Promote Lung Development Research Articles

Neonatal Survival After Serial Amnioinfusions for Bilateral Renal Agenesis

Early anhydramnios during pregnancy, resulting from fetal bilateral renal agenesis, causes lethal pulmonary hypoplasia in neonates. Restoring amniotic fluid via serial amnioinfusions may promote lung development, enabling survival. To assess neonatal outcomes of serial amnioinfusions initiated before 26 weeks' gestation to mitigate lethal pulmonary hypoplasia. Prospective, nonrandomized clinical trial conducted at 9 US fetal therapy centers between December 2018 and July 2022. Outcomes are reported for 21 maternal-fetal pairs with confirmed anhydramnios due to isolated fetal bilateral renal agenesis without other identified congenital anomalies. Enrolled participants initiated ultrasound-guided percutaneous amnioinfusions of isotonic fluid before 26 weeks' gestation, with frequency of infusions individualized to maintain normal amniotic fluid levels for gestational age. The primary end point was postnatal infant survival to 14 days of life or longer with dialysis access placement. The trial was stopped early based on an interim analysis of 18 maternal-fetal pairs given concern about neonatal morbidity and mortality beyond the primary end point despite demonstration of the efficacy of the intervention. There were 17 live births (94%), with a median gestational age at delivery of 32 weeks, 4 days (IQR, 32-34 weeks). All participants delivered prior to 37 weeks' gestation. The primary outcome was achieved in 14 (82%) of 17 live-born infants (95% CI, 44%-99%). Factors associated with survival to the primary outcome included a higher number of amnioinfusions (P = .01), gestational age greater than 32 weeks (P = .005), and higher birth weight (P = .03). Only 6 (35%) of the 17 neonates born alive survived to hospital discharge while receiving peritoneal dialysis at a median age of 24 weeks of life (range, 12-32 weeks). Serial amnioinfusions mitigated lethal pulmonary hypoplasia but were associated with preterm delivery. The lower rate of survival to discharge highlights the additional mortality burden independent of lung function. Additional long-term data are needed to fully characterize the outcomes in surviving neonates and assess the morbidity and mortality burden. ClinicalTrials.gov Identifier: NCT03101891.

The administration of a pre-digested fat-enriched formula prevents necrotising enterocolitis-induced lung injury in mice.

Necrotising enterocolitis (NEC) is a devastating gastrointestinal disease of prematurity that typically develops after the administration of infant formula, suggesting a link between nutritional components and disease development. One of the most significant complications that develops in patients with NEC is severe lung injury. We have previously shown that the administration of a nutritional formula that is enriched in pre-digested Triacylglyceride that do not require lipase action can significantly reduce the severity of NEC in a mouse model. We now hypothesise that this 'pre-digested fat (PDF) system' may reduce NEC-associated lung injury. In support of this hypothesis, we now show that rearing newborn mice on a nutritional formula based on the 'PDF system' promotes lung development, as evidenced by increased tight junctions and surfactant protein expression. Mice that were administered this 'PDF system' were significantly less vulnerable to the development of NEC-induced lung inflammation, and the administration of the 'PDF system' conferred lung protection. In seeking to define the mechanisms involved, the administration of the 'PDF system' significantly enhanced lung maturation and reduced the production of reactive oxygen species (ROS). These findings suggest that the PDF system protects the development of NEC-induced lung injury through effects on lung maturation and reduced ROS in the lung and also increases lung maturation in non-NEC mice.

Influence of Nutrition and Maternal Bonding on Postnatal Lung Development in the Newborn Pig.

BackgroundMicrobial colonization and immune cell maturation coincide at mucosal sites and are decisive for postnatal lung development. How external factors influence neonatal pulmonary immune development is poorly understood.ObjectiveTo elucidate the impact of key determinants in early life, nutrition, and maternal bonding, on postnatal lung maturation in a human-relevant animal model. To investigate the underlying immunological changes of impaired lung maturation and study the mechanisms of conversion.MethodsNewborn piglets were kept with or without isolation from their mothers and fed bovine milk-based infant formula or received milk of sow. Lung growth, histomorphology, respiratory immune responses, and lung microbiota were analyzed. Mother- and sow-milk-deprived piglets received maternal material or were reintroduced to the maternal environment at varying intervals to study options for reversal.ResultsFormula feeding combined with isolation of newborn piglets resulted in disturbed postnatal lung maturation. Reduced lung growth correlated with dampened IL-33 expression, impaired lung myeloid cell activation, and decreased Th1 differentiation, along with diminished richness and diversity of the lung microbiota. Transfer of bacteria-enriched maternal material reversed the negative effects on pulmonary immune maturation. Early (within 3 days) but not late (within 7 days) reintroduction to the mother allowed restoration of normal lung development.ConclusionOur findings reveal that lung growth, respiratory immunity, and microbial lung colonization in newborns depend on postnatal diet and maternal contact, and targeting these key regulators could promote lung development during this critical life stage.SummaryDisturbances in natural diet and reduced maternal contact during the neonatal period impair postnatal lung maturation. In pediatrics, timely breast milk feeding and intensive maternal bonding represent valuable intervention measures to promote early postnatal lung development.

MSC Based Therapies to Prevent or Treat BPD-A Narrative Review on Advances and Ongoing Challenges.

Bronchopulmonary dysplasia (BPD) remains one of the most devastating consequences of preterm birth resulting in life-long restrictions in lung function. Distorted lung development is caused by its inflammatory response which is mainly provoked by mechanical ventilation, oxygen toxicity and bacterial infections. Dysfunction of resident lung mesenchymal stem cells (MSC) represents one key hallmark that drives BPD pathology. Despite all progress in the understanding of pathomechanisms, therapeutics to prevent or treat BPD are to date restricted to a few drugs. The limited therapeutic efficacy of established drugs can be explained by the fact that they fail to concurrently tackle the broad spectrum of disease driving mechanisms and by the huge overlap between distorted signal pathways of lung development and inflammation. The great enthusiasm about MSC based therapies as novel therapeutic for BPD arises from the capacity to inhibit inflammation while simultaneously promoting lung development and repair. Preclinical studies, mainly performed in rodents, raise hopes that there will be finally a broadly acting, efficient therapy at hand to prevent or treat BPD. Our narrative review gives a comprehensive overview on preclinical achievements, results from first early phase clinical studies and challenges to a successful translation into the clinical setting.

Lung megakaryocytes display distinct transcriptional and phenotypic properties.

Megakaryocytes (MKs) are responsible for platelet biogenesis, which is believed to occur canonically in adult bone marrow (BM) and in the fetal liver during development. However, emerging evidence highlights the lung as a previously underappreciated residence for MKs that may contribute significantly to circulating platelet mass. Although a diversity of cells specific to the BM is known to promote the maturation and trafficking of MKs, little investigation into the impact of the lung niche on the development and function of MKs has been done. Here, we describe the application of single-cell RNA sequencing, coupled with histological, ploidy, and flow cytometric analyses, to profile primary MKs derived from syngeneic mouse lung and hematopoietic tissues. Transcriptional profiling demonstrated that lung MKs have a unique signature distinct from their hematopoietic counterparts, with lung MKs displaying enrichment for maturation markers, potentially indicating a propensity for more efficient platelet production. Reciprocally, fetal lung MKs also showed the robust expression of cytokines and growth factors that are known to promote lung development. Lastly, lung MKs possess an enrichment profile skewed toward roles in immunity and inflammation. These findings highlight the existence of a lung-specific MK phenotype and support the notion that the lung plays an independent role in the development and functional maturation of MKs. The immune phenotype displayed by lung MKs also introduces their potential role in microbial surveillance and antigen presentation.

Expert consensus on nutritional management of preterm infants with bronchopulmonary dysplasia

Inadequate nutrition supply in the early stage after birth is a risk factor for the development of bronchopulmonary dysplasia (BPD) in preterm infants, and it is also closely associated with the progression and clinical outcome of BPD. Optimized nutritional support is of great importance to reduce the incidence and severity of BPD and promote lung development and neurological prognosis. Based on the relevant studies in China and overseas, the expert consensus on BPD nutrition management is developed by the Grading of Recommendations Assessment, Development and Evaluation (GRADE) method. The consensus includes the following seven aspects: the importance of nutrition in BPD, fluid intake, energy intake, enteral nutrition, parenteral nutrition, post-discharge nutrition, and nutrition monitoring and evaluation.

Involvement of YAP and LATS1 in lung development in a rat model of nitrofen-induced congenital diaphragmatic hernia

Purpose: To investigate the role of Hippo pathway in lung development in congenital diaphragmatic hernia (CDH).
 Methods: One oral dose of nitrofen was maternally administered for induction of CDH on embryonic day 9.5 (E 9.5). Sildenafil was administered intragastrically at a dose of 100 mg/kg on E 11.5. Three rat groups were used: control, CDH, and CDH + sildenafil. Cesarean section was used for fetal delivery on E 21.5. Fetuses with left diaphragmatic hernia (except in control rats) were chosen for investigations. Fetal body weight and weight of lung tissue were recorded, and lung histological evaluation, western blot and PCR were carried out after lung processing.
 Results: There was markedly higher expression of YAP in CDH rats than in control rats was unaffected by antenatal sildenafil administration (p < 0.05). However, prenatal sildenafil intervention significantly increased LATS1 expression in the lung of CDH fetuses (p < 0.05).
 Conclusion: These results indicate that increased pulmonary YAP expression in CDH rat model might contribute to pulmonary vascular remodeling and suppression of lung development. Thus, antenatal sildenafil administration not only mitigates abnormal vascular remodeling but also promotes lung development, most likely via increased expression of LATS1.
 Keywords: Congenital diaphragmatic hernia (CDH), Hippo pathway, Lung development, Sildenafil

Ex Vivo MRI Analytical Methods and Brain Pathology in Preterm Lambs Treated with Postnatal Dexamethasone †.

Postnatal glucocorticoids such as dexamethasone are effective in promoting lung development in preterm infants, but are prescribed cautiously due to concerns of neurological harm. We developed an analysis pipeline for post-mortem magnetic resonance imaging (MRI) to assess brain development and hence the neurological safety profile of postnatal dexamethasone in preterm lambs. Lambs were delivered via caesarean section at 129 days’ (d) gestation (full term ≈ 150 d) with saline-vehicle control (Saline, n = 9), low-dose tapered dexamethasone (cumulative dose = 0.75 mg/kg, n = 8), or high-dose tapered dexamethasone (cumulative dose = 2.67 mg/kg, n = 8), for seven days. Naïve fetal lambs (136 d gestation) were used as end-point maturation controls. The left-brain hemispheres were immersion-fixed in 10 % formalin (24 h), followed by paraformaldehyde (>6 months). Image sequences were empirically optimized for T1- and T2-weighted MRI and analysed using accessible methods. Spontaneous lesions detected in the white matter of the frontal cortex, temporo-parietal cortex, occipital lobe, and deep to the parahippocampal gyrus were confirmed with histology. Neither postnatal dexamethasone treatment nor gestation showed any associations with lesion incidence, frontal cortex (total, white, or grey matter) or hippocampal volume (all p > 0.05). Postnatal dexamethasone did not appear to adversely affect neurodevelopment. Our post-mortem MRI analysis pipeline is suitable for other animal models of brain development.

An Artificial Placenta Protects Against Lung Injury and Promotes Continued Lung Development in Extremely Premature Lambs.

An artificial placenta (AP) utilizing extracorporeal life support (ECLS) could protect premature lungs from injury and promote continued development. Preterm lambs at estimated gestational age (EGA) 114-128 days (term = 145) were delivered by Caesarian section and managed in one of three groups: AP, mechanical ventilation (MV), or tissue control (TC). Artificial placenta lambs (114 days EGA, n = 3; 121 days, n = 5) underwent venovenous (VV)-ECLS with jugular drainage and umbilical vein reinfusion for 7 days, with a fluid-filled, occluded airway. Mechanical ventilation lambs (121 days, n = 5; 128 days, n = 5) underwent conventional MV until failure or maximum 48 hours. Tissue control lambs (114 days, n = 3; 121 days, n = 5; 128 days, n = 5) were sacrificed at delivery. At the conclusion of each experiment, lungs were procured and sectioned. Hematoxylin and eosin (H&E) slides were scored 0-4 in seven injury categories, which were summed for a total injury score. Slides were also immunostained for platelet-derived growth factor receptor (PDGFR)-α and α-actin; lung development was quantified by the area fraction of double-positive tips of secondary alveolar septa. Support duration of AP lambs was 163 ± 9 (mean ± SD) hours, 4 ± 3 for early MV lambs, and 40 ± 6 for late MV lambs. Total injury scores at 121 days were 1.7 ± 2.1 for AP vs. 5.5 ± 1.6 for MV (p = 0.02). Using immunofluorescence, double-positive tip area fraction at 121 days was 0.017 ± 0.011 in AP lungs compared with 0.003 ± 0.003 in MV lungs (p < 0.001) and 0.009 ± 0.005 in TC lungs. At 128 days, double-positive tip area fraction was 0.012 ± 0.007 in AP lungs compared with 0.004 ± 0.004 in MV lungs (p < 0.001) and 0.016 ± 0.009 in TC lungs. The AP is protective against lung injury and promotes lung development compared with mechanical ventilation in premature lambs.

Upregulated EFNB2 and EPHB4 promotes lung development inanitrofen-induced congenital diaphragmatic hernia rat model.

Congenital diaphragmatic hernia (CDH) is a common congenital malformation associated with high mortality rates, mainly due to pulmonary hypoplasia and persistent pulmonary hypertension following birth. The present study aimed to investigate abnormal lung development in a rat CDH model, and examine temporal and spatial changes in the expression of ephrin type-B receptor 4 (EPHB4) and ephrin-B2 (EFNB2) during fetal lung development, to elucidate the role of these factors during lung morphogenesis. Pregnant rats received nitrofen on embryonic day (E) 8.5 to induce CDH, and fetal lungs were collected on E13.5, E15.5, E17.5, E19.5, and E21.5. The mean linear intercept (MLI) and mean alveolar number (MAN) were observed in fetal lung tissue at E21.5 following hematoxylin and eosin staining. E13.5 fetal lungs were cultured for 96 h in serum-free medium and branch development was observed under a microscope. The gene and protein expression levels of EPHB4 and EFNB2 were assessed by reverse transcription-quantitative polymerase chain reaction analysis, and immunoblotting and immunohistochemistry, respectively. The fetal rat lungs were treated with EFNB2 and the activity of key signaling pathways was assessed. The lung index (lung weight/body weight) at E21.5 was significantly lower in the CDH rats, compared with that in the control fetal rats. The MLI and MAN were also lower in the CDH group. The number of lung terminal buds at E13.5 (embryonic stage), and the lung-explant perimeter and surface were all smaller in the CDH group rats than in the control group at the same age. Pulmonary hypoplasia was observed following 96 h of in vitro culture. No significant differences were found in the expression levels of EFNB2 and EPHB4 between the CDH and control groups at E13.5 (embryonic stage) or E15.5 (pseudoglandular stage), however, EFNB2 and EPHB4 were significantly upregulated at E17.5 (canalicular stage), and at E19.5 and E21.5 (saccular/alveolar stages). EFNB2 stimulated pulmonary branching and EFNB2 supplementation decreased the activity of p38, c-Jun NH2-terminal kinase, extracellular signal-regulated kinase, and signal transducer and activator of transcription. The CDH fetal rats developed pulmonary dysplasia at an early stage of fetal pulmonary development. Upregulated expression of EFNB2 and EPHB4 was observed in the rat lung of nitrofen-induced CDH, and the increased expression of EFNB2 promoted rat lung development in the nitrofen-induced CDH model.

Perfluorocarbons Prevent Lung Injury and Promote Development during Artificial Placenta Support in Extremely Premature Lambs

Background: Extremely premature neonates suffer high morbidity and mortality. An artificial placenta (AP) using extracorporeal life support (ECLS) is a promising therapy. Objectives: We hypothesized that intratracheal perfluorocarbon (PFC) instillation during AP support would reduce lung injury and promote lung development relative to intratracheal amniotic fluid or crystalloid. Methods: Lambs at an estimated gestational age (EGA) 116–121 days (term 145 days) were placed on venovenous ECLS with jugular drainage and umbilical vein reinfusion and intubated. Airways were managed by the instillation of amniotic fluid and tracheal occlusion (TO; n = 4), or lactated Ringer’s (LR; n = 4) or perfluorodecalin (a PFC) without occlusion (n = 4). After 7 days, the animals were sacrificed. Early (EGA 116–121 days) and late (EGA 125–131 days) tissue control lambs were delivered and sacrificed. Lungs were formalin-inflated to 30 cm H₂O and sectioned for histology. Injury was scored by an unbiased pathologist. Slides were immunostained for PDGFR-α and α-actin; development was quantified by the area fraction of double-positive tips. Surfactant protein-C (SP-C) concentration in bronchoalveolar lavage fluid was quantified using ELISA. Results: Total injury scores were lower in PFC lungs (1.8 ± 1.7) than in TO (6.5 ± 2.1; p = 0.01) and LR lungs (5.5 ± 2.4; p = 0.01). The area fraction of double-positive alveolar tips appeared higher in PFC lungs than in TO lungs (0.18 ± 0.007 vs. 0.008 ± 0.004; p = 0.07). SP-C concentration was higher in PFC lungs than in TO lungs (37.9 ± 7.6 vs. 20.0 ± 5.4 pg/mL; p = 0.005), and both early (12.4 ± 1.7 g/mL; p = 0.007) and late tissue control lungs (15.1 ± 5.0 pg/mL; p = 0.0008). Conclusion: During AP support, intratracheal PFC prevents lung injury and promotes normal lung development better than crystalloid or amniotic fluid with TO.

Knockout of microRNA‑26a promotes lung development and pulmonary surfactant synthesis.

Normal formation and function of the lungs are essential for the transition of the fetus to an air‑breathing environment at birth. The synthesis of pulmonary surfactant (PS), which is produced by type II alveolar epithelial cells (AECIIs), is required for proper lung development. Previous invitro studies have suggested that PS synthesis is regulated by microRNA (miR)‑26a in fetal rat AECIIs. The present study explored the potential role of miR‑26a in lung development and PS synthesis by using a miR‑26a‑1/miR‑26a‑2 double knockout mouse model. Hematoxylin and eosin staining and transmission electron microscopy were used to observe the morphology of fetal lungs. Reverse transcription‑quantitative polymerase chain reaction and western blot analysis were performed to examine the mRNA and protein levels of surfactant‑associated proteins. The results demonstrated that the lung formation in the knockout mice was more mature, and that there were more mature lamellar bodies inside AECIIs in miR‑26a knockout mice at late stages of lung development. The findings further demonstrated that knockout of miR‑26a increased surfactant‑associated mRNA and protein expression levels. The results indicated that knockout of miR‑26a promotes lung development and PS synthesis.

A Randomized Trial of Low-Flow Oxygen versus Nasal Continuous Positive Airway Pressure in Preterm Infants

Background: Nasal continuous positive airway pressure (nCPAP) stabilizes the residual volume and may decrease the risk of ‘atelectotrauma', potentially promoting lung development in neonates. Objectives: To assess whether replacing nCPAP by low-flow O₂ by nasal cannula affects lung function expressed as the arterial/alveolar oxygen tension ratio (a/A pO₂ ratio) on postnatal day 28. Methods: Preterm infants (birth weight <1,500 g and gestational age, GA >26 + 0 weeks) stable on nCPAP between postnatal days 4 and 7 were randomized to nCPAP or low-flow O₂ by nasal cannula (<0.2 liters/min). Study criteria defined how to wean/restart respiratory support or change from low-flow O₂ to nCPAP and vice versa. Transcutaneous monitoring was used for the assessment of the a/A pO₂ ratio on day 28 using a head box for all infants for accurate measurement and to eliminate possible effects from nCPAP or low-flow O₂ on oxygen requirement. Results: We enrolled 52 infants (nCPAP group n = 30 and low-flow O₂ group n = 22). The a/A pO₂ ratio at 28 days was 0.43 ± 0.17 (nCPAP group) versus 0.48 ± 0.18 (p = 0.36). The duration of nCPAP was 16.4 (low-flow group) versus 41.1 days (nCPAP group), p < 0.001. There was no difference between groups in the fraction needing any respiratory support at 36 weeks' corrected age, length of stay, weight at discharge, and relative weight gain. Conclusions: Replacing nCPAP by low-flow O₂ in preterm infants with GA >26 weeks at the end of the first week of life did not seem to affect the a/A pO₂ ratio or weight gain negatively. Thus, prolonged nCPAP seems not to have a positive effect on lung function at 28 days of life and replacement by low-flow O₂ could reduce the cost of equipment and increase the ease of nursing.

Akt Activation and Inhibition of Cytochrome C Release: Mechanistic Insights into Leptin-promoted Survival of Type II Alveolar Epithelial Cells.

Fetal growth restriction (FGR) increases the risk of perinatal death, partly due to defects in lung development. Leptin, a polypeptide hormone, is involved in fetal lung development. We previously demonstrated that treatment with exogenous leptin during gestation significantly promotes fetal lung maturity in the rat model of FGR. In this study, to delineate the molecular pathways through which leptin may enhance fetal lung development, we investigated the impact of leptin treatment on the survival of type II alveolar epithelial cells (AECs), essential leptin-responsive cells involved in lung development, in a rat model of FGR. The rat model of FGR was induced in pregnant Sprague-Dawley rats by partial uterine artery and vein ligation. In vivo and in vitro analyses of fetal lung tissues and freshly-isolated cultured AECs, respectively, showed that leptin protects type II AECs from hypoxia-induced apoptosis. Further molecular studies revealed the role of Akt activation in the leptin-mediated promotion of survival of type II AECs. The data also showed that the anti-apoptotic effects of leptin are dependent on phosphoinositol 3-kinase (PI3K) activation, and involve the down-regulation of caspases 3 and 9, upregulation of pro-survival proteins Bcl-2, and p-Bad, and inhibition of the release of cytochrome c from mitochondria. Taken together, our data suggested that leptin enhances the maturity of fetal lungs by mediating the regulation of caspase-3 and -9 during hypoxia-induced apoptosis of type II AECs and provide support for the potential of leptin as a therapeutic agent for promoting lung development in FGR.

Prenatal administration of neuropeptide bombesin promotes lung development in a rat model of nitrofen-induced congenital diaphragmatic hernia

Background/purposeFetal medical treatment to improve lung hypoplasia in congenital diaphragmatic hernia (CDH) has yet to be established. The neuropeptide bombesin (BBS) might play an important role in lung development. The present study aims to determine whether prenatally administered BBS could be useful to promote fetal lung development in a rat model of nitrofen-induced CDH. MethodsPregnant rats were administered with nitrofen (100mg) on gestation day 9.5 (E9.5). BBS (50mg/kg/day) was then daily infused intraperitoneally from E14, and fetal lungs were harvested on E21. The expression of PCNA was assessed by both immunohistochemical staining and RT-PCR to determine the amount of cell proliferation. Lung maturity was assessed as the expression of TTF-1, a marker of alveolar epithelial cell type II. ResultsThe lung-body-weight ratio was significantly increased in CDH/BBS(+) compared with CDH/BBS(−) (p<0.05). The number of cells stained positive for PCNA and TTF-1 was significantly decreased in CDH/BBS(+) compared with CDH/BBS(−) (p<0.01). The TTF-1 mRNA expression levels were significantly decreased in CDH/BBS(+) compared with CDH/BBS(−) (p<0.05). ConclusionsPrenatally administered BBS promotes lung development in a rat model of nitrofen-induced CDH. Neuropeptide BBS could help to rescue lung hypoplasia in fetal CDH.

Role of neuregulin‐1β in dexamethasone‐enhanced surfactant synthesis in fetal type II cells

It is well established that glucocorticoids elevate the production of fibroblast-pneumocyte factor (FPF), which induces type II cells to synthesize surfactant phospholipids. FPF, however, has not been identified and it is not clear whether it is a single factor or a complex mixture of factors. In this study it has been shown that, when lung fibroblasts are exposed to dexamethasone, the concentration of neuregulin-1β (NRG1β) in conditioned medium is elevated 2-fold (P<0.05), even though NRG1β gene expression is unaffected. This, together with the finding that exposure of type II cells to NRG1β directly stimulates by 3-fold the rate of phospholipid synthesis (P<0.05), suggests that NRG1β is a component of FPF that promotes lung development.

Connective tissue lung: different composition between male and female

Abnormal elasticity of lung tissue has a major impact on the clinical progression and outcome of many lung diseases including respiratory distress syndrome, asthma, emphysema and pneumothorax post thoracic injury. The major components of lung connective tissue are elastin, collagen and together provide the lung with its elasticity and tensile strength. Elastin is composed of flexible cross-linked polypeptides and has a linear stress-strain relation up to 200% strain; collagen has a more organized structure with both crystalline and amorphous phases and exhibits a highly unlinear stress-strain curve. The thoracic injury is responsible for about 25% of death for injury and in particular it represent 12% of all death in man in comparison to 7% of dealt in female. This different in death rate is due to the different hormone levels between man and female. On the other hand female sex hormones, estrogens (e.g. 17b-estradiol) exert important biological actions, both protective and undesirable. In particular in addition to their primary function as reproductive hormones, estrogens (e.g. 17b-estradiol) influence a wide range of other physiological processes in humans and other mammals, including cardiovascular, respiratory, and neuronal function, and bone density.. With special respect to the cardiorespiratory system, estrogens promote lung development and differentiation and exhibit pulmonary and cardiovascular protective properties. However, very little is known about the different composition of the two major constituents of the connective tissue of the female and men lung. We have studied by CLSM the elastin an collagen in man and female lung. All proteins tested are present in lung tissue of male and female and they are localized in the peribronchiolar and in the interalveolar septum. The reaction to elastin shows very differences staining in the basal laminae of small and medium bronchioles in two sexes, in particular these structures stained very strongly in female but not in male. Opposite behavior we have showed to collagen type IV reactions. In male the collagen type IV is localized as a continuous network throughout the lung parenchymal tissue and in the basal laminae it shows strongly staining.

Hypoxia-Inducible Factors Promote Alveolar Development and Regeneration

Understanding how alveoli and the underlying capillary network develop and how these mechanisms are disrupted in disease states is critical for developing effective therapies for lung regeneration. Recent evidence suggests that lung angiogenesis promotes lung development and repair. Vascular endothelial growth factor (VEGF) preserves lung angiogenesis and alveolarization in experimental O2-induced arrested alveolar growth in newborn rats, but combined VEGF+angiopoietin 1 treatment is necessary to correct VEGF-induced vessel leakiness. Hypoxia-inducible factors (HIFs) are transcription factors that activate multiple O2-sensitive genes, including those encoding for angiogenic growth factors, but their role during postnatal lung growth is incompletely understood. By inducing the expression of a range of angiogenic factors in a coordinated fashion, HIF may orchestrate efficient and safe angiogenesis superior to VEGF. We hypothesized that HIF inhibition impairs alveolarization and that HIF activation regenerates irreversible O2-induced arrested alveolar growth. HIF inhibition by intratracheal dominant-negative adenovirus (dnHIF-1α)-mediated gene transfer or chetomin decreased lung HIF-1α, HIF-2α, and VEGF expression and led to air space enlargement and arrested lung vascular growth. In experimental O2-induced arrested alveolar growth in newborn rats, the characteristic features of air space enlargement and loss of lung capillaries were associated with decreased lung HIF-1α and HIF-2α expression. Intratracheal administration of Ad.HIF-1α restored HIF-1α, endothelial nitric oxide synthase, VEGF, VEGFR2, and Tie2 expression and preserved and rescued alveolar growth and lung capillary formation in this model. HIFs promote normal alveolar development and may be useful targets for alveolar regeneration.

M2 macrophage polarisation is associated with alveolar formation during postnatal lung development

BackgroundMacrophages are traditionally associated with inflammation and host defence, however a greater understanding of macrophage heterogeneity is revealing their essential roles in non-immune functions such as development, homeostasis and regeneration. In organs including the brain, kidney, mammary gland and pancreas, macrophages reside in large numbers and provide essential regulatory functions that shape organ development and maturation. However, the role of macrophages in lung development and the potential implications of macrophage modulation in the promotion of lung maturation have not yet been ascertained.MethodsEmbryonic day (E)12.5 mouse lungs were cultured as explants and macrophages associated with branching morphogenesis were visualised by wholemount immunofluorescence microscopy. Postnatal lung development and the correlation with macrophage number and phenotype were examined using Colony-stimulating factor-1 receptor-enhanced green fluorescent protein (Csf1r-EGFP) reporter mice. Structural histological examination was complemented with whole-body plethysmography assessment of postnatal lung functional maturation over time.Flow cytometry, real-time (q)PCR and immunofluorescence microscopy were performed to characterise macrophage number, phenotype and localisation in the lung during postnatal development. To assess the impact of developmental macrophage modulation, CSF-1 was administered to neonatal mice at postnatal day (P)1, 2 and 3, and lung macrophage number and phenotype were assessed at P5. EGFP transgene expression and in situ hybridisation was performed to assess CSF-1R location in the developing lung.ResultsMacrophages in embryonic lungs were abundant and densely located within branch points during branching morphogenesis. During postnatal development, structural and functional maturation of the lung was associated with an increase in lung macrophage number. In particular, the period of alveolarisation from P14-21 was associated with increased number of Csf1r-EGFP+ macrophages and upregulated expression of Arginase 1 (Arg1), Mannose receptor 1 (Mrc1) and Chemokine C-C motif ligand 17 (Ccl17), indicative of an M2 or tissue remodelling macrophage phenotype. Administration of CSF-1 to neonatal mice increased trophic macrophages during development and was associated with increased expression of the M2-associated gene Found in inflammatory zone (Fizz)1 and the growth regulator Insulin-like growth factor (Igf)1. The effects of CSF-1 were identified as macrophage-mediated, as the CSF-1R was found to be exclusively expressed on interstitial myeloid cells.ConclusionsThis study identifies the presence of CSF-1R+ M2-polarised macrophages localising to sites of branching morphogenesis and increasing in number during the alveolarisation stage of normal lung development. Improved understanding of the role of macrophages in lung developmental regulation has clinical relevance for addressing neonatal inflammatory perturbation of development and highlights macrophage modulation as a potential intervention to promote lung development.

Exposure to dexamethasone during late gestation causes female-specific decreases in core body temperature and prepro-thyrotropin-releasing hormone expression in the paraventricular nucleus of the hypothalamus in rats

Synthetic glucocorticoids (GC) have been used to promote lung development in preterm infants, thereby decreasing respiratory distress syndrome and mortality, yet, concern has arisen from reports that such treatment predisposes individuals to disease in adulthood. Given the variety of preclinical studies that show metabolic and behavioral abnormalities in adulthood following fetal exposure to synthetic GC, we examined the effect of in utero exposure to the synthetic GC, dexamethasone (DEX), on hypothalamic expression of thyrotropin-releasing hormone (TRH) a central neuropeptide involved in mediating behavior and metabolic balance. Pregnant Sprague–Dawley rats were administered 0.4mg/kg DEX on gestational days 18–21. As adults (postnatal day (PD) 60), the offspring were fitted with temperature sensing transmitters allowing real-time monitoring of core body temperature (CBT) across the 24h light dark period. This revealed a significant decrease in CBT throughout the day in prenatal DEX-treated females on estrus and diestrus, but not in male offspring. The reduction in CBT by prenatal DEX exposure was accompanied by a significant decrease in the expression of Trh transcript in the paraventricular nucleus of the hypothalamus (PVN) of female rats at PD 60 and this effect was also present on PD7. There was also a female-specific reduction in the number of preproTRH-immunoreactive (ir) neurons in the PVN, with ppTRH-ir nerve fibers decreases that were present in both male and female offspring. No changes in thyroid hormone (triiodothyronine, T3; thyroxine, T4) were observed in adult offspring, but during development, both males and females (PD14) had lower T3 and T4 levels. These data indicate abnormal expression of TRH results from fetal DEX exposure during late gestation, possibly explaining the decreased CBT observed in the female offspring.