Naringenin Impairs Two-Pore Channel 2 Activity And Inhibits VEGF-Induced Angiogenesis

Irene Pafumi,Cristina Giunta,Antonio Filippini,Margherita Festa,Franco Gambale,Armando Carpaneto,Laura Cornara,Fioretta Palombi,Francesca Papacci,Laura Lagostena,Annarita Favia,Vijay Gutla

doi:10.1038/s41598-017-04974-1

Abstract

Our research introduces the natural flavonoid naringenin as a novel inhibitor of an emerging class of intracellular channels, Two-Pore Channel 2 (TPC2), 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, we demonstrated that naringenin dampens intracellular calcium responses of human endothelial cells stimulated with VEGF, histamine or NAADP-AM, but not with ATP or Angiopoietin-1 (negative controls). 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. Overall, the 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. TPC2 inhibition is emerging as a key therapeutic step in a range of important pathological conditions including the progression and metastatic potential of melanoma, Parkinson’s disease, and Ebola virus infection. The identification of naringenin as an inhibitor of TPC2-mediated signaling provides a novel and potentially relevant tool for the advancement of this field of research.

Highlights

IntroductionSince TRPM3-deficient mice display an impaired perception of noxious heat, the inhibition of TRPM3 may represent a novel tool for analgesic therapy[19]
We transiently transformed protoplasts isolated from a mutant of Arabidopsis thaliana lacking the endogenous two-pore channels (TPCs) with hTPC2 equipped with an EGFP fused to its C-terminus[35]
In this work we show that intracellular TPC channels are a target for Nar and that this flavonoid can inhibit the ability of Two-Pore Channel 2 (TPC2) to elicit Ca2+ release from acidic compartments in Nicotinic Acid Adenine Dinucleotide Phosphate (NAADP)-mediated responses to different agonists i.e. VEGF and histamine

Summary

Introduction

Since TRPM3-deficient mice display an impaired perception of noxious heat, the inhibition of TRPM3 may represent a novel tool for analgesic therapy[19] Another member of the TRP family, TRPP2 or polycystin-2, a Ca2+-permeable non-selective cation channel located in the endoplasmic reticulum and in the primary cilium, is modulated by Nar concentrations ranging from 50 to 200 μM20. The present study investigates the ability of Nar to directly bind TPC2 and inhibit its activation as well as to abolish the NAADP/TPC2-dependent mobilization of Ca2+ induced by VEGF. This inhibition is paralleled by an impairment in angiogenic responses to VEGF both in vitro and in an established in vivo murine model. These data identify TPC2 as a novel and important target for Nar, shedding new light on its mechanism of action

Methods

Results

Conclusion