title: VEGF-induced neoangiogenesis is mediated by NAADP and two-pore channel-2-dependent Ca2+ signaling.
abstract: Vascular endothelial growth factor (VEGF) and its receptors VEGFR1/VEGFR2 play major roles in controlling angiogenesis, including vascularization of solid tumors. Here we describe a specific Ca(2+) signaling pathway linked to the VEGFR2 receptor subtype, controlling the critical angiogenic responses of endothelial cells (ECs) to VEGF. Key steps of this pathway are the involvement of the potent Ca(2+) mobilizing messenger, nicotinic acid adenine-dinucleotide phosphate (NAADP), and the specific engagement of the two-pore channel TPC2 subtype on acidic intracellular Ca(2+) stores, resulting in Ca(2+) release and angiogenic responses. Targeting this intracellular pathway pharmacologically using the NAADP antagonist Ned-19 or genetically using Tpcn2(-/-) mice was found to inhibit angiogenic responses to VEGF in vitro and in vivo. In human umbilical vein endothelial cells (HUVECs) Ned-19 abolished VEGF-induced Ca(2+) release, impairing phosphorylation of ERK1/2, Akt, eNOS, JNK, cell proliferation, cell migration, and capillary-like tube formation. Interestingly, Tpcn2 shRNA treatment abolished VEGF-induced Ca(2+) release and capillary-like tube formation. Importantly, in vivo VEGF-induced vessel formation in matrigel plugs in mice was abolished by Ned-19 and, most notably, failed to occur in Tpcn2(-/-) mice, but was unaffected in Tpcn1(-/-) animals. These results demonstrate that a VEGFR2/NAADP/TPC2/Ca(2+) signaling pathway is critical for VEGF-induced angiogenesis in vitro and in vivo. Given that VEGF can elicit both pro- and antiangiogenic responses depending upon the balance of signal transduction pathways activated, targeting specific VEGFR2 downstream signaling pathways could modify this balance, potentially leading to more finely tailored therapeutic strategies.
authors.affiliation: Department of Anatomy, Histology, Forensic Medicine and Orthopaedics, Unit of Histology and Medical Embryology, Sapienza University of Rome, 00161 Rome, Italy;
authors.forename: Annarita
authors.initials: A
authors.lastname: Favia
authors.affiliation: Experimental Chemotherapy Laboratory, Regina Elena National Cancer Institute, 00128 Rome, Italy;
authors.forename: Marianna
authors.initials: M
authors.lastname: Desideri
authors.affiliation: Department of Anatomy, Histology, Forensic Medicine and Orthopaedics, Unit of Histology and Medical Embryology, Sapienza University of Rome, 00161 Rome, Italy;
authors.forename: Guido
authors.initials: G
authors.lastname: Gambara
authors.affiliation: Department of Anatomy, Histology, Forensic Medicine and Orthopaedics, Unit of Histology and Medical Embryology, Sapienza University of Rome, 00161 Rome, Italy; Institute of Histology and Embryology, Catholic University of the Sacred Heart, 00168 Rome, Italy; and.
authors.forename: Alessio
authors.initials: A
authors.lastname: D'Alessio
authors.affiliation: Department of Pharmacology, University of Oxford, Oxford OX1 3QT, United Kingdom.
authors.forename: Margarida
authors.initials: M
authors.lastname: Ruas
authors.affiliation: Department of Anatomy, Histology, Forensic Medicine and Orthopaedics, Unit of Histology and Medical Embryology, Sapienza University of Rome, 00161 Rome, Italy;
authors.forename: Bianca
authors.initials: B
authors.lastname: Esposito
authors.affiliation: Experimental Chemotherapy Laboratory, Regina Elena National Cancer Institute, 00128 Rome, Italy;
authors.forename: Donatella
authors.initials: D
authors.lastname: Del Bufalo
authors.affiliation: Department of Pharmacology, University of Oxford, Oxford OX1 3QT, United Kingdom.
authors.forename: John
authors.initials: J
authors.lastname: Parrington
authors.affiliation: Department of Anatomy, Histology, Forensic Medicine and Orthopaedics, Unit of Histology and Medical Embryology, Sapienza University of Rome, 00161 Rome, Italy;
authors.forename: Elio
authors.initials: E
authors.lastname: Ziparo
authors.affiliation: Department of Anatomy, Histology, Forensic Medicine and Orthopaedics, Unit of Histology and Medical Embryology, Sapienza University of Rome, 00161 Rome, Italy;
authors.forename: Fioretta
authors.initials: F
authors.lastname: Palombi
authors.affiliation: Department of Pharmacology, University of Oxford, Oxford OX1 3QT, United Kingdom antonio.filippini@uniroma1.it antony.galione@pharm.ox.ac.uk.
authors.forename: Antony
authors.initials: A
authors.lastname: Galione
authors.affiliation: Department of Anatomy, Histology, Forensic Medicine and Orthopaedics, Unit of Histology and Medical Embryology, Sapienza University of Rome, 00161 Rome, Italy; antonio.filippini@uniroma1.it antony.galione@pharm.ox.ac.uk.
authors.forename: Antonio
authors.initials: A
authors.lastname: Filippini
mesh_terms: D000818:Animals; D015220:Calcium Channels / Q000235:genetics / Q000378:metabolism*; D020013:Calcium Signaling / Q000187:drug effects / Q000502:physiology*; D002243:Carbolines / Q000494:pharmacology; D061307:Human Umbilical Vein Endothelial Cells / Q000166:cytology / Q000378:metabolism*; D006801:Humans; D051379:Mice; D018345:Mice, Knockout; D009249:NADP / Q000031:analogs & derivatives / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D018919:Neovascularization, Physiologic / Q000187:drug effects / Q000502:physiology*; D010879:Piperazines / Q000494:pharmacology; D042461:Vascular Endothelial Growth Factor A / Q000235:genetics / Q000378:metabolism*; D040301:Vascular Endothelial Growth Factor Receptor-2 / Q000235:genetics / Q000378:metabolism*; D000097903:Two-Pore Channels
chemical_list: D015220:Calcium Channels; D002243:Carbolines; D009249:NADP; D010879:Piperazines; D042461:Vascular Endothelial Growth Factor A; D040301:Vascular Endothelial Growth Factor Receptor-2; C539827:1-(3-((4-(2-fluorophenyl)piperazin-1-yl)methyl)-4-methoxyphenyl)-2,3,4,9-tetrahydro-1H-pyrido(3,4-b)indole-3-carboxylic acid; C407357:TPCN1 protein, human; D000097903:Two-Pore Channels; C546784:TPCN1 protein, mouse; C530224:TPCN2 protein, human; C546785:TPCN2 protein, mouse; C467484:VEGFA protein, human; C467480:vascular endothelial growth factor A, mouse; C024376:NAADP; C000588866:KDR protein, human; C000592875:Kdr protein, mouse
keywords: NAADP receptors; TPC2; antiangiogenic strategies; calcium signaling; endothelial cells