Hypoxia-Induced Endothelial Apoptosis Through Nuclear Factor-κB (NF-κB)–Mediated bcl-2 Suppression

Author:

Matsushita Hidetsugu1,Morishita Ryuichi1,Nata Toshie1,Aoki Motokuni1,Nakagami Hironori1,Taniyama Yoshiaki1,Yamamoto Kei1,Higaki Jitsuo1,Yasufumi Kaneda1,Ogihara Toshio1

Affiliation:

1. From the Department of Geriatric Medicine (H.M., R.M., M.A., H.N., Y.T., K. Yamamoto, J.H., T.O.) and Division of Gene Therapy Science (R.M., T.N., K. Yasafumi), Osaka University Medical School, Suita 565, Japan.

Abstract

Abstract —The transcription factor nuclear factor-κB (NF-κB) 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-κB to apoptosis, it is still controversial. Especially, the role of NF-κB 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-κB activation. Decrease in total cell number and increase in apoptotic cells induced by hypoxia were significantly attenuated by NF-κB decoy, but not by scrambled decoy, oligodeoxynucleotides (ODNs) ( P <0.01). Increase in DNA fragmentation induced by hypoxia was also significantly inhibited by NF-κB decoy ODNs as compared with scrambled decoy ODNs ( P <0.01). Moreover, transfection of NF-κB 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-κB 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-κB decoy ODNs significantly attenuated decrease in bcl-2 mRNA. These results demonstrate that activation of NF-κB by hypoxia induced endothelial apoptosis in a bcl-2–dependent manner. The importance of NF-κB in endothelial apoptosis was confirmed by the observation that pyrrolidine dithiocarbamate, a potent NF-κB inhibitor, prevented endothelial apoptosis, caspase 3–like activity, and bcl-2 downregulation induced by hypoxia. To test this hypothesis in vivo, we transfected NF-κB 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-κB 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-κB as a decoy approach, we demonstrated that activation of NF-κB by hypoxia caused aortic endothelial cell death and apoptosis through the suppression of bcl-2. NF-κB–mediated endothelial apoptosis induced by hypoxia may be involved in the pathogenesis of endothelial dysfunction observed in cardiovascular ischemic diseases.

Publisher

Ovid Technologies (Wolters Kluwer Health)

Subject

Cardiology and Cardiovascular Medicine,Physiology

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