Tumor necrosis factor-alpha enhances hypoxia-reoxygenation-mediated apoptosis in cultured human coronary artery endothelial cells: critical role of protein kinase C

Background: Ischemia and tumor necrosis factor-alpha (TNF alpha) released d uring ischemia both cause apoptosis and necrosis of myocardial tissues. Sin ce endothelium may be critically important in determination of cardiac func tion, we examined the interaction between TNF alpha and hypoxia-reoxygenati on with regard to induction of apoptosis and underlying signaling pathway i n cultured human coronary artery endothelial cells (HCAECs). Methods and re sults: HCAECs were cultured and exposed to hypoxia alone, hypoxia-reoxygena tion, TNF alpha alone, TNF alpha plus hypoxia-reoxygenation, or TNF alpha o nly during the period of reoxygenation. Apoptosis was evaluated by transmis sion electron microscopy, DNA nick-end labeling and DNA laddering. Hypoxia alone caused modest time-dependent apoptosis of cultured HCAECs, and reoxyg enation increased the number of apoptotic cells (P<0.01 vs. hypoxia alone). TNF alpha induced concentration-dependent apoptosis, and enhanced reoxygen ation-mediated apoptosis in cultured HCAECs (P<0.01 vs, hypoxia-reoxygenati on alone). As expected, monoclonal antibody to TNF alpha significantly bloc ked the pro-apoptotic effect of TNF alpha-induced apoptosis.(P<0.01). TNF a lpha-induced apoptosis was found to be associated with marked activation of protein kinase C (PKC), and pretreatment of cells with a specific PKC inhi bitor markedly reduced TNF alpha-mediated PKC activity and apoptosis. Concl usion: These observations indicate that hypoxia alone causes modest apoptos is, reoxygenation increases apoptosis beyond that caused by hypoxia in cult ured HCAECs. TNF alpha alone causes apoptosis, and further enhances apoptos is caused by hypoxia-reoxygenation. The activation of PKC plays a critical role in TNF alpha-induced apoptosis of cultured HCAECs. (C) 1999 Elsevier S cience B.V. All rights reserved.