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 18

Buvinic et al. who demonstrated that functional expression levels of P2Y receptors
along the cord, superficial chorionic vessels and cotyledons of the human placenta
differ [20]. P2Y1 and P2Y2 receptors were not only unevenly distributed along the
human placental vascular tree but also were coupled to different signaling pathways
in the cord/chorionic vessels versus the cotyledon leading to opposing vasomotor
responses.

2.3 Extracellular Nucleotide-Initiated Signaling Pathways

2.3.1 Extracellular Nucleotides Activate FAK and Induce
Cytoskeletal Changes and EC Migration

We have demonstrated that stimulation of HUVECs with ATP or UTP induces
phosphorylation of FAK, paxillin and p130 cas , molecules that were reported to
regulate cell adhesion, spreading and migration [95, 97]. Indeed, we have fur-
ther shown that exposure of HUVECs to extracellular UTP or ATP induces actin
filament formation and cytoskeletal rearrangements resulting in the spreading of
HUVECs, and enhances cell migration [62]. Since chelation of intracellular cal-
cium with BAPTA inhibited UTP-induced FAK phosphorylation, as well as EC
migration, these responses are likely linked to Ca 2+ originating from intracellu-
lar stores, and therefore to activation of P2Y2 and possibly P2Y4 receptors.
Migration of HUVECs in response to ADP, a ligand for P2Y1 receptors, was much
less significant than that observed with ATP or UTP. However, in another study,
involvement of P2Y1 receptors (via MAPK activation) in EC migration has been
documented [109]. Moreover, we showed that the mechanism of nucleotide-induced
migration of HUVECs depends upon PI3K activation, as inhibitors of PI3K, wort-
mannin and LY294002, attenuate migration of HUVEC in response to ATP or
UTP [62].
It has been reported that α v β 3 integrin promotes cell adhesion to the extracel-

lular matrix, cell migration and angiogenesis [67]. Integrins are also involved in
activation of the FAK signaling pathway [108], and an increase in [Ca 2+ ] i is nec-
essary to induce the interaction of integrins with the cytoskeleton [84]. Moreover,
the integrin-binding motif RGD has been identified in P2Y2 receptors and shown
to promote co-localization of this receptor with

α v β 3 integrin, enabling nucleotides
to activate integrin signaling pathways, cytoskeletal rearrangements and cell migra-
tion [8, 43, 72]. All these data prompted us to investigate effects of extracellular
nucleotides on integrin expression. Indeed, we have observed an increase in the
expression of α v integrin in HUVECs stimulated with UTP or ATP. The mecha-

nism of this up-regulation of

α v integrin expression by extracellular nucleotides still
needs to be elucidated. A scheme of extracellular nucleotide effects on FAK, PI3K
and integrin activation is presented in Fig. 2.1.
Phosphorylation of FAK and paxillin has been observed previously in ECs treated
with VEGF [1, 7], and P2Y2 receptors have been recently reported to transactivate