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Two Pore Channels (TPCs) are an emerging family of intracellular channels, expressed on acidic compartments, which mediate calcium signaling evoked by NAADP. In particular, we demonstrated that TPC2 isoform has a main role in angiogenesis (Favia et al. PNAS 2014 Nov 4;111(44):E4706-15). TPC2 inhibition is emerging as a key therapeutic step in a range of important pathological conditions including the progression and metastatic potential of cancer, Parkinson’s disease, and Ebola virus infection. We introduce naringenin, a natural flavonoid, as a novel TPC2 inhibitor as shown by electrophysiological evidence in a heterologous system, i.e. Arabidopsis vacuoles lacking endogenous TPCs. In view of the control exerted by TPC2 on intracellular calcium signaling and angiogenesis, we demonstrate that naringenin dampens intracellular calcium responses of human endothelial cells stimulated with VEGF, histamine or NAADP-AM, but not with ATP or Angiopoietin-1. The ability of naringenin to impair TPC2-dependent biological activities was further explored in an established in vivo model in which VEGF-containing matrigel plugs implanted in mice failed to be vascularized in the presence of naringenin. Our present data suggest that naringenin inhibition of TPC2 activity and the observed inhibition of angiogenic response to VEGF are linked by impaired intracellular calcium signaling. The relationship we describe here between naringenin and TPC2 is therefore likely to have wider implications in systems other than the vascular system, thus representing a novel tool for experimental, and possibly even clinical, research purposes., Italian Journal of Anatomy and Embryology, Vol. 122, No. 1 (Supplement) 2017

We have recently identified a novel transduction pathway through which Vascular Endothelial Growth Factor (VEGF) controls experimentally induced neoangiogenesis, specifically involving endothelial VEGF receptor subtype 2 and the release of intracellular calcium from NAADP (Nicotinic Acid Adenosine Dinucleotide Phosphate) responsive acidic stores (1). We have now extended this research to an in vivo model of tumor angiogenesis and show that the pharmacologic NAADP inhibitor Ned-19 (2) impairs the vascularization, growth and metastatic spreading of the very aggressive VEGF producing murine tumor, B16 melanoma. In parallel in vitro experiments, we tested whether Ned-19 could directly affect the production of VEGF by the tumor cells, and found that treatment of B16 cells with Ned-19 unexpectedly results in increased VEGF release. These observations indicate that in our model 1) tumor angiogenesis is impaired by Ned-19 even in the presence of increased exposure to VEGF and 2) that NAADP system is active also in B16 melanoma cells. On the basis of this second observation further possible direct effects of Ned-19 on melanoma cell aggressiveness such as growth and invasivity are presently investigated and preliminary results suggest that NAADP system inhibition could potentially represent a twofold therapeutic strategy, directly targeting both tumor angiogenesis and tumor cell growth., Italian Journal of Anatomy and Embryology, Vol. 120, No. 1 (Supplement) 2015