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

Ischemia and tumor necrosis factor-alpha (TNF alpha) released during ischemia both cause apoptosis and necrosis of myocardial tissues. Since endothelium may be critically important in determination of cardiac function, we examined the interaction between TNF alpha and hypoxia-reoxygenation with regard to induction of apoptosis and underlying signaling pathway in cultured human coronary artery endothelial cells (HCAECs). HCAECs were cultured and exposed to hypoxia alone, hypoxia-reoxygenation, TNF alpha alone, TNF alpha plus hypoxia-reoxygenation, or TNF alpha only during the period of reoxygenation. Apoptosis was evaluated by transmission electron microscopy, DNA nick-end labeling and DNA laddering. Hypoxia alone caused modest time-dependent apoptosis of cultured HCAECs, and reoxygenation increased the number of apoptotic cells (P < 0.01 vs. hypoxia alone). TNF alpha induced concentration-dependent apoptosis, and enhanced reoxygenation-mediated apoptosis in cultured HCAECs (P < 0.01 vs. hypoxia-reoxygenation alone). As expected, monoclonal antibody to TNF alpha significantly blocked the pro-apoptotic effect of TNF alpha-induced apoptosis (P < 0.01). TNF alpha-induced apoptosis was found to be associated with marked activation of protein kinase C (PKC), and pretreatment of cells with a specific PKC inhibitor markedly reduced TNF alpha-mediated PKC activity and apoptosis. These observations indicate that hypoxia alone causes modest apoptosis, reoxygenation increases apoptosis beyond that caused by hypoxia in cultured HCAECs. TNF alpha alone causes apoptosis, and further enhances apoptosis caused by hypoxia-reoxygenation. The activation of PKC plays a critical role in TNF alpha-induced apoptosis of cultured HCAECs.