Alveolarization is the process by which the alveoli, the principal gas exchange units of the lung, are formed. Along with the maturation of the pulmonary vasculature, alveolarization is the objective of late lung development. The terminal airspaces that were formed during early lung development are divided by the process of secondary septation, progressively generating an increasing number of alveoli that are of smaller size, which substantially increases the surface area over which gas exchange can take place. Disturbances to alveolarization occur in bronchopulmonary dysplasia (BPD), which can be complicated by perturbations to the pulmonary vasculature that are associated with the development of pulmonary hypertension. Disturbances to lung development may also occur in persistent pulmonary hypertension of the newborn in term newborn infants, as well as in patients with congenital diaphragmatic hernia. These disturbances can lead to the formation of lungs with fewer and larger alveoli and a dysmorphic pulmonary vasculature. Consequently, affected lungs exhibit a reduced capacity for gas exchange, with important implications for morbidity and mortality in the immediate postnatal period and respiratory health consequences that may persist into adulthood. It is the objective of this Perspectives article to update the reader about recent developments in our understanding of the molecular mechanisms of alveolarization and the pathogenesis of BPD.

Progress in developing new therapies for bronchopulmonary dysplasia (BPD) is sometimes complicated by the lack of a standardised animal model. Our objective was to develop a robust hyperoxia-based mouse model of BPD that recapitulated the pathological perturbations to lung structure noted in infants with BPD. Newborn mouse pups were exposed to a varying fraction of oxygen in the inspired air (FiO2) and a varying window of hyperoxia exposure, after which lung structure was assessed by design-based stereology with systemic uniform random sampling. The efficacy of a candidate therapeutic intervention using parenteral nutrition was evaluated to demonstrate the utility of the standardised BPD model for drug discovery. An FiO2 of 0.85 for the first 14 days of life decreased total alveoli number and concomitantly increased alveolar septal wall thickness, which are two key histopathological characteristics of BPD. A reduction in FiO2 to 0.60 or 0.40 also caused a decrease in the total alveoli number, but the septal wall thickness was not impacted. Neither a decreasing oxygen gradient (from FiO2 0.85 to 0.21 over the first 14 days of life) nor an oscillation in FiO2 (between 0.85 and 0.40 on a 24 h:24 h cycle) had an appreciable impact on lung development. The risk of missing beneficial effects of therapeutic interventions at FiO2 0.85, using parenteral nutrition as an intervention in the model, was also noted, highlighting the utility of lower FiO2 in selected studies, and underscoring the need to tailor the model employed to the experimental intervention. Thus, a state-of-the-art BPD animal model that recapitulates the two histopathological hallmark perturbations to lung architecture associated with BPD is described. The model presented here, where injurious stimuli have been systematically evaluated, provides a most promising approach for the development of new strategies to drive postnatal lung maturation in affected infants.

Bronchopulmonary dysplasia (BPD) is the most common complication of preterm birth, with appreciable morbidity and mortality in a neonatal intensive care setting. Much interest has been shown in the identification of pathogenic pathways that are amenable to pharmacological manipulation (1) to facilitate the development of novel therapeutic and medical management strategies and (2) to identify the basic mechanisms of late lung development, which remains poorly understood. A number of animal models have therefore been developed and continue to be refined with the aim of recapitulating pathological pulmonary hallmarks noted in lungs from neonates with BPD. These animal models rely on several injurious stimuli, such as mechanical ventilation or oxygen toxicity and infection and sterile inflammation, as applied in mice, rats, rabbits, pigs, lambs and nonhuman primates. This review addresses recent developments in modeling BPD in experimental animals and highlights important neglected areas that demand attention. Additionally, recent progress in the quantitative microscopic analysis of pathology tissue is described, together with new in vitro approaches of value for the study of normal and aberrant alveolarization. The need to examine long-term sequelae of damage to the developing neonatal lung is also considered, as is the need to move beyond the study of the lungs alone in experimental animal models of BPD.

The gasotransmitter hydrogen sulfide (H2S) is emerging as a mediator of lung physiology and disease. Recent studies revealed that H2S administration limited perturbations to lung structure in experimental animal models of bronchopulmonary dysplasia (BPD), partially restoring alveolarization, limiting pulmonary hypertension, limiting inflammation, and promoting epithelial repair. No studies have addressed roles for endogenous H2S in lung development. H2S is endogenously generated by cystathionine β-synthase (Cbs) and cystathionine γ-lyase (Cth). We demonstrate here that the expression of Cbs and Cth in mouse lungs is dynamically regulated during lung alveolarization and that alveolarization is blunted in Cbs(-/-) and Cth(-/-) mouse pups, where a 50% reduction in the total number of alveoli was observed, without any impact on septal thickness. Laser-capture microdissection and immunofluorescence staining indicated that Cbs and Cth were expressed in the airway epithelium and lung vessels. Loss of Cbs and Cth led to a 100-500% increase in the muscularization of small- and medium-sized lung vessels, which was accompanied by increased vessel wall thickness, and an apparent decrease in lung vascular supply. Ablation of Cbs expression using small interfering RNA or pharmacological inhibition of Cth using propargylglycine in lung endothelial cells limited angiogenic capacity, causing a 30-40% decrease in tube length and a 50% decrease in number of tubes formed. In contrast, exogenous administration of H2S with GYY4137 promoted endothelial tube formation. These data confirm a key role for the H2S-generating enzymes Cbs and Cth in pulmonary vascular development and homeostasis and in lung alveolarization.

Caffeine modulated TGF-β signaling in vitro and in vivo. Caffeine administration was well-tolerated by newborn mice, but did not influence the course of blunted postnatal lung maturation in a hyperoxia-based experimental mouse model of BPD.

MicroRNA are emerging as powerful regulators of cell differentiation and tissue and organ development. Several microRNA have been described to play a role in branching morphogenesis, a key step in early lung development. However, considerably less attention has been paid to microRNA as regulators of the process of secondary septation, which drives lung alveolarization during late lung development. Secondary septation is severely perturbed in bronchopulmonary dysplasia (BPD), a common complication of preterm birth characterized by blunted alveolarization. A number of studies to date have reported microRNA microarray screens in animal models of BPD; however, only two studies have attempted to demonstrate causality. Although the expression of miR-150 was altered in experimental BPD, a miR-150−/− knockout mouse did not exhibit appreciable protection in a BPD animal model. Similarly, while the expression of miR-489 in the lung was reduced in clinical and experimental BPD, antagomiR and over-expression approaches could not validate a role for miR-489 in the impaired alveolarization associated with experimental BPD. This mini-review aims to highlight microRNA that have been revealed by multiple microarray studies to be potential causal players in normal and pathological alveolarization. Additionally, the challenges faced in attempting to demonstrate a causal role for microRNA in lung alveolarization are discussed. These include the tremendous variability in the animal models employed, and the limitations and advantages offered by the available tools, including antagomiRs and approaches for the validation of a specific microRNA-mRNA interaction during lung alveolarization.

Bronchopulmonary dysplasia (BPD) is a common complication of preterm birth characterized by arrested lung alveolarization, which generates lungs that are incompetent for effective gas exchange. We report here deregulated expression of miR‐34a in a hyperoxia‐based mouse model of BPD, where miR‐34a expression was markedly increased in platelet‐derived growth factor receptor (PDGFR)α‐expressing myofibroblasts, a cell type critical for proper lung alveolarization. Global deletion of miR‐34a; and inducible, conditional deletion of miR‐34a in PDGFRα+ cells afforded partial protection to the developing lung against hyperoxia‐induced perturbations to lung architecture. Pdgfra mRNA was identified as the relevant miR‐34a target, and using a target site blocker in vivo, the miR‐34a/Pdgfra interaction was validated as a causal actor in arrested lung development. An antimiR directed against miR‐34a partially restored PDGFRα+ myofibroblast abundance and improved lung alveolarization in newborn mice in an experimental BPD model. We present here the first identification of a pathology‐relevant microRNA/mRNA target interaction in aberrant lung alveolarization and highlight the translational potential of targeting the miR‐34a/Pdgfra interaction to manage arrested lung development associated with preterm birth.

Background: The laboratory mouse is widely used in preclinical models of bronchopulmonary dysplasia, where lung alveolarization is stunted by exposure of pups to hyperoxia. Whether the diverse genetic backgrounds of different inbred mouse strains impacts lung development in newborn mice exposed to hyperoxia has not been systematically assessed.Methods: Hyperoxia (85% O 2 , 14 days)-induced perturbations to lung alveolarization were assessed by design-based stereology in C57BL/6J, BALB/cJ, FVB/NJ, C3H/HeJ, and DBA/2J inbred mouse strains. The expression of components of the lung antioxidant machinery was assessed by real-time reverse transcriptase polymerase chain reaction and immunoblot.Results: Hyperoxia-reduced lung alveolar density in all five mouse strains to different degrees (C57BL/6J, 64.8%; FVB/NJ, 47.4%; BALB/cJ, 46.4%; DBA/2J, 45.9%; and C3H/HeJ, 35.9%). Hyperoxia caused a 94.5% increase in mean linear intercept in the C57BL/6J strain, whilst the C3H/HeJ strain was the least affected (31.6% increase).In contrast, hyperoxia caused a 65.4% increase in septal thickness in the FVB/NJ strain, where the C57BL/6J strain was the least affected (30.3% increase). The expression of components of the lung antioxidant machinery in response to hyperoxia was strain dependent, with the C57BL/6J strain exhibiting the most dramatic engagement. Baseline expression levels of components of the lung antioxidant systems were different in the five mouse strains studied, under both normoxic and hyperoxic conditions. Conclusion: The genetic background of laboratory mouse strains dramatically influenced the response of the developing lung to hyperoxic insult. This might be explained, at least in part, by differences in how antioxidant systems are engaged by different mouse strains after hyperoxia exposure. K E Y W O R D S alveolarization, bronchopulmonary dysplasia, hyperoxia, strain