Hypoxia-induced endothelial apoptosis through nuclear factor-κB (NF-κB)-mediated bcl-2 suppression -: In vivo evidence of the importance of NF-κB in endothelial cell regulation

The transcription factor nuclear factor-kappa B (NF-kappa B) plays a pivotal role in the coordinated transactivation of cytokine and adhesion molecule genes involved in endothelial activation. Although recent reports have documented the contribution of NF-kappa B to apoptosis, it is still controversial. Especially, the role of NF-kappa B in endothelial apoptosis is largely unknown. Hypoxia significantly induced human aortic endothelial cell death and apoptosis in a time-dependent manner (P<0.01), accompanied by NF-kappa B activation. Decrease in total cell number and increase in apoptotic cells induced by hypoxia were significantly attenuated by NF-kappa B decoy, but not by scrambled decoy, oligodeoxynucleotides (ODNs) (P<0.01). Increase in DNA fragmentation induced by hypoxia was also significantly inhibited by NF-kappa B decoy ODNs as compared with scrambled decoy ODNs (P<0.01). Moreover, transfection of NF-kappa B decoy ODNs resulted in a significant decrease in caspase-3-like activity, which is a common pathway for apoptosis, compared with scrambled decoy ODNs. Importantly, transfection of NF-kappa B decoy ODNs significantly increased protein of bcl-2, an inhibitor of apoptosis, and did not alter bax, a promoter of apoptosis, thereby resulting in a significant increase in the ratio of bcl-2 to bax (P<0.01). bcl-2 mRNA was also decreased by hypoxia, whereas transfection of NF-kappa B decoy ODNs significantly attenuated decrease in bcl-2 mRNA. These results demonstrate that activation of NF-kappa B by hypoxia induced endothelial apoptosis in a bcl-2-dependent manner. The importance of NF-kappa B in endothelial apoptosis was confirmed by the observation that pyrrolidine dithiocarbamate, a potent NF-kappa B inhibitor, prevented endothelial apoptosis, caspase 3-like activity, and bcl-2 downregulation induced by hypoxia. To test this hypothesis in vivo, we transfected NF-kappa B decoy ODNs into rat intact carotid artery after reperfusion injury. Reperfusion injury was associated with a significant increase in endothelial apoptosis at 24 hours, whereas NF-kappa B decoy ODN treatment markedly decreased terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL)-positive endothelial cells at 24 hours after reperfusion (P<0.01), Here, using synthetic double-stranded DNA with high affinity for NF-kappa B as a decoy approach, we demonstrated that activation of NF-kappa B by hypoxia caused aortic endothelial cell death and apoptosis through the suppression of bcl-2. NF-kappa B-mediated endothelial apoptosis induced by hypoxia may be involved in the pathogenesis of endothelial dysfunction observed in cardiovascular ischemic diseases.