The role of P2Y2 nucleotide receptors in vascular inflammation

Extracellular nucleotides and nucleosides can regulate vascular tone. Extracellular nucleotides are released at the site of tissue injury and may play an important role in the atherogenesis process by promoting monocyte/macrophage adherence and infiltration, impairing endothelial barrier, and stimulating migration and proliferation of vascular smooth muscle cells, thereby contributing to the development of intimal thickening. In vascular injury models, including collared rabbit carotid arteries and rat aorta balloon angioplasty, P2Y2 mRNA was significantly up-regulated while P2Y4 mRNA was unaffected and P2Y6 mRNA was undetectable. This dissertation concerns the specific roles played by the P2Y2 receptor in pathophysiological vascular development. Promotion of monocyte adherence to vascular endothelium by nucleotides was first suggested by the observation that activation of up-regulated P2Y2 receptors in the collared rabbit carotid artery increased monocyte/macrophage infiltration and intimal hyperplasia. We addressed the hypothesis that activation of the P2Y2 receptor modulates the expression of VCAM-1 in vascular endothelial cells that is important for monocyte recruitment. Suppression of P2Y2 receptor expression by P2Y2 anti-sense oligonucleotide inhibited UTP-induced VCAM-1 expression. UTP-induced VCAM-1 expression in human coronary artery endothelial cells (HCAEC) is dependent on intracellular calcium stores, p38 mitogen-activated protein kinase (MAPK) and Rho kinase, but independent of MEK1/2. Expression of VCAM-1 in HCAEC increased the adherence of monocytic xiii U937 cells to HCAEC monolayers. We report here that P2Y2 receptor-induced VCAM-1 expression is mediated by rapid tyrosine phosphorylation of VEGFR-2 in HCAEC. RNA interference (RNAi) targeting of VEGFR-2 expression or inhibition of VEGFR-2 tyrosine kinase activity abolished P2Y2 receptor-mediated VCAM-1 expression. Furthermore, VEGFR-2 association with the P2Y2 receptor upon UTP stimulation is dependent on the integrity of SH3 binding sites in the P2Y2 receptor C-terminal tail and Src kinase activity, and is required for VCAM-1 expression. We also discovered that the P2Y2 receptor is linked to the cytoskeleton through direct interaction with the actin-binding protein filamin A (FLNa), which is a large protein of 280 kD and serves as a cross-linker of actin polymers and as a scaffolding protein for various signaling molecules. This interaction was mapped to the C-terminal tail of the P2Y2 receptor (amino acids 322 to 333) and is required for FLNa phosphorylation induced by extracellular nucleotides. P2Y2 receptor activation causes spreading and migration of vascular smooth muscle cells (SMC) in vitro and ex vivo. Disruption of this interaction impairs the ability of the receptor to induce the spreading and migration of SMC. Taken together, these results demonstrated that extracellular nucleotides and the P2Y2 receptor have several important functions in vascular endothelial cells and smooth muscle cells and may be a key contributor to the progression of cardiovascular diseases, such as atherosclerosis and hypertension. These results encourage drug design targeting the P2Y2 receptor as a means to prevent and/or treat arterial disease.