Hyperoxic Lung Injury Research Articles

Effect of caffeine on hyperoxic lung injury of premature rats and its relationship with p38 motigen-activated protein kinase signal pathway

Objective To study the effect of caffeine on hyperoxic lung injury of premature rats and its relationship with p38 motigen-activated protein kinase(MAPK) signal pathway. Methods Sixty Wistar premature rats were divided into 4 groups(n=15) according to the random number table: air + normal saline group(A+ N group), air + caffeine group(A+ C group), hyperoxia + normal saline group(H+ N group), and hyperoxia + caffeine group(H+ C group). Among them, H+ N group and H+ C group were continually exposed to hyperoxia(oxygen concentration was 60%~70%). For A+ C group and H+ C group, the premature rats were injected with caffeine of 29 mg/(kg·d) into their peritoneal cavities every day after birth.For A+ N group and H+ N group, the premature rats were injected with normal saline of the same volume into their peritoneal cavities.In each group, the lung tissues of 5 premature rats were randomly selected on the third, seventh and fourteenth day respectively.The pathological changes of lung tissue, radiated alveolar count(RAC) and collagen content in lung tissue were observed under a light microscope.The wet/dry ratio(W/D) was measured.Two-step immunohistochemistry was used to detect the distribution of p38MAPK in lung tissue.The content of phosphorylated p38MAPK(p-p38MAPK) protein was detected by western blot. Results Compared with the air groups, the lung tissues of premature rats in high oxygen exposure groups showed different degree of inflammatory changes on the third, seventh, and fourteenth day.The changes were more obvious with the prolonged exposure to hyperoxia.Pulmonary fibrosis was visible on the fourteenth day, which was improved after caffeine intervention.The RAC value of premature rats in hyperoxia exposure groups was significantly lower than that in air-exposure groups(P<0.05), and the W/D ratio and collagen content in lung tissue increased significantly(P<0.05), which were improved after caffeine intervention(P<0.05). The results of two-step immunohistochemistry showed that the number of p-p38MAPK positive cells in the lung tissue of premature rats in hyperoxia exposure groups increased and widely distributed, but decreased after caffeine intervention.The results of western blot showed that the content of p-p38MAPK protein in lung tissue of premature rats in hyperoxia exposure groups was significantly higher than that of air groups(P<0.05), but it decreased after caffeine intervention(P<0.05). Conclusion Hyperoxia can promote the formation of pulmonary fibrosis by activating p38MAPK signal pathway.Caffeine can interdict the expression of p38MAPK to alleviate the fibrosis of lung tissue exposed to hyperoxia and thus protects the lung tissue. Key words: Hyperoxia; Lung injury; Caffeine; p38 motigen-activated protein kinase; Premature rats

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.

Notch signaling pathway and bronchopulmonary dysplasia

Bronchopulmonary dysplasia(BPD)is a chronic lung disease caused by various physical and chemical factors, which causes immature lung injury and abnormal repair after injury.In recent years, BPD has become one of the most difficult problems in neonatal intensive care unit.Notch signaling pathway is a conservative pathway that plays a regulatory role in embryonic development.Notch signaling pathways have a wide range of biological effects.By mediating cell-to-cell interactions, Notch signals can finely regulate the growth, development and apoptosis of cells.This article reviews the relationship between Notch signaling pathway and BPD in lung development, hyperoxia-induced lung injury and lung regeneration. Key words: Notch signaling pathway; Bronchopulmonary dysplasia; Hyperoxia lung injury

Mesenchymal Stem Cells Increase Alveolar Differentiation in Lung Progenitor Organoid Cultures

Lung epithelial cell damage and dysfunctional repair play a role in the development of lung disease. Effective repair likely requires the normal functioning of alveolar stem/progenitor cells. For example, we have shown in a mouse model of bronchopulmonary dysplasia (BPD) that mesenchymal stem cells (MSC) protect against hyperoxic lung injury at least in part by increasing the number of Epcam+ Sca-1+ distal lung epithelial cells. These cells are capable of differentiating into both small airway (CCSP+) and alveolar (SPC+) epithelial cells in three-dimensional (3D) organoid cultures. To further understand the interactions between MSC and distal lung epithelial cells, we added MSC to lung progenitor 3D cultures. MSC stimulated Epcam+ Sca-1+ derived organoid formation, increased alveolar differentiation and decreased self-renewal. MSC-conditioned media was sufficient to promote alveolar organoid formation, demonstrating that soluble factors secreted by MSC are likely responsible for the response. This work provides strong evidence of a direct effect of MSC-secreted factors on lung progenitor cell differentiation.

HIF‐1 alpha Mediates Sex‐specific miR‐30a Expression in Neonatal Hyperoxic Lung Injury: Implications for Bronchopulmonary Dysplasia

BackgroundBronchopulmonary Dysplasia (BPD) is characterized by an arrest in lung development with severe impairment of alveolar septation and vascular development. Premature male neonates are at a greater risk of developing bronchopulmonary dysplasia (BPD). The reasons underlying sexually dimorphic outcomes in premature neonates are not known. Neonatal female mice have improved alveolarization and pulmonary vascular development compared to male littermates in a murine model of BPD. Analysis of the pulmonary transcriptome suggests that miR‐30a, may modulate these sex‐specific differences in gene expression. HIF‐1α plays an important role in post‐natal lung development, especially in recovery from hyperoxic injury and acute lung injury. We hypothesized that differential sex‐specific expression of miR‐30a is mediated through HIF‐1α.MethodsMale and female neonatal WT (C57BL6) mice were exposed to hyperoxia (P1–P5; saccular stage of lung development) and euthanized on P7 or P21 after recovery in normoxia. Human pulmonary microvascular endothelial cells (HPMECs; from 18–24 weeks gestation donors; 3 male and 3 female) were obtained from ScienCell (Carlsbad, California). They were cultured in endothelial cell media per protocol. Cells from passage from 3 to 6 were used for experiments. For hyperoxic exposure, cells were incubated in 95% O2 and 5% CO2 at 37 °C for upto 72 hours. Cells in normoxia were maintained in air and 5% CO2. miR‐30a and Dll4 (miR‐30a target) expression was measured. Hif‐1α binding to its targets genes (by ChIP‐qPCR) and expression was measured in vivo. To further establish potential regulation of miR‐30a by Hif‐1α, miR‐30a expression after Hif‐1α inhibition was measured in neonatal HPMECs.ResultsExpression of miR‐30a was increased and Dll4 decreased on PND21 in hyperoxia‐exposed neonatal female mice compared to males. On PND21, hyperoxia‐ exposed female mice showed a significant increase in Hif‐1α binding to target genes compared to room air controls and hyperoxia‐exposed male mice. In contrast to male mice, females did not show a decrease in Hif‐1α expression at PND21 after early hyperoxia exposure and this was accompanied by a significant decrease in Phd2 expression in female mice, which metabolizes Hif‐1α. Motif analysis revealed a hypoxia response element (HRE) [AG]CGTG site 428 base pairs upstream of miR‐30a. siRNA mediated loss of Hif‐1α expression in human pulmonary microvascular endothelial cells decreased miR‐30a and increased Dll4 mRNA and protein expression.ConclusionsHif‐1α mediated differential sex‐specific miR‐30a expression may contribute to protection from hyperoxic lung injury in female neonatal mice and contribute to the sex‐specific differences seen in BPD.Support or Funding InformationK08 HL‐127103R03 HL‐141572This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Thioredoxin Reductase-1 Inhibition Augments Endogenous Glutathione-Dependent Antioxidant Responses in Experimental Bronchopulmonary Dysplasia.

Background Aurothioglucose- (ATG-) mediated inhibition of thioredoxin reductase-1 (TXNRD1) improves alveolarization in experimental murine bronchopulmonary dysplasia (BPD). Glutathione (GSH) mediates susceptibility to neonatal and adult oxidative lung injury. We have previously shown that ATG attenuates hyperoxic lung injury and enhances glutathione- (GSH-) dependent antioxidant defenses in adult mice. Hypothesis The present studies evaluated the effects of TXNRD1 inhibition on GSH-dependent antioxidant defenses in newborn mice in vivo and lung epithelia in vitro. Methods Newborn mice received intraperitoneal ATG or saline prior to room air or 85% hyperoxia exposure. Glutamate-cysteine ligase (GCL) catalytic (Gclc) and modifier (Gclm) mRNA levels, total GSH levels, total GSH peroxidase (GPx) activity, and Gpx2 expression were determined in lung homogenates. In vitro, murine transformed club cells (mtCCs) were treated with the TXNRD1 inhibitor auranofin (AFN) or vehicle in the presence or absence of the GCL inhibitor buthionine sulfoximine (BSO). Results In vivo, ATG enhanced hyperoxia-induced increases in Gclc mRNA levels, total GSH contents, and GPx activity. In vitro, AFN increased Gclm mRNA levels, intracellular and extracellular GSH levels, and GPx activity. BSO prevented AFN-induced increases in GSH levels. Conclusions Our data are consistent with a model in which TXNRD1 inhibition augments hyperoxia-induced GSH-dependent antioxidant responses in neonatal mice. Discrepancies between in vivo and in vitro results highlight the need for methodologies that permit accurate assessments of the GSH system at the single-cell level.

The effects of omega-3 fatty acids on the newborn rat hyperoxic lung injury

Objective: Bronchopulmonary dysplasia (BPD) is an important cause of morbidity in preterms. Inflammation plays a central role in the pathogenesis of the disease while omega-3 fatty acids are known to have anti-inflammatory effects. In this study, we examined the effects of supplementary omega-3 fatty acids on hyperoxic lung injury.Methods: Experimental hyperoxic lung injury induced newborn 3-day-old rats were monitored in a confined hyperoxic environment with an oxygen concentration of 90–95% for a 2-week period. Rats were divided into three groups as placebo, low-dose Omega-3, and high-dose Omega-3. During the 2-week study period, low and high-dose Omega-3 groups were given 300 and 600 mg/kg/day omega-3 docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) respectively, while those in placebo received the same amount of serum physiologic. At the end of the 2-week study, lungs of all the rats were removed and morphologic evaluation under light microscopy was performed. Mean cord length (Lm), alveolar surface area (SA), and alveolar wall thickness (Wt) were calculated to find out whether a statistically significant difference between groups existed.Results: Similar alveolar development was observed between groups. No difference was seen between mean Lm values. Although the alveolar surface area was found to be higher in high-dose omega-3 group, the difference was not considered to be statistically significant. While the widest alveolar wall thickness was observed in the placebo group, alveolar wall thickness difference between high-dose omega-3 group and placebo group was found to be statistically significant (placebo Wt=17,8 ± 2.3 µm, low-dose omega −3 Wt=15,6 ± 2,5 µm, high-dose omega −3 Wt=14,2 ± 2 µm) (p < .05).Conclusions: Omega-3 fatty acids were observed to prevent alveolar wall thickness to some extent, though with no noticeable effect on hyperoxic lung injury.

Protective effect of Rhubarb on newborn rats with bronchopulmonary dysplasia

Objective To investigate the effect of Rhubarb on bronchopulmonary dysplasia(BPD) and its potential mechanism. Methods Neonatal Sprague-Dawley rats (postnatal day 4) were exposed to 600 mL/L O2 to induce BPD.The experimental rats were randomly divided into 4 groups with 16 in each group: air+ saline group, air+ Rhubarb(600 mg/kg)group, and hyperoxia+ saline group, and hyperoxia+ Rhubarb group.The rats were sacrificed and lung tissues were obtained on day 14 and 21 after birth.Hematoxylin-easin staining was used to detect the pathomorphology of the lungs.Apoptosis of the lung tissue was detected by means of TdT-mediated dUTP nick-end labeling(TUNEL). The expression of Fas was detected by adopting Western blot.The activity of Caspase-8 and Caspase-3 was detected by using spectrophotometer. Results The lung structure of rats was markedly abnormal (decreased, enlarged and simplified alveoli) after being exposed to hyperoxia at any time point.The apoptosis indexes (39.91±1.91 vs.10.11±1.64, 48.80±4.51 vs.12.90±3.18), the expression levels of Fas (0.47±0.02 vs.0.21±0.01, 0.55±0.02 vs.0.22±0.01) and the activities of Caspase-8 (52.59±1.23 vs.40.74±1.08, 60.20±3.48 vs.40.39±2.47) and Caspsase 3 (57.17±1.88 vs.42.00±1.19, 64.57±2.79 vs.41.54±1.27) were all increased in the hyperoxia+ saline group compared with those in the air+ saline group on day 14 and 21, and the differences were statistically significant(all P<0.001). However, compared with the hyperoxia+ saline group, Rhubarb administration dramatically decreased pulmonary apoptosis index (26.49±2.65 vs.22.97±3.66), Fas protein level (0.27±0.03 vs.0.31±0.01) and the activity of Caspase-8 (32.70±2.69 vs.30.66±4.48) and Caspase-3 (44.94±1.60 vs.44.59±1.66), and the differences were statistically significant(all P<0.001). Meanwhile, lung damage after hyperoxia was significantly attenuated in the hyperoxia+ Rhubarb group. Conclusion Rhubarb can reduce the hyperoxic lung injury of BPD by reducing the apoptosis of newborn rat lung tissue cells, and the mechanism may involve the apoptosis pathway of Fas. Key words: Bronchopulmonary dysplasia; Rhubarb; Apoptosis

Reactive oxygen and nitrogen species induce cell apoptosis via a mitochondria-dependent pathway in hyperoxia lung injury.

Hyperoxia-induced lung injury limits the application of mechanical ventilation on rescuing the lives of premature infants and seriously ill and respiratory failure patients, and its mechanisms are not completely understood. In this article, we focused on the relationship between hyperoxia-induced lung injury and reactive oxygen species (ROS), reactive nitrogen species (RNS), mitochondria damage, as well as apoptosis in the pulmonary epithelial II cell line RLE-6TN. After exposure to hyperoxia, the cell viability was significantly decreased, accompanied by the increase in ROS, nitric oxide (NO), inflammatory cytokines, and cell death. Furthermore, hyperoxia triggered the loss of mitochondrial membrane potential (▵Ψm), thereby promoting cytochrome c to release from mitochondria to cytoplasm. Further studies conclusively showed that the Bax/Bcl-2 ratiowas enlarged to activate the mitochondria-dependent apoptotic pathway after hyperoxia treatment. Intriguingly, the effects of hyperoxia on the level of ROS, NO and inflammation, mitochondrial damage, as well as cell death were reversed by free radical scavengers N-acetylcysteine and hemoglobin. In addition, a hyperoxia model of neonatal Sprague-Dawley (SD) rats presented the obvious characteristics of lung injury, such as a decrease in alveolar numbers, alveolar mass edema, and disorganized pulmonary structure. The effects of hyperoxia on ROS, RNS, inflammatory cytokines, and apoptosis-related proteins in lung injury tissues of neonatal SD rats were similar to that in RLE-6TN cells. In conclusion, mitochondria are a primary target of hyperoxia-induced free radical, whereas ROS and RNS are the key mediators of hyperoxia-induced cell apoptosis via the mitochondria-dependent pathway in RLE-6TN cells.

Effects of nebulized N--acetylcystein on the expression of HMGB1 and RAGE in rats with hyperoxia--induced lung injury.

To investigate the role of high mobility group box 1 (HMGB1) and receptor for advanced glycation end product (RAGE) in the lungs of hyperoxia-induced rats and the effect of N--acetlycystein (NAC). A model of hyperoxic lung injury was established, rats in the NAC intervention, and control, hyperoxia group were given nebulized NAC aerosol, nebulized same volume of saline once a day for 7 consecutive days, respectively. Wet/dry ( W/ D) ratio of the lungs was determined to evaluate the edema of the lung tissues. Conventional hematoxylin-eosin (HE) staining was used to observe the pathological changes of lung tissues. Immunohistochemical staining was used to investigate the expression of HMGB1 and RAGE in the lung tissues. Quantitative reverse-transcription polymerase chain reaction and western blot analysis were used to measured the changes in the messenger RNA (mRNA) and protein expression of HMGB1 and RAGE, respectively. Weight gain of the rats in the hyperoxia group was significantly slower than that in the control group and intervention group (p < 0.05). HE staining results showed lung tissues in the hyperoxia group were severely damaged compared with control group. W/D ratio in hyperoxia group was significantly higher than that in control group and intervention group (p < 0.05). Protein and mRNA expression of HMGB1 and RAGE in the hyperoxia group were significantly higher than control group and intervention group (p < 0.05). HMGB1 and RAGE were involved in the pathogenesis of hyperoxia-induced lung injury, inhalation of NAC might alleviate hyperoxia-induced lung injury by regulating the expression of HMGB1 and RAGE.

Alterations in VASP phosphorylation and profilin1 and cofilin1 expression in hyperoxic lung injury and BPD

BackgroundHyperoxia is a frequently employed therapy for prematurely born infants, induces lung injury and contributes to development of bronchopulmonary dysplasia (BPD). BPD is characterized by decreased cellular proliferation, cellular migration, and failure of injury repair systems. Actin binding proteins (ABPs) such as VASP, cofilin1, and profilin1 regulate cell proliferation and migration via modulation of actin dynamics. Lung mesenchymal stem cells (L-MSCs) initiate repair processes by proliferating, migrating, and localizing to sites of injury. These processes have not been extensively explored in hyperoxia induced lung injury and repair.MethodsABPs and CD146+ L-MSCs were analyzed by immunofluorescence in human lung autopsy tissues from infants with and without BPD and by western blot in lung tissue homogenates obtained from our murine model of newborn hyperoxic lung injury.ResultsDecreased F-actin content, ratio of VASPpS157/VASPpS239, and profilin 1 expression were observed in human lung tissues but this same pattern was not observed in lungs from hyperoxia-exposed newborn mice. Increases in cofilin1 expression were observed in both human and mouse tissues at 7d indicating a dysregulation in actin dynamics which may be related to altered growth. CD146 levels were elevated in human and newborn mice tissues (7d).ConclusionAltered phosphorylation of VASP and expression of profilin 1 and cofilin 1 in human tissues indicate that the pathophysiology of BPD involves dysregulation of actin binding proteins. Lack of similar changes in a mouse model of hyperoxia exposure imply that disruption in actin binding protein expression may be linked to interventions or morbidities other than hyperoxia alone.

11 - Lung epithelial specific heme oxygenase-1 deletion does not worsen hyperoxic lung injury in adult mice

Thioredoxin reductase-1 (TXNRD1) inhibition activates nuclear factor (erythroid-derived 2)-like 2(Nrf2) responses and prevents acute lung injury (ALI). Heme oxygenase-1 (HO-1) induction following TXNRD1 inhibition is Nrf2-dependent in airway epithelial (club) cells in vitro. To evaluate the contribution of HO-1 toward the protective effects of TXNRD1 inhibitors in vivo, a club cell specific HO-1 knockout (KO) mouse is needed. The present studies characterized the effects of hyperoxia on club cell specific HO-1 KO mice that were generated by crossing HO-1 floxed mice with transgenic mice expressing cre recombinase under control of a club cell specific promoter. Twelve-week old wildtype (WT) and KO mice were continuously exposed to >95% hyperoxia (HYP) or room air (RA) for 72h. Following exposure, mice were sacrificed and weighed. The left lung was inflation fixed and the right lung was collected and weighed. Injury was quantitatively assessed by calculating right lung/body weight ratios (g/kg) and qualitatively assessed in HE features that appeared to be more prominent in KO/HYP mice. In conclusion, lung epithelial cell HO-1 deletion does not alter hyperoxic lung injury in adult mice. Future studies will utilize this unique transgenic model to evaluate the contribution HO-1 toward the protective effects of TXNRD1 inhibitors in vivo.

MicroRNA-30a as a candidate underlying sex-specific differences in neonatal hyperoxic lung injury: implications for BPD.

Premature male neonates are at a greater risk of developing bronchopulmonary dysplasia (BPD). The reasons underlying sexually dimorphic outcomes in premature neonates are not known. The role of miRNAs in mediating sex biases in BPD is understudied. Analysis of the pulmonary transcriptome revealed that a large percentage of angiogenesis-related differentially expressed genes are miR-30a targets. We tested the hypothesis that there is differential expression of miR-30a in vivo and in vitro in neonatal human pulmonary microvascular endothelial cells (HPMECs) upon exposure to hyperoxia. Neonatal male and female mice (C57BL/6) were exposed to hyperoxia [95% fraction of inspired oxygen (FiO2), postnatal day ( PND) 1-5] and euthanized on PND 7 and 21. HPMECs (18-24-wk gestation donors) were subjected to hyperoxia (95% O2 and 5% CO2) or normoxia (air and 5% CO2) up to 72 h. miR-30a expression was increased in both males and females in the acute phase ( PND 7) after hyperoxia exposure. However, at PND 21 (recovery phase), female mice showed significantly higher miR-30a expression in the lungs compared with male mice. Female HPMECs showed greater expression of miR-30a in vitro upon exposure to hyperoxia. Delta-like ligand 4 (Dll4) was an miR-30a target in HPMECs and showed sex-specific differential expression. miR-30a increased angiogenic sprouting in vitro in female HPMECs. Lastly, we show decreased expression of miR-30a and increased expression of DLL4 in human BPD lung samples compared with controls. These results support the hypothesis that miR-30a could, in part, contribute to the sex-specific molecular mechanisms in play that lead to the sexual dimorphism in BPD.

Interleukin-3 plays a vital role in hyperoxic acute lung injury in mice via mediating inflammation

BackgroundInterleukin (IL)-3 amplifies inflammation. However, the effect of IL-3 in acute lung injury (ALI), an acute inflammatory disease, is unclear. The aim of this study was to test the hypothesis that IL-3 plays an important role in hyperoxia-induced ALI.MethodsHyperoxic ALI was induced in wild-type (WT) and IL-3 gene disrupted (IL-3−/−) mice by exposure to 100% O2 for 72 h.ResultsHyperoxia increased IL-3 levels in plasma and lung tissues in WT mice. Pulmonary inflammation and edema were detected by histological assay in WT mice exposed to 100% O2 for 72 h. However, the hyperoxia-induced lung histological changes were improved in IL-3−/− mice. The hyperoxia-induced elevation of neutrophils in bronchoalveolar lavage fluids and circulation were reduced in IL-3−/− mice. Meanwhile, the levels of tumor necrosis factor-α and IL-6 were suppressed in IL-3−/− mice compared with WT mice. Moreover, the hyperoxia-induced the activation of IκBα kinase (IKK) β, IκBα phosphorylation, and nuclear factor-κB translocation were inhibited in IL-3−/− mice compared with WT mice.ConclusionsOur results suggest IL-3 is a potential therapeutic target for hyperoxia-induced ALI.

Intratracheal transplantation of mesenchymal stem cells attenuates hyperoxia-induced lung injury by down-regulating, but not direct inhibiting formyl peptide receptor 1 in the newborn mice.

Formyl peptide receptor 1 (FPR1) has been shown to be a key regulator of inflammation. However, its role in bronchopulmonary dysplasia (BPD) has not been delineated yet. We investigated whether FPR1 plays a pivotal role in regulating lung inflammation and injuries, and whether intratracheally transplanted mesenchymal stem cells (MSCs) attenuate hyperoxic lung inflammation and injuries by down-regulating FPR1. Newborn wild type (WT) or FPR1 knockout (FPR1-/-) C57/BL6 mice were randomly exposed to 80% oxygen or room air for 14 days. At postnatal day (P) 5, 2×105 MSCs were intratracheally transplanted. At P14, mice were sacrificed for histopathological and morphometric analyses. Hyperoxia significantly increased lung neutrophils, macrophages, and TUNEL-positive cells, while impairing alveolarization and angiogenesis, along with a significant increase in FPR1 mRNA levels in WT mice. The hyperoxia-induced lung inflammation and lung injuries were significantly attenuated, with the reduced mRNA level of FPR1, in WT mice with MSC transplantation and in FPR1-/- mice, irrespective of MSCs transplantation. However, only MSC transplantation, but not the FPR1 knockout, significantly attenuated the hyperoxia-induced increase in TUNEL-positive cells. Our findings indicate that FPR1 play a critical role in regulating lung inflammation and injuries in BPD, and MSCs attenuate hyperoxic lung inflammation and injuries, but not apoptosis, with down regulating, but not direct inhibiting FPR1.

Transforming growth factor-β downregulates sGC subunit expression in pulmonary artery smooth muscle cells via MEK and ERK signaling.

TGFβ activation during newborn lung injury decreases the expression of pulmonary artery smooth muscle cell (PASMC)-soluble guanylate cyclase (sGC), a critical mediator of nitric oxide signaling. Using a rat PASMC line (CS54 cells), we determined how TGFβ downregulates sGC expression. We found that TGFβ decreases sGC expression through stimulating its type I receptor; TGFβ type I receptor (TGFβR1) inhibitors prevented TGFβ-1-mediated decrease in sGCα1 subunit mRNA levels in the cells. However, TGFβR1-Smad mechanisms do not regulate sGC; effective knockdown of Smad2 and Smad3 expression and function did not protect sGCα1 mRNA levels during TGFβ-1 exposure. A targeted small-molecule kinase inhibitor screen suggested that MEK signaling regulates sGC expression in TGFβ-stimulated PASMC. TGFβ activates PASMC MEK/ERK signaling; CS54 cell treatment with TGFβ-1 increased MEK and ERK phosphorylation in a biphasic, time- and dose-dependent manner. Moreover, MEK/ERK activity appears to be required for TGFβ-mediated sGC expression inhibition in PASMC; MEK and ERK inhibitors protected sGCα1 mRNA expression in TGFβ-1-treated CS54 cells. Nuclear ERK activity is sufficient for sGC regulation; heterologous expression of a nucleus-retained, constitutively active ERK2-MEK1 fusion protein decreased CS54 cell sGCα1 mRNA levels. The in vivo relevance of this TGFβ-MEK/ERK-sGC downregulation pathway is suggested by the detection of ERK activation and sGCα1 protein expression downregulation in TGFβ-associated mouse pup hyperoxic lung injury, and the determination that ERK decreases sGCα1 protein expression in TGFβ-1-treated primary PASMC obtained from mouse pups. These studies identify MEK/ERK signaling as an important pathway by which TGFβ regulates sGC expression in PASMC.

Mice Lacking the Cytochrome P450 1B1 Gene Are Less Susceptible to Hyperoxic Lung Injury Than Wild Type

Supplemental oxygen is a life-saving intervention administered to individuals suffering from respiratory distress, including adults with acute lung injury (ALI) and acute respiratory distress syndrome (ARDS). Despite the clinical benefit, supplemental oxygen can create a hyperoxic environment that increases reactive oxygen species, oxidative stress, and lung injury. We have previously shown that cytochrome P450 (CYP)1A enzymes decrease susceptibility to hyperoxia-induced lung injury. In this investigation, we determined the role of CYP1B1 in hyperoxic lung injury in vivo. Eight- to ten-week old C57BL/6 wild type (WT) and Cyp1b1-/- mice were exposed to hyperoxia (>95% O2) for 24-72 h or maintained in room air (21% O2). Lung injury was assessed by histology and lung weight to body weight (LW/BW) ratios. Extent of inflammation was determined by assessing pulmonary neutrophil infiltration and cytokine levels. Lipid peroxidation markers were quantified by gas chromatography mass spectrometry, and oxidative DNA adducts were quantified by 32P-postlabeling as markers of oxidative stress. We found that Cyp1b1-/- mice displayed attenuation of lung weight and pulmonary edema, particularly after 48-72 h of hyperoxia compared with WT controls. Further, Cyp1b1-/- mice displayed decreased levels of pulmonary oxidative DNA adducts and pulmonary isofurans after 24 h of hyperoxia. Cyp1b1-/- mice also showed increased pulmonary CYP1A1 and 1A2 and mRNA expression. In summary, our results support the hypothesis that Cyp1b1-/- mice display decreased hyperoxic lung injury than wild type counterparts and that CYP1B1 may act as a pro-oxidant during hyperoxia exposure, contributing to increases in oxidative DNA damage and accumulation of lipid hydroperoxides.

The therapeutic effect of mesenchymal stem cells on pulmonary myeloid cells following neonatal hyperoxic lung injury in mice

BackgroundExposure to high levels of oxygen (hyperoxia) after birth leads to lung injury. Our aims were to investigate the modulation of myeloid cell sub-populations and the reduction of fibrosis in the lungs following administration of human mesenchymal stem cells (hMSC) to neonatal mice exposed to hyperoxia.MethodNewborn mice were exposed to 90% O2 (hyperoxia) or 21% O2 (normoxia) from postnatal days 0–4. A sub-group of hyperoxia mice were injected intratracheally with 2.5X105 hMSCs. Using flow cytometry we assessed pulmonary immune cells at postnatal days 0, 4, 7 and 14. The following markers were chosen to identify these cells: CD45+ (leukocytes), Ly6C+Ly6G+ (granulocytes), CD11b+CD11c+ (macrophages); macrophage polarisation was assessed by F4/80 and CD206 expression. hMSCs expressing enhanced green fluorescent protein (eGFP) and firefly luciferase (fluc) were administered via the trachea at day 4. Lung macrophages in all groups were profiled using next generation sequencing (NGS) to assess alterations in macrophage phenotype. Pulmonary collagen deposition and morphometry were assessed at days 14 and 56 respectively.ResultsAt day 4, hyperoxia increased the number of pulmonary Ly6C+Ly6G+ granulocytes and F4/80lowCD206low macrophages but decreased F4/80highCD206high macrophages. At days 7 and 14, hyperoxia increased numbers of CD45+ leukocytes, CD11b+CD11c+ alveolar macrophages and F4/80lowCD206low macrophages but decreased F4/80highCD206high macrophages. hMSCs administration ameliorated these effects of hyperoxia, notably reducing numbers of CD11b+CD11c+ and F4/80lowCD206low macrophages; in contrast, F4/80highCD206high macrophages were increased. Genes characteristic of anti-inflammatory ‘M2’ macrophages (Arg1, Stat6, Retnla, Mrc1, Il27ra, Chil3, and Il12b) were up-regulated, and pro-inflammatory ‘M1’ macrophages (Cd86, Stat1, Socs3, Slamf1, Tnf, Fcgr1, Il12b, Il6, Il1b, and Il27ra) were downregulated in isolated lung macrophages from hyperoxia-exposed mice administered hMSCs, compared to mice without hMSCs. Hydroxyproline assay at day 14 showed that the 2-fold increase in lung collagen following hyperoxia was reduced to control levels in mice administered hMSCs. By day 56 (early adulthood), hMSC administration had attenuated structural changes in hyperoxia-exposed lungs.ConclusionsOur findings suggest that hMSCs reduce neonatal lung injury caused by hyperoxia by modulation of macrophage phenotype. Not only did our cell-based therapy using hMSC induce structural repair, it limited the progression of pulmonary fibrosis.

Abstract P-623: CALCITONIN GENE-RELATED PEPTIDE PROTECTS TYPE II ALVEOLAR EPITHELIAL CELLS FROM HYPEROXIA-INDUCE DNA DAMAGE AND CELL DEATH

Aims & Objectives: The aim of this study is to investigate the effects of CGRP on DNA damage and cell death of alveolar epithelial type II cells (AEC II) exposed to 60% oxygen. Methods AEC II were isolated from 19–20 d fetal rat lung and exposed to air or 60% oxygen with CGRP or CGRP8-37. The cells were evaluated with SP-C immunofluorescence and ROS levels were detected by probing with DCFH-DA. Apoptosis rate and cell cycle of AEC II were analyzed by flow cytometry, and apoptosis was determined by immunblot of actived caspase 3. The DNA damage was confirmed by immunofluorescence of H2AX using High-Content Analysis. Results Results showed that the levels of ROS levels, apoptosis cell number and the expression of γH2AX were markedly increased in hyperoxia group compared with those in air group. Reversely, ROS levels, apoptosis cell number and the expression of γH2AX were significantly lower with a significant arrest of S and G2/M phases in CGRP/O2 group than in hyperoxia or CGRP8-37/O2 group. Conclusions We concluded that CGRP could protect lung epithelium cells against hyperoxic insult and up-regulation of CGRP is probably a potential approach to hyperoxic lung injury.

P-63 - Activation of NF-KB in Airway Epithelia Protects Against Hyperoxia-Induced Proinflammatory Lung Injury

Hyperoxia-induced acute lung injury (HALI) during mechanical ventilation can lead to excessive proinflammatory responses, endothelial and epithelial cell damage, and alveolar edema. We and others have demonstrated that NF-kB activation has a protective effect on cultured lung epithelial cells. The present study investigates the effects of NF-kB activation on ALI using CC10-I- κBαSR transgenic mice that are deficient in the NF-kB activation in airway epithelial cells. These mice showed significantly more severe lung injury than wild type mice after being exposed to 95% oxygen for 3 d, characterized by higher wet/dry weight ratios of lung tissue and increased total protein content in bronchoalveolar lavage fluids (BALF). Severe lung injury was associated with elevated neutrophil infiltration and extracellular HMGB1 accumulation in BALF. In addition, prolonged exposure of cultured human bronchial epithelial (HBE) cells to hyperoxia induced the release of HMGB1. Higher levels of extracellular HMGB1 were observed in HBE cells cultured with BAY 11–7082, an inhibitor of NF- kB activation. These results indicate that the NF-kB signaling pathway in airway epithelial cells plays an important protective role against HMGB1-mediated hyperoxic inflammatory lung injury.