Model Of Bronchopulmonary Dysplasia Research Articles

Human β-Defensin-2 Improves Hyperoxia-Induced Lung Structural and Functional Injury in Neonatal Rats.

BackgroundBronchopulmonary dysplasia (BPD) is a major complication of extreme prematurity, characterized by alveolar simplification and pulmonary malfunction. Hyperoxia-induced lung injury in neonatal rats has been used as a model of BPD, as indicated by lung architectural change and alveolar simplification that resembles clinical feature of BPD. β-defensin-2 (BD2) plays an important role in lung diseases by inhibiting inflammation response. However, little is known about its role in BPD. The aim of this study was to determine the effect of human BD2 (hBD2) gene on hyperoxia-induced animal model of BPD.Material/MethodsThe neonatal rats were exposed to 90% oxygen (O2) continuously for 14 days to mimic the BPD-like lung injury. These rats were then randomly assigned to the following four groups: in room air (air), in 90% O2, in 90% O2 with null adenovirus vector infection (O2+Ad), and in 90% O2 with gene therapy through adenovirus transfected hBD2 (O2+Ad-hBD2). Morphology of lungs, pulmonary function and expression of inflammatory cytokines on P7, P10, P14, and P21 were documented and compared across the 4 groups.ResultsThe overexpression of hBD2 mediated by the adenovirus vector was successfully constructed. hBD2 gene therapy increased hBD2 mRNA expression, increased radial alveolar count (RAC), lung volume and compliance, decreased mean linear intercept (MLI), tissue damping, and elastance. Furthermore, pro-inflammatory cytokines IL-1β, IL-6, and TNF-α were inhibited and anti-inflammatory cytokines IL-10 was increased in the lungs of rats in O2+Ad-hBD2 group.ConclusionsIn hyperoxia-induced rat models of BPD, hBD2 promotes alveolarization and improves pulmonary function. The mechanism may contribute in alleviating inflammation response and inhibiting pro-inflammatory factors including IL-1β, IL-6, and TNF-α.

Bronchopulmonary dysplasia: risk prediction models for very-low- birth-weight infants.

Our objective is to develop risk prediction models for moderate/severe bronchopulmonary dysplasia (BPD) and BPD and/or death in very-low-birth-weight infants (VLBWI) at birth, 3, 7, and 14 postnatal days. It is a multicenter study including 16,407 infants weighing 500-1500 g (2001-2015) from the Neocosur Network. BPD was defined as oxygen dependency at 36 weeks. Variables were selected using forward logistic regression models. Predictive values were evaluated using the ROC curve. In total, 2580 (15.7%) presented BPD and 6121 (37.3%) BPD/death. The AUC values for the BPD models were 0.788, 0.818, 0.827, and 0.894 respectively. For BPD/death, the AUC values were 0.860, 0.869, 0.867, and 0.906. BW and gestational age had higher contribution at birth; at later ages, the length of oxygen therapy and ventilation had the highest contribution. All AUC values were statistically significant when compared with a neutral value of 0.5 (p-value < 0.001). We developed high predictive power models for moderate/severe BPD and BPD/death at four postnatal ages.

Mesenchymal Stem/Stromal Cell Therapy for Bronchopulmonary Dysplasia in the Neonatal Intensive Care Unit

Clinical trials of mesenchymal stem/stromal cell (MSC) therapy for bronchopulmonary dysplasia (BPD) are already underway. A thorough understanding of the preclinical work that underpins these trials is critical for neonatal practitioners to properly evaluate them. The more significant advances have been in investigating the mechanisms of action by which MSCs are thought to ameliorate BPD, and the recognition that this therapeutic effect is largely contained within the non-cellular exosome fraction of MSCs. In rodent hyperoxia models of BPD, MSCs have a pro-angiogenic effect mediated largely by vascular endothelial growth factor (VEGF) and shift the balance of endogenous lung cells from a pro-inflammatory to a pro-healing phenotype. MSC-derived exosomes can recapitulate these effects.

Lipoxin A4 reduces hyperoxia-induced lung injury in neonatal rats through PINK1 signaling pathway

Bronchopulmonary dysplasia (BPD) is a common chronic lung disease in premature infants and is mainly caused by hyperoxia exposure and mechanical ventilation. Alveolar simplification, pulmonary vascular abnormalities and pulmonary inflammation are the main pathological changes in hyperoxic lung injury animals. Lipoxin A4 (LXA4) is an important endogenous lipid that can mediate the regression of inflammation and plays a role in acute lung injury and asthma. The purpose of this study was to evaluate the effects of LXA4 on inflammation and lung function in neonatal rats with hyperoxic lung injury and to explore the mechanism of the PINK1 pathway. After 85% oxygen exposure in newborn rats for 7 days, the BPD model was established. We found that LXA4 could significantly reduce cell and protein infiltration and oxidative stress in rat lungs, improve pulmonary function and alveolar simplification, and promote weight gain. LXA4 inhibited the expression of TNF-α, MCP-1 and IL-1β in serum and BALF from hyperoxic rats. Moreover, we found that LXA4 could reduce the expression of the PINK1 gene and down-regulate the expression of PINK1, Parkin, BNIP3L/Nix and the autophagic protein LC3B.These protective effects of LXA4 could be partially reversed by addition of BOC-2.Thus, we concluded that LXA4 can alleviate the airway inflammatory response, reduce the severity of lung injury and improve lung function in a hyperoxic rat model of BPD partly through the PINK1 signaling pathway.

MicroRNA-421 inhibition alleviates bronchopulmonary dysplasia in a mouse model via targeting Fgf10.

Bronchopulmonary dysplasia (BPD) is a common and refractory disease affecting newborn children and infants with alveolar dysplasia and declined pulmonary function. Several microRNAs (miRNAs) have been found to be differentially expressed in BPD progression. This study further explores the role of miR-421 via fibroblast growth factor 10 (Fgf10) in mice with BPD. A mouse model of BPD was established through the induction of hyperoxia, in which the expression pattern of miR-421 and Fgf10 was identified. Furthermore, adenovirus-packed vectors were injected in mice to intervene miR-421 and Fgf10 expression, including miR-421 mimics or inhibitors, and si-Fgf10 to explore the role of miR-421 and Fgf10 in BPD. The target relationship between miR-421 and Fgf10 was investigated. Inflammatory response and cell apoptosis were observed in the mice, with inflammatory cytokines and apoptosis-related factors detected by applying Reverse transcription quantitative polymerase chain reaction, Western blot analysis, and enzyme-linked immunosorbent assay. Fgf10 was confirmed as a target gene of miR-421. Elevated expression of miR-421 was evident, while Fgf10 was poorly expressed in BPD. upregulation of miR-421 and silence of Fgf10 aggravated inflammatory response in lung tissue and promoted lung cell apoptosis in BPD. The aforementioned alterations could be reversed by downregulation of miR-421. Collectively, inhibition of miR-421 can assist in the development of BPD in mice BPD by upregulating Fgf10. Therefore, the present study provides a probable target for the treatment of BPD.

Human amnion cell-derived extracellular vesicles in the treatment of bronchopulmonary dysplasia and associated pulmonary hypertension

Background & Aim We previously showed that human amnion epithelial cells (hAECs) are a viable source of cell therapy for established bronchopulmonary dysplasia (BPD). This work has culminated in a first-in-human clinical trial in babies with established BPD. In this current study, we sought to assess the therapeutic potential of hAEC-derived EVs (hAEC-EVs) in an experimental model of BPD. Methods, Results & Conclusion Methods Lipopolysaccharide (LPS) was introduced to C57bl6 mouse fetuses intra-amniotically at E16 prior to exposure to 65% oxygen (hyperoxia) at birth. hAEC-EVs were injected intravenously on postnatal day (PND) 4. One cohort of mice were culled on either postnatal day PND7 or PND14, while another cohort were kept in hyperoxia until PND 28, following which they were recovered under normoxia conditions for a further 2 weeks or 6 weeks for cardiovascular assessment. Results The isolated hAEC-EVs had distinct cup shaped morphology with average size of 80-140nm. ALIX, Grp94 and HLA-G were expressed in EVs, and Pathway enrichment analysis showed that endothelin signaling pathway, Wnt signaling pathway, and inflammation mediated by chemokine and cytokine signaling pathway were enriched in hAEC-EVs. In the mouse model of experimental BPD, hAEC-EV administration improved tissue-to-airspace ratio and septal crest density in a dose-dependent manner. hAEC-EVs reduced the levels of inflammatory cytokines such as interleukin (IL)-1β and tumour necrosis factor-alpha (TNF-α) on PND7. The improvement of lung injury was associated with the increase of the percentage of type II alveolar cells (AT2s) at both PND7 and 14. Surprisingly, neonatal hAEC-EV delivery reduced airway hyper-responsiveness, mitigated pulmonary hypertension and prevented right ventricle hypertrophy that associated with BPD-like lung injury at week 6. These improvements persisted till week 10. All these observations showed that HAEC-EVs mitigate the BPD lung injury through decreasing inflammation, activating local AT2 niche and reversing pulmonary artery remodeling. Conclusions hAEC-derived EVs had similar therapeutic effects to hAECs in this mouse model of experimental BPD. hAEC-derived EVs improved lung structure and reduced lung inflammation in a manner similar to that achieved using hAECs. EVs repaired lung injury partly through the activation of local stem/progenitor cells. Furthermore, hAEC-EV administration had long term benefit in improving lung function, preventing pulmonary hypertension and right ventricle hypertrophy.

EPO enhances the protective effects of MSCs in experimental hyperoxia-induced neonatal mice by promoting angiogenesis.

Bronchopulmonary dysplasia (BPD) is the most common type of chronic lung disease in infancy; however, there is no effective treatment for it. In the present study, a neonatal mouse BPD model was established by continuous exposure to high oxygen (HO) levels. Mice were divided randomly into 5 groups: control, BPD, EPO, MSCs, and MSCs+EPO. At 2 weeks post-treatment, vessel density and the expression levels of endothelial growth factor (VEGF), stromal cell-derived factor-1 (SDF-1), and its receptor C-X-C chemokine receptor type 4 (CXCR4) were significantly increased in the MSC+EPO group compared with the EPO or MSCs group alone; moreover, EPO significantly enhanced MSCs proliferation, migration, and anti-apoptosis ability in vitro. Furthermore, the MSCs could differentiate into cells that were positive for the type II alveolar epithelial cell (AECII)-specific marker surfactant protein-C, but not positive for the AECI-specific marker aquaporin 5. Our present results suggested that MSCs in combination with EPO could significantly attenuate lung injury in a neonatal mouse model of BPD. The mechanism may be by the indirect promotion of angiogenesis, which may involve the SDF-1/CXCR4 axis.

The expression and effect of long non-coding RNA H19 in BPD neonatal rats

Objective To investigate the expression of long non-coding RNA H19(LncRNA H19)and its regulation of histone methyltransferase 2(enhancer of zeste homolog 2, EZH2)in the lung tissue of neonatal rats with bronchopulmonary dysplasia(BPD), and to lay a foundation for elucidating the pathogenesis of BPD lung epithelium-interstitial transformation(EMT). Methods The BPD model of SD neonatal rats was induced by hyperoxia(inhalation oxygen concentration was 85%)(n=50), and oxygen inhalation concentration of the control group was 21%(n=50). The two groups were collected at 1d, 3d, 7d, 14d and 21d after birth in lung tissue.Immunohistochemistry, Western blot, real-time quantitative PCR and other techniques were used to detect the intracellular localization, and the expression level of EZH2 protein and the mRNA expression level of H19 and EZH2. Results Immunohistochemistry showed that EZH2 protein was located in the nucleus and cytoplasm of alveolar epithelial cells, and the expression of EZH2 protein in the model group was significantly enhanced compared with the control group.Similarly, the results of Western blot demonstrated that the expression of EZH2 protein in the model group increased from 1d(control group: 0.196±0.030, model group 0.650±0.149)to 21d(control group 0.934±0.215, model group 1.785±0.298)rather than the control group(P<0.05). Compared with the control group, the mRNA expression level of H19 in the model group increased from 7d(control group 2.591±0.211, model group 3.558±0.093, P<0.05)and the expression level of EZH2 mRNA started to increase from 3d(control group 1.246±0.015, model group 2.148±0.215, P<0.05). Moreover, the differences between the two groups were obvious with the time of hyperoxia exposure. Conclusion In the development of BPD, the expression levels of H19 and EZH2 protein in lung tissue is up-regulated, and the peak of H19 expression precedes EZH2, which suggest that H19 might be involved in the pathogenesis of pulmonary dysplasia induced by EZH2-mediated EMT. Key words: Long non-coding RNA H19; Enhancer of zeste homolog 2; Bronchopulmonary dysplasia; Newborn rat

Acute and chronic changes in the control of breathing in a hyperoxia‐induced model of bronchopulmonary dysplasia

Infants born very prematurely (&lt;28 weeks gestation) have immature lungs and often require supplemental oxygen. However, long‐term hyperoxia exposure can arrest lung development leading to bronchopulmonary dysplasia (BPD), which increases acute and long‐term respiratory morbidity and mortality. The neural mechanisms controlling breathing are highly plastic during development. Whether the ventilatory control system adapts to pulmonary disease associated with hyperoxia exposure in infancy remains unclear. Here, we tested the hypothesis that there would be age‐dependent adaptations in the control of breathing in an established rat model of hyperoxia‐induced BPD. Hyperoxia exposure (FIO2: 0.9 from 0–10 days of life) led to a BPD‐like lung phenotype, including sustained reductions in alveolar surface area and counts, and modest increases in airway resistance. Hyperoxia exposure also led to chronic increases in room air and acute hypoxic minute ventilation (VE) and age‐dependent changes in breath‐to‐breath tidal volume and breathing frequency variability. Hyperoxia‐exposed rats had normal hypoxic ventilatory responses, oxygen saturation (SpO2) in room air, but greater reductions in SpO2 during acute hypoxia (12% O2) indicating reduced hypoxic sensitivity and/or hypoventilation due to diseased lungs. However, VE was increased indicating an increase in respiratory drive. Perinatal hyperoxia led to greater glial fibrillary acidic protein expression and an increase in neuron counts within 6 of 8 and 1 of 8 key brainstem regions controlling breathing, respectively, suggesting astrocytic expansion. In conclusion, perinatal hyperoxia in rats induced a BPD‐like phenotype and age‐dependent adaptations in VE that may be mediated through changes to the neural architecture of the ventilatory control system. Our results suggest chronically altered ventilatory control in BPD.Support or Funding InformationChildren's Research Hospital of Wisconsin Research Institute, NIH R01 HL 122358, &amp; Parker B. Francis FoundationThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Effect of Montelukast on Bronchopulmonary Dysplasia (BPD) and Related Mechanisms.

BackgroundBronchopulmonary dysplasia (BPD) is a chronic lung disease common in preterm infants. Montelukast, an effective cysteinyl leukotriene (cysLT) receptor antagonist, has a variety of pharmacological effects and has protective effects against a variety of diseases. Currently, the efficacy and safety of montelukast sodium in treating BPD has been revealed, however, the precise molecular mechanism of the effect of montelukast on BPD development remain largely unclear. Therefore, this study aimed to investigate the effect and mechanism of montelukast on BPD in vivo and in vitro.Material/MethodsA mouse BPD model and hyperoxia-induced lung cell injury model were established and treated with montelukast. Then mean linear intercept (MLI), radial alveolar count (RAC), lung weight/body weight (LW/BW) ratio, pro-inflammatory factors, and oxidative stress-related factors in lung tissues were determined. Cell viability and apoptosis were detected using MTT assay and flow cytometer respectively.ResultsThe results showed that montelukast treatment relieved mouse BPD, evidenced by increased RAC and decreased MLI and LW/BW ratios. We also found that montelukast treatment reduced pro-inflammatory factors (TNF-α, IL-6, and IL-1β) production, enhanced superoxide dismutase (SOD) activity, and reduced malondialdehyde (MDA) content in the lung tissues of BPD mice. Besides, montelukast eliminated the reduced cell viability and enhanced cell apoptosis induced by hyperoxia exposure in vitro. Moreover, the upregulated pro-inflammatory factors production and p-p65 protein level in lung cells caused by hyperoxia were decreased by montelukast treatment.ConclusionsMontelukast protected against mouse BPD induced by hyperoxia through inhibiting inflammation, oxidative stress, and lung cell apoptosis.

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

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

Acute and chronic changes in the control of breathing in a rat model of bronchopulmonary dysplasia.

Infants born very prematurely (<28 wk gestation) have immature lungs and often require supplemental oxygen. However, long-term hyperoxia exposure can arrest lung development, leading to bronchopulmonary dysplasia (BPD), which increases acute and long-term respiratory morbidity and mortality. The neural mechanisms controlling breathing are highly plastic during development. Whether the ventilatory control system adapts to pulmonary disease associated with hyperoxia exposure in infancy remains unclear. Here, we assessed potential age-dependent adaptations in the control of breathing in an established rat model of BPD associated with hyperoxia. Hyperoxia exposure ( ; 0.9 from 0 to 10 days of life) led to a BPD-like lung phenotype, including sustained reductions in alveolar surface area and counts, and modest increases in airway resistance. Hyperoxia exposure also led to chronic increases in room air and acute hypoxic minute ventilation (V̇e) and age-dependent changes in breath-to-breath variability. Hyperoxia-exposed rats had normal oxygen saturation ( ) in room air but greater reductions in during acute hypoxia (12% O2) that were likely due to lung injury. Moreover, acute ventilatory sensitivity was reduced at P12 to P14. Perinatal hyperoxia led to greater glial fibrillary acidic protein expression and an increase in neuron counts within six of eight or one of eight key brainstem regions, respectively, controlling breathing, suggesting astrocytic expansion. In conclusion, perinatal hyperoxia in rats induced a BPD-like phenotype and age-dependent adaptations in V̇e that may be mediated through changes to the neural architecture of the ventilatory control system. Our results suggest chronically altered ventilatory control in BPD.

Pulmonary vascular disease is evident in gene regulation of experimental bronchopulmonary dysplasia

Objective: To examine the gene expression regarding pulmonary vascular disease in experimental bronchopulmonary dysplasia in young mice. Premature delivery puts babies at risk of severe complications. Bronchopulmonary dysplasia (BPD) is a common complication of premature birth leading to lifelong affection of pulmonary function. BPD is recognized as a disease of arrested alveolar development. The disease process is not fully described and no complete cure or prevention is known. The focus of interest in the search for treatment and prevention of BPD has traditionally been at airspace level; however, the pulmonary vasculature is increasingly acknowledged in the pathology of BPD. The aim of the investigation was to study the gene expression in lungs with BPD with regards to pulmonary vascular disease (PVD).Methods: We employed a murine model of hyperoxia-induced BPD and gene expression microarray technique to determine the mRNA expression in lung tissue from young mice. We combined gene expression pathway analysis and analyzed the biological function of multiple single gene transcripts from lung homogenate to study the PVD relevant gene expression.Results: There were n = 117 significantly differentially regulated genes related to PVD through down-regulation of contractile elements, up- and down-regulation of factors involved in vascular tone and tissue-specific genes. Several genes also allowed for pinpointing gene expression differences to the pulmonary vasculature. The gene Nppa coding for a natriuretic peptide, a potent vasodilator, was significantly down-regulated and there was a significant up-regulation of Pde1a (phosphodiesterase 1A), Ptger3 (prostaglandin e receptor 3), and Ptgs1 (prostaglandin-endoperoxide synthase one).Conclusion: The pulmonary vasculature is affected by the arrest of secondary alveolarization as seen by differentially regulated genes involved in vascular tone and pulmonary vasculature suggesting BPD is not purely an airspace disease. Clues to prevention and treatment may lie in the pulmonary vascular system.

272 - Impairment of thioredoxin 1 disrupts perinatal alveolar development

Bronchopulmonary Dysplasia (BPD) is a chronic lung disease that affects pre-term infants whose lungs are unable to support life outside of the womb. These babies receive therapeutic oxygen which compensates for poor lung function but also causes oxidative damage to lung epithelium, resulting in a simplified lung marked by larger and fewer alveoli. We identified thioredoxin-1 (Trx1) as a protein of interest in the pathogenesis of BPD since Trx1 is cytoprotective against hyperoxic cell death. Using a perinatal hyperoxic mouse model of BPD, pulmonary Trx1 activity inversely correlates with atmospheric oxygen tension following birth and hyperoxic treatment. Therefore, we tested if impairment of lung epithelial Trx1 promotes BPD pathogenesis. Trx1 floxed mice (Trx1fl) were bred to mice expressing cre recombinase driven by the surfactant protein C promoter (sftpc-cre) to generate a mouse with Trx1 deficient lung epithelial cells. Although expected genotypic ratios were detected at birth, lethality was noted in Trx1fl/fl;sftpc-cre mice as early as 14 days of age. The LD50 for Trx1 deficient mice was 20 days old, while Trx1 heterozygote littermates appeared normal (p

Intratracheal administration of clinical-grade mesenchymal stem cell-derived extracellular vesicles reduces lung injury in a rat model of bronchopulmonary dysplasia.

Mesenchymal stem cells (MSCs) prevent the onset of bronchopulmonary dysplasia (BPD) in animal models, an effect that seems to be mediated by their secreted extracellular vesicles (EVs). The aim of this study was to compare the protective effects of intratracheally (IT) administered MSCs versus MSC-EVs in a hyperoxia-induced rat model of BPD. At birth, rats were distributed as follows: animals raised in ambient air for 2 wk ( n = 10), and animals exposed to 60% oxygen for 2 wk and treated with IT-administered physiological solution ( n = 10), MSCs ( n = 10), or MSC-EVs ( n = 10) on postnatal days 3, 7, and 10. The sham-treated hyperoxia-exposed animals showed reductions in total surface area of alveolar air spaces, and total number of alveoli ( Nalv), and an increased mean alveolar volume (Valv). EVs prompted a significant increase in Nalv ( P < 0.01) and a significant decrease in Valv ( P < 0.05) compared with sham-treated animals, whereas MSCs only significantly improved Nalv ( P < 0.05). Small pulmonary vessels of the sham-treated hyperoxia-exposed rats also showed an increase in medial thickness, which only EVs succeeded in preventing significantly ( P < 0.05). In conclusion, both EVs and MSCs reduce hyperoxia-induced damage, with EVs obtaining better results in terms of alveolarization and lung vascularization parameters. This suggests that IT-administered EVs could be an effective approach to BPD treatment.

Lung omics signatures in a bronchopulmonary dysplasia and pulmonary hypertension-like murine model.

Bronchopulmonary dysplasia (BPD), the most common chronic lung disease in infants, is associated with long-term morbidities, including pulmonary hypertension (PH). Importantly, hyperoxia causes BPD and PH; however, the underlying mechanisms remain unclear. Herein, we performed high-throughput transcriptomic and proteomic studies using a clinically relevant murine model of BPD with PH. Neonatal wild-type C57BL6J mice were exposed to 21% oxygen (normoxia) or 70% oxygen (hyperoxia) during postnatal days (PNDs) 1-7. Lung tissues were collected for proteomic and genomic analyses on PND 7, and selected genes and proteins were validated by real-time quantitative PCR and immunoblotting analysis, respectively. Hyperoxia exposure dysregulated the expression of 344 genes and 21 proteins. Interestingly, hyperoxia downregulated genes involved in neuronal development and maturation in lung tissues. Gene set enrichment and gene ontology analyses identified apoptosis, oxidoreductase activity, plasma membrane integrity, organ development, angiogenesis, cell proliferation, and mitophagy as the predominant processes affected by hyperoxia. Furthermore, selected deregulated proteins strongly correlated with the expression of specific genes. Collectively, our results identified several potential therapeutic targets for hyperoxia-mediated BPD and PH in infants.

The thioredoxin reductase inhibitor auranofin induces heme oxygenase-1 in lung epithelial cells via Nrf2-dependent mechanisms.

Thioredoxin reductase-1 (TXNRD1) inhibition effectively activates nuclear factor (erythroid-derived 2)-like 2 (Nrf2) responses and attenuates lung injury in acute respiratory distress syndrome (ARDS) and bronchopulmonary dysplasia (BPD) models. Upon TXNRD1 inhibition, heme oxygenase-1 (HO-1) is disproportionally increased compared with Nrf2 target NADPH quinone oxidoreductase-1 (Nqo1). HO-1 has been investigated as a potential therapeutic target in both ARDS and BPD. TXNRD1 is predominantly expressed in airway epithelial cells; however, the mechanism of HO-1 induction by TXNRD1 inhibitors is unknown. We tested the hypothesis that TXNRD1 inhibition induces HO-1 via Nrf2-dependent mechanisms. Wild-type (WT), Nrf2KO1.3, and Nrf2KO2.2 cells were morphologically indistinguishable, indicating that Nrf2 can be deleted from murine-transformed club cells (mtCCs) using CRISPR/Cas9 gene editing. Hemin, a Nrf2-independent HO-1-inducing agent, significantly increased HO-1 expression in WT, Nrf2KO1.3, and Nrf2KO2.2. Auranofin (AFN) (0.5 µM) inhibited TXNRD1 activity by 50% and increased Nqo1 and Hmox1 mRNA levels by 6- and 24-fold, respectively, in WT cells. Despite similar levels of TXNRD1 inhibition, Nqo1 mRNA levels were not different between control and AFN-treated Nrf2KO1.3 and Nrf2KO2.2. AFN slightly increased Hmox1 mRNA levels in Nrf2KO1.3 and Nrf2KO2.2 cells compared with controls. AFN failed to increase HO-1 protein in Nrf2KO1.3 and Nrf2KO2.2 compared with a 36-fold increase in WT mtCCs. Our data indicate that Nrf2 is the primary mechanism by which TXNRD1 inhibitors increase HO-1 in lung epithelia. Future studies will use ARDS and BPD models to define the role of HO-1 in attenuation of lung injury by TXNRD1 inhibitors.

Iloprost attenuates hyperoxia-mediated impairment of lung development in newborn mice.

Cyclooxygenase-2 (COX-2/PTGS2) mediates hyperoxia-induced impairment of lung development in newborn animals and is increased in the lungs of human infants with bronchopulmonary dysplasia (BPD). COX-2 catalyzes the production of cytoprotective prostaglandins, such as prostacyclin (PGI2), as well as proinflammatory mediators, such as thromboxane A2. Our objective was to determine whether iloprost, a synthetic analog of PGI2, would attenuate hyperoxia effects in the newborn mouse lung. To test this hypothesis, newborn C57BL/6 mice along with their dams were exposed to normoxia (21% O2) or hyperoxia (85% O2) from 4 to 14 days of age in combination with daily intraperitoneal injections of either iloprost 200 µg·kg-1·day-1, nimesulide (selective COX-2 antagonist) 100 mg·kg-1·day-1, or vehicle. Alveolar development was estimated by radial alveolar counts and mean linear intercepts. Lung function was determined on a flexiVent, and multiple cytokines and myeloperoxidase (MPO) were quantitated in lung homogenates. Lung vascular and microvascular morphometry was performed, and right ventricle/left ventricle ratios were determined. We determined that iloprost (but not nimesulide) administration attenuated hyperoxia-induced inhibition of alveolar development and microvascular density in newborn mice. Iloprost and nimesulide both attenuated hyperoxia-induced, increased lung resistance but did not improve lung compliance that was reduced by hyperoxia. Iloprost and nimesulide reduced hyperoxia-induced increases in MPO and some cytokines (IL-1β and TNF-α) but not others (IL-6 and KC/Gro). There were no changes in pulmonary arterial wall thickness or right ventricle/left ventricle ratios. We conclude that iloprost improves lung development and reduces lung inflammation in a newborn mouse model of BPD.

Early gestational mesenchymal stem cell secretome attenuates experimental bronchopulmonary dysplasia in part via exosome-associated factor TSG-6

BackgroundMesenchymal stem cells (MSCs) are promising tools for the treatment of human lung disease and other pathologies relevant to newborn medicine. Recent studies have established MSC exosomes (EXO), as one of the main therapeutic vectors of MSCs in mouse models of multifactorial chronic lung disease of preterm infants, bronchopulmonary dysplasia (BPD). However, the mechanisms underlying MSC-EXO therapeutic action are not completely understood. Using a neonatal mouse model of human BPD, we evaluated the therapeutic efficiency of early gestational age (GA) human umbilical cord (hUC)-derived MSC EXO fraction and its exosomal factor, tumor necrosis factor alpha-stimulated gene-6 (TSG-6).MethodsConditioned media (CM) and EXO fractions were isolated from 25 and 30 weeks GA hUC-MSC cultures grown in serum-free media (SFM) for 24 h. Newborn mice were exposed to hyperoxia (> 95% oxygen) and were given intraperitoneal injections of MSC-CM or MSC-CM EXO fractions at postnatal (PN) day 2 and PN4. They were then returned to room air until PN14 (in a mouse model of severe BPD). The treatment regime was followed with (rh)TSG-6, TSG-6-neutralizing antibody (NAb), TSG-6 (si)RNA-transfected MSC-CM EXO and their appropriate controls. Echocardiography was done at PN14 followed by harvesting of lung, heart and brain for assessment of pathology parameters.ResultsSystemic administration of CM or EXO in the neonatal BPD mouse model resulted in robust improvement in lung, cardiac and brain pathology. Hyperoxia-exposed BPD mice exhibited pulmonary inflammation accompanied by alveolar-capillary leakage, increased chord length, and alveolar simplification, which was ameliorated by MSC CM/EXO treatment. Pulmonary hypertension and right ventricular hypertrophy was also corrected. Cell death in brain was decreased and the hypomyelination reversed. Importantly, we detected TSG-6, an immunomodulatory glycoprotein, in EXO. Administration of TSG-6 attenuated BPD and its associated pathologies, in lung, heart and brain. Knockdown of TSG-6 by NAb or by siRNA in EXO abrogated the therapeutic effects of EXO, suggesting TSG-6 as an important therapeutic molecule.ConclusionsPreterm hUC-derived MSC-CM EXO alleviates hyperoxia-induced BPD and its associated pathologies, in part, via exosomal factor TSG-6. The work indicates early systemic intervention with TSG-6 as a robust option for cell-free therapy, particularly for treating BPD.

Autophagy inducers restore impaired autophagy, reduce apoptosis, and attenuate blunted alveolarization in hyperoxia-exposed newborn rats.

Autophagy is a common process during development. Abnormal autophagy can impact cell apoptosis. Previous studies have shown that apoptosis is present during bronchopulmonary dysplasia (BPD). However, there is no consensus on the level of coexisting autophagy. This study was designed to investigate the role of autophagy and the effects of autophagy inducers in a BPD model. A total of 100 newborn Sprague-Dawley rats were randomly assigned to model and control groups. BPD models were established by hyperoxic induction(FiO2 0.80). Some of them were treated with autophagy-inducing agents. As compared to the control group, more autophagic bodies were found within Type II alveolar epithelial cells (AT-II cells) under transmission electron microscopy (TEM) in the model group at 3 d . These autophagic bodies were also accompanied by apoptotic bodies and expression of both bodies peaked at 7 d. As shown by TdT-mediated dUTP nick end labeling (TUNEL), there were more apoptotic cells in the model group than in the control group. Protein expression levels of LC3B-II, p62, Lamp1, and cleaved Caspase-3 increased with increased hyperoxic exposure time. No significant differences were observed in the mRNA expression levels of LC3B, p62, and Lamp1. After introducing an autophagy inducer, either rapamycin or lithium chloride, the radial alveolar count (RAC) value of BPD model group increased as compared with placebo group, the thickness of alveolar septum decreased, while apoptosis decreased. Reduced autophagy resulting from blocked autophagy flow may be a key link in the pathogenesis of BPD. By enhancing repressed autophagy, apoptosis could be reduced and alveolar development improved.