Uridine adenosine tetraphosphate acts as a proangiogenic factor in vitro through purinergic P2Y receptors

Uridine adenosine tetraphosphate (Up(4)A), a dinucleotide, exerts vascular influence via purinergic receptors (PR). We investigated the effects of Up(4)A on angiogenesis and the putative PR involved. Tubule formation assay was performed in a three-dimensional system, in which human endothelial cells were cocultured with pericytes with various Up(4)A concentrations for 5 days. Expression of PR subtypes and angiogenic factors was assessed in human endothelial cells with and without P2Y(6)R antagonist. No difference in initial tubule formation was detected between Up(4)A stimulation and control conditions at day 2. In contrast, a significant increase in vascular density in response to Up(4)A was observed at day 5. Up(4)A at an optimal concentration of 5 mu M promoted total tubule length, number of tubules, and number of junctions, all of which were inhibited by the P2Y(6)R antagonist MRS2578. Higher concentrations of Up(4)A (10 mu(M)) had no effects on angiogenesis parameters. Up(4)A increased mRNA level of P2YRs (P2Y(2)R, P2Y(4)R, and P2Y(6)R) but not P2XR (P2X(4)R and P2X(7)R) or P1R (A(2A)R and A(2B)R), while Up(4)A upregulated VEGFA and ANGPT1, but not VEGFR(2), ANGPT2, Tie1, and Tie2. In addition, Up(4)A increased VEGFA protein levels. Transcriptional upregulation of P2YRs by Up(4)A was inhibited by MRS2578. In conclusion, Up(4)A is functionally capable of promoting tubule formation in an in vitro coculture system, which is likely mediated by pyrimidine-favored P2YRs but not P2XRs or P1Rs, and involves upregulation of angiogenic factors.