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2.3.4 Effects of Extracellular Nucleotides on ECs Exposed to High
Glucose
Maintenance of eNOS activity and NO bioavailability in the endothelium has been
proved to be of great importance. In certain diseases, including atherosclerosis and
diabetes mellitus, NO production is impaired as a result of attenuation of eNOS
expression and activity [105]. Reduced NO production, resulting from high glucose-
induced inactivation of eNOS, is one of the key pathophysiological causes of EC
dysfunction and vascular complications in diabetic patients.
It is documented that NO exerts anti-inflammatory effects in ECs through inhibi-
tion of nuclear factor kappa B (NF-
κ
B) activation by various mechanisms, including
stabilization of inhibitor
), nitrosylation of p50, and quenching of reac-
tive oxygen species (ROS) [25]. NO also protects ECs from apoptosis by reducing
oxidative damage [60], promotes angiogenesis by increasing VEGF production [41]
and inhibits platelet aggregation leading to homeostasis of thrombotic events [92].
Relevant to decreased NO bioavailability in diabetes, high glucose/ROS decrease
eNOS activity by activation of PKC
κ
B
α
(I
κ
B
α
II (which negatively regulates eNOS activity
through its phosphorylation at Thr-495 [23, 76]), post-translational O-glycosylation
of the Ser-1177 residue (which decreases phosphorylation of this residue that is
required for eNOS activity [38, 44]), and inhibition of the PI3K/Akt pathway
upstream of eNOS [42].
We hypothesized that the Akt-independent activation of eNOS by PKC
β
in ECs
treated with extracellular nucleotides [29] may be resistant to the damaging effects
of high glucose. Indeed, we verified this hypothesis by showing that, in contrast
to PI3K/Akt-dependent eNOS activation by VEGF that is inhibited by high glu-
cose, 20-30 mM D-glucose did not affect nucleotide-induced phosphorylation and
activation of eNOS (data not shown).
δ
2.3.5 Effects of ATP in Diabetic ApoE-Knockout Mice
To evaluate the in vivo effects of extracellular nucleotides, we carried out pharma-
cokinetic studies in C57BL/6 mice. First, we verified the bioavailability of ATP
in mouse blood after a single intraperitoneal (i.p.) injection of this nucleotide
(1 mmol/kg body weight). ATP levels in the serum showed a 7-fold increase (from
0.7
M) 4 hours after the ATP injection, in agreement with
previously published data [69].
Then, we investigated the effects of ATP administration in atherosclerosis-
prone apolipoprotein E (ApoE)-deficient mice (ApoE-KO) rendered diabetic with
streptozotocin. ApoE-KO mice, control and diabetic, were injected daily with saline
or ATP (1 mmol/kg) i.p. from 12 to 20 weeks of age. Twenty-week old mice were
sacrificed and their aortic arches and hearts were stained with hematoxilin/eosin
(H/E) or analyzed by immunohistochemistry (IHC). Our preliminary data for a small
animal group (n
±
0.13 to 5.3
±
1.1
μ
=
3) show greater atherosclerotic lesion areas in aortic arches of
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