VEGF-induced neoangiogenesis is mediated by NAADP + and two-pore channel-2–dependent Ca2 signaling Annarita Faviaa, Marianna Desiderib, Guido Gambaraa, Alessio D’Alessioa,c, Margarida Ruasd, Bianca Espositoa, Donatella Del Bufalob, John Parringtond, Elio Ziparoa, Fioretta Palombia, Antony Galioned,1,2, and Antonio Filippinia,1,2 aDepartment of Anatomy, Histology, Forensic Medicine and Orthopaedics, Unit of Histology and Medical Embryology, Sapienza University of Rome, 00161 Rome, Italy; bExperimental Chemotherapy Laboratory, Regina Elena National Cancer Institute, 00128 Rome, Italy; cInstitute of Histology and Embryology, Catholic University of the Sacred Heart, 00168 Rome, Italy; and dDepartment of Pharmacology, University of Oxford, Oxford OX1 3QT, United Kingdom Edited* by Michael J. Berridge, The Babraham Institute, Cambridge, United Kingdom, and approved September 24, 2014 (received for review April 3, 2014) Vascular endothelial growth factor (VEGF) and its receptors VEGFR1/ strategies nullify the success of such interventions (5, 7, 8). Re- VEGFR2 play major roles in controlling angiogenesis, including sistance to anti-VEGF therapies may occur through a variety of + vascularization of solid tumors. Here we describe a specific Ca2 mechanisms, including evocation of alternative compensatory signaling pathway linked to the VEGFR2 receptor subtype, control- factors, selection of hypoxia-resistant tumor cells, action of ling the critical angiogenic responses of endothelial cells (ECs) to proangiogenic circulating cells, and increased circulating VEGF. Key steps of this pathway are the involvement of the potent nontumor proangiogenic factors. Moreover, cross-interactions + Ca2 mobilizing messenger, nicotinic acid adenine-dinucleotide (both cellular and humoral) between ECs and other environ- phosphate (NAADP), and the specific engagement of the two-pore mental cues have to be taken into account for the ultimate aim of channel TPC2 subtype on acidic intracellular Ca2+ stores, resulting tailoring therapeutic interventions according to the specific + in Ca2 release and angiogenic responses. Targeting this intracel- pattern of the angiogenic microenvironment and EC conditions lular pathway pharmacologically using the NAADP antagonist (5–7). The search for novel key downstream effectors is there- Ned-19 or genetically using Tpcn2−/− mice was found to inhibit fore of potential significance in the perspective of angiogenesis angiogenic responses to VEGF in vitro and in vivo. In human um- control in cancer progression. bilical vein endothelial cells (HUVECs) Ned-19 abolished VEGF- Autophosphorylation of VEGFR2 upon binding VEGF results in + induced Ca2 release, impairing phosphorylation of ERK1/2, Akt, the activation of downstream signaling cascades through complex eNOS, JNK, cell proliferation, cell migration, and capillary-like tube and manifold molecular interactions that transmit signals leading formation. Interestingly, Tpcn2 shRNA treatment abolished VEGF- to angiogenic responses. Stimulation of different EC types via + induced Ca2 release and capillary-like tube formation. Impor- VEGFR2 results in increases in intracellular free calcium 2+ tantly, in vivo VEGF-induced vessel formation in matrigel plugs concentrations [Ca ]i (9, 10) and the crucial role of this signaling in mice was abolished by Ned-19 and, most notably, failed to occur element in the regulation of EC functions and angiogenesis is −/− −/− in Tpcn2 mice, but was unaffected in Tpcn1 animals. These recognized (11, 12), and thought to be largely mediated by the 2+ results demonstrate that a VEGFR2/NAADP/TPC2/Ca signaling phospholipase Cγ (PLCγ)/inositol 1,4,5 trisphosphate (IP3) sig- 2+ pathway is critical for VEGF-induced angiogenesis in vitro and in naling pathway (10). It has been reported that IP3 releases Ca 2+ vivo. Given that VEGF can elicit both pro- and antiangiogenic from intracellular stores in ECs, increasing [Ca ]i, and is aug- + responses depending upon the balance of signal transduction mented by store-operated Ca2 influx (13). This signaling primes pathways activated, targeting specific VEGFR2 downstream signal- the endothelium for angiogenesis through the activation of ing pathways could modify this balance, potentially leading to downstream effectors such as endothelial nitric oxide synthase more finely tailored therapeutic strategies. Significance endothelial cells | calcium signaling | antiangiogenic strategies | NAADP receptors | TPC2 The formation of new blood vessels (neoangiogenesis) ac- companies tissue regeneration and healing, but is also crucial n the adult the formation of new capillaries is an uncommon for tumor growth, hence understanding how capillaries are Ioccurrence mostly restricted to pathological rather than phys- stimulated to grow in response to local cues is essential for the iological conditions, the majority of blood vessels remaining much sought-after aim of controlling this process. We have quiescent once organ growth is accomplished (1). Physiological elucidated a Ca2+ signaling pathway involving NAADP, TPCs, neoangiogenesis is generally restricted to body sites undergoing and lysosomal Ca2+ release activated in vascular endothelial regeneration or restructuring (e.g., tissue lesion repair and cor- cells by VEGF, the main angiogenic growth factor, and we pus luteum formation), whereas pathological neoangiogenesis show that the angiogenic response can be abolished, in cul- takes place in different diseases ranging from macular de- tured cells and in vivo, by inhibiting components of this sig- generation to atherosclerosis, and is vital for the highly noxious naling cascade. The specificity of this pathway in terms of VEGF development of solid tumors, thus representing a promising receptor subtype, intracellular messengers, target channels and 2+ target for therapeutic strategies (2). Vascular endothelial growth Ca storage organelles, offers new targets for novel anti- factors (VEGF), and in particular the family member VEGF-A, angiogenic therapeutic strategies. are major regulators of angiogenesis and regulate ECs, mainly Author contributions: A. Favia, A.G., and A. Filippini designed research; A. Favia, M.D., G.G., through the stimulation of VEGF receptor-2 (VEGFR2), a re- M.R., and B.E. performed research; A. Favia, M.D., G.G., A.D., M.R., D.D.B., E.Z., F.P., A.G., and ceptor tyrosine kinase, to induce cell proliferation, migration, A. Filippini analyzed data; and A. Favia, J.P., F.P., A.G., and A. Filippini wrote the paper. and sprouting in the early stages of angiogenesis (3, 4). Anti- The authors declare no conflict of interest. angiogenic agents that target VEGF signaling have become an *This Direct Submission article had a prearranged editor. important component of therapies in multiple cancers, but their 1A.G. and A. Filippini contributed equally to this work. use is limited by acquisition of resistance to their therapeutic 2To whom correspondence may be addressed. Email: [email protected] or effects (5, 6). When overall VEGF receptor (VEGFR) signaling [email protected]. is experimentally impaired by the use of blocking antibodies or of This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. specific tyrosine kinase inhibitors, alternative cellular and tissue 1073/pnas.1406029111/-/DCSupplemental. E4706–E4715 | PNAS | Published online October 20, 2014 www.pnas.org/cgi/doi/10.1073/pnas.1406029111 Downloaded by guest on September 29, 2021 + PNAS PLUS (eNOS), protein kinases C (PKC), and mitogen-activated protein ER-mediated Ca2 release as rationalized by the “trigger” kinases (MAPKs). Indeed, it has been reported that the interplay hypothesis (or two-pool model) (17). 2+ 2+ between IP3-dependent Ca mobilization and store-operated Ca NAADP has been described in different cell types as an impor- + + entry produces Ca2 signals whose inhibition impairs the angio- tant Ca2 mobilizing messenger for different agonists (32–37) and genic effect of VEGF (14, 15). Given the complexity of both VEGF has been characterized using the selective membrane-permeant + and Ca2 signaling, and the crucial finding that VEGF evokes pro- noncompetitive antagonist Ned-19, which blocks NAADP-induced + and antiangiogenic responses, it is clear that the specificity of Ca2 release (38). Pretreatment of HUVECs for 30 min with + + VEGF-evoked Ca2 signatures deserves further investigation. 100 μmol/L Ned-19 inhibits VEGF-induced Ca2 release (Fig. 1E + + Differences in Ca2 signatures, which are key to determining and Fig. S1A), and partially blocks histamine-evoked Ca2 release + + specific Ca2 -dependent cellular responses, rely upon often as previously shown (31) (Fig. 1F),butfailedtoblockCa2 responses 2+ complex spatiotemporal variations in [Ca ]i (16). A major de- to thrombin (Fig. 1G), known to be independent of NAADP (31). terminant of these are based on functionally distinct intracellular These data demonstrate that NAADP likely mediates VEGFR2- 2+ 2+ Ca -mobilizing messengers, namely IP3 and cyclic adenosine evoked Ca signaling in HUVECs. + diphosphoribose (cADPR), which mobilize Ca2 from the en- doplasmic reticulum (ER) stores, and nicotinic acid adenine VEGF Induces NAADP-Dependent Ca2+ Mobilization Through the + dinucleotide phosphate (NAADP), which triggers Ca2 release Phosphorylation of VEGFR2. To investigate whether phosphoryla- + from acidic organelles, such as lysosomes and endosomes (17, tion of VEGFR2 is necessary to induce NAADP-dependent Ca2 18). NAADP likely targets