67 results on '"Poletto, V."'
Search Results
52. Increased plasma nicotinamide phosphoribosyltransferase is associated with a hyperproliferative phenotype and restrains disease progression in MPN-associated myelofibrosis.
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Rosti V, Campanelli R, Massa M, Viarengo G, Villani L, Poletto V, Bonetti E, Catarsi P, Magrini U, Grolla AA, Travelli C, Genazzani AA, and Barosi G
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- C-Reactive Protein analysis, Case-Control Studies, Cell Proliferation, Female, Hemoglobins analysis, Humans, Leukocyte Count, Male, Phenotype, Platelet Count, Polycythemia Vera, Prognosis, Thrombocythemia, Essential, Cytokines blood, Disease Progression, Myeloproliferative Disorders pathology, Nicotinamide Phosphoribosyltransferase blood, Primary Myelofibrosis pathology
- Abstract
Myeloproliferative neoplasm (MPN)-associated myelofibrosis is a clonal, neoplastic disorder of the hematopoietic stem cells, in which inflammation and immune dysregulation play an important role. Extracellular nicotinamide phosphoribosyltransferase (eNAMPT), also known as visfatin, is a cytokine implicated in a number of inflammatory and neoplastic diseases. Here plasma levels of eNAMPT in patients with MPN-associated myelofibrosis and their effects on disease phenotype and outcomes were examined. The concordance of eNAMPT levels with the marker of general inflammation high-sensitivity C-reactive protein (hs-CRP) was also studied. A total of 333 MPN-associated myelofibrosis patients (187 males and 146 females) and 31 age- and gender-matched normal-weight healthy subjects were enrolled in the study main body. Levels of eNAMPT and hs-CRP were simultaneously assayed in 209 MPN-associated myelofibrosis patients. Twenty-four polycythemia vera or essential thrombocythemia patients were used as controls. eNAMPT was over expressed in MPN-associated myelofibrosis, and eNAMPT expression was correlated with higher white blood cell count, higher hemoglobin, and higher platelet count, suggesting that eNAMPT is an indispensable permissive agent for myeloproliferation of MPN-associated myelofibrosis. The lack of correlation between eNAMPT and hs-CRP revealed that eNAMPT in MPN-associated myelofibrosis does not behave as a canonical inflammatory cytokine. In addition, higher levels of eNAMPT predicted longer time to blast transformation, and protected against progression toward thrombocytopenia and large splenomegaly. In conclusion, in MPN-associated myelofibrosis high levels of eNAMPT mark the myeloproliferative potential and, at variance with a high number of cancers, are protective against disease progression. Am. J. Hematol. 91:709-713, 2016. © 2016 Wiley Periodicals, Inc., (© 2016 Wiley Periodicals, Inc.)
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- 2016
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53. Constitutive Store-Operated Ca(2+) Entry Leads to Enhanced Nitric Oxide Production and Proliferation in Infantile Hemangioma-Derived Endothelial Colony-Forming Cells.
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Zuccolo E, Bottino C, Diofano F, Poletto V, Codazzi AC, Mannarino S, Campanelli R, Fois G, Marseglia GL, Guerra G, Montagna D, Laforenza U, Rosti V, Massa M, and Moccia F
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- Anilides pharmacology, Cell Proliferation drug effects, Child, Child, Preschool, Demography, Endothelial Cells drug effects, Endothelial Progenitor Cells drug effects, Female, Gene Expression Regulation drug effects, Gentamicins pharmacology, Humans, Indoles pharmacology, Intracellular Space metabolism, Lanthanum pharmacology, Male, RNA, Messenger genetics, RNA, Messenger metabolism, Thiadiazoles pharmacology, Calcium metabolism, Colony-Forming Units Assay, Endothelial Cells pathology, Endothelial Progenitor Cells pathology, Hemangioma pathology, Nitric Oxide biosynthesis
- Abstract
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.
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- 2016
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54. Targeting Stim and Orai Proteins as an Alternative Approach in Anticancer Therapy.
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Moccia F, Zuccolo E, Poletto V, Turin I, Guerra G, Pedrazzoli P, Rosti V, Porta C, and Montagna D
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- Antineoplastic Agents chemistry, Antineoplastic Agents pharmacology, Calcium Channel Blockers chemistry, Calcium Channel Blockers pharmacology, Calcium Channel Blockers therapeutic use, Calcium Signaling drug effects, Humans, Neoplasm Proteins antagonists & inhibitors, Neoplasms metabolism, Neoplasms pathology, ORAI1 Protein antagonists & inhibitors, Stromal Interaction Molecule 1 antagonists & inhibitors, Antineoplastic Agents therapeutic use, Neoplasm Proteins metabolism, Neoplasms drug therapy, ORAI1 Protein metabolism, Stromal Interaction Molecule 1 metabolism
- Abstract
An increase in intracellular Ca2+ concentration plays a key role in the establishment of many cancer hallmarks, including aberrant proliferation, migration, invasion, resistance to apoptosis and angiogenesis. The dysregulation of Ca2+ entry is one of the most subtle mechanisms by which cancer cells overwhelm their normal counterparts and gain the adaptive advantages that result in tumour growth, vascularisation and dissemination throughout the organism. Both constitutive and agonist-induced Ca2+ influx may be mediated by store-dependent as well as store-independent Ca2+ entry routes. A growing body of evidences have shown that different isoforms of Stromal Interaction Molecules (Stim1) and Orai proteins, i.e. Stim1, Stim2, Orai1 and Orai3, underlie both pathways in cancer cells. The alteration in either the expression or the activity of Stim and Orai proteins has been linked to the onset and maintenance of tumour phenotype in many solid malignancies, including prostate, breast, kidney, esophageal, skin, brain, colorectal, lung and liver cancers. Herein, we survey the existing data in support of Stim and Orai involvement in tumourigenesis and provide the rationale to target them in cancer patients. Besides, we summarize the most recent advances in the identification of novel pharmacological tools that could be successfully used in clinical therapy.
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- 2016
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55. Dysregulation of VEGF-induced proangiogenic Ca2+ oscillations in primary myelofibrosis-derived endothelial colony-forming cells.
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Dragoni S, Reforgiato M, Zuccolo E, Poletto V, Lodola F, Ruffinatti FA, Bonetti E, Guerra G, Barosi G, Rosti V, and Moccia F
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- Cells, Cultured, Endothelial Cells pathology, Female, Humans, Neovascularization, Pathologic drug therapy, Neovascularization, Pathologic pathology, Primary Myelofibrosis drug therapy, Primary Myelofibrosis pathology, Stem Cells pathology, Calcium Signaling, Endothelial Cells metabolism, Neovascularization, Pathologic metabolism, Primary Myelofibrosis metabolism, Stem Cells metabolism, Vascular Endothelial Growth Factor A metabolism
- Abstract
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., (Copyright © 2015 ISEH - International Society for Experimental Hematology. Published by Elsevier Inc. All rights reserved.)
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- 2015
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56. May the remodeling of the Ca²⁺ toolkit in endothelial progenitor cells derived from cancer patients suggest alternative targets for anti-angiogenic treatment?
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Moccia F and Poletto V
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- Animals, Calcium Channels metabolism, Endothelial Cells pathology, Humans, Neoplasm Proteins metabolism, Stem Cells pathology, Calcium metabolism, Calcium Signaling, Endothelial Cells metabolism, Neoplasms blood supply, Neoplasms metabolism, Neoplasms pathology, Neoplasms therapy, Neovascularization, Pathologic metabolism, Neovascularization, Pathologic pathology, Neovascularization, Pathologic therapy, Stem Cells metabolism
- Abstract
Endothelial progenitor cells (EPCs) may be recruited from bone marrow to sustain the metastatic switch in a number of solid cancers, including breast cancer (BC) and renal cellular carcinoma (RCC). Preventing EPC mobilization causes tumor shrinkage. Novel anti-angiogenic treatments have been introduced in therapy to inhibit VEGFR-2 signaling; unfortunately, these drugs blocked tumor angiogenesis in pre-clinical murine models, but resulted far less effective in human patients. Understanding the molecular mechanisms driving EPC proliferation and tubulogenesis in cancer patients could outline novel targets for alternative anti-angiogenic treatments. Store-operated Ca²⁺ entry (SOCE) regulates the growth of human EPCs, and it is mediated by the interaction between the endoplasmic reticulum Ca²⁺-sensor, Stim1, and the plasmalemmal Ca²⁺ channels, Orai1 and TRPC1. EPCs do not belong to the neoplastic clone: thus, unlike tumor endothelium and neoplastic cells, they should not remodel their Ca²⁺ toolkit in response to tumor microenvironment. However, our recent work demonstrated that EPCs isolated from naïve RCC patients (RCC-EPCs) undergo a dramatic remodeling of their Ca²⁺ toolkit by displaying a remarkable drop in the endoplasmic reticulum Ca²⁺ content, by down-regulating the expression of inositol-1,4,5-receptors (InsP3Rs), and by up-regulating Stim1, Orai1 and TRPC1. Moreover, EPCs are dramatically less sensitive to VEGF stimulation both in terms of Ca²⁺ signaling and of gene expression when isolated from tumor patients. Conversely, the pharmacological abolition of SOCE suppresses proliferation in these cells. These results question the suitability of VEGFR-2 as a therapeutically relevant target for anti-angiogenic treatments and hint at Orai1 and TRPC1 as more promising alternatives. This article is part of a Special Issue entitled: 13th European Symposium on Calcium., (Copyright © 2014 Elsevier B.V. All rights reserved.)
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- 2015
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57. Endothelial progenitor cells support tumour growth and metastatisation: implications for the resistance to anti-angiogenic therapy.
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Moccia F, Zuccolo E, Poletto V, Cinelli M, Bonetti E, Guerra G, and Rosti V
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- Calcium metabolism, Drug Resistance, Neoplasm genetics, Endothelial Progenitor Cells metabolism, Gene Expression Regulation, Neoplastic drug effects, Humans, Neoplasms drug therapy, Neoplasms pathology, Neovascularization, Pathologic drug therapy, Neovascularization, Pathologic pathology, Signal Transduction drug effects, Vascular Endothelial Growth Factor A antagonists & inhibitors, Vascular Endothelial Growth Factor A genetics, Endothelial Progenitor Cells pathology, Neoplasms genetics, Neovascularization, Pathologic genetics, Vascular Endothelial Growth Factor A biosynthesis
- Abstract
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.
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- 2015
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58. JAK2 exon 14 skipping in patients with primary myelofibrosis: a minor splice variant modulated by the JAK2-V617F allele burden.
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Catarsi P, Rosti V, Morreale G, Poletto V, Villani L, Bertorelli R, Pedrazzini M, Zorzetto M, and Barosi G
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- Cell Line, Computational Biology, Exons, Gene Expression Profiling, Humans, Janus Kinase 2 chemistry, Mutation, Regression Analysis, Sequence Deletion, Janus Kinase 2 genetics, Primary Myelofibrosis genetics, RNA Splicing
- Abstract
Background: Primary myelofibrosis (PMF) is an acquired clonal disease of the hematopoietic stem cell compartment, characterized by bone marrow fibrosis, anemia, splenomegaly and extramedullary hematopoiesis. About 60% of patients with PMF harbor a somatic mutation of the JAK2 gene (JAK2-V617F) in their hematopoietic lineage. Recently, a splicing isoform of JAK2, lacking exon 14 (JAK2Δ14) was described in patients affected by myeloproliferative diseases., Materials and Methods: By using a specific RT-qPCR method, we measured the ratio between the splicing isoform and the JAK2 full-length transcript (JAK2+14) in granulocytes, isolated from peripheral blood, of forty-four patients with PMF and nine healthy donors., Results: We found that JAK2Δ14 was only slightly increased in patients and, at variance with published data, the splicing isoform was also detectable in healthy controls. We also found that, in patients bearing the JAK2-V617F mutation, the percentage of mutated alleles correlated with the observed increase in JAK2Δ14. Homozygosity for the mutation was also associated with a higher level of JAK2+14. Bioinformatic analysis indicates the possibility that the G>T transversion may interfere with the correct splicing of exon 14 by modifying a splicing regulatory sequence., Conclusions: Increased levels of JAK2 full-length transcript and a small but significant increase in JAK2 exon 14 skipping, are associated with the JAK2-V617F allele burden in PMF granulocytes. Our data do not confirm a previous claim that the production of the JAK2Δ14 isoform is related to the pathogenesis of PMF.
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- 2015
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59. A functional transient receptor potential vanilloid 4 (TRPV4) channel is expressed in human endothelial progenitor cells.
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Dragoni S, Guerra G, Fiorio Pla A, Bertoni G, Rappa A, Poletto V, Bottino C, Aronica A, Lodola F, Cinelli MP, Laforenza U, Rosti V, Tanzi F, Munaron L, and Moccia F
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- Adult, Anilides pharmacology, Calcium metabolism, Cation Transport Proteins biosynthesis, Cell Proliferation drug effects, Cells, Cultured, Endothelial Cells cytology, Humans, Leucine analogs & derivatives, Leucine pharmacology, RNA, Messenger biosynthesis, Ruthenium Red pharmacology, Stem Cells cytology, Sulfonamides pharmacology, TRPV Cation Channels agonists, TRPV Cation Channels antagonists & inhibitors, TRPV Cation Channels genetics, Tetradecanoylphorbol Acetate pharmacology, Thiadiazoles pharmacology, Young Adult, Endothelial Cells metabolism, Endothelium, Vascular metabolism, Neovascularization, Physiologic physiology, Stem Cells metabolism, TRPV Cation Channels biosynthesis
- Abstract
Endothelial progenitor cells (EPCs) are mobilized into circulation to replace damaged endothelial cells and recapitulate the vascular network of injured tissues. Intracellular Ca(2+) signals are key to EPC activation, but it is yet to be elucidated whether they are endowed with the same blend of Ca(2+) -permeable channels expressed by mature endothelial cells. For instance, endothelial colony forming cells (ECFCs), the only EPC subset truly committed to acquire a mature endothelial phenotype, lack canonical transient receptor potential channels 3, 5 and 6 (TRPC3, 5 and 6), which are widely distributed in vascular endothelium; on the other hand, they express a functional store-operated Ca(2+) entry (SOCE). The present study was undertaken to assess whether human circulating EPCs possess TRP vanilloid channel 4 (TRPV4), which plays a master signalling role in mature endothelium, by controlling both vascular remodelling and arterial pressure. We found that EPCs express both TRPV4 mRNA and protein. Moreover, both GSK1016790A (GSK) and phorbol myristate acetate and, two widely employed TRPV4 agonists, induced intracellular Ca(2+) signals uniquely in presence of extracellular Ca(2+). GSK- and PMA-induced Ca(2+) elevations were inhibited by RN-1734 and ruthenium red, which selectively target TRPV4 in mature endothelium. However, TRPV4 stimulation with GSK did not cause EPC proliferation, while the pharmacological blockade of TRPV4 only modestly affected EPC growth in the presence of a growth factor-enriched culture medium. Conversely, SOCE inhibition with BTP-2, La(3+) and Gd(3+) dramatically decreased cell proliferation. These data indicate that human circulating EPCs possess a functional TRPV4 protein before their engraftment into nascent vessels., (© 2014 Wiley Periodicals, Inc.)
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- 2015
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60. Hydrogen sulphide triggers VEGF-induced intracellular Ca²⁺ signals in human endothelial cells but not in their immature progenitors.
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Potenza DM, Guerra G, Avanzato D, Poletto V, Pareek S, Guido D, Gallanti A, Rosti V, Munaron L, Tanzi F, and Moccia F
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- Adult, Cell Proliferation drug effects, Cells, Cultured, Cytoplasm metabolism, Endothelial Progenitor Cells cytology, Endothelial Progenitor Cells drug effects, Endothelium, Vascular cytology, Endothelium, Vascular drug effects, Healthy Volunteers, Humans, Type C Phospholipases metabolism, Wound Healing drug effects, Young Adult, Endothelial Progenitor Cells metabolism, Endothelium, Vascular metabolism, Hydrogen Sulfide pharmacology, Inositol 1,4,5-Trisphosphate metabolism, Signal Transduction drug effects, Vascular Endothelial Growth Factor A metabolism
- Abstract
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.)
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- 2014
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61. Enhanced expression of Stim, Orai, and TRPC transcripts and proteins in endothelial progenitor cells isolated from patients with primary myelofibrosis.
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Dragoni S, Laforenza U, Bonetti E, Reforgiato M, Poletto V, Lodola F, Bottino C, Guido D, Rappa A, Pareek S, Tomasello M, Guarrera MR, Cinelli MP, Aronica A, Guerra G, Barosi G, Tanzi F, Rosti V, and Moccia F
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- Adenosine Triphosphate pharmacology, Adult, Aged, Anilides pharmacology, Calcium metabolism, Calcium Channels genetics, Cell Proliferation drug effects, Cell Separation, Colony-Forming Units Assay, Endoplasmic Reticulum drug effects, Endoplasmic Reticulum metabolism, Endothelial Progenitor Cells drug effects, Female, Gadolinium pharmacology, Humans, Indoles pharmacology, Inositol 1,4,5-Trisphosphate metabolism, Intracellular Space drug effects, Intracellular Space metabolism, Lanthanum pharmacology, Male, Membrane Potentials drug effects, Membrane Proteins genetics, Middle Aged, RNA, Messenger genetics, RNA, Messenger metabolism, Signal Transduction drug effects, TRPC Cation Channels genetics, Thiadiazoles pharmacology, Young Adult, Calcium Channels metabolism, Endothelial Progenitor Cells metabolism, Membrane Proteins metabolism, Primary Myelofibrosis genetics, Primary Myelofibrosis pathology, TRPC Cation Channels metabolism
- Abstract
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.
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- 2014
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62. Orai1 and transient receptor potential channels as novel molecular targets to impair tumor neovascularization in renal cell carcinoma and other malignancies.
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Moccia F, Dragoni S, Poletto V, Rosti V, Tanzi F, Ganini C, and Porta C
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- Angiogenesis Inhibitors pharmacology, Animals, Calcium metabolism, Carcinoma, Renal Cell blood supply, Carcinoma, Renal Cell metabolism, Endothelial Cells physiology, Humans, Kidney Neoplasms blood supply, Kidney Neoplasms metabolism, Molecular Targeted Therapy, ORAI1 Protein, Signal Transduction, Stem Cells physiology, Angiogenesis Inhibitors therapeutic use, Calcium Channels metabolism, Carcinoma, Renal Cell drug therapy, Kidney Neoplasms drug therapy, Neovascularization, Pathologic drug therapy, Transient Receptor Potential Channels metabolism
- Abstract
The term "angiogenic switch" describes one of the earlier events of tumorigenesis, that occurs when the balance between pro-and anti-angiogenic factors shifts towards a pro-angiogenic outcome. This leads to the transition from a microscopic indolent lesion to a macroscopic and vascularised primary tumor, that may eventually metastasize and spread to distant sites. The molecular mechanisms underlying such a critical step in the carcinogenetic process have been extensively investigated. Both local endothelial cells (ECs) and endothelial progenitor cells (EPCs), recruited from bone marrow, have been implicated in the angiogenic switch, which is ultimately triggered by a plethora of growth factors released by cancer cells, pivotal among which is vascular endothelial growth factor (VEGF); indeed, VEGF both activates ECs nearby the growing tumor, and leads to EPC mobilization into the circulation. In kidney, in particular, the frequent mutation of the Von Hippel Lindau tumor suppressor gene leads to an overproduction of pro-angiogenic factors which makes this neoplasm quite sensitive to antiangiogenic drugs. However, it is now evident that the use of VEGF(Rs) inhibitors in everyday clinical practice is not as effective as observed in murine models. The investigation of alternative signaling pathways involved in the angiogenic switch is, therefore, imperative in order to induce tumor regression whereby preventing harmful drawback consequences. Ca(2+) entry across the plasma membrane has long been known to stimulate mature ECs to undergo angiogenesis. Recent work from several groups worldwide has then outlined that members of the Transient Receptor Potential (TRP) super-family of cationic channels and Orai1 provide the pathway for such proangiogenic Ca(2+) signal. In addition, Canonical TRP 1 (TRPC1) and Orai1 channels control proliferation and tubulogenesis in both normal EPCs and EPCs isolated from peripheral blood of tumor patients. As a consequence, TRP channels and Orai1 might serve as novel molecular targets to develop alternative and more effective strategies of angiogenesis inhibition.
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- 2014
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63. Spleen endothelial cells from patients with myelofibrosis harbor the JAK2V617F mutation.
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Rosti V, Villani L, Riboni R, Poletto V, Bonetti E, Tozzi L, Bergamaschi G, Catarsi P, Dallera E, Novara F, Massa M, Campanelli R, Fois G, Peruzzi B, Lucioni M, Guglielmelli P, Pancrazzi A, Fiandrino G, Zuffardi O, Magrini U, Paulli M, Vannucchi AM, and Barosi G
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- Aged, Cell Separation, Comparative Genomic Hybridization, Female, Flow Cytometry, Humans, In Situ Hybridization, Fluorescence, Laser Capture Microdissection, Male, Middle Aged, Mutation, Reverse Transcriptase Polymerase Chain Reaction, Endothelial Cells pathology, Janus Kinase 2 genetics, Primary Myelofibrosis genetics, Spleen pathology
- Abstract
Increased microvessel density contributes to abnormal BM and spleen microenvironment in myelofibrosis (MF). Taking advantage of the JAK2V617F mutation as a marker of malignancy, in the present study, we investigated whether splenic endothelial cells (ECs) obtained from capillaries by laser microdissection or from fresh spleen tissue by cell culture or cell sorting harbored such mutation in patients bearing the mutation in their granulocytes and undergoing splenectomy for therapeutical reasons. To extend the analysis to the ECs of large vessels, endothelial tissue from the splenic vein was also studied. We found JAK2V617F(+) ECs in 12 of 18 patients also bearing the mutation in their granulocytes. In 3 patients, the mutation was found in at least 2 different EC samples obtained by laser microdissection, cell culture, or cell sorting. The mutation was detected in the splenic vein ECs of 1 of 6 patients investigated. In conclusion, we provide evidence that some ECs from the spleen and splenic veins of patients with MF bear the JAK2V617F mutation. We suggest that splenic ECs are involved in the process of malignant transformation in MF.
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- 2013
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64. JAK2 V617F genotype is a strong determinant of blast transformation in primary myelofibrosis.
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Barosi G, Poletto V, Massa M, Campanelli R, Villani L, Bonetti E, Viarengo G, Catarsi P, Klersy C, and Rosti V
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- Adolescent, Adult, Age Factors, Aged, Aged, 80 and over, Alleles, Bone Marrow Cells, Child, Cohort Studies, Female, Genotype, Heterozygote, Homozygote, Humans, Male, Middle Aged, Mutation, Proportional Hazards Models, Retrospective Studies, Treatment Outcome, Young Adult, Janus Kinase 2 genetics, Primary Myelofibrosis genetics, Primary Myelofibrosis mortality
- Abstract
Purpose: The influence of JAK2 V617F mutation on blast transformation (BT) and overall survival (OS) in primary myelofibrosis (PMF) is controversial. In a large cohort of patients we applied competing risks analysis for studying the influence of JAK2V617F mutation on BT in PMF., Patients and Methods: In 462 PMF-fibrotic type patients (bone marrow [BM] fibrosis grade >0) we computed the incidence of BT and death in the framework of Cox regression analysis and of Fine and Gray competing risks analysis for BT., Results: At the Cox regression analysis, having either a wild-type (wt) or a homozygous JAK2V617F genotype were factors for BT (HR, 1.98 and 2.04, respectively, with respect to the heterozygous genotype), but not for OS. At the competing risks regression analysis, the risk for BT in wt and homozygous V617F patients increased with respect to Cox analysis, giving a sHR of 2.17 and 2.12, respectively. Correcting the results for the variables that could have influence on BT, JAK2V617F wt and homozygous genotypes remained independently associated with BT. In a validation cohort of 133 independent cases with PMF-prefibrotic type (BM fibrosis grade = 0), the BT predictive model including JAK2V617F genotype and older age retained high discriminant capacity (C statistics, 0.70; 95% CI, 0.47 to 0.92)., Conclusion: The accumulation of mutated alleles in the JAK2V617F clone or the selective acquisition of a proliferative advantage in the wt clone are two relevant routes to BT in PMF. The influence of these results on treatment decisions with anti-JAK2 agents should be tested.
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- 2013
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65. A3669G polymorphism of glucocorticoid receptor is a susceptibility allele for primary myelofibrosis and contributes to phenotypic diversity and blast transformation.
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Poletto V, Rosti V, Villani L, Catarsi P, Carolei A, Campanelli R, Massa M, Martinetti M, Viarengo G, Malovini A, Migliaccio AR, and Barosi G
- Subjects
- Adolescent, Adult, Aged, Aged, 80 and over, Case-Control Studies, Child, Cohort Studies, Female, Follow-Up Studies, Humans, Janus Kinase 2 genetics, Male, Middle Aged, Phenotype, Primary Myelofibrosis mortality, Prognosis, Survival Rate, Young Adult, Genetic Predisposition to Disease, Lymphocyte Activation genetics, Mutation genetics, Polymorphism, Single Nucleotide genetics, Primary Myelofibrosis genetics, Receptors, Glucocorticoid genetics
- Abstract
The frequency of A3669G single nucleotide polymorphism (SNP) of human glucocorticoid receptor has been reported increased in polycythemia vera. We investigated the frequency of A3669G SNP and its impact on disease phenotype and progression in 499 patients with primary myelofibrosis (PMF). The distribution of the A3669G allele differed between PMF patients and 2 healthy control populations (odds ratio, 1.6 and 1.8). The variant allele at the homozygous state (G/G) was associated with higher white blood cell count, larger spleen index, and higher frequency of circulating CD34(+) cells at diagnosis. The latter association remained significant after correction for the JAK2V617F genotype. In patients JAK2V617F mutated, the G/G genotype was associated with shorter overall survival (77.6 months vs 298 months, P = .049) and blast transformation (BT)-free survival (76.7 months vs 261 months; P = .018). The latter association remained significant after correction for the known BT risk factors, such as age, sex, white blood cell count, percentage of blasts, IPSS prognostic score, and homozygosity for JAK2V617F (hazard ratio = 3.3; P = .006). In conclusion, the glucocorticoid receptor A3669G is a susceptibility allele for PMF: it contributes to confer the phenotype of excess myeloproliferation, and it cooperates with the JAK2V617F mutation in determining BT.
- Published
- 2012
- Full Text
- View/download PDF
66. JAK2 46/1 haplotype predisposes to splanchnic vein thrombosis-associated BCR-ABL negative classic myeloproliferative neoplasms.
- Author
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Villani L, Bergamaschi G, Primignani M, Rosti V, Carolei A, Poletto V, Catarsi P, Spolverini A, Vannucchi AM, and Barosi G
- Subjects
- Bone Marrow Neoplasms genetics, Case-Control Studies, Fusion Proteins, bcr-abl genetics, Gene Frequency, Haplotypes, Humans, Janus Kinase 2 physiology, Mesenteric Veins, Mutation, Phenylalanine genetics, Polymorphism, Genetic physiology, Polymorphism, Single Nucleotide, Splanchnic Circulation genetics, Valine genetics, Genetic Predisposition to Disease, Janus Kinase 2 genetics, Mesenteric Vascular Occlusion genetics, Myeloproliferative Disorders genetics, Venous Thrombosis genetics
- Published
- 2012
- Full Text
- View/download PDF
67. Evidence that prefibrotic myelofibrosis is aligned along a clinical and biological continuum featuring primary myelofibrosis.
- Author
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Barosi G, Rosti V, Bonetti E, Campanelli R, Carolei A, Catarsi P, Isgrò AM, Lupo L, Massa M, Poletto V, Viarengo G, Villani L, and Magrini U
- Subjects
- Adolescent, Adult, Aged, Aged, 80 and over, Child, Cohort Studies, DNA Mutational Analysis, Female, Humans, Janus Kinase 2 genetics, Kaplan-Meier Estimate, Logistic Models, Male, Middle Aged, Mutation, Missense, Phenotype, Primary Myelofibrosis genetics, Primary Myelofibrosis mortality, Prognosis, World Health Organization, Young Adult, Primary Myelofibrosis classification, Primary Myelofibrosis diagnosis
- Abstract
Purpose: In the WHO diagnostic classification, prefibrotic myelofibrosis (pre-MF) is included in the category of primary myelofibrosis (PMF). However, strong evidence for this position is lacking., Patients and Methods: We investigated whether pre-MF may be aligned along a clinical and biological continuum in 683 consecutive patients who received a WHO diagnosis of PMF., Results: As compared with PMF-fibrotic type, pre-MF (132 cases) showed female dominance, younger age, higher hemoglobin, higher platelet count, lower white blood cell count, smaller spleen index and higher incidence of splanchnic vein thrombosis. Female to male ratio and hemoglobin steadily decreased, while age increased from pre-MF to PMF- fibrotic type with early and to advanced bone marrow (BM) fibrosis. Likely, circulating CD34+ cells, LDH levels, and frequency of chromosomal abnormalities increased, while CXCR4 expression on CD34+ cells and serum cholesterol decreased along the continuum of BM fibrosis. Median survival of the entire cohort of PMF cases was 21 years. Ninety-eight, eighty-one and fifty-six percent of patients with pre-MF, PMF-fibrotic type with early and with advanced BM fibrosis, respectively, were alive at 10 years from diagnosis., Conclusion: Pre-MF is a presentation mode of PMF with a very indolent phenotype. The major consequences of this contention is a new clinical vision of PMF, and the need to improve prognosis prediction of the disease.
- Published
- 2012
- Full Text
- View/download PDF
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