Dysregulation of VEGF-induced proangiogenic Ca2+ oscillations in primary myelofibrosis-derived endothelial colony-forming cells
2015; Elsevier BV; Volume: 43; Issue: 12 Linguagem: Inglês
10.1016/j.exphem.2015.09.002
ISSN1873-2399
AutoresSilvia Dragoni, Marta Reforgiato, Estella Zuccolo, Valentina Poletto, Francesco Lodola, Federico Alessandro Ruffinatti, Elisa Bonetti, Germano Guerra, Giovanni Barosi, Vittorio Rosti, Francesco Moccia,
Tópico(s)Sphingolipid Metabolism and Signaling
Resumo•Endothelial progenitor cells are implicated in primary myelofibrosis (PMF).•Anti-vascular endothelial growth factor (VEGF) therapy is almost ineffective in PMF.•We studied VEGF-induced proangiogenic Ca2+ oscillations in PMF-derived endothelial progenitor cells.•VEGF induces weak Ca2+ activity, proliferation, and angiogenesis in PMF-derived endothelial progenitor cells.•These results might explain the modest effect of anti-VEGF therapies in PMF. 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 Ca2+ oscillations could be impaired in endothelial progenitor cells of subjects with malignancies. Therefore, we employed Ca2+ imaging, wavelet analysis, and functional assays to assess whether and how VEGF-induced Ca2+ 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 Ca2+ 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 Ca2+ oscillations are patterned by the interaction between inositol-1,4,5-trisphosphate-dependent Ca2+ mobilization and store-operated Ca2+ entry. However, in most PMF-ECFCs, Ca2+ oscillations are triggered by a store-independent Ca2+ entry pathway. We found that diacylglycerol gates transient receptor potential canonical 1 channel to trigger VEGF-dependent Ca2+ spikes by recruiting the phospholipase C/inositol-1,4,5-trisphosphate signaling pathway, reflected as a decrease in endoplasmic reticulum Ca2+ content. Finally, we found that, apart from being less robust and dysregulated as compared with N-ECFCs, VEGF-induced Ca2+ oscillations modestly stimulate PMF-ECFC growth and in vitro angiogenesis. These results may explain the modest effect of anti-VEGF therapies in PMF. 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 Ca2+ oscillations could be impaired in endothelial progenitor cells of subjects with malignancies. Therefore, we employed Ca2+ imaging, wavelet analysis, and functional assays to assess whether and how VEGF-induced Ca2+ 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 Ca2+ 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 Ca2+ oscillations are patterned by the interaction between inositol-1,4,5-trisphosphate-dependent Ca2+ mobilization and store-operated Ca2+ entry. However, in most PMF-ECFCs, Ca2+ oscillations are triggered by a store-independent Ca2+ entry pathway. We found that diacylglycerol gates transient receptor potential canonical 1 channel to trigger VEGF-dependent Ca2+ spikes by recruiting the phospholipase C/inositol-1,4,5-trisphosphate signaling pathway, reflected as a decrease in endoplasmic reticulum Ca2+ content. Finally, we found that, apart from being less robust and dysregulated as compared with N-ECFCs, VEGF-induced Ca2+ oscillations modestly stimulate PMF-ECFC growth and in vitro angiogenesis. These results may explain the modest effect of anti-VEGF therapies in PMF. Tumor growth and metastasis depend on the development of an intricate vascular network supported by several mechanisms, including sprouting angiogenesis and vasculogenesis, that is, de novo neovessel formation by circulating endothelial progenitor cells (EPCs) [1Carmeliet P. Jain R.K. Molecular mechanisms and clinical applications of angiogenesis.Nature. 2011; 473: 298-307Crossref PubMed Scopus (3554) Google Scholar, 2Gao D.C. Nolan D. McDonnell K. et al.Bone marrow-derived endothelial progenitor cells contribute to the angiogenic switch in tumor growth and metastatic progression.Biochim Biophys Acta. 2009; 1796: 33-40PubMed Google Scholar, 3Moccia F. Poletto V. May the remodeling of the Ca(2+) toolkit in endothelial progenitor cells derived from cancer patients suggest alternative targets for anti-angiogenic treatment?.Biochim Biophys Acta. 2015; 1853: 1958-1973Crossref PubMed Scopus (37) Google Scholar]. Therefore, a number of antiangiogenic therapies have been introduced to target vascular endothelial growth factor (VEGF), which is the main stimulator of both endothelial cells (ECs) and EPCs in healthy individuals [1Carmeliet P. Jain R.K. Molecular mechanisms and clinical applications of angiogenesis.Nature. 2011; 473: 298-307Crossref PubMed Scopus (3554) Google Scholar, 4Koch S. Claesson-Welsh L. Signal transduction by vascular endothelial growth factor receptors.Cold Spring Harbor Perspect Med. 2012; 2: a006502Crossref Scopus (560) Google Scholar], in cancer patients [1Carmeliet P. Jain R.K. Molecular mechanisms and clinical applications of angiogenesis.Nature. 2011; 473: 298-307Crossref PubMed Scopus (3554) Google Scholar, 3Moccia F. Poletto V. May the remodeling of the Ca(2+) toolkit in endothelial progenitor cells derived from cancer patients suggest alternative targets for anti-angiogenic treatment?.Biochim Biophys Acta. 2015; 1853: 1958-1973Crossref PubMed Scopus (37) Google Scholar]. Likewise, recent studies have suggested that angiogenesis also plays a key role in the pathogenesis of such hematologic disorders as Philadelphia-negative (Ph–) myeloproliferative neoplasms (MPNs), for example, primary myelofibrosis (PMF) [5Medinger M. Passweg J. Angiogenesis in myeloproliferative neoplasms: New markers and future directions.Memo. 2014; 7: 206-210Crossref PubMed Scopus (24) Google Scholar, 6Le Bousse-Kerdilès M.C. Primary myelofibrosis and the "bad seeds in bad soil" concept.Fibrogenesis Tissue Repair. 2012; 5: S20Crossref PubMed Scopus (25) Google Scholar]. PMF arises as a consequence of somatic mutation in either the JAK2 gene, leading to a valine-to-phenylalanine substitution at position 617 (JAK2–V617F) in about 60% of patients, or the calreticulin (CALR) gene in 25% of patients, or the MPL gene in 5% of patients; about 10% of patients do not harbor any of these mutations in their hematopoietic cells [7Rumi E. Pietra D. Pascutto C. et al.Clinical effect of driver mutations of JAK2, CALR, or MPL in primary myelofibrosis.Blood. 2014; 124: 1062-1069Crossref PubMed Scopus (286) Google Scholar]. A marked increase in microvessel density (MVD) has been observed in both the bone marrow and spleen of patients with high JAK2–V617F mutant allele burden [8Barosi G. Rosti V. Massa M. et al.Spleen neoangiogenesis in patients with myelofibrosis with myeloid metaplasia.Br J Haematol. 2004; 124: 618-625Crossref PubMed Scopus (40) Google Scholar]. This observation paved the way for the launch of a number of clinical trials aimed at treating MPF patients with anti-VEGF monotherapies. However, both valatanib, which specifically inhibits the tyrosine kinase activity of VEGFR-2, and bevacizumab, a monoclonal anti-VEGF antibody, have a modest and transient effect only in a minority of the subjects [5Medinger M. Passweg J. Angiogenesis in myeloproliferative neoplasms: New markers and future directions.Memo. 2014; 7: 206-210Crossref PubMed Scopus (24) Google Scholar]. Interestingly, we recently reported that VEGF does not trigger proangiogenic oscillations in intracellular Ca2+ concentration ([Ca2+]i) in renal cellular carcinoma (RCC)-derived EPCs [9Lodola F. Laforenza U. Bonetti E. et al.Store-operated Ca2+ entry is remodelled and controls in vitro angiogenesis in endothelial progenitor cells isolated from tumoral patients.PLoS One. 2012; 7: e42541Crossref PubMed Scopus (49) Google Scholar], which might help explain the relative failure of anti-VEGF treatments in these patients [3Moccia F. Poletto V. May the remodeling of the Ca(2+) toolkit in endothelial progenitor cells derived from cancer patients suggest alternative targets for anti-angiogenic treatment?.Biochim Biophys Acta. 2015; 1853: 1958-1973Crossref PubMed Scopus (37) Google Scholar, 10Moccia F. Dragoni S. Poletto V. et al.Orai1 and Transient Receptor Potential Channels as novel molecular targets to impair tumor neovascularisation in renal cell carcinoma and other malignancies.Anticancer Agents Med Chem. 2014; 14: 296-312Crossref PubMed Scopus (42) Google Scholar]. EPCs might be instrumental in sustaining the aberrant bone marrow and spleen vascularization that characterizes PMF, but no study has hitherto directly assessed the proangiogenic effect of VEGF on these cells. Intracellular Ca2+ signaling is essential for EPC activation [11Moccia F. Tanzi F. Munaron L. Endothelial remodelling and intracellular calcium machinery.Curr Mol Med. 2014; 14: 457-480Crossref PubMed Scopus (62) Google Scholar, 12Moccia F. Lodola F. Dragoni S. et al.Ca2+ signalling in endothelial progenitor cells: A novel means to improve cell-based therapy and impair tumour vascularisation.Curr Vasc Pharmacol. 2014; 12: 87-105Crossref PubMed Scopus (64) Google Scholar]. VEGF stimulates EPC proliferation through an oscillatory increase in [Ca2+]i that is shaped by the concerted interaction between Ca2+ release from the endoplasmic reticulum (ER), the largest intracellular Ca2+ store, and Ca2+ influx from the extracellular milieu [13Dragoni S. Laforenza U. Bonetti E. et al.Vascular endothelial growth factor stimulates endothelial colony forming cells proliferation and tubulogenesis by inducing oscillations in intracellular Ca2+ concentration.Stem Cells. 2011; 29: 1898-1907Crossref PubMed Scopus (57) Google Scholar, 14Potenza D.M. Guerra G. Avanzato D. et al.Hydrogen sulphide triggers VEGF-induced intracellular Ca2+ signals in human endothelial cells but not in their immature progenitors.Cell Calcium. 2014; 56: 225-234Crossref PubMed Scopus (52) Google Scholar]. VEGF binds to VEGFR-2, thereby stimulating phospholipase Cγ (PLCγ) to generate inositol-1,4,5-triphosphate (InsP3) and diacylglycerol (DAG); InsP3, in turn, mobilizes ER-stored Ca2+ on the activation of InsP3 receptors (InsP3Rs). The subsequent drop in ER Ca2+ levels is detected by Stim1, an ER Ca2+ sensor, which translocates in close (≈20 nm) proximity to the plasma membrane to gate the store-operated Ca2+ channels (SOCCs), Orai1 and transient receptor potential canonical 1 (TRPC1) [9Lodola F. Laforenza U. Bonetti E. et al.Store-operated Ca2+ entry is remodelled and controls in vitro angiogenesis in endothelial progenitor cells isolated from tumoral patients.PLoS One. 2012; 7: e42541Crossref PubMed Scopus (49) Google Scholar, 13Dragoni S. Laforenza U. Bonetti E. et al.Vascular endothelial growth factor stimulates endothelial colony forming cells proliferation and tubulogenesis by inducing oscillations in intracellular Ca2+ concentration.Stem Cells. 2011; 29: 1898-1907Crossref PubMed Scopus (57) Google Scholar, 15Sánchez-Hernández Y. Laforenza U. Bonetti E. et al.Store-operated Ca(2+) entry is expressed in human endothelial progenitor cells.Stem Cells Dev. 2010; 19: 1967-1981Crossref PubMed Scopus (60) Google Scholar, 16Li J. Cubbon R.M. Wilson L.A. et al.Orai1 and CRAC channel dependence of VEGF-activated Ca2+ entry and endothelial tube formation.Circ Res. 2011; 108: 1190-1198Crossref PubMed Scopus (150) Google Scholar]. The Ca2+ machinery is, however, dysregulated in patients affected by malignant diseases. For instance, a lower ER Ca2+ concentration and the downregulation of InsP3Rs prevent VEGF from triggering Ca2+ oscillations in RCC-derived EPCs [3Moccia F. Poletto V. May the remodeling of the Ca(2+) toolkit in endothelial progenitor cells derived from cancer patients suggest alternative targets for anti-angiogenic treatment?.Biochim Biophys Acta. 2015; 1853: 1958-1973Crossref PubMed Scopus (37) Google Scholar, 9Lodola F. Laforenza U. Bonetti E. et al.Store-operated Ca2+ entry is remodelled and controls in vitro angiogenesis in endothelial progenitor cells isolated from tumoral patients.PLoS One. 2012; 7: e42541Crossref PubMed Scopus (49) Google Scholar, 10Moccia F. Dragoni S. Poletto V. et al.Orai1 and Transient Receptor Potential Channels as novel molecular targets to impair tumor neovascularisation in renal cell carcinoma and other malignancies.Anticancer Agents Med Chem. 2014; 14: 296-312Crossref PubMed Scopus (42) Google Scholar]. More recently, we found that the Ca2+ toolkit undergoes a dramatic rearrangement also in EPCs derived from PMF patients bearing the JAK2 mutation [17Dragoni S. Laforenza U. Bonetti E. et al.Enhanced expression of Stim, Orai, and TRPC transcripts and proteins in endothelial progenitor cells isolated from patients with primary myelofibrosis.PLoS One. 2014; 9: e91099Crossref PubMed Scopus (25) Google Scholar]. These cells exhibited a reduction in InsP3-depedent Ca2+ release and two pharmacologically distinguishable types of SOCCs: one is stimulated by passive ER depletion, is inhibited by BTP-2 and La3+, and is resistant to Gd3+, whereas the other is tuned by the InsP3-sensitive Ca2+ pool and inhibited by BTP-2, La3+, and Gd3+ [17Dragoni S. Laforenza U. Bonetti E. et al.Enhanced expression of Stim, Orai, and TRPC transcripts and proteins in endothelial progenitor cells isolated from patients with primary myelofibrosis.PLoS One. 2014; 9: e91099Crossref PubMed Scopus (25) Google Scholar]. Unlike EPCs derived from peripheral blood of healthy donors and from umbilical cord blood (UCB) [15Sánchez-Hernández Y. Laforenza U. Bonetti E. et al.Store-operated Ca(2+) entry is expressed in human endothelial progenitor cells.Stem Cells Dev. 2010; 19: 1967-1981Crossref PubMed Scopus (60) Google Scholar, 18Dragoni S. Laforenza U. Bonetti E. et al.Canonical transient receptor potential 3 channel triggers vascular endothelial growth factor-induced intracellular Ca2+ oscillations in endothelial progenitor cells isolated from umbilical cord blood.Stem Cells Dev. 2013; 22: 2561-2580Crossref PubMed Scopus (77) Google Scholar], the pharmacologic blockade of store-operated Ca2+ entry (SOCE) did not block PMF-derived EPC proliferation when the cells were grown in an endothelial growth medium enriched with growth factors, including VEGF [17Dragoni S. Laforenza U. Bonetti E. et al.Enhanced expression of Stim, Orai, and TRPC transcripts and proteins in endothelial progenitor cells isolated from patients with primary myelofibrosis.PLoS One. 2014; 9: e91099Crossref PubMed Scopus (25) Google Scholar]. These findings lead to two important questions: (i) Is VEGF capable of triggering Ca2+ oscillations in EPCs harvested from PMF patients? (ii) Does VEGF stimulate proliferation in these cells? Elucidating whether and how the remodeling of the Ca2+ toolkit affects the pathogenesis of PMF becomes more relevant in the light of the report that a mutation in the gene encoding for calreticulin, the most important ER Ca2+-binding protein, occurs in 35% of these patients [19Klampfl T. Gisslinger H. Harutyunyan A.S. et al.Somatic mutations of calreticulin in myeloproliferative neoplasms.N Engl J Med. 2013; 369: 2379-2390Crossref PubMed Scopus (1391) Google Scholar]. The present investigation was therefore undertaken to analyze the molecular mechanisms and physiologic consequences of VEGF-induced Ca2+ oscillations in JAK2-mutated PMF. As in our previous studies, we focused on endothelial colony-forming cells (ECFCs), the only EPC subtype truly belonging to the endothelial phenotype, endowed with clonal ability, and capable of engrafting within the endothelial lining of neovessels [20Yoder M.C. Human endothelial progenitor cells.Cold Spring Harbor Perspect Med. 2012; 2: a006692Crossref Scopus (86) Google Scholar, 21Moccia F. Ruffinatti F.A. Zuccolo E. Intracellular Ca2+ signals to reconstruct a broken heart: Still a theoretical approach?.Curr Drug Targets. 2015; 16: 793-815Crossref PubMed Google Scholar]. We used Ca2+ imaging and functional assays to establish that the oscillatory activity of JAK2-mutated PMF-derived ECFCs (PMF-ECFCs) is reduced as compared with that of control cells (N-ECFCs). Moreover, albeit Ca2+ spikes are patterned by the interplay between InsP3-dependent Ca2+ release and SOCE, they are triggered by TRPC1-mediated Ca2+ entry and do not stimulate cell proliferation. These results shed light on the molecular mechanisms involved in the pathogenesis of PMF and could help explain why anti-VEGF treatment is ineffective in patients with JAK2-mutated PMF. Three patients with PMF were enrolled in the study (two females). All three were out of treatment and harbored a JAKV617F mutation in their granulocytes, whereas they lacked the recently reported calreticulin mutation [19Klampfl T. Gisslinger H. Harutyunyan A.S. et al.Somatic mutations of calreticulin in myeloproliferative neoplasms.N Engl J Med. 2013; 369: 2379-2390Crossref PubMed Scopus (1391) Google Scholar]. Three healthy subjects (two females) volunteered to participate in the study. All patients and healthy subjects signed an informed consent. The study was approved by the institutional review board of the IRCCS Policlinico San Matteo Foundation, Pavia, Italy. All technical details of ECFC isolation and cultivation, measurement of intracellular Ca2+ dynamics, and proliferation assays have been described elsewhere [9Lodola F. Laforenza U. Bonetti E. et al.Store-operated Ca2+ entry is remodelled and controls in vitro angiogenesis in endothelial progenitor cells isolated from tumoral patients.PLoS One. 2012; 7: e42541Crossref PubMed Scopus (49) Google Scholar, 17Dragoni S. Laforenza U. Bonetti E. et al.Enhanced expression of Stim, Orai, and TRPC transcripts and proteins in endothelial progenitor cells isolated from patients with primary myelofibrosis.PLoS One. 2014; 9: e91099Crossref PubMed Scopus (25) Google Scholar, 22Piaggio G. Rosti V. Corselli M. et al.Endothelial colony-forming cells from patients with chronic myeloproliferative disorders lack the disease-specific molecular clonality marker.Blood. 2009; 114: 3127-3130Crossref PubMed Scopus (66) Google Scholar] and are detailed in the Supplementary Methods (online only, available at www.exphem.org). Measurement of ER Ca2+ in PMF-ECFCs was carried out using the ER-entrapped low-affinity Ca2+ dye mag-Fura-2 according to the protocol previously described [23Dedkova E.N. Blatter L.A. Nitric oxide inhibits capacitative Ca2+ entry and enhances endoplasmic reticulum Ca2+ uptake in bovine vascular endothelial cells.J Physiol. 2002; 539: 77-91Crossref PubMed Scopus (120) Google Scholar]. Briefly, cells were loaded with mag-Fura-2-acetoxymethyl ester (mag-Fura-2-AM, 2 μmol/L) in physiologic saline solution for 2 hours at 37°C. The membrane-permeable intracellular Ca2+ buffer BAPTA/AM (30 μmol/L) was added during the last 30 min of fluorochrome loading to prevent any contamination from changes in cytosolic Ca2+ concentration Accordingly, control experiments (not shown) confirmed that the presence of BAPTA in the cytosol eliminated contamination of the mag-Fura-2 signal by changes in cytosolic Ca2+ levels. Similar to Fura-2 measurements, PMF-ECFCs were excited alternately at 340 and 380 nm, and the emitted light was detected at 510 nm. From 10 up to 30 regions of interests (ROIs) were drawn around each cell to collect the signal from single ECFCs. ER Ca2+ concentration was monitored by measuring, for each ROI, the ratio of the mean fluorescence emitted at 510 nm when exciting alternatively at 340 and 380 nm (termed ratio). Under the described experimental conditions, any change in the ratio reflects a change in ER Ca2+ levels. Ratio measurements were performed and plotted online every 2 s. The experiments were carried out at room temperature (22°C). The tube formation assay was carried out as previously described [13Dragoni S. Laforenza U. Bonetti E. et al.Vascular endothelial growth factor stimulates endothelial colony forming cells proliferation and tubulogenesis by inducing oscillations in intracellular Ca2+ concentration.Stem Cells. 2011; 29: 1898-1907Crossref PubMed Scopus (57) Google Scholar]. Early passage (P1 or P2) PMF-ECFCs were detached by trypsinization and resuspended in EBM-2 medium containing 2% fetal calf serum. ECFC-derived cells were plated at 1.5 × 104 per well in Cultrex (Trevigen)-coated 96-well plates, in the presence of EBM-2 ± 10 ng/mL VEGF. Capillary network formation was assessed starting from 4 to 24 hours later. Three different sets of experiments, each performed in duplicate, were carried out. Experiments were repeated a minimum of three times, and the vasculogenic response was measured by evaluating both dimensional and topologic parameters. As illustrated in [24Jian J. Zheng Z. Zhang K. et al.Fibromodulin promoted in vitro and in vivo angiogenesis.Biochem Biophys Res Commun. 2013; 436: 530-535Crossref PubMed Scopus (40) Google Scholar], we analyzed the length of endothelial tubelike structures (TLSs), the number of the polygon structures established by TLS, which are referred to as complex meshes and are indicative of endothelial cell migration, and the number of junctions between TLSs per area. These analyses were performed using the Angiogenesis Analyzer plugin of ImageJ (Gilles Carpentier, Faculté des Sciences et Technologie, Université Paris Est, Creteil Val de Marne, France) [24Jian J. Zheng Z. Zhang K. et al.Fibromodulin promoted in vitro and in vivo angiogenesis.Biochem Biophys Res Commun. 2013; 436: 530-535Crossref PubMed Scopus (40) Google Scholar, 25Fortunato T.M. Vara D.S. Wheeler-Jones C.P. Pula G. Expression of protease-activated receptor 1 and 2 and anti-tubulogenic activity of protease-activated receptor 1 in human endothelial colony-forming cells.PLoS One. 2014; 9: e109375Crossref PubMed Scopus (9) Google Scholar]. All Ca2+ data were collected from ECFCs isolated from peripheral blood of at least three healthy volunteers or PMF patients. In every figure, each trace is representative of at least three independent experiments conducted on cells isolated from three distinct healthy donors and three PMF patients. Pooled data are given as means ± SE, and statistical significance (p < 0.05) was evaluated with Student's t test for unpaired observations. The amplitude of the initial peak Ca2+ response to VEGF was measured as the difference between the ratio at the peak and the mean ratio of 1-min baseline before the peak. To give a quantitative evaluation of the differences in calcium oscillatory activity between N- and PMF-ECFCs after VEGF administration, an approach based on wavelet analysis was employed, using KYM 0.5 software (http://sourceforge.net/projects/kym/) as described in [26Ruffinatti F.A. Lovisolo D. Distasi C. Ariano P. Erriquez J. Ferraro M. Calcium signals: Analysis in time and frequency domains.J Neurosci Methods. 2011; 199: 310-320Crossref PubMed Scopus (5) Google Scholar]. See Supplementary Methods (online only, available at www.exphem.org) for further details. Data regarding ECFC proliferation and tubulogenic activity are also presented as means ± SE, and statistical significance (p < 0.05) was evaluated with Student's t-test for unpaired observations. EBM and EGM-2 were purchased from Clonetics (Cell System, St. Katharinen, Germany). Fura-2/AM and mag-Fura-2 AM were obtained from Molecular Probes (Molecular Probes Europe, Leiden, Netherlands). N-(4-[3,5-Bis(trifluoromethyl)-1H-pyrazol-1-yl]phenyl)-4-methyl-1,2,3-thiadiazole-5-carboxamide (BTP-2) was purchased from Calbiochem (La Jolla, CA, USA). VEGF was provided by PeproTech (UK). All other chemicals were obtained from Sigma Chemical Company (St. Louis, MO). The most suitable concentration of VEGF for triggering intracellular Ca2+ oscillations in ECFCs is 10 ng/mL [13Dragoni S. Laforenza U. Bonetti E. et al.Vascular endothelial growth factor stimulates endothelial colony forming cells proliferation and tubulogenesis by inducing oscillations in intracellular Ca2+ concentration.Stem Cells. 2011; 29: 1898-1907Crossref PubMed Scopus (57) Google Scholar, 18Dragoni S. Laforenza U. Bonetti E. et al.Canonical transient receptor potential 3 channel triggers vascular endothelial growth factor-induced intracellular Ca2+ oscillations in endothelial progenitor cells isolated from umbilical cord blood.Stem Cells Dev. 2013; 22: 2561-2580Crossref PubMed Scopus (77) Google Scholar]. Therefore, we used this dose to compare VEGF-induced Ca2+ spikes between N- and PMF-ECFCs. Similar to their control counterparts [13Dragoni S. Laforenza U. Bonetti E. et al.Vascular endothelial growth factor stimulates endothelial colony forming cells proliferation and tubulogenesis by inducing oscillations in intracellular Ca2+ concentration.Stem Cells. 2011; 29: 1898-1907Crossref PubMed Scopus (57) Google Scholar], Fura-2/AM-loaded PMF-ECFCs did not exhibit a spontaneous increase in [Ca2+]i (data not shown). The addition of 10 ng/mL VEGF elicited intracellular Ca2+ oscillations in 286 of 372 cells (76.8%); the remaining 86 cells (23.2%) produced only a transient Ca2+ spike (Figure 1, Figure 2B ). As illustrated in Figure 1A, neighboring PMF-ECFCs recorded from the same microscopic field displayed asynchronous Ca2+ spikes in response to VEGF. Such heterogeneity had already been observed in both N-ECFCs and UCB-derived ECFCs [13Dragoni S. Laforenza U. Bonetti E. et al.Vascular endothelial growth factor stimulates endothelial colony forming cells proliferation and tubulogenesis by inducing oscillations in intracellular Ca2+ concentration.Stem Cells. 2011; 29: 1898-1907Crossref PubMed Scopus (57) Google Scholar, 18Dragoni S. Laforenza U. Bonetti E. et al.Canonical transient receptor potential 3 channel triggers vascular endothelial growth factor-induced intracellular Ca2+ oscillations in endothelial progenitor cells isolated from umbilical cord blood.Stem Cells Dev. 2013; 22: 2561-2580Crossref PubMed Scopus (77) Google Scholar] and represents the hallmark of growth factor-induced Ca2+ signals in ECs [27Moccia F. Berra-Romani R. Tritto S. Signorelli S. Taglietti V. Tanzi F. Epidermal growth factor induces intracellular Ca2+ oscillations in microvascular endothelial cells.J Cell Physiol. 2003; 194: 139-150Crossref PubMed Scopus (58) Google Scholar], as well other nonexcitable cell types [28Fewtrell C. Ca2+ oscillations in non-excitable cells.Annu Rev Physiol. 1993; 55: 427-454Crossref PubMed Scopus (202) Google Scholar]. Each baseline Ca2+ transient was preceded by a gradual increase in [Ca2+]i (dashed box in Fig. 1), the so-called pacemaker Ca2+ rise, which had already been observed in N- and UCB-ECFCs challenged with VEGF and is a feature of InsP3-dependent Ca2+ mobilization [13Dragoni S. Laforenza U. Bonetti E. et al.Vascular endothelial growth factor stimulates endothelial colony forming cells proliferation and tubulogenesis by inducing oscillations in intracellular Ca2+ concentration.Stem Cells. 2011; 29: 1898-1907Crossref PubMed Scopus (57) Google Scholar, 18Dragoni S. Laforenza U. Bonetti E. et al.Canonical transient receptor potential 3 channel triggers vascular endothelial growth factor-induced intracellular Ca2+ oscillations in endothelial progenitor cells isolated from umbilical cord blood.Stem Cells Dev. 2013; 22: 2561-2580Crossref PubMed Scopus (77) Google Scholar] (see also below). Moreover, VEGF-elicited Ca2+ spikes immediately ceased on agonist washout and resumed on VEGF re-addition to the perfusate (Fig. 2A). These preliminary data indicate that VEGF activates repetitive Ca2+ spikes in PMF-ECFCs. We then carried out a thorough comparison of VEGF-induced Ca2+ oscillations in N- and PMF-ECFCs (Fig. 2B). There was no statistical difference in the percentage of busting cells between the two ECFC types (Fig. 2C). However, the latency to the spiking response was significantly longer in PMF-ECFCs (Fig. 2D), whereas the amplitude of their initial Ca2+ transient was significantly lower (Fig. 2E). Because of the complexity of VEGF-induced Ca2+ oscillations [29Moccia F. Dragoni S. Lodola F. et al.Store-dependent Ca(2+) entry in endothelial progenitor cells as a perspective tool to enhance cell-based therapy and adverse tumour vascularization.Curr Med Chem. 2012; 19: 5802-5818Crossref PubMed Scopus (115) Google Scholar], extrapolation of clear-cut quantitative information from Ca2+ traces is not an easy task. Therefore, we further exploited homemade software based on wavelet analysis to finely compare the trains of Ca2+ spikes arising in the 2 cell types, as previously described [21Moccia F. Ruffinatti F.A. Zuccolo E. Intracellular Ca2+ signals to reconstruct a broken heart: Still a theoretical approach?.Curr Drug Targets. 2015; 16: 793-815Crossref PubMed Google Scholar, 26Ruffinatti F.A. Lovisolo D. Distasi C. Ariano P. Erriquez J. Ferraro M. Calcium signals: Analysis in time and frequency domains.J Neurosci Methods. 2011; 199: 310-320Crossref PubMed Scopus (5) Google Scholar]. In agreement with the previous results, the oscillatory index was significantly (p < 0.05) reduced in patient-derived ECFCs as compared with control cells despite the fact that VEGFR-2 is normally expressed in these cells [17Dragoni S. Laforenza U. Bonetti E. et al.Enhanced expression of Stim, Orai, and TRPC transcripts and proteins in endothelial progenitor cells isolated from patients with primary myelofibros
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