Bronchopulmonary Dysplasia Mice Research Articles

Endothelial progenitor cells derived from embryonic stem cells prevent alveolar simplification in a murine model of bronchopulmonary dysplasia.

Introduction: Vascular remodeling and compromised alveolar development are hallmarks of chronic pulmonary diseases such as bronchopulmonary dysplasia (BPD). Despite advances in neonatal healthcare the number of BPD cases worldwide continues to increase. One approach to overcoming the premature arrest in lung development seen in BPD is to stimulate neonatal angiogenesis via delivery and engraftment of endothelial progenitor cells (EPCs). One such population is resident to the pulmonary microvasculature and expresses both FOXF1 and c-KIT. Previous studies have shown that c-KIT+FOXF1+ EPCs are highly sensitive to elevated levels of oxygen (hyperoxia) and are decreased in premature infants with BPD and hyperoxia-induced BPD mouse models. We hypothesize that restoring EPCs through transplantation of c-KIT+FOXF1+ EPCs derived in vitro from pluripotent embryonic stem cells (ESCs), will stimulate neonatal angiogenesis and alveolarization in mice with hyperoxia-induced lung injury. Methods: Utilizing a novel ESC line with a FOXF1:GFP reporter, we generated ESC-derived c-KIT+FOXF1+ EPCs in vitro. Using a second ESC line which contains FOXF1:GFP and tdTomato transgenes, we differentiated ESCs towards c-KIT+FOXF1+ EPCs and tracked them in vivo after injection into the neonatal circulation of hyperoxia-injured mice. After a recovery period in room air conditions, we analyzed c-KIT+FOXF1+ EPC engraftment and quantified the number of resident and circulating endothelial cells, the size of alveolar spaces, and the capillary density after EPC transplantations. Results and conclusion: Herein, we demonstrate that addition of BMP9 to the directed endothelial differentiation protocol results in very efficient generation of c-KIT+FOXF1+ EPCs from pluripotent ESCs. ESC-derived c-KIT+FOXF1+ EPCs effectively engraft into the pulmonary microvasculature of hyperoxia-injured mice, promote vascular remodeling in alveoli, increase the number of resident and circulating endothelial cells, and improve alveolarization. Altogether, these results provide a proof-of-principle that cell therapy with ESC-derived c-KIT+FOXF1+ EPCs can prevent alveolar simplification in a hyperoxia-induced BPD mouse model.

Nesfatin-1 alleviates hyperoxia-induced lung injury in newborn mice by inhibiting oxidative stress through regulating SIRT1/PGC-1α pathway

Bronchopulmonary dysplasia (BPD) is a pulmonary disease commonly observed in premature infants and it is reported that oxidative stress is a critical induction factor in BPD and is considered as a promising target for treating BPD. Nesfatin-1 is a brain-gut peptide with inhibitory effects on food intake, which is recently evidenced to show suppressive effect on oxidative stress. The present study aims to explore the therapeutic effect and mechanism of Nesfatin-1 in BPD mice. AECIIs were extracted from newborn rats and exposed to hyperoxia for 24 h, followed by treatment with 5 and 10 nM Nesfatin-1. Declined cell viability, increased apoptotic rate, upregulated Bax, downregulated Bcl-2, increased release of ROS and MDA, and suppressed SOD activity were observed in hyperoxia-treated AECIIs, which were extremely reversed by Nesfatin-1. Newborn rats were exposed to hyperoxia, followed by treated with 10 μg/kg Nesfatin-1 and 20 μg/kg Nesfatin-1. Severe pathological changes, elevated MDA level, and declined SOD activity were observed in lung tissues of BPD mice, which were rescued by Nesfatin-1. Furthermore, the protective effect of Nesfatin-1 on hyperoxia-challenged AECIIs was abolished by silencing SIRT1. Collectively, Nesfatin-1 alleviated hyperoxia-induced lung injury in newborn mice by inhibiting oxidative stress through regulating SIRT1/PGC-1α pathway.

ILC2 regulates hyperoxia-induced lung injury via an enhanced Th17 cell response in the BPD mouse model

BackgroudRecent research has focused on the role of immune cells and immune responses in the pathogenesis of bronchopulmonary dysplasia (BPD), but the exact mechanisms have not yet been elucidated. Previously, the key roles of type 2 innate lymphoid cells (ILC2) in the lung immune network of BPD were explored. Here, we investigated the role Th17 cell response in hyperoxia-induced lung injury of BPD, as well as the relationship between ILC2 and Th17 cell response.MethodsA hyperoxia-induced BPD mouse model was constructed and the pathologic changes of lung tissues were evaluated by Hematoxylin-Eosin staining. Flow cytometry analysis was conducted to determine the levels of Th17 cell, ILC2 and IL-6+ILC2. The expression levels of IL-6, IL-17 A, IL-17 F, and IL-22 in the blood serum and lung tissues of BPD mice were measured by ELISA. To further confirm the relationship between ILC2 and Th17 cell differentiation, ILC2 depletion was performed in BPD mice. Furthermore, we used immunomagnetic beads to enrich ILC2 and then flow-sorted mouse lung CD45+Lin-CD90.2+Sca-1+ILC2. The sorted ILC2s were injected into BPD mice via tail vein. Following ILC2 adoptive transfusion, the changes of Th17 cell response and lung injury were detected in BPD mice.ResultsThe expression levels of Th17 cells and Th17 cell-related cytokines, including IL-17 A, IL-17 F, and IL-22, were significantly increased in BPD mice. Concurrently, there was a significant increase in the amount of ILC2 and IL-6+ILC2 during hyperoxia-induced lung injury, which was consistent with the trend for Th17 cell response. Compared to the control BPD group, ILC2 depletion was found to partially abolish the Th17 cell response and had protective effects against lung injury after hyperoxia. Furthermore, the adoptive transfer of ILC2 enhanced the Th17 cell response and aggravated lung injury in BPD mice.ConclusionsThis study found that ILC2 regulates hyperoxia-induced lung injury by targeting the Th17 cell response in BPD, which shows a novel strategy for BPD immunotherapy.

Umbilical cord blood exosomes from very preterm infants with bronchopulmonary dysplasia aggravate lung injury in mice

Bronchopulmonary dysplasia (BPD) is characterized by abnormal development of the blood vessels and alveoli in lungs, which largely occurs in premature infants. Exosomes (EXO) from very preterm infants (VPI) with BPD (BPD-EXO) impair angiogenic activities of human umbilical vein endothelial cells (HUVECs) via EXO-miRNAs cargo. This study aimed to determine whether and how BPD-EXO affect the development of BPD in a mouse model. We showed that treating BPD mice with BPD-EXO chronically and irreversibly aggravated lung injury. BPD-EXO up-regulated 139 and down-regulated 735 genes in the mouse lung tissue. These differentially expressed genes were enriched to the MAPK pathway (e.g., Fgf9 and Cacna2d3), which is critical to angiogenesis and vascular remodeling. BPD-EXO suppressed expression of Fgf9 and Cacna2d3 in HUVECs and inhibited migration, tube formation, and increased cell apoptosis in HUVECs. These data demonstrate that BPD-EXO aggravate lung injury in BPD mice and impair lung angiogenesis, plausibly leading to adverse outcomes of VPI with BPD. These data also suggest that BPD-EXO could serve as promising targets for predicting and treating BPD.

The NEB-P2ry1 lung-brainstem axis and the control of breathing in a mouse model of bronchopulmonary dysplasia

Breathing control problems, like apnea of prematurity, are a common complication in preterm infants with bronchopulmonary dysplasia (BPD), but the underlying factors remain incompletely understood. A subpopulation of pulmonary vagal afferent nerve fibers (P2ry1+) innervate CO2 and O2 sensitive clusters of lung neuroendocrine cells (neuroepithelial bodies (NEBs)) located along conducting airways. P2ry1 fibers provide direct sensory input to the brainstem and inhibit breathing upon stimulation. NEBs are hyperplastic in BPD infants and may increase activity of inhibitory P2ry1 vagal fibers. Herein, we sought to determine if the NEB-P2ry1 lung-brainstem axis is altered in association with breathing control in BPD. BPD in mice was induced by exposure to chronic neonatal (postnatal day (P) 0-14) hyperoxia (Hx; 70% O2; n=20) or normoxia (Nx; n=17). Consistent with human BPD, Hx caused alveolar simplification supported by a reduction in alveolar density (p<0.05) and alveoli (p<0.05) in P22 H&E-stained lung sections. Hx mice spent more time apneic at P14 and P17 (p<0.05) in room air and acute hypoxia. Overall (main effect), room air minute ventilation (VE) was reduced in HX mice (p<0.05). Further, VE was reduced during acute hypoxia at P14 and P17 (not P21) with an overall reduction of breathing in Hx mice (p<0.05). From a subset of mice (n=4-5/group) VCO2, VO2, and heat production at rest were greater in P21 Hx mice during room air, suggesting the Hx mice were hypoventilating. Immunofluorescent staining of CGRP (hallmark NEB marker), Ki67 (proliferation), and SEZ6L2 (new NEB cell-membrane marker) in Hx and Nx lungs were imaged and analyzed by a blinded investigator. Consistent with clinical findings, Hx mice had hyperplastic NEBs (8.6 ± 0.45 vs 10.7 ± 1.3 cells/NEB; p<0.05) while only 2 of 143 NEBs expressed Ki67, suggesting no proliferation at P22. CGRP co-localized with SEZ6L2 in both Nx and Hx lungs. A subset of mice received intratracheal delivery of 1ug SEZ6L2-Saporin toxin to lesion NEBs and assess their function. 4 days after SEZ-Saporin treatment breathing frequency was elevated by 11% ± 5% in room air and by 7% ± 5% during acute hypoxia but not in PBS-treated, adult mice (n=3/group) suggesting NEBs have a mild inhibitory influence under normal conditions. Lastly, wireless optogenetic stimulation of P2ry1 vagal fibers in unanesthetized adult control mice (n=3) increased room air breathing by 20% ± 1% and drive to breathe by 36% ± 19% compared to pre-stim breathing. Together, our data demonstrate that hyperoxia-induced BPD alters the control of breathing which may in part be mediated via a unique lung-brainstem axis comprised of hyperplastic NEBs causing greater inhibition of excitatory P2ry1 vagal fibers. The NEB-P2ry1 axis may serve as a novel target for improving breathing in BPD. GCM: Advancing Healthier Wisconsin REAC Award, AHA 20CDA35310121; MRH: HL122358; JLG: DK133121, HL134850, and HL084207; JLS: DK133121 This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Inhibition of hippocampal cyclin-dependent kinase 5 activity ameliorates learning and memory dysfunction in a mouse model of bronchopulmonary dysplasia.

Oxygen therapy plays a vital role in the development of bronchopulmonary dysplasia (BPD), which is the independent risk factor for neurodevelopment deficits in premature infants. However, the effect of hippocampal cyclin-dependent kinase 5 (CDK5) on BPD-associated neurodevelopment deficits is not fully understood. Mice were placed in a hyperoxia chamber from postnatal Day 1 to Day 7. Hematoxylin and eosin staining was used to evaluate the lung histomorphological characteristics. Learning and memory functions of mice were detected by Morris water maze. TUNEL staining was applied to measure the number of apoptotic cells. The expression of CDK5, apoptosis-related protein, and neuroplasticity-related proteins were analyzed by Western blot. Golgi staining was used to assess the structure of dendritic spines. Hyperoxia-induced BPD mice showed a long-term learning and memory dysfunction, more severe neuronal apoptosis, and a decline of synaptic plasticity. Inhibition of CDK5 overactivation ameliorated cognitive deficits, neuronal apoptosis, and synaptic plasticity disorders in BPD mice. This study first found a vital role of CDK5 in BPD-associated neurodevelopmental disorders. Inhibition of CDK5 overexpression could effectively improve cognitive dysfunctions in BPD mice, which indicated that hippocampal CDK5 may be a new target for prevention and treatment in learning and memory dysfunction of BPD.

Pollen-Inspired Shell-Core Aerosol Particles Capable of Brownian Motion for Pulmonary Vascularization.

Nebulization is the most widely used respiratory delivery technique with non-invasive properties. However, nebulized drugs often fail to function due to the excretion and immune clearance of the respiratory system. Inthiswork, inspired by pollen in nature, novel shell-core aerosol particles (APs) capable of Brownian motion are constructed for respiratory delivery. Drugs-loaded poly(lactic-co-glycolic acid) nanoparticles are prepared by emulsification to form the inner core, and the membranes of macrophages are extracted to form the outer shell. The optimized size and the shell-core structure endow APs with Brownian motion and atomization stability, thus enabling the APs to reach the bronchi and alveoli deeply for effective deposition. Camouflaging themacrophage membranes equips the APs with immune evasion. In vitro experiments prove that deferoxamine (DFO)-loaded APs (DFO@APs) can promote the angiogenesis of human umbilical vein endothelial cells. A hyperoxia-induced bronchopulmonary dysplasia (BPD) model is constructed to validate the efficiency of DFO@APs. In BPD mice, DFO@APs can release DFO in the alveolar interstitium, thus promoting the reconstruction of microvasculature, ultimately inducing lung development for treating BPD. In conclusion, this study develops "pollen"-inspired shell-core aerosol particles capable of Brownian motion, which provides a novel idea and theoretical basis for respiratory administration.

Expression of long noncoding RNA uc.375 in bronchopulmonary dysplasia and its function in the proliferation and apoptosis of mouse alveolar epithelial cell line MLE 12.

Background: According to our previous gene ChIP results, long noncoding RNA uc.375 was down-regulated in lung tissue of bronchopulmonary dysplasia (BPD) mice induced by hyperoxia. FoxA1 gene showed higher levels in lung tissue of BPD mice and is reported to promote the apoptosis of alveolar epithelial cells. We aimed to clarify the expression pattern of uc.375 in BPD and explore the interaction between uc.375 and FoxA1. Methods: Newborn mice were placed in a 95% high-oxygen environment for 7 days. Lung tissue samples from mice were used for lncRNA microarray to screen BPD related lncRNAs. Mouse alveolar epithelial cell line MLE 12 was stably transfected with uc.375 and FoxA1 silencing or overexpression lentiviral vectors. The proliferation activity of MLE 12 cells was detected by a cell counting kit 8 (CCK-8) assay. MLE 12 cell apoptosis was determined by Hoechst/PI staining and flow cytometry analysis. The protein levels of Cleaved Caspase-3, FoxA1, SP-C and UCP2 were investigated by western blot. The relative mRNA expression levels were detected by quantitative real-time PCR. Results: uc.375 is mainly distributed in the nucleus of alveolar epithelial cells, as revealed by In Situ Hybridization assay results. uc.375 was lowly expressed in the lung tissues of BPD mice. According to the results of CCK-8 assay, analysis of Hoechst/PI staining and western blotting, uc.375 silencing inhibited cell proliferation, facilitated apoptosis of MLE 12 cells, promoted caspase 3 and FoxA1 expression, and inhibited the expression of SP-C and UCP2. On the contrary, after overexpressing uc.375, the opposite results were obtained. Silencing FoxA1 inhibited MLE 12 apoptosis, promoted proliferation, inhibited apoptosis-related factor caspase 3, and promoted the expression of SP-C and UCP2. FoxA1 silencing also reversed the effect induced by uc.375 knockdown on the proliferation and apoptosis of MLE 12 cells. Conclusion: Based on the biomedical images-derived analysis results, uc.375 negatively regulates FoxA1 expression, affects alveolar development, and plays an important role in the initiation and progression of BPD, providing a new molecular target for the prevention and treatment of BPD.

Deficiency in DDR1 Induces Pulmonary Hypertension and Impaired Alveolar Development.

Pulmonary hypertension (PH) is a multifaceted condition characterized by elevated pulmonary arterial pressure, which can result in right ventricular dysfunction and failure. Disorders of lung development can present with secondary PH, which is a leading cause of mortality in infants with bronchopulmonary dysplasia (BPD). DDR1 (discoidin domain receptor 1) is a collagen-binding receptor that regulates tissue fibrosis and inflammation and controls cellular growth and migration. However, the roles of DDR1 in lung development or the pathogenesis of PH are unknown. Studying mice with a DDR1 deletion (Ddr1-/-), we have noted 35% mortality between 1 and 4 months of age, and we demonstrate that DDR1 deficiency results in reduced right ventricular contractility and muscularization of distal pulmonary arteries, consistent with PH. Pathology analysis revealed enlarged alveolar spaces in Ddr1-/- mice by Postnatal Day 7, consistent with impaired alveolar development. Gene expression analysis showed that Ddr1-/- mice have reduced concentrations of alveologenesis factors and epithelial-to-mesenchymal transition markers. Mechanistic studies invitro confirmed that DDR1 mediated epithelial-to-mesenchymal transition, migration, and growth of alveolar epithelial cells. Taken together, these data suggest that DDR1 plays important roles mediating alveolarization during lung development. Our studies also describe a new model of spontaneous PH and bronchopulmonary dysplasia in mice.

Extracellular Signal-Regulated Kinase 1 Alone Is Dispensable for Hyperoxia-Mediated Alveolar and Pulmonary Vascular Simplification in Neonatal Mice.

Bronchopulmonary dysplasia (BPD) is a morbid lung disease distinguished by lung alveolar and vascular simplification. Hyperoxia, an important BPD causative factor, increases extracellular signal-regulated kinases (ERK)-1/2 expression, whereas decreased lung endothelial cell ERK2 expression reduces angiogenesis and potentiates hyperoxia-mediated BPD in mice. However, ERK1′s role in experimental BPD is unclear. Thus, we hypothesized that hyperoxia-induced experimental BPD would be more severe in global ERK1-knockout (ERK1-/-) mice than their wild-type (ERK1+/+ mice) littermates. We determined the extent of lung development, ERK1/2 expression, inflammation, and oxidative stress in ERK1-/- and ERK1+/+ mice exposed to normoxia (FiO2 21%) or hyperoxia (FiO2 70%). We also quantified the extent of angiogenesis and hydrogen peroxide (H2O2) production in hyperoxia-exposed neonatal human pulmonary microvascular endothelial cells (HPMECs) with normal and decreased ERK1 signaling. Compared with ERK1+/+ mice, ERK1-/- mice displayed increased pulmonary ERK2 activation upon hyperoxia exposure. However, the extent of hyperoxia-induced inflammation, oxidative stress, and interrupted lung development was similar in ERK1-/- and ERK1+/+ mice. ERK1 knockdown in HPMECs increased ERK2 activation at baseline, but did not affect in vitro angiogenesis and hyperoxia-induced H2O2 production. Thus, we conclude ERK1 is dispensable for hyperoxia-induced experimental BPD due to compensatory ERK2 activation.

Long non‑coding RNA Rian protects against experimental bronchopulmonary dysplasia by sponging miR‑421

Bronchopulmonary dysplasia (BPD) is a frequent complication characterized by accelerated lung alveolarization in newborns. Long non-coding RNAs (lncRNAs) and microRNAs (miRs) are regarded as essential regulators in various diseases, including BPD. However, the detailed mechanism of the functions of RNA imprinted and accumulated in nucleus (Rian) lncRNA in the progression of BPD have remained elusive. The aim of the present study was to illustrate the interaction between miR-421 and Rian in BPD models and MLE-12 cells. The ability of Rian to protect neonatal lungs from hyperoxia-induced lung damage was examined. A mouse model of BPD and a hyperoxia-stimulated MLE-12 cell damage model were generated and treated with specific plasmid/mimics for the overexpression of Rian/miR-421. The interaction between miR-421 and Rian was predicted and verified using StarBase and a dual-luciferase reporter assay, respectively. The expression levels of miR-421 or Rian in both tissues and the MLE-12 alveolar epithelial cell line were assessed using reverse transcription-quantitative (RT-q)PCR. As parameters of alveolarization, the mean linear intercept (MLI), radial alveolar count (RAC) and the lung weight/body weight (LW/BW) ratio were measured. Furthermore, RT-qPCR was used to measure mRNA levels of pro-inflammatory cytokines (TNF-α, IL-6 and IL-1β) in the lung tissue of mice, and ELISAs were performed to determine the levels of pro-inflammatory cytokines (TNF-α, IL-6 and IL-1β) in the supernatant of MLE-12 cells. Cell growth and apoptosis were evaluated using an MTT assay and flow cytometry, respectively. Furthermore, caspase-3 activity was assessed using a caspase-3 activity detection kit. Prediction with StarBase and the dual-luciferase reporter assay revealed that miR-421 directly targeted Rian. RT-qPCR analysis confirmed that Rian was downregulated and miR-421 was upregulated in lung tissues of the mouse model of BPD and in hyperoxia-induced MLE-12 cells. However, the expression of miR-421 was decreased by Rian-overexpression, an effect that was reversed by miR-421 mimics. In addition, BPD was alleviated by Rian-plasmid, as confirmed by the enhanced RAC and reduced MLI and LW/BW ratio. The present results also indicated that Rian-plasmid inhibited the secretion of pro-inflammatory cytokines (TNF-α, IL-6 and IL-1β) in BPD mouse serum and hyperoxia-induced MLE-12 cells. In addition, Rian-plasmid eliminated the effect of hyperoxia to inhibit cell viability and induce apoptosis in MLE-12 cells. However, all of these effects of Rian were markedly reversed by miR-421 mimics. The present results indicated that Rian may attenuate hyperoxic damage in neonatal lungs and may serve as a novel molecular target for BPD treatment.

Bronchopulmonary Dysplasia and Pulmonary Hypertension. The Role of Smooth Muscle adh5.

Bronchopulmonary dysplasia (BPD) is characterized by alveolar simplification, airway hyperreactivity, and pulmonary hypertension. In our BPD model, we have investigated the metabolism of the bronchodilator and pulmonary vasodilator GSNO (S-nitrosoglutathione). We have shown the GSNO catabolic enzyme encoded by adh5 (alcohol dehydrogenase-5), GSNO reductase, is epigenetically upregulated in hyperoxia. Here, we investigated the distribution of GSNO reductase expression in human BPD and created an animal model that recapitulates the human data. Blinded comparisons of GSNO reductase protein expression were performed in human lung tissues from infants and children with and without BPD. BPD phenotypes were evaluated in global (adh5-/-) and conditional smooth muscle (smooth muscle/adh5-/-) adh5 knockout mice. GSNO reductase was prominently expressed in the airways and vessels of human BPD subjects. Compared with controls, expression was greater in BPD smooth muscle, particularly in vascular smooth muscle (2.4-fold; P = 0.003). The BPD mouse model of neonatal hyperoxia caused significant alveolar simplification, airway hyperreactivity, and right ventricular and vessel hypertrophy. Global adh5-/- mice were protected from all three aspects of BPD, whereas smooth muscle/adh5-/- mice were only protected from pulmonary hypertensive changes. These data suggest adh5 is required for the development of BPD. Expression in the pulmonary vasculature is relevant to the pathophysiology of BPD-associated pulmonary hypertension. GSNO-mimetic agents or GSNO reductase inhibitors, both of which are currently in clinical trials for other conditions, could be considered for further study in BPD.

Adiponectin ameliorates hyperoxia-induced lung endothelial dysfunction and promotes angiogenesis in neonatal mice.

Bronchopulmonary dysplasia (BPD) is a common respiratory disease of preterm infants. Lower circulatory/intrapulmonary levels of the adipokine, adiponectin (APN), occur in premature and small-for-gestational-age infants and at saccular/alveolar stages of lung development in the newborn rat. However, the role of low intrapulmonary APN during hyperoxia exposure in developing lungs is unknown. We test the hypothesis that treatment of hyperoxia-exposed newborn mice with recombinant APN protein attenuates the BPD phenotype characterized by inflammation, impaired alveolarization, and dysregulated vascularization. We used developmentally appropriate in vitro and in vivo BPD modeling systems as well as human lung tissue. We observed reduced levels of intrapulmonary APN in experimental BPD mice and human BPD lungs. APN-deficient (APN-/-) newborn mice exposed to moderate (60% O2) hyperoxia showed a worse BPD pulmonary phenotype (inflammation, enhanced endothelial dysfunction, impaired pulmonary vasculature, and alveolar simplification) as compared to wild-type (WT) mice. Treatment of hyperoxia-exposed newborn WT mice with recombinant APN protein attenuated the BPD phenotype (diminished inflammation, decreased pulmonary vascular injury, and improved pulmonary alveolarization) and improved pulmonary function tests. Low intrapulmonary APN is associated with disruption of lung development during hyperoxia exposure, while recombinant APN protein attenuates the BPD pulmonary phenotype. Intrapulmonary APN levels were significantly decreased in lungs of experimental BPD mice and human BPD lung tissue at various stages of BPD development. Correlative data from human lung samples with decreased APN levels were associated with increased lung adhesion markers (intercellular adhesion molecule-1, vascular cell adhesion molecule-1, and E-selectin). Decreased APN levels were associated with endothelial dysfunction and moderate BPD phenotype in APN-deficient, as compared to WT, experimental BPD mice. WT experimental BPD mice treated with recombinant APN protein had an improved pulmonary structural and functional phenotype. Exogenous APN may be considered as a potential therapeutic agent to prevent BPD.

Integrated MicroRNA-mRNA Analyses of Distinct Expression Profiles in Hyperoxia-Induced Bronchopulmonary Dysplasia in Neonatal Mice.

Bronchopulmonary dysplasia (BPD) is a common chronic lung disease of preterm neonates; the underlying pathogenesis is not fully understood. Recent studies suggested microRNAs (miRNAs) may be involved in BPD. miRNA and mRNA microarrays were performed to analyze the expression profiles of miRNA and mRNA in BPD and control lung tissues after oxygen and air exposure on day 21. Bioinformatics methods, including Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG), were performed to predict the potential functions of differentially expressed genes. Then, miRNA-mRNA regulatory network was constructed by protein-protein interaction (PPI) data and TarBase data. Our results showed that a total of 192 differentially expressed miRNAs (74 downregulated and 118 upregulated) and 1,225 differentially expressed mRNAs (479 downregulated and 746 upregulated) were identified between BPD mice and normoxia-control mice. GO and KEGG analysis showed that for downregulated genes, the top significant enriched GO terms and KEGG pathways were both mainly related to immune and inflammation processes; for upregulated genes, the top significant enriched GO terms and KEGG pathways were both mainly related to extracellular matrix (ECM) remodeling. PPI network and miRNA-mRNA regulatory network construction revealed that the key genes and pathways associated with inflammation and immune regulation. Our findings revealed the integrated miRNA-mRNA data of distinct expression profiles in hyperoxia-induced BPD mice, and may provide some clues of the potential biomarkers for BPD, and provide novel insights into the development of new promising biomarkers for the treatment of BPD. · Integrated advanced bioinformatics methods may offer a better way to understand the molecular expression profiles involved in BPD.. · ECM remodeling, inflammation, and immune regulation may be essential to BPD.. · The miRNA-mRNA regulatory network construction may contribute to develop new biomarkers for the treatment of BPD..

MicroRNA 219-5p inhibits alveolarization by reducing platelet derived growth factor receptor-alpha

BackgroundMicroRNA (miR) are small conserved RNA that regulate gene expression post-transcription. Previous genome-wide analysis studies in preterm infants indicate that pathways of miR 219-5p are important in infants with Bronchopulmonary Dysplasia (BPD).MethodsHere we report a prospective cohort study of extremely preterm neonates wherein infants diagnosed with severe BPD expressed increased airway miR-219-5p and decreased platelet derived growth factor receptor alpha (PDGFR-α), a target of mir-219-5p and a key regulator of alveolarization, compared to post-conception age-matched term infants.ResultsmiR-219-5p was highly expressed in the pulmonary epithelial lining in lungs of infants with BPD by in situ hybridization of human infant lungs. In both in vitro and in vivo (mouse) models of BPD, miR-219-5p was increased on exposure to hyperoxia compared with the normoxia control, with a complementary decrease of PDGFR-α. To further confirm the target relationship between miR‐219 and PDGFR-α, pulmonary epithelial cells (MLE12) and lung primary fibroblasts were treated with a mimic of miR-219-5p and a locked nucleic acid (LNA) based inhibitor of miR-219-5p. In comparison with the control group, the level of miR‐219 increased significantly after miR‐219 mimic treatment, while the level of PDGFR-α declined markedly. LNA exposure increased PDGFR-α. Moreover, in BPD mouse model, over-expression of miR-219-5p inhibited alveolar development, indicated by larger alveolar spaces accompanied by reduced septation.ConclusionsTaken together, our results demonstrate that increased miR-219-5p contributes to the pathogenesis of BPD by targeting and reducing PDGFR-α. The use of specific miRNA antagonists may be a therapeutic strategy for preventing the development of BPD.

Acetate Downregulates the Activation of NLRP3 Inflammasomes and Attenuates Lung Injury in Neonatal Mice With Bronchopulmonary Dysplasia.

Background: Bronchopulmonary dysplasia (BPD) is a common pulmonary complication in preterm infants. Acetate is a metabolite produced by the gut microbiota, and its anti-inflammatory function is well known. The role of acetate in BPD has not been studied. Here, we investigate the effects of acetate on lung inflammation and damage in mice model of BPD.Objective: To investigate the role of acetate in the development of BPD.Methods: C57BL/6 mice were randomly divided into three groups on the 3rd day after birth: room air group, hyperoxia group, and hyperoxia + acetate (250 mM, 0.02 ml/g) group. The expression of inflammatory factors was determined by ELISA and RT-PCR, and NLRP3 and caspase-1 were detected by Western blot. High-throughput sequencing was used to detect bacterial communities in the mice intestines.Results: After acetate treatment, the expression levels of TNF-α, IL-1β, IL-18, NLRP3, and caspase-1 were significantly reduced, while the expression of GPR43 was increased. In the BPD mice treated with acetate, the proportion of Escherichia-Shigella was lower than in placebo-treated BPD mice, while the abundance of Ruminococcus was increased.Conclusions: These results indicate that acetate may regulate intestinal flora and reduce inflammatory reactions and lung injury in BPD. Therefore, acetate may be an effective drug to protect against neonatal BPD.

Mechanism of lncRNA H19 in Regulating Pulmonary Injury in Hyperoxia-Induced Bronchopulmonary Dysplasia Newborn Mice.

Bronchopulmonary dysplasia (BPD) is a pulmonary injury related to inflammation and is a major cause of premature infant death. Long noncoding RNAs (lncRNAs) are important regulators in pulmonary injury and inflammation. We investigated the molecular mechanism of lncRNA H19 in pulmonary injury and inflammation in hyperoxia (Hyp)-induced BPD mice. The BPD newborn mouse model was established and intervened with H19 to evaluate the pathologic conditions and radial alveolar count (RAC) in lung tissues of mice in the room air (RA) and Hyp group on the 4th, 7th, and 14th days after birth. The levels of BPD-related biomarkers vascular endothelial growth factor (VEGF), transforming growth factor β1 (TGF-β1), and surfactant protein C (SPC) in lung tissues were detected on the 14th day after birth. The expression of and relationships among H19 and miR-17, miR-17, and STAT3 were detected and verified. Levels of interleukin (IL)-6, IL-1β, p-STAT3, and STAT3 levels in mouse lung tissues were detected on the 14th day after birth. Hyp-induced mice showed increased alveolar diameter, septum, and hyperemia and inflammatory cell infiltration, upregulated H19, decreased overall number and significantly reduced RAC on the 7th and 14th days after birth, which were reversed in the si-H19-treated mice. VEGF was upregulated and TGF-β1 and SPC was decreased in si-H19-treated mice. Moreover, H19 competitively bound to miR-17 to upregulate STAT3. IL-6 and IL-1β expressions and p-STAT3 and STAT3 levels were downregulated after inhibition of H19. Downregulated lncRNA H19 relieved pulmonary injury via targeting miR-17 to downregulate STAT3 and reduced inflammatory response caused by p-STAT3 in BPD newborn mice. · lncRNA H19 was highly expressed in Hyp-induced BPD newborn mice.. · si-H19 relieved pulmonary injury in Hyp-induced BPD newborn mice.. · si-H19 upregulated miR-17 and downregulated STAT3 expression..

LncRNA CASC2 targets CAV1 by competitively binding with microRNA-194-5p to inhibit neonatal lung injury

Long non-coding RNAs (lncRNAs) are vital regulators of different biological processes during bronchopulmonary dysplasia (BPD). This study was conducted to probe the biological roles of lncRNA CASC2 in the pathogenesis of BPD and neonatal lung injury. Firstly, a hyperoxia-induced mouse model with BPD was established. LncRNAs with differential expression in lung tissues of normal and BPD mice were analyzed by microarray. An adenovirus vector overexpressing CASC2 was constructed and its functions on BPD symptoms in model mice were analyzed. Gain- and loss-of function studies of CASC2 were performed in a bronchial epithelial cell line BEAS-2B to determine its role in cell apoptosis and proliferation under normoxic and hyperoxic conditions. The downstream mechanical molecules of lncRNA CASC2 were predicted on bioinformatics systems and confirmed by luciferase assays. The functional interactions among lncRNA CASC2, miR-194-5p, and CAV1 in BPD were determined by rescue experiments. Consequently, lncRNA CASC2 was found to be poorly expressed in BPD mice. Besides, overexpressed CASC2 was found to relieve the symptoms of BPD in neonatal mice and suppress apoptosis as well as promote proliferation in hyperoxia-induced BEAS-2B cells. Importantly, CASC2 was found to regulate CAV1 expression by competitively binding to miR-194-5p and downregulate the activity of the TGF-β1 signaling pathway, thereby suppressing lung injury. Either miR-194-5p upregulation or CAV1 downregulation blocked the roles of CASC2. To sum up, this study evidenced that CASC2 alleviates hyperoxia-induced lung injury in mouse and cell models with the involvement of a miR-194-5p-CAV1 crosstalk and the TGF-β1 inactivation.

Involvement of Hdac3-mediated inhibition of microRNA cluster 17-92 in bronchopulmonary dysplasia development

BackgroundThe incidence of bronchopulmonary dysplasia (BPD), a chronic lung disease of newborns, has been paradoxically rising despite medical advances. Histone deacetylase 3 (Hdac3) has been reported to be a crucial regulator in alveologenesis. Hence, this study aims to investigate the mechanism of Hdac3 in the abnormal pulmonary angiogenesis and alveolarization of BPD.MethodsA hyperoxia-induced BPD model of was developed in newborn mice, and primary lung fibroblasts were isolated from adult mice. Hdac3 was knocked out in vivo and knocked down in vitro, while microRNA (miR)-17 was downregulated in vivo and in vitro to clarify their roles in abnormal pulmonary angiogenesis and alveolarization. Mechanistic investigations were performed on the interplay of Hdac3, miR-17-92 cluster, enhancer of zeste homolog 1 (EZH1), p65 and placental growth factor (Pgf).ResultsHdac3 was involved in abnormal alveolarization and angiogenesis in BPD mice. Further, the expression of the miR-17-92 cluster in BPD mice was downregulated by Hdac3. miR-17 was found to target EZH1, and Hdac3 rescued the inhibited EZH1 expression by miR-17 in lung fibroblasts. Additionally, EZH1 augmented Pgf expression by recruiting p65 thus enhancing the progression of BPD. Hdac3 augmented the recruitment of p65 in the Pgf promoter region through the miR-17/EZH1 axis, thus enhancing the transcription and expression of Pgf, which elicited abnormal angiogenesis and alveolarization of BPD mice.ConclusionsAltogether, the present study revealed that Hdac3 activated the EZH1-p65-Pgf axis through inhibiting miR-17 in the miR-17-92 cluster, leading to accelerated abnormal pulmonary angiogenesis and alveolarization of BPD mice.

Tissue-Resident Type 2 Innate Lymphoid Cells Arrest Alveolarization in Bronchopulmonary Dysplasia.

Bronchopulmonary dysplasia (BPD) is a severe complication of the respiratory system associated with preterm birth. Type 2 innate lymphoid cells (ILC2s) play a major role in tissue homeostasis, inflammation, and wound healing. However, the role in BPD remains unclear. The present study showed that ILC2s, interleukin-4 (IL-4), IL-13, and anti-inflammatory (M2) macrophages increased significantly in BPD mice as compared to the control mice. Administration with recombinant mouse IL-33 amplified the above phenomena and aggravated the alveolar structural disorder and functional injury in mice subjected to BPD, and the opposite was true with anti-ST2 antibody. In addition, the depletion of ILC2s in BPD mice with anti-CD90.2 antibody substantially abolished the destructive effect on BPD. In the treatment of BPD with dexamethasone, the number of ILC2s and M2 macrophages and levels of IL-4 and IL-13 decreased with remission as compared to the control group. This study identified a major destructive role of the ILC2s in BPD that could be attenuated as a therapeutic strategy.