state IL-10 levels are known to be suppressed in experimental BPD, differences in plasma IL-10 levels have been associated with different patterns of BPD (118), and reductions in IL-10 levels have been associated with aging and increased susceptibility to infection (563). By way of developments, one recent in vitro study has suggested that IL-10 may revert to normal the Janus kinase (JAK) and tyrosine kinase 2 (TYK2) signaling that is suppressed in fetal rat ATII cells under hyperoxic conditions (281). The relevance of this observation for lung development in vivo remains to be documented.
Proteases and protease inhibitors.
Proteases and protease inhibitors have recognized roles in mammalian lung development (273, 354), which also extends to air sac development in invertebrates such as Drosophila melanogaster (137). A protease/protease inhibitor imbalance is currently regarded as a key contributing factor to arrested alveolarization associated with BPD (354), although much remains to be clarified about how various proteases together with the respective cognate inhibitors may impact normal and aberrant lung development. Proteases play an important role in both the production and maturation of components of the ECM as well as the degradation of the ECM and, as such, are well placed to be pivotal mediators of structural changes to the developing lung. In addition, protease activity regulates the abundance of other proteinaceous mediators of lung development, such as receptors, and proteases both generate matrikines and other signaling molecules and regulate (through degradation) the abundance of these signaling molecules, which orchestrate lung development (265, 354). To date, both neutrophil elastase and matrix metalloproteinase (MMP) members have received—and continue to receive—attention, with one recent report suggesting that low serum levels of tissue inhibitor of metalloproteinases inhibitor-2 (TIMP-2) at birth may be associated with subsequent development of BPD (278). Along similar lines, another recent study generated an Adamts18 [encoding a disintegrin-like and metallopeptidase (reprolysin type) with thrombospondin type 1 motif, 18]-knockout mouse (38), where decreased cellularity of septa was evident by visual inspection of lung sections, suggesting that ADAMTS18 plays a causal role in alveolarization. However, there was no difference in lung architecture by 8 wk of age, and no studies in BPD models were reported. The authors also suggested that ADAMTS18 may play a role in airway branching, where adjacent airways noted in lung sections from adult mice appeared to be in closer proximity to one another in Adamts18−/− mice; however, a three-dimensional analysis, such as bronchial casts or micro computed tomography (microCT) imaging would provide much better supporting data.
A closely related member of the ADAMTS (a disintegrin and metalloproteinase with thrombospondin motifs) family, ADAMTS-like 2 (ADAMTSL2), which lacks a protease domain, has also been implicated in lung and airway development. Mutations in ADAMTSL2 are associated with acromicric dysplasia, which in addition to short stature and brachydactyly, includes tracheal-bronchial stenosis. Dirk Hubmacher and coworkers (222) deleted exon 5 of the Adamtsl2 gene in mice, where a pronounced bronchial epithelial dysplasia was noted that occluded the bronchial lumen, causing perinatal lethality. Bronchial epithelial dysplasia was accompanied by a dramatic increase in fibrillin 2 (FBN2) and microfibrillar-associated protein 2 (MFAP2; also called microfibril-associated glycoprotein-1, MAGP1). While Adamtsl2−/− mice exhibited increased TGF-β signaling, administration of a pan-neutralizing anti-TGF-β1,2,3 antibody did not revert or reduce bronchial epithelial dysplasia, leading the authors to conclude that mechanisms other than an impact on TGF-β signaling underlay the perturbations to the bronchial epithelium noted in Adamtsl2−/− mice. It remains of interest to explore the impact of ADAMTSL2 on normal and aberrant postnatal lung development.
Mechanisms of deregulation of protease expression other than hyperoxia have also been explored. For example, in adult mice that underwent unilateral pneumonectomy, a pronounced increase in steady-state levels of chymotrypsin-like elastase family member 1 (CELA1) was noted, which was further demonstrated to exhibit stretch-dependent binding to the lung ECM (246). These data are in line with another report from the same group that documented the exponential increase in lung elastase activity commensurate with increased physical strain (582). These data contribute new information to our understanding of how physical strain induces lung matrix remodeling. It remains of interest to test these ideas in preclinical BPD models.
Cysteine proteinases have also received attention in the context of aberrant lung alveolarization, where overexpression of cathepsin K (CTSK) in mice led to the formation of enlarged air spaces under normoxic conditions (258). Despite slightly enlarged distal air spaces in CTSK-overexpressing mice, the hyperoxic environment was initially better tolerated in comparison with wild-type mice. The mechanisms explaining the actions of CTSK under hyperoxic conditions remain to be elucidated.
Extracellular matrix.
The elastic theory of lung development (75) places the elastic properties of elastin fibers at the center of lung development. Indeed, any perturbations to elastin expression and elastin fiber assembly generally result in defective alveologenesis. Anne Hilgendorff and coworkers (208) employed elastin haploinsufficient Eln+/− mice to explore the role of elastin gene dose on lung alveolarization in mechanically ventilated mouse pups. While Eln+/− mouse pups exhibited 50% less tropoelastin and approximately doubled the amount of collagen I and lysyl oxidase protein, no impact of elastin haploinsufficiency on lung alveolarization was noted. However, some disturbances to the lung vasculature were evident, including a reduction in the capillary density, as well as alterations to the medial walls of intra-acinar vessels.
Observations such as these have increased interest in the molecular machinery that directs elastin fiber formation, such as lysyl oxidases. Ivana Mižíková and coworkers (356) inhibited lysyl oxidases by daily administration of the pan-lysyl oxidase inhibitor β-aminopropionitrile (BAPN) in developing mouse pups in the background of hyperoxia exposure. While BAPN administration was without effect at P9.5, and worsened lung structure at P19.5, an improvement in elastin fiber morphology was noted, suggesting that while BAPN administration alone was not sufficient to improve alveolarization, some benefit for elastin fiber assembly was achieved. It would be interesting to explore the impact of BAPN administration in preclinical BPD models with less severe injurious insults (<85% O2), in the event that the potential benefit of the intervention was lost because of the extremely harsh level of oxygen toxicity. Remaining with lysyl oxidases, it is noteworthy that, in addition to a role in ECM maturation, lysyl oxidases have also recently been described to influence the lung fibroblast transcriptome (355), hinting at gene-regulatory matrix-independent roles for lysyl oxidases that may be relevant to lung development.
Gene ablation studies have also been undertaken to clarify roles for lysyl oxidases in lung development. Mice deficient in lysyl oxidase-like 3 (LOXL3) exhibited cleft palate and spinal deformity (591), as well as blunted embryonic lung development (590), with perturbations to the size of saccular structures. Additionally, lung volume was also decreased, perhaps because of a reduction in the size of the thoracic cavity (590). To date, Loxl3−/− mice have not been employed in preclinical models of BPD.
While lysyl oxidases have attracted much attention as mediators of elastic fiber assembly in arrested alveolarization in animal models of BPD, other mediators have also been identified, where hyperoxia exposure of newborn mice drove increased steady-state levels of the αv integrin, which was suggested to recruit elastin and disturb elastin fiber formation (191). However, no hierarchy was established in the study, and changes in integrin expression may be epiphenomenal or may be downstream of disturbed elastin fiber assembly. These issues remain to be clarified.
Perturbations to the expression of components of the lung ECM, including elastin, as well as components of the ECM assembly and remodeling machinery are not only induced by hyperoxia but also may be induced by mechanical stress. Kroon and colleagues (262) documented the impact of room air mechanical ventilation with different tidal volumes on gene regulation in rat pups. A pronounced increase in steady-state mRNA levels of the elastogenic genes Tnc (encoding tenascin C) Eln, Loxl1 (encoding lysyl oxidase-like 1), and Fbln5 (encoding fibulin 5) was noted after 8 h of high (defined as 25 ml/kg)-tidal-volume ventilation, while elevated steady-state levels of Eln, Loxl1, and Fbln5 mRNA were also increased after 12 h of medium (defined as 8.5 ml/kg)-tidal-volume ventilation. Interestingly, Pdgfra (encoding platelet-derived growth factor receptor α), which is critically important for alveolarization, was exquisitely sensitive to mechanical ventilation and was dramatically upregulated after 8 h of low (defined as 3.5 ml/kg)-, medium-, and high-tidal-volume ventilation with room air, which also impacted Vegfr1 mRNA steady-state levels in selected instances. Thus, similar to hyperoxia, mechanical ventilation may uncouple elastin synthesis and assembly, resulting in defective lung alveolarization and vascular development. In rodent models of CDH based on nitrofen administration to pregnant rats, the resultant embryos carrying diaphragmatic hernias also exhibited reduced lysyl oxidase mRNA steady-state levels, both in hypoplastic lungs and in the diaphragm muscle (516). These data are consistent with the presence of diaphragmatic hernias in lysyl oxidase-knockout mice (213, 322).
Collagens also continue to receive attention as pivotal elements of lung developmental processes (354). In addition to fibrillar collagens, the basement membrane collagens, namely, collagen type IV, have also received attention: dramatic changes in steady-state levels of Col4a1 (encoding collagen IVα1) and Col4a2 (encoding collagen IVα2) mRNA were noted during avian (Gallus gallus) blood-gas barrier development. Modulation of COL4A1 and COL4A2 expression with transgenic mice revealed a role for basement membrane collagen in epithelial-endothelial association, as well as septal myofibroblast proliferation, differentiation, and migration (307).
Noncoding RNA.
Noncoding RNAs, in particular, microRNAs, have emerged as pivotal regulators of organogenesis, and the lung is almost certainly no exception (26, 116, 245, 368). While the expression of many microRNA species is known to be deregulated in animal models of BPD (570), very few studies have addressed causal roles for microRNA species in normal and aberrant alveolarization (382, 384). Among the latest microRNA species reported to be deregulated by hyperoxia is miR-196a, where exposure of newborn but not adult mice to hyperoxia (179) caused a pronounced decrease in steady-state miR-196a levels in the lung, which was accompanied by increased hemoxygenase 1 (HMOX1) steady-state mRNA levels. Using Bach1 (encoding BTB and CNC homology 1, basic leucine zipper transcription factor 1)-knockout mice [which are more resistant to the effects of hyperoxia than wild-type mice (230)], the authors convincingly demonstrate that BACH1 mediated the impact of hyperoxia on steady-state miR-196a levels. Unfortunately, no lung morphology studies were undertaken; therefore, it remains unclear whether the hyperoxia/BACH1/miR-196a axis plays any causal role in normal or aberrant lung alveolarization. However, the study is most instructive, as it is one of the very few studies that address upstream regulation of microRNA expression rather than downstream targets of microRNA.
Data continue to be generated that implicate roles for microRNA in lung cell differentiation, a process that is most relevant to lung development. Among recent reports is a study that identified the regulation of miR-29a, miR-200b, miR-200c, and miR-21 expression during lung epithelial cell differentiation in human fetal lung explants (55), where a regulatory link between NK2 homeobox 1 (NKX2-1), miR-200c, and the transcription factors nuclear factor I B (NFIB) and myeloblastosis oncogene (MYB) in lung epithelial cells has also been established (513). It has been suggested (55) that the miR-200 family of microRNA, in a negative feedback loop with zinc finger E-box binding homeobox 1 (ZEB1) and TGF-β, regulate ATII cell differentiation in embryonic lungs. This interesting in vitro observation requires further development in vivo, to demonstrate a functional role for miR-200 family members during both normal and, perhaps, aberrant lung development. In a second study, a progressive reduction in miR-124 steady-state levels in the lung was noted over the course of lung development in rats (559). Additionally, NFIB, which is a target of oncofetal microRNA (52, 513) and is a regulator of lung development, was identified as a direct target of miR-124. Expression of miR-124 was predominant in the airway epithelium, and adenoviral overexpression of miR-124 in rat fetal lung explants inhibited lung maturation and ATII cell transdifferentiation (559). Thus both the miR-200 family and miR-124 have been implicated in distal epithelial cell differentiation in the developing lung; however, causal studies confirming the participation of either or both microRNAs in alveolarization have not yet been undertaken.
Concerning the regulation of microRNA expression in the developing lung, it is noteworthy that the expression of selected microRNA species in fetal lungs is androgen responsive in mice (70) and is glucocorticoid responsive in rats (584). Furthermore, in fetal lungs of macaques the expression of miR-155–5p (which targets Fgf9 mRNA, encoding fibroblast growth factor 9) was induced by group B Streptococcus inoculation into the choriodecidual space (341). These data are interesting, since a role for miR-140-Fgf9 interaction has been proposed in the developing lung epithelium (580) that might underlie the development of pleuropulmonary blastoma in mice after genetic ablation of Dicer1 (encoding dicer 1, ribonuclease III) in the epithelium (551, 580). Furthermore, this event has been linked with YY1 transcription factor (YY1) in lung branching morphogenesis (68).
Perturbed expression of several microRNA species has been described in lung tissues from patients with BPD. By screening tissues from BPD subjects, Lynette Rogers, Trent Tipple, and coworkers described decreased circulating plasma levels of miR-17, a member of the miR-17/92 cluster, in infants who subsequently developed BPD, and the magnitude of the decrease correlated with BPD severity (450, 451). Building on these important observations, reduced steady-state levels of miR-17, miR-18a, miR-19a, miR-20a, and miR-92 were noted in the lungs of mouse pups exposed to hyperoxia, where the nursing dams had also received intraperitoneal E. coli LPS (450). The reduced steady-state levels of members of the miR-17/92 cluster correlated with increased steady-state levels of three DNA methyltransferases, DNA methyltransferase 1 (DNMT1), DNA methyltransferase 3A (DNMT3A), and DNA methyltransferase 3A (DNMT3B), leading these investigators to propose that microRNA-mediated deregulation of DNA methyltransferase expression was a pathogenic contributor to BPD.
A second key study by Namasivayam Ambalavanan and coworkers on steady-state microRNA levels in BPD subjects revealed lower steady-state miR-489 levels in the lungs of BPD patients compared with control subjects (404). This trend was preserved in a preclinical model, where exposure of mice to hyperoxia resulted in reduced steady-state miR-489 levels in mouse lungs. These data led the authors to propose that the modulation of miR-489 targets, including Igf1 and Tnc, led to arrested lung alveolarization associated with BPD. Follow-up work in miR-489 conditional knockout mice is eagerly awaited, to further dissect this exciting pathogenic pathway.
In addition to the miR17/92 cluster and miR-489, miR-150 has also received attention as a mediator of aberrant lung alveolarization in experimental BPD. Steady-state miR-150 levels are reduced in the lungs of mouse pups exposed to hyperoxia (381). The steady-state levels of glycoprotein nonmetastatic melanoma protein B (GPNMB), a miR-150 target, were correspondingly increased when miR-150 levels were decreased. No causal role for miR-150 in arrested alveolarization could be demonstrated, since the lungs of miR-150−/− mice exhibited structure similar to wild-type mice, although some evidence of dysmorphic capillary structure in the lungs of miR-150−/− mice was presented (381).
Most recently, reduced circulating levels of miR-29b have been noted in plasma obtained from preterm infants during the first week of life who subsequently developed BPD, and decreased circulating miR-29b levels were inversely correlated with BPD severity (142). These observations were supported with a preclinical BPD model in mice in which lung alveolarization was arrested by intraperitoneal injection of E. coli LPS in pregnant dams followed by exposure of newborn mice to hyperoxia. Administration of miR-29b to mouse pups with an adeno-associated virus vector attenuated the impact of hyperoxia on lung alveolarization and improved ECM protein production and deposition.
Apart from BPD, perturbations to microRNA steady-state levels have also been reported in preclinical models of CDH, where administration of nitrofen to pregnant rats resulted in increased and decreased steady-state microRNA levels of 11 and 14 microRNA species, respectively, in the lungs of fetuses, with miR-33 being the most dramatically impacted microRNA (601). This study remains descriptive, with causal studies still to be performed. Remaining with CDH, miR-449a has been noted to regulate lung epithelial cell proliferation during mammalian and avian lung development, and reduced steady-state levels of miR-449a were noted in a CDH fetus, with correspondingly increased steady-state levels of the miR-449a target Mycn (encoding v-myc avian myelocytomatosis viral related oncogene, neuroblastoma derived; also called N-Myc) (468). Thus a role for reduced miR-449a steady-state levels in lung hypoplasia associated with CDH was proposed; however, this remains to be causally validated in an animal model.
Moving beyond microRNA, studies are starting to emerge that highlight roles for long noncoding (lnc) RNA in late lung development and BPD. These studies include a recent lncRNA microarray screen of lungs from mice exposed to hyperoxia, where increased steady-state levels of 882 and reduced steady-state levels of 887 lncRNA species, respectively, were noted. Furthermore, these authors predicted a role for the interaction between the AK033210 lncRNA and Tnc in experimental BPD; however, the causality of this interaction in terms of alveolarization remains to be validated (47).
Epithelial cells and epithelial cell plasticity.
The mechanisms by which injurious stimuli such as hyperoxia stunt alveolarization include perturbations to epithelial cell transdifferentiation, notably, the ATII↔ATI cell transdifferentiation process. Exposure of rats to hyperoxia caused increased transdifferentiation of ATII to ATI cells in vivo as well as in vitro (214). These data are noteworthy, given a recent exciting report by Jun Yang and coworkers that implicated ATI cells as key players in the development of the alveolar units (576). This was believed to involve a conformational “flattening” change in topology and a role for ATI cells in directing angiogenesis through VEGF-A production. These findings are most interesting, given the recent report of regulation of VEGF production in alveolar epithelial cells by nemo-like kinase (NLK), where Nlk−/− mice died with cyanosis within 36 h of birth and exhibited alveolar simplification (254). Thus these authors identified NLK as a regulator of epithelial-endothelial interactions in the developing lung.
The regulation of epithelial cell junction proteins during lung development has also received attention; for example, with the doxycycline-driven overexpression of claudin 6 (CLDN6) using a surfactant associated protein C (Sftpc)-rtTA-based system mice appeared to “slow” late lung development, where mouse lung development appeared to be retarded in the canalicular stage, with time-matched control mice having entered the saccular stage. These data indicate that CLDN6 either modulated or accompanied transition from the canalicular to the saccular lung (236). Remaining with junction proteins, hyperoxia exposure of ATII cells in vitro was recently demonstrated to disturb tight junctions through modulation of tight junction protein 1 (TJP1, also called ZO-1), occluding (OCLN), claudin 4 (CLDN4), and caveolin 1 (CAV1) (572). The in vivo relevance of this observation remains to be investigated.
The unfolded protein response (UPR) and endoplasmic reticulum (ER) stress have received recent attention as potential pathogenic contributors and candidate interventional targets in arrested lung development. Using a lung epithelium-specific knockout mouse that did not express 78-kDa glucose-regulated protein (GRP78), a master regulator of ER homeostasis and the UPR, Per Flodby and coworkers (157) documented that the absence of GRP78 in the epithelium disrupted lung alveolarization, generated altered ER structures, triggered UPR activation, and increased apoptosis. These data are supported by the observed increase in steady-state levels of proteins involved in ER stress and the UPR in a hyperoxia-based preclinical model of BPD (310).
In addition to key roles in the mesenchyme and fibroblasts discussed below, wingless-type MMTV integration site family (WNT) signaling has also been credited with roles in the epithelium during lung development (287, 539, 548), where WNT signaling was documented to play a role in bud formation by inducing apical constriction during the pseudoglandular stage. However, genetic constitutive activation of WNT signaling negatively regulated air sac formation, leading the investigators to conclude that loss of WNT signaling—to relieve apical constriction—was required to facilitate progression to the canalicular and saccular stages, allowing air sac formation (162). These data are interesting, considering that activation of WNT signaling results in the expansion of ATII cells, whereas inhibition of WNT signaling inhibits ATII cell development and also shunts alveolar epithelial development toward the ATI cell lineage. These findings revealed a wave of WNT-dependent ATII cell expansion that was required for lung alveologenesis and maturation (159). WNT signaling has also been implicated in ATI cell differentiation, where mesenchymal histone deacetylase 3 (HDAC3) impacted WNT/β-catenin (CTNNB1) signaling in the lung epithelium, and the absence of Hdac3 correlated with decreased WNT signaling and defective ATI cell differentiation (558). Recently described putative roles for WNT/CTNNB1 signaling in lung alveolarization are summarized in Fig. 1.
Fig. 1.Recently described roles for WNT/CTNNB1 activity in late lung development: schematic illustration integrating several recently identified functions of WNT/CTNNB1 signaling in late lung development, where WNT regulators (216, 440, 558) and WNT target genes (452) have been identified in the context of lung development and WNT proteins have been suggested to be maternally delivered to nursing neonates (360, 361). Furthermore, several physiological effects have been ascribed to WNT signaling, including the regulation of 1) alveolar type (AT)II cell expansion, 2) transdifferentiation of ATII cells to ATI cells (159, 162), and 3) myofibroblast progenitor differentiation (288). BMP-4, bone morphogenetic protein-4; HDAC3, histone deacetylase 3; HOX, homeobox.
More broadly considering bronchiolar-to-alveolar developmental transition, Emma Rawlins and coworkers have addressed whether lung epithelial progenitors are intrinsically programmed or alveolar cell identity is determined by environmental factors (274). In that study, it was demonstrated that epithelial identity is extrinsically determined, with both glucocorticoid and signal transducer and activator of transcription 3 (STAT3) signaling controlling the timing of alveolar initiation. Both glucocorticoids (57, 69, 207, 387, 397, 406, 469) and STAT3 (80, 98, 438, 594, 598) continue to receive attention as mediators of lung physiology and development. It is tempting to speculate that some of the effects of glucocorticoids on preparation of the lung for the transition into extrauterine life might relate to glucocorticoid regulation of chloride channels such as cystic fibrosis transmembrane conductance regulator (CFTR) (275), given that these and other channels are key mediators of alveolo-capillary barrier function (76, 336, 342, 476). It would additionally be most exciting to assess whether pharmacological modulation of these progenitor cell programming pathways might present an opportunity for alveolar regeneration, or for protecting the structural development of the lung from injurious insults.
The role of Notch signaling has also received recent attention, where Notch signaling was demonstrated to play a key role in epithelial-mesenchymal interactions required for alveolar formation. In that study, ATII cells were identified as a major site of NOTCH2 activation, and Notch signaling was required for ATII cell induction of platelet-derived growth factor α (PDGFA) ligands and paracrine activation of PDGFRA signaling in alveolar myofibroblast progenitors. A role for Notch signaling in the developing airways was also hinted at in another study (536).
Fibroblasts and fibroblast plasticity.
Fibroblasts and the lung mesenchyme continue to receive attention as key players that direct lung development (290, 344, 589). The WNT/CΤΝΝΒ1 signaling pathway was highlighted as a mediator of lung fibroblast biology, in the context of BPD, where Jennifer Sucre and coworkers reported nuclear phosphorylation of CTNNB1 at Tyr489 in epithelial and mesenchymal cells from fetal lungs as well as the lungs of patients who died with BPD but not in the lungs of term-born infants (504). In an in vitro organoid model, exposure of organoids to cyclic hypoxia/hyperoxia promoted nuclear localization of phospho-CΤΝΝΒ1(Tyr489), which was blocked by dasatinib, a tyrosine kinase inhibitor. Thus these authors suggested that CTNNB1 may play a role in arrested alveolarization associated with BPD.
The inhibition of CTNNB1 signaling has also been documented to protect against alveolar and vascular pathology in neonatal mouse lungs driven by connective tissue growth factor (CTGF) (452), where overexpression of CTGF in mouse lung epithelial cells (with the conditional, inducible Sftpc-rtTA driver line) caused alveolar simplification and reduced vascular density. Inhibition of CTNNB1 signaling with ICG001 limited the effects of CTGF overexpression on lung alveolar and vascular structure, possibly by normalizing cylin D1 (CCND1), fibronectin (FN1), and collagen Iα1 (COL1A1) expression.
Canonical WNT/CTNNB1 signaling has also been highlighted to be temporally regulated in myofibroblast progenitors during the transition from the pseudoglandular to the saccular phase of lung development (288). In this key study from Parviz Minoo and coworkers, progenitors of secondary crest myofibroblasts were demonstrated to be developmentally committed in the early lung mesoderm. The related observation that homeobox protein (HOX) transcription factors regulate a WNT2/WNT2b/BMP4 signaling axis in early lung development also highlighted HOX transcription factors as potentially important upstream regulators of WNT/CTNNB1 signaling during lung development (216). These data further highlight the diverse roles of canonical WNT/CTNNB1 signaling in late lung development (283).
The functional and phenotypic diversity of fibroblast subtypes in the developing and adult lung continues to draw attention (532), in particular, the delineation and overlap of myofibroblasts, lipofibroblasts, and fibroblasts marked by PDGFRA (394, 457). In general, myofibroblasts express ACTA2 (actin, α2, smooth muscle, aorta; often called α-smooth muscle actin, α-SMA). The pathways that regulate the lineage commitment that generates myofibroblasts and lipofibroblasts have received recent attention (286). Notably, TGF-β signaling via TGFBR1 was demonstrated to control an early pathway that regulated the commitment and differentiation of myofibroblasts vs. lipofibroblasts. This pathway included roles for PDGFRA, peroxisome proliferator-activated receptor-γ (PPARG), paired related homeobox 1 (PRRX1), and zinc finger protein 423 (ZFP423). Along similar lines, evidence has been provided implicating fibroblast growth factor (FGF)10 in the formation of lipofibroblasts during lung organogenesis (14).
The functional diversity of PDGFRA+ fibroblasts has been addressed by using PDGFRA+ fibroblasts that express levels of green fluorescent protein (GFP) from the Pdgfra promoter and are thus designated either GFPhigh or GFPlow cells (183). In a mouse model of lung regeneration based on unilateral pneumonectomy, GFPhigh and GFPlow cells were sorted by flow cytometry and subjected to microarray analysis, which identified GFPhigh cells as matrix lipofibroblasts, with the ECM as the dominant cellular component, while GFPlow cells were identified as myofibroblasts, with plasma membrane and cell junctions as the major cellular component. Loss-of-function (using the pharmacological inhibitor nilotinib) and gain-of-function (using a constitutively active D842V PDGFRA variant) studies revealed PDGFRA as important for matrix (lipo) fibroblast activation and differentiation but not for myofibroblast differentiation (183). The origin of PDGFRA+ fibroblasts has also received attention in lineage-tracing studies, where PDGFRA+ cells were documented to give rise to alveolar myofibroblasts and lipofibroblasts during lung alveolarization (396). The recent observation of opposing lipogenic-to-myogenic switches in fibroblastic phenotype during the development or resolution of lung fibrosis (148) raises the exciting possibility that this type of fibroblast phenotypic plasticity may also contribute to pathways that direct pathogenic lung development or lung regeneration and repair. On a different note, the detection of circulating fibrocytes in the blood of BPD patients and the correlation of circulating fibrocyte number with the presence of PH in BPD patients are noteworthy (289) and demand further study in experimental models.
Nerves and pulmonary neuroendocrine cells.
The role of nerves in lung development remains a neglected area of lung developmental biology; however, some data on this neglected topic are starting to emerge, where neuropeptide Y, coneurotransmitter of the sympathetic nerve system, has been documented to be a mediator of neuro-immune interactions that regulates lung growth in intrauterine growth restriction (IUGR) (525). Moving to the parasympathetic nerve system, parasympathetic innervation has also been implicated in defective airway branching, and hence lung hypoplasia, in experimental CDH (448).
Pulmonary neuroendocrine cells have also received attention. These cells are widely distributed throughout the airway mucosa of the lung, both as solitary cells and collected together in innervated clusters called neuroepithelial bodies (NEBs). One particularly noteworthy recent development reported by Christin Kuo and Mark Krasnow (266) was the mapping of the formation of NEBs, where cell clusters arise not from local proliferation but rather from directed migration of progenitors to branch junctions, promoting formation of the NEB. This occurs by transient EMT and the crawling of the migrating cells over neighbor cells by a process christened “slithering.” Other recent work, reviewed in Reference 117, has described the differentiation and temporal changes in the abundance of pulmonary neuroendocrine cells as lung development proceeds. While these cell types have received much attention in early lung development and airway branching (392), much work remains to be done to address roles for pulmonary neuroendocrine cells in normal or aberrant late lung development.
Vasculature, ACD, PPHN, and BPD-associated PH.
PH and right ventricular hypertrophy affect up to 25% of premature infants with appreciable BPD (27, 43); preterm birth, congenital diaphragm herniation, congenital heart defects, acute pulmonary disease, and chromosomal disorders are recognized risk factors (385), and pulmonary vein stenosis is associated with increased risk for worse outcomes (510). There is much interest in understanding the physiological basis of PH and of congenital malformation of lung vessels in neonates, as well as the contribution of early pulmonary vascular disease to the development of BPD and BPD-associated cardiac complications (22, 42, 173, 370, 453, 478). Furthermore, angiogenesis and the development of the pulmonary vasculature is often considered to be a driver of lung alveolarization (565).
The pulmonary circulation in BPD has been the subject of much observational study, and abnormal vascular growth, which includes perturbed vessel branching and spatial organization as well as precapillary arteriovenous anastomotic vessels, has been described (128, 165, 369). Intrapulmonary vascular shunts have also been described in alveolar capillary dysplasia with misalignment of pulmonary veins (ACDMPV) (166). Additionally, florid intussusceptive-like microvascular dysangiogenesis (126) and a reduction in the alveolar capillary surface area in preterm infants with BPD have been noted (45). These are all important potential contributing factors to the development of PH in BPD patients. Daniela Laux and coworkers have now added to this list of possible contributing factors, with the description of pulmonary vein stenosis in two French cohorts of BPD patients (277).
Several preclinical approaches have been taken to manage PH in animal models of BPD, where arginase inhibition with amino-2-borono-6-hexanoic acid resulted in a pronounced improvement in vascular structure and function in a bleomycin-based model of PH in rat pups (182). Along with increased l-arginine bioavailability, arginase inhibition restored normal pulmonary vascular resistance, normalized medial wall thickness, and normalized cardiac remodeling.
In a related study, right ventricle cyclic nucleotide signaling was demonstrated to be decreased in mouse pups exposed to hyperoxia (199). Right ventricular remodeling was noted, and when mouse pups recovered in room air right ventricular remodeling was still evident but had resolved by adulthood. These authors documented that hyperoxia drove increased phosphodiesterase 5A (PDE5A) expression and activity in the right ventricle but not in the left ventricle or septum. Overexpression of Pde5a in cardiomyocytes caused right ventricular hypertrophy at baseline under room air conditions, leading these authors to suggest that PDE5A is a key modulator of right ventricular remodeling in neonates. However, given that right ventricular hypertrophy develops as a consequence of pulmonary vascular occlusion, the authors have not clarified how the findings in the right ventricle relate to BPD-associated PH.
Retinoic acid has also been studied in the context of vascular development during alveolarization, where retinoic acid was identified as a lung angiocrine that could regulate lung alveolarization through autocrine regulation of endothelial development (587). Additionally, evidence of paracrine regulation of elastin synthesis by retinoic acid through induction of FGF18 in mesenchymal cells was also reported (587), which is relevant given the impact of FGF signaling on lung fibroblast function (242).
Genomic studies have identified some key players in pathological lung vascular development, where SNPs in the FOXF1 gene and genomic deletion upstream of the FOXF1 gene have been noted in patients with ACDMPV, recently reviewed in Reference 132. Building on these studies, inducible overexpression of FOXF1 in mice in endothelial and hematopoietic cells yielded mouse pups with immature lungs and disordered vascular networks, as well as disturbances to gas exchange physiology (131). Chip-Seq analyses identified Sox11 (encoding SRY-box 11), Ghr (encoding growth hormone receptor), Ednrb (encoding endothelin receptor type B), and Slit2 (encoding slit guidance ligand 2) as downstream targets of FOXF1. The potential utility of these observations in managing the devastating clinical consequences of ACDMPV remains to be established.
Maternal factors that modulate lung maturation.
Recent work from Kevin Nicholas and coworkers has explored the presence of factors that direct lung development, in the milk of the tammar wallaby (Macropus eugenii). These marsupials have a comparatively short gestation (~30 days), and the altricial young are underdeveloped and largely resemble a fetus. Furthermore, milk ingested by the altricial young during lactation (which may proceed for up to 300 days) changes in composition over the lactation period to meet the nutritional demands of the young; however, milk may also contain factors that promote specific developmental processes in the young (72). To address this possibility, mouse embryonic lungs were exposed to skim milk from tammar wallabies that were harvested at different time points over lactation. Milk harvested between days 40 and 100 of lactation stimulated branching morphogenesis of mouse lung explants, where milk harvested at day 60 of lactation also promoted expression of SFTPC (encoding surfactant protein C), SFTPB (encoding surfactant protein B), WNT7B, BMP4, and ID2 (encoding inhibitor of DNA binding 2, HLH protein), stimulated proliferation and Matrigel invasion of mesenchymal cells, and stimulated proliferation of lung epithelial cells (361). Building on these observations, a cross-fostering approach was adopted, where tammar pouch young (technically termed “joeys”) were restricted to milk composition not extending beyond day 25, for the first 45 days of postnatal life (360). The cross-fostered young had significantly smaller lungs at P45 compared with control young, with a reduced cross-sectional airway lumen size and a higher percentage of parenchymal tissue. The steady-state levels of BMP4, WNT11, AQP4 (encoding aquaporin 4), HOPX (encoding HOP homeobox), and SFTPB mRNA were significantly reduced in lungs from cross-fostered young. The authors built on these studies, using a proteomic analysis of tammar milk and documented dynamic changes in tammar milk composition comparing day 20, day 60, and day 120 of lactation, where changes in the abundance of several proteins pertinent to the regulation of organogenesis were noted, including PDGFA, insulin-like growth factor binding protein 5 (IGFBP5), insulin-like growth factor binding protein-like 1 (IGFBPL1), and EGF-like domain multiple 6 (EGFL6) (360). These data provide compelling evidence that postnatal lung maturation in the altricial young may be regulated by maternal factors supplied to the developing young through milk. Along these lines, it is worth mentioning a recent report that compared the growth and neonatal complications of very low-birth-weight infants who received either exclusively breast milk or exclusively formula (495). Exclusively breast milk-fed neonates exhibited a lower incidence of BPD, as well as a lower incidence of necrotizing enterocolitis or retinopathy of prematurity (ROP). Whether this observation can be attributed to specific maternal factors transferred during lactation, or generally to nutrition per se, remains to be determined.
Maternal nutrition and maternal obesity.
In addition to maternal factors transmitted during lactation that may influence lung development, the role of maternal obesity (346) and maternal nutrition during lactation in lung development has also been addressed. In a study on maternal protein restriction, where nursing rats were provided with either a 20% protein diet (control) or a 6% protein diet (protein restriction diet) (153), maternal protein restriction resulted in a pronounced decrease in body mass and lung mass. The authors also reported a persistent (up to 2 mo of postnatal life) decrease in alveolus density and alveolar septal wall thickness and cite lung stereology literature for the method employed (402). However, the lung tissue sections illustrated bear no resemblance to stereological studies performed in plastic resin, and the morphometric approach that was taken to acquire these data was not clarified in the report. Remaining with maternal protein restriction, it is noteworthy that the resultant increases in glucocorticoid levels have been implicated in arrested lung alveolarization associated with maternal protein restriction (409), although glucocorticoids have also been implicated in protection of alveolarization, which was perturbed after exposure to the environmental contaminant bisphenol A (206). Clearly, there is still much to be learned about glucocorticoid function in the developing lung (62), and studies on the deleterious impact on extrapulmonary systems of glucocorticoids when used to promote lung development have not been conducted in preclinical models. The importance of these studies is underscored by the correlation of a greater probability of severe metabolic bone disease, due to bone demineralization, with cumulative duration of hydrocortisone and prednisolone therapy in severe BPD patients (234).
Maternal nutrition has also been addressed in transcriptomic studies in rats subjected to IUGR (23% protein diet vs. an isocaloric 9% protein diet), where IUGR resulted in changes in the steady-state levels of several genes (588) that were dynamically regulated over the first 21 days of postnatal life and included genes implicated in cell adhesion (313), cardiac muscle contraction, and peroxisome proliferator-activated receptor pathways.
Lipids and lipid metabolites not only have received attention as disease mediators via lipid signaling (282) but have also been considered in the context of maternal nutrition. The impact of a maternal high-fat diet on lung development in embryos and offspring was also assessed, where pregnant mice were fed with high-fat (42% of calories from fat) or regular (21.2% of calories from fat) chow before mating and continued on a high-fat diet throughout lactation. Male animals received regular chow. Maternal nutrition with high-fat diet caused placental inflammation, leading to placental insufficiency, and also caused fetal growth restriction (340). Additionally, a pronounced inhibition of fetal lung development was noted. The alveolar simplification persisted into adolescence (340). Along similar lines, in a second study female rats were fed a high-fat diet (45% of calories from fat) and were mated with male rats that were fed a standard diet (10% of calories from fat) (493). Pregnant rats continued with the high-fat diet throughout pregnancy and during lactation. Offspring from mothers that received the high-fat diet exhibited increased body mass at birth, as well as increased inflammatory cell infiltration and increased lung collagen deposition, compared with offspring from dams that received the standard diet (493). Interestingly, the exact opposite was observed in a third study in rats where pregnant dams were fed a high-fat diet (40% of calories from fat) vs. a standard diet (18% of calories from fat) 1 mo before mating and up to gestational day 14, where no impact on alveolar structure was noted in newborns or in adolescents, suggesting no impact of high-fat maternal nutrition on lung alveolarization in offspring with that protocol. The induction of late gestational diabetes with streptozotocin, additive to the high-fat diet, was also without any impact on alveolar structure (40). The reason for these discordant results comparing the first two studies with the third study are not immediately apparent; however, these reports generally provide support for an association between maternal high-fat diet and lung inflammation and arrested lung alveolarization in offspring. High-fat diets are thought to promote a systemic inflammatory state with increased oxidative stress, leading Lyda Williams and coworkers to supplement a high-fat diet with N-acetylcysteine (564) in a study in mice. While lung structure was not quantified, visual inspection of lung sections suggests that N-acetylcysteine supplementation worsened lung structure and was also associated with increased mortality. It remains to be established whether this was perhaps an effect of N-acetylcysteine dose.
The use of ω-3 polyunsaturated fatty acid (PUFA) preparations has been widely explored in clinical setting as a basis of parenteral nutrition (see Enteral and parenteral nutrition). Concerning maternal nutrition in preclinical models, a comparison of maternal nutritional supplementation with ω-3 vs. ω-6 PUFA revealed that ω-3 PUFA prevented the impaired alveolarization associated with hyperoxia exposure (480). In that study, pregnant rats received daily PUFA enteral nutritional supplementation and pups were exposed to hyperoxia, where improvements in lung structure were noted. These data might suggest that maternal nutritional supplementation with ω-3 PUFA may be useful to promote lung alveolarization. A stereological study to this end is recommended.
Studies examining the impact of maternal docosahexaenoic acid (DHA) supplementation in a perinatal BPD model on inflammatory, oxidative, and apoptotic events in adult lungs have also been undertaken. Pregnant mice were treated with intraperitoneal E. coli 0111:B4 LPS, and pups were exposed to hyperoxia followed by room air recovery (20). Pregnant and nursing dams received DHA nutritional supplementation. Maternal DHA nutritional supplementation prevented the sustained inflammation, oxidation, and apoptosis that were observed in adult mice nursed by dams that did not receive DHA (20). Building on these studies, a mechanistic explanation has been offered, whereby macrophages from mice with DHA-supplemented nutrition exhibit altered NOTCH1 and jagged 1 (JAG1) levels, and these macrophages exhibited a reduced ability to stimulate high-mobility group box 1 (HMGB1) release from the MLE-12 lung epithelial cell line (19). This is noteworthy given the recent report that HMGB1 contributes causally to arrested lung alveolarization in experimental BPD (583). No doubt, the use of DHA supplementation to promote proper structural development of the lung will continue to be explored (302).
Moving from rat to sheep models of arrested lung development (192), interestingly, IUGR induced in pregnant sheep at days 103–105 of gestation (where term is 148 days) by ligation of one umbilical artery did not reveal any perturbations to lung structure assessed after IUGR (21).
Enteral and parenteral nutrition.
In addition to maternal nutrition, nutrition of the neonate plays a pivotal role in lung development and in the clinical management of BPD patients (317, 422). This idea continues to be validated in preclinical models, most notably a recent report documenting that postnatal growth restriction in rats by milk intake restriction augmented the PH and right ventricular remodeling provoked by exposure to hyperoxia (561).
Lipid emulsions are widely employed for parenteral nutrition in preterm infants at risk for BPD. There continues to be much interest at the clinical level in establishing whether particular lipid emulsions offer advantages over others. Little work in this regard has been done in a preclinical setting. A recent Cochrane review suggested that alternative lipid emulsions do not offer any particular benefit over the classical pure soybean oil-based lipid emulsions for parenterally fed preterm infants (250). However, a recent meta-analysis documented the potential utility of fish oil lipid emulsions to prevent severe ROP (542), and another report suggested that a medium-chain triglyceride ω-3 PUFA-enriched intravenous fat emulsion was associated with a more favorable cytokine and fatty acid profile compared with a soybean oil-based intravenous fat emulsion (487). In that study, a more favorable profile was regarded as having higher α-tocopherol, eicosapentaenoic acid, docosahexaenoic acid, and ω-3 PUFA levels and lower linolenic acid and total PUFA levels, with a lower ω-6-to-ω-3 PUFA ratio. It remains of interest to explore how different nutrition supplementation protocols impact lung development in preclinical models, in particular whether the impact on lung development is attributable to nutrition (caloric intake) per se or whether particular nutritional supplementation protocols exert an effect on lung development by specifically modulating distinct biochemical pathways that direct lung organogenesis. Given current interest in exploring the extrapulmonary consequences of BPD in animal models, together with the studies cited above, it seems useful to also examine the impact of nutritional supplementation on pathological extrapulmonary events.
Vitamin A and related retinoids.
Vitamin A and retinoids in lung development represent something of a never-ending story, where vitamin A and retinoid supplementation have long been—and continue to be—explored as a meaningful pharmacological intervention to drive lung growth (331). This theme continues to be supported by recent observations, such as the finding that plasma vitamin A levels at birth were lower in infants who went on to develop BPD (3). Key recent developments include the observation that hyperoxia exposure downregulated the expression of the retinoid receptors retinoic acid receptor-α (RARA), retinoic acid receptor-γ (RARG), and retinoid X receptor-γ (RXRG) in the lungs of newborn mice, where hyperoxia exposure inhibited both proliferation and expression of retinoid receptors in A549 cells in vitro (95). The identification of an evolutionarily conserved (across Xenopus, mouse, and human) retinoic acid-hedgehog signaling cascade that is required for respiratory specification by regulating WNT2/WNT2B generation in the mesoderm consolidates the central role of retinoid signaling in the development of gas exchange organs (440). This pathway also regulated WNT responsiveness of the endoderm, for example, by activating relevant NK2 homeobox 1 (NKX2-1) programs (440). These data are interesting, given the exciting recent observation that the forkhead box P1 (FOXP1)/forkhead box P2 (FOXP2)/forkhead box P3 (FOXP3) transcription factors—which are known to be regulated by retinoids—have been identified as regulators of NKX2-1, as well as SRY (sex determining region Y)-box 2 (SOX2) and SRY-box 9 (SOX9), by repressing expression of transcriptional regulators not normally expressed in the developing lung. These regulators include Pax2 (encoding paired box 2), Pax8 (encoding paired box 2), Pax9 (encoding paired box 2), and the Hoxa9–13 cluster (encoding a group of homeobox A genes). In short, FOXP1/FOXP2/FOXP3 are essential for promotion of lung endoderm development by repressing expression of nonpulmonary transcription factors (294).
Returning to retinoids, in vitro retinoic acid has been documented to promote fetal ATII cell proliferation by promoting S-phase entry and inhibiting apoptosis and also promoted fetal ATII to ATI transdifferentiation (170). Vitamin A signaling has also been implicated in lung hypoplasia associated with CDH, where disruptions to retinoid signaling pathways were evident by increased expression of aldehyde dehydrogenase 1 family, member A2 (ALDH1A2; also called retinaldehyde dehydrogenase 2) in lung tissue from human CDH patients and from rabbits with surgically induced CDH (111). Additionally, in nitrofen-induced CDH in rats, reduced levels of CYP26B1 (encoding cytochrome P-450, family 26, subfamily b, polypeptide 1) and LRAT (encoding lecithin-retinol acyltransferase) were noted, where CYP26B1 is involved in the specific inactivation of all-trans-retinoic acid and LRAT catalyzes the esterification of all-trans-retinol.
Vitamin D.
Hypovitaminosis D is prevalent during pregnancy, and this observation has led to growing interest in the role of vitamin D signaling in fetal growth and, in particular, lung alveolarization (97) as well as the utility of vitamin D supplementation to manage arrested alveolarization and BPD (89, 316, 325). This idea, however, is not without controversy (248). Recently, high-intensity solar ultraviolet (UV)B-radiation doses during pregnancy have been reported to be associated with risk reduction of early mortality in preterm infants, possibly because of effects on vitamin D synthesis during pregnancy (464). This idea has stimulated the exploration of vitamin D supplementation in preterm infants in a recent small clinical trial (158), and the outcomes of larger trials are eagerly awaited.
Recent exciting preclinical developments in this area include the observation that the vitamin D receptor (VDR) cytochrome P-450, family 27, subfamily b, polypeptide 1 (CYP27B1; also called 25-hydroxyvitamin-D3-1-α-hydroxylase) and cytochrome P-450 family 24 subfamily A member 1 (CYP24A1) increase in expression in the fetal lung before birth. Antenatal administration of E. coli O55:B5 LPS to mimic chorioamnionitis in rats reduced VDR-increased CYP24A1 steady-state levels (326). Administration of LPS concurrently with vitamin D prevented the impact of LPS on VDR and CYP24A1 steady-state levels. In fetal pulmonary artery endothelial cells, LPS blunted cell proliferation and tube formation, while concomitant administration of LPS with vitamin D partially restored proliferative and angiogenic capacity. In a related study (324), the same investigators demonstrated that the impaired right ventricle-pulmonary artery coupling caused by antenatal LPS was partially restored. These data highlight an exciting opportunity to manage the disordered lung vascular development associated with chorioamnionitis with vitamin D.
Other studies have recently examined the impact of prenatal vs. postnatal vitamin D deficiency on lung structure in mice (460), where vitamin D-deficient pups exhibited reduced tracheal diameter with decreased tracheal cartilage minimal width. Vitamin D deficiency also increased airway resistance, reduced lung compliance, and led to alveolar simplification. Postnatal vitamin D nutritional supplementation improved lung function and alveolar development but did not correct tracheal narrowing. Beyond nutritional supplementation, nebulization of 1,25-dihydroxycholecalciferol (the biologically active form of vitamin D) as well as 25-hydroxyvitamin D (a vitamin D precursor) to rat pups (breathing room air) increased postnatal lung alveolarization (522).
Caffeine.
Since the first use of caffeine to manage apnea of prematurity in 1977 (30), the use of caffeine in preterm infants continues to be an exciting and controversial (390) area of neonatal medicine, where early caffeine use is reported to reduce BPD (2, 305, 410). Caffeine therapy during the first 10 days of life in very low-birth-weight preterm infants is associated with a reduction in both BPD and the duration of mechanical ventilation (473). Whether caffeine can drive lung alveolarization or protect secondary septation in developing lungs during injury remains controversial (485). Several studies continue to address this intriguing question. Caffeine administration to preterm rabbits exposed to hyperoxia from the day of delivery led to improved lung structure and improved lung compliance (377). Two studies in rats (by the same group) have also alluded to the utility of caffeine to drive lung alveolarization under hyperoxic conditions (523), where caffeine was suggested to protect immature lungs from hyperoxia-induced damage, either by modulating ER stress and the UPR (523), which is relevant to hyperoxia-induced arrest of alveolarization (310), or by improving endothelial nitric oxide synthase (eNOS) activity through increased tetrahydrobiopterin bioavailability (241). These studies contrast with a recent report that evaluated caffeine use in the hyperoxia-based BPD model in mice (using C57BL/6J mice), where Philipp Rath and coworkers applied caffeine concomitant with hyperoxia exposure (443) and no beneficial effect of caffeine administration on lung alveolarization was noted. These data are in line with the reports of a lack of any effect, or indeed a deleterious effect on lung structure, of caffeine administration to mice (in that study, FVB/N mice) in a comparable hyperoxia-exposure model. As discussed in a recent editorial in this journal (442), with these data, together with other data that predate the scope of publications reviewed in this review, caffeine appears to have a beneficial effect on lung alveolarization in all preclinical animal models of BPD except mouse models. It may be that this represents an important difference between the mouse models and models that employ other mammals (including rats), which would be an important consideration in translational studies on drug discovery for BPD. Nevertheless, work that continues to emerge in in vitro studies, such as a recent report about caffeine modulating glucocorticoid-induced expression of CTGF in lung epithelial cells and fibroblasts (155), continues to tantalize us with the potential utility of caffeine to drive alveolarization in BPD patients. With this in mind, ongoing work in clinical settings continues to explore new methods of caffeine (and related methylxanthine derivatives) administration, such as nebulized pentoxifylline, which was recently reported to be well tolerated but did not reduce the duration of oxygen supplementation in extremely preterm infants at high risk for BPD (474).
Cigarette smoke and nicotine.
The impact of cigarette smoke components on lung alveolarization and the respiratory health of children continues to be a subject of interest (74, 178, 232, 345, 454, 496, 552). Exposure of mouse pups to e-cigarette vapors using e-cigarette cartridges (containing 1.8% nicotine in propylene glycol, where propylene glycol served as control) caused arrested lung alveolarization (347). These data indicate that exposure to nicotine in e-cigarette vapors impacts lung development in mice. The impact of maternal smoke exposure has also been addressed, where exposure of pregnant mice to sidestream tobacco smoke (486) appeared to increase alveolar volume, although the morphometric approach in that study was not clear. This effect was blunted by the nonselective, noncompetitive antagonist of nicotinic acetylcholine receptors mecamylamine. The impact of sidestream cigarette smoke exposure during pregnancy was attributed to elevated levels of proapoptotic and antiangiogenic factors and reduced levels of antiapoptotic factors in the lungs of neonates, as well as increased expression of bombesin (415). One particularly unusual recent study suggested that electroacupuncture applied to a particular acupuncture point (the “Zusanli” point, ST36) limited the impact of maternal nicotine exposure on lung alveolarization in offspring (235).
Placental insufficiency.
There is currently much interest in addressing a potential role for placental insufficiency in the development of BPD and BPD-associated PH. This idea is highlighted by placental complications with fatal consequences being associated with moderate to severe BPD in very preterm infants in the EPIPAGE-2 cohort study (531), although severe BPD has not been associated with placental pathology in one recent prospective cohort study (546). Similarly, placental villous vascularity was decreased in infants with BPD-associated PH (573).
Placental insufficiency has also been addressed in preclinical BPD models, where maternal high-fat diet was associated with placental inflammation (340) (discussed elsewhere in this review). Uteroplacental insufficiency induced by bilateral uterine vessel ligation, which caused IUGR, resulted in increased RARB steady-state levels and perturbed saccular and alveolar structures (220). These studies addressing the role of the placenta in disturbances to lung alveolarization represent a welcome emergence of a new and important area in lung developmental pathobiology.
The microbiome.
The microbiome is emerging as a key player in disease and responses to disease, including lung disease (133, 426), and preliminary studies have already revealed similarities and differences in the airway microbiome comparing preterm and term infants and infants with BPD (271). Furthermore, longitudinal changes in the airway microbiome of ventilated preterm-born infants might be associated with BPD severity, although it is not yet clear whether these changes are causal or are a response to clinical management of affected infants (553). Along similar lines, specific bacterial pathogens such as Corynebacterium species are reportedly more likely to be present in severe BPD infants with longer duration of endotracheal ventilation (228). These clinical studies underscore the need for preclinical studies in animal models that address the contribution of the microbiome to disturbances to lung development.
Comorbidities.
While the role of chorioamnionitis continues to be intensively studied as a risk factor or protective factor for BPD (104) and forms the basis of some clinical BPD models, the presence of pulmonary infections in preterm-born infants with or at risk for BPD remains of serious clinical concern (435). These infections include Ureaplasma colonization of the respiratory tract in BPD patients (547), postnatal cytomegalovirus infection, which is associated with increased risk for BPD (255), as well as rhinovirus (417), influenza (194, 446), and respiratory syncytial virus (RSV) infection (446). Despite the tremendous capacity for bacterial and viral infections to impact lung alveolarization, very few preclinical studies have addressed this important issue. Among notable reports that have recently emerged was the important observation that exposure of mouse pups to hyperoxia exaggerated the inflammatory response to subsequent rhinovirus infection. Lung structure was not quantitatively assessed in that study, which lays important groundwork for follow-up preclinical studies on the interactions between pulmonary infections and lung development in the neonatal period. These studies are important in light of a recent report that describes the ability of influenza viruses to infect epithelial stem cells of the lung and hence modulate FGF2B-driven lung repair (436).
New mediators of alveologenesis or cell differentiation, survival, and repair.
Several recent reports have highlighted novel mediators of lung development in knockout mouse studies (Table 2). Ion channels are credited with important roles in lung homeostasis and development (336), and recent developments include the identification of the potassium voltage-gated channel subfamily J member 13 (KCNJ13; also called Kir7.1) inwardly rectifying K+ channel, abrogation of which was associated with cleft palate as well as blunted lung development and thickened alveolar septa (545). The transcriptional regulator LIM-domain only protein 4 (LMO4) has also received attention, where epithelial deletion of Lmo4 using Shh-Cre and Scgb1a1-CreERT2 lines (background strain not declared) was undertaken (211). The authors maintained that epithelial deletion of Lmo4 did not impact lung development; however, only embryonic stages were examined in that study. In another study, neuralized E3 ubiquitin protein ligase 3 (NEURL1; also called lung-inducible neutralized-related C3HC4 RING domain protein, LINCR) was induced in the mouse lung epithelium in Sftpc-rtTA/(tetO)-Neurl1 transgenic mice (306). Mice homozygous for the transgene developed hypoplastic lungs with apparently decreased numbers of alveoli and large cysts. These data imply a role for Neurl1 in lung alveolarization, by an as-yet-undetermined mechanism. A role for serine (or cysteine) peptidase inhibitor, clade F, member 1 (SERPINF1; also called pigment epithelial-derived factor, PEDF) as a mediator of lung alveolarization has also been noted (101). In mouse pups exposed to hyperoxia, elevated expression of SERPINF1 was noted in the lung epithelium. Genetic ablation of SERPINF1 expression in Serpinf1−/− mice protected against the impact of hyperoxia in lung alveolarization. These data validate SERPINIF1 as a pathogenic factor in experimental BPD. A second growth factor, FGF10, which is known to impact lung development (92, 93), has also received attention, where Fgf10+/− heterozygotes exhibited increased sensitivity to hyperoxia exposure and further potentiated hyperoxia-induced disturbances to lung development, possibly by adversely impacting ATII cell function and transdifferentiation (94). The pathological effects of FGF10 overexpression are interesting, given other reports that highlight suppression of FGF10 expression in patients with idiopathic pulmonary fibrosis (90).
| Transgenic Modification and Target Cell/Tissue (if applicable) | Strain | Injury Stimulus | Molecular/Pathway Target | Lung Pathological Findings | References |
|---|---|---|---|---|---|
| Adamts18 (global KO) | C56BL/6J × 129/OlaHsd | None | Zn-dependent metalloproteases | Increased septal granularity | 38 |
| Adamtsl2 (global KO) | C57BL/6N | None | Zn-dependent metalloproteases | Bronchial epithelial dysplasia | 222 |
| Alk5 (mesoderm progenitor KO) | C57BL/6 | None | TGF-β signaling | Disturbances or mesenchymal cell dynamics | 286 |
| Apc (mesoderm KO) | 129S6 × SvEvTac | None | β-catenin | Disturbances to air space structure | 288 |
| Axin2 reporter | Not declared | None | Wnt signaling | None | 159 |
| Bach1 (global KO) | C57BL/6 | 95% O2, P1–P3 | Transcription regulator | Not assessed | 179 |
| Bach1 (global KO) | C57BL/6 | 95% O2, P1–P3; 21% O2, P4–P14 | Transcription regulator | Disordered alveolarization improved | 230 |
| Bmpr1a (epithelial KO) | C57BL/6 | None | BMP signaling | Disturbances to air space structure | 315 |
| CatK (global OE) | FVB/N | 90% O2, P1–P14 | Cysteine proteases | Disordered alveolarization improved | 258 |
| Cbs (global KO) | C57BL/6 | None | Cystathionine β-synthase | Disturbances to air space and vascular structure | 318 |
| Cth (global KO) | C57BL/6 | None | Cystathionine γ-lyase | Disturbances to air space and vascular structure | 318 |
| Cldn6 (epithelial OE) | C57BL/6 | None | Claudin 6 | Disturbances to air space structure | 236 |
| Col4a1+/G394V and Col4a2+/G646D | C3H/HeJ | None | Type IV collagen | Disturbances to air space and vascular structure | 307 |
| Creb1 (global KO) | C57BL/6 | None | cAMP signaling | None | 29 |
| Ctgf (epithelial OE) | FVB | 90% O2, P1–P14 | Connective tissue growth factor | Disturbances to air space structure | 452 |
| Cyp1a (global KO) | C57BL/6J | 85% O2, P2–P14 | Cytochrome P-4501A | Disturbances to air space structure worsened | 337 |
| Eln (global heterozygous KO) | C57BL/6J | MV with RA | Elastin | Disturbances to capillary structure worsened | 208 |
| Ezh2 (epithelial KO) | C57BL/6J | None | Histone-lysine N-methyltransferase | Disturbances to lung lineage specification and basal cell fate; lung structure not assessed | 168, 212 |
| Ezh2 (mesothelium or endoderm progenitor KO) | Not declared | None | Histone-lysine N-methyltransferase | Restricts smooth muscle gene expression and basal cell lineage; lung structure not assessed | 488, 489 |
| Fgf10 (heterozygous KO) | C57BL/6 | 85% O2, P1–fP8 | FGF signaling | Disturbances to air space structure worsened | 94 |
| Foxf1 (global OE) | Not declared | None | Forkhead box F1 transcription factors | Disturbances to air space and vessel structure | 131 |
| Foxm1 (myeloid KO) | C57/B6 | 85% O2; P1-P21 | Forkhead box M transcription factors | Disturbances to air space structure worsened | 569 |
| Foxp1/Foxp2/Foxp4 (global KO) | C57BL/6J | None | Forkhead box P transcription factors | Disturbances to endoderm development | 294 |
| Fstl1 (epithelial OE) | Not declared | None | BMP signaling | None | 295 |
| Gli2 and Gli3 (global KO) | Not declared | None | GLI family zinc finger | Required for respiratory specification; lung structure not assessed | 440 |
| Grp78 (Epithelial KO) | Not declared | None | 78-kDa glucose-regulated protein | Disturbances to air space structure | 157 |
| Hdac3 (developing mesenchyme KO) | C57BL/6 × 129SVJ | None | Histone deacetylases, regulating lung epithelial development | Disturbances to air space structure | 558 |
| HIF1a (CA epithelial OE) | C57BL/6 × SJL | 85% O2, P1–P28 | HIF-1α | Disordered alveolarization improved | 527 |
| Hopx (CreER driver) and others | FVB/NJ | None | Many | ATI cells can be reprogrammed | 576 |
| Hox5a/Hox5b/Hox5c (global KO) | C57BL/6 | None | Homeodomain-containing transcription factors | Hypoplastic embryonic lungs developed | 216 |
| Ikbkb (epithelial OE) | Not declared | None | NF-κB signaling | Disturbances to air space structure | 54 |
| Itgam (DT-based depletion) | FVB/NJ | 90% O2; P1–P3 | CD11b cell depletion with DT | Inflammation improved; lung structure not assessed | 150 |
| Kcnj13 (global KO) | C57BL/6NTac | None | Kir7.1 | Disturbances to air space structure | 545 |
| Lmo4 (epithelial KO) | Not declared | None | LIM-domain only protein family | None | 211 |
| Loxl3 (global KO) | Not declared | None | Lysyl oxidase | Disturbances to air space structure | 590 |
| Lpar1 (global KO) | C57BL/6 × 129Sv/J | None | lysophosphatidic acid signaling | Disturbances to air space structure | 163 |
| Lpar1 (global KO) | Rat Wistar | 90% O2, P1–P8 + 75% O2, P9 + RA, P10–7 wk + LPS | lysophosphatidic acid signaling | Disturbances to air space structure | 100 |
| Lrp4 (global KO) | C57BL/6J | None | Low-density lipoprotein receptor-related protein 4 | Polyhydramnios; lung structure not assessed | 520 |
| Ltbp4S (global KO) Fbln5 (global KO) | C57BL/6N | None | Latent TGF-β-binding protein 4 and fibulin-5 | Disturbances to air space structure in Ltbp4S−/− background were attenuated in Ltbp4S−/−/Fbln5−/− mice | 120 |
| miR-150 (global KO) | C57Bl/6J | 95% O2, P1–P6 | miR-150 | Dysmorphic capillaries noted | 381 |
| Neurl3 (epithelial OE) | FVB/N | None | Neuralized E3 ubiquitin protein ligase 3 | Disturbances to air space structure | 306 |
| Nfe2l2 (global KO) | CD-1 | 85–95% O2, P2–P4 | Oxidative stress | None | 348 |
| Nlk (global KO) | Not declared | None | Nemo-like kinase | Disturbances to air space structure | 254 |
| Nlrp3 (global KO) | C57BL/6 | 85% O2, P3–P14 | NLRPS inflammasome | Disordered alveolarization improved | 297 |
| Nos3 (global heterozygous and homozygous KO) | C57BL/6J | None | Endothelial nitric oxide synthase | Disturbances to air space structure | 477 |
| Nr3c1 (global KO) | C57Bl/6J | None | Glucocorticoid receptor | Lung development delayed | 274 |
| Pde5 (cardiac OE) | C57BL/6 | None | Phosphodiesterase 5 | Disturbances to cardiac remodeling | 199 |
| Pdgfra (reporter) | C57BL/6 | None | PDGFRα | Studies on fibroblast dynamics | 396 |
| Pdgfra (reporter) Pdgfra (CA OE) | Not declared | None | PDGFRα signaling | Fibroblast dynamics and differentiation addressed; lung structure not assessed | 183 |
| Serpinf1 (global KO) | C57BL/6J | 95% O2, P5–P13 | Pigment epithelium-derived factor | Disordered alveolarization improved | 101 |
| Smad1 (epithelial KO) | C57BL/6 | None | BMP signaling | Disturbances to air space structure | 315 |
| Sod2 (global KO) | C57BL/6 | 75% O2, P2–P10 | Superoxide dismutase 2 | None | 188 |
| Sod3 (global KO) | Not declared | Bleomycin | Extracellular superoxide dismutase | Disturbances to air space and vascular structure, and pulmonary hemodynamics worsened | 129 |
| Tgfbi (global KO) | C57BL/6J | None | TGF-β signaling | Disturbances to air space structure | 8 |
| Tnf (global KO) | C57B/6J | Mechanical ventilation (40% O2) | TNF-α | None | 147 |
| Vegfa (epithelial KO) | CD-1 | None | Vascular endothelial growth factor A | Disturbances to air space structure | 587 |
| Yy1 (epithelial KO) | 129/Sv | None | GLI-Kruppel class of zinc finger proteins. | Pleuropulmonary blastoma | 68 |
The NOS3/VEGF axis has also received attention as a mediator of aberrant lung alveolarization, where exogenous intramuscular VEGF administration to Nos3−/− mice after hyperoxia exposure resulted in better alveolarized lungs than those from control mice that received vehicle instead of VEGF (477).
The gasotransmitter H2S has also been implicated in normal postnatal lung alveolarization, where in knockout mice for the endogenous H2S-generating enzymes cystathionine β-synthase and cystathionine γ-lyase both enzymes were implicated in lung alveolarization as well as lung vascular development, assessed by stereological analysis of alveolar structures as well as mean lung vascular wall thickness (318). To date, the third member of the H2S-generating enzymes, mercaptopyruvate sulfurtransferase, has not received attention.
One particularly “out of the box” study using knockout mice to study lung development was the report that Lrp4 deletion caused unilateral or bilateral kidney agenesis, resulting in polyhydramnios (520), despite defects in fetal urine production. Usually, bilateral renal agenesis or obstruction of the urinary tract leads to oligohydramnios, which results in perinatal infant death due to lung hypoplasia. The unexpected finding of polyhydramnios was attributed to loss of two water channels, aquaporin 9 (AQP9) in the fetal membrane and aquaporin 1 (AQP1) in the placenta, in Lrp4−/− mice. Additionally, fetal breathing and swallowing movements were compromised in Lrp4−/− mice. Interest in the area has also led to the development of a mouse model of pulmonary hypoplasia induced by oligohydramnios, where amniotic sacs are punctured at embryonic day (E)14.5 (378). With this model, both cell size and epithelial and endothelial development were revealed to be impacted by oligohydramnios, where ATI cell differentiation was proposed to be impacted by mechanical signals lost because of oligohydramnios. To date, the mechanisms of oligohydramnios-induced lung hypoplasia remain unknown (568). These studies highlight the need to examine the impact of genetic alterations not only on fetal lungs and the fetus but also on the maternal-placental-fetal unit. Along these lines, the observation that soluble endoglin (sENG) levels are elevated in the amniotic fluid of pregnant mothers with chorioamnionitis led to a study that explored the causal impact of elevated amniotic fluid levels of sENG in pregnant rats on lung development of offspring (492). Most interestingly, elevated amniotic fluid levels of sENG resulted in decreased lung VEGF and NOS3 expression, which was accompanied by aberrant alveolarization, aberrant vascular development, and disturbances to pulmonary hemodynamics and cardiac remodeling.
New mediators of alveolar development have also been identified by pharmacological approaches (Table 3). With the use of the transient receptor potential cation channel, subfamily V, member 4 (TRPV4) agonist GSK1016790A in fetal mouse lung epithelial cells, TRPV4 was identified as a candidate modulator of both inflammation and the transduction of mechanical signals in the lung epithelium that promote epithelial cell differentiation (386).
| Drug (dose and frequency) | Species and Strain | Injury Stimulus | Molecular/Pathway Target | Lung Pathological Finding | References |
|---|---|---|---|---|---|
| 101.10 (1 mg/kg sc), preinjury | Mouse CD-1 | IL-1β | IL-1R antagonist | Alveolarization improved | 375 |
| 1D11 (10 mg/kg ip) on E13.5 and E17.5 | Mouse C57BL/6N | Transgenic (Adamtsl2deletion) | Pan-TGFβ1,2,3-neutralizing antibody | Bronchial epithelial dysplasia was not attenuated | 222 |
| AAV9-miR29b.eGFP (1 × 109 AAV particles, intranasal) on P3 | Mouse C3H/HeN | E. coli LPS + 85% O2, P1–P14 | miR-29b | Alveolarization improved | 142 |
| N-acetylcysteine (route and dose not declared) from E0.5, throughout lactation | Mouse CD1 | High-fat diet | Oxidative stress | Alveolarization delayed | 564 |
| ActRIIB-Fc (5 mg/kg ip) on P4, P7, P10, and P13 | Mouse C57BL/6 | 85% O2, P1–P14 | Activin receptor antagonist | Alveolarization improved | 299 |
| All-trans-retinoic acid: retinyl palmitate (10:1) (dose unclear, oral) daily P3–P14 | Rat S-D | SU5416 | Vitamin A | Alveolarization, vascularization, and cardiac remodeling improved | 587 |
| AMD3100 (240 μg/kg sc) daily, P5–P15 | Rat S-D | 90% O2, P2–P16 | CXCR4 antagonist | Alveolarization, vascularization, pulmonary hemodynamics, and cardiac remodeling improved | 139 |
| Amino-2-borono-6-hexanoic acid (3.5 mg/kg ip) daily, P1–P14 | Rat S-D | Bleomycin | Arginase inhibitor | Pulmonary hemodynamics, vascular and cardiac remodeling improved | 182 |
| β-Aminopropionitrile (15 mg/kg ip) daily, P9–P19 | Mouse C57BL/6 | 85% O2, P1–P14 | Lysyl oxidase inhibitor | No improvement | 356 |
| antagomiR-489 (5 μg·g−1·day−1, intranasal) daily, P4–P14 | Mouse C57BL/6 | 85% O2, P4–P14 | miR-489 | Alveolarization improved | 404 |
| Anti-EMAP II antibody (dose undeclared) every 3 days, P3–P15 | Mouse C57BL/6 | 85% O2, P3–P15 | Endothelial monocyte-activating polypeptide II | Alveolarization, vascularization, pulmonary hemodynamics, and cardiac remodeling improved | 279 |
| Anti-HMGB1 antibody (10 mg/kg/day sc) every 3 days, P1–P21 | Mouse C57BL/6 | 85% O2, P1–P28 | Neutralizes high-mobility group box-1 | Alveolarization improved, elastin deposition improved | 583 |
| Anti-MIP-2 antibody (10 μg/g ip) on P6 | Mouse C57BL/6 | E. coli LPS | Neutralizes MIP-2 | Improved alveolarization | 215 |
| Aurothioglucose (25 mg/kg ip) on P1 | Mouse C3H/HeN | 85% O2, P1–P14 | Thioredoxin-1 inhibitor | Alveolarization improved | 293 |
| BAY 11-7082 (10 mg/kg ip) on P6 | Mouse C57BL/6 | E. coli LPS | IKK-α and IKK-β inhibitor | Alveolarization worsened | 215 |
| Caffeine (10 mg/kg ip) loading dose P1, followed by 5 mg/kg ip daily as maintenance dose | Rabbit | 95% O2, P1–P5 | Caffeine | Alveolarization improved, lung function improved | 377 |
| Caffeine (25 mg·kg−1·day−1 ip) daily, P1–P14 | Mouse C57BL/6 | 85% O2, P1–P14 | Caffeine | No improvement | 443 |
| Carbachol (25 nM in amniotic fluid) intra-amniotic at E10.5 | Mouse CD1 | Nitrofen | Cholinergic agonist | Airway branching improved | 448 |
| Deferoxamine (150 mg·kg−1·day−1 ip) daily, P1–P14 | Rat S-D | 85% O2, P1–P7, then 21% O2, P8–P14 | Prolyl-4-hydroxylase inhibitor; stabilizes HIF-1α | Alveolarization and vascularization improved | 103 |
| Dexpanthenol (500 mg/kg ip) daily, P1–P4 | Rat S-D | 95% O2, P1–P14 | Tumor necrosis factor-α and interleukin-1β antagonist | Alveolarization improved | 408 |
| 1,25-Dihydroxycholecalciferol (100, 500, or 1,000 ng/kg) nebulization daily, P1–P14 | Rat S-D | None | Vitamin D | Alveolarization improved | 522 |
| Disulfiram (25 mg/kg ip) daily, P2–P14 | Mouse CD1 | None | Retinaldehyde dehydrogenase inhibitor | Alveolarization worsened | 587 |
| Docosahexaenoic acid in maternal food | Mouse C3H/HeN | E. coli LPS, E16 | Fatty acid metabolism | Anti-inflammatory effects | 19 |
| Docosahexaenoic acid, in maternal food, E16 until weaning | Mouse C3H/HeN | E. coli LPS, E16 + 85% O2, P1–P14 | Fatty acid metabolism | Prevent inflammation | 20 |
| Electroacupuncture at “Zusanli” point (2/15 Hz, 20 min) daily, E6–P21 | Rat S-D | Nicotine | Unknown | Alveolarization improved | 235 |
| EMAP II (80 μg/kg sc) every 3 days, P3–P15 | Mouse C57BL/6 | 85% O2, P3–P15 | Endothelial monocyte-activating polypeptide II | Alveolarization, vascularization, pulmonary hemodynamics, and cardiac remodeling worsened | 279 |
| Endoglin, soluble (2 × 108 PFU viral vector) intra-amniotic at E17 | Rat S-D | None | eNOS signaling | Impaired lung development | 492 |
| Erlotinib (50 μg) intra-amniotic at E16.5 | Rat S-D | E. coli LPS | EGFR inhibitor | Alveolarization improved | 290 |
| Erythropoietin (1,200 U/kg sc) P1–P2–P3 | Rat S-D | 85% O2, P1–P14 | Erythropoiesis | Alveolarization improved | 114 |
| Fluoxetine (7 mg/kg) by gastric gavage, daily, E0–E21 | Rat Wistar | None | Serotonin | Vascularization worsened | 512 |
| Follistatin (2 ng ip) on P4, P7, P10, and P13 | Mouse C57BL/6 | 85% O2, P1–P14 | Receptor type IIB-Fc antagonist, activin-binding protein | Alveolarization improved | 299 |
| GCSF (50 µg iv) daily from E125–E129 | Sheep | E. coli LPS + mechanical ventilation | Inflammation | Alveolarization worsened | 192 |
| GGA (500 mg/kg/day, oral) P1, P2, and P3 | Mouse C57BL/6J | 90% O2; P1–P3 | HSP70 inducer | Alveolarization improved | 530 |
| GSSG (dose not declared) | Guinea pig Hartley | Ascorbylperoxide | Oxidative stress | Alveolarization improved | 151 |
| 25-Hydroxyvitamin D (100, 500, or 1,000 ng/kg) nebulization daily, P1–P14 | Rat S-D | None | Vitamin D | Alveolarization improved | 522 |
| ICG001 (10 mg/kg ip) daily, P5–P10 | Mouse FVB | 90% O2; P1–P14 | Wnt/β-catenin inhibitor | Alveolarization improved | 452 |
| IL-1Ra (10 or 100 mg/kg sc) daily, P3–P28 | Mouse C57BL/6J | Maternal LPS + neonatal 60% or 85% O2, P3–P28 | IL-1β signaling | Alveolarization improved (in selected protocols) | 456 |
| IL-1Ra (10 mg/kg sc) (protocol not declared) | Mouse C57BL/6J | 85% O2; P1–P14 | IL-1β signaling | Alveolarization improved | 557 |
| IL-1Rra (1 mg/kg sc) daily, P3–P14 | Mouse C57BL/6 | 85% O2; P3–P14 | IL-1β signaling | Alveolarization improved | 297 |
| Kineret (4 mg/kg sc), preinjury | Mouse CD-1 | IL-1β | IL-1R antagonist | Alveolarization improved | 375 |
| LA1 (1 mg/kg ip) twice daily, P2–P14 | Rat S-D | 85% O2; P2–P14 | CD11b/CD18 agonist | Alveolarization and vascular remodeling improved | 231 |
| Leptin (1 mg/kg ip) E19–E20 | Rat S-D | Uterine artery ligation | Leptin | Fetal lung maturation enhanced | 96 |
| Liraglutide (0.05 g/kg sc) daily, E24–E31 | Rabbit | Surgical CDH | Stable GLP-1 derivative | Vascular remodeling improved | 145 |
| Metformin (25 or 100 mg/kg sc) daily, P1–P10 | Rat Wistar | 100% O2; P1–P10 | Potent antidiabetic; lower glucose levels in the circulation | Alveolarization and vascularization improved | 99 |
| Metyrapone (0.5 mg/ml, ad libitum in drinking water), E10–P0 | Rat S-D | 50% Food-restricted diet | Glucocorticoid pathways | Alveolarization improved | 409 |
| MK-801 (Dizocilipine; 0.05 mg/kg ip) daily, E15.5–E20.5 | Rat S-D | 10.5% O2 8 h/day, E15.5–E20.5 | N-Methyl-d-aspartate receptor agonist | Alveolarization improved | 298 |
| Montelukast (10 mg/kg ip) daily, P1–P14 | Rat S-D | Bleomycin | Leukotriene receptor antagonist | No effect | 146 |
| N6022 (1 mg/kg ip) single injection at P21 | Mouse C57BL/6 | 60% O2; P1–P21 | Alcohol-dehydrogenase 5 inhibitor | Airway function improved | 437 |
| NSC23766 (5 mg/kg ip) daily, P2–P12 | Rat S-D | 90% O2; P2–P12 | Rac1 inhibitor | Alveolarization, vascularization, pulmonary hemodynamics, and cardiac remodeling worsened | 224 |
| Omeprazole (10 or 25 mg/kg ip) daily, P1–P14 | Mouse C57BL/6J | 85% O2; P1–P14 | Proton pump inhibitor | Alveolarization and vascularization worsened | 483 |
| ONO-1301SR (30 mg/kg sc) single injection at E9.5 | Rat S-D | Nitrofen | Prostacyclin agonist | Alveolarization improved | 538 |
| pCMV-miR-489 (0.7 mg·kg−1·day−1, intranasal) every second day, P4–P14 | Mouse C57BL/6 | 85% O2; P4–P14 | miR-489 | Alveolarization worsened | 404 |
| Protein C (1,200 IU/kg sc) daily, P3–P28 | Mouse C57BL/6J | Maternal LPS + neonatal 60% or 85% O2 P3–P28 | IL-1β signaling | Alveolarization improved (in selected protocols) | 456 |
| PUFA ω-3 (po) maternal diet supplement, E14–P10 | Rat Wistar | 85% O2; P3–P10 | Polyunsaturated fatty acid | Alveolarization improved | 480 |
| PUFA ω-6 (po) maternal diet supplement, E14–P10 | Rat Wistar | 85% O2; P3–P10 | Polyunsaturated fatty acid | No effect | 480 |
| SB431542 (4.2 µg/g ip) daily | Rat S-D | 60% O2; P1–P14 | TGFBR1 inhibitor | Alveolarization improved | 53 |
| SC57461A (10 mg/kg ip) daily, P1–P14 | Rat S-D | Bleomycin | LTA4 hydrolase inhibitor | Vascular remodeling improved | 146 |
| Sulforaphane (5 μmol ip) E13, E15, and E17 | CD-1 | 85–95% O2; P2–P4 | NFE2L2 activator | No effect | 348 |
| Vitamin D (in diet, po), via nutrition | Mouse C57BL/6J | UV light free and vitamin D-depleted diet | Vitamin D | Airway function improved | 460 |
| Zileuton (10 mg/kg ip) daily P1–P14 | Rat S-D | Bleomycin | 5-LPO inhibitor | Pulmonary hemodynamics improved | 146 |
The elevated levels of HMGB1 proteins in tracheal aspirates from BPD patients has stimulated interest in HMGB1 as a regulator of lung alveolarization. Neutralization of HMGB1 with an anti-HMGB1 IgY antibody in mouse pups maintained under hyperoxic conditions attenuated the deleterious impact of hyperoxia on lung alveolarization (583). Improvements in lung structural development after anti-HMGB1 IgY treatment were attributed to attenuation of TGF-β1 and IL1Β levels in the lung and improved septal elastin deposition.
The utility of omeprazole, a proton pump inhibitor, to potentiate aryl hydrocarbon receptor (AHR) activity in experimental BPD has also been studied (483), where mouse pups received omeprazole concomitant with exposure to hyperoxia. Administration of omeprazole was associated with worse lung structure, leading these authors to suggest that omeprazole-mediated reductions in AHR function exacerbated hyperoxia-induced perturbations to lung development, ostensibly by reduced expression of the AHR target genes Nqo1 as well as Cyp1a1 (encoding cytochrome P-450 family 1 subfamily A member 1) (483). It is important to note that these studies were correlative, and the impact of omeprazole directly on AHR function was not assessed; nor was a causal link between omeprazole, AHR, and lung development validated. The utility of omeprazole was validated in a second independent study in preterm-delivered rabbits exposed to hyperoxia, where omeprazole blunted the impact of hyperoxia on lung structure and also improved respiratory function. Both studies highlight the utility of omeprazole as an exciting candidate interventional agent in preterm birth.
Geranylgeranylacetone (GGA) has also been demonstrated to promote lung alveolarization in mouse pups exposed to hyperoxia with concomitant administration of GGA, and lung structure was assessed (530). Improvements in lung alveolarization in the GGA-treated, hyperoxia-exposed mice were attributed to decreased apoptosis, possibly through GGA-driven increased heat shock protein 1B (HSPA1; also called HSP70) expression; however, the activation of the GGA/HSPA1B pathway as a causal player in lung alveolarization remains to be established.
Apart from transgenic and knockout mouse studies and pharmacological interventions, some correlative studies have been undertaken in which changes in the expression of selected genes and protein of interest that accompany normal or disordered lung development have highlighted possible mediators of lung alveolarization.
Several correlative studies have noted changes in gene or protein expression associated with aberrant late lung development, but no causal studies with pharmacological agents or transgenic mice have yet been undertaken. In the nitrofen-based CDH model, reduced expression of PAS-domain transcription factor (NPAS3), which is known to regulate Drosophila tracheogenesis, has been associated with lung hypoplasia (414). Similarly, the bombesin and ghrelin systems have been proposed to sensitize the lungs to the action of retinoids in nitrofen-induced CDH (415), and ventilation of preterm-delivered rat fetuses with nitrofen-induced CDH deregulated nitric oxide synthase and VEGF receptor expression in the lung (167). It was thus proposed that these factors may contribute to lung hypoplasia in experimental CDH. Other notable changes in protein expression in experimental CDH include alterations to T-box transcription factors 2, 4, and 5 steady-state levels in the branching airways (517) and to β-carotene-15,15′-oxygenase-1 and -2 steady-state levels (515).
Dynamic changes in the expression of versican (VCAN) over the course of embryonic mouse lung development have suggested a role for VCAN in lung development, and the observation that Pseudomonas aeruginosa infection can increase VCAN expression in adult mice hints at a possible deregulation of VCAN expression in neonates in a background of chorioamnionitis. However, a causal role for VCAN in lung alveolarization remains to be experimentally demonstrated (491). Histological studies on lung tissue from human patients who died at risk of BPD as well as premature-delivered baboons that were exposed to hyperoxia revealed a reduced abundance of the large neutral amino acid transporter solute carrier family 7 member 5 (SLC7A5; also called CD98 and LAT1) in affected lungs, suggesting that impaired amino acid transport may contribute to arrested lung development or that blunted S-nitrosothiol import might impair inhaled NO therapy. These possibilities remain to be validated in vivo (46). The long-form leptin receptor (124), SFTPA and SFTPB (87), adrenomedullin (ADM), calcitonin receptor-like receptor (CALCRL), and receptor activity modifying protein 2 (RAMP2) (350) have also received attention.
Signaling pathways relevant to lung development continue to be studied in cell culture. In vitro studies on cytochrome P-450 family 1 subfamily B member 1 (CYP1B1) identified a functional role for CYP1B1 in human lung epithelial cell responses to hyperoxia exposure in vitro (134), where overexpression of the CYP1B1 gene potentiated hyperoxia-driven cytotoxicity in human H358 human airway epithelial cells while siRNA-mediated knockdown of CYP1B1 expression protected human BEAS-2B airway epithelial cells from hyperoxia-driven cytotoxicity. Thus these authors proposed that CYP1B1 may represent an interventional target in BPD. In analogous studies by the same group, growth differentiation factor 15 (GDF15) was important for the viability of lung epithelial cells (529), and the AHR was important for the viability of lung endothelial cells (595) under hyperoxic conditions. This group has further indicated that AHR may modulate hyperoxic lung injury by regulating genes responsible for cell proliferation and inflammation (482). New insights into leptin function have also been reported, where leptin has been documented to promote ATII cell survival in a phosphoinositide-3-kinase regulatory subunit 1 (PIK3R1)-dependent fashion, leading the investigators to propose that leptin promotes fetal lung maturity (96). These in vitro studies now require in vivo validation.
Role of sex in aberrant lung development.
The sex bias in favor of male preterm infants developing BPD (501) has stimulated much interest in addressing how sex may impact normal and aberrant lung development. The importance of this idea is underscored by the observations that regulatory factors such as glucocorticoids can program pathways that generate sex-specific effects that may subsequently be inherited over multiple generations (110). Several studies have recently examined sex bias in preclinical models of BPD. Krithika Lingappan and coworkers report that male mice are more susceptible to the effects of hyperoxia on the structural development of the lung than female mice (301). In that study, mouse pups were exposed to hyperoxia and allowed to recover in room air, after which lung structure was assessed. The male bias in that study was attributed to accentuated inflammatory cell infiltration into the lungs, provoked by hyperoxia, in male animals. These data contrast with other studies, however, where a stereological assessment of the lung structure in mouse pups exposed to hyperoxia did not reveal any sex bias of the disturbances to lung structure provoked by hyperoxia (383). The same mouse strain was employed in both studies. It is noteworthy that the latter study may not have been powered to detect the impact of sex on lung responses to hyperoxia. Additionally, these two studies utilized different levels of oxygen injury (85% O2 vs. 95% O2) and different durations of oxygen exposure (P1–P5 vs. P5–P14), and the assessment of lung structure was made at different postnatal ages (P14 vs. P21). Along these lines, the impact of sex on the progress of normal lung development has also been assessed by stereology in normally developing mice (430), where lung volume and total number of alveoli in the lung were similar in male and female mice up to P10, after which lung volume in male mice was larger, leading to a higher total number of alveoli being noted in the male mouse lungs from P10 onwards.
One recent study, which employed administration of the antiandrogen flutamide to pregnant mice, demonstrated that the expression of selected microRNA species in fetal lungs was influenced by androgens (70). To date, this study remains descriptive, since no lung structure analyses were performed in that study and causality between sex hormones, microRNA expression, and lung development was not addressed. Several other avenues have been explored to explain sex differences in aberrant postnatal lung maturation, including studies on the metabolism of estradiol, which has recently been acknowledged to impact sex differences in the cardiac consequences of PH (160, 267) and impact influenza virus infection in a sex-specific manner (416), highlighted CYP1A1 as a possible mediator of the protective effect of estradiol in hyperoxia-induced perturbations to lung alveolarization (333). This idea remains to be causally validated in vivo.
In human subjects, the early developing fetal lung has been interrogated for transcriptomic correlates of maturation and sex, using 61 female lungs and 78 male lungs of ages between 54 and 127 days of gestation (256). In that study, before gestational day 96, male lungs appeared more mature than female lungs, based on transcriptomic profiling, with the trend reversed after gestational day 96. Additionally, while sex differences were noted (primarily in the olfactory transduction, DNA replication, and the tricarboxylic acid cycle pathways) in the transcriptome of the developing lung, gestational age was a more dominant factor than was sex, for disease risk, when assessed with asthma and BPD genes (256).
In the context of stem cells, sex-relevant differences have also been noted, where female bone marrow-derived MSCs have greater therapeutic efficacy than male MSCs in reducing neonatal hyperoxia-induced lung inflammation and vascular remodeling. Taking this idea further, the beneficial effects of female MSCs were more pronounced in male animals. Thus female MSCs may be the most potent bone marrow-derived MSC population for lung repair in severe BPD complicated by PH (466).
New Interventional Strategies to Drive Alveolarization
Several recent reports have highlighted novel efforts to drive proper lung development in preclinical models utilizing pharmacological interventions (including synthetic modulators of gene expression) to promote lung alveolarization (Table 3). Among these are treatment of mouse pups with either activin A receptor type IIB-Fc (ActRIIB-Fc) antagonist or follistatin during exposure to hyperoxia (299). Both ActRIIB-Fc and follistatin antagonize activin A, a member of the TGF-β superfamily of growth factors. Neutralization of activin A activity with either interventional agent protected against the hyperoxia-induced arrest of lung alveolarization. These studies highlight the potential utility of antagonizing activin A and/or TGF-β signaling to promote proper lung alveolarization, and they dovetail nicely with two other studies that document the requirement for apical secretion of follistatin-like 1 (FSTL1), a secreted BMP antagonist, for normal lung development (295) as well as decreased epithelial follistatin-1 expression in hypoplastic lungs in nitrofen-induced CDH in rats (518). The maternal administration of another peptide growth factor, glucagon-like peptide 1, to pregnant rabbits in a surgical model of CDH (145) improved lung vessel structure and was without impact on proximal airway structure but had significant fetal and maternal side effects. Moving from growth factors to cytokines, antagonism of tumor necrosis factor (TNF)-α signaling has also been explored, where the TNF-α antagonist etanercept was demonstrated to protect lung alveolarization when administered to rats exposed to hyperoxia (253). An identical study was performed by a second group, also in Turkey, reporting the same results, where etanercept administration to rat pups in the hyperoxia model protected lung alveolarization (405). These studies are difficult to reconcile with recent clinical data that describe lower levels of TNF-α in tracheal aspirate of mechanically ventilated preterm infants who went on to develop BPD, where increased lung inflammatory cell recruitment, increased lung apoptosis, and increased TGF-β signaling in the lung were noted in Tnfa−/− mouse pups that underwent mechanical ventilation (147). These effects were attributed to complex interplay between TNF-α and TGF-β signaling systems in the developing lung. Clearly, there remains much work to do in addressing TNF-α function during aberrant lung alveolarization.
Cellular energy homeostasis has also been targeted in an effort to promote lung alveolarization, where metformin was applied as an inhibitor of protein kinase, AMP-activated, α1 catalytic subunit (PRKAA1; also called 5′-AMP-activated protein kinase, AMPK) to rats maintained in a hyperoxia environment (99). While metformin administration prolonged life and reduced pulmonary inflammation, coagulation, and fibrosis, metformin did not limit the injurious effects of oxygen on alveolar development or prevent PH and right ventricular hypertrophy. A second report by the same group (100) explored the impact of Ki16425 administration [to inhibit lysophosphatidic acid (LPA) receptors (LPARs) 1 and 3] to newborn rats exposed to hyperoxia. The basis of this study was a report that expression levels of LPAR-1 and LPAR-3, as well as autotaxin, which generates the LPA ligand, are increased in rat lungs in vivo in response to hyperoxia exposure (481). Furthermore, with Lpar1−/− mice LPA-LPAR1 signaling was documented to be critical for lung alveolarization (163). The Ki16425 intervention limited lung inflammation and PH and reduced fibrosis but, however, was without effect on lung alveolarization or lung vascular development (100). These data were further supported with rats carrying a missense mutation in the Lpar1 gene.
Stabilization of hypoxia-inducible factor-1α (HIF-1α) has also received attention in lung alveolarization, where rat pups received the HIF-1α stabilizer deferoxamine, in an experimental BPD model that relied on intra-amniotic LPS administration, followed by hyperoxia exposure and subsequent room-air recovery (103). Deferoxamine administration attenuated the detrimental impact of hyperoxia on alveolarization and lung vascular development. These data contrast with a genetic approach to the same question (527), where a stable, constitutively active, HIF-1α-subunit (called HIF-1αΔODD) was expressed in the distal lung epithelium of transgenic mice exposed to hyperoxia. In that system, overexpression of HIF-1αΔODD in the lung epithelium did not protect lung alveolarization from the effects of hyperoxia. The reason for the different findings in the two studies is not immediately apparent; however, it is noteworthy that different experimental animals (mouse vs. rat) and different oxygen injury protocols were employed, and the deferoxamine would have globally stabilized HIF-1α, while the transgenic approach stabilized HIF-1α in a specific cell type (the lung epithelium).
Given the role of oxidative stress in hyperoxia-based BPD models, thioredoxin reductase has also received attention as a therapeutic target, where administration of aurothioglucose, a thioredoxin reductase-1 inhibitor, to mouse pups protected against hyperoxia-induced disturbances to lung alveolarization (293). These effects were attributed to Nfe2l2 activation, evident by increased steady-state levels of NQO1 and HMOX1. In a related study, the mitochondrion-specific antioxidant (2-(2,2,6,6-tetramethylpiperidin-1-oxyl-4-ylamino)-2-oxoethyl)triphenylphosphonium chloride (mitoTEMPO) was administered to mouse pups with concomitant exposure hyperoxia (122). Exposure to hyperoxia elicited an increase in Nox1 expression, and mitoTEMPO administration protected the developing lungs from the deleterious impact of hyperoxia on alveolarization. The data suggest the possible utility of targeting mitochondrial reactive oxygen species (ROS) to promote normal lung alveolarization.
The use of molecular hydrogen (H2) as a lung-protective agent has also been applied to rats in which chorioamnionitis was induced with intrauterine E. coli O55:B5 LPS. Pregnant dams were supplied with H2 ad libitum in drinking water (374). Pups from dams that received H2 in drinking water exhibited improved alveolarization at early investigated time points, but these improvements were not evident at later time points. The H2 treatment improved DNA oxidation status, assessed with 8-hydroxy-2′-deoxyguanosine, and restored normal FGF receptor 4 (FGFR4), VEGF receptor 2 (VEGFR2), and HMOX1 expression in pup lungs. The protective effects of H2 are consistent with a report that treatment of A549 human lung adenocarcinoma epithelial cells with 10% hydrogen gas for 24 h decreased production of ROS in both LPS-treated and untreated cells (195).
Using rats exposed to hyperoxia, Zhang and coworkers described the accumulation of SFTPC in ATII cells (593). Inhibition of autophagy, with either 3-methyladenine or knockdown of Atg7, prevented the hyperoxia-induced accumulation of SP-C in the MLE-12 mouse lung epithelial cell line, suggesting that autophagy mediated elements of hyperoxia-driven pathology. Along similar lines, Vineet Bhandari and coworkers (507) elegantly demonstrated the utility of blocking expression or function of regulatory-associated protein of MTOR, complex 1 (RPTOR; part of the MTORC1 complex that is a negative regulator of autophagy) with the pharmacological agent Torin2, Rptor+/− mice, or intranasal delivery of Rptor-silencing siRNA, in mouse pups exposed to hyperoxia. All three interventions improved lung structure and cardiac remodeling. These beneficial effects of RPTOR antagonism were accompanied by increased autophagy. Thus these two reports add aberrant lung development to the list of lung pathologies in which autophagy has been recently implicated (6), including diffuse parenchymal lung disease (12, 64, 197, 227, 233, 398), influenza virus infection (184, 578), space radiation-induced lung injury (106), sepsis (379), PH (267), cigarette smoke pathology (296, 600), and emphysema (509). This study dovetails nicely with a related study that documents increased expression of autophagy-related microtubule-associated protein 1 light chain 3α (MAP1LC3A) in the lung in response to hyperoxia and implicates MAP1LC3A as a negative regulator of ATII cell transdifferentiation into ATI cells (592). There is therefore tremendous scope for the further study of autophagy in relation to normal and aberrant lung development.
Other novel interventional strategies to promote lung alveolarization during stunted lung development include dexpanthenol (408), fluoxetine (512), and ONO-1301SR, a slow-release form of a novel synthetic prostacyclin agonist with thromboxane inhibitory activity (538). In addition to parenteral pharmacological interventions, the use of mouse models of BPD has also been extended to the study of the effects of continuous positive airway pressure (CPAP; 6 cm H2O, 3 h/day) during exposure of mouse pups to hyperoxia, followed by recovery in room air. In addition to perturbed alveolarization, hyperoxia exposure elevated respiratory system resistance (Rrs) and decreased compliance (Crs), while CPAP limited the impact of hyperoxia on lung alveolarization, Rrs, and Crs (447). It remains noteworthy that preclinical studies in animal models that address prenatal interventions to limit or reverse perturbations to postnatal alveolarization are very much lacking (238).
The –omics Cluster of Approaches
Transcriptomics.
The plasticity of the lung transcriptome continues to form the basis of many studies in lung development (329), with one recent meta-analysis identifying 83 lung-specific genes, including 62 protein-coding genes, 5 pseudogenes, and 16 noncoding RNA genes (571), where an in silico analysis suggested that ≈50% of lung-specific genes were implicated in lung pathology and ≈25% of lung-specific genes were implicated in lung development.
Transcriptomic studies have been conducted in animal models of arrested lung development, including preterm rabbits exposed to hyperoxia (463), where 2,217 deregulated transcripts were noted, including genes implicated in inflammation, vascular development, lung development, and oxidative stress pathways. The studies complement the transcriptome-wide screening described above for IUGR studies in rabbits (588).
Transcriptomic studies have also addressed mouse lung development, where a genomewide analysis of gene expression was undertaken at 26 pre- and postnatal time points between E9.5 and P56), in three common inbred strains of laboratory mouse: the C57BL76J, A/J, and C3H/HeJ strains (51). With principal component analysis and least-squares regression modeling, strain-independent and strain-dependent patterns of gene expression were noted. Particularly noteworthy was that the gene expression patterns noted supported the canonical stages of mammalian lung development. Similar studies have been undertaken during lung development in rhesus macaques, to highlight cross talk of dynamic functional modules (groups of genes in regulatory networks) during primate lung development (586). Pathological changes to transcriptome were assessed in lungs from rat pups that received intraperitoneal E. coli 026:B6 followed by a period of mechanical ventilation (130). A microarray analysis of the lung transcriptome revealed alternations to MMP expression and the complement system, identifying MMP and complement-mediated pathways as candidate pathogenic factors in the associated lung pathology.
In human subjects, the early developing fetal lung has been interrogated for transcriptomic correlates of maturation and sex, using 61 female lungs and 78 male lungs of ages between 54 and 127 days of gestation (256). In that study, before gestational day 96 male lungs appeared more mature than female lungs, based on transcriptomic profiling, with the trend reversed after gestational day 96. Additionally, while sex differences were noted (primarily in the olfactory transduction, DNA replication, and the tricarboxylic acid cycle pathways), in the transcriptome of the developing lung, gestational age was a more dominant factor than was sex, for disease risk, when assessed with asthma and BPD genes (256). Along similar lines, exome sequencing has been employed in a pilot study to identify novel genes relevant for BPD susceptibility in 26 patients with severe BPD in an Italian cohort (85). These genes included NOS2 (encoding nitric oxide synthase 2) MMP1 (encoding matrix metallopeptidase 1) CRP (encoding C-reactive protein), LBP (encoding lipopolysaccharide binding protein), and members of the Toll-like receptor family (85). Exome sequencing has also been performed on blood spots from BPD patients, where 258 genes were identified with rare nonsynonymous mutations in BPD patients (291). The identified genes were related to lung development-relevant pathways, including collagen fibril organization, epithelial morphogenesis, and WNT signaling. Many of the genes identified have already been implicated in aberrant lung development in experimental BPD. This study raised the very novel idea that rare genetic variants may be important causal contributors to increased risk for BPD (5).
Genomics.
Genomic changes, including SNPs and copy number variants (585), as well as gene-environment interactions (61) continue to be considered as underlying genetic predisposition factors for BPD. As such, there is much interest in the detection of SNPs associated with BPD or BPD susceptibility (Table 4). Genomewide association studies have also been undertaken in an effort to link SNPs with susceptibility to BPD. One recent study that employed 751 infants, of whom 428 developed BPD or died, was unable to identify any SNP significantly associated with BPD susceptibility (24). However, multiple SNPs in CD44, ADAR (encoding adenosine deaminase), miR-219, and genes encoding phosphorus oxygen lyase enzymes (including adenylate and guanylate cyclases) suggested that these molecules may play a role in genetic predisposition to BPD, with increased expression of CD44 and miR-219 noted in the lung tissue from patients who died with BPD.
| Study | Population and Location of Study | Gene Symbol | Gene Name | rs ID | Common Variant Name | Variant Type | Association | OR | 95% CI | P Value |
|---|---|---|---|---|---|---|---|---|---|---|
| 175 | <32 wk neonates with or without BPD, Greece | NQO1 | NAD(P)H Quinone Dehydrogenase 1 | rs1800566 | 609C>T | Missense, Pro187Ser | Higher risk of BPD | NR | NR | 0.026 |
| 323 | Preterm neonates, Egypt | TIRAP | Toll-Interleukin 1 Receptor (TIR) Domain-Containing Adaptor Protein | rs8177374 | 2054C>T | Missense, Ser180Leu | Higher risk of severe BPD | NR | NR | 0.044 |
| TLR5 | Toll-like Receptor 5 | rs5744168 | 1174C>T | Nonsense, Arg392Stop | No association with BPD nor BPD severity | 0.051 | ||||
| 421 | Gestational age ≤28 wk, caucasian, Italy | VEGFA | Vascular Endothelial Growth Factor A | rs1547651 | −8339A>T | Substitution in 5′ flanking region | No association with BPD | 0.9 | ||
| rs833058 | C>T | No association with BPD | 0.4 | |||||||
| rs833061 | −460T>C | 5′ Near gene substitution | No association with BPD | 0.3 | ||||||
| rs3025039 | 936C>T | Substitution in 3′ UTR | No association with BPD | 0.07 | ||||||
| eNOS | Endothelial Nitric Oxide Synthase | rs2070744 | −786T>C | Substitution in promoter | Association with BPD development | 1.89 | 1.03–3.46 | 0.04 | ||
| rs61722009 | 4a4b | 27-bp repeats in intron 4 | No association with BPD | 0.7 | ||||||
| rs1799983 | 894G>T | Missense, Glu298Asp | Association with BPD development | 1.92 | 1.09–3.39 | 0.02 | ||||
| AGT | Angiotensinogen | rs699 | 803T>C | Missense, Met268Thr | No association with BPD | 0.08 | ||||
| AGTR1 | Angiotensinogen Type 1 Receptor | rs5186 | 1166A>C | Substitution in 3′UTR | No association with BPD | 0.4 | ||||
| ACE | Angiotensin-Converting Enzyme | rs4291 | −240A>T | Substitution in promoter | No association with BPD | 0.06 | ||||
| rs1799752 | Alu (I/D) | Nonsense substitution in intron 16 | No association with BPD | 0.5 | ||||||
| HMOX1 | Heme Oxygenase-1 | rs3074372 | (GT)n L | GT repeat in promoter | No association with BPD | 0.3 | ||||
| 467 | VLBW infants (<1.5 kg), USA | NFE2L2 | NAD(P)H Quinone Dehydrogenase 1 | rs6721961 | −617C>A | Substitution in promoter | Association with decreased BPD severity | 0.6 | 0.4–0.9 | 0.023 |
| SOD2 | Superoxide Dismutase 2 | rs4880 | c.47C>T | Missense, Val16Ala | No association with BPD nor BPD severity | NR | ||||
| GCLC | Glutamate-Cysteine Ligase Catalytic Subunit | rs17883901 | −129C>T | 5′ Near gene substitution | No association with BPD | NR | ||||
| GSTP1 | Glutathione S-Transferase Pi 1 | rs1695 | c.313A>G | Missense, Ile104Val | No association with BPD | NR | ||||
| HMOX1 | Heme Oxygenase 1 | rs2071747 | c.19G>C | Missense, Asp7His | No association with BPD | NR | ||||
| NQO1 | NAD(P)H Quinone Dehydrogenase 1 | rs1800566 | 609C>T | Missense, Pro187Ser | Homozygous state was associated with higher risk of BPD | 3 | 1.4–6.8 | 0.007 | ||
| 596 | Preterm infants (<32 wk, 500–1,499 g), Han population, China | SFTPB | Surfactant Protein B | rs2077079 | −18C>A | Substitution in 5′ UTR | Higher risk of BPD | 1.71 | 1.228–2.389 | 0.001 |
| rs1130866 | 1580C>T | Missense, Thr131Ile | Lower risk of BPD | 0.752 | 0.593–0.954 | 0.016 | ||||
| rs762548 | 4564T>C | Intronic substitution | No association with BPD | 0.183 | ||||||
| 321* | Preterm singleton infants <30 wk without major congenital malformations, Finland | KDR (VEGFR2) | Kinase Insert Domain Receptor | rs4576072 | C>T | Intronic substitution | Higher risk of BPD grade 2 or 3† | 3.15 | 1.62–6.12 | 0.0005 |
| 534 | BPD patients, USA | DDAH1 | Dimethylarginine Dimethylaminohydrolase 1 | rs480414 | G>A | Intronic substitution | Lower risk of PH in BPD patients | 0.39 | 0.18–0.88 | 0.01 |
| 418‡ | Preterm infants (<32 wk), Denmark | UGT1A1 | UDP Glucuronosyltransferase family1 member A1 | rs3064744 | UGT1A1*28 | TA insertion | Higher risk of BPD. | 1.71 | 1.23–2.39 | NR |
| Higher risk of needing supplementary oxygen | 1.25 | 1.05–1.50 | NR | |||||||
| Longer duration of oxygen therapy | 6.38 | 1.87–10.89 | NR |
Copy number variants have also been addressed as contributors to BPD susceptibility, where three loci, at 11q13.2, 16p13.3, and 22q11.23–q12.1, that exhibit copy number variants with increased frequency in BPD patients were identified (9). Genes residing within these regions included 15 genes that undergo temporal changes in expression during transition from the pseudoglandular to the canalicular stage, which is a most relevant period of lung development in BPD. Pathway analysis of these genes highlighted AHR signaling (482, 483, 595), xenobiotic metabolism signaling, and glutathione-mediated detoxification (151, 152), all of which have also been highlighted elsewhere in this review, as have genomic studies on FOXF1 SNPs in ACDMPV (132).
Proteomics.
While some interest in the utility of proteomics to study childhood disease has been discussed (413), no studies have been generated to date that address BPD or preclinical studies on BPD. Such studies are long overdue, but encouraging methodological developments, including the proteomic analysis of laser capture microdissected alveolar tissue samples (107), suggest that such studies are in the pipeline.
Epigenetics.
Epigenetic control of lung development and epigenetic contributions to the early origins of adult lung disease (247) are an emerging area of interest in lung developmental biology. The importance of this emerging area is emphasized, given increasing recent evidence of perturbations to DNA and histone acetylation and methylation in lung diseases, including emphysema, which has histopathological characteristics not dissimilar to those of BPD (506). Epigenetic control of SOD3 expression in PH has also been reported (395), and it is noteworthy that SOD3 has also been associated with aberrant alveolarization (129). Epigenetic effects have also been implicated in the regulation of airway smooth muscle remodeling in asthma (108, 431), and control of IL-22 receptor subunit α1 (IL22RA1) expression during childhood in the lung in nonhuman primates (141).
Attempts have also been made to correlate epigenetic changes to the chromatin, such as DNA methylation, with alterations to the transcriptome, in the context of alveolarization. Genomewide DNA methylation and steady-state mRNA levels were assessed in preseptation (P3) and postseptation (P42) lungs from normally developing mice and in lung tissue from human preterm infants with BPD compared with preterm stillbirths, and correlations were drawn between DNA methylation and gene expression (115). These data identified 20 genes that may be regulated by DNA methylation during mouse and human lung development, including genes implicated in basal cell carcinoma signaling, sonic hedgehog signaling, and axonal guidance pathways. The causality of methylation-regulated expression for these gene sets remains to be demonstrated in animal models of lung development. The pioneering work of Trent Tipple and coworkers on correlating steady-state levels of members of the microRNA-17/92 cluster with promoter methylation in severe BPD (450) is discussed in Noncoding RNA above.
More targeted approaches to understanding the role of histone and DNA methylation in lung development have also been undertaken, where the histone methyl transferase called enhancer of zeste 2 polycomb repressive complex 2 subunit (EZH2) has been documented to play a critical role in lung lineage specification and survival at birth (212), ostensibly by repression of IGF-I signaling to prevent basal cell differentiation in the developing lung (168). In addition to data indicating that EZH2 restricts the basal cell lineage during normal lung endoderm development to allow the proper patterning of epithelial lineages during lung formation (489), EZH2 has also been reported to restrict the smooth muscle lineage during mouse lung mesothelial development (488). Specifically in the context of experimental BPD, Zhu and coworkers (602), using rats exposed to hyperoxia, documented that runt-related transcription factor 3 (RUNX3), a transcription factor known to play a role in ATII cell apoptosis (169), was downregulated in the lung epithelium after hyperoxia exposure. This was correlated with increased expression of DNMT3B and EZH2 in lung tissues and ATII cells, and high DNA methylation and H3K27me3 modifications were present in the RUNX3 promoter region. In vitro, the H3K27me3 inhibitor JMJD3 (Jumonji domain containing 3) and the EZH2 inhibitor 3-deazaneplanocin A (DZNep) as well as the DNA methylation inhibitor 5-aza-2′-deoxycytidine (5-aza-CdR) appreciably reversed the hyperoxia-induced downregulation of RUNX3 expression in ATII cells. Recently described roles for EZH2 in lung alveolarization are summarized in Fig. 2.
Fig. 2.Proposed roles for the histone-lysine N-methyltransferase enhancer of zeste homolog 2 (EZH2) in late lung development. EZH2 is pivotally placed to impact multiple processes central to late lung development. These processes are integrated in the scheme, where numbers in parentheses direct the reader to the source article. ATII, alveolar type II.
In vitro investigations employing 5-aza-CdR, a methyltransferase inhibitor, revealed that 5-aza-CdR inhibited the growth of lung fibroblasts isolated from the lungs of rat pups exposed to hyperoxia, ostensibly by inhibiting the methylation of the Cdkn2a gene (encoding cyclin-dependent kinase inhibitor 2A, also called P16) (599), suggesting other (new) pathways to explore in vivo.
Stem Cells
The potential utility of exogenous stem cell application or recruitment of endogenous stem cells to protect lung development or drive lung repair continues to be a very active area of preclinical and clinical neonatal medicine, as well as basic research. Several key reviews have emerged in this area over the past 30 months (10, 13, 59, 66, 144, 176, 177, 181, 198, 200, 276, 284, 357–359, 371, 372, 399, 400, 411, 432, 461, 471, 490, 503), with the number of reviews far exceeding the number of original research articles!
Perturbations to endogenous progenitor cells function under pathological conditions remains a matter of interest, for example, the recently described impairment of cord blood endothelial colony-forming cells (ECFCs), a type of endothelial progenitor cell, in CDH (161). However, the bulk of current interest in lung stem cells lies in the derivation of lung cells from pluripotent cells, and recent developments in this direction include the generation of ATII cells from human urinary induced pluripotent stem cells with a four-step induction protocol (556); the generation of early pulmonary endoderm cells (which then formed cells that are representative of distal airway epithelium) from human foregut stem cells (193); and the directed generation of airway epithelial cells from human bone marrow MSCs (292). Step-by-step protocols describing either the in vitro generation of lung and airway progenitor cells from human pluripotent stem cells (221) or the generation of functional CFTR-expressing airway epithelial cells from human pluripotent stem cells (566) have been published. Recent stem cell-based preclinical studies include the combined application of endothelial progenitor cells and inhaled NO in rats, exposed to hyperoxia, where combination therapy resulted in improved lung alveolarization (309). In a related study (11), the in vivo therapeutic efficacies and paracrine potencies of human umbilical cord blood-derived mesenchymal stromal cells, human adipose tissue-derived mesenchymal stromal cells, and human umbilical cord blood mononuclear cells were compared, where human umbilical cord blood-derived mesenchymal stromal cells exhibited the best therapeutic efficacy and paracrine potency in protecting against neonatal hyperoxic lung injury when applied via the intratracheal route to rat pups exposed to hyperoxia. Similarly, intratracheal administration of GFP-tagged bone marrow-derived MSCs to newborn rats exposed to hyperoxia improved alveolarization (187). Related studies have also been undertaken in the hyperoxia-based mouse model of BPD with intravenous administration of bone marrow-derived MSCs, where a beneficial effect on lung alveolarization was noted, without (311) and with (312, 597) concomitant erythropoietin administration, ostensibly by therapeutic modulation of TGF-β signaling. The utility of placenta-derived human MSCs to manage arrested alveolarization in rats induced by combination of intraperitoneal E. coli LPS administration to pregnant dams followed by hyperoxia exposure was also recently documented (105). In that study, intratracheal administration of MSCs to rats improved lung alveolarization and increased lung vessel density. Interestingly, the intratracheal administration of human umbilical cord blood-derived MSCs was recently documented to attenuate both lung and brain injuries in rat pups exposed to hyperoxia, where brain weight and myelin basic protein expression were inversely correlated with lung mean linear intercept (MLI) and inflammatory cytokines, and the number of terminal deoxynucleotidyl transferase dUTP nick end-labeling (TUNEL)-positive brain cells was also correlated with lung MLI (257). This study represents a very welcome extension of experimental BPD studies to include consideration of the brain alongside the lung. A number of observations made using stem cells to manage hypoxia-induced adult lung disease, including an impact on EMT (540), hypoxia-induced proliferation of tissue-resident endothelial progenitor cells in the lung (389), and the ability to modify stem cells by microRNA transfection (223), remain to be implemented in studies on neonatal lung disease.
Development and Refinement of Models
There is currently much discussion about the proper modeling of BPD in experimental animals. A tremendous wealth of information about both normal lung development as well as aberrant lung alveolarization has been gleaned through the use of existing animal models of BPD (209). Arrested alveolarization and perturbed respiratory function associated with preterm birth and BPD can be modeled in mice (56), rats (401), rabbits (119, 328), preterm lambs (15, 121, 528), preterm pigs (32, 81), and baboons (581). Inroads continue to be made in assessing the utility of these models (243, 244, 319, 388), with the aim of developing and using very translational models in mind. Several recent studies have considered the levels of oxygen used in hyperoxia-based BPD models, with a recent systematic comparison of several oxygen injury protocols in newborn mice, including 40% O2, 60% O2, and 85% O2, as well as intermittent and gradient exposures being evaluated for an impact on lung alveolarization (383). In a complementary study (149), the impact of intermittent hypoxia and hyperoxia was addressed, where alveolar simplification was also noted after exposure to intermittent hyperoxia (4 h of 50% O2 alternated with room air for 20 h; until P7) or intermittent hypoxia/hyperoxia (2 h of 12% O2 followed by 2 h of 50% O2 followed by 20 h of room air; cycle repeated until P7).
In general, the translational relevance of a model, in terms of modeling human disease, improves as the size of the experimental animal increases. For this reason, despite concerns about costs and ethical challenges, development of the rabbit, lamb, pig, and baboon models remains important. To this end, for example, Julio Jiménez and coworkers continue to refine the preterm rabbit model and recently reported parallel changes in the development of PH and arrest of lung development compared with clinical subjects (237). Also, a limited amount of work continues to be generated with nonhuman primates, for example, a recent RNA-Seq where a developmental stage-specific protein-protein interaction network was proposed, which highlighted functions for Notch signaling, the cell cycle, NOD-like receptor signaling, and Toll-like receptor signaling pathways as “bridging pathways” during embryonic and postnatal lung development in rhesus macaques (586). Pulmonary systems in BPD models continue to be characterized in detail, for example, the recent report that SFTPB rather than SFTPC is a more reliable marker to evaluate surfactant maturation in fetal sheep lungs (304), which has also received attention in the mouse lung epithelium (332). Beyond the widely used rodent, rabbit, porcine, ovine, and nonhuman primate models, descriptive work describing lung development in bovine fetuses has also recently appeared (138).
Important developments have also been made with the artificial placenta (63, 351), where Benjamin Bryner and coworkers documented the survival of very premature lambs (110–120 days of gestation, where term lambs reach 145 days of gestation) using an artificial placenta (77). The lambs, which correspond to 22–24 wk of gestation in humans in terms of lung developmental stages, survived for 1 wk with an artificial placenta (77).
Interventional studies continue to be refined for the application of inducible, conditional, transgenic mice in preclinical models of BPD. Among recent developments are the refinements of a tamoxifen administration protocol that maximizes tamoxifen responsiveness while titrating tamoxifen dose against the increased sensitivity of hyperoxia-exposed mouse pups to hyperoxia (458). These are important developments, and future directions should include attention to the role of litter size in studies on lung development (litter size is often not reported in methods sections of papers, and small vs. large litter size almost certainly impacts pup nutrition, which would have consequences for lung development).
Several alternative models have been—and continue to be—developed to study alveolarization and associated angiogenesis both in vivo and in vitro. Among these is the engraftment of human fetal lung material in the renal subcapsular space of severe combined immunodeficient (SCID) mice, where plexus-forming angiogenesis was noted 4 wk after engraftment, suggestive of intussusceptive-type angiogenesis (127). At the junction of in vivo and in vitro systems, decellularized lung scaffolds have been used to derive airway epithelia from embryonic stem cells (484), with utility for the study of cell-matrix interactions during lung organogenesis and for disease modeling or drug discovery platforms of airway-related pathologies such as cystic fibrosis, and there is much interest in the utility of decellularized lung scaffolds to study lung vascularization (498). At the purely in vitro level, air-liquid interface and complex cell cultures (23), organoids, and lung on a chip (471) continue to be developed and refined.
The use of three-dimensional organoids to study lung structure and physiology continues to be developed (39, 376). The stepwise differentiation of human pluripotent stem cells (embryonic and induced) into lung organoids has been described, and RNA-Seq has revealed that these organoids were strikingly similar to human fetal lungs on the basis of global transcriptional profiles. These data add support to the idea that human lung organoids are an appropriate model to study human lung development, maturation, and disease (143). Alternative methods for organoid or organoid-like structure generation are also under development and include the culture of fetal human lung fibroblasts on sodium alginate beads (504, 505). Along these lines, the generation of three-dimensional cultures of lung epithelial cells or cell lines with photodegradable microsphere templates has also been described (285).
Lung explants are widely used to study early lung development, notably, lung branching, in vitro and may be particularly useful when pharmacological inhibitors that are toxic to living, intact organisms are employed. For example, lung explants have recently been used to document that angiogenesis is not required for airway branching in vitro (196). These explant systems have not been widely used to study lung alveolarization yet.
Relating BPD to animal models.
Referring to a term-born mouse exposed to hyperoxia as having BPD is simply not correct. Mouse and rat pups have no requirement for postnatal oxygen supplementation. It is important that basic scientists addressing the pathogenic mechanisms of BPD appreciate the distinction between BPD as a clinical syndrome that is defined by the level and duration of oxygen supplementation required and hyperoxia-induced perturbations to lung alveolarization in experimental animals, which are often considered as the same as BPD. While term-born mice and rats are born in the saccular stage of lung development, these mice have lungs that are properly adapted and competent for efficient gas exchange, while preterm-born infants have immature lungs that are exposed to an oxygen-containing environment too early, and these lungs are not yet properly adapted and thus are not competent for the level of atmospheric gas exchange demanded by preterm-born infants.
Bridging the translational divide that exists between clinical neonatal respiratory medicine and lung developmental biology has never been more urgent. To this end, it is increasingly important to recognize that BPD is a syndrome, not a single clinical entity, with consideration of different patterns of BPD now emerging (118, 391). With this in mind, Vineet Bhandari and coworkers have also recently suggested consideration of the exact nature of acute respiratory distress syndrome that may develop in patents with a history of BPD (60).
Perturbed lung development in BPD is often considered synonymously with arrested lung alveolarization, with associated pulmonary vascular disease neglected by comparison. Indeed, pulmonary vascular disease associated with BPD has been described as the new frontier in BPD (123). As pointed out this year by Colby Day and Rita Ryan, we need to focus on more appropriate, pathology-driven nomenclature that appropriately describes the different disease patterns that now constitute the syndrome of BPD (123). It is very likely that the different pathological pictures that emerge in different patterns of BPD will have different etiologies, which will need to be recapitulated by the refinement of existing animal models, or the development of new animal models, to facilitate the proper translational analysis and to validate candidate new intervention strategies tailored to these new emerging patterns of BPD.
The time is also ripe for a reconsideration of how BPD is modeled in experimental animals. A review of the prevailing literature reveals important discrepancies that warrant discussion within the community. One important question to ask is: How well do prevailing animal models recapitulate BPD lung pathology? This is particularly important considering the evolution of the lung histopathological picture of BPD patients over the past 50 years. The medical management of preterm-born infants has changed a lot over that time, but BPD models have not “moved with the times.” Do we know how the lung architecture looks in today’s BPD patients? Most studies that have reported lung pathology in BPD patients rely on material harvested 20–30 years ago. Given the evolution of the histopathology of BPD since 1967, and the emergence of different patterns of BPD, the delineation of the pathological features, in terms of lung parenchymal, vascular, and airway structure, appears to be very urgent indeed. Until basic investigations know what to model, it is most likely that the prevailing models will continue to be used, even if these models do not accurately recapitulate the current pathology of the syndrome that we are trying to understand and treat.
A second important question to ask is: How well do our injurious stimuli align with pathological stimuli that cause BPD? The clinical causes of BPD relate to a combination of several injurious events and stimuli including, but not limited to, preterm birth, infection and inflammation, comorbidities, oxygen toxicity, and baro- and/or volutrauma. One or more of these pathogenic factors are employed in experimental animal models to simulate lung (and extrapulmonary) damage. However, there is widespread criticism of the very high concentrations of oxygen that are sometimes employed (up to 100% O2 for 14 days). These very high levels of inspired oxygen do not compare well with current clinical practice, which aims to use inspired oxygen levels as low as possible (in the region of 40–60% O2). Related to the point raised above regarding exactly how lung histopathology looks today in BPD patients, it would be very worthwhile to ascertain what structural perturbations to the lung architecture need to be mimicked to assess what the lowest inspired oxygen levels are that recapitulate these changes in experimental animals. It is time to engage the pulmonary pediatric pathology community to answer these important questions!
At least two sets of recent studies (383, 393, 455, 456) have highlighted how the beneficial impact of a candidate intervention might be missed when the injurious stimulus is too high. These observations also make a strong case for using as low an inspired oxygen level as possible in experimental animal models of BPD.
Biomarkers
Several advances have been made identifying candidate biomarkers that are predictive for the development in BPD in preterm infants (Table 5), and this idea has been the subject of several recent thought-provoking reviews (174, 270, 314, 420, 449), with associated reviews addressing biomarkers that predict the clinical course and severity of RSV infection (73). In a pathobiology context, biomarkers are interesting because biomarkers may themselves be novel pathological players in disease onset or progression or they may reveal the activity of new pathogenic pathways. As such, biomarkers may facilitate understanding of disease pathogenesis.
| Biomarker | Source | Association | Correlation | Reference |
|---|---|---|---|---|
| Absolute nucleated red blood cells | Blood | BPD | No | 330 |
| Acylcarnitine | Plasma and dried blood spots | BPD | No | 186 |
| ADMA | Plasma | BPD | Yes | 252 |
| ADMA | Plasma | BPD, PH in BPD | Yes | 33, 535 |
| Antioxidants (vitamin A, vitamin C, catalase) | Plasma | BPD, NEC | Yes | 3 |
| Blood cytokine profile | Blood spots | Classic vs. atypical BPD | Yes | 118 |
| BNP | Serum | Tricuspid regurgitation | Yes | 259 |
| C20:4-PC-to-C22:6-PC ratio | Cord plasma, parturient serum | BPD severity | Yes | 58 |
| CEACAM6 | Biopsy | BPD | Yes | 180 |
| Complex lipid profile | ebc metabolome | BPD | Yes | 84 |
| Growth factors | Amniotic fluid | CDH | Yes | 82 |
| Isofuranes | Urine samples | BPD | Yes | 263 |
| LOOH, GSH | BALF | BPD | Yes | 152 |
| miR-17/92 cluster | Plasma | BPD | Yes | 450 |
| Neutrophil elastase | Amniotic fluid | BPD | No | 226 |
| NTproBNP and BNP | Arterial blood | PH in preterm infants | No | 260 |
| NTproBNP, citrulline | Plasma | PH in BPD | Yes | 363 |
| Plasma proendothelin-1 | Plasma | BPD | Yes | 50 |
| Platelet mass index | Blood | BPD, NEC, ROP, IVH | Yes | 403 |
| SPARC | Tracheal aspirates | BPD | Yes | 425 |
| TIMP-2 | Blood | BPD | Yes | 278 |
| VEGF-to-PlGF ratio | Plasma | BPD | Yes | 434 |
Given the pathogenic roles ascribed to MMPs and MMP inhibitors, it is noteworthy that low TIMP-2 serum levels at birth have recently been described to be associated with the subsequent development of BPD in preterm infants (278). Furthermore, secreted protein, acidic, rich in cysteine (SPARC; also called osteonectin) was also recently been documented to predict the development of BPD (425). The antioxidant systems of neonates have also received attention, where decreased plasma levels of vitamin A, vitamin E, and catalase at birth were lower in patients who went on to develop BPD (3). Similarly, lower platelet mass has also been associated with BPD, as well as ROP, intraventricular hemorrhage, and necrotizing enterocolitis (403). In contrast, both absolute nucleated red blood cell counts (330) and amniotic fluid levels of neutrophil elastase (226) do not appear to have any utility as biomarkers for BPD in preterm infants. The identification of biomarkers to assess nutritional status of preterm infants has also received attention, where acylcarnitine profiles that reflect fatty acid metabolism have been suggested to reflect nutritional status in preterm infants (186).
Concerning the isoprostane, isofuran, neuroprostane, and neurofuran lipid peroxidation by-products in urine, isofuran in particular was highlighted as a potentially useful predictive marker for the development of BPD when assessed during the first 4 days of postnatal life in preterm infants at <32 wk of gestational age (263). In terms of lipid analyses, BAL fluids have also received attention, where increased lipid hydroperoxide levels and decreased levels of reduced glutathione were noted in BAL fluids from BPD patients, suggesting that lung biochemical monitoring of preterm infants might predict BPD (152). The phospholipid composition of maternal and cord plasma has also received attention, where a low arachidonic acid-phosphatidylcholine-to-docosahexaenoic-phosphatidylcholine ratio was associated with BPD severity in preterm infants at <28 wk of gestational age (58). Plasma proendothelin-1 has also demonstrated utility as a predictor for the development of BPD in preterm infants at <32 wk of gestational age when measured at the end of the first week of postnatal life (50), and VEGF/placental growth factor heterodimer levels have been associated with BPD and death in preterm infants at <32 wk of gestational age (434), while placental growth factor in cord blood is considered to predict the development of BPD in preterm infants (219, 577). Furthermore, increased plasma levels of asymmetric dimethylarginine (ADMA) (33, 535) and N-terminal pro-B-type natriuretic peptide (NTpro-BNP) and citrulline (363) have been associated with the development of PH in BPD patients. Biomarker studies have not been restricted to BPD patients; changes in levels of various growth factors in amniotic fluid have also been noted in human CDH cases (82).
Not all “biomarkers” are biomarkers alone; several reports have identified candidate functional roles in pathology. For example, increased steady-state mRNA levels of carcinoembryonic antigen-related cell adhesion molecule 6 (CEACAM6) as well as elevated levels of CEACAM6 protein in the lungs have been described in infants with chronic lung disease (180). However, this marker localized to hyperplastic epithelial cells that exhibited a sevenfold elevated proliferation rate (assessed by PCNA staining), which may indicate a yet-to-be-determined role for CECAM6 in regulating the proliferation of human epithelial cells. The same is true for the miR-17/92 cluster, which appears to function as both biomarker and pathogenic mediator of BPD (450).
Extrapulmonary Considerations
To date, animal models of BPD have carefully examined aberrant alveolarization and vascular development in the lung. However, prematurity, and BPD as a syndrome, encompass not only disturbances to lung development but also pathological involvement of the retina (in ROP) (203) and the musculature (541) and acute kidney injury (34, 35) and are complicated by adverse neurodevelopmental outcomes (28, 37, 524, 579), where hyperoxia per se impacts the development of the central nervous system (444). The liver has recently been recognized as a player in parturition and preterm birth (338), and iatrogenic skin disorders have been noted in preterm infants with BPD (113).
It is acknowledged here and elsewhere (25, 382) that more attention should be paid to extrapulmonary systems in experimental animal models of BPD. Along these lines, recent reports have described interruptions to retinal vascular development and severe and irreversible neurovascular disruption in newborn mice exposed to hyperoxia, with various periods of posthyperoxia recovery in room air (269). Similarly, exposure of rats to hyperoxia followed by recovery in room air caused a decrease in the cross-sectional surface area of the anterior and posterior brain, a decrease in the cross-sectional surface area of the anterior commissure, and a decrease in the thickness of the motor cortex (424). These changes were also accompanied by a decrease in the total retinal thickness as well as a decrease in the thickness of the outer segment, the inner nuclear layer, and the inner and outer plexiform layers of the retina. These changes in the brain and the eye were correlated with disturbed alveolarization (424). Also in rats, intra-amniotic administration of LPS followed first by exposure of newborn pups to hyperoxia and then by room-air recovery caused increased apoptosis in the hippocampus (579). This was accompanied by poorer performance in the Morris water maze test, suggesting impaired cognitive function (579).
Longitudinal and Long-Term Studies
There is much interest in how preterm birth and lung disease in the immediate postnatal period may cause or predispose patients to lung disease in adult life (67, 78, 135, 207, 334, 407, 433). Recent clinical reports continue to provide evidence that adult preterm birth survivors—in particular, those who developed BPD—experience respiratory symptoms and exhibit clinically relevant pulmonary impairment during adolescence and into adult life (86).
The early origin of adult lung disease has been recently addressed in several studies in preclinical models, including in newborn mice exposed to hypoxia or hyperoxia after which animals were maintained under room air conditions until adulthood (439). These studies documented that hyperoxia, but not hypoxia, caused increased muscularization of the walls of small (up to 100-μm diameter) pulmonary arteries in adult mice; additionally, exposure of neonates to hypoxia and hyperoxia resulted in an emphysema-like histopathological phenotype in adult mice as well as increased airway reactivity and left ventricular dysfunction in adult mice (439). Along similar lines, but using lower oxygen concentrations, exposure of rat pups to hyperoxia, followed by room-air recovery to adulthood, revealed no persistent impact on lung alveolar structure (327). In contrast, exposure of hyperoxia-treated pups to intermittent hypoxia (10% O2, for 10 min every 6 h) between P14 and P28 did perturb lung alveolar structure as assessed in adult mice. Perturbations to lung vascular and heart structure and function were also noted after intermittent hypoxia exposure.
Other studies have addressed the long-term effects of early exposure to hyperoxia, where mouse pups were exposed to hyperoxia and then recovered in room air to adulthood (264). Early exposure to hyperoxia caused increased airway reactivity in adult mice, assessed by metacholine challenge, where increased thickness of the airway smooth muscle layer was noted in lung sections. The authors further report that perturbations to lung alveolarization persisted in adulthood (264). These data indicated that early exposure to hyperoxia results in persistent changes in lung structure and functions. Along these lines, and given that long-term sequelae of BPD include lifelong obstructive lung disease and refractory bronchospasm, exposure of mouse pups to hyperoxia increased the abundance of S-nitrosoglutathione reductase [also called alcohol dehydrogenase 5 (class III), chi polypeptide; encoded by Adh5], a mediator of endogenous bronchoconstriction (437). This was attributed to decreased abundance of miR-342–3p, which targets the Adh5 mRNA. Treatment of adolescent or adult mice that had been exposed to hyperoxia as neonates with S-nitrosoglutathione or with a ADH5 inhibitor attenuated airway hyperresponsiveness. These data highlight a possible mechanism and management strategy for the airway hyperresponsiveness noted in BPD survivors. As described in Lung Function and Structure-Function Correlates, exposure to hyperoxia in the immediate postnatal period also leads to compromised lung function in adult mice (7, 112). It is noteworthy that early hyperoxia exposure can also create predisposition to respiratory virus infection; it has recently been documented that exposure of newborn mice to hyperoxia followed by room air recovery to adulthood resulted in reduced host resistance to influenza A virus infection in adulthood (445). As such, hyperoxia exposure in the neonatal period may predispose affected individuals to viral susceptibility later in life.
Longitudinal studies have also been performed in nonhuman primates, where 30-day-old rhesus macaques were sensitized to house-dust mites and exposed either to house-dust mite allergen with or without concomitant ozone exposure or to ozone alone and lung structure was then evaluated by stereology either 6 or 30 mo after exposure (205). Early-life exposure to house-dust mite allergen with or without allergen caused more alveoli to be generated in the lungs, with higher capillary density, indicating that early-life exposure to house-dust mite allergen influences postnatal lung development. Interestingly, house-dust mite exposure during the immediate postnatal period in mice was demonstrated to skew Th2 immunity, in an IL33-dependent manner (125); however, the relevance of this observation to lung alveolarization is not yet clear.
Long-term follow-up studies remain of interest in a clinical context as well, where BPD survivors are now being followed into adolescence to assess what impact having BPD as a neonate continues to have on lung health and lung function in later life. Among recent reports in this area is a metabolomics analysis of exhaled breath condensates that was able to distinguish between BPD survivors and age-matched individuals with no history of pediatric lung disease. The investigators suggested that this study indicated altered surfactant composition that persisted beyond infancy (84). In a separate study (574) using a nitrogen multiple-breath washout approach in children (aged 6–16 yr) who had been born either preterm or term, the Sacin score suggested a functionally normal alveolar compartment in all both groups while the Scond score suggested functionally impaired upper airways in the preterm group. These investigators suggested that these data indicated functional evidence for continued (catch-up) alveolarization in preterm-born children (574). The area of metabolomics is clearly emerging in studies on the effects of preterm birth on lung development, with a metabolomic analysis of amniotic fluid revealing that such analyses may be useful in the prediction of preterm delivery and BPD (48). However, metabolomics studies in preclinical models are entirely lacking to date.
Lung Function and Structure-Function Correlates
Despite the plethora of studies on the development of methodological approaches for the assessment of lung structure, few studies to date have assessed lung function over the course of normal or aberrant lung development. One recent study to address this reported the expression profiling of lung function genes over the course of lung development in human lungs, where TMEM163 (encoding transmembrane protein 163) steady-state mRNA levels increased and CDC123 (encoding cell division cycle 123) steady-state mRNA levels decreased with fetal lung age, where SNPs in TMEM163 and CDC123 impact forced vital capacity (FVC) and forced expiratory volume in the first second (FEV1) and FEV1/FVC, respectively (352). These expression trends were confirmed by immunohistochemistry. These data provide the first evidence that lung function genes are differentially expressed over the course of lung development.
Two recent reports (7, 112) have highlighted structure-function correlates in animal models, where elements of lung structure, including MLI and microvascular volume, which were determined morphometrically, not stereologically, were correlated with lung function assessed by the diffusion factor for carbon monoxide (DfCO). Studies using mouse pups exposed to hyperoxia that subsequently recovered in room air highlighted that exposure to hyperoxia in the immediate neonatal period resulted in compromised lung function (assessed by DfCO) in adult mice. These preliminary studies are an important step forward, which highlights the need to assess lung function alongside lung structure. It is worth noting that reduced DlCO in infants with BPD has been noted, which might reflect structural perturbations to the lungs of these patients (91), with the caveat that these studies were performed on BPD survivors as stable outpatients <3 yr of age, who did not require oxygen therapy when DlCO measurements were made (41). Related studies employing DlCO have also been conducted that indicate that preterm birth (27–35 wk of gestational age, without BPD) per se does not impair lung parenchymal development (36). These studies relied entirely on DlCO data, and not on direct studies on lung parenchymal structure.
Air pollution continues to be an important consideration for effects on lung development (83, 261, 543). The impact of lung structure on lung function over the course of lung development has practical implications for studies on particle deposition in the lung, where nanoparticle deposition in infant lungs can be impacted by changes in the structure of the pulmonary acinus as the lung develops (204); this led Frank Henry and Akira Tsuda to predict that human infants at the age of ≈2 yr might be most at risk for the harmful effects of air pollution. These studies also have implications for the deposition of pharmacological agents with inhalers, where dose estimates used to determine inhaler use might overestimate particle deposition by up to 55% for newborns and, conversely, might underestimate particle deposition by up to 17% for 2-year-old infants (204). Such studies are likely to be greatly facilitated by mathematical modeling, such as a recent exciting report of spatially localized and time-resolved characterization of oxygen transport in the acinus, spanning the period from infancy to adulthood (210), and the development of computational models that describe inhalation therapy relevant to the therapeutic application of aerosols in the developing acinus (251).
To date, despite the plethora of work on lung function assessment in adult animals and published protocols for the determination of pressure-volume loops in experimental mice (300), very few studies to date report lung function studies in neonatal mice and rats. This is clearly an area that warrants further attention by investigators, where perturbations to the lung architecture must be correlated with lung function if the alterations to lung structure are to have physiological meaning.
Methodological Advances
Microscopic and radiological imaging.
The quantitative assessment of elements of the lung architecture in developing lungs has made great strides with the increased application of stereological approaches to the study of normal and aberrant lung alveolarization. Several detailed guidelines have recently been published outlining the correct use of stereological approaches for the study of alveolar architecture (373). Most stereological studies to date address the estimations of, inter alia, alveoli number, septal wall thickness, and gas-exchange surface area. These studies have included the stereological monitoring of saccular and alveolar development between P5 and P669 (430). To date, stereological studies on capillaries have proved problematic, mostly because of the “sheetlike” nature of the alveolar capillary network. However, progress continues to be made in addressing this complex issue, and a recent publication by Christian Mühlfeld and coworkers has developed a methodological approach for the estimation of alveolar capillaries by design-based stereology based on the Euler-Poincaré characteristic, by counting topological constellations such as “islands,” “bridges,” and “holes,” which can be easily recognized in lung sections (562a). These advances in stereological analysis of lung structure complement other advances in microscopy, which include microscopic approaches (423), such as optical cryoimaging of mitochondrial redox states (335).
Given that stereological analysis of lung structure is a laborious process, there are efforts underway to automate the analysis of lung structure. These efforts are admirable, although plagued by formidable barriers. Yves Tremblay’s group recently reported the development of an automated high-performance analysis of lung morphometry (465), describing morphometric measurements, including the MLI, and the number of septal tips in a defined area. While the approach is “pixel perfect,” and the investigators can measure the parameters that they are measuring with very high precision, what element of the lung architecture these parameters represent remains unclear. This is always known with the stereology approach. A drawback of the study is that there was no side-by-side comparison to alternative approaches. For example, can a given intervention that disturbs the lung architecture be detected with the same accuracy with the automated morphometry method as with the stereological approach? Presently, there appears to be no realistic substitute for the stereological approach if robust quantitative data that can describe the total number of alveoli in a lung, the mean septal wall thickness, the gas-exchange surface area, and a multitude of other parameters is desired. In their current state, automated approaches to the analysis of lung architecture may be suitable for a “quick and dirty” assessment of generalized lung structure, which may provide a high-throughput means of analysis to test the impact of, for example, chemical agents or mutations on the lung architecture. However, in its current state, the automated analysis of lung structure is unlikely to identify comparatively minor or subtle changes in lung structure, nor will it identify specific pathological transformations of lung tissue. Every journey needs a destination, and this destination is admirable; however, we are barely beyond the start of this particular road. Future initiatives to develop automated analysis of lung structure might be supported by fractal analysis, as elegantly illustrated in a recent exciting report from the De Caro group in Padova (427).
There is increasing enthusiasm for visualizing the lung structure in three dimensions, the utility of which has been highlighted by the discovery of intrapulmonary anastomoses in the lungs of patients with BPD with three-dimensional reconstructions of histological sections (165). Similarly, prominent intrapulmonary bronchopulmonary anastomoses have been detected in infants and children with Down syndrome with an identical approach (79, 526). This idea has been expanded in several recent reports. The Sun group in San Diego has beautifully documented the developing alveolus in three dimensions with confocal microscopy of 70-μm sections of mouse lungs, illustrated the spatial organization of ECM and ECM-producing cells, and proposed that the elastin network is organized into a “fishnet” structure, which directs the protrusion of alveolar ridges into the alveolar lumen, thereby generating nascent alveoli (71). Three-dimensional reconstructions have also been employed to study lung vascular structure by Christian Mühlfeld and coworkers, using digital reconstruction of serial sections to study the alveolar capillary network (185).
Radiological methods offer the best approach to the analysis of lung structure in three dimensions, with the caveat that the resolution of parenchymal structures remains poor. Several new clinical approaches to studying the infant lungs by radiological means are now entering widespread use to allow for longitudinal assessment of BPD patients. These approaches include low-dose computerized tomographic techniques that lessen radiation burden, and nonionizing ultrashort echo time, and hyperpolarized gas magnetic resonance imaging (MRI) techniques (555).
The use of hyperpolarized gas in MRI is finding some application for the study of lung growth under normal and aberrant conditions, including long-term follow-up studies on CDH patients. With the use of 3He MRI and 1H MRI for lung volume measurements, the 3He apparent diffusion coefficient was calculated separately for the ipsilateral and contralateral lungs in nine young adult (18–31 yr old) CDH patients. These radiological studies indicated that functional and microstructural changes in affected lungs persisted into adulthood (497).
Radiological approaches continue to be refined in preclinical models to study the lung vasculature, where robust high-resolution segmentation is achieved, permitting quantitative analysis of postnatal lung development using radiopaque casting of the pulmonary artery vascular tree (419). These studies could be performed in mice as young as 2 wk of age, which is the end point in most studies addressing lung alveolarization in mice.
Polymerase chain reaction.
Several recent studies have addressed useful modifications to routine laboratory approaches to study changes in gene expression, and these include suggestions for the consideration of RNA integrity in lungs, which are notoriously rich in RNase (280), and suggestions for suitable reference genes for real-time RT-PCR studies in RNA pools from healthy and diseased mouse lungs, which include Hprt (encoding hypoxanthine guanine phosphoribosyl transferase), Eef2 (eukaryotic translation elongation factor 2), and Rpl13a (encoding ribosomal protein L13A) (280) and Tub1a (encoding alpha tubulin) (349). A related study in cattle lungs revealed 5.8S rRNA and PPIA (encoding peptidylprolyl isomerase A) to be suitable reference genes in bovine material (303).
Lessons from Other Vertebrates and Invertebrates
In 1866, the German embryologist and physician Ernst Häckel, who was a great proponent of recapitulation theory, famously implied that “ontogeny recapitulates phylogeny” (189, 190). The Häckelian form of recapitulation theory is now considered defunct (308); however, much may still be learned about lung development in mammals by examining lung development in lower vertebrates and invertebrates. In addition to the well-studied respiratory system development in insects such as Drosophila (339), resources are now becoming available that describe the development of the respiratory system in other vertebrates, including amphibians such as Xenopus (441). Even the development of book lungs, the respiration organ used for atmospheric gas exchange in arachnids, currently receives attention (154), although book lungs are not evolutionarily related to mammalian lungs. Along these same lines, in one particularly noteworthy report, molecular studies revealed that lung development in the extant basal actinopterygian fish, the Senegal bichir (Polypterus senegalus), is very similar to that of tetrapods (521). Similar observations were made with the coelacanth Latimeria chalumnae, and these data provide the first molecular evidence that the developmental program for lungs was already established in the common ancestor of actinopterygians and sarcopterygians.
Technical Concerns About Reporting
As highlighted elsewhere in this review, a number of concerns about terminology persist in the reporting of research on lung development and BPD. Among these is the incorrect assumption that mice exposed to hyperoxia “have BPD.” These mice have hyperoxia-induced perturbations to alveolarization, not BPD. BPD is a clinical definition related to the need for oxygen supplementation at a particular level and for a particular duration in the postnatal period. While this may be the case for preterm-delivered nonhuman primates and lambs, it is unlikely to be the case for term-born mouse or rat pups.
Another frequently encountered misnomer is contained in the statement that “lungs from hyperoxia-exposed mice are emphysematous.” While it is true that the histopathological picture of both emphysema and clinical and experimental emphysema includes alveolar simplification, emphysema implies the destruction of preexisting alveolar units, while in BPD patients and in animal models of BPD the formation of the alveoli is blocked or stunted. These are different concepts, and until it is demonstrated that a protease/antiprotease imbalance in developing lungs causes the destruction of preexisting alveolar units and as such is a pathophysiological component of arrested alveolarization noted in clinical or experimental BPD, it remains incorrect to refer to aberrantly developing lungs as having emphysema or being emphysematous.
As highlighted in a recent editorial (365), the word “septum” is increasingly incorrectly pluralized to “septae.” In Latin, neuter nominative and accusative nouns of the second declension such as septum, which end in –um, are made plural by the ending –a. Thus the correct plural form of septum is septa.
Perhaps more politically charged is the interchangeable use of “sex differences” vs. “gender differences.” This idea has been the subject of an Institute of Medicine report, where it was clarified that sex refers to the classification of living things, generally as male or female, according to their reproductive organs and functions assigned by chromosomal complement, while gender refers to a person’s self-representation as male or female, where gender is rooted in biology and shaped by environment and experience (229). The World Health Organization takes a similar position, suggesting that sex refers to the biological and physiological characteristics that define men and women and “male” and “female” are sex categories, while gender refers to the socially constructed roles, behaviors, activities, and attributes that a given society considers appropriate for men and women and “masculine” and “feminine” are gender categories (320, 567). With these ideas in mind, it appears appropriate to describe preclinical studies comparing effects in male and female mice as “sex differences.” We—as authors, reviewers, and editors—must prevent these neologisms and incorrect terminology from becoming accepted through increasing and widespread use in the scientific literature.
In addition, declaration of the strain of the experimental animal employed continues to be an important omission in many preclinical studies. The notorious impact of strain differences on the responses of mouse lungs to injurious stimuli cannot be ignored. This not only extends to strains (for example, C57BL/6 vs. C3H/HeN) but also to substrains (for example, C57BL/6J vs. C57BL/6N). This is not a trivial concern, since there are demonstrable differences in the response of the retina to 75% O2 from P1 to P14 between C57BL/6J and C57BL/6N mouse pups (268). These effects are not limited to hyperoxia, since the gut metabolome and microbiome differ between C57BL/6J and C57BL/6N mice maintained on a high-fat diet, an area of interest in lung alveolarization (554). These reports highlight a need for the careful consideration—and reporting—of the strains and substrains of experimental animals that are employed for studies on lung alveolarization. This review serves as a plea to investigators to include vitally important strain information in a manner that is easy to locate!
New Resources Available to Investigators
New online resources to assist lung developmental biologists and neonatologists in their studies on alveolarization are becoming increasingly available and adapted to increasingly user-friendly platforms. Among these are interrogatable databases housed at the Jackson Laboratory and available through the LungMAP consortium. Both facilities are briefly described here to highlight their availability to the community.
Data from a recent transcriptome-wide analysis of gene expression during pre- and postnatal lung development in three common inbred strains of laboratory mouse, the C57BL76J, A/J, and C3H/HeJ strains, are publicly available (51) at the Jackson Laboratory via the link http://lungdevelopment.jax.org/. An example of data output from this database is illustrated in Fig. 3, where the data sets were interrogated for Sftpb expression over the period between E9.5 and P56 in all three strains.
Fig. 3.Example of data output from the lung development gene expression database at The Jackson Laboratory. As an example, Sftpb gene expression for A/J (blue inverted triangle), C57BL/6J (filled circles), and C3H/HeJ (orange triangle) over the course of mouse lung development between embryonic day (E)9 and postnatal day (P)56 is illustrated.
Similarly, as part of the LungMAP consortium (https://www.lungmap.net/), a searchable database via the Lung Gene Expression Analysis (LGEA) portal, available at https://research.cchmc.org/pbge/lunggens/mainportal.html, has been made available to the community for mapping single-cell gene expression in the developing lung. This has now been expanded to include transcriptomics data from lung tissues and cells from humans and mice at various stages of lung development and is useful for the analysis, display, and interpretation of gene expression patterns that have been obtained from single cells, as well as sorted cell populations and, indeed, whole lung samples (31, 140). An example of data output, taking Pgdfra gene expression as a candidate, is illustrated in Fig. 4. No doubt, the utility of this valuable portal will be extended in the future.
Fig. 4.An example of data output form the LGEA portal. A: screen capture from the lung sorted cells analysis section of the LGEA web portal. A query for the expression profiles of mouse PDGRFα+ fibroblasts at different time points was selected. B: expression profiles of PDGFRα+ fibroblasts sorted at days E16, E18, P7, and P28 obtained with Short Time-series Expression Miner (STEM) software as displayed in the LGEA portal. C: screen capture in which profile 18 was selected. Profile 18 clustered genes in which gene expression increased transiently during bulk alveolarization (P7) and decreased by P28. D: expression pattern of genes comprising profile 18. E: list of genes in profile 18. F: the most-enriched functions identified for genes of profile 18. Images in B, D, and F were downloaded from the LGEA portal. Original data from Dr. Anne K. Perl.
Conclusions
This review of the recent (published between 1 January 2015 and 30 June 2017) literature dealing with mechanisms of lung alveolarization and BPD has highlighted a broad spectrum of developments that include the identification of novel pathways relevant to normal and aberrant lung alveolarization, the development and refinement of new technologies to study lung development, and the increasing availability of new resources to assist investigators in the field. Some noticeable shifts in attention to particular areas were evident in our review of the literature. Noticeable among these shifts was fewer original studies (but more reviews) on the utility of MSC to restore lung development under pathological conditions. In contrast, a flurry of activity was detected in the areas of maternal, enteral, and parenteral nutrition and how nutrition impacts lung development. Methodological approaches continue to be developed to study lung structure, in particular, concerning the stereological analysis of the lung architecture, where new methodology is now being developed to address the complicated question of how to address the “amount” of capillaries in the lung. Other welcome emerging areas include consideration of extrapulmonary systems in preclinical studies as well as correlates of disturbed lung structure with lung function, which is not trivial in small animals such as mouse pups in the immediate postnatal period. Some areas are also noticeable by their absence; prominent among these is the dearth of studies on the interaction between pulmonary infection and lung development, an area that is most relevant in a translation sense, considering children with developing lungs with coexistent respiratory virus infection. It is clear that much challenging and exciting work lies ahead.
GRANTS
This study was supported by the Max Planck Society; Rhön Klinikum (D. E. Surate Solaligue, J. A. Rodríguez-Castillo, K. Ahlbrecht, and R. E. Morty) Rhön Klinikum AG Grants FI_66 (R. E. Morty) and FI_71 (K. Ahlbrecht); University Hospital Giessen and Marburg Grant UKGM62589135 (R. E. Morty); the Federal Ministry of Higher Education, Research and the Arts of the State of Hessen “LOEWE Programme” (R. E. Morty); the German Center for Lung Research (Deutsches Zentrum für Lungenforschung) (R. E. Morty); and by German Research Foundation (Deutsche Forschungsgemeinschaft) through Excellence Cluster EXC147 (R. E. Morty), Collaborative Research Center SFB1213/1 (R. E. Morty), Clinical Research Unit KFO309/1 (R. E. Morty), and individual research grant Mo 1789/1 (R. E. Morty).
DISCLOSURES
No conflicts of interest, financial or otherwise, are declared by the authors.
AUTHOR CONTRIBUTIONS
D.E.S.S., J.A.R.C., K.A., and R.E.M. drafted manuscript; D.E.S.S., J.A.R.C., K.A. prepared figures; D.E.S.S., J.A.R.C., K.A., and R.E.M. edited and revised manuscript; D.E.S.S., J.A.R.C., K.A., and R.E.M. approved final version of manuscript.
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