N. Fujita et al. / Inhibition of ACE protects endothelial cells
265
manner of endothelial cell death (apoptosis or necrosis) and the specific protective effects of ACE in-
hibitors may have important clinical implications.
The present study has shown that LDH release by HAEC, which means cell membrane damage as-
sociated with necrosis, increased in a time-dependent manner during reoxygenation. In contrast, DNA
fragmentation (indicating apoptosis) was induced by hypoxia without reoxygenation and reached a peak
after 4 hours. The difference in the manner of cell death was determined by reoxygenation. In fact, re-
oxygenation after hypoxia is well known to activate xanthine oxidase and the production of the ROS,
including superoxide, resulting in cell membrane injury [15,16]. The present study also showed that in-
tracellular ROS increased after reoxygenation. Though intracellular glutathione and thioredoxin are able
to scavenge oxygen radicals, a marked increase of ROS that exceeds the capacity of these scavengers will
cause cellular injury. Some studies have shown that low concentrations of NO might scavenge superox-
ide radicals, and our study showed that NO release by treatment with bradykinin or an ACE inhibitor
could prevent the increase of ROS.
A previous study showed that oxidative stress on cells caused by hypoxia leads to release of cy-
tochrome c from the mitochondria [15] and this may trigger activation of the caspase family that results
in apoptosis. We demonstrated that caspase-3, which is activated in the last stage of induction of apop-
tosis, was increased by hypoxia. It is well known that high concentrations of NO are proapoptotic and
cytotoxic to various cell lines [17,18], while low concentrations of NO are protective against apoptosis.
NO influences various proteins and enzymes associated with apoptosis, and the inhibition of caspases by
NO has attracted attention [19–22]. Therefore, the mechanism by which quinaprilat prevented apoptosis
in the present study is suggested to be inhibition of caspase-3 activation secondary to accumulation of
bradykinin and a resulting increase in the production of NO in hypoxic endothelial cells. In our another
study, we measured NO release from HAEC by using diaminofluorescein (DAF)-2, which is NO-reactive
fluorescence indicator [23]. In the result, the fluorescence intensity by NO release was decreased in hy-
poxia as well as reoxygenation, however it was increased in the presence of quinaprilat (data not shown).
In addition, H/R decreased cNOS mRNA, protein and activity in cultured human coronary artery en-
dothelial cells (HCAEC) in a previous study [24], we also have found that there was little iNOS mRNA
expression in normoxia, hypoxia or H/R detected by RT-PCR method (data not shown).
In conclusion, we demonstrated that quinaprilat has a cytoprotective effect against hypoxia- or H/R-
induced endothelial cell injury, preventing both apoptosis and necrosis, possibly as a result of the ac-
cumulation of bradykinin secondary to ACE inhibition and subsequent increased production of NO via
activation of cNOS.
Acknowledgement
We wish to thank Yoshitomi Pharma. Co. for their technical advice and for providing quinaprilat.
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