Capillary Changes Precede Disordered Alveolarization in a Mouse Model of Bronchopulmonary Dysplasia.

Bronchopulmonary dysplasia (BPD), the most common sequela of preterm birth, is a severe disorder of the lung that is often associated with long-lasting morbidity. A hallmark of BPD is the disruption of alveolarization, whose pathogenesis is incompletely understood. Here, we tested the vascular hypothesis that disordered vascular development precedes the decreased alveolarization associated with BPD. Neonatal mouse pups were exposed to 7, 14, or 21 days of normoxia (21% O2) or hyperoxia (85% O2) with n = 8-11 for each group. The right lungs were fixed by vascular perfusion and investigated by design-based stereology or three-dimensional reconstruction of data sets obtained by serial block-face scanning EM. The alveolar capillary network of hyperoxia-exposed mice was characterized by rarefaction, partially altered geometry, and widening of capillary segments as shown by three-dimensional reconstruction. Stereology revealed that the development of alveolar epithelium and capillary endothelium was decreased in hyperoxia-exposed mice; however, the time course of these effects was different. That the surface area of the alveolar epithelium was smaller in hyperoxia-exposed mice first became evident at Day 14. In contrast, the surface area of the endothelium was reduced in hyperoxia-exposed mouse pups at Day 7. The thickness of the air-blood barrier decreased during postnatal development in normoxic mice, whereas it increased in hyperoxic mice. The endothelium and the septal connective tissue made appreciable contributions to the thickened septa. In conclusion, the present study provides clear support for the idea that the stunted alveolarization follows the disordered microvascular development, thus supporting the vascular hypothesis of BPD.

A comparison of airway pressures for inflation fixation of developing mouse lungs for stereological analyses.

The morphometric analysis of lung structure using the principles of stereology has emerged as a powerful tool to describe the structural changes in lung architecture that accompany the development of lung disease that is experimentally modelled in adult mice. These stereological principles are now being applied to the study of the evolution of the lung architecture over the course of prenatal and postnatal lung development in mouse neonates and adolescents. The immature lung is structurally and functionally distinct from the adult lung, and has a smaller volume than does the adult lung. These differences have raised concerns about whether the inflation fixation of neonatal mouse lungs with the airway pressure (Paw) used for the inflation fixation of adult mouse lungs may cause distortion of the neonatal mouse lung structure, leading to the generation of artefacts in subsequent analyses. The objective of this study was to examine the impact of a Paw of 10, 20 and 30 cmH2O on the estimation of lung volumes and stereologically assessed parameters that describe the lung structure in developing mouse lungs. The data presented demonstrate that low Paw (10 cmH2O) leads to heterogeneity in the unfolding of alveolar structures within the lungs, and that high Paw (30 cmH2O) leads to an overestimation of the lung volume, and thus, affects the estimation of volume-dependent parameters, such as total alveoli number and gas-exchange surface area. Thus, these data support the use of a Paw of 20 cmH2O for inflation fixation in morphometric studies on neonatal mouse lungs.

Minoxidil Cannot Be Used To Target Lysyl Hydroxylases during Postnatal Mouse Lung Development: A Cautionary Note.

The lysyl hydroxylases (procollagen-lysine 5-dioxygenases) PLOD1, PLOD2, and PLOD3 have been proposed as pathogenic mediators of stunted lung development in bronchopulmonary dysplasia (BPD), a common complication of preterm birth. In affected infants, pulmonary oxygen toxicity stunts lung development. Mice lacking Plod1 exhibit 15% mortality, and mice lacking Plod2 or Plod3 exhibit embryonic lethality. Therefore, to address any pathogenic role of lysyl hydroxylases in stunted lung development associated with BPD, minoxidil was administered to newborn mice in an oxygen toxicity-based BPD animal model. Minoxidil, which has attracted much interest in the management of systemic hypertension and androgenetic alopecia, can also be used to reduce lysyl hydroxylase activity in cultured cells. An in vivo pilot dosing study established 50 mg⋅kg-1⋅day-1 as the maximum possible minoxidil dose for intraperitoneal administration in newborn mouse pups. When administered at 50 mg⋅kg-1⋅day-1 to newborn mouse pups, minoxidil was detected in the lungs but did not impact lysine hydroxylation, collagen crosslinking, or lysyl hydroxylase expression in the lungs. Consistent with no impact on mouse lung extracellular matrix structures, minoxidil administration did not alter the course of normal or stunted lung development in newborn mice. At doses of up to 50 mg⋅kg⋅day-1, pharmacologically active concentrations of minoxidil were not achieved in neonatal mouse lung tissue; thus, minoxidil cannot be used to attenuate lysyl hydroxylase expression or activity during mouse lung development. These data also highlight the need for new and specific lysyl hydroxylase inhibitors. SIGNIFICANCE STATEMENT: Extracellular matrix crosslinking is mediated by lysyl hydroxylases, which generate hydroxylated lysyl residues in procollagen peptides. Deregulated collagen crosslinking is a pathogenic component of a spectrum of diseases, and thus, there is interest in validating lysyl hydroxylases as pathogenic mediators of disease and potential "druggable" targets. Minoxidil, administered at the maximum possible dose, did not inhibit lysyl hydroxylation in newborn mouse lungs, suggesting that minoxidil was unlikely to be of use in studies that pharmacologically target lysyl hydroxylation in vivo.

Mouse genetic background impacts susceptibility to hyperoxia-driven perturbations to lung maturation.

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% O2 , 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.

Targeting miR-34a/Pdgfra interactions partially corrects alveologenesis in experimental bronchopulmonary dysplasia.

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.

Control Interventions Can Impact Alveolarization and the Transcriptome in Developing Mouse Lungs.

There is currently much interest in understanding the mechanisms of normal and aberrant lung alveolarization, particularly in the context of bronchopulmonary dysplasia, a common complication of preterm birth where alveolarization is impeded. To this end, the parenteral administration of pharmacological agents that modulate biochemical pathways, or facilitate modulation of gene expression in transgenic animals, has facilitated the discovery and validation of mechanisms that direct lung development. Such studies include control interventions, where the solvent vehicle, perhaps containing an inactive form of the agent applied, is administered; thereby providing a well-controlled point of reference for the analysis of the partner experiment. In the present study, the impact of several widely used control interventions in developing C57Bl/6J mouse pups was examined for effects on lung structure and the lung transcriptome. Parenteral administration of scrambled microRNA inhibitors (called antagomiRs) that are used to control in vivo microRNA neutralization studies, impacted lung volume, septal thickness, and the transcriptome of developing mouse lungs; with some effects dependent upon nucleotide sequence. Repeated intraperitoneal isotonic saline injections altered lung volume, with limited impact on the transcriptome. Parenteral administration of the tamoxifen solvent Miglyol accelerated mouse pup growth, and changed the abundance of 73 mRNA transcripts in the lung. Tamoxifen applied in Miglyol-in the absence of Cre recombinase-decreased pup growth, lung volume, and lung alveolarization and changed the abundance of 298 mRNA transcripts in the lung. These data demonstrate that widely used control interventions can directly impact lung alveolarization and the lung transcriptome in studies on lung development. Anat Rec, 302:346-363, 2019. © 2018 Wiley Periodicals, Inc.

Targeting transglutaminase 2 partially restores extracellular matrix structure but not alveolar architecture in experimental bronchopulmonary dysplasia.

The generation, maturation and remodelling of the extracellular matrix (ECM) are essential for the formation of alveoli during lung development. Alveoli formation is disturbed in preterm infants that develop bronchopulmonary dysplasia (BPD), where collagen fibres are malformed, and perturbations to lung ECM structures may underlie BPD pathogenesis. Malformed ECM structures might result from abnormal protein cross-linking, in part attributable to the increased expression and activity of transglutaminase 2 (TGM2) that have been noted in affected patient lungs, as well as in hyperoxia-based BPD animal models. The objective of the present study was to assess whether TGM2 plays a causal role in normal and aberrant lung alveolarization. Targeted deletion of Tgm2 in C57BL/6J mice increased septal thickness and reduced gas-exchange surface area in otherwise normally developing lungs. During aberrant lung alveolarization that occurred under hyperoxic conditions, collagen structures in Tgm2-/- mice were partially protected from the impact of hyperoxia, where normal dihydroxylysinonorleucine and hydroxylysylpiridinoline collagen cross-link abundance was restored; however, the lung alveolar architecture remained abnormal. Inhibition of transglutaminases (including TGM2) with cysteamine appreciably reduced transglutaminase activity in vivo, as assessed by Nε -(γ-l-glutamyl)-l-lysine abundance and TGM catalytic activity, and restored normal dihydroxylysinonorleucine and hydroxylysylpiridinoline collagen cross-link abundance under pathological conditions. Furthermore, a moderate improvement in alveoli size and gas-exchange surface density was noted in cysteamine-treated mouse lungs in which BPD was modelled. These data indicate that TGM2 plays a role in normal lung alveolarization, and contributes to the formation of aberrant ECM structures during disordered lung alveolarization.

Transmission of microRNA antimiRs to mouse offspring via the maternal-placental-fetal unit.

The emergence of microRNA as regulators of organogenesis and tissue differentiation has stimulated interest in the ablation of microRNA expression and function during discrete periods of development. To this end, inducible, conditional modulation of microRNA expression with doxycycline-based tetracycline-controlled transactivator and tamoxifen-based estrogen receptor systems has found widespread use. However, the induction agents and components of genome recombination systems negatively impact pregnancy, parturition, and postnatal development; thereby limiting the use of these technologies between late gestation and the early postnatal period. MicroRNA inhibitor (antimiR) administration also represents a means of neutralizing microRNA function in vitro and in vivo. To date, these studies have used direct (parenteral) administration of antimiRs to experimental animals. As an extension of this approach, an alternative means of regulating microRNA expression and function is described here: the maternal-placental-fetal transmission of antimiRs. When administered to pregnant dams, antimiRs were detected in offspring and resulted in a pronounced and persistent reduction in detectable steady-state free microRNA levels in the heart, kidney, liver, lungs, and brain. This effect was comparable to direct injection of newborn mouse pups with antimiRs, although maternal delivery resulted in fewer off-target effects. Furthermore, depletion of steady-state microRNA levels via the maternal route resulted in concomitant increases in steady-state levels of selected microRNA targets. This novel methodology permits the temporal regulation of microRNA function during late gestation and in neonates, without recourse to conventional approaches that rely on doxycycline and tamoxifen, which may confound studies on developmental processes.

Stereological analysis of individual lung lobes during normal and aberrant mouse lung alveolarisation.

The quantitative assessment of the lung architecture forms the foundation of many studies on lung development and lung diseases, where parameters such as alveoli number, alveolar size, and septal thickness are quantitatively influenced by developmental or pathological processes. Given the pressing need for robust data that describe the lung structure, there is currently much enthusiasm for the development and refinement of methodological approaches for the unbiased assessment of lung structure with improved precision. The advent of stereological methods highlights one such approach. However, design-based stereology is both expensive and time-demanding. The objective of this study was to examine whether 'limited' stereological analysis, such as the stereological analysis of a single mouse lung lobe, may serve as a surrogate for studies on whole, intact mouse lungs; both in healthy lungs and in diseased lungs, using an experimental animal model of bronchopulmonary dysplasia (BPD). This served the dual-function of exploring BPD pathobiology, asking whether there are regional (lobar) differences in the responses of developing mouse lungs to oxygen injury, by examining each mouse lung lobe separately in the BPD model. Hyperoxia exposure resulted in decreased alveolar density, alveoli number, and gas-exchange surface area in all five mouse lung lobes, and increased the arithmetic mean septal thickness in all mouse lung lobes except the lobus cardialis. The data presented here suggest that - in healthy developing mice - a single mouse lung lobe might serve as a surrogate for studies on whole, intact mouse lungs. This is not the case for oxygen-injured developing mouse lungs, where a single lobe would not be suitable as a surrogate for the whole, intact lung. Furthermore, as the total number of alveoli can only be determined by an analysis of the entire lung, and given regional differences in lung structure, particularly under pathological conditions, the stereological assessment of the whole, intact lung remains desirable.

Recent advances in our understanding of the mechanisms of late lung development and bronchopulmonary dysplasia.

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.

Stereological monitoring of mouse lung alveolarization from the early postnatal period to adulthood.

Postnatal lung maturation generates a large number of small alveoli, with concomitant thinning of alveolar septal walls, generating a large gas exchange surface area but minimizing the distance traversed by the gases. This demand for a large and thin gas exchange surface area is not met in disorders of lung development, such as bronchopulmonary dysplasia (BPD) histopathologically characterized by fewer, larger alveoli and thickened alveolar septal walls. Diseases such as BPD are often modeled in the laboratory mouse to better understand disease pathogenesis or to develop new interventional approaches. To date, there have been no stereology-based longitudinal studies on postnatal mouse lung development that report dynamic changes in alveoli number or alveolar septal wall thickness during lung maturation. To this end, changes in lung structure were quantified over the first 22 mo of postnatal life of C57BL/6J mice. Alveolar density peaked at postnatal day (P)39 and remained unchanged at 9 mo (P274) but was reduced by 22 mo (P669). Alveoli continued to be generated, initially at an accelerated rate between P5 and P14, and at a slower rate thereafter. Between P274 and P669, loss of alveoli was noted, without any reduction in lung volume. A progressive thinning of the alveolar septal wall was noted between P5 and P28. Pronounced sex differences were observed in alveoli number in adult (but not juvenile) mice, when comparing male and female mouse lungs. This sex difference was attributed exclusively to the larger volume of male mouse lungs.

Caffeine administration modulates TGF-ß signaling but does not attenuate blunted alveolarization in a hyperoxia-based mouse model of bronchopulmonary dysplasia.

BACKGROUND: Caffeine is widely used to manage apnea of prematurity, and reduces the incidence of bronchopulmonary dysplasia (BPD). Deregulated transforming growth factor (TGF)-ß signaling underlies arrested postnatal lung maturation in BPD. It is unclear whether caffeine impacts TGF-ß signaling or postnatal lung development in affected lungs. METHODS: The impact of caffeine on TGF-ß signaling in primary mouse lung fibroblasts and alveolar epithelial type II cells was assessed in vitro. The effects of caffeine administration (25 mg/kg/d for the first 14 d of postnatal life) on aberrant lung development and TGF-ß signaling in vivo was assessed in a hyperoxia (85% O2)-based model of BPD in C57BL/6 mice. RESULTS: Caffeine downregulated expression of type I and type III TGF-ß receptors, and Smad2; and potentiated TGF-ß signaling in vitro. In vivo, caffeine administration normalized body mass under hyperoxic conditions, and normalized Smad2 phosphorylation detected in lung homogenates; however, caffeine administration neither improved nor worsened lung structure in hyperoxia-exposed mice, in which postnatal lung maturation was blunted. CONCLUSION: Caffeine modulated TGF-ß 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.