Pathogenesis of chronic myelogenous leukemia.

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Vascular endothelial growth factor promoted endothelial progenitor cell mobilization into the peripheral blood of a patient with POEMS syndrome

We assessed the percentage of endothelial progenitor cells (EPCs) in the peripheral blood of a patient with POEMS with elevated VEGF plasma levels. High VEGF plasma levels were associated with increased EPC concentration and treatment with an anti-VEGF antibody induced a consensual decrease of both parameters. In vitro cultures of the patient BM cells suggested that the stromal compartment could be responsible for VEGF overproduction

Cell fusion in tumor progression: the isolation of cell fusion products by physical methods

<p>Abstract</p> <p>Background</p> <p>Cell fusion induced by polyethylene glycol (PEG) is an efficient but poorly controlled procedure for obtaining somatic cell hybrids used in gene mapping, monoclonal antibody production, and tumour immunotherapy. Genetic selection techniques and fluorescent cell sorting are usually employed to isolate cell fusion products, but both procedures have several drawbacks.</p> <p>Results</p> <p>Here we describe a simple improvement in PEG-mediated cell fusion that was obtained by modifying the standard single-step procedure. We found that the use of two PEG undertreatments obtains a better yield of cell fusion products than the standard method, and most of these products are bi- or trinucleated polykaryocytes. Fusion rate was quantified using fluorescent cell staining microscopy. We used this improved cell fusion and cell isolation method to compare giant cells obtained in vitro and giant cells obtained in vivo from patients with Hodgkin's disease and erythroleukemia.</p> <p>Conclusions</p> <p>In the present study we show how to improve PEG-mediated cell fusion and that cell separation by velocity sedimentation offers a simple alternative for the efficient purification of cell fusion products and to investigate giant cell formation in tumor development.</p

MULTILAYER ANTERIOR SKULL BASE RECONSTRUCTION WITH CORTICAL RIB BONE GRAFT: PRELIMINARY EXPERIENCE

Objective: During the past decades, different methods have been described for anterior skull base reconstruction. Regarding larger skull base defects, few investigators have described the use of bone grafts to foster support and prevent frontal lobe sagging, herniation, or falling. The aim of this study is to describe the use of a rib bone graft, which could be an option in these cases due to its rigidity and dimensions. Methods: We retrospectively collected preoperative, intraoperative, and postoperative data at the last follow-up of 10 patients who underwent multilayer anterior skull base reconstruction, including rib bone graft, for large anterior cranial base defects at 2 tertiary care academic hospitals. Results: Eight patients underwent endoscopic craniectomy for sinonasal malignancies, and the other two underwent transnasal endoscopic surgery for congenital meningoencephalocele. Anterior skull base defects measured on average 3.8 cm ± 0.9 SD antero-posteriorly (range 2.5-5 cm) and 2.3 ± 0.9 SD latero-laterally (range 0.9-4 cm). Multilayer reconstruction was performed in all cases, including a rib bone graft positioned as intracranial extradural layer. No patient experienced thoracic complications during the postoperative period. No side effects related to the bone graft or meningoencephalocele occurrence were reported after a mean follow-up of 8.0 ± 6.3 months. Conclusions: The use of a cortical rib bone graft could be a safe and effective option in skull base reconstruction when managing large defects after cancer removal

Liposomes as a Putative Tool to Investigate NAADP Signaling in Vasculogenesis

none8noNicotinic acid adenine dinucleotide phosphate (NAADP) is the newest discovered intracellular second messengers, which is able to release Ca(2+) stored within endolysosomal (EL) vesicles. NAADP-induced Ca(2+) signals mediate a growing number of cellular functions, ranging from proliferation to muscle contraction and differentiation. Recently, NAADP has recently been shown to regulate angiogenesis by promoting endothelial cell growth. It is, however, still unknown whether NAADP stimulates proliferation also in endothelial progenitor cells, which are mobilized in circulation after an ischemic insult to induce tissue revascularization. Herein, we described a novel approach to prepare NAADP-containing liposomes, which are highly cell membrane permeable and are therefore amenable for stimulating cell activity. Accordingly, NAADP-containing liposomes evoked an increase in intracellular Ca(2+) concentration, which was inhibited by NED-19, a selective inhibitor of NAADP-induced Ca(2+) release. Furthermore, NAADP-containing liposomes promoted EPC proliferation, a process which was inhibited by NED-19 and BAPTA, a membrane permeable intracellular Ca(2+) buffer. Therefore, NAADP-containing liposomes stand out as a promising tool to promote revascularization of hypoxic/ischemic tissues by favoring EPC proliferation. J. Cell. Biochem. 9999: 1-8, 2017. © 2017 Wiley Periodicals, Inc.openDi Nezza, Francesca; Zuccolo, Estella; Poletto, Valentina; Rosti, Vittorio; De Luca, Antonio; Moccia, Francesco; Guerra, Germano; Ambrosone, LuigiDi Nezza, Francesca; Zuccolo, Estella; Poletto, Valentina; Rosti, Vittorio; De Luca, Antonio; Moccia, Francesco; Guerra, Germano; Ambrosone, Luig

Phenotypic and functional characterization of endothelial progenitor cells isolated from peripheral blood of renal cell carcinoma patients

Endothelial progenitor cells (EPCs) are mobilized from either bone marrow or arterial walls to restore blood perfusion to ischemic organs and establish the vascular network within growing tumors [1]. The Ca2+ machinery plays a key role in EPC activation and might serve a molecular target for novel therapies of highly angiogenic tumors, such as renal cell carcinoma (RCC) [1]. The Ca2+ toolkit is remodelled in EPCs isolated from RCC patients (RCC-EPCs) as respect to healthy donors [2]. The present study was undertaken to evaluate for the first time the functional properties of EPCs isolated from tumor patients by focusing on RCC-EPCs. We extended our analysis at microscopic level by monitoring the sub-cellular structure of RCC-EPCs relative to their Ca2+ signalling fingerprint. Our results showed a striking functional and ultrastructural difference between RCC-EPCs and their normal counterparts, which might be the basis for designing novel, more specific anti-angiogenic treatments

“VEGF induces human endothelial progenitor cells proliferations by eliciting oscillations in intracellular Ca2+ concentration”

Endothelial progenitor cells (EPCs) traffic from the bone marrow to the site of tissue regeneration and sustain neo-vascularization after acute vascular injury and upon the angiogenic switch in solid tumors. Therefore, they represent a suitable tool for cell-based therapy in regenerative medicine and provide a novel promising target in the fight against cancer. The main stimulus responsible for EPC egression from the bone marrow and engraftment within neovessels is vascular endothelial growth factor (VEGF). Intracellular Ca2+ signals regulate numerous endothelial functions, such as proliferation, migration, and differentiation, and underpin VEGF effect on mature endothelium. We have recently shown that EPC growth is governed by a store-dependent Ca2+ entry (SOCE) pathway on the plasma membrane, which is activated by depletion of the inositol-1,4,5-trisphosphate (InsP3)-sensitive Ca2+ pools1. The present study aimed at investigating the nature and the role of VEGF-elicited Ca2+ signals in EPCs. All the putative SOCE mediators (i.e. TRPC1, TRPC4, Orai1 and Stim1) were present in EPCs. VEGF induced long lasting Ca2+ oscillations, however, removal of external Ca2+ (0Ca2+) and SOCE inhibition with BTP-2 reduced the number of Ca2+ spikes. Blockade of phospholipase C-? (PLC-?) with U73122 and emptying the InsP3-sensitive Ca2+ pools with cyclopiazonic acid (CPA) prevented the Ca2+ response to VEGF. Accordingly, the Ca2+ response to VEGF was inhibited by superfusing CPA during the ongoing oscillations. Notably, VEGF induced EPC was abrogated by SOCE inhibition with BTP-2. Similarly, VEGF promoted NF-kB translocation into the nucleus in a BTP-2-sensitive manner. Thus, VEGF causes an initial InsP3-dependent Ca2+ discharge followed by SOCE-mediated Ca2+ entry in cEPCs. SOCE, in turn, controls store refilling and induces cell proliferation by recruiting NF-kB

Evidence that Prefibrotic Myelofibrosis Is Aligned along a Clinical and Biological Continuum Featuring Primary Myelofibrosis

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

miR-494-3p overexpression promotes megakaryocytopoiesis in primary myelofibrosis hematopoietic stem/progenitor cells by targeting SOCS6

Primary myelofibrosis (PMF) is a chronic Philadelphia-negative myeloproliferative neoplasm characterized by hematopoietic stem cell-derived clonal myeloproliferation, involving especially the megakaryocyte lineage. To better characterize how the altered expression of microRNAs might contribute to PMF pathogenesis, we have previously performed the integrative analysis of gene and microRNA expression profiles of PMF hematopoietic stem/progenitor cells (HSPCs), which allowed us to identify miR- 494-3p as the upregulated microRNA predicted to target the highest number of downregulated mRNAs. To elucidate the role of miR-494-3p in hematopoietic differentiation, in the present study we demonstrated that miR-494-3p enforced expression in normal HSPCs promotes megakaryocytopoiesis. Gene expression profiling upon miR-494-3p overexpression allowed the identification of genes commonly downregulated both after microRNA overexpression and in PMF CD34+ cells. Among them, suppressor of cytokine signaling 6 (SOCS6) was confirmed to be a miR-494-3p target by luciferase assay. Western blot analysis showed reduced level of SOCS6 protein as well as STAT3 activation in miR-494-3p overexpressing cells. Furthermore, transient inhibition of SOCS6 expression in HSPCs demonstrated that SOCS6 silencing stimulates megakaryocytopoiesis, mimicking the phenotypic effects observed upon miR-494-3p overexpression. Finally, to disclose the contribution of miR-494-3p upregulation to PMF pathogenesis, we performed inhibition experiments in PMF HSPCs, which showed that miR-494-3p silencing led to SOCS6 upregulation and impaired megakaryocyte differentiation. Taken together, our results describe for the first time the role of miR-494- 3p during normal HSPC differentiation and suggest that its increased expression, and the subsequent downregulation of its target SOCS6, might contribute to the megakaryocyte hyperplasia commonly observed in PMF patients