Effect of Short-term Exposure of Different Concentrations of Hyperoxia on Fetal Alveolar Type II Cell Death

PURPOSE: Lung injury imposed by hyperoxia is the most important cause of bronchopulmonary dysplasia (BPD) in premature lungs, and hyperoxia has the chief biological effect of inducing cell death. The objective of this study was to investigate the response of cell death in fetal alveolar type II cells (FATIICs) exposed to different concentrations of hyperoxia for 36 h. METHODS: FATIICs were isolated on embryonic day 19 and exposed to 65%- or 85%-oxygen for 36 h. Cells in room air were used as controls. FACScan was performed in hyperoxic and control samples at 0/6/12/24/36 h, and the patterns of cell death were compared at each time point using flow-cytometry. RESULTS: Cell necrosis as measured by selective propidium iodide staining increased significantly from 12 h of 65%-hyperoxia and 6 h of 85%-hyperoxia, respectively. Cell necrosis increased 1.6-fold, 3.0-fold and 4.6-fold after 12 h, 24 h, and 36 h, respectively during 65%-hyperoxia and increased by 2.4-fold, 3.1-fold, 6.3-fold, and 8.8-fold after 6 h, 12 h, 24 h, and 36 h, respectively during 85%-hyperoxia compared to controls. Apoptotic cells as measured by selective Annexin-V staining peaked at 1.3% at 24 h of 65%-hyperoxia and peaked at 1.2% at 6 h of 85%-hyperoxia, respectively and then decreased rapidly. CONCLUSION: This study shows that exposure to sublethal and lethal hyperoxia increases necrosis of FATIICs remarkably from the early stage of hyperoxia. These findings support the idea that short-term exposure to oxygen from birth may contribute to the evolution of "new" BPD in preterm lungs.

Interleukin-10 Down-Regulates Cathepsin B Expression in Fetal Rat Alveolar Type II Cells Exposed to Hyperoxia

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.

Fetal Alveolar Type II Cell Injury Induced by Short-term Exposure to Hyperoxia

A High concentration of oxygen (>40%) is used as a life-saving therapy in preterm newborns since birth. By generating excess reactive oxygen species, however, hyperoxia can cause lung injury leading to bronchopulmonary dysplasia (BPD). Although hyperoxia-induced lung injury contributes to the evolution of BPD, the mechanisms by which hyperoxia contributes to the genesis of lung injury in preterm lungs are not yet fully defined, and there are no specific measures for the protection of preterm lungs against injury secondary to hyperoxia. Alveolar type II cells are key components of the alveolar structure, and they are responsible for the restoration of normal alveolar epithelium after acute lung injury. However, hyperoxia is primarily delivered to the alveolar epithelium and alveolar type II cells can be the main target for the injury secondary to hyperoxia. To date, my researches have been focused on injury of fetal alveolar type II cells exposed to hyperoxia and the role of anti-inflammatory cytokine, IL-10 minimizing fetal type II cell injury induced by hyperoxia. Based on my previous studies, this article summarizes the cellular and molecular mechanisms of fetal type II cell injury induced in the early stage of hyperoxia and the protective potency of IL-10 in fetal alveolar type II cells and neonatal lungs injured by hyperoxia.