TGF-α/EGFR signaling promotes lipopolysaccharide-induced abnormal elastin deposition and alveolar simplification.

Bronchopulmonary dysplasia (BPD) is characterized by shortened secondary septa and fewer, larger alveoli. Elastin deposition to the distal tips of the secondary septa is critical for elongation of the secondary septa. Alveolar myofibroblasts, which are thought to migrate to the septal tips during alveolarization, are mainly responsible for elastin production and deposition. Antenatal exposure to inflammation induces abnormal elastin deposition, thereby increasing the risk of developing BPD. Here, we found that lipopolysaccharide (LPS) significantly increased the expression of transforming growth factor-α (TGF-α) in an LPS-induced rat model of BPD and in LPS-treated human pulmonary epithelial cells (BEAS-2B). In addition, in vitro experiments suggested that LPS upregulated TGF-α expression via toll-like receptor 4 (TLR4)/tumor necrosis factor α-converting enzyme (TACE) signaling. Increased TGF-α levels via its receptor epidermal growth factor receptor (EGFR)-induced lysyl oxidase (LOX) overactivation and cell division cycle 42 (Cdc42) activity inhibition of myofibroblasts. Similarly, in vivo LOX overactivation and inhibition of Cdc42 activity were observed in the lungs of LPS-exposed pups. LOX overactivation led to abnormal elastin deposition, and inhibition of Cdc42 activity disturbed the directional migration of myofibroblasts and disrupted elastin localization. Most importantly, the EGFR inhibitor erlotinib partially rescued LOX overactivation and Cdc42 activity inhibition, and improved elastin deposition and alveolar development in antenatal LPS-treated rats. Taken together, our data suggest that TGF-α/EGFR signaling is critically involved in the regulation of elastin deposition and represents a novel therapeutic target.

Systemic postnatal corticosteroid use for the prevention of bronchopulmonary dysplasia and its relationship to early neurodevelopment in extremely preterm infants.

BACKGROUND: Systemic postnatal corticosteroid use in extremely preterm infants poses a risk of adverse neurodevelopmental outcomes. This study explores their use beyond seven days of age with early neurodevelopmental assessments during the fidgety period (9-20 weeks postterm age). METHODS: This retrospective single-center cohort study included inborn extremely preterm infants from 1 January 2014 to 31 December 2018. Outborn infants, those with congenital or genetic abnormalities, and those who received postnatal corticosteroids for nonrespiratory reasons were excluded. The cohort was dichotomized based on the status of corticosteroid receipt. Early neurodevelopmental outcomes were reported using Prechtl's General Movements Assessment. RESULTS: Of the 282 infants, 67 (23.75%) received corticosteroids. Of these, 34 (50.75%) received them for dependency on invasive ventilation (intermittent positive-pressure ventilation), and the remainder received them for dependency on non-invasive ventilation continuous positive airway pressure (CPAP) or bi-level positive airway pressure (BiPAP). Abnormal or absent fidgety movements were observed in 13% of infants (7/54) who received corticosteroids compared to 2% of infants (3/146) who did not. An increased odds for an abnormal general movements assessment from corticosteroid use after adjusting for gestational age [adjusted odds ratio (aOR) = 5.5, 95% confidence interval (CI) = 1.14-26.56] was observed. The motor optimality scores differed between the two groups [corticosteroid group: 25.5 (23-26) versus no-corticosteroid group: 26 (24-28); z = - 2.02]. A motor optimality score < 20 was observed in 14.8% of infants (8/54) in the corticosteroid group compared to 2% of infants (3/146) in the noncorticosteroid group. This difference was significant after adjustment for gestational age (aOR 5.96, 95% CI 1.28-27.74). CONCLUSIONS: Abnormal early neurodevelopment was observed in infants who received systemic postnatal corticosteroids. The relationship between these findings and other factors influencing early neurodevelopment needs further exploration.

Systemic Steroids in Preventing Bronchopulmonary Dysplasia (BPD): Neurodevelopmental Outcome According to the Risk of BPD in the EPICE Cohort.

BACKGROUND: Postnatal steroids (PNS) have been used to prevent bronchopulmonary dysplasia (BPD) in preterm infants but have potential adverse effects on neurodevelopment. These effects might be modulated by their risk of BPD. We aimed to compare patients' neurodevelopment with PNS treatment according to their risk of BPD in a European cohort. METHODS: We developed a prediction model for BPD to classify infants born between 24 + 0 and 29 + 6 weeks of gestation in three groups and compared patients' neurological outcome at two years of corrected age using the propensity score (PS) method. RESULTS: Of 3662 neonates included in the analysis, 901 (24.6%) were diagnosed with BPD. Our prediction model for BPD had an area under the ROC curve of 0.82. In the group with the highest risk of developing BPD, PNS were associated with an increased risk of gross motor impairment: OR of 1.95 after IPTW adjustment (95% CI 1.18 to 3.24, p = 0.010). This difference existed regardless of the type of steroid used. However, there was an increased risk of cognitive anomalies for patients treated with dexa/betamethasone that was no longer observed with hydrocortisone. CONCLUSIONS: This study suggests that PNS might be associated with an increased risk of gross motor impairment regardless of the group risk for BPD. Further randomised controlled trials exploring the use of PNS to prevent BPD should include a risk-based evaluation of neurodevelopmental outcomes. This observation still needs to be confirmed in a randomised controlled trial.

Glucocorticoids in a Neonatal Hyperoxic Lung Injury Model: Pulmonary and Neurotoxic effects.

BACKGROUND: We aimed to compare the effect of dexamethasone (Dex), hydrocortisone (Hc), and methylprednisolone (Mpz) at equivalent doses on somatic growth, lung healing, and neurotoxicity in a hyperoxic rat model. We hypothesized that Mpz and Hc would be superior to Dex with less neurotoxicity by exerting similar therapeutic efficacy on the injured lung. METHODS: Neonatal rats were randomized to control, bronchopulmonary dysplasia (BPD), Dex, Hc, and Mpz groups. All drugs were administered daily following day 15 over 7 days. Histopathological and immunohistochemical analyses of the lung and brain were performed on day 22. RESULTS: All types had much the same impact on lung repair. Oxidative markers in the lung were similar in the steroid groups. While nuclear factor erythroid 2-related factor and heat-shock protein 70 dropped following steroid treatment, no difference was noted in other biochemical markers in the brain between the study groups. Apoptotic activity and neuron loss in the parietal cortex and hippocampus were noted utmost in Dex, but alike in other BPD groups. CONCLUSIONS: Mpz does not appear to be superior to Dex or Hc in terms of pulmonary outcomes and oxidative damage in the brain, but safer than Dex regarding apoptotic neuron loss. IMPACT: This is the first study that compared the pulmonary efficacy and neurotoxic effects of Dex, Hc, and Mpz simultaneously in an established BPD model. This study adds to the literature on the importance of possible antioxidant and protective effects of glucocorticoid therapy in an oxidative stress-exposed brain. Mpz ended up with no more additional neuron loss or apoptosis risk by having interchangeable effects with others for the treatment of established BPD. Mpz and Hc seem safe as a rescue therapy in terms of adverse outcomes for established BPD in which lung and brain tissue is already impaired.

Adrenomedullin Deficiency Potentiates Lipopolysaccharide-Induced Experimental Bronchopulmonary Dysplasia in Neonatal Mice.

Lung inflammation interrupts alveolarization and causes bronchopulmonary dysplasia (BPD). Besides mechanical ventilation and hyperoxia, sepsis contributes to BPD pathogenesis. Adrenomedullin (Adm) is a multifunctional peptide that exerts anti-inflammatory effects in the lungs of adult rodents. Whether Adm mitigates sepsis-induced neonatal lung injury is unknown. The lung phenotype of mice exposed to early postnatal lipopolysaccharide (LPS) was recently shown to be similar to that in human BPD. This model was used to test the hypothesis that Adm-deficient neonatal mice will display increased LPS-induced lung injury than their wild-type (WT) littermates. Adm-deficient mice or their WT littermates were intraperitoneally administered 6 mg/kg of LPS or vehicle daily on postnatal days (PNDs) 3 to 5. The lungs were harvested at several time points to quantify inflammation, alveolarization, and vascularization. The extent of LPS-induced lung inflammation in Adm-deficient mice was 1.6-fold to 10-fold higher than their WT littermates. Strikingly, Adm deficiency induced STAT1 activation and potentiated STAT3 activation in LPS-exposed lungs. The severity of LPS-induced interruption of lung development was also greater in Adm-deficient mice at PND7. At PND14, LPS-exposed WT littermates displayed substantial improvement in lung development, whereas LPS-exposed Adm-deficient mice continued to have decreased lung development. These data indicate that Adm is necessary to decrease lung inflammation and injury and promote repair of the injured lungs in LPS-exposed neonatal mice.

VitA or VitD ameliorates bronchopulmonary dysplasia by regulating the balance between M1 and M2 macrophages.

PURPOSE: To investigate the therapeutic effects of vitamin A (VitA) or vitamin D (VitD) against bronchopulmonary dysplasia (BPD) and the underlying mechanism from the perspective of macrophage polarization. METHODS: A BPD model was established on rats. Hematoxylin and eosin staining was used to evaluate the pathological state of lung tissues. The expression of CD68 was determined by immunohistochemistry assay. The infiltration of M1 and M2 macrophages was marked by immunofluorescence. The expression levels of tumor necrosis factor (TNF)-α, interleukin (IL)-10, nitric oxide synthase (NOS), and arginase-1 (Arg-1) were evaluated by quantitative reverse transcription polymerase chain reaction assay, and the ratio of M1/M2 in the bronchoalveolar lavage fluid was determined by flow cytometry. RESULTS: Disordered alveolar structure in the lung tissue, thickened alveolar septa, and infiltration of inflammatory cells around the alveolar cavity and pulmonary septa were observed in lipopolysaccharide (LPS)-treated rats. On day 21 post-natal (PN21), the pathological state was aggravated, alveolar simplification was observed, and the expression level of CD68 in the lung tissues was significantly elevated, and these results were dramatically alleviated in the VitA, VitD, and VitA+VitD groups. However, no significant synergistic effect was observed between VitA+VitD and VitA or VitD groups. After the administration with VitA or VitD, IL-10 and Arg-1 were up-regulated on PN10. TNF-α and NOS were up-regulated on PN21. The ratio of macrophage polarization and M2 macrophages increased considerably after the stimulation with LPS, and this result was significantly reversed by VitA or VitD. A significant difference was observed on the effect of different dosages of VitA or VitD on macrophage polarization, among which moderate dosages of VitA or VitD exerted the most significant influence on macrophage polarization. CONCLUSION: The BPD-linked pulmonary injury stimulated by LPS can be ameliorated by the introduction of VitA or VitD.

Hyperpolarized 129Xe magnetic resonance spectroscopy in a rat model of bronchopulmonary dysplasia.

Premature infants often require mechanical ventilation and oxygen therapy, which can result in bronchopulmonary dysplasia (BPD), characterized by developmental arrest and impaired lung function. Conventional clinical methods for assessing the prenatal lung are not adequate for the detection and assessment of long-term health risks in infants with BPD, highlighting the need for a noninvasive tool for the characterization of lung microstructure and function. Theoretical diffusion models, like the model of xenon exchange (MOXE), interrogate alveolar gas exchange by predicting the uptake of inert hyperpolarized (HP) 129Xe gas measured with HP 129Xe magnetic resonance spectroscopy (MRS). To investigate HP 129Xe MRS as a tool for noninvasive characterization of pulmonary microstructural and functional changes in vivo, HP 129Xe gas exchange data were acquired in an oxygen exposure rat model of BPD that recapitulates the fewer and larger distal airways and pulmonary vascular stunting characteristics of BPD. Gas exchange parameters from MOXE, including airspace mean chord length (Lm), apparent hematocrit in the pulmonary capillaries (HCT), and pulmonary capillary transit time (tx), were compared with airspace mean axis length and area density (MAL and ρA) and percentage area of tissue and air (PTA and PAA) from histology. Lm was significantly larger in the exposed rats (P = 0.003) and correlated with MAL, ρA, PTA, and PAA (0.59<|ρ|<0.66 and P < 0.05). Observed increase in HCT (P = 0.012) and changes in tx are also discussed. These findings support the use of HP 129Xe MRS for detecting fewer, enlarged distal airways in this rat model of BPD, and potentially in humans.

FOSL1 is a novel mediator of endotoxin/lipopolysaccharide-induced pulmonary angiogenic signaling.

Systemic sepsis is a known risk factor for bronchopulmonary dysplasia (BPD) in premature infants, a disease characterized by dysregulated angiogenesis and impaired vascular and alveolar development. We have previoulsy reported that systemic endotoxin dysregulates pulmonary angiogenesis resulting in alveolar simplification mimicking BPD in neonatal mice, but the underlying mechanisms remain unclear. We undertook an unbiased discovery approach to identify novel signaling pathways programming sepsis-induced deviant lung angiogenesis. Pulmonary endothelial cells (EC) were isolated for RNA-Seq from newborn C57BL/6 mice treated with intraperitoneal lipopolysaccharide (LPS) to mimic systemic sepsis. LPS significantly differentially-regulated 269 genes after 6 h, and 1,934 genes after 24 h. Using bioinformatics, we linked 6 h genes previously unknown to be modulated by LPS to 24 h genes known to regulate angiogenesis/vasculogenesis to identify pathways programming deviant angiogenesis. An immortalized primary human lung EC (HPMEC-im) line was generated by SV40 transduction to facilitate mechanistic studies. RT-PCR and transcription factor binding analysis identified FOSL1 (FOS like 1) as a transcriptional regulator of LPS-induced downstream angiogenic or vasculogenic genes. Over-expression and silencing studies of FOSL1 in immortalized and primary HPMEC demonstrated that baseline and LPS-induced expression of ADAM8, CXCR2, HPX, LRG1, PROK2, and RNF213 was regulated by FOSL1. FOSL1 silencing impaired LPS-induced in vitro HPMEC angiogenesis. In conclusion, we identified FOSL1 as a novel regulator of sepsis-induced deviant angiogenic signaling in mouse lung EC and human fetal HPMEC.

Halogen exposure injury in the developing lung.

Owing to a high-volume industrial usage of the halogens chlorine (Cl2 ) and bromine (Br2 ), they are stored and transported in abundance, creating a risk for accidental or malicious release to human populations. Despite extensive efforts to understand the mechanisms of toxicity upon halogen exposure and to develop specific treatments that could be used to treat exposed individuals or large populations, until recently, there has been little to no effort to determine whether there are specific features and or the mechanisms of halogen exposure injury in newborns or children. We established a model of neonatal halogen exposure and published our initial findings. In this review, we aim to contrast and compare the findings in neonatal mice exposed to Br2 with the findings published on adult mice exposed to Br2 and the neonatal murine models of bronchopulmonary dysplasia. Despite remarkable similarities across these models in overall alveolar architecture, there are distinct functional and apparent mechanistic differences that are characteristic of each model. Understanding the mechanistic and functional features that are characteristic of the injury process in neonatal mice exposed to halogens will allow us to develop countermeasures that are appropriate for, and effective in, this unique population.

Neonatal therapy with PF543, a sphingosine kinase 1 inhibitor, ameliorates hyperoxia-induced airway remodeling in a murine model of bronchopulmonary dysplasia.

Hyperoxia (HO)-induced lung injury contributes to bronchopulmonary dysplasia (BPD) in preterm newborns. Intractable wheezing seen in BPD survivors is associated with airway remodeling (AWRM). Sphingosine kinase 1 (SPHK1)/sphingosine-1-phosphate (S1P) signaling promotes HO-mediated neonatal BPD; however, its role in the sequela of AWRM is not known. We noted an increased concentration of S1P in tracheal aspirates of neonatal infants with severe BPD, and earlier, demonstrated that Sphk1-/- mice showed protection against HO-induced BPD. The role of SPHK1/S1P in promoting AWRM following exposure of neonates to HO was investigated in a murine model. Therapy using PF543, the specific SPHK1 inhibitor, during neonatal HO reduced alveolar simplification followed by reduced AWRM in adult mice. This was associated with reduced airway hyperreactivity to intravenous methacholine. Neonatal HO exposure was associated with increased expression of SPHK1 in lung tissue of adult mice, which was reduced with PF543 therapy in the neonatal stage. This was accompanied by amelioration of HO-induced reduction of E-cadherin in airway epithelium. This may be suggestive of arrested partial epithelial mesenchymal transition (EMT) induced by HO. In vitro studies using human primary airway epithelial cells (HAEpCs) showed that SPHK1 inhibition or deletion restored HO-induced reduction in E-cadherin and reduced formation of mitochondrial reactive oxygen species (mtROS). Blocking mtROS with MitoTempo attenuated HO-induced partial EMT of HAEpCs. These results collectively support a therapeutic role for PF543 in preventing HO-induced BPD in neonates and the long-term sequela of AWRM, thus conferring a long-term protection resulting in improved lung development and function.

Sex difference in bronchopulmonary dysplasia of offspring in response to maternal PM2.5 exposure.

The adverse effects of fine particulate matters (PM2.5) on respiratory diseases start in utero. In order to investigate whether maternal PM2.5 exposure could lead to bronchopulmonary dysplasia (BPD) in offspring, PM2.5 was collected in Taiyuan, Shanxi, China during the annual heating period. Mice were mated and gestation day 0 (GD0) was considered the day on which a vaginal plug was observed. The plug-positive mice received 3 mg/kg b.w. PM2.5 by oropharyngeal aspiration every other day starting on GD0 and throughout the gestation period. Offspring were sacrificed at postnatal days (PNDs) 1, 7, 14 and 21. We assessed some typical BPD-like symptoms in offspring. The results showed that maternal PM2.5 exposure caused low birth weight, hypoalveolarization, decreased angiogenesis, suppressed production of secretory and surfactant proteins, and increased inflammation in the lungs of male offspring. However, maternal PM2.5 exposure induced only hypoalveolarization and inflammation in the lungs of female offspring. Furthermore, these alterations were reversed during postnatal development. Our results demonstrated that maternal exposure to PM2.5 caused reversible BPD-related consequences in offspring, and male offspring were more sensitive than females. However, these alterations were reversed during postnatal development.

Neonatal ibuprofen exposure and bronchopulmonary dysplasia in extremely premature infants.

OBJECTIVE: To evaluate the association of ibuprofen exposure with the risk of bronchopulmonary dysplasia (BPD) in extremely premature infants. STUDY DESIGN: This was a retrospective study of all extremely premature infants admitted to a tertiary unit from 2016 to 2018. RESULTS: A total of 203 extremely premature infants were included in this study. The rate of BPD was significantly higher in infants with early exposure to ibuprofen (42.5%) compared to infants with no exposure (21.6%, P = 0.001). After adjusting for covariates, the risk of BPD was associated independently with ibuprofen exposure (odds ratios (OR) 2.296, 95% confidence interval (CI): 1.166-4.522, p = 0.016). Further analysis showed a trend towards higher risk of BPD in infants with successful patent ductus arteriosus (PDA) closure after ibuprofen treatment (32.3%) compared to non-treated infants (20.2%, p = 0.162). CONCLUSION: Our findings suggest that ibuprofen exposure may contribute to the occurrence of BPD in extremely preterm infants.

Aerosol therapy in relation to retinopathy of prematurity in mechanically ventilated preterm infants.

BACKGROUND: Aerosol administration is increasingly being used as a therapeutic intervention for mechanically ventilated preterm infants. However, the effects of inhalation therapy on retinopathy of prematurity (ROP) have not yet been explored. METHODS: A retrospective cohort study was conducted in a tertiary level neonatal intensive care unit (NICU) from 2011 to 2013. All preterm infants with a gestational age (GA) of 24~29 weeks receiving invasive intubation for more than 1 week in the NICU were included. Infants with severe congenital anomalies were excluded. ROP was defined as stage II or greater according to medical records by ophthalmologists. A multivariate logistic regression model was used to estimate the risk of ROP in relation to inhalation therapy after adjusting for confounders. RESULTS: In total, 205 infants were enrolled in this study, including 154 with inhalation therapy and 51 without inhalation therapy. Univariate analyses showed an association of inhalation with the following characteristics: sex (p = 0.047), GA (p = 0.029), sepsis (p = 0.047), bronchopulmonary dysplasia (BPD) (p < 0.001), and ROP (p = 0.001). Furthermore, logistic regression analysis indicated that inhalation therapy was an independent risk factor for ROP (odds ratio (OR) = 2.639; 95% confidence interval (CI) = 1.050~6.615). In addition, infants with a GA of 24~25 weeks (OR = 6.063; 95% CI = 2.482~14.81) and 26~27 weeks (OR = 3.825; 95% CI = 1.694~8.638) were at higher risk of ROP than those with a GA of 28~29 weeks. Other factors - including sex, sepsis, BPD, and delivery mode - did not carry significant risk. CONCLUSION: Aerosol therapy with pure oxygen delivery is associated with ROP. Clinicians should exercise great caution when conducting aerosol therapy with excess oxygen in mechanically ventilated preterm infants.

Effect of Intranasal Instillation of Lipopolysaccharide on Lung Development and Its Related Mechanism in Newborn Mice.

Premature infants are prone to repeated lung infections after birth, which can disrupt the development of lung structure and function. However, the effects of postnatal pulmonary inflammation on lung development in newborn mice have not been reported and may play an important role in the development of bronchopulmonary dysplasia (BPD). This study aimed to establish a BPD model of postnatal pulmonary inflammation in premature infants and to explore its role and possible mechanisms in the pathogenesis of BPD. We exposed postnatal day 1 mice to lipopolysaccharide (LPS) and normal saline for 14 days. Pulmonary inflammation and alveolar microvascular development were assessed by histology. In addition, we also examined the expression of vascular endothelial growth factor (VEGF), VEGFR2, nuclear factor-kappa-B (NF-κB) and related inflammatory mediators [interleukin-1ß (IL-1ß), tumor necrosis factor-alpha (TNF-α), macrophage inflammatory protein-1α (MIP-1α), monocyte chemoattractant protein-1 (MCP-1)] in the lungs. Lung histology revealed inflammatory cell infiltration, alveolar simplification, and decreased microvascular density in LPS-exposed lungs. VEGF and VEGFR2 expression was decreased in the lungs of LPS-exposed neonatal mice. Furthermore, we detected elevated levels of the inflammatory mediators IL-1ß, TNF-α, MIP-1α, and MCP-1 in the lungs, which are associated with the activation of NF-κB. Intranasal instillation of LPS inhibits lung development in newborn mice, and postnatal pulmonary inflammation may participate in the pathogenesis of BPD. The mechanism is related to the inhibition of VEGF and VEGFR2 and the upregulation of inflammatory mediators through activation of NF-κB.

Broad spectrum antibiotics in newborns increase multi-drug resistant infections.

Our objective was to determine if broad spectrum antibiotics (BSA) are associated with multi-resistant bacterial (MRB) infections in neonatal patients. We conducted a case-control study with two groups of patients: those with and without a MRB infection. We included 43 cases and 43 controls. MRB strains were: 21 S. maltophila (49%), 11 ESBL-producing Enterobacteriae (25%), 8 P. aeruginosa (19%) and 3 MRSA (7%). Odds ratio (OR) for MRB after seven days of carbapenems was 4.25 (95% confidence interval (CI) 1.4-17.4) and OR for MRB after seven days of third generation cephalosporin was 8 (95% CI 1.1-34.9). BSA longer than seven days, increases MRB infections 22.5 times in patients with bronchopulmonary dysplasia (BPD). Our data show a clear association between the use of BSA and the development of MRB infections, especially in BPD. Although we cannot state this is a causal relationship, we can recommend avoiding prolonged treatment with these antibiotics in preterm babies at risk of BPD.

Pulmonary endothelial cells exhibit sexual dimorphism in their response to hyperoxia.

Abnormal pulmonary vascular development is a critical factor in the pathogenesis of bronchopulmonary dysplasia (BPD). Despite the well-established sex-specific differences in the incidence of BPD, the molecular mechanism(s) behind these are not completely understood. Exposure to a high concentration of oxygen (hyperoxia) contributes to BPD and creates a profibrotic environment in the lung. Our objective was to elucidate the sex-specific differences in neonatal human pulmonary microvascular endothelial cells (HPMECs) in normoxic and hyperoxic conditions, including the propensity for endothelial-to-mesenchymal transition. HPMECs (18- to 24-wk gestation donors, 6 male donors and 5 female donors) were subjected to hyperoxia (95% O2 and 5% CO2) or normoxia (air and 5% CO2) up to 72 h. We assessed cell migration and angiogenesis at baseline. Cell proliferation, viability, and expression of endothelial (CD31) and fibroblast markers (α-smooth muscle actin) were measured upon exposure to hyperoxia. Female HPMECs had significantly higher cell migration when assessed by the wound healing assay (40.99 ± 4.4%) compared with male HPMECs (14.76 ± 3.7%) and showed greater sprouting (1710 ± 962 µm in female cells vs. 789 ± 324 in male cells) compared with male endothelial cells in normoxia. Hyperoxia exposure decreased cell viability (by 9.8% at 48 h and 11.7% at 72 h) and proliferation (by 26.7% at 72 h) markedly in male HPMECs, whereas viability was sustained in female endothelial cells. There was greater expression of α-smooth muscle actin (2.5-fold) and decreased expression (5-fold) of CD31 in male HPMECs upon exposure to hyperoxia. The results indicate that cellular sex affects response in HPMECs in normoxia and hyperoxia. NEW & NOTEWORTHY Cellular sex affects response in human neonatal pulmonary microvascular endothelial cells in normoxia and hyperoxia. Under normoxic conditions, female human neonatal pulmonary microvascular endothelial cells display greater migration and angiogenic sprouting compared with male endothelial cells. Compared with female endothelial cells, hyperoxia exposure decreased cell viability and proliferation and increased α-smooth muscle actin and decreased CD31 expression in male endothelial cells, indicating an increased endothelial-mesenchymal transition.

Peptidome analysis of lung tissues from a hyperoxia-induced bronchopulmonary dysplasia mouse model: Insights into the pathophysiological process of bronchopulmonary dysplasia.

The aim of this study was to identify and compare the peptidomic profiles of lung tissues from neonatal mice with and without bronchopulmonary dysplasia (BPD). Hyperoxia was used to establish the BPD mouse model. Lung tissues obtained on postnatal day (PND) 9 were processed for analysis via histological staining and label-free liquid chromatography-mass spectrometry (LC-MS/MS). Histological analysis of the lung sections from the BPD group showed significant alveolar simplification and aberrant pulmonary vascularization. We identified 3,704 total peptides, of which 63 were differentially expressed in the lung tissues from the BPD group compared with those from the control group. Within this subset, 31 peptides were downregulated, and 32 peptides were upregulated. Bioinformatics analysis suggested several potential roles of the differentially expressed peptides in the pathophysiological process of BPD. In summary, this study highlights novel peptide candidates, and provides new insights for further understanding the molecular mechanism of BPD development.

Intrauterine growth restriction decreases NF-κB signaling in fetal pulmonary artery endothelial cells of fetal sheep.

Intrauterine growth restriction (IUGR) in premature newborns increases the risk for bronchopulmonary dysplasia, a chronic lung disease characterized by disrupted pulmonary angiogenesis and alveolarization. We previously showed that experimental IUGR impairs angiogenesis; however, mechanisms that impair pulmonary artery endothelial cell (PAEC) function are uncertain. The NF-κB pathway promotes vascular growth in the developing mouse lung, and we hypothesized that IUGR disrupts NF-κB-regulated proangiogenic targets in fetal PAEC. PAECs were isolated from the lungs of control fetal sheep and sheep with experimental IUGR from an established model of chronic placental insufficiency. Microarray analysis identified suppression of NF-κB signaling and significant alterations in extracellular matrix (ECM) pathways in IUGR PAEC, including decreases in collagen 4α1 and laminin α4, components of the basement membrane and putative NF-κB targets. In comparison with controls, immunostaining of active NF-κB complexes, NF-κB-DNA binding, baseline expression of NF-κB subunits p65 and p50, and LPS-mediated inducible activation of NF-κB signaling were decreased in IUGR PAEC. Although pharmacological NF-κB inhibition did not affect angiogenic function in IUGR PAEC, angiogenic function of control PAEC was reduced to a similar degree as that observed in IUGR PAEC. These data identify reductions in endothelial NF-κB signaling as central to the disrupted angiogenesis observed in IUGR, likely by impairing both intrinsic PAEC angiogenic function and NF-κB-mediated regulation of ECM components necessary for vascular development. These data further suggest that strategies that preserve endothelial NF-κB activation may be useful in lung diseases marked by disrupted angiogenesis such as IUGR.

Serotonin 2A receptor inhibition protects against the development of pulmonary hypertension and pulmonary vascular remodeling in neonatal mice.

Pulmonary hypertension (PH) complicating bronchopulmonary dysplasia (BPD) worsens clinical outcomes in former preterm infants. Increased serotonin (5-hydroxytryptamine, 5-HT) signaling plays a prominent role in PH pathogenesis and progression in adults. We hypothesized that increased 5-HT signaling contributes to the pathogenesis of neonatal PH, complicating BPD and neonatal lung injury. Thus, we investigated 5-HT signaling in neonatal mice exposed to bleomycin, previously demonstrated to induce PH and alveolar simplification. Newborn wild-type mice received intraperitoneal PBS, ketanserin (1 mg/kg), bleomycin (3 U/kg) or bleomycin (3 U/kg) plus ketanserin (1 mg/kg) three times weekly for 3 wk. Following treatment with bleomycin, pulmonary expression of the rate-limiting enzyme of 5-HT synthesis, tryptophan hydroxylase-1 (Tph1), was significantly increased. Bleomycin did not affect pulmonary 5-HT 2A receptor (R) expression, but did increase pulmonary gene expression of the 5-HT 2BR and serotonin transporter. Treatment with ketanserin attenuated bleomycin-induced PH (increased RVSP and RVH) and pulmonary vascular remodeling (decreased vessel density and increased muscularization of small vessels). In addition, we found that treatment with ketanserin activated pulmonary MAPK and Akt signaling in mice exposed to bleomycin. We conclude that 5-HT signaling is increased in a murine model of neonatal PH and pharmacological inhibition of the 5-HT 2AR protects against the development of PH in neonatal lung injury. We speculate this occurs through restoration of MAPK signaling and increased Akt signaling.

Exposure of neonatal mice to bromine impairs their alveolar development and lung function.

The halogen bromine (Br2) is used extensively in industry and stored and transported in large quantities. Its accidental or malicious release into the atmosphere has resulted in significant casualties. The pathophysiology of Br2-induced lung injury has been studied in adult animals, but the consequences of Br2 exposure to the developing lung are completely unknown. We exposed neonatal mouse littermates on postnatal day 3 (P3) to either Br2 at 400 ppm for 30 min (400/30), to Br2 at 600 ppm for 30 min (600/30), or to room air, then returned them to their dams and observed until P14. Mice exposed to Br2 had decreased survival (S) and had decreased weight (W) at P14 in the 400/30 group (S = 63.5%, W = 6.67 ± 0.08) and in the 600/30 group (S = 36.1%, W = 5.13 ± 0.67) as compared with air breathing mice (S = 100%, W = 7.96 ± 0.30). Alveolar development was impaired, as evidenced by increased mean linear intercept at P14. At P14, Br2 exposed mice also exhibited a decrease of arterial partial pressure of oxygen, decreased quasi-static lung compliance, as well as increased alpha smooth muscle actin mRNA and protein and increased mRNA for IL-1ß, IL-6, CXCL1, and TNFα. Global gene expression, evaluated by RNA sequencing and Ingenuity Pathway Analysis, revealed persistent abnormalities in gene expression profiles at P14 involving pathways of "formation of lung" and "pulmonary development." The data indicate that Br2 inhalation injury early in life results in severe lung developmental consequences, wherein persistent inflammation and global altered developmental gene expression are likely mechanistic contributors.