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.

Cationic liposomes have been intensively studied both in basic and applied research because of their promising potential as non-viral molecular vehicles. This work was aimed to gain more information on the interactions between the plasmamembrane and liposomes formed by a natural phospholipid and a cationic surfactant of the gemini family. The present work was conducted with the synergistic use of diverse experimental approaches: electro-rotation measurements, atomic force microscopy, ζ-potential measurements, laser scanning confocal microscopy and biomolecular/cellular techniques. Electro-rotation measurements pointed out that the interaction of cationic liposomes with the cell membrane alters significantly its dielectric and geometric parameters. This alteration, being accompanied by significant changes of the membrane surface roughness as measured by atomic force microscopy, suggests that the interaction with the liposomes causes locally substantial modifications to the structure and morphology of the cell membrane. However, the results of electrophoretic mobility (ζ-potential) experiments show that upon the interaction the electric charge exposed on the cell surface does not vary significantly, pointing out that the simple adhesion on the cell surface of the cationic liposomes or their fusion with the membrane is to be ruled out. As a matter of fact, confocal microscopy images directly demonstrated the penetration of the liposomes inside the cell and their diffusion within the cytoplasm. Electro-rotation experiments performed in the presence of endocytosis inhibitors suggest that the internalization is mediated by, at least, one specific pathway. Noteworthy, the liposome uptake by the cell does not cause a significant biological damage.

Angiopoietins are vascular factors essential for blood vessel assembly and correct organization and maturation. This study describes a novel calcium-dependent machinery activated through Angiopoietin-1/2-Tie receptor system in HUVECs monolayer. Both cytokines were found to elicit intracellular calcium mobilization. Targeting intracellular Ca2+ signaling, antagonizing IP3 with 2-APB or cADPR with 8Br-cADPR, was found to modulate in vitro angiogenic responses to Angiopoietins in a specific way. 2-APB and 8Br-cADPR impaired the phosphorylation of AKT and FAK induced by Ang-1 and Ang-2. On the other hand, phosphorylation of ERK1/2 and p38, as well as cell proliferation, was not affected by either inhibitor. The ability of ECs to migrate following Angs stimulation, evaluated by “scratch assay,” was reduced by either 2-APB or 8Br-cADPR following Ang-2 stimulation and only slightly affected by 2-APB in cells stimulated with Ang-1. These results identify a novel calcium-dependent machinery involved in the complex interplay regulating angiogenic processes showing that IP3- and cADPR-induced Ca2+ release specifically regulates distinct Angs-mediated angiogenic steps.

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