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

Uridine adenosine tetraphosphate (Up₄A), a dinucleotide, exerts vascular influence via purinergic receptors (PR). We investigated the effects of Up₄A 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 Up₄A concentrations for 5 days. Expression of PR subtypes and angiogenic factors was assessed in human endothelial cells with and without P2Y₆R antagonist. No difference in initial tubule formation was detected between Up₄A stimulation and control conditions at day 2. In contrast, a significant increase in vascular density in response to Up₄A was observed at day 5. Up₄A at an optimal concentration of 5 μM promoted total tubule length, number of tubules, and number of junctions, all of which were inhibited by the P2Y₆R antagonist MRS2578. Higher concentrations of Up₄A (10 μM) had no effects on angiogenesis parameters. Up₄A increased mRNA level of P2YRs (P2Y₂R, P2Y₄R, and P2Y₆R) but not P2XR (P2X₄R and P2X7R) or P1R (A_2AR and A_2BR), while Up₄A upregulated VEGFA and ANGPT1, but not VEGFR₂, ANGPT2, Tie1, and Tie2. In addition, Up4A increased VEGFA protein levels. Transcriptional upregulation of P2YRs by Up₄A was inhibited by MRS2578. In conclusion, Up₄A 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.