Anales RANF

P.87 THE ENDOTHELIAL P2Y2 RECEPTOR MODULATES SPROUTING. S. Mühleder 1,2,3 , C. Fuchs 2,4 , J. Basílio 5 , D. Szwarc 2,4 , K. Pill 1,2 , K. Labuda 1,2 , P. Slezak 1,2 , C. Siehs 6 , J. Pröll 7 , E. Priglinger 1,2 , C. Hoffmann 8 , W. Junger 1,9 , J. Grillari 1,2 , H. Redl 1,2 , W. Holnthoner 1,2 1 Ludwig Boltzmann Institute for Experimental and Clinical Traumatology, AUVA Research Center, Vienna, Austria; 2 Austrian Cluster for Tissue Regeneration, Vienna, Austria; 3 Kompetenzzentrum für MechanoBiologie (INTERREG V-A AT-CZ ATCZ133), Vienna, Austria; 4 University of Applied Sciences Technikum Wien, Vienna, Austria; 5 Medical University of Vienna, Vienna, Austria; 6 Mag. Dipl.-Ing. Dr. Christian Siehs, IT-Services, GLN 9110002040261, Vienna, Austria; 7 Center for Medical Research, Johannes Kepler University, Linz, Austria; 8 Center for Molecular Biomedicine, Universitätsklinikum Jena, Friedrich-Schiller-Universität, Jena, Germany 9 Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts, USA. Purinergic P2Y receptors are G protein-coupled surface receptors that respond to extracellular nucleotides and are critical regulators of several functions within the vascular system, including platelet aggregation and vascular inflammation. While endothelial P2Y receptors in particular have been shown to be required for vasodilatation and barrier function, little is known about their role in angiogenesis and endothelial sprouting. Here, we demonstrate that the purinergic receptor P2Y2 is a key regulator of endothelial sprouting. Enhanced expression of P2Y2 in HUVEC and in human iPSC-derived endothelial colony forming cells leads to increased sprouting through an autocrine mechanism in a VEGF-independent manner and to tubulogenesis in fibrin matrices in absence of supporting cells. Overexpression of P2Y11 on HUVEC as a control did not influence sprouting. Conversely, the use of a selective antagonist or siRNA-mediated knock-down of P2Y2 impairs sprouting and tubulogenesis. Mechanistically, overexpression of P2Y2 in endothelial cells results in a pro-angiogenic phenotype, confirmed by the induced expression of the pro-angiogenic genes ANGPT2, CXCR4 and CD34 and constitutive phosphorylation of ERK1/2 and VEGFR-2, suggesting that P2Y2-overexpressing cells acquire an angiogenic tip-cell phenotype. Moreover, stimulation of P2Y2 with its ligand uracil triphosphate does not influence sprouting in wild type endothelial cells unless P2Y2 was constitutively expressed, indicating that a ligand-independent mechanism may be responsible for the observed effects. Additionally, spontaneous vascular network formation induced by P2Y2 overexpression is impaired upon VEGFR-2 inhibition. Our data suggest an essential function of P2Y2 in blood vessel growth, presumably through a cross-talk of P2Y2 with VEGFR-2.