Hyperoxic Lung Injury In Mice Research Articles

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.

Probucol attenuates hyperoxia-induced lung injury in mice

Hyperoxic lung injury is pathologically characterized by alveolar edema, interlobular septal edema, hyaline membrane disease, lung inflammation, and alveolar hemorrhage. Although the precise mechanism by which hyperoxia causes lung injury is not well defined, oxidative stress, epithelial cell death, and proinflammatory cytokines are thought to be involved. Probucol—a commercially available drug for treating hypercholesterolemia—has been suggested to have antioxidant and antiapoptotic effects. This study aimed to assess whether probucol could attenuate hyperoxic lung injury in mice. Mice were exposed to 95% O2 for 72 h, with or without pre-treatment with 130 μg/kg probucol intratracheally. Probucol treatment significantly decreased both the number of inflammatory cells in the bronchoalveolar lavage fluid and the degree of lung injury in hyperoxia-exposed mice. Probucol treatment reduced the number of cells positive for 8-hydroxyl-2′-deoxyguanosine or terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) and suppressed NF-κB activation, Bax expression, and caspase-9 activation in lung tissues from hyperoxia-exposed mice. These results suggest that probucol can reduce oxidative DNA damage, apoptotic cell death, and inflammation in lung tissues. Intratracheal administration of probucol may be a novel treatment for lung diseases induced by oxidative stress, such as hyperoxic lung injury and acute respiratory distress syndrome.

Targeting complement anaphylatoxin C5a receptor in hyperoxic lung injury in mice.

Receptor binding of complement anaphylatoxin C5a results in proinflammatory activation of numerous diseases, but the role of receptor-mediated action during hyperoxic lung injury has, to the best of our knowledge, not yet been investigated. The contribution of the C5a receptor (C5aR) to hyperoxic lung injury in mice was investigated in this study. The effect of C5aR on hyperoxic lung injury in Balb/c mice was examined employing a C5aR antagonist (C5aRA). The mice were ventilated with 100% oxygen for 36 h with or without the administration of C5aRA. C5aR expression levels in non-treated or 100% oxygen-treated mice were assessed by reverse transcription polymerase chain reaction (RT-PCR) and flow cytometry. The body weight and the relative lung weight of the mice, and the morphological changes in the lung were then determined. The total cell counts and the number of macrophages, neutrophils and lymphocytes in bronchoalveolar lavage fluid (BALF) were determined using cytocentrifuge slides and a hemocytometer. The levels of interleukin-6 (IL-6), monocyte chemotactic protein (MCP-1) and tumor necrosis factor-α (TNF-α) in BALF and the myeloperoxidase (MPO) activity in the lung tissue were measured by enzyme-linked immunosorbent assay. The relative levels of CD68 and F4/80 messenger ribonucleic acid (mRNA) expression in the lung tissue were detected by RT-PCR. The TNF-α, IL-6 and MCP-1 protein expression levels in the lung tissue were assessed by western blot analysis. The results revealed hyperoxia-induced morphological changes, lung injury and increased expression levels of C5aR in lung tissue. The hyperoxia-induced increases in the total cell count and the number of macrophages, neutrophils and lymphocytes in the BALF were all significantly reduced in the mice receiving C5aRA. Treatment with C5aRA also attenuated the morphological changes and reduced MPO activity, and CD68 and F4/80 mRNA expression levels in the lung tissue, as well as the levels of IL-6, MCP-1 and TNF-α in BALF and lung tissue. In conclusion, C5a-C5aR action accelerated hyperoxia-induced lung injury, but this hyperoxic lung injury was attenuated by treatment with C5aRA.

Therapeutic effects of fibroblast grow factor 10 (FGF 10) after hyperoxic lung injury in mice

Therapeutic effects of fibroblast grow factor 10 (FGF 10) after hyperoxic lung injury in mice

Evidence that Fgf10 offers therapeutic opportunities after hyperoxic lung injury in mice

Bronchopulmonary dysplasia (BPD), a chronic lung disease of preterm infants, is characterized by impaired alveolar growth and pathologic vascularization.

Sex-specific differences in hyperoxic lung injury in mice: Implications for acute and chronic lung disease in humans

Sex-specific differences in pulmonary morbidity in humans are well documented. Hyperoxia contributes to lung injury in experimental animals and humans. The mechanisms responsible for sex differences in the susceptibility towards hyperoxic lung injury remain largely unknown. In this investigation, we tested the hypothesis that mice will display sex-specific differences in hyperoxic lung injury. Eight week-old male and female mice (C57BL/6J) were exposed to 72h of hyperoxia (FiO2>0.95). After exposure to hyperoxia, lung injury, levels of 8-iso-prostaglandin F2 alpha (8-iso-PGF 2α) (LC–MS/MS), apoptosis (TUNEL) and inflammatory markers (suspension bead array) were determined. Cytochrome P450 (CYP)1A expression in the lung was assessed using immunohistochemistry and western blotting. After exposure to hyperoxia, males showed greater lung injury, neutrophil infiltration and apoptosis, compared to air-breathing controls than females. Pulmonary 8-iso-PGF 2α levels were higher in males than females after hyperoxia exposure. Sexually dimorphic increases in levels of IL-6 (F>M) and VEGF (M>F) in the lungs were also observed. CYP1A1 expression in the lung was higher in female mice compared to males under hyperoxic conditions. Overall, our results support the hypothesis that male mice are more susceptible than females to hyperoxic lung injury and that differences in inflammatory and oxidative stress markers contribute to these sex-specific dimorphic effects. In conclusion, this paper describes the establishment of an animal model that shows sex differences in hyperoxic lung injury in a temporal manner and thus has important implications for lung diseases mediated by hyperoxia in humans.

Omeprazole Attenuates Hyperoxic Lung Injury in Mice via Aryl Hydrocarbon Receptor Activation and Is Associated with Increased Expression of Cytochrome P4501A Enzymes

Hyperoxia contributes to lung injury in experimental animals and bronchopulmonary dysplasia (BPD) in preterm infants. Cytochrome P4501A (CYP1A) enzymes, which are regulated by the aryl hydrocarbon receptor (AhR), have been shown to attenuate hyperoxic lung injury in rodents. Omeprazole, a proton pump inhibitor, used in humans to treat gastric acid-related disorders, induces hepatic CYP1A in vitro. However, the mechanism by which omeprazole induces CYP1A and its impact on CYP1A expression in vivo and hyperoxic lung injury are unknown. Therefore, we tested the hypothesis that omeprazole attenuates hyperoxic lung injury in adult wild-type (WT) C57BL/6J mice by an AhR-mediated induction of pulmonary and hepatic CYP1A enzymes. Accordingly, we determined the effects of omeprazole on pulmonary and hepatic CYP1A expression and hyperoxic lung injury in adult WT and AhR dysfunctional (AhRd) mice. We found that omeprazole attenuated lung injury in WT mice. Attenuation of lung injury by omeprazole paralleled enhanced pulmonary CYP1A1 and hepatic CYP1A2 expression in the omeprazole-treated mice. On the other hand, omeprazole failed to enhance pulmonary CYP1A1 and hepatic CYP1A2 expression and protect against hyperoxic lung injury in AhRd mice. In conclusion, our results suggest that omeprazole attenuates hyperoxic lung injury in mice by AhR-mediated mechanisms, and this phenomenon is associated with induction of CYP1A enzymes. These studies have important implications for the prevention and/or treatment of hyperoxia-induced disorders such as BPD in infants and acute respiratory distress syndrome in older children and adults.

Vitamin A and Retinoic Acid Act Synergistically to Increase Lung Retinyl Esters During Normoxia and Reduce Hyperoxic Lung Injury in Newborn Mice

We have shown that vitamin A (VA) and retinoic acid (RA) synergistically increase lung retinyl ester content in neonatal rats. To confirm whether this biochemical synergism attenuates early neonatal hyperoxic lung injury in mice, we exposed newborn C57BL/6 mice to 95% O2 or air from birth to 4 d. The agent [vehicle, VA, RA, or the combination vitamin A+retinoic acid (VARA)] was given orally daily. Lung and liver retinyl ester content was measured, and lung injury and development were evaluated. We observed that lung, but not liver, retinyl ester levels were increased more by VARA than by VA or RA alone. Hyperoxic lung injury was reduced by VA and RA, and more so by VARA. VARA attenuated the hyperoxia-induced increases in macrophage inflammatory protein (MIP)-2 mRNA and protein expression, but did not alter hyperoxia-induced effects on peptide growth factors (PDGF, VEGF, and TGF-beta1). The 4-d exposure to hyperoxia or retinoids did not lead to observable differences in lung development. We conclude that the VARA combination has synergistic effects on lung retinyl ester concentrations and on the attenuation of hyperoxia-induced lung injury in newborn mice, possibly by modulation of inflammatory mediators.

Genetic ablation of the Bach1 gene reduces hyperoxic lung injury in mice: Role of IL-6

Bach1 is a transcriptional repressor of the heme oxygenase (HO)-1 gene. Bach1-null (Bach1-/-) mice are reported to be protected from myocardial ischemia/reperfusion injury; however, the effect of Bach1 disruption on another oxidative stress model of hyperoxic lung injury has yet to be determined. To investigate the role of Bach1 in hyperoxic lung injury, Bach1-/- mice and wild-type (WT) mice were exposed to 90% O2. During hyperoxic exposure, the survival of Bach1-/- mice was significantly longer than that of WT mice. However, the administration of zinc protoporphyrin, an inhibitor of HO-1 activity, did not change the mortality in either of the mice, thus suggesting that this protective effect was not mediated by an HO-1 overexpression in Bach1-/- mice. The indices of lung injury in the lungs of Bach1-/- mice were lower than those of WT mice; unexpectedly, however, the levels of IL-6 in bronchoalveolar lavage (BAL) fluid from Bach1-/- mice were significantly higher than those of WT mice. Interestingly, the intrapulmonary administration of small interfering RNA against IL-6 was shown to reduce the IL-6 levels in BAL fluids and shorten the survival in Bach1-/- mice during hyperoxic exposure. In addition, a chromatin immunoprecipitation analysis revealed the binding of Bach1 to the IL-6 promoter and its detachment after oxidative stress. Considering the previous observation that the transgenic mice overexpressing IL-6 are protected from hyperoxic lung injury, these results therefore indicate that IL-6 mediates an increased survival in Bach1-/- mice during hyperoxic exposure.

Thioredoxin-related mechanisms in hyperoxic lung injury in mice.

Reduction of glutathione disulfide (GSSG) to glutathione (GSH) by glutathione reductase (GR) enhances the efficiency of GSH-dependent antioxidant activities. However, GR-deficient (a1Neu) mice are less susceptible to acute lung injury from continuous exposure to > 95% O(2) (96 h: 6.9 +/- 0.1 g right lung/kg body versus room air 3.6 +/- 0.3) than are C3H/HeN control mice (10.6 +/- 1.3 versus 4.2 +/- 0.3, P < 0.001). a1Neu mice have greater hepatic thioredoxin (Trx)1 and Trx2 levels than do C3H/HeN mice, suggesting compensation for the absence of GR. a1Neu mice exposed to hyperoxia for 96 hours showed lower levels of inflammatory infiltrates in lungs than did similarly exposed C3H/HeN mice. Pretreatment with aurothioglucose (ATG), a thioredoxin reductase (TrxR) inhibitor, exacerbated the effects of hyperoxia on lung injury in a1Neu mice (11.6 +/- 0.8, P < 0.001), but attenuated hyperoxic lung edema and inflammation in C3H/HeN mice (6.3 +/- 0.4, P < 0.001). No consistent alterations were observed in lung GSH contents or liver GSH or GSSG levels after ATG pretreatment. The data suggest that modulation of Trx/TrxR systems might provide therapeutically useful alterations of cellular resistance to oxidant stresses. The protective effects of ATG against hyperoxic lung injury could prove to be particularly useful therapeutically.

A novel potent inhibitor of inducible nitric oxide synthase, ONO-1714, reduces hyperoxic lung injury in mice

High-concentration oxygen therapy is used to treat tissue hypoxia, but hyperoxia causes lung injury. Overproduction of nitric oxide by nitric oxide synthase (NOS) is thought to promote hyperoxic lung injury. The present study was conducted to examine the role of inducible nitric oxide synthase (iNOS) in hyperoxic lung injury in mice. Mice were exposed to >98% oxygen for 72 h, and ONO-1714 (0.05 mg/kg) (ONO) was subcutaneously administered to block iNOS. Hyperoxia significantly increased total cell count, protein concentration, and nitrites/nitrates in the bronchoalveolar lavage fluid and proinflammatory cytokines in the lung tissue. ONO significantly prevented the increases in all of these variables. ONO suppressed histologic evidence of lung injury. ONO markedly inhibited iNOS protein expression and nitrotyrosine production in lung homogenates. After exposure to hyperoxia, alveolar epithelial cells stained positively for 8-hydroxy-2'-deoxyguanosine, a proper marker of oxidative DNA damage by reactive oxygen species. ONO attenuated this finding. NOS play important roles in the pathogenesis of hyperoxic lung injury. Selective iNOS inhibitors may be useful for the treatment of hyperoxic lung injury.

P21Waf1/Cip1 and the prevention of oxidative stress.

pulmonary dysfunction resulting from acute oxygen toxicity is due at least in part to the death of lung epithelial cells. Pulmonary epithelial cells are essential to the maintenance of the alveolar-capillary barrier, and compromise may lead to leakage of fluid and proteins in the airways and alveoli

Deficiency in the c-Jun NH2-terminal kinase signaling pathway confers susceptibility to hyperoxic lung injury in mice.

Hyperoxia generates an oxidative stress in the mouse lung, which activates the major stress-inducible kinase pathways, including c-Jun NH2-terminal kinase (JNK). We examined the effect of Jnk1 gene deletion on in vivo responses to hyperoxia in mice. The survival of Jnk1-/- mice was reduced relative to wild-type mice after exposure to continuous hyperoxia. Jnk1-/- mice displayed higher protein concentration in bronchoalveolar lavage (BAL) fluid and increased expression of heme oxygenase-1, a stress-inducible gene, after 65 h of hyperoxia. Contrary to other markers of injury, the leukocyte count in BAL fluid of Jnk1-/- mice was markedly diminished relative to that of wild-type mice. The decrease in BAL leukocyte count was not associated with any decrease in lung myeloperoxidase activity at baseline or after hyperoxia treatment. Pretreatment with inhaled lipopolysaccharide increased BAL neutrophil content and extended hyperoxia survival time to a similar extent in Jnk1-/- and wild-type mice. Associated with increased mortality, Jnk1-/- mice had increased pulmonary epithelial cell apoptosis after exposure to hyperoxia compared with wild-type mice. These results indicate that JNK pathways participate in adaptive responses to hyperoxia in mice.

Role of NRF2 in protection against hyperoxic lung injury in mice.

NRF2 is a transcription factor important in the protection against carcinogenesis and oxidative stress through antioxidant response element (ARE)-mediated transcriptional activation of several phase 2 detoxifying and antioxidant enzymes. This study was designed to determine the role of NRF2 in the pathogenesis of hyperoxic lung injury by comparing pulmonary responses to 95-98% oxygen between mice with site-directed mutation of the gene for NRF2 (Nrf2-/-) and wild-type mice (Nrf2+/+). Pulmonary hyperpermeability, macrophage inflammation, and epithelial injury in Nrf2-/- mice were 7.6-fold, 47%, and 43% greater, respectively, compared with Nrf2+/+ mice after 72 h hyperoxia exposure. Hyperoxia markedly elevated the expression of NRF2 mRNA and DNA-binding activity of NRF2 in the lungs of Nrf2+/+ mice. mRNA expression for ARE- responsive lung antioxidant and phase 2 enzymes was evaluated in both genotypes of mice to identify potential downstream molecular mechanisms of NRF2 in hyperoxic lung responses. Hyperoxia-induced mRNA levels of NAD(P)H:quinone oxidoreductase 1 (NQO1), glutathione-S-transferase (GST)-Ya and -Yc subunits, UDP glycosyl transferase (UGT), glutathione peroxidase-2 (GPx2), and heme oxygenase-1 (HO-1) were significantly lower in Nrf2-/- mice compared with Nrf2+/+ mice. Consistent with differential mRNA expression, NQO1 and total GST activities were significantly lower in Nrf2-/- mice compared with Nrf2+/+ mice after hyperoxia. Results demonstrated that NRF2 has a significant protective role against pulmonary hyperoxic injury in mice, possibly through transcriptional activation of lung antioxidant defense enzymes.

Accumulation of p21(Cip1/WAF1) during hyperoxic lung injury in mice.

Hyperoxic lung injury results in decreased cell proliferation, DNA damage, and cell death. Because the cyclin-dependent kinase inhibitor p21(Cip1/WAF1) (p21) inhibits cell proliferation in G1/S, enhances DNA repair, and regulates apoptosis in some cells, we hypothesized that the expression of p21 would increase in lungs of C57Bl/6J male mice exposed to and recovered from > 95% oxygen. A low level of p21 messenger RNA (mRNA) expression was detected by Northern blot analysis of room air-exposed lungs. Exposure to hyperoxia resulted in a modest increase in p21 mRNA expression by 24 h, followed by a marked induction by 48 to 72 h. In situ hybridization revealed that p21 mRNA abundance increased in bronchiolar epithelium and in resident alveolar cells, but not in smooth-muscle cells or large airway epithelium. Hyperoxia increased the expression of p21 protein by 24 h and continued to increase at 48 and 72 h. Immunohistochemical staining showed that p21 protein accumulated in the bronchiolar epithelium and in alveolar regions that had increased p21 mRNA expression. In contrast, the expression of the cyclin-dependent kinase inhibitor p27(Kip1) was not altered by hyperoxia. To determine whether p21 expression was altered during the repair process, mice were exposed to hyperoxia for 64 h and allowed to recover for up to 4 d in room air. The abundance of p21 mRNA and protein decreased by 1 to 2 d of recovery and returned to room air-exposed levels by 3 to 4 d of recovery. These findings support the concept that bronchiolar epithelial and alveolar cells damaged by hyperoxia express molecules such as p21, which may participate in regulating cell proliferation, DNA repair, and cell death.

P-selectin is upregulated early in the course of hyperoxic lung injury in mice

While treatment with supplemental oxygen is often essential in patients with lung disease, prolonged therapy may cause lung injury by itself. Although the mechanisms responsible for initiating hyperoxic lung damage almost certainly involve primary oxidative transformations, the possible contributions of inflammation to the tissue injury have been attracting increasing research activity. Increases in intercellular adhesion molecule-1 (ICAM-1) coincide with the inflammation, but in other models of inflammation transient adhesion mediated by members of the Selectin gene family was found to be essential before ICAM-1/β 2 interactions could occur. We, therefore, wondered whether a similar sequence of initial transient adhesion followed by subsequent responses would be observed in hyperoxic lung inflammation. We, therefore, determined the effects of hyperoxia exposure on lung mRNA for P-and E-Selectin in mouse lungs. We found that there was no detectable mRNA for E-Selectin through 72 h of hyperoxia exposure by Northern blotting, but that mRNA for P-Selectin was detectable as early as 48 h after initiation of hyperoxia. To determine the location of P-Selectin upregulation we examined hyperoxia-exposed mouse lungs by in situ hybridization and found that the upregulation of P-Selectin at 48 h was localized to large muscularized vessels, at 72 h expression was detected in some medium size muscularized vessels, and at 96 h abundant expression was observed also on nonmuscularized small vessels. In conclusion, increases in mRNA for P-Selectin early in the course of hyperoxia exposure suggest that P-Selectin expression in hyperoxic lungs increases in parallel with upregulation of ICAM-1, leading to the accumulation of neutrophils in hyperoxic lungs, and that interventions targeting these two adhesion molecules may lead to a diminution in hyperoxic lung inflammation and lung injury.

Effect of Fasting on Hyperoxic Lung Injury in Mice: The Role of Glutathione

Fasted mice exposed to 100% oxygen have more lung damage and die sooner than do fed mice. The mechanism responsible for this phenomenon has not been identified. We performed the following experiments to test the hypothesis that reduced glutathione content in lung tissue of fasted mice contributes to the increased susceptibility to hyperoxic lung damage. First, air-exposed mice were fasted for as long as 3 days. They had little change in lung levels of superoxide dismutase (SOD) or catalase, but they had a 41% decrease in glutathione by Day 3 (p less than 0.001). Second, fed mice and fasted mice were exposed to 100% oxygen for as long as 4 days. Both groups had nearly identical values of lung SOD and catalase, but the fasted mice had lower levels of glutathione (p less than 0.001). Third, fed mice received the glutathione synthesis inhibitor buthionine sulfoximine (BSO; 20 mM) in their drinking water for 2 wk and were then exposed to either air or 100% oxygen. Air-exposed mice receiving BSO for 14 days had no change in lung SOD content, a 43% increase in catalase (p less than 0.001), and a 41% decrease in glutathione (p less than 0.01). Oxygen-exposed, BSO-treated mice had no change in SOD and an increase in catalase, but lower glutathione levels, more deaths, and increased lung damage on Day 3 (BAL protein: 1.72 +/- 0.21 versus 0.94 +/- 0.08 mg/ml; p less than 0.01) than did diluent-treated mice. Fourth, fasted mice were given liposomes containing glutathione intratracheally.(ABSTRACT TRUNCATED AT 250 WORDS)