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Arachidonic acid (AA) stimulates endothelial cell (EC) proliferation through an increase in intracellular Ca2 + concentration ([Ca2 +]i), that, in turn, promotes nitric oxide (NO) release. AA-evoked Ca2 + 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 [Ca2 +]i and the following activation of the endothelial NO synthase (eNOS). AA induced a dose-dependent [Ca2 +]i raise that was mimicked by its non-metabolizable analogue eicosatetraynoic acid. AA-evoked Ca2 + signals required both intracellular Ca2 + release and external Ca2 + inflow. AA-induced Ca2 + 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 Ca2 + entry was, in turn, mediated by TRPV4, while it did not involve store-operated Ca2 + entry. Moreover, AA caused an increase in NO levels which was blocked by preventing the concomitant increase in [Ca2 +]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 Ca2 +- and NO-dependent manner. Therefore, AA-evoked Ca2 + signals emerge as an additional target to prevent cancer vascularisation, which may be sustained by ECFC recruitment.

Endothelial progenitor cells could be implicated in the aberrant neoangiogenesis that occurs in bone marrow and spleen in patients with primary myelofibrosis (PMF). However, antivascular endothelial growth factor (VEGF) monotherapy had only a modest and transient effect in these individuals. Recently it was found that VEGF-induced proangiogenic intracellular Ca 2+ oscillations could be impaired in endothelial progenitor cells of subjects with malignancies. Therefore, we employed Ca 2+ imaging, wavelet analysis, and functional assays to assess whether and how VEGF-induced Ca 2+ oscillations are altered in PMF-derived endothelial progenitor cells. We focused on endothelial colony-forming cells (ECFCs), which are the only endothelial progenitor cell subtype capable of forming neovessels both in vivo and in vitro. VEGF triggers repetitive Ca 2+ spikes in both normal ECFCs (N-ECFCs) and ECFCs obtained from PMF patients (PMF-ECFCs). However, the spiking response to VEGF is significantly weaker in PMF-ECFCs. VEGF-elicited Ca 2+ oscillations are patterned by the interaction between inositol-1,4,5-trisphosphate-dependent Ca 2+ mobilization and store-operated Ca 2+ entry. However, in most PMF-ECFCs, Ca 2+ oscillations are triggered by a store-independent Ca 2+ entry pathway. We found that diacylglycerol gates transient receptor potential canonical 1 channel to trigger VEGF-dependent Ca 2+ spikes by recruiting the phospholipase C/inositol-1,4,5-trisphosphate signaling pathway, reflected as a decrease in endoplasmic reticulum Ca 2+ content. Finally, we found that, apart from being less robust and dysregulated as compared with N-ECFCs, VEGF-induced Ca 2+ oscillations modestly stimulate PMF-ECFC growth and in vitro angiogenesis. These results may explain the modest effect of anti-VEGF therapies in PMF.

BackgroundAn 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 findingsWe 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.ConclusionsTwo 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.