Abstract 13187: C/EBP Homologous Protein (CHOP) Inhibition Protects Against Hyperoxia-induced Endothelial Cell Apoptosis
Objectives: Prolonged oxygen therapy induces endothelial cell death and causes lung injury, which play critical roles in the development of acute respiratory distress syndrome (ARDS). C/EBP homologous protein (CHOP) is a transcription factor that is activated by endoplasmic reticulum (ER) stress and is a key mediator in ER stress-induced apoptosis. The aim of this study was to investigate if CHOP inhibition could decrease hyperoxia-induced endothelial cell death.
Methods: Cultured human endothelial cells (EA.hy926) were exposed to normoxia (room air) or hyperoxia (95% O2) for 24 and 48 hours. CHOP expression and caspase-3 activation were measured by Western blot. Endothelial cell apoptosis was analyzed with Annexin V by flow-cytometry.
Results: Western blotting analysis showed that CHOP expression in cultured human endothelial cells was absent under normoxic condition; however, its expression was increased significantly after 24- and 48-hour hyperoxic exposure compared with normoxic culture (Fig). When cultured human endothelial cells were transfected with CHOP siRNA, CHOP expression induced by tunicamycin (5μg/ml) for 16 hours was decreased by approximately 70% compared with the cells transfected with control (scrambled) siRNA (Fig). Tunicamycin-induced activation of caspase-3 was also reduced by approximately 60% in CHOP knockdown cells compared with control cells (Fig). When exposed to hyperoxia for 48 hours, 11.4±0.82% of endothelial cells underwent apoptosis (Fig), while CHOP knockdown by siRNA reduced hyperoxia-induced endothelial cell apoptosis from 11.4% to 1.03% (n=3, p<0.001) (Fig).
Conclusions: These results indicate that hyperoxia causes ER stress leading to increased CHOP expression; and CHOP knockdown attenuates hyperoxia-induced apoptosis in human endothelial cells. CHOP inhibition may potentially be used as a therapeutic strategy against ER stress-mediated apoptosis and development of ARDS under hyperoxic conditions.
- © 2010 by American Heart Association, Inc.