© 2000 American Heart Association, Inc.
Integrative Physiology |
Hypoxia-Induced Endothelial Apoptosis Through Nuclear Factor-
B (NF-B)–Mediated bcl-2 Suppression
In Vivo Evidence of the Importance of NF-B in Endothelial Cell Regulation
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.
Correspondence to Ryuichi Morishita, Associate Professor, Division of Gene Therapy Science, Osaka University Medical School, 2-2 Yamada-oka, Suita 565, Japan. E-mail morishit{at}geriat.med.osaka-u.ac.jp
Abstract—The transcription factor
nuclear factor-
B (NF-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-B to
apoptosis, it is still controversial. Especially, the role of
NF-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-B
activation. Decrease in total cell number and increase in
apoptotic cells induced by hypoxia were significantly
attenuated by NF-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-B decoy ODNs as compared with
scrambled decoy ODNs (P<0.01). Moreover, transfection
of NF-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-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-B decoy ODNs
significantly attenuated decrease in bcl-2 mRNA. These results
demonstrate that activation of NF-B by hypoxia induced
endothelial apoptosis in a bcl-2–dependent
manner. The importance of NF-B in endothelial
apoptosis was confirmed by the observation that pyrrolidine
dithiocarbamate, a potent NF-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-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-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-B as a
decoy approach, we demonstrated that activation of NF-B by
hypoxia caused aortic endothelial cell death
and apoptosis through the suppression of bcl-2.
NF-B–mediated endothelial apoptosis induced
by hypoxia may be involved in the pathogenesis of
endothelial dysfunction observed in
cardiovascular ischemic diseases.
Key Words: hypoxia • nuclear factor-B • bcl-2 • bax • decoy
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