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Arachidonic acid (AA) stimulates endothelial cell (EC) proliferation through an increase in intracellular Ca 2+ concentration ([Ca 2+ ] i ), that, in turn, promotes nitric oxide (NO) release. AA-evoked Ca 2+ signals are mainly mediated by Transient Receptor Potential Vanilloid 4 (TRPV4) channels. Circulating endothelial colony forming cells (ECFCs) represent the only established precursors of ECs. In the present study, we, therefore, sought to elucidate whether AA promotes human ECFC (hECFC) proliferation through an increase in [Ca 2+ ] i and the following activation of the endothelial NO synthase (eNOS). AA induced a dose-dependent [Ca 2+ ] i raise that was mimicked by its non-metabolizable analogue eicosatetraynoic acid. AA-evoked Ca 2+ signals required both intracellular Ca 2+ release and external Ca 2+ inflow. AA-induced Ca 2+ release was mediated by inositol-1,4,5-trisphosphate receptors from the endoplasmic reticulum and by two pore channel 1 from the acidic stores of the endolysosomal system. AA-evoked Ca 2+ entry was, in turn, mediated by TRPV4, while it did not involve store-operated Ca 2+ entry. Moreover, AA caused an increase in NO levels which was blocked by preventing the concomitant increase in [Ca 2+ ] i and by inhibiting eNOS activity with NG-nitro-l-arginine methyl ester (l-NAME). Finally, AA per se did not stimulate hECFC growth, but potentiated growth factors-induced hECFC proliferation in a Ca 2+ - and NO-dependent manner. Therefore, AA-evoked Ca 2+ signals emerge as an additional target to prevent cancer vascularisation, which may be sustained by ECFC recruitment., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Truly endothelial progenitor cells (EPCs) can be mobilized from bone marrow to support the vascular network of growing tumors, thereby sustaining the metastatic switch. Endothelial colony forming cells (ECFCs) are the only EPC subtype belonging to the endothelial phenotype and capable of incorporating within neovessels. The intracellular Ca(2+) machinery plays a key role in ECFC activation and is remodeled in renal cellular carcinoma-derived ECFCs (RCC-ECFCs). Particularly, RCC-ECFCs seems to undergo a drop in endoplasmic reticulum (ER) Ca(2+) concentration ([Ca(2+) ]ER ). This feature is remarkable when considering that inositol-1,4,5-trisphosphate (InsP3 )-dependent ER-to-mitochondria Ca(2+) transfer regulates the intrinsic apoptosis pathway. Herein, we sought to assess whether: (1) the [Ca(2+) ]ER and the InsP3 -induced ER-mitochondria Ca(2+) shuttle are reduced in RCC-ECFCs; and (2) the dysregulation of ER Ca(2+) handling leads to apoptosis resistance in tumor-derived cells. RCC-ECFCs displayed a reduction both in [Ca(2+) ]ER and in the InsP3 -dependent mitochondrial Ca(2+) uptake, while they expressed normal levels of Bcl-2 and Bak. The decrease in [Ca(2+) ]ER was associated to a remarkable ER expansion in RCC-ECFCs, which is a hallmark of ER stress, and did not depend on the remodeling of the Ca(2+) -transporting and the ER Ca(2+) -storing systems. As expected, RCC-ECFCs were less sensitive to rapamycin- and thapsigargin-induced apoptosis; however, buffering intracellular Ca(2+) levels with BAPTA dampened apoptosis in both cell types. Finally, store-operated Ca(2+) entry was seemingly uncoupled from the apoptotic machinery in RCC-ECFCs. Thus, the chronic underfilling of the ER Ca(2+) pool could confer a survival advantage to RCC-ECFCs and underpin RCC resistance to pharmacological treatment. J. Cell. Biochem. 117: 2260-2271, 2016. © 2016 Wiley Periodicals, Inc., (© 2016 Wiley Periodicals, Inc.)

Clonal endothelial progenitor cells (EPCs) have been implicated in the aberrant vascular growth that features infantile hemangioma (IH), the most common benign vascular tumor in childhood that may cause ulceration, bleeding, and/or permanent disfigurement. Endothelial colony-forming cells (ECFCs), truly endothelial EPCs endowed with clonal ability and capable of forming patent vessels in vivo, remodel their Ca(2+) toolkit in tumor-derived patients to acquire an adaptive advantage. Particularly, they upregulate the proangiogenic store-operated Ca(2+) entry (SOCE) pathway due to the overexpression of its underlying components, that is, stromal interaction molecule 1 (Stim1), Orai1, and transient receptor potential canonical 1 (TRPC1). The present work was undertaken to assess whether and how the Ca(2+) signalosome is altered in IH-ECFCs by employing Ca(2+) and nitric oxide (NO) imaging, real-time polymerase chain reaction, western blotting, and functional assays. IH-ECFCs display a lower intracellular Ca(2+) release in response to either pharmacological (i.e., cyclopiazonic acid) or physiological (i.e., ATP and vascular endothelial growth factor) stimulation. Conversely, Stim1, Orai1, and TRPC1 transcripts and proteins are normally expressed in these cells and mediate a constitutive SOCE, which is sensitive to BTP-2, La(3+), and Pyr6 and recharges the intracellular Ca(2+) pool. The resting SOCE in IH-ECFCs is also associated to an increase in their proliferation rate and the basal production of NO compared to normal cells. Likewise, the pharmacological blockade of SOCE and NO synthesis block IH-ECFC growth. Collectively, these data indicate that the constitutive SOCE activation enhances IH-ECFC proliferation by augmenting basal NO production and sheds novel light on the molecular mechanisms of IH.

Endothelial progenitor cells (EPCs) have recently been shown to promote the angiogenic switch in solid neoplasms, thereby promoting tumour growth and metastatisation. The genetic suppression of EPC mobilization from bone marrow prevents tumour development and colonization of remote organs. Therefore, it has been assumed that anti-angiogenic treatments, which target vascular endothelial growth factor (VEGF) signalling in both normal endothelial cells and EPCs, could interfere with EPC activation in cancer patients. Our recent data, however, show that VEGF fails to stimulate tumour endothelial colony-forming cells (ECFCs), i.e. the only EPC subtype truly belonging to the endothelial lineage. The present article will survey current evidence about EPC involvement in the angiogenic switch: we will focus on the controversy about EPC definition and on the debate around their actual incorporation into tumour neovessels. We will then discuss how ECFC insensitivity to VEGF stimulation in cancer patients could underpin their well-known resistance to anti-VEGF therapies.

Hydrogen sulphide (H2S) is a newly discovered gasotransmitter that regulates multiple steps in VEGF-induced angiogenesis. An increase in intracellular Ca(2+) concentration ([Ca(2+)]i) is central to endothelial proliferation and may be triggered by both VEGF and H2S. Albeit VEGFR-2 might serve as H2S receptor, the mechanistic relationship between VEGF- and H2S-induced Ca(2+) signals in endothelial cells is unclear. The present study aimed at assessing whether and how NaHS, a widely employed H2S donor, stimulates pro-angiogenic Ca(2+) signals in Ea.hy926 cells, a suitable surrogate for mature endothelial cells, and human endothelial progenitor cells (EPCs). We found that NaHS induced a dose-dependent increase in [Ca(2+)]i in Ea.hy926 cells. NaHS-induced Ca(2+) signals in Ea.hy926 cells did not require extracellular Ca(2+) entry, while they were inhibited upon pharmacological blockade of the phospholipase C/inositol-1,4,5-trisphosphate (InsP3) signalling pathway. Moreover, the Ca(2+) response to NaHS was prevented by genistein, but not by SU5416, which selectively inhibits VEGFR-2. However, VEGF-induced Ca(2+) signals were suppressed by dl-propargylglycine (PAG), which blocks the H2S-producing enzyme, cystathionine γ-lyase. Consistent with these data, VEGF-induced proliferation and migration were inhibited by PAG in Ea.hy926 cells, albeit NaHS alone did not influence these processes. Conversely, NaHS elevated [Ca(2+)]i only in a modest fraction of circulating EPCs, whereas neither VEGF-induced Ca(2+) oscillations nor VEGF-dependent proliferation were affected by PAG. Therefore, H2S-evoked elevation in [Ca(2+)]i is essential to trigger the pro-angiogenic Ca(2+) response to VEGF in mature endothelial cells, but not in their immature progenitors., (Copyright © 2014. Published by Elsevier Ltd.)

Background: An increase in the frequency of circulating endothelial colony forming cells (ECFCs), the only subset of endothelial progenitor cells (EPCs) truly belonging to the endothelial phenotype, occurs in patients affected by primary myelofibrosis (PMF). Herein, they might contribute to the enhanced neovascularisation of fibrotic bone marrow and spleen. Store-operated Ca2+ entry (SOCE) activated by the depletion of the inositol-1,4,5-trisphosphate (InsP3)-sensitive Ca2+ store drives proliferation in ECFCs isolated from both healthy donors (N-ECFCs) and subjects suffering from renal cellular carcinoma (RCC-ECFCs). SOCE is up-regulated in RCC-ECFCs due to the over-expression of its underlying molecular components, namely Stim1, Orai1, and TRPC1., Methodology/principal Findings: We utilized Ca2+ imaging, real-time polymerase chain reaction, western blot analysis and functional assays to evaluate molecular structure and the functional role of SOCE in ECFCs derived from PMF patients (PMF-ECFCs). SOCE, induced by either pharmacological (i.e. cyclopiazonic acid or CPA) or physiological (i.e. ATP) stimulation, was significantly higher in PMF-ECFCs. ATP-induced SOCE was inhibited upon blockade of the phospholipase C/InsP3 signalling pathway with U73111 and 2-APB. The higher amplitude of SOCE was associated to the over-expression of the transcripts encoding for Stim2, Orai2-3, and TRPC1. Conversely, immunoblotting revealed that Stim2 levels remained constant as compared to N-ECFCs, while Stim1, Orai1, Orai3, TRPC1 and TRPC4 proteins were over-expressed in PMF-ECFCs. ATP-induced SOCE was inhibited by BTP-2 and low micromolar La3+ and Gd3+, while CPA-elicited SOCE was insensitive to Gd3+. Finally, BTP-2 and La3+ weakly blocked PMF-ECFC proliferation, while Gd3+ was ineffective., Conclusions: Two distinct signalling pathways mediate SOCE in PMF-ECFCs; one is activated by passive store depletion and is Gd3+-resistant, while the other one is regulated by the InsP3-sensitive Ca2+ pool and is inhibited by Gd3+. Unlike N- and RCC-ECFCs, the InsP3-dependent SOCE does not drive PMF-ECFC proliferation.