Unravelling the role of NAADP/TPC2 Ca2+ signalling during angiogenesis and tumor progression

Two pore channels (TPCs) represent an emerging class of ion channels principally involved in calcium (Ca2+) mobilization elicited by nicotinic acid adenine dinucleotide phosphate (NAADP) [1, 2]. TPCs localize in the endo-lysosomal compartments and they exist in three isoforms. In particular, we have previously demonstrated the pivotal role of NAADP/TPC2 pathway in angiogenesis and mouse melanoma [3, 4]. In this study, we have explored and characterized the potentiality of Naringenin (Nar), a natural flavonoid, with the aim to investigate a novel pharmacological tool able to interfere with TPC2 activity both in endothelium and in B16 melanoma cells. We demonstrated that Nar impairs intracellular Ca2+ responses of endothelial cells stimulated with VEGF, histamine or NAADP-AM, but not with ATP or Angiopoietin-1 (known to elicit NAADP-independent responses). In addition, we have observed that Nar is able to inhibit angiogenesis in vitro and in an established in vivo murine model. Taken as a whole the present data suggest that Nar inhibition of TPC2 activity and the observed inhibition of angiogenic response to VEGF are linked by impaired intracellular Ca2+ signalling. Several experimental evidence have shown that Ca2+ is involved in fundamental cancer cell activities such as proliferation, migration, invasiveness, metastasis dissemination and survival of tumor cells [5]. Based on this evidence we have focused our attention on the study of VEGF-dependent Ca2+ signalling in melanoma cancer cells [4]. In this work, we also demonstrated that Nar inhibits VEGF and NAADP-dependent Ca2+ release also in B16 melanoma cells. Moreover, Nar impairment of B16 migration/ proliferation and vasculogenic mimicry strengthens the hypothesis that TPC2 represent a potential target for control of the progression of melanoma and possibly other tumors. References: 1. Calcraft, P.J., et al., NAADP mobilizes calcium from acidic organelles through two-pore channels. Nature, 2009. 459(7246): p. 596-600. 2. Pitt, S.J., B. Reilly-O'Donnell, and R. Sitsapesan, Exploring the biophysical evidence that mammalian two-pore channels are NAADP-activated calcium-permeable channels. J Physiol, 2016. 594(15): p. 4171-9. 3. Favia, A., et al., VEGF-induced neoangiogenesis is mediated by NAADP and two-pore channel-2- dependent Ca2+ signaling. Proc Natl Acad Sci U S A, 2014. 111(44): p. E4706-15. 4. Favia, A., et al., NAADP-Dependent Ca(2+) Signaling Controls Melanoma Progression, Metastatic Dissemination and Neoangiogenesis. Sci Rep, 2016. 6: p. 18925. 5. Monteith, G.R., N. Prevarskaya, and S.J. Roberts-Thomson, The calcium-cancer signalling nexus. Nat Rev Cancer, 2017. 17(6): p. 367-38

Regulation of Angiogenic Functions by Angiopoietins through Calcium-Dependent Signaling Pathways

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

Cationic liposomes formulated with DMPC and a gemini surfactant traverse the cell membrane without causing a significant bio-damage

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. © 2014 Elsevier B.V

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

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\u2019s 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

Clinical outcomes.

<p>Clinical outcomes.</p

Distribution of study population

<p>Distribution of study population</p

IGF-1 plasma level distribution at 32 weeks of PMA.

<p>The figure shows the individual values of IGF-1 in all newborns of group A and group B at 32 weeks of PMA: IGF-1 distribution and range values are similar in the two study groups (p 0.48).</p

Nutritional data.

<p>Nutritional data.</p

Feasibility of transferring intensive cared preterm infants from incubator to open crib at 1600 grams

Background: Ability to maintain a normal body temperature in an open crib is an important physiologic competency generally requested to discharge preterm infants from the hospital. The aim of this study is to assess the feasibility of an early weaning protocol from incubator in preterm newborns in a Neonatal Intensive Care Unit. Methods. 101 infants with birth weight < 1600 g were included in this feasibility study. We compared 80 newborns successfully transferred from an incubator to open crib at 1600 g with 21 infants transferred at weight 65 1700 g. The primary outcome was to evaluate feasibility of the protocol and the reasons for the eventual delay. Secondary outcomes were the identification of factors that would increase the likelihood of early weaning, the impact of an earlier weaning on discharge timing, and the incidence of adverse outcomes. Newborns in the study period were then compared with an historical control group with similar characteristics. Results: Early weaning was achieved in 79.2% of infants without significant adverse effects on temperature stability or weight gain. Delayed weaning was mainly due to the need of respiratory support. Gestational age affected the likelihood of early weaning (OR 1.7282 95% CI: 1.3071 - 2.2850). In the multivariate linear regression, early weaning reduced length of stay (LOS) by 25.8 days (p < 0.0001). Conclusions: Preterm infants can be weaned successfully from an incubator to an open crib at weight as low as 1600 grams without significant adverse effect. Early weaning significantly reduces LOS in preterm newborns. \ua9 2014 Barone et al.; licensee BioMed Central Ltd