OBJECTIVES: To study the role of adrenomedullin (ADM) in hyperoxia-induced lung injury by examining the effect of ADM on the expression of calcitonin receptor-like receptor (CRLR), receptor activity-modifying protein 2 (RAMP2), extracellular signal-regulated kinase (ERK), and protein kinase B (PKB) in human pulmonary microvascular endothelial cells (HPMECs) under different experimental conditions. METHODS: HPMECs were randomly divided into an air group and a hyperoxia group ( RESULTS: Compared with the air group, the hyperoxia group had significant increases in the mRNA and protein expression levels of ADM, CRLR, RAMP2, ERK1/2, and PKB ( CONCLUSIONS ERK1/2 and PKB may be the downstream targets of the ADM signaling pathway. ADM mediates the ERK/PKB signaling pathway by regulating CRLR/RAMP2 and participates in the protection of hyperoxia-induced lung injury.
Adrenomedullin/genetics* ; Endothelial Cells ; Humans ; Hyperoxia/complications* ; Lung Injury ; Receptor Activity-Modifying Proteins

OBJECTIVES: To study the role of the low-density lipoprotein receptor-related protein 1 (LRP1)-proline-rich tyrosine kinase 2 phosphorylation (pPyk2)-matrix metalloproteinases 9 (MMP9) pathway in hyperoxia-induced lung injury in neonatal rats. METHODS: A total of 16 neonatal rats were randomly placed in chambers containing room air (air group) or 95% medical oxygen (hyperoxia group) immediately after birth, with 8 rats in each group. All of the rats were sacrificed on day 8 of life. Hematoxylin and eosin staining was used to observe the pathological changes of lung tissue. ELISA was used to measure the levels of soluble LRP1 (sLRP1) and MMP9 in serum and bronchoalveolar lavage fluid (BALF). Western blot was used to measure the protein expression levels of LRP1, MMP9, Pyk2, and pPyk2 in lung tissue. RT-PCR was used to measure the mRNA expression levels of LRP1 and MMP9 in lung tissue. RESULTS: The hyperoxia group had significantly higher levels of sLRP1 and MMP9 in serum and BALF than the air group ( CONCLUSIONS The activation of the LRP1-pPyk2-MMP9 pathway is enhanced in hyperoxia-induced lung injury in neonatal rats, which may be involved in the pathogenesis of bronchopulmonary dysplasia.
Animals ; Animals, Newborn ; Hyperoxia/complications* ; Lung ; Lung Injury/etiology* ; Matrix Metalloproteinase 9/genetics* ; Rats

High concentrations of oxygen, indispensable for the treatment of severe hypoxemia from neonatal as well as adult respiratory distress syndrome, increase the risk of oxygen toxicity. Biochemical mechanisms are lipid peroxidation, protein sulfhydryl oxidation, enzyme inactivation, and DNA damage. Recent reports suggest that cytokines might be involved in free radical injury as well as in adaptive response to hyperoxic injury. However, actual signal transduction pathways involving cytokines have not yet been clarified. In this study we exposed cultured human umbilical vein endothelial cells (HUVECs) to either ambient air or 100% oxygen, and compared for the rate of DNA synthesis ([3H]thymidine uptake) at different time points up to 72 h. After exposing the cells to each treatment condition, we extracted RNA, constructed complementary DNA using reverse transcriptase, amplified the specific DNA segments of cytokines by polymerase chain reaction (PCR), and used the PCR products for gel electrophoresis to examine the bands which signified mRNA levels of corresponding cytokines. There was a significant decrease in the rate of DNA synthesis as early as 24 h. The mRNA expression of IL-1 beta and TNFa seemed less influenced by hyperoxia, while IL-8 and TGF beta showed marked increase in mRNA levels at 6 h of 100% oxygen exposure.
Cells, Cultured ; Cytokines/genetics* ; DNA/biosynthesis ; Endothelium, Vascular/metabolism* ; Human ; Hyperoxia/metabolism* ; RNA, Messenger/analysis* ; Umbilical Veins

PURPOSE: Hyperoxia has the chief biological effect of cell death. We have previously reported that cathepsin B (CB) is related to fetal alveolar type II cell (FATIIC) death and pretreatment of recombinant IL-10 (rIL-10) attenuates type II cell death during 65%-hyperoixa. In this study, we investigated what kinds of changes of CB expression are induced in FATIICs at different concentrations of hyperoxia (65%- and 85%-hyperoxia) and whether pretreatment with rIL-10 reduces the expression of CB in FATIICs during hyperoxia. MATERIALS AND METHODS: Isolated embryonic day 19 fetal rat alveolar type II cells were cultured and exposed to 65%- and 85%-hyperoxia for 12 h and 24 h. Cells in room air were used as controls. Cytotoxicity was assessed by lactate dehydrogenase (LDH) released into the supernatant. Expression of CB was analyzed by fluorescence-based assay upon cell lysis and western blotting, and LDH-release was re-analyzed after preincubation of cathepsin B-inhibitor (CBI). IL-10 production was analyzed by ELISA, and LDH-release was re-assessed after preincubation with rIL-10 and CB expression was re-analyzed by western blotting and real-time PCR. RESULTS: LDH-release and CB expression in FATIICs were enhanced significantly in an oxygen-concentration-dependent manner during hyperoxia, whereas caspase-3 was not activated. Preincubation of FATIICs with CBI significantly reduced LDH-release during hyperoxia. IL-10-release decreased in an oxygen-concentration-dependent fashion, and preincubation of the cells with rIL-10 significantly reduced cellular necrosis and expression of CB in FATIICs which were exposed to 65%- and 85%-hyperoxia. CONCLUSION: Our study suggests that CB is enhanced in an oxygen-concentration-dependent manner, and IL-10 has an inhibitory effect on CB expression in FATIICs during hyperoxia.
Animals ; Cathepsin B/*genetics/metabolism ; *Down-Regulation ; Gene Expression Regulation ; Hyperoxia/*genetics ; Interleukin-10/*pharmacology/physiology ; L-Lactate Dehydrogenase/metabolism ; Necrosis/chemically induced ; Oxygen/metabolism ; Rats

OBJECTIVETo explore the effect of silence of Pin1 expression on hyperoxia-induced apoptosis in alveolar epithelial cells A549.

METHODSA549 cells were divided into four groups: control, hyperoxia, negative lentivirus and Pin1-shRNA hyperoxia. The hyperoxia group was exposed to a mixture of 95%O2 and 5%CO2 for 10 minutes. Then cells were cultured in a closed environment. After 24 hours, the changes of morphology were observed under an inverted microscope. Cell apoptosis was detected by flow cytometry (FCM). The expression of X-linked inhibitor of apoptosis protein (XIAP) and Caspase-9 were detected by immunohistochemistry. The production of reactive oxygen species (ROS) and cellular mitochondria membrane potential (△Ψm) were determined by fluorescence microscopy.

RESULTSUnder the inverted microscope, the A549 cells grew slowly and the changes in morphology of the cells were most obvious in the hyperoxia and negative lentivirus groups. The changes in morphology of A549 cells were obviously improved in the Pin1-shRNA hyperoxia group. The FCM results showed that the apoptosis rate of A549 cells increased, Caspase-9 expression increased, XIAP expression decreased, mitochondrial ROS production increased and mitochondrial membrane potential decreased in the hyperoxia and negative lentivirus groups compared with the control group (P<0.05). Compared with the hyperoxia and negative lentivirus groups, the apoptosis rate of A549 cells decreased, Caspase-9 expression decreased, XIAP expression increased, mitochondrial ROS production decreased and mitochondrial membrane potential increased in the Pin1-shRNA hyperoxia group (P<0.05), although the levels of the indexes did not reach to those of the control group.

CONCLUSIONSSilencing of Pin1 could suppress hyperoxia-induced apoptosis of A549 cells.

Apoptosis ; Caspase 9 ; genetics ; Humans ; Hyperoxia ; pathology ; Membrane Potential, Mitochondrial ; NIMA-Interacting Peptidylprolyl Isomerase ; Peptidylprolyl Isomerase ; physiology ; Reactive Oxygen Species ; metabolism ; X-Linked Inhibitor of Apoptosis Protein ; genetics

BACKGROUNDExposure of adult mice to more than 95% O2 produces a lethal injury by 72 hours. Nuclear factor kappa B (NF-κB) is a transcriptional factor that plays a key role in the modulation of cytokine networks during hyperoxia-induced acute lung injury (ALI). Osteopontin (OPN) is a phosphorylated glycoprotein produced principally by macrophages. Studies have reported that exogenous OPN can maintain the integrity of the cerebral microvascular basement membrane and reduce brain damage through inhibiting NF-κB activities in the brain after subarachnoid hemorrhage. However, it is not clear whether OPN can reduce lung injury during ALI by inhibiting transcriptional signal pathways of NF-κB and consequent inhibition of inflammatory cytokines. Thus we examined the effects and mechanisms of recombinant OPN (r-OPN) on ALI.

METHODSNinety-six mice were randomly divided into phosphate buffered saline (PBS) and r-OPN groups. Mice were put in an oxygen chamber (>95% O2) and assessed for lung injury at 24, 48, and 72 hours. Expressions of NF-κB, matrix metalloproteinases 2 and 9 (MMP-2 and MMP-9), and tissue inhibitors of MMP-2 and MMP-9 (TIMP-1, TIMP-2) mRNA in lungs were examined with RT-PCR. Expression and distribution of NF-κB protein in lungs were measured with immunohistochemistry.

RESULTSExposure to hyperoxia for 72 hours induced more severe lung injury in the PBS group compared with the r-OPN group. Expression of NF-κB mRNA in the PBS group exposed to hyperoxia for 48 and 72 hours was significantly higher than the r-OPN group (P < 0.05). With 72-hour exposure, expression of TIMP-1 mRNA in the r-OPN group was significantly higher than that of the PBS group (P < 0.05). Expression of TIMP-2 mRNA in the r-OPN group at 48 and 72 hours was significantly higher than those in the PBS group (P < 0.05). After 72-hour exposure, expression of NF-κB protein in airway epithelium in the PBS group was significantly higher than that in the r-OPN group (P < 0.05).

CONCLUSIONr-OPN can inhibit the release and activation of MMPs through inhibition of the expression of NF-κB and promotion of the expression of TIMPs, and alleviate hyperoxia-induced ALI.

Acute Lung Injury ; genetics ; metabolism ; Animals ; Hyperoxia ; metabolism ; physiopathology ; Matrix Metalloproteinase 2 ; genetics ; metabolism ; Matrix Metalloproteinase 9 ; genetics ; metabolism ; Mice ; NF-kappa B ; genetics ; metabolism ; Osteopontin ; genetics ; metabolism ; Tissue Inhibitor of Metalloproteinase-1 ; genetics ; metabolism ; Tissue Inhibitor of Metalloproteinase-2 ; genetics ; metabolism

BACKGROUNDExposure of adult mice to more than 95% O(2) produces a lethal injury by 72 hours. Nitric oxide synthase (NOS) is thought to contribute to the pathophysiology of murine hyperoxia-induced acute lung injury (ALI). Osteopontin (OPN) is a phosphorylated glycoprotein produced principally by macrophages. OPN inhibits inducible nitric oxide synthase (iNOS), which generates large amounts of nitric oxide production. However, the relationship between nitric oxide and endogenous OPN in lung tissue during hyperoxia-induced ALI has not yet been elucidated, thus we examined the role that OPN plays in the hyperoxia-induced lung injury and its relationships with NOS.

METHODSOne hundred and forty-four osteopontin knock-out (KO) mice and their matched wild type background control (WT) were exposed in sealed cages > 95% oxygen or room air for 24- 72 hours, and the severity of lung injury was assessed; expression of OPN, endothelial nitric oxide synthase (eNOS) and iNOS mRNA in lung tissues at 24, 48 and 72 hours of hyperoxia were studied by reverse transcription-polymerase chain reaction (RT-PCR); immunohistochemistry (IHC) was performed for the detection of iNOS, eNOS, and OPN protein in lung tissues.

RESULTSOPN KO mice developed more severe acute lung injury at 72 hours of hyperoxia. The wet/dry weight ratio increased to 6.85 +/- 0.66 in the KO mice at 72 hours of hyperoxia as compared to 5.31 +/- 0.92 in the WT group (P < 0.05). iNOS mRNA (48 hours: 1.04 +/- 0.08 vs. 0.63 +/- 0.09, P < 0.01; 72 hours: 0.89 +/- 0.08 vs. 0.72 +/- 0.09, P < 0.05) and eNOS mRNA (48 hours: 0.62 +/- 0.08 vs. 0.43 +/- 0.09, P < 0.05; 72 hours: 0.67 +/- 0.08 vs. 0.45 +/- 0.09, P < 0.05) expression was more significantly increased in OPN KO mice than their matched WT mice when exposed to hyperoxia. IHC study showed higher expression of iNOS (20.54 +/- 3.18 vs. 12.52 +/- 2.46, P < 0.05) and eNOS (19.83 +/- 5.64 vs. 9.45 +/- 3.82, P < 0.05) in lung tissues of OPN KO mice at 72 hours of hyperoxia.

CONCLUSIONOPN can protect against hyperoxia-induced lung injury by inhibiting NOS.

Animals ; Hyperoxia ; genetics ; physiopathology ; Immunohistochemistry ; Lung ; metabolism ; Lung Injury ; etiology ; genetics ; metabolism ; Mice ; Mice, Knockout ; Nitric Oxide Synthase ; genetics ; metabolism ; Nitric Oxide Synthase Type II ; genetics ; Nitric Oxide Synthase Type III ; genetics ; Osteopontin ; genetics ; physiology ; Reverse Transcriptase Polymerase Chain Reaction

OBJECTIVEVascular endothelial growth factor (VEGF) contributes to lung development and recovery of lung structure from lung injury. This study aimed to explore the changes of expression of VEGF protein and mRNA in neonatal rats following hyperoxic lung injury.

METHODSForty-eight Sprague-Dawley neonatal rats were randomly continually exposed to hyperoxia (FiO2=95%) or to room air (FiO2=21%, control group) 30 minutes after birth. VEGF protein and mRNA expression in the lungs was determined by immunohistochemical methods and reverse tanscription polymerasechain reaction (RT-PCR) respectively 3,7 and 14 days after birth.

RESULTSVEGF protein and mRNA expression increased with increasing postnatal age in the control group. In the hyperoxia exposure group VEGF protein expression decreased markedly at 7 days (7.79+/-5.23 vs 12.67+/-3.82; P<0.01) and 14 days of hyperoxia exposure (5.85+/-3.37 vs 15.10+/-8.91; P<0.01) compared with the controls. VEGF mRNA expression in the hyperoxia exposure group was significantly reduced from 3 days (0.78+/-0.22 vs 1.19+/-0.63) through 14 days of hyperoxia exposure (0.48+/-0.12 vs 1.89+/-0.81) compared with the controls (P<0.01).

CONCLUSIONSVEGF is associated with lung development in neonatal rats. Hyperoxia exposure can decrease VEGF protein and VEGF mRNA expression in the lungs of neonatal rats. VEGF might be involved in the pathogenesis of hyperoxic lung injury.

Animals ; Animals, Newborn ; Female ; Hyperoxia ; complications ; Lung ; chemistry ; metabolism ; Lung Diseases ; etiology ; metabolism ; Male ; RNA, Messenger ; analysis ; Rats ; Rats, Sprague-Dawley ; Vascular Endothelial Growth Factor A ; analysis ; genetics

OBJECTIVETo study the effect of hyperoxia exposure on high mobility group protein-B1 (HMGB1) expression in neonatal mice and the role of HMGB1 in the pathogenesis of bronchopulmonary dysplasia (BPD).

METHODSC57BL/6 mice were randomly exposed to 60% O2 or air 1 day after birth. BPD was induced by 60% O2 exposure. The pulmonary tissue samples were harvested 3, 7 and 14 days after exposure. The pathologic changes of pulmonary tissues were observed by hematoxylin and eosin staining, Masson staining and radical alveolar count. The expression of HMGB1 protein in lungs was detected by immunofluorescence. The expression of HMGB1 mRNA was detected by real-time fluorescent quantitative PCR.

RESULTSIn the BPD group, the lungs developed decreased alceolar septation, swollen alveolar epithelium, stroma edema, interstitial fibrosis and developmental lag when compared with the control group. These changes became more obvious with more prolonged hyperoxia exposure. The expression of HMGB1 protein and mRNA 7 and 14 days after exposure increased significantly in the BPD group compared with that in the control group.

CONCLUSIONSHyperoxia exposure results in an increase in lung HMGB1 expression. The increased HMGB1 expression may be associated with the development of BPD.

Animals ; Bronchopulmonary Dysplasia ; etiology ; HMGB1 Protein ; analysis ; genetics ; physiology ; Humans ; Hyperoxia ; complications ; Infant, Newborn ; Lung ; pathology ; Mice ; Mice, Inbred C57BL ; RNA, Messenger ; analysis

OBJECTIVETo study the effects of prolonged 85% oxygen exposure on lung vascular development and the expression of angiopoietin-1 (Ang-1) in the neonatal rat lungs.

METHODSNinety-six Sprague-Dawley rat pups were randomly exposed to air (control group) and 85% oxygen (experimental group) 6 hrs after birth. The rats were sacrificed 3, 7 and 14 days after exposure and their lungs were sampled. The lung sections were stained with hematoxylin and eosin for histological evaluation and analysis of vessel volume density. Expressions of angiopoietin-1 (Ang-1) in lung tissue were measured by immunohistochemistry. Expression of Ang-1 protein and mRNA was detected by Western Blot and Real time-PCR.

RESULTSAfter being exposed to 85% oxygen for 14 days, lung tissues had pathological changes as "new" bronchopulmonary dysplasia (BPD). The RAC on day 7 and day 14 in experimental group decreased significantly as compared with the control group [(10.55 ± 0.13) vs. (11.74 ± 0.19), (12.47 ± 0.05) vs. (15.03 ± 0.16), P < 0.05]. The X-ray showed that the diameter of lung vessel was much smaller and the vessels had less branches in experimental group compared with the control group on day 14. The vessel volume density on day 14 in experimental group decreased significantly as compared with the control group [(3.55 ± 0.09) vs. (6.03 ± 0.16), P < 0.05]. Immunohistochemistry and Western blotting showed that the expressions of Ang-1 protein on day 7 and day 14 in the experimental group decreased significantly as compared with the control group [(4.27 ± 0.34) vs. (3.10 ± 0.29), P < 0.05, (5.65 ± 0.49) vs. (3.21 ± 0.28), P < 0.01], [(0.88 ± 0.31) vs. (0.41 ± 0.12), P < 0.05, (0.90 ± 0.29) vs. (0.21 ± 0.06), P < 0.01]. The expressions of Ang-1 mRNA on day 7 and day 14 in the experimental group also decreased significantly as compared with the control group [(0.85 ± 0.14) vs. (0.44 ± 0.21), P < 0.05, (0.87 ± 0.24) vs. (0.24 ± 0.05), P < 0.01].

CONCLUSIONSProlonged exposure of high concentration of oxygen may cause impairment of lung vascular development by inhibiting expression of Ang-1 in neonatal rats, which is likely to contribute to pathogenesis of BPD.

Angiopoietin-1 ; metabolism ; Animals ; Animals, Newborn ; Hyperoxia ; Lung ; blood supply ; metabolism ; Pulmonary Artery ; growth & development ; RNA, Messenger ; genetics ; Rats ; Rats, Sprague-Dawley ; Vascular Endothelial Growth Factor A ; metabolism