H2O2 Induces Apoptosis in Calf Pulmonary Artery Endothelial Cells

Kim, Bum Shik; Chung, Joo Ho

Abstract

BACKGROUND: Although the pathophysiology of pulmonary vascular injury has been reported, exact its mechanism has not yet been elucidated. Recent studies have reported that reactive oxygen species, including hydrogen peroxide(H2O2) and superoxide, formed in association with a variety of oxidative stress-related diseases, may play an important role in cell injury and apoptosis. In this study, we investigated whether H2O2 induces apoptosis, a possible mechanism of pulmonary vascular injury. MATERIAL AND METHOD: The calf pulmonary artery endothelial cell line(CPAE) was used in the experiment. For the measurement of cytotoxicity of H2O2, 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide(MTT) assay was performed. To find out whether H2O2 affects cell morphology, cells were examined by phase-contrast microscopy. To confirm the induction of apoptosis, H2O2-treated cells were analyzed by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling(TUNEL) assay, 4',6-diamidino-2-phenylindole(DAPI) staining and flow cytometric analysis. RESULT: Compared to the untreated control(100%), viabilities of cells treated with H2O2 of concentrations of 10 microM, 50 microM, 100 microM and 500 microM for 3 hours were about 70%, 33%, 26% and 28%, respectively. Morphologically, H2O2-treated cells(50 microM) showed retraction of processes, cell shrinkage, cytoplasm condensation and irregularity of shape. Both TUNEL assay and DAPI staining also showed typical apoptotic features of nuclear condensation and nuclear fragmentation in some nuclei of H2O2-treated cells. Through flow cytometric analysis, it was determined that there was an increased number of cells in the sub-G1 phase and a decreased number of cells in the G1 phase among the H2O2-treated cells, compared to those of the control.
CONCLUSION
Through morphological and biochemical analyses, it was first demonstrated that cell death induced by H2O2 in CPAE showed classic apoptotic features. These results suggest the possibility that H2O2-induced apoptosis may, in part, be involved in the molecular mechanism of pulmonary vascular injury.