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 , 00168 Rome, Italy; and dDepartment of , , 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 | | 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), 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 a channel distinct from IP3 and rya- release in VEGF signaling, we used TSU-68 (39), which inhibits nodine receptors (RyRs), known as two-pore channels (TPCs) tyrosine phosphorylation of VEGFR2 in VEGF stimulated + (19–25), and the resulting localized NAADP-evoked Ca2 signals HUVECs (Fig. S2A). As shown in Fig. S2 B and C, VEGF-induced, 2+ 2+ may in some cases be globalized via IP3 and RyRs through Ca but not histamine-induced, Ca release, is reduced by pretreatment + -induced Ca2 release (26, 27). However, in a few cell types, with TSU-68. In line with VEGFR2 phosphorylation being upstream + + direct activation of RyRs and Ca2 influx channels by NAADP of NAADP-mediated Ca2 release, we found that the VEGF- have also been proposed as alternative mechanisms (28, 29). It induced phosphorylation of this receptor at Tyr1175 is not affected + has been demonstrated that NAADP-sensitive Ca2 stores are by Ned-19 (Fig. S2D). present in the endothelium, and that NAADP is capable of + regulating vascular smooth muscle contractility and blood pres- Involvement of NAADP-Induced Ca2 Mobilization in VEGFR2- sure by EC-dependent mechanisms (30). In addition, we have Dependent Signaling Pathways. VEGF signaling via VEGFR2 previously demonstrated that NAADP is a specific and essential results in essential EC responses and angiogenesis, comprising intracellular mediator of ECs histamine H1 receptors, evoking production of nitric oxide (NO), increase in endothelial perme- 2+ [Ca ]i release and secretion of von Willebrand factor, which ability, cell proliferation, cell survival and migration (3, 10), and requires the functional expression of TPCs (31). involves a complex network of intracellular transduction path- In the present work, we identify a novel pathway for VEGFR2 ways and various downstream targets. With the aim of identifying signal transduction whereby receptor activation leads to NAADP the involvement of NAADP in VEGF-dependent signaling + + and TPC2-dependent Ca2 release from acidic Ca2 stores, events, we evaluated the activation of known protein targets such which in turn controls angiogenic response in vitro and in vivo. as ERK1/2 MAPK, Akt, JNK, eNOS, and p38 MAPK. It is CELL BIOLOGY These findings demonstrate, to our knowledge for the first time, known that in ECs VEGF-induced phosphorylation of ERK1/2 + the direct relationship between NAADP-mediated Ca2 release MAPK (40), Akt (41), JNK (42), and eNOS (43) requires in- + and the signaling mechanisms underlying ECs angiogenesis me- tracellular Ca2 mobilization. We found that the phosphoryla- diated by VEGF. tion of ERK1/2 MAPK, JNK, Akt, eNOS (Fig. 2A) stimulated by VEGF is reduced by treatment with Ned-19, suggesting that this Results posttranslational modification of these requires + + Evaluation of VEGF-Induced Ca2 Release via VEGFR2. Stimulation of NAADP-dependent Ca2 release. In contrast, Ned-19 failed to A ECs with VEGF is known to enhance production of IP3 and inhibit p38 MAPK phosphorylation (Fig. 2 ). It has been pro- 2+ elevate [Ca ]i (9). To characterize the possible contribution of posed (44) that activation of p38 MAPK negatively regulates + each subtype of VEGFR to Ca2 signaling, primary cultures VEGF-induced angiogenesis by reducing phosphorylation of of HUVECs were stimulated with different concentrations of ERK1/2 MAPK and increasing vascular permeability but the various VEGFR agonists (Fig. 1): VEGF-A165 (also termed mechanisms are unknown. Consistent with this, the inhibition of VEGF), which principally binds to VEGFR2 but may also in- p38 MAPK by SB203580 causes a significant increase in phos- teract with VEGFR1, and VEGF-B that activates VEGFR1 se- phorylation of ERK1/2 MAPK (Fig. 2B) and enhances angio- + lectively. This analysis shows that Ca2 mobilization is triggered genesis in vitro (Fig. 2C), confirming that VEGF activates both only through VEGFR2 (Fig. 1 A and B), the main transducer of proangiogenic and antiangiogenic pathways. VEGF effects on EC differentiation, proliferation, migration, and formation of vascular tubes (10). On the basis of the con- Ned-19 Inhibits VEGF-Dependent Cell Proliferation. As a part of the centration-response curve, 100 μg/L VEGF-A165 was used for all process of angiogenic sprouting induced by VEGF, ECs undergo subsequent experiments. proliferation (45). The involvement of NAADP in this process + To evaluate the involvement of different intracellular Ca2 was evaluated using the NAADP antagonist Ned-19 by mea- + storage organelles in VEGF-induced Ca2 release, we adopted a suring different indicators of cell proliferation. Cell number, pharmacological approach using bafilomycin A1, which inhibits assessed by flow cytometry, appeared to be increased by VEGF + pH-dependent Ca2 uptake into acidic stores by inhibition of the treatment, and Ned-19 markedly reduced this (Fig. 3A and Fig. + vacuolar-type H -ATPase pump, and thapsigargin, which inhib- S1B). In addition, in cells treated for 24 h with VEGF, the ex- + its ER SERCA pumps (17). VEGF-induced Ca2 release was pression of proliferating cellular nuclear antigen α (PCNAα), a significantly impaired not only by thapsigargin (Fig. 1C), but nuclear protein involved in the control of DNA replication, is even more so by bafilomycin A1 (Fig. 1D), showing a major in- reduced by Ned-19 (Fig. 3B), as was the number of viable cells + volvement of acidic Ca2 stores in response to this agonist. These assessed by the thiazolyl blue tetrazolium bromide (MTT) assay data are in accordance with the widely observed cross-talk be- for spectrophotometric determination of cell viability (Fig. 3C). 2+ tween NAADP and IP3/cADPR pathways by which Ca release EC proliferation behavior induced by VEGF is therefore de- + initiated from NAADP-sensitive acidic stores triggers further pendent upon NAADP-mediated Ca2 release. Accordingly,

Favia et al. PNAS | Published online October 20, 2014 | E4707 Downloaded by guest on September 29, 2021 Fig. 1. VEGF mobilizes calcium through VEGFR2, activating Ca2+ release from acidic stores. Live imaging in single FURA-2-AM loaded cells. Intracellular Ca2+ levels

in HUVECs after stimulation with different concentrations (10, 50, 100, 200, 300, and 400 μg/L) of VEGF-A165, also known as VEGF (activator of both receptors, 2+ more selective for VEGFR2) (A) and VEGF-B (VEGFR1 selective agonist) (B). (C and D) Identification of VEGF activated intracellular Ca stores. (C1 and D1)Traces 2+ representing Ca release in cells stimulated with 100 μg/L VEGF after treatment with either vehicle alone (control) or 1 μmol/L thapsigargin for 15 min (C1)or0.5 2+ μmol/L bafilomycin A1 for 1 h (D1). (C2 and D2) Maximum Ca concentrations after stimulation with 100 μg/L VEGF. (E–G) Cells were pretreated with 100 μmol/L − Ned-19 (selective antagonist of NAADP) for 30 min, then stimulated with either 100 μg/L VEGF (E)or100μmol/L histamine (F,positivecontrol)or210 3 U/L 2+ 2+ thrombin (G, negative control). Changes in Ca levels are shown as representative traces (E1, F1,andG1) and as maximum Ca concentrations in bar charts (E2, F2, and G2). Arrow indicates time of agonist addition. Each data point in bar charts represent mean ± SEM from three to five independent experiments, n = 41–180 cells. **P < 0.01; ***P < 0.0002.

+ disruption of acidic Ca2 stores by bafilomycin A1 similarly (Fig. 4D), which clearly showed that the capacity for VEGF- results in decreased cell proliferation (Fig. S3A). induced migration is reduced in cells treated with Ned-19. + NAADP-mediated Ca2 release is thus required for VEGF-de- VEGF-Dependent Cell Migration Is Modulated by NAADP-Dependent pendent migratory capabilities of HUVECs. Ca2+ Release. EC migration, an essential angiogenic process, involves degradation of the extracellular matrix by metal- The Role of NAADP in the Angiogenic Process in Vitro and in Vivo. lopeptidases and the activation of several signaling pathways The formation of capillary-like tubes in vivo is regarded as rep- which converge on cytoskeletal remodeling, resulting in cell ex- resentative of later, differentiative, stages of angiogenesis, and is tension, contraction and forward progression (46). The activity commonly assayed to test compounds for pro- or antiangiogenic of the secreted matrix metallopeptidase 9 (MMP9) is associated effects. Plating onto matrigel matrices stimulates EC attachment, with conversion of pro-MMP9 to MMP9. This activation was migration and differentiation into tubular structures simulating tested by gelatin zymography in culture media. VEGF-dependent the in vivo process (48). When plated on matrigel matrices at secretion and activation of this metallopeptidase was markedly high densities, HUVECs form cord-like capillary structures reduced by Ned-19 (Fig. 4A). Western blot analysis showed that within a few hours, and this process is enhanced by VEGF. VEGF-stimulated phosphorylated focal adhesion kinase (FAK), Importantly, we found that this key step of angiogenesis was known to control cell motility within the extracellular matrix inhibited by Ned-19 pretreatment (Fig. 5A). An approximate (47), was reduced by Ned-19 (Fig. 4B), again implying NAADP estimate of the efficiency of this process can be inferred by the involvement. Further direct demonstration of the NAADP de- extent of cellular network formation, whereby cells first align to pendence of VEGF-induced cell motility came from Boyden form linear segments, and subsequently interconnect to form chamber assays (Fig. 4C) and by scratch wound healing assays closed polygonal structures (49). As shown in Fig. 5B, the

E4708 | www.pnas.org/cgi/doi/10.1073/pnas.1406029111 Favia et al. Downloaded by guest on September 29, 2021 their responses to VEGF 48 h later. The efficiency and specificity PNAS PLUS of TPC2 knock down was evaluated by quantitative RT-PCR; the expression of Tpcn2 was found to be significantly reduced (Fig. S4), whereas Tpcn1 expression was unaffected. As shown in Fig. + 6A, the specific VEGF-evoked Ca2 mobilization was equally inhibited in ECs treated with either Tpcn2-shRNA. Further- more, anti-Tpcn2 shRNA-treated HUVECs, in contrast to those transfected with scrambled shRNA, failed to generate closed polygonal structures (Fig. 6 B and C). The effects of knocking down Tpcn2 expression thus recapitulate the effects of Ned-19 + on VEGF-evoked Ca2 release (Fig. 1) and the formation of tubular structures (Fig. 5 A and B). To further test whether the NAADP putative targets TPC1/TPC2 are involved in the observed angiogenic responses to VEGF, we −/− −/− performed in vivo angiogenesis assays in Tpcn1 (50) and Tpcn2 mice (19), using the matrigel plug assay. Over 5 days, VEGF pro- duced an intense vascularization of the plugs (assessed both by macroscopic observations and by the measure of Hb content) in −/− wild-type (WT) and Tpcn1 mice (Fig. 7 A and B) but not in −/− Tpcn2 mice (Fig. 7 A and C). This finding suggests that TPC2 is essential for VEGF-induced angiogenesis, and importantly dem- onstrates an isoform specific role for TPC2 in this process. A schematic representation based on our experimental evi- Fig. 2. The involvement of Ned-19 in VEGFR-2 mediated signaling. (A) The dence described indicates how the control of VEGF-induced activation of downstream targets after stimulation of HUVECs with VEGF for angiogenesis may occur through the mediation of the VEGFR2/ + 15 min was studied. p-ERK1/2 MAPK, p-JNK, p-Akt, p-eNOS and p-p38 MAPK NAADP/TPC2/Ca2 signaling pathway (Fig. 8). were tested by Western blotting and probed with specific antibodies. (B and C) HUVECs were preincubated for 1 h with p38 inhibitor SB203580, followed Discussion by treatment with VEGF. The effect was evaluated by Western blot as The formation of new vascular capillaries, in physiological, in- phosphorylation of ERK1/2 MAPK (B) and as formation of tubes on Matrigel- coated dishes in the angiogenesis assay (C). Data are representative of at flammatory, or cancer processes, proceeds through a defined least three independent experiments. β-actin was used as a loading control sequence of steps as diverse as cell proliferation, migration, in Western blots. differentiation and morphogenesis, all of which involve control by VEGF-linked signaling cascades. We have shown here, using in vitro and in vivo models of angiogenesis, that the basic steps number of closed polygons formed in cells stimulated with

through which ECs form new capillaries rely upon signaling CELL BIOLOGY VEGF in the presence of Ned-19 is reduced by 73% (P < 0.01) compared with samples stimulated with VEGF only. This indi- + cates the involvement of NAADP-mediated Ca2 signaling at the level of capillary-like formation in vitro. Furthermore, bafilo- + mycin A1 disruption of acidic Ca2 stores also resulted in im- pairment of capillary-like network formation in response to VEGF (Fig. S3B). + The role of NAADP-mediated Ca2 signaling in the overall angiogenic process in vivo was then analyzed by different approaches in murine models. To assess the inhibitory effect of Ned-19 on in vivo angiogenesis, matrigel plug assays were per- formed. Five days after s.c. injections of plugs in C57BL/6 mice, the extent of plug vascularization under different experimental conditions was evaluated by measuring the hemoglobin (Hb) content, and by histological hematoxylin and eosin (H&E) analysis (Fig. 5 C–E). As macroscopically apparent (Fig. 5C), the plugs containing VEGF, but not those containing VEGF plus Ned-19 or vehicle only, can be seen to undergo intense vascu- larization. Hb content in the plugs containing VEGF plus Ned-19 was significantly lower than in the plugs containing VEGF alone (Fig. 5D), and histological analysis of plug vascularization confirmed these results (Fig. 5E). These findings indicate an es- + sential role for NAADP-mediated Ca2 release in both in vitro and in vivo VEGF-induced angiogenic processes.

Involvement of TPC2 Channel in the Response to VEGF in Vitro and in Vivo. Because TPCs have been proposed as principal targets Fig. 3. Ned-19 inhibits VEGF-dependent cell proliferation of HUVECs. Cells + for NAADP in its action to mobilize Ca2 from endolysosomal were treated as indicated for 24 h. (A) Number of cells evaluated by flow cytometry. (B) PCNAα immunoblotting of total cell lysates. β-actin was used stores (19–25), we assessed whether Tpnc2 silencing mimics 2+ as loading control. (C) Cell viability quantified as OD (570 nm) by MTT assay. the effects of Ned-19 in inhibiting VEGF-induced Ca release Data shown in B are representative of three independent experiments. and in vitro angiogenesis. First we transfected HUVECs with two Where applicable, values are expressed as mean ± SEM from three to five different anti-human Tpcn2 shRNA constructs and analyzed independent experiments. **P < 0.01; ***P < 0.0002.

Favia et al. PNAS | Published online October 20, 2014 | E4709 Downloaded by guest on September 29, 2021 Fig. 4. Ned-19 inhibits VEGF-dependent cell migration of HUVECs. (A) Cell lysates were collected and subjected to gelatin zymography to measure MMP9 activity. (B) Cell lysate tested by Western blotting with a p-FAK specific antibody. β-actin was used as loading control. (C) Cells treated as indicated were allowed to migrate for 24 h across the membrane in Boyden chambers. Cells that migrated into the filter were counted in 20 fields per well. Results are expressed as % of control cell migration. (D) Scratch assay to evaluate the cell migration capacity in the indicated experimental conditions. (Upper) Wounded monolayer at the time of manual damage. (Lower) Degree of wound healing in 24 h. Data shown in A, B, and D are representative of three independent experiments. Where applicable, values are expressed as mean ± SEM from three to five independent experiments. *P < 0.05; **P < 0.01.

+ pathways involving VEGFR2, NAADP, TPC2, and Ca2 release responses to VEGF, including proliferation, migration, and from acidic stores. The inhibition of this signaling pathway sig- formation of capillary-like structures (Figs. 3–5). Taken together, nificantly decreases the activation of the known VEGFR2 these data indicate that angiogenic responses to VEGF are de- downstream targets ERK1/2 MAPK, JNK, Akt, and eNOS with pendent on NAADP signaling, and confirm that the p38 MAPK the exception of p38 MAPK, and blocks angiogenesis in both in pathway is not involved in these angiogenic responses. Because vitro and in vivo models. VEGF has the potential to elicit both pro- and antiangiogenic In line with previous reports (51, 52) our data using HUVECs biological responses, depending upon the balance of intracellular + confirm that intracellular Ca2 increases in response to VEGF signaling pathways activated by this agonist, the identification of are not mediated by VEGFR1 signaling, but selectively involve a pathway apparently restricted to its proangiogenic effects VEGFR2, the major receptor subtype mediating angiogenic without weakening antiangiogenic pathways offers a new responses (10). The role of each subtype of VEGFR is a crucial conceptual tool to manipulate the balance between these two issue for the development of targeted strategies aimed at mod- antagonistic responses. + ulating angiogenesis. VEGFR1 and VEGFR2 are differentially The involvement of Ca2 in the responses to VEGFR2 stim- expressed in normal cells (53), but their absolute and relative ulation is well recognized, although the available information can expression levels can considerably vary in different tumors hardly be framed into a comprehensive picture, given the mul- + (54), and they are known to couple to different intracellular tiplicity of the pathways involved. The role played by specific Ca2 signaling pathways although they are also subject to extensive signatures are pivotal: a wealth of data demonstrates that cross-talk (10). Specific antiangiogenic pathways are also known cellular processes are controlled by spatiotemporally complex + to be activated by VEGF via specific pathways operating not only Ca2 signaling patterns which are decoded to elicit different through VEGFR1 (52, 55) but also through VEGFR2. In par- biological responses (16, 17). In ECs, we have previously ticular, inhibition of p38 MAPK has been shown to enhance in demonstrated that the secretion of von Willebrand factor is + vitro and in vivo angiogenesis induced by VEGF (44). Impor- differentially regulated by different Ca2 signals, dependent on + tantly, internalization of the VEGFR2 is necessary for acti- the recruitment of specific Ca2 mobilizing messengers. NAADP + vation and downstream signaling involving ERK1/2 MAPK and was found to mediate the release of intracellular Ca2 triggered Akt proteins, whereas p38 MAPK activation is independently specifically through histamine H1 receptor, resulting in secretion + regulated and is characterized by a temporally and spatially of von Willebrand factor, whereas thrombin-mediated Ca2 sig- + distinct pathway (56, 57). Our Western blot experiments char- naling was NAADP-independent (31). In our study of Ca2 signals acterizing the effects of the NAADP antagonist Ned-19 in triggered by VEGF, we have focused on the most ancient yet most + HUVECs show that various downstream effector kinases, but recently discovered Ca2 mobilizing second messenger, NAADP, not p38 MAPK, are ultimately dependent on NAADP-evoked and its putative target channels, TPCs on acidic stores (58). Our + Ca2 release. Parallel in vitro experiments show that under results show that inhibition of the NAADP pathway abolishes + similar conditions Ned-19 treatment strongly reduces angiogenic intracellular Ca2 increases following VEGF stimulation without

E4710 | www.pnas.org/cgi/doi/10.1073/pnas.1406029111 Favia et al. Downloaded by guest on September 29, 2021 PNAS PLUS

Fig. 5. NAADP pathway inhibition impairs of VEGF-induced vessel formation in vitro and in vivo. (A) Representative images of one of three independent experiments. HUVECs were plated in Matrigel-coated dishes and incubated in EBM-2 with vehicle alone (Ctr), in medium supplemented with VEGF or Ned-19, or in medium containing both VEGF and Ned-19. Each condition was tested in triplicate for each individual experiment. (B) Quantitative evaluation of tube formation as the number of closed polygons formed in five fields for each experimental condition. (C–E) In vivo vessel formation was assessed after s.c. injection of C57BL/6 mice with Matrigel plugs containing either vehicle or VEGF or VEGF and 50, 100, or 150 μmol/L Ned-19. After 5 d, mice were killed and vascularization was evaluated both macroscopically as shown in two representative images (C) and as Hb content expressed as absorbance (OD)/1 g matrigel plug (D). (E) Representative images of H&E stained paraffin sections of VEGF-containing plugs in presence or absence of Ned-19. n = 15 plugs for each condition. Where applicable values from three independent experiments are expressed as mean ± SEM *P < 0.05; **P < 0.01.

interfering with the phosphorylation of Y1175 residue of induce tubular structures as a hallmark of angiogenic progression CELL BIOLOGY + VEGFR2. The biphasic Ca2 response to NAADP and the de- (Fig. 6B). These experiments were translated into an in vivo + pendence of the sustained phase of Ca2 release on the ER are model using the matrigel plug assay. Paralleling the HUVEC + consistent with the idea that NAADP-induced Ca2 signals are experiments, Ned-19 inhibited VEGF-induced vascularization in −/− small and localized, but act as a trigger for larger and global wild type mice, whereas Tpcn2 mice treated with VEGF alone + intracellular Ca2 mobilization through coupling to the ER sys- phenocopied the effects of Ned-19 in WT animals. Importantly, −/− tem (17). Our data, obtained through pharmacological impair- normal VEGF-induced vascularization was seen in Tpcn1 + + ment of Ca2 increase from different Ca2 store compartments, mice, showing a clear difference in roles for TPC1 and TPC2 are consistent with this model. Indeed, treatment of ECs with in angiogenic responses to VEGF. Recent work has shown that + both thapsigargin and bafilomycin, inhibited VEGF-induced Ca2 TPC1, although regulated by NAADP, has marked differences + release. These data indicate that VEGFR2-mediated Ca2 in ion conductances to TPC2 and may be largely a proton signaling requires the recruitment and interactions of both ER channel under certain conditions (60). This, in addition to their + and acidic Ca2 stores. differential endolysosomal distribution with TPC2 being the + The downstream role of NAADP-mediated Ca2 signaling in predominant late endosomal/lysosomal isoform and with TPC1 the angiogenic responses to VEGF was assessed in two principal being more broadly expressed across the endosomal system, ways. First, the selective membrane-permeant NAADP anat- may indicate distinct roles in cell physiology as exemplified gonist, Ned-19 was used. This molecule was developed by a here (58, 59). computational ligand-based drug discovery program (38), and Although it has been reported that under certain conditions has been widely tested in many cellular systems and validated TPCs are PI(3,5)P2 regulated but NAADP-insensitive lysosomal + + as a selective inhibitor of NAADP-evoked Ca2 release, and as a Na channels (61), a large amount of accumulating data pub- modulator of NAADP-sensitive TPC2 channels reconsituted in lished by others firmly suggests a major, if not direct, role for + lipid bilayer studies (59). Ned-19 treatment was found to be TPCs in endolysosomal Ca2 release and NAADP-mediated profoundly antiangiogenic in an in vivo model of neoangiogenesis responses (21–25, 57–59). Although additional studies are using matrigel plug assays (48). In this model, plug neo- needed to resolve this controversy, our findings reported here vascularization stimulated by VEGF was observed in the control are supportive of the role of TPC2 in mediating NAADP-evoked + samples, but was inhibited by Ned-19 treatment in a concentra- Ca2 release and downstream responses in ECs and angiogenesis tion-dependent manner (Fig. 5). both in vitro and in vivo. The second approach was to ablate expression of TPCs as We are aware that the inability of VEGF to induce vascular- −/− putative endolysomal targets for NAADP. In ization of plugs in Tpcn2 mice is not easily reconciled with the HUVEC cells transfected with anti-Tpcn2 shRNA, VEGF- fact that in these animals a functioning circulatory system + evoked Ca2 signals were much reduced, consistent with a key develops, whereas genetic ablation of VEGF or VEGFR2 results + role for these proteins in NAADP-mediated Ca2 release. Anti- in dramatic defects of blood island formation and development Tpcn2 shRNA also specifically impaired the ability of VEGF to (reviewed in ref. 62). These data might indicate differences in

Favia et al. PNAS | Published online October 20, 2014 | E4711 Downloaded by guest on September 29, 2021 devised to overturn the unfavorable balance between proangio- genic and antiangiogenic signaling, requiring that specific path- ways for these responses are identified (6). We believe that a thorough knowledge of the basic signaling machineries of EC in response to VEGF is fundamental to any further integration into more accurate and detailed pictures of pathological neoangiogenesis. In our experimental models, EC responses to VEGF were an- alyzed independently of other angiogenic signals; under these controlled conditions we have identified a master VEGFR2/ + NAADP/TPC2/Ca2 signaling pathway controlling the angiogenic response of ECs to VEGF. This specific intracellular pathway appears to be obligatory because its pharmacological and genetic ablation at different points in the pathway abolishes angiogenic responses both in vitro and in vivo. Moreover, our data indicate that two different VEGF-activated pathways operate: one + proangiogenic, involving acidic Ca2 stores and requiring NAADP as second messenger, and the other antiangiogenic, involving the p38 MAPK pathway and activated independently of NAADP- + evoked Ca2 release. Our exploration of specific VEGFR2- blocking strategies could lead to finely tailored therapies capable of discriminating between different signal trans- duction pathways activated by VEGF, and so potentiate current treatments by overturning the balance between proangiogenic and antiangiogenic VEGF effects. Understanding how to best + exploit the distinct Ca2 signaling pathways in angiogenesis could contribute to identifying new targets for antiangiogenic therapeutic strategies. Materials and Methods Cell Culture. Human umbilical vein endothelial cells (HUVECs) were obtained from Lonza Sales Ldt (Switzerland) and were cultured in EGM-2 Endothelial Cell Growth Medium-2 (Endothelial Basal Medium EBM-2 + EGM-2 BulletKit, Lonza), + 100 mM Penicillin/Streptomycin (Sigma). They were maintained at

37 °C in a humidified 5% (vol/vol) CO2 incubator and used at passage number 2–6.

+ Fig. 6. TPC2 silencing inhibits both VEGF-induced Ca2 release and in vitro + angiogenesis. (A) Intracellular Ca2 levels in HUVECs transfected with anti- human Tpcn2 shRNA constructs and stimulated with VEGF. n = 45 cells. (B) Representative images of one of three independent experiments in which HUVECs transfected with scramble or with two different anti-human Tpcn2 shRNA constructs were plated in Matrigel-coated dishes and incubated in EBM-2 with vehicle alone or VEGF. (C) Quantitative evaluation of capillary- like tube formation as the number of closed polygons formed in five fields for each experimental condition from three independent experiments. N.D. (not detectable) indicates that TPC2 silenced cells failed to form closed polygons. Where applicable, values from three independent experiments are expressed as mean ± SEM; *P < 0.05; ***P < 0.0002.

intra- and/or intercellular signaling between the process of an- giogenesis, based on the recruitment of embryonic precursors, and that of neoangiogenesis observed in the adult. The heterogeneity of conditions and sites for neoangiogenesis no doubt introduces a range of variables so that any simplistic model of responses to angiogenic factors turns out to be un- satisfactory. Angiogenesis is regulated by a balance of factors which, upon the switch of tumor cells to an angiogenic pheno- Fig. 7. Experiments in transgenic mice lacking NAADP putative targets − − − − type, leads to tumor growth and progression (63). The charac- TPC1 and TPC2. Tpcn1 / mice (A and B) and Tpcn2 / mice (A–C) were s.c. terization of these factors, the regulation of their production, injected with matrigel plugs containing or not VEGF. 5 d later the mice were killed and the plugs analyzed for vascularization. (A) Macroscopic analysis of and mechanisms of action have long been subject to intense in- − − − − the matrigel plugs from Tpcn1 / and Tpcn2 / mice. (B and C) Measure of − − vestigation, but the ambitious aim of finely controlling angio- Hb content expressed as absorbance (OD)/1 gr matrigel plug in Tpcn1 / mice − − genesis is not yet in sight (4). The notable challenge represented (B) and Tpcn2 / mice (C)(n = 10–24 plugs for each condition). Where by resistance to anti-VEGF therapies point to a high level of applicable, values from three independent experiments are expressed as complexity. Apparently, a more comprehensive strategy has to be mean ± SEM *P < 0.05; **P < 0.01; ***; P < 0.0002.

E4712 | www.pnas.org/cgi/doi/10.1073/pnas.1406029111 Favia et al. Downloaded by guest on September 29, 2021 (1 ratio image per s) using Metafluor software (Universal Imaging Corpo- PNAS PLUS ration). Calibration was obtained at the end of each experiment by maxi- mally increasing intracellular Ca2+-dependent FURA-2-AM fluorescence with 5 μmol/L ionomycin (ionomycin calcium salt from Strepotimyces conglobatus, Sigma) followed by recording minimal fluorescence in a Ca2+-free medium. 2+ [Ca ]i was calculated according to the formulas previously described (64).

Flow Cytometric Assessment of Cell Proliferation. Cells serum-starved in EBM-2 for 4 h were treated with VEGF and Ned-19. After 24 h, cells were harvested by trypsin/EDTA (Sigma), rinsed with PBS + 1% BSA (Sigma) and incubated with 1 μg/mL propidium iodide (PI, Sigma) plus 0,1 U/L of RNase for 3 h at room temperature. Cells were then analyzed using a Coulter Epics XL flow cytometer (Beckman Coulter) and data were analyzed using FCS3 express Software (De Novo Software) to determine the cell number for each experimental condition.

MTT Test. To perform the methylthiazolyldiphenyl-tetrazolium (MTT) test as a measurement of cell proliferation 5 mg/mL MTT (Sigma) was dissolved in PBS and filtered. HUVECs were cultured in EGM-2 to 90% confluence, detached using trypsin/EDTA, then added onto 96-well plates at a concentration of 10 × 104 cells in 200 μL of EGM-2 per well and incubated in the specific experimental condition for 24 h. As a control for background absorption, cells were omitted in some of the wells. After incubation the complete medium was removed, 100 μL of serum-free medium (EBM-2) containing 0.5 mg/mL MTT solution was added to each well and the plate was incubated in a humidified

5% (vol/vol) CO2 incubator at 37 °C for 3 h to allow MTT to be metabolized. Then, 100 μL of DMSO (Sigma) was added to each well, pipetting up and down to dissolve crystals, and optical density was read at 550 nm.

Gelatin Zymography. The MMP-9 in the media and in the cells was quantified separately by gelatin-substrate zymography. After 24 h of treatment, equal amounts of proteins from the cell-conditioned medium were separated on 8% (vol/vol) SDS/PAGE containing 1 mg/mL (final concentration) gelatin B. Electrophoresis was performed in nonreducing conditions, and SDS was re- moved by incubating the gel in 2% (vol/vol) Triton X-100 at 37 °C for 30 min. The gel was then soaked in 0.05 mol/L Tris·HCl buffer (pH 8.0) containing

5 mmol/L CaCl2 for about 18 h at 37 °C and stained with 1% Coomassie brilliant blue R-250. The gelatinolytic activity was identified as a clear band.

Molecular weights of the bands were estimated through the use of pre- CELL BIOLOGY

+ stained molecular-weight markers. Pictures of the gel were taken and Fig. 8. Schematic representation of VEGFR2/NAADP/TPC2/Ca2 signaling densitometry was performed using Image J software. pathway in the control of VEGF-induced angiogenic responses.

Boyden Chamber Assay. Cell ability to migrate was evaluated by the Boyden chamber assay, which makes use of a chamber composed of two medium- Biochemistry. The following antibodies were used: Phospho-44/42 MAPK filled compartments separated by a micro porous membrane (8 μm pore size, (T202/Y204: E10, Cell Signaling), Phospho-p38 MAPK (Thr180/Tyr182, Cell Signaling) and Phospho-Akt (Ser-473, Cell Signaling), Purified Mouse Anti- BD Bioscences). The lower well contained medium with 5% (vol/vol) serum eNOs (Ps1177, BD Transduction Laboratories), Rabbit pAb to PCNA (Abcam), and the chemoattractant agent (in our study: VEGF). Cells were placed in the Phospho-FAK (Tyr925, Cell Signaling), β-Actin HRP-conjugated (Sigma), upper chamber in complete medium containing the drug to assay (such as Stabilized Goat Anti-Mouse HRP-conjugated (Pierce), Stabilized Peroxidase- Ned-19) or the vehicle alone and allowed to migrate through the pores of conjugated Goat Anti-Rabbit [(H + L), Thermo Scientific], p-VEGFR2 [(Tyr- the membrane into the lower compartment. After 24 h the membrane be- 1175), Santa Cruz Biotechnology]. tween the two compartments was fixed and stained, and the number of cells that had migrated to the lower side of the membrane was evaluated in Reagents used are: Histamine (Sigma), Thrombin (Calbiochem), VEGF-A165 a Zeiss Axioscope microscope. and VEGF-B (Peprotech), Ned-19 (Tocris Bioscences), Bafilomycin (Sigma), Thapsigargin (Sigma), TSU-68 (SU668, Selleckchem), SB203580 (Calbiochem). In the in vitro assays, Ned-19 treatment was initiated 30 min before VEGF Scratch Assay. Confluent EC monolayers plated in 35-mm dishes were scraped “ ” stimulation. Concentrations of 25, 50, and 100 μmol/L Ned-19 were found in a straight line with a p10 pipette tip to create a scratch. The scratch + to inhibit VEGF-induced Ca2 release substantially; 100 μmol/L Ned-19 was created was of a similar size in the different experimental conditions to chosen for in vitro biological assays involving longer treatments. minimize any possible variation caused by the difference in its width. Debris was removed by a wash with PBS, which was then replaced with fresh me- Western Blot. HUVECs were first serum-starved in EBM-2 for 4 h and then were dium in the presence or absence of VEGF and Ned-19. First images were incubated with Ned-19 for 30 min or with TSU-68 for 1 h or with SB203580 for acquired at time 0 before incubating cells at 37 °C for 24 h. 1 h before VEGF stimulation for 15 min. The intensity of Western blot bands was quantified by Image J software (NIH) from at least three independent Silencing of TPC2. Two shRNA constructs (based on the pRS vector) targeting experiments, normalized to β-actin content, and compared with vehicle- human Tpcn2 and a control scramble sequence (noneffective 29-mer treated controls (set as 1). scrambled shRNA cassette, TR30012), all from OriGene, were used for cell transfection. Calcium Imaging. HUVECs cultured on 35-mm dishes were incubated in culture The targeting sequences were: ATCAGGCTGTGGTCTTCATCGAAGATGCT medium containing 3.5 μmol/L FURA-2-AM (Invitrogen) for 1 h at 37 °C, and (TI303526) and CGTCATTGTGGCTCTTCCTGGAAACAGCA (TI303528). Trans- then rinsed with HBSS (Sigma). Each dish was placed into a culture chamber fection was carried out according to the manufacturer’s instructions for at 37 °C on the stage of an inverted fluorescence microscope (Nikon transient transfection of adherent cells (QIAGEN). Efficiency of knockdown TE2000E), connected to a cooled CCD camera (512B Cascade, Roper Scien- was tested by quantitative RT-PCR 48 h after transfection. tific). Samples were illuminated alternately at 340 and 380 nm using a ran- dom access monochromator (Photon Technology International) and In Vitro Matrigel Assay. EC tube formation was evaluated by an angiogenesis emission was detected using a 510 nm emission filter. Images were acquired in vitro assay. Briefly, 130 μL of Matrigel Basement Membrane Matrix (BD

Favia et al. PNAS | Published online October 20, 2014 | E4713 Downloaded by guest on September 29, 2021 − − − − − − Biosciences) was added to each well of precooled 24-well tissue culture Tpcn1 / and Tpcn2 / Mice. Generation of Tpcn2 / mice was described plate. Pipette tips and Matrigel solution were kept cold throughout to avoid elsewhere (19); these animals (Tpcn2Gt(YHD437)Byg) carry an insertional mu- −/− solidification. The plate was incubated at 37 °C for 1 h to allow the matrix tation from a gene trap vector in Tpcn2 between exons 1 and 2. The Tpcn1 tm1Dgen solution to solidify. A total of 4 × 104 cells in a final volume of 500 μL of basal line used in this study (Tpcn1 ) was obtained from EMMA (The Euro- medium (EBM-2) were seeded onto the surface of each well containing the pean Mouse Mutant Archive). These mice have a targeted disruption of – polymerized matrix. ECs were pretreated with the inhibitor or with vehicle exons 4 5 that leads to absence of Tpcn1 mRNA expression (50). The animals were housed at the University of Rome Histology Unit alone and stimulated with the specific agonist (VEGF) for 4–24 h at 37 °C. accredited animal facility, in individual cages in an environmentally con- Tube formation was inspected under an inverted microscope (Nikon Eclipse trolled room (23 °C, 12 h light–dark cycle) and provided with food and water TS100) at 20× magnification and images were acquired by a digital camera ad libitum. All of the procedures were approved by the Italian Ministry (Nikon Coolpix995). The number of closed formed polygons from three in- for Health and conducted according to the US National Institutes of dependent experiments were counted in five random view-fields per well Health guidelines. and the values averaged. − − − − In Vivo Matrigel Plug Assay in Tpcn1 / , Tpcn2 / , and WT Mice. Matrigel In Vivo Matrigel Plug Assay in C57BL/6 Mice. To evaluate the ability of Ned-19 (600 μL, BD Biosciences) supplemented with heparin (0.032 U/L, Schwarz to modulate the neovascularization within matrigel plugs, matrigel (600 μL, Pharma S.p.A), VEGF (100 μg/L, Reliatech), TNFα (2 μg/L, R&D Systems) was − − − − BD Biosciences) supplemented with heparin (0.032 U/L, Schwarz Pharma S.p.A), injected s.c. into the flank of (4–5 wk old) WT and Tpcn1 / and Tpcn2 / VEGF (100 μg/L, Reliatech), TNFα (2 μg/L, R&D Systems), and Ned-19 at different mice as described above. After 5 d, the angiogenic response was evaluated doses(50,100,and150μmol/L, Tocris Bioscience) was injected s.c. into the by macroscopic analysis at autopsy, and by measurement of the Hb content flank of C57BL/6 mice (4–5 wk old), where it rapidly formed a gel. The negative in the matrigel plug as described before. The values were expressed as controls contained heparin alone, the positive controls heparin plus VEGF and optical density/100 mg matrigel. TNFα. Within days, cells from the surrounding tissues have the opportunity to migrate into the matrigel and to form vascular structures connected to the Statistical Analysis. Data are presented as the mean ± SEM of results from at least three independent experiments. A Student t test was used for statis- mousebloodvessels.After5d,themicewerekilledbyCO2 asphyxia and the < < angiogenic response was evaluated by macroscopic analysis at autopsy, and by tical comparison between means where applicable. *P 0.05; **P 0.01; < measurement of the hemoglobin content in the matrigel plug. Hemoglobin ***P 0.0002. was mechanically extracted from the pellets in water and measured using the Drabkin method by spectrophotometrical analysis (Sigma) at 540 nm. The ACKNOWLEDGMENTS. This work was supported by grants from Ministero dell’ Istruzione, dell’Università e della Ricerca and Agenzia Spaziale Italiana values were expressed as optical density/100 mg matrigel. Histological analysis (to A. Filippini), from Fondazione Roma (to E.Z.), and from Associazione of fixed and paraffin embedded matrigel plugs was also performed using Italiana Ricerca sul Cancro (to D.D.B.). A.G. and J.P. were in receipt of Well- H&E stain. come Trust programme Grant 084102/Z/07/Z.

1. Carmeliet P (2005) Angiogenesis in life, disease and medicine. 438(7070): 22. Zong X, et al. (2009) The two-pore channel TPCN2 mediates NAADP-dependent 932–936. Ca(2+)-release from lysosomal stores. Pflugers Arch 458(5):891–899. 2. Chung AS, Ferrara N (2011) Developmental and pathological angiogenesis. Annu Rev 23. Davis LC, et al. (2012) NAADP activates two-pore channels on T cell cytolytic granules Cell Dev Biol 27:563–584. to stimulate exocytosis and killing. Curr Biol 22(24):2331–2337. 3. Olsson AK, Dimberg A, Kreuger J, Claesson-Welsh L (2006) VEGF receptor signalling - 24. Zhang ZH, Lu YY, Yue J (2013) Two pore channel 2 differentially modulates neural in control of vascular function. Nat Rev Mol Cell Biol 7(5):359–371. differentiation of mouse embryonic stem cells. PLoS ONE 8(6):e66077. 4. Carmeliet P, Jain RK (2011) Molecular mechanisms and clinical applications of an- 25. Jha A, Ahuja M, Patel S, Brailoiu E, Muallem S (2014) Convergent regulation of the 2+ giogenesis. Nature 473(7347):298–307. lysosomal two-pore channel-2 by Mg , NAADP, PI(3,5)P2 and multiple protein 5. Abdullah SE, Perez-Soler R (2012) Mechanisms of resistance to vascular endothelial kinases. EMBO J 33(5):501–511. growth factor blockade. Cancer 118(14):3455–3467. 26. Patel S, Churchill GC, Galione A (2001) Coordination of Ca2+ signalling by NAADP. 6. Claesson-Welsh L, Welsh M (2013) VEGFA and tumour angiogenesis. J Intern Med Trends Biochem Sci 26(8):482–489. 273(2):114–127. 27. Galione A, Petersen OH (2005) The NAADP receptor: New receptors or new regula- – 7. Chung AS, et al. (2013) An interleukin-17-mediated paracrine network promotes tion? Mol Interv 5(2):73 79. tumor resistance to anti-angiogenic therapy. Nat Med 19(9):1114–1123. 28. Hohenegger M, Suko J, Gscheidlinger R, Drobny H, Zidar A (2002) Nicotinic acid- 8. Shojaei F, et al. (2010) HGF/c-Met acts as an alternative angiogenic pathway in adenine dinucleotide phosphate activates the skeletal muscle . – sunitinib-resistant tumors. Cancer Res 70(24):10090–10100. Biochem J 367(Pt 2):423 431. + 9. Moccia F, Berra-Romani R, Tanzi F (2012) Update on vascular endothelial Ca(2+) sig- 29. Guse AH, Lee HC (2008) NAADP: A universal Ca2 trigger. Sci Signal 1(44):re10. nalling: A tale of ion channels, pumps and transporters. World J Biol Chem 3(7): 30. Brailoiu GC, et al. (2010) Acidic NAADP-sensitive calcium stores in the endothelium: Agonist-specific recruitment and role in regulating blood pressure. J Biol Chem 127–158. 285(48):37133–37137. 10. Koch S, Claesson-Welsh L (2012) Signal transduction by vascular endothelial growth 31. Esposito B, et al. (2011) NAADP links histamine H1 receptors to secretion of von factor receptors. Cold Spring Harbor Perspect Med 2(7):a006502. Willebrand factor in human endothelial cells. Blood 117(18):4968–4977. 11. Munaron L (2006) Intracellular calcium, endothelial cells and angiogenesis. Recent 32. Gambara G, et al. (2008) NAADP-induced Ca(2+ signaling in response to endothelin Patents Anticancer Drug Discov 1(1):105–119. is via the receptor subtype B and requires the integrity of lipid rafts/caveolae. J Cell 12. Patton AM, Kassis J, Doong H, Kohn EC (2003) Calcium as a molecular target in Physiol 216(2):396–404. angiogenesis. Curr Pharm Des 9(7):543–551. 33. Churchill GC, et al. (2003) Sperm deliver a new second messenger: NAADP. Curr Biol 13. Andrikopoulos P, et al. (2011) Ca2+ influx through reverse mode Na+/Ca2+ exchange 13(2):125–128. is critical for vascular endothelial growth factor-mediated extracellular signal-regu- 34. Masgrau R, Churchill GC, Morgan AJ, Ashcroft SJ, Galione A (2003) NAADP: A new lated kinase (ERK) 1/2 activation and angiogenic functions of human endothelial cells. second messenger for glucose-induced Ca2+ responses in clonal pancreatic beta cells. – J Biol Chem 286(44):37919 37931. Curr Biol 13(3):247–251. + 14. Li J, et al. (2011) Orai1 and CRAC channel dependence of VEGF-activated Ca2 entry 35. Yamasaki M, et al. (2004) Organelle selection determines agonist-specific Ca2+ signals – and endothelial tube formation. Circ Res 108(10):1190 1198. in pancreatic acinar and beta cells. J Biol Chem 279(8):7234–7240. 15. Abdullaev IF, et al. (2008) Stim1 and Orai1 mediate CRAC currents and store-operated 36. Kim BJ, et al. (2008) Generation of nicotinic acid adenine dinucleotide phosphate and – calcium entry important for endothelial cell proliferation. Circ Res 103(11):1289 1299. cyclic ADP-ribose by glucagon-like peptide-1 evokes Ca2+ signal that is essential for 16. Berridge MJ, Bootman MD, Roderick HL (2003) Calcium signalling: Dynamics, insulin secretion in mouse pancreatic islets. Diabetes 57(4):868–878. – homeostasis and remodelling. Nat Rev Mol Cell Biol 4(7):517 529. 37. Macgregor A, et al. (2007) NAADP controls cross-talk between distinct Ca2+ stores 17. Galione A, Churchill GC (2002) Interactions between calcium release pathways: Mul- in the heart. J Biol Chem 282(20):15302–15311. tiple messengers and multiple stores. Cell Calcium 32(5-6):343–354. 38. Naylor E, et al. (2009) Identification of a chemical probe for NAADP by virtual 18. Morgan AJ, Platt FM, Lloyd-Evans E, Galione A (2011) Molecular mechanisms of en- screening. Nat Chem Biol 5(4):220–226. dolysosomal Ca2+ signalling in health and disease. Biochem J 439(3):349–374. 39. Laird AD, et al. (2000) SU6668 is a potent antiangiogenic and antitumor agent that 19. Calcraft PJ, et al. (2009) NAADP mobilizes calcium from acidic organelles through induces regression of established tumors. Cancer Res 60(15):4152–4160. two-pore channels. Nature 459(7246):596–600. 40. Liao WX, et al. (2009) Compartmentalizing VEGF-induced ERK2/1 signaling in pla- 20. Brailoiu E, et al. (2009) Essential requirement for two-pore channel 1 in NAADP- cental artery endothelial cell caveolae: A paradoxical role of caveolin-1 in placental mediated calcium signaling. J Cell Biol 186(2):201–209. angiogenesis in vitro. Mol Endocrinol 23(9):1428–1444. 21. Tugba Durlu-Kandilci N, et al. (2010) TPC2 proteins mediate nicotinic acid adenine 41. Banumathi E, et al. (2011) VEGF-induced retinal angiogenic signaling is critically de- + + dinucleotide phosphate (NAADP)- and agonist-evoked contractions of smooth pendent on Ca2 signaling by Ca2 /calmodulin-dependent protein kinase II. Invest muscle. J Biol Chem 285(32):24925–24932. Ophthalmol Vis Sci 52(6):3103–3111.

E4714 | www.pnas.org/cgi/doi/10.1073/pnas.1406029111 Favia et al. Downloaded by guest on September 29, 2021 42. Pedram A, Razandi M, Levin ER (1998) Extracellular signal-regulated protein kinase/ 53. Imoukhuede PI, Popel AS (2011) Quantification and cell-to-cell variation of vascular PNAS PLUS Jun kinase cross-talk underlies vascular endothelial cell growth factor-induced endothelial growth factor receptors. Exp Cell Res 317(7):955–965. endothelial cell proliferation. J Biol Chem 273(41):26722–26728. 54. Zhang Z, Neiva KG, Lingen MW, Ellis LM, Nör JE (2010) VEGF-dependent tumor an- 43. Duda DG, Fukumura D, Jain RK (2004) Role of eNOS in neovascularization: NO for giogenesis requires inverse and reciprocal regulation of VEGFR1 and VEGFR2. Cell endothelial progenitor cells. Trends Mol Med 10(4):143–145. Death Differ 17(3):499–512. 44. Issbrucker K, et al. (2003) p38 MAP kinase–a molecular switch between VEGF-induced 55. Rahimi N, Dayanir V, Lashkari K (2000) Receptor chimeras indicate that the vascular angiogenesis and vascular hyperpermeability. FASEB J 17(2):262–264. endothelial growth factor receptor-1 (VEGFR-1) modulates mitogenic activity of 45. Muñoz-Chápuli R, Quesada AR, Angel Medina M (2004) Angiogenesis and signal VEGFR-2 in endothelial cells. J Biol Chem 275(22):16986–16992. transduction in endothelial cells. Cell Mol Life Sci 61(17):2224–2243. 46. Lamalice L, Le Boeuf F, Huot J (2007) Endothelial cell migration during angiogenesis. 56. Lampugnani MG, Orsenigo F, Gagliani MC, Tacchetti C, Dejana E (2006) Vascular Circ Res 100(6):782–794. endothelial cadherin controls VEGFR-2 internalization and signaling from in- 47. Lechertier T, Hodivala-Dilke K (2012) Focal adhesion kinase and tumour angiogenesis. tracellular compartments. J Cell Biol 174(4):593–604. J Pathol 226(2):404–412. 57. Sawamiphak S, et al. (2010) Ephrin-B2 regulates VEGFR2 function in developmental 48. Staton CA, Reed MW, Brown NJ (2009) A critical analysis of current in vitro and in vivo and tumour angiogenesis. Nature 465(7297):487–491. angiogenesis assays. Int J Exp Pathol 90(3):195–221. 58. Galione A (2011) NAADP receptors. Cold Spring Harb Perspect Biol 3(1):a004036. 49. Passaniti A, et al. (1992) A simple, quantitative method for assessing angiogenesis and 59. Pitt SJ, et al. (2010) TPC2 is a novel NAADP-sensitive Ca2+ release channel, operating antiangiogenic agents using reconstituted basement membrane, heparin, and as a dual sensor of luminal pH and Ca2+. J Biol Chem 285(45):35039–35046. fibroblast growth factor. Lab Invest 67(4):519–528. 60. Pitt SJ, Lam AK, Rietdorf K, Galione A, Sitsapesan R (2014) Reconstituted human TPC1 50. Ruas M, et al. (2014) TPC1 has two variant isoforms and their removal has different is a proton-permeable ion channel and is activated by NAADP or Ca2+. Sci Signal effects on endo-lysosomal functions compared to loss of TPC2. Mol Cell Biol, 10.1128/ 7(326):ra46. MCB.00113-14. 61. Wang X, et al. (2012) TPC proteins are phosphoinositide- activated sodium-selective 51. Xiong Y, et al. (2009) Vascular endothelial growth factor (VEGF) receptor-2 tyrosine ion channels in endosomes and lysosomes. Cell 151(2):372–383. 1175 signaling controls VEGF-induced von Willebrand factor release from endothelial 62. Tammela T, Enholm B, Alitalo K, Paavonen K (2005) The biology of vascular endo- cells via phospholipase C-gamma 1- and protein kinase A-dependent pathways. J Biol thelial growth factors. Cardiovasc Res 65(3):550–563. Chem 284(35):23217–23224. 63. Sakurai T, Kudo M (2011) Signaling pathways governing tumor angiogenesis. 52. Zeng H, Dvorak HF, Mukhopadhyay D (2001) Vascular permeability factor (VPF)/vas- – cular endothelial growth factor (VEGF) peceptor-1 down-modulates VPF/VEGF Oncology 81(Suppl 1):24 29. + receptor-2-mediated endothelial cell proliferation, but not migration, through 64. Grynkiewicz G, Poenie M, Tsien RY (1985) A new generation of Ca2 indicators with phosphatidylinositol 3-kinase-dependent pathways. J Biol Chem 276(29):26969–26979. greatly improved fluorescence properties. J Biol Chem 260(6):3440–3450. CELL BIOLOGY

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