VEGF-induced neoangiogenesis is mediated by NAADP and two-pore channel-2–dependent Ca²⁺ signaling

• Published in: Proceedings of the National Academy of Sciences - PNAS 2014-11, Vol.111 (44), p.E4706-E4715
• Main Authors: Favia, Annarita, Desideri, Marianna, Gambara, Guido, D'Alessio, Alessio, Ruas, Margarida, Esposito, Bianca, Del Bufalo, Donatella, Parrington, John, Ziparo, Elio, Palombi, Fioretta, Galione, Antony, Filippini, Antonio
• Format: Article
• Language: English
• Subjects: Animals; Biological Sciences; Calcium Channels - genetics; Calcium Channels - metabolism; Calcium Signaling - drug effects; Calcium Signaling - physiology; Carbolines - pharmacology; Human Umbilical Vein Endothelial Cells - cytology; Human Umbilical Vein Endothelial Cells - metabolism; Humans; Mice; Mice, Knockout; NADP - analogs & derivatives; NADP - antagonists & inhibitors; NADP - genetics; NADP - metabolism; Neovascularization, Physiologic - drug effects; Neovascularization, Physiologic - physiology; Piperazines - pharmacology; PNAS Plus; Vascular Endothelial Growth Factor A - genetics; Vascular Endothelial Growth Factor A - metabolism; Vascular Endothelial Growth Factor Receptor-2 - genetics; Vascular Endothelial Growth Factor Receptor-2 - metabolism
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.1406029111

Significance The formation of new blood vessels (neoangiogenesis) accompanies tissue regeneration and healing, but is also crucial for tumor growth, hence understanding how capillaries are stimulated to grow in response to local cues is essential for the much sought-after aim of controlling this process. We have elucidated a Ca ²⁺ signaling pathway involving NAADP, TPCs, and lysosomal Ca ²⁺ release activated in vascular endothelial cells by VEGF, the main angiogenic growth factor, and we show that the angiogenic response can be abolished, in cultured cells and in vivo, by inhibiting components of this signaling cascade. The specificity of this pathway in terms of VEGF receptor subtype, intracellular messengers, target channels and Ca ²⁺ storage organelles, offers new targets for novel antiangiogenic therapeutic strategies. 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 downstr