VEGF-induced neoangiogenesis is mediated by NAADP and two-pore channel-2 -dependent ca2+signaling

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 Ca²⁺ 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 Ca²⁺ mobilizing messenger, nicotinic acid adenine-dinucleotide phosphate (NAADP), and the specific engagement of the two-pore channel TPC2 subtype on acidic intracellular Ca²⁺ stores, resulting in Ca²⁺ release and angiogenic responses. Targeting this intracellular pathway pharmacologically using the NAADP antagonist Ned-19 or genetically using Tpcn^2-/- mice was found to inhibit angiogenic responses to VEGF in vitro and in vivo. In human umbilical vein endothelial cells (HUVECs) Ned-19 abolished VEGFinduced Ca²⁺ release, impairing phosphorylation of ERK1/2, Akt, eNOS, JNK, cell proliferation, cell migration, and capillary-like tube formation. Interestingly, Tpcn2 shRNA treatment abolished VEGFinduced Ca²⁺ release and capillary-like tube formation. Importantly, in vivo VEGF-induced vessel formation in matrigel plugs in mice was abolished by Ned-19, most notably, failed to occur in Tpcn^{2-/ -} mice, but was unaffected in Tpcn^1-/- animals. These results demonstrate that a VEGFR2/NAADP/TPC2/Ca²⁺ 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.