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Fig. 5.4
The effects of purine and pyrimidine nucleotides and non-hydrolysable nucleotide
analogs on VVEC DNA synthesis and migration. (
a
) Growth arrested VVEC (72 h, serum-free
DMEM) were stimulated with indicated nucleotides (100
Ci
[methyl-
3
H] thymidine for 24 h. Incorporated radioactivity was determined in total cell lysates.
(
b
) Growth arrested VVEC (72 h, serum-free DMEM) were plated in permeable inserts (Costar,
8.0
μ
M) in the presence of 0.125
μ
pore size) in serum-free DMEM. The lower transwell compartment contained indicated
nucleotides (100
μ
M). After 24 h, the amount of cells migrated through the filter was determined
as described in [29]. Data represents means S.E. (as % of nonstimulated control) from three to
five independent experiments, conducted on distinct VVEC populations;
∗
p
<0.05,
∗∗
p
<0.01 vs.
nonstimulated control
μ
nucleotides in VVEC. Determining the purinergic receptor profile in VVEC will be
an important step in further understanding the angiogenic characteristics of these
cells.
5.10 Intracellular Signaling Pathways Associated
with Extracellular ATP-Induced Vasa Vasorum
Angiogenesis
Based on findings in other cell types possessing high proliferative potential, empha-
sis was placed on the Phosphoinositide 3-kinase (PI3K), mTOR (
m
ammalian
T
arget
o
f
R
apamycin), and ERK1/2. These pathways play key role in the regulation
of metastatic cell growth, tumor progression and angiogenesis [31, 37, 84, 87].
However, there is a lack of evidence demonstrating the activation of these path-
ways in response to extracellular ATP, as well as evidence linking ATP-dependent
activation of these pathways and angiogenic responses in endothelial cells. It was
found that extracellular ATP mediates its angiogenic effects in VVEC through
dramatic and prolonged activation of PI3K/mTOR and ERK1/2 pathways. The
responses to extracellular ATP might be particularly important in the hypoxic and
inflammed adventitial microenvironment where increased extracellular ATP level
can be expected. In our recent studies we demonstrated that in contrast to VVEC,
extracellular ATP had much lesser effect on mTOR, Akt, and ERK1/2 signaling
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