title: Hypoxia-induced endothelial apoptosis through nuclear factor-kappaB (NF-kappaB)-mediated bcl-2 suppression: in vivo evidence of the importance of NF-kappaB in endothelial cell regulation.
abstract: The transcription factor nuclear factor-kappaB (NF-kappaB) 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-kappaB to apoptosis, it is still controversial. Especially, the role of NF-kappaB 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-kappaB activation. Decrease in total cell number and increase in apoptotic cells induced by hypoxia were significantly attenuated by NF-kappaB decoy, but not by scrambled decoy, oligodeoxynucleotides (ODNs) (P<0.01). Increase in DNA fragmentation induced by hypoxia was also significantly inhibited by NF-kappaB decoy ODNs as compared with scrambled decoy ODNs (P<0.01). Moreover, transfection of NF-kappaB 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-kappaB 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-kappaB decoy ODNs significantly attenuated decrease in bcl-2 mRNA. These results demonstrate that activation of NF-kappaB by hypoxia induced endothelial apoptosis in a bcl-2-dependent manner. The importance of NF-kappaB in endothelial apoptosis was confirmed by the observation that pyrrolidine dithiocarbamate, a potent NF-kappaB inhibitor, prevented endothelial apoptosis, caspase 3-like activity, and bcl-2 downregulation induced by hypoxia. To test this hypothesis in vivo, we transfected NF-kappaB 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-kappaB 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-kappaB as a decoy approach, we demonstrated that activation of NF-kappaB by hypoxia caused aortic endothelial cell death and apoptosis through the suppression of bcl-2. NF-kappaB-mediated endothelial apoptosis induced by hypoxia may be involved in the pathogenesis of endothelial dysfunction observed in cardiovascular ischemic diseases.
authors.affiliation: Department of Geriatric Medicine, Division of Gene Therapy Science, Osaka University Medical School, Suita 565, Japan.
authors.forename: H
authors.initials: H
authors.lastname: Matsushita
authors.forename: R
authors.initials: R
authors.lastname: Morishita
authors.forename: T
authors.initials: T
authors.lastname: Nata
authors.forename: M
authors.initials: M
authors.lastname: Aoki
authors.forename: H
authors.initials: H
authors.lastname: Nakagami
authors.forename: Y
authors.initials: Y
authors.lastname: Taniyama
authors.forename: K
authors.initials: K
authors.lastname: Yamamoto
authors.forename: J
authors.initials: J
authors.lastname: Higaki
authors.forename: K
authors.initials: K
authors.lastname: Yasufumi
authors.forename: T
authors.initials: T
authors.lastname: Ogihara
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D002339:Carotid Arteries / Q000503:physiopathology; D002478:Cells, Cultured; D015536:Down-Regulation; D004730:Endothelium, Vascular / Q000503:physiopathology*; D006801:Humans; D000860:Hypoxia / Q000503:physiopathology*; D008297:Male; D016328:NF-kappa B / Q000235:genetics / Q000502:physiology*; D009841:Oligonucleotides / Q000235:genetics / Q000494:pharmacology; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000037:antagonists & inhibitors* / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D015427:Reperfusion Injury / Q000503:physiopathology; D014162:Transfection
chemical_list: D016328:NF-kappa B; D009841:Oligonucleotides; D019253:Proto-Oncogene Proteins c-bcl-2