Changes in respiratory management and the impact on bronchopulmonary dysplasia.

OBJECTIVE: Noninvasive respiratory support has reduced the need for mechanical ventilation and surfactant administration in very premature neonates. We sought to determine how the increased use of noninvasive ventilation and less surfactant instillation has impacted the development of bronchopulmonary dysplasia (BPD) using four currently used BPD definitions. STUDY DESIGN: This is a retrospective, single-center cohort study of neonates born at less than 28 weeks gestation between 2010 and 2018. A respiratory practice change (less surfactant and more noninvasive ventilation) occurred in 2014 following participation in the Surfactant Positive Airway Pressure and Pulse Oximetry trial. Therefore, patients were divided into two epochs to compare postnatal respiratory and clinical course and BPD outcomes across four currently relevant definitions (Vermont Oxford Network, National Institute of Child Health and Human Development, Canadian, and Neonatal Research Network). RESULTS: Clinical and demographic variables were similar between epochs. Despite significant differences in maternal and infant characteristics and clinical course, the incidence of BPD was not significantly different between the two epochs regardless of the BPD definition utilized. There was a wide range in the incidence of BPD depending on the definition used. CONCLUSIONS: Despite decreased use of surfactant administration and invasive mechanical ventilation between the two epochs, the incidence of BPD did not change and there was wide variation depending on the definition used. A better understanding of the risk factors associated with BPD and a consensus definition is urgently needed to: 1) more accurately compare various studies, 2) help facilitate the conduct of clinical trials, and 3) enhance the development of novel therapeutic interventions to improve outcome.

Expressed Breast Milk Analysis: Role of Individualized Protein Fortification to Avoid Protein Deficit After Preterm Birth and Improve Infant Outcomes.

Background: Expressed breast milk (EBM) protein content is highly variable between mothers and often below published values that are still used for EBM protein fortification strategies. This approach may result in significant protein deficit and suboptimal protein energy (P/E) ratio. The study aim was to determine whether individualized EBM protein analysis and fortification will reduce preterm infant protein deficits and improve growth and neurodevelopmental outcome. Study Methods: In a single-center randomized, blinded study of infants born at 24 0/7-29 6/7 weeks, mother-specific protein values measured by a milk analyzer were used to individualize infant-specific protein intake (interventional group, IG), and compared this to a standardized protein fortification scheme based on published values of EBM protein content of 1.4 g/dL (control group, CG). For IG, milk analyzer protein values of mother's EBM were used to adjust protein content of the EBM. The CG EBM protein content was adjusted using the standard published value of 1.4 g/dL and not based on milk analyzer values. EBM protein content, protein intake, protein/energy (P/E) ratio, weight (WT), head circumference (HC), length (L), growth velocity (GV) from 2 to 6 weeks of age, WT, HC and L Z-Scores at 32- and 35-weeks PMA, and lean body mass (35 weeks PMA skin fold thickness) were measured. Neurodevelopment was assessed by Bayley III at average 24 months corrected gestational age (CGA). Results: EBM protein content before fortification was significantly below published values of 1.4 g/dL at all time points in both CG and IG. CG protein deficit was significantly decreased and progressively worsened throughout the study. Individualized protein fortification in IG avoided protein deficit and optimized P/E ratio. Although no significant change in short-term GV (at 6 weeks of age) was seen between groups, IG infants born at <27 weeks had significant improvements in WT and L z-scores, and leaner body mass at 32 and 35 weeks PMA. IG exhibited significantly improved cognitive scores at 24 months CGA. Conclusions: Infant-specific protein supplementation of mother's EBM optimized P/E ratio by eliminating protein deficit and improved growth z scores at 32- and 35-weeks PMA and neurocognitive testing at 24 months.

A Pathogenic Relationship of Bronchopulmonary Dysplasia and Retinopathy of Prematurity? A Review of Angiogenic Mediators in Both Diseases.

Bronchopulmonary dysplasia (BPD) and retinopathy of prematurity (ROP) are common and significant morbidities of prematurely born infants. These diseases have in common altered and pathologic vascular formation in the face of incomplete organ development. Therefore, it is reasonable to question whether factors affecting angiogenesis could have a joint pathogenic role for both diseases. Inhibition or induced expression of a single angiogenic factor is unlikely to be 100% causative or protective of either of BPD or ROP. It is more likely that interactions of multiple factors leading to disordered angiogenesis are present, increasing the likelihood of common pathways in both diseases. This review explores this possibility by assessing the evidence showing involvement of specific angiogenic factors in the vascular development and maldevelopment in each disease. Theoretical interactions of specific factors mutually contributing to BPD and ROP are proposed and, where possible, a timeline of the proposed relationships between BPD and ROP is developed. It is hoped that future research will be inspired by the theories put forth in this review to enhance the understanding of the pathogenesis in both diseases.

Oxygen differentially affects the hox proteins Hoxb5 and Hoxa5 altering airway branching and lung vascular formation.

Hoxb5 and Hoxa5 transcription factor proteins uniquely impact lung morphogenesis at the developmental time point when extremely preterm infants are born. The effect of O2 exposure (0.4 FiO2) used in preterm infant care on these Hox proteins is unknown. We used ex vivo fetal mouse lung organ cultures to explore the effects of 0.4 FiO2 on lung airway and vascular formation in the context of Hoxb5 and Hoxa5 expression and regulation. Compared to room air, 48 h (h) 0.4 FiO2 adversely attenuated airway and microvasculature formation while reducing lung growth and epithelial cell volume, and increasing mesenchymal volume. 0.4 FiO2 decreased pro-angiogenic Hoxb5 and VEGFR2 while not altering protein levels of angiostatic Hoxa5. Lungs returned to RA after 24 h 0.4FiO2 had partial structural recovery but remained smaller and less developed. Mesenchymal cell apoptosis increased and proliferation decreased with time in O2 while epithelial cell proliferation significantly increased. Hoxb5 overexpression led to prominent peri-airway VEGFR2 expression and promoted lung vascular and airway patterning. Hoxa5 overexpression had the opposite effects. We conclude that 0.4 FiO2 exposure causes a profound loss of airway and lung microvascular development that occurs partially via reduction in pro-angiogenic Hoxb5 while angiostatic Hoxa5 expression is maintained.

Regulatory interactions between androgens, Hoxb5, and TGF ß signaling in murine lung development.

Androgens enhance airway branching but delay alveolar maturation contributing to increased respiratory morbidity in prematurely born male infants. Hoxb5 protein positively regulates airway branching in developing lung. In other organs, androgen regulation intersects with Hox proteins and TGF ß -SMAD signaling, but these interactions have not been studied in the lung. We hypothesized that androgen alteration of airway branching early in lung development requires Hoxb5 expression and that these androgen-Hoxb5 interactions occur partially through regional changes in TGF ß signaling. To evaluate acute effects of androgen and TGF ß on Hoxb5, E11 whole fetal mouse lungs were cultured with dihydrotestosterone (DHT) with/without Hoxb5 siRNA or TGF ß inhibitory antibody. Chronic in utero DHT exposure was accomplished by exposing pregnant mice to DHT (subcutaneous pellet) from E11 to E18. DHT's ability to enhance airway branching and alter phosphorylated SMAD2 cellular localization was partially dependent on Hoxb5. Hoxb5 inhibition also changed the cellular distribution of SMAD7 protein. Chronic in utero DHT increased Hoxb5 and altered SMAD7 mesenchymal localization. TGF ß inhibition enhanced airway branching, and Hoxb5 protein cellular localization was more diffuse. We conclude that DHT controls lung airway development partially through modulation of Hoxb5 protein expression and that this level of regulation involves interactions with TGF ß signaling.

miRNA regulated pathways in late stage murine lung development.

BACKGROUND: MicroRNAs play important roles in regulating biological processes, including organ morphogenesis and maturation. However, little is known about specific pathways regulated by miRNA during lung development. Between the canalicular and saccular stages of the developing lung several important cellular events occur, including the onset of surfactant synthesis, microvascular remodeling and structural preparation for subsequent alveolarization. The miRNAs that are actively regulated, and the identity of their targets during this important developmental interval in the lung remain elusive. RESULTS: Using TLDA low density real-time PCR arrays, the expression of 376 miRNAs in male and female fetal mouse lungs of gestational days E15 - E18 were profiled. Statistical analyses identified 25 and 37 miRNAs that changed significantly between sexes and with gestation, respectively. In silico analysis using Ingenuity Pathway Analysis (IPA) identified specific pathways and networks known to be targets of these miRNAs which are important to lung development. Pathways that are targeted by sex regulated miRNAs include retinoin, IGFR1, Tp53 and Akt. Pathways targeted by gestation-regulated miRNAs include VEGFA and mediators of glucose metabolism. CONCLUSION: MiRNAs are differentially regulated across time and between sexes during the canalicular and saccular stages of lung development. Sex-associated differential miRNA expression may regulate the differences in structural and functional male and female lung development, as shown by networks generated using in silico analysis. These data provide a valuable resource to further enhance the understanding of miRNA control of lung development and maturation.

MiR-221 and miR-130a regulate lung airway and vascular development.

Epithelial-mesenchymal interactions play a crucial role in branching morphogenesis, but very little is known about how endothelial cells contribute to this process. Here, we examined how anti-angiogenic miR-221 and pro-angiogenic miR-130a affect airway and vascular development in the fetal lungs. Lung-specific effects of miR-130a and miR-221 were studied in mouse E14 whole lungs cultured for 48 hours with anti-miRs or mimics to miR-130a and miR-221. Anti-miR 221 treated lungs had more distal branch generations with increased Hoxb5 and VEGFR2 around airways. Conversely, mimic 221 treated lungs had reduced airway branching, dilated airway tips and decreased Hoxb5 and VEGFR2 in mesenchyme. Anti-miR 130a treatment led to reduced airway branching with increased Hoxa5 and decreased VEGFR2 in the mesenchyme. Conversely, mimic 130a treated lungs had numerous finely arborized branches extending into central lung regions with diffusely localized Hoxa5 and increased VEGFR2 in the mesenchyme. Vascular morphology was analyzed by GSL-B4 (endothelial cell-specific lectin) immunofluorescence. Observed changes in airway morphology following miR-221 inhibition and miR-130a enhancement were mirrored by changes in vascular plexus formation around the terminal airways. Mouse fetal lung endothelial cells (MFLM-91U) were used to study microvascular cell behavior. Mimic 221 treatment resulted in reduced tube formation and cell migration, where as the reverse was observed with mimic 130a treatment. From these data, we conclude that miR-221 and miR-130a have opposing effects on airway and vascular morphogenesis of the developing lung.

Presenilin-1 processing of ErbB4 in fetal type II cells is necessary for control of fetal lung maturation.

Maturation of pulmonary fetal type II cells to initiate adequate surfactant production is crucial for postnatal respiratory function. Little is known about specific mechanisms of signal transduction controlling type II cell maturation. The ErbB4 receptor and its ligand neuregulin (NRG) are critical for lung development. ErbB4 is cleaved at the cell membrane by the γ-secretase enzyme complex whose active component is either presenilin-1 (PSEN-1) or presenilin-2. ErbB4 cleavage releases the 80kDa intracellular domain (4ICD), which associates with chaperone proteins such as YAP (Yes-associated protein) and translocates to the nucleus to regulate gene expression. We hypothesized that PSEN-1 and YAP have a development-specific expression in fetal type II cells and are important for ErbB4 signaling in surfactant production. In primary fetal mouse E16, E17, and E18 type II cells, PSEN-1 and YAP expression increased at E17 and E18 over E16. Subcellular fractionation showed a strong cytosolic and a weaker membrane location of both PSEN-1 and YAP. This was enhanced by NRG stimulation. Co-immunoprecipitations showed ErbB4 associated separately with PSEN-1 and with YAP. Their association, phosphorylation, and co-localization were induced by NRG. Confocal immunofluorescence and nuclear fractionation confirmed these associations in a time-dependent manner after NRG stimulation. Primary ErbB4-deleted E17 type II cells were transfected with a mutant ErbB4 lacking the γ-secretase binding site. When compared to transfection with wild-type ErbB4, the stimulatory effect of NRG on surfactant protein mRNA expression was lost. We conclude that PSEN-1 and YAP have crucial roles in ErbB4 signal transduction during type II cell maturation.

Aberrant cell adhesion molecule expression in human bronchopulmonary sequestration and congenital cystic adenomatoid malformation.

In many organs, integrins and cadherins are partly regulated by Hox genes, but their interactions in airway morphogenesis and congenital lung diseases are unknown. We previously showed that the Hox protein HoxB5 is abnormally increased in bronchopulmonary sequestration (BPS) and congenital cystic adenomatoid malformation (CCAM), congenital lung lesions with abnormal airway branching. We now report on alpha(2)-, alpha(3)-, and beta(1)-integrin and E-cadherin expression in normal human lung and in BPS and CCAM tissue previously shown to have abnormal HoxB5 expression and on the relationship of cell adhesion molecule expression to Hoxb5 regulation. alpha(2)-, alpha(3)-, and beta(1)-integrins and E-cadherin expression in normal human lung and BPS and CCAM were evaluated using Western blot and immunohistochemistry. Fetal mouse lung fibroblasts with Hoxb5-specific siRNA downregulation were evaluated for alpha(2)-integrin protein levels by Western blot. Compared with normal human lung, a previously undetected alpha(2)-integrin isoform potentially lacking essential cytoplasmic sequences was significantly increased in BPS and CCAM, and alpha(2)-integrin spatial and cellular expression was more intense. E-cadherin protein levels were also significantly increased, whereas alpha(3) increased in CCAM compared with canalicular, but not with alveolar, stage lung. beta(1)-integrin levels were unchanged. We conclude that in BPS and CCAM, altered alpha(2)-integrin cytoplasmic signaling contributes to abnormal cellular behavior in these lung lesions. Aberrant cell adhesion molecule and Hox protein regulation are likely part of the mechanism involved in the development of BPS and CCAM.

Morphologic analysis of the neuromuscular development of the anorectal unit in fetal rats with retinoic acid induced myelomeningocele.

To investigate whether myelomeningocele (MMC) is associated with a global neuromuscular maldevelopment of the lower gastrointestinal (GI) tract and anorectum, the distribution and staining intensity of non-neuronal (alpha-smooth-muscle-actin), neural crest cell (NCC, [Hoxb5]), and neuronal markers (PGP-9.5, synaptophysin, neurotubulin-beta-III) within the distal colon, rectum, and anal sphincters were analyzed by immunohistochemistry and Western blot in rat fetuses with retinoic acid (RA) induced MMC. At term (E22), no gross-morphological differences of the anorectal unit of OIL (n=21) MMC (n=31), and RA-exposed-non MMC (RA, n=19) fetuses were found. Smooth muscle cells were evenly distributed within the muscle layers of the rectum and the internal anal sphincter in OIL, MMC, and RA fetuses. Density and staining intensity of NCC and mature enteric neurons within the myenteric plexus of the distal colon and rectum and innervation pattern within anal sphincters in MMC fetuses were analogous to RA and OIL controls. Normal smooth muscle and myenteric plexus development of the rectum and normal innervation of the anal sphincters and pelvic floor suggests that MMC is not associated with a global neuromuscular maldevelopment of lower GI structures in this short-gestational model.

ErbB4 regulates fetal surfactant phospholipid synthesis in primary fetal rat type II cells.

Insufficient fetal surfactant production leads to respiratory distress syndrome among preterm infants. Neuregulin signals the onset of fetal surfactant phospholipid synthesis through formation of erbB receptor dimers. We hypothesized that erbB4 downregulation in fetal type II epithelial cells will downregulate not only fetal surfactant phospholipid synthesis, but also affect proliferation and erbB receptor localization. We tested these hypotheses using small interfering RNA (siRNA) directed against the erbB4 gene to silence erbB4 receptor function in cultures of primary day 19 fetal rat lung type II cells. ErbB4 siRNA treatment inhibited erbB4 receptor protein expression, fibroblast-conditioned medium induced erbB4 phosphorylation, and fetal surfactant phospholipid synthesis. Cell proliferation, measured as thymidine incorporation, was also inhibited by erbB4 siRNA treatment. Downregulation of erbB4 receptor protein changed erbB1 localization at baseline and after stimulation, as determined by confocal microscopy and subcellular fractionation. We conclude that erbB4 is an important receptor in the control of fetal lung type II cell maturation.

Hoxb-5 down regulation alters Tenascin-C, FGF10 and Hoxb gene expression patterns in pseudoglandular period fetal mouse lung.

Organ-specific patterning is partly determined by Hox gene regulatory interactions with the extracellular matrix (ECM), cell adhesion and fibroblast growth factor (FGFs) signaling pathways but coordination of these mechanisms in lung development is unknown. We have previously shown that Hoxb-5 affects airway patterning during lung morphogenesis. Hoxb-5 regulation in fetal lung affects ECM expression of tenascin-C and alters FGF10 spatial and cellular expression. To test this hypothesis, gestational day 13.5 (Gd13.5) fetal mouse lung fibroblasts and whole lungs were cultured with Hoxb-5-specific small interfering RNA (siRNA). Western blots showed that siRNA-down regulation of Hoxb-5 led to decreased tenascin-C and FGF10 and was associated with increased Hoxb-4 and decreased Hoxb-6 protein levels. Hoxa-5 protein levels were not affected. Hoxb-5 siRNA-treated whole lung cultures had a significant decrease in total lung and peripheral branching region surface area. Immunostaining showed negligible levels of Hoxb-5 protein and tenascin-C, and loss of FGF10 spatial restriction. We conclude that Hoxb-5 helps regulate lung airway development through modulation of ECM expression of tenascin-C. ECM changes induced by Hoxb-5 may affect mesenchymal-epithelial cell signaling to alter spatial and cellular restriction of FGF10. Hoxb-5 may also affect lung airway branching indirectly by cross regulation of other Hoxb genes.

Expression of Hoxb-5 during human lung development and in congenital lung malformations.

BACKGROUND: We have previously shown that the Hox gene Hoxb-5 is necessary for normal mouse lung branching morphogenesis. Abnormal Hoxb-5 regulation causes specific alterations in airway branching. We hypothesized that Hoxb-5 is similarly involved in human lung branching morphogenesis, and is abnormally expressed in bronchopulmonary sequestration (BPS) and congenital cystic adenomatoid malformation (CCAM), both of which are congenital lung malformations with abnormal airway development. METHODS: The temporal, spatial, and cellular expression of the Hoxb-5 protein was evaluated in normal human lung and BPS and CCAM tissue using Western blot analysis and immunocytochemistry. RESULTS: The expression of Hoxb-5 during human lung development showed strong similarities to that during mouse lung development. Western blots showed high Hoxb-5 protein levels in the pseudoglandular period (PSG), decreased but sustained levels in the canalicular period (CAN), and negligible levels during the alveolar period (ALV). Immunocytochemistry showed Hoxb-5 protein expression in mesenchymal cells around branching airways in the pseuodglandular period, subepithelial fibroblast localization (especially at airway branch points) in the CAN and minimal expression in the ALV. In BPS and CCAM tissue, Hoxb-5 protein levels were increased compared to age- and developmentally-matched lung tissue, and were more similar to the PSG and CAN with Hoxb-5-positive cells in mesenchyme surrounding abnormally branched airways. CONCLUSIONS: Hoxb-5 expression during human lung branching morphogenesis, which is similar to that observed in mouse lung development, indicates that it plays a role in controlling airway patterning. This notion is supported by results from BPS and CCAM tissue, in which Hoxb-5 is maintained in a manner typical of an earlier developmental stage and is associated with development of abnormal lung tissue.

Thyroid hormone affects distal airway formation during the late pseudoglandular period of mouse lung development.

We recently showed that T3 treatment of cultured gestational day 11.5 early pseudoglandular period mouse lungs, accelerated terminal airway development at the expense of decreased branching morphogenesis. As the ability of T3 to influence epithelial cell differentiation increases with advancing development, we hypothesized that in the late pseudoglandular period, T3 would cause further premature changes in the morphology of the distal airways leading to abnormal saccular development. Gestational day 13.5 embryonic mouse lungs were cultured for 3 and 7 days without or with added T3. Increasing T3 dose and time in culture resulted in progressive development of thin walled, abnormal saccules, an increase in cuboidal and flattened epithelia and airway space with a concomitant decrease in mesenchymal cell volume. Consistent with increased cuboidal and flattened epithelial cell volume identified by morphometry, immunostaining suggested increased cell proliferation detected by localization of proliferating cell nuclear antigen (PCNA) in epithelial cells of T3 treated lungs. T3 decreased mesenchymal expression of Hoxb-5 protein and caused progressive localization of Nkx2.1 and SP-C proteins to distal cuboidal epithelia of early abnormal saccules, evidence that T3 prematurely and abnormally advanced mesenchymal and epithelial cell differentiation. Western blot showed a T3-dependent decrease in Hoxb-5 and a trend towards decreased Nkx2.1 and SP-C, after 3 and 7 days of culture, respectively. We conclude that exogenous T3 treatment during the late pseudoglandular period prematurely and abnormally accelerates terminal saccular development. This may lead to abnormal mesenchymal and epithelial cell fate.

Mesenchymal nuclear transcription factors in nitrofen-induced hypoplastic lung.

BACKGROUND: Nitrofen-induced pulmonary hypoplasia (PH) with or without coexistent congenital diaphragmatic hernia (CDH) in animals mimics the human condition. We have demonstrated reduced steroid-thyroid-retinoid receptors in hypoplastic lungs. Therefore, we hypothesize that expression of two additional mesenchymally derived nuclear transcription factors, retinoid X receptor alpha (RXR-alpha) and homeobox b-5 (Hoxb-5), would also be altered in hypoplastic lungs. MATERIALS AND METHODS: We used timed-pregnant CD-1 mice either untreated or gavaged with nitrofen on gestational day (Gd) 8. Normal lungs were compared with hypoplastic lungs with severe left PH and CDH at Gd 14, 16, and 19 and from neonates. We performed immunoblotting for RXR-alpha and Hoxb-5 proteins and immunohistochemistry for Hoxb-5 protein. RESULTS: Almost 70-80% of nitrofen-exposed mice had no apparent external phenotypic abnormalities, such as craniofacial defects. Fetal body and lung weights were reduced. RXR-alpha and Hoxb-5 proteins were highest at Gd 14 and decreased as development progressed. Densitometric analysis of RXR-alpha or Hoxb-5 proteins in normal and hypoplastic lungs showed no significant difference; however, the immunolocalization pattern of Hoxb-5 protein differed. Hoxb-5 protein was primarily in mesenchymal cells of normal lungs on Gd 14; however, by Gd 19, it appeared to be mainly in the epithelial cells of prealveolar structures and in adjacent subepithelial mesenchymal cells. In hypoplastic lungs no significant changes were observed in Hoxb-5 staining in mesenchymal cells at Gd 14 nor at Gd 16; however, Hoxb-5 expression persisted in mesenchyme and epithelium at Gd 19 and in neonatal hypoplastic lungs, unlike normal lungs. CONCLUSIONS: (1) Unaltered RXR-alpha protein implies that despite altered retinoic acid receptors (RARs) in hypoplastic lungs, the mechanisms of nitrofen-induced PH may be independent of RXR-alpha pathways. (2) In hypoplastic lungs, the persistent mesenchymal expression of Hoxb-5, in later stages of development and at birth, suggests delayed development and maturation compared to normal lungs. We speculate that nitrofen induces PH via RAR-dependent but RXR-independent interactions, which may be downstream of the Hoxb-5 gene or may involve other more anteriorly expressed Hox genes.

Thyroid hormone affects embryonic mouse lung branching morphogenesis and cellular differentiation.

Although thyroid hormone (T(3)) influences epithelial cell differentiation during late fetal lung development, its effects on early lung morphogenesis are unknown. We hypothesized that T(3) would alter embryonic lung airway branching and temporal-spatial differentiation of the lung epithelium and mesenchyme. Gestational day 11.5 embryonic mouse lungs were cultured for 72 h in BGJb serum-free medium without or with added T(3) (0.2, 2.0, 10.0, or 100 nM). Evaluation of terminal bud counts showed a dose- and time-dependent decrease in branching morphogenesis. Cell proliferation was also significantly decreased with higher doses of T(3). Morphometric analysis of lung histology showed that T(3) caused a dose-dependent decrease in mesenchyme and increase in cuboidal epithelia and airway space. Immunocytochemistry showed that with T(3) treatment, Nkx2.1 and surfactant protein SP-C proteins became progressively localized to cuboidal epithelial cells and mesenchymal expression of Hoxb5 was reduced, a pattern resembling late fetal lung development. We conclude that exogenous T(3) treatment during early lung development accelerated epithelial and mesenchymal cell differentiation at the expense of premature reduction in new branch formation and lung growth.