Abstract
Alveolar type II cells are main target of hyperoxia-induced lung injury. The authors investigated whether lysosomal protease, cathepsin B (CB), is activated in fetal alveolar type II cells in the transitional period from the canalicular to saccular stages during 65%-hyperoxia and whether CB is related to fetal alveolar type II cell (FATIIC) death secondary to hyperoxia. FATIICs were isolated from embryonic day 19 rats and exposed to 65%-oxygen for 24 h and 36 h. The cells exposed to room air were used as controls. Cell cytotoxicity was assessed by lactate dehydrogenase-release and flow cytometry, and apoptosis was analyzed by TUNEL assay and flow cytometry. CB activity was assessed by colorimetric assay, qRT-PCR and western blots. 65%-hyperoxia induced FATIIC death via necrosis and apoptosis. Interestingly, caspase-3 activities were not enhanced in FATIICs during 65%-hyperoxia, whereas CB activities were greatly increased during 65%-hyperoxia in a time-dependent manner, and similar findings were observed with qRT-PCR and western blots. In addition, the preincubation of CB inhibitor prior to 65%-hyperoxia reduced FATIIC death significantly. Our studies suggest that CB activation secondary to hyperoxia might have a relevant role in executing the cell death program in FATIICs during the acute stage of 65%-hyperoxia.
Highlights
A high concentration of oxygen is a main stay in preterm infants with respiratory distress since birth
Caspase-3 activity was analyzed by colorimetric activity assay upon cell lysis. 65%hyperoxia did not increase cleaved caspase-3 in fetal alveolar type II cell (FATIIC) at 24 h (Control = 0.12 Abs/mg protein/h ± 0.12 vs. Hyperoxia = 0.10 Abs/mg protein/h ± 0.01) and 36 h (Control = 0.20 Abs/mg protein/h ± 0.07 vs. Hyperoxia = 0.21 Abs/mg protein/h ± 0.01) compared to the controls (Figure 3A)
The main findings of the present study is that FATIIC death was induced via necrosis and apoptosis during the acute stage of 65%-hyperoxia, and only cathepsin B (CB), not caspase-3, was enhanced remarkably in a time-dependent manner in FATIICs during 65%-hyperoxia, and preincubaiton of FATIICs with CB inhibitor (CBI) prior to hyperoxia attenuated FATIIC death significantly
Summary
A high concentration of oxygen is a main stay in preterm infants with respiratory distress since birth. Exposure to a high concentration of oxygen (> 40%) for prolonged periods causes lung injuries, which is a major contributing factor to the development of bronchopulmonary dysplasia (Barazzone et al, 2000; O’Reilly et al, 2000; Ward et al, 2000). Hyperoxia has the major biological effect of cell death, which contributes to the genesis of hyperoxia-induced lung injuries (Lee and Choi, 2003). Apoptosis of alveolar type II cells has been regarded as a critical event in the development of lung injuries (Perl et al, 2005). More attention is required to define the mechanisms that induce alveolar type II cell death during hyperoxia
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