Nucleotides and Novel Signaling Pathways in Endothelial Cells: Possible Roles in Angiogenesis, Endothelial Dysfunction and Diabetes Mellitus - Extracellular ATP and Adenosine as Regulators of Endothelial Cell Function - page 24

saline-treated diabetic ApoE-KO mice than ATP-treated animals. Atherosclerotic
lesions in ATP-treated diabetic ApoE-KO mice exhibited a decrease in immunos-
taining for the pro-inflammatory proteins, p65/NF- B, VCAM-1, and P-selectin, as

compared to saline-treated ApoE-KO mice. Administration of ATP also attenuated
expression of the NADPH oxidase subunit p22 phox , which should result in decreased
NADPH oxidase activity and reduced ROS formation (data not shown). These data
need further confirmation in a larger animal group of experimental animals, how-
ever they are very promising, and suggest that administration of P2 receptor agonists
could represent a novel therapeutic approach to protect vascular endothelium from
accelerated atherosclerosis in diabetes.
In a similar, but not diabetic in vivo model, atherosclerotic lesions were ana-
lyzed in ApoE-KO mice and mice deficient in both ApoE and the P2Y1 receptor
[59]. Atherosclerotic lesions in the aortic sinus and entire aorta were smaller in
ApoE/P2Y1-KO mice, compared with ApoE-KO mice. Moreover, in ApoE/P2Y1-
KO mouse aorta, pro-inflammatory VCAM-1 expression and macrophage infil-
tration were reduced, compared with ApoE-KO mice. However, detailed experi-
ments with bone marrow transplantation into ApoE-KO and ApoE/P2Y1-KO mice
indicated that non-hematopoietic-derived P2Y1 receptors, most likely originat-
ing from the endothelial or smooth muscle cells, contribute to development of
atherosclerotic lesions. These data are contradictory to our results, showing rather
anti-atherosclerotic and anti-inflammatory effects of administered ATP in diabetic
ApoE-KO mice. However, as these two animal systems differ, direct comparisons
of the data are difficult.

κ

2.3.6 Other Signaling Pathways Activated by Extracellular
Nucleotides in ECs

We have characterized in detail signaling pathways activated by extracellular
nucleotides, leading to activation of FAK, AMPK and eNOS. However, published
and unpublished results indicate that extracellular nucleotides also activate other
signaling pathways in ECs. These include ERK1/2 [4, 86, 109], p38 MAPK [68,
107, 109] and JNK [109], a family of Ser/Thr protein kinases that transduces sig-
nals from the cell membrane to the nucleus in response to various stimuli and are
implicated in the expression of genes that regulate EC proliferation and apoptosis. In
one study, P2Y receptors were shown to activate ERK in HUVECs, independent of
calcium, through a pathway involving PI3K, 3-phosphoinositide-dependent protein
kinase-1 (PDK1), and PKC was shown to be respon-

sible for ERK activation in response to ATP and UTP in the EAhy926 endothelial
cell line [51].
It was also reported that ATP released by oxidative stress induces P2Y receptor-
dependent activation of cell survival proteins, PI3K and ERK1/2 in human lung
microvascular endothelial cells (HLMVECs), a prime target of hyperoxic injury.
Moreover, under hypoxia, ATP activates mTOR-dependent pathways and increases

ζ [79]. In another study, PKC α