Publications by authors named "Vittorio Rosti"

133 Publications

Increased Plasma Levels of lncRNAs , and Correlate with Negative Prognostic Factors in Myelofibrosis.

Cancers (Basel) 2021 Sep 22;13(19). Epub 2021 Sep 22.

Department of Medical and Surgical Sciences, University of Modena and Reggio Emilia, AOU Policlinico, 41124 Modena, Italy.

Long non-coding RNAs (lncRNAs) have been recently described as key mediators in the development of hematological malignancies. In the last years, circulating lncRNAs have been proposed as a new class of non-invasive biomarkers for cancer diagnosis and prognosis and to predict treatment response. The present study is aimed to investigate the potential of circulating lncRNAs as non-invasive prognostic biomarkers in myelofibrosis (MF), the most severe among Philadelphia-negative myeloproliferative neoplasms. We detected increased levels of seven circulating lncRNAs in plasma samples of MF patients ( = 143), compared to healthy controls ( = 65). Among these, high levels of , or correlate with detrimental clinical variables, such as high count of leukocytes and CD34+ cells, severe grade of bone marrow fibrosis and presence of splenomegaly. Strikingly, high plasma levels of = 0.0018) ( = 0.0008) or ( = 0.0348) are also associated with a poor overall-survival while high levels of correlate with a shorter leukemia-free-survival. Finally, multivariate analysis demonstrated that the plasma level of is an independent prognostic variable, suggesting that, if confirmed in future in an independent patients' cohort, it could be used for further studies to design an updated classification model for MF patients.
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http://dx.doi.org/10.3390/cancers13194744DOI Listing
September 2021

Clinical Relevance of (rs3025039) +936 C>T Polymorphism in Primary Myelofibrosis: Susceptibility, Clinical Co-Variates, and Outcomes.

Genes (Basel) 2021 Aug 20;12(8). Epub 2021 Aug 20.

Center for the Study of Myelofibrosis, Laboratory of Biochemistry, Biotechnology and Advanced Diagnostics, Istituto di Ricovero e Cura a Carattere Scientifico Policlinico S. Matteo Foundation, 27100 Pavia, Italy.

We evaluated the association of rs3025039 polymorphism with clinical co-variates and outcomes in 849 subjects with primary myelofibrosis (PMF) and 250 healthy controls. Minor T-allele frequency was higher in subjects with compared with those without (18% vs. 13%; = 0.014). In subjects with , the TT genotype was associated at diagnosis with lower platelet concentrations ( = 0.033), higher plasma LDH concentration ( = 0.005), higher blood CD34-positive cells ( = 0.027), lower plasma cholesterol concentration ( = 0.046), and higher concentration of high-sensitivity C-reactive protein ( = 0.018). These associations were not found in subjects with PMF without . In subjects with the TT genotype, risk of death was higher compared with subjects with CC/CT genotypes (HR = 2.12 [1.03, 4.35], = 0.041). Finally, the TT genotype was associated with higher frequency of deep vein thrombosis in typical sites (12.5% vs. 2.5%; OR = 5.46 [1.51, 19.7], = 0.009). In conclusion, in subjects with PMF, the rs3025039 CT or TT genotypes are more common in those with than in those without mutation and are associated with disease severity, poor prognosis, and risk of deep vein thrombosis.
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http://dx.doi.org/10.3390/genes12081271DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8393853PMC
August 2021

The human amniotic fluid stem cell secretome triggers intracellular Ca oscillations, NF-κB nuclear translocation and tube formation in human endothelial colony-forming cells.

J Cell Mol Med 2021 Aug 20;25(16):8074-8086. Epub 2021 Jul 20.

Department of Biology and Biotechnology "Lazzaro Spallanzani", Laboratory of General Physiology, University of Pavia, Pavia, Italy.

Second trimester foetal human amniotic fluid-derived stem cells (hAFS) have been shown to possess remarkable cardioprotective paracrine potential in different preclinical models of myocardial injury and drug-induced cardiotoxicity. The hAFS secretome, namely the total soluble factors released by cells in their conditioned medium (hAFS-CM), can also strongly sustain in vivo angiogenesis in a murine model of acute myocardial infarction (MI) and stimulates human endothelial colony-forming cells (ECFCs), the only truly recognized endothelial progenitor, to form capillary-like structures in vitro. Preliminary work demonstrated that the hypoxic hAFS secretome (hAFS-CM ) triggers intracellular Ca oscillations in human ECFCs, but the underlying mechanisms and the downstream Ca -dependent effectors remain elusive. Herein, we found that the secretome obtained by hAFS undergoing hypoxic preconditioning induced intracellular Ca oscillations by promoting extracellular Ca entry through Transient Receptor Potential Vanilloid 4 (TRPV4). TRPV4-mediated Ca entry, in turn, promoted the concerted interplay between inositol-1,4,5-trisphosphate- and nicotinic acid adenine dinucleotide phosphate-induced endogenous Ca release and store-operated Ca entry (SOCE). hAFS-CM -induced intracellular Ca oscillations resulted in the nuclear translocation of the Ca -sensitive transcription factor p65 NF-κB. Finally, inhibition of either intracellular Ca oscillations or NF-κB activity prevented hAFS-CM -induced ECFC tube formation. These data shed novel light on the molecular mechanisms whereby hAFS-CM induces angiogenesis, thus providing useful insights for future therapeutic strategies against ischaemic-related myocardial injury.
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http://dx.doi.org/10.1111/jcmm.16739DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8358861PMC
August 2021

Clonal Megakaryocyte Dysplasia with Normal Blood Values Is a Distinct Myeloproliferative Neoplasm.

Acta Haematol 2021 Jul 19:1-8. Epub 2021 Jul 19.

Department of Immunology and Inflammation, Center for Haematology Research, Imperial College London, London, United Kingdom.

Introduction: In 1991, we reported 18 persons with a clinical-pathologic entity and termed atypical myeloproliferative disorder because they did not meet the contemporary diagnostic criteria for a myeloproliferative neoplasm. We sought to gain further knowledge on this disease entity.

Methods: This retrospective cohort study included consecutive subjects registered in the database of the Center for the Study of Myelofibrosis in Pavia, Italy, from 1998 to 2020 (June), and diagnosed with atypical myeloproliferative disorder according to our adjudicated criteria. We studied clinical, histological, cytogenetic, and molecular covariates and risks of thrombosis, disease progression, and death. Data were compared with those of concurrent subjects with prefibrotic myelofibrosis.

Results: Fifteen new subjects with atypical myeloproliferative disorder were identified. Seven were male. Median age was 50 years (IQR, 41-54 years). Thirteen were diagnosed with a synchronous symptomatic or incidentally detected thrombotic event. The bone marrow showed megakaryocyte hyperplasia with dysplasia. JAK2V617F was present in 10 subjects and CALR mutation in one. No other somatic mutations were identified in next generation sequencing. After a median follow-up of 101 months (IQR, 40-160 months), no subject had disease progression or blast transformation. Incidence of post-diagnosis or recurrent thrombosis was 3.9 events (95% confidence interval, 3.5-4.0) and 5.0 events (4.6-5.6) per 100 person-years. Features of subjects with atypical myeloproliferative disorder differed markedly from those of 546 subjects with prefibrotic myelofibrosis.

Conclusion: Our data indicate that these 15 persons have a distinct myeloproliferative neoplasm. We propose naming this new disorder clonal megakaryocyte dysplasia with normal blood values.
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http://dx.doi.org/10.1159/000517207DOI Listing
July 2021

Impact of the rs1024611 Polymorphism of on the Pathophysiology and Outcome of Primary Myelofibrosis.

Cancers (Basel) 2021 May 22;13(11). Epub 2021 May 22.

Department of Medicine and Surgery, Anatomy Unit, University of Parma, 43126 Parma, Italy.

Single nucleotide polymorphisms (SNPs) can modify the individual pro-inflammatory background and may therefore have relevant implications in the MPN setting, typified by aberrant cytokine production. In a cohort of 773 primary myelofibrosis (PMF), we determined the contribution of the rs1024611 SNP of CCL2-one of the most potent immunomodulatory chemokines-to the clinical and biological characteristics of the disease, demonstrating that male subjects carrying the homozygous genotype G/G had an increased risk of PMF and that, among PMF patients, the G/G genotype is an independent prognostic factor for reduced overall survival. Functional characterization of the SNP and the CCL2-CCR2 axis in PMF showed that i) homozygous PMF cells are the highest chemokine producers as compared to the other genotypes; ii) PMF CD34+ cells are a selective target of CCL2, since they uniquely express CCR2 (CCL2 receptor); iii) activation of the CCL2-CCR2 axis boosts pro-survival signals induced by driver mutations via Akt phosphorylation; iv) ruxolitinib effectively counteracts CCL2 production and down-regulates CCR2 expression in PMF cells. In conclusion, the identification of the role of the CCL2/CCR2 chemokine system in PMF adds a novel element to the pathophysiological picture of the disease, with clinical and therapeutic implications.
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http://dx.doi.org/10.3390/cancers13112552DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8196972PMC
May 2021

Long-term safety and efficacy of givinostat in polycythemia vera: 4-year mean follow up of three phase 1/2 studies and a compassionate use program.

Blood Cancer J 2021 03 6;11(3):53. Epub 2021 Mar 6.

Clinical R&D Department, Italfarmaco S.p.A, Cinisello Balsamo, Italy.

Polycythemia vera (PV) is a BCR-ABL1-negative myeloproliferative neoplasm (MPN) characterized by excessive proliferation of erythroid, myeloid, and megakaryocytic components in the bone marrow, mainly due to a Janus kinase 2 gene mutation (JAK2). Givinostat, a histone-deacetylase inhibitor that selectively targets JAK2 cell growth, has demonstrated good efficacy and safety in three phase 1/2 studies in patients with PV. This manuscript focuses on the 4-year mean (2.8 year median) follow-up of an open-label, long-term study that enrolled 51 patients with PV (out of a total of 54 with MPN) who received clinical benefit from givinostat in these previous studies or on compassionate use, and who continued to receive givinostat at the last effective and tolerated dose. The primary objectives are to determine givinostat's long-term safety and tolerability, and efficacy evaluated by the investigators according to internationally recognized response criteria. During follow-up, only 10% of PV patients reported Grade 3 treatment-related adverse events (AEs), while none had Grade 4 or 5 treatment-related AEs. The overall response rate for the duration of follow-up was always greater than 80% in patients with PV. In conclusion, givinostat demonstrated a good safety and efficacy profile in patients with PV, data supporting long-term use in this population.
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http://dx.doi.org/10.1038/s41408-021-00445-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7936975PMC
March 2021

Gene expression profile correlates with molecular and clinical features in patients with myelofibrosis.

Blood Adv 2021 03;5(5):1452-1462

Center for Genome Research.

Myelofibrosis (MF) belongs to the family of classic Philadelphia-negative myeloproliferative neoplasms (MPNs). It can be primary myelofibrosis (PMF) or secondary myelofibrosis (SMF) evolving from polycythemia vera (PV) or essential thrombocythemia (ET). Despite the differences, PMF and SMF patients are currently managed in the same way, and prediction of survival is based on the same clinical and genetic features. In the last few years, interest has grown concerning the ability of gene expression profiles (GEPs) to provide valuable prognostic information. Here, we studied the GEPs of granulocytes from 114 patients with MF, using a microarray platform to identify correlations with patient characteristics and outcomes. Cox regression analysis led to the identification of 201 survival-related transcripts characterizing patients who are at high risk for death. High-risk patients identified by this gene signature displayed an inferior overall survival and leukemia-free survival, together with clinical and molecular detrimental features included in contemporary prognostic models, such as the presence of high molecular risk mutations. The high-risk group was enriched in post-PV and post-ET MF and JAK2V617F homozygous patients, whereas pre-PMF was more frequent in the low-risk group. These results demonstrate that GEPs in MF patients correlate with their molecular and clinical features, particularly their survival, and represent the proof of concept that GEPs might provide complementary prognostic information to be applied in clinical decision making.
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http://dx.doi.org/10.1182/bloodadvances.2020003614DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7948267PMC
March 2021

Co-mutation pattern, clonal hierarchy, and clone size concur to determine disease phenotype of SRSF2-mutated neoplasms.

Leukemia 2021 08 21;35(8):2371-2381. Epub 2020 Dec 21.

Department of Molecular Medicine, University of Pavia, Pavia, Italy.

Somatic mutations in splicing factor genes frequently occur in myeloid neoplasms. While SF3B1 mutations are associated with myelodysplastic syndromes (MDS) with ring sideroblasts, SRSF2 mutations are found in different disease categories, including MDS, myeloproliferative neoplasms (MPN), myelodysplastic/myeloproliferative neoplasms (MDS/MPN), and acute myeloid leukemia (AML). To identify molecular determinants of this phenotypic heterogeneity, we explored molecular and clinical features of a prospective cohort of 279 SRSF2-mutated cases selected from a population of 2663 patients with myeloid neoplasms. Median number of somatic mutations per subject was 3. Multivariate regression analysis showed associations between co-mutated genes and clinical phenotype, including JAK2 or MPL with myelofibrosis (OR = 26.9); TET2 with monocytosis (OR = 5.2); RAS-pathway genes with leukocytosis (OR = 5.1); and STAG2, RUNX1, or IDH1/2 with blast phenotype (MDS or AML) (OR = 3.4, 1.9, and 2.1, respectively). Within patients with SRSF2-JAK2 co-mutation, JAK2 dominance was invariably associated with clinical feature of MPN, whereas SRSF2 mutation was dominant in MDS/MPN. Within patients with SRSF2-TET2 co-mutation, clinical expressivity of monocytosis was positively associated with co-mutated clone size. This study provides evidence that co-mutation pattern, clone size, and hierarchy concur to determine clinical phenotype, tracing relevant genotype-phenotype associations across disease entities and giving insight on unaccountable clinical heterogeneity within current WHO classification categories.
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http://dx.doi.org/10.1038/s41375-020-01106-zDOI Listing
August 2021

Shared and Distinctive Ultrastructural Abnormalities Expressed by Megakaryocytes in Bone Marrow and Spleen From Patients With Myelofibrosis.

Front Oncol 2020 16;10:584541. Epub 2020 Nov 16.

Biomedical and Neuromotor Sciences, Alma Mater University Bologna, Bologna, Italy.

Numerous studies have documented ultrastructural abnormalities in malignant megakaryocytes from bone marrow (BM) of myelofibrosis patients but the morphology of these cells in spleen, an important extramedullary site in this disease, was not investigated as yet. By transmission-electron microscopy, we compared the ultrastructural features of megakaryocytes from BM and spleen of myelofibrosis patients and healthy controls. The number of megakaryocytes was markedly increased in both BM and spleen. However, while most of BM megakaryocytes are immature, those from spleen appear mature with well-developed demarcation membrane systems (DMS) and platelet territories and are surrounded by platelets. In BM megakaryocytes, paucity of DMS is associated with plasma (thick with protrusions) and nuclear (dilated with large pores) membrane abnormalities and presence of numerous glycosomes, suggesting a skewed metabolism toward insoluble polyglucosan accumulation. By contrast, the membranes of the megakaryocytes from the spleen were normal but these cells show mitochondria with reduced crests, suggesting deficient aerobic energy-metabolism. These distinctive morphological features suggest that malignant megakaryocytes from BM and spleen express distinctive metabolic impairments that may play different roles in the pathogenesis of myelofibrosis.
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http://dx.doi.org/10.3389/fonc.2020.584541DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7701330PMC
November 2020

Increased B4GALT1 expression is associated with platelet surface galactosylation and thrombopoietin plasma levels in MPNs.

Blood 2021 04;137(15):2085-2089

Department of Molecular Medicine, University of Pavia, Pavia, Italy.

Aberrant megakaryopoiesis is a hallmark of the myeloproliferative neoplasms (MPNs), a group of clonal hematological malignancies originating from hematopoietic stem cells, leading to an increase in mature blood cells in the peripheral blood. Sialylated derivatives of the glycan structure β4-N-acetyllactosamine (Galβ1,4GlcNAc or type-2 LacNAc, hereafter referred to as LacNAc) regulate platelet life span, hepatic thrombopoietin (TPO) production, and thrombopoiesis. We found increased TPO plasma levels in MPNs with high allele burden of the mutated clones. Remarkably, platelets isolated from MPNs had a significant increase in LacNAc expression that correlated with the high allele burden regardless of the underlying identified mutation. Megakaryocytes derived in vitro from these patients showed an increased expression of the B4GALT1 gene encoding β-1,4-galactosyltransferase 1 (β4GalT1). Consistently, megakaryocytes from MPN showed increased LacNAc expression relative to healthy controls, which was counteracted by the treatment with a Janus kinase 1/2 inhibitor. Altered expression of B4GALT1 in mutant megakaryocytes can lead to the production of platelets with aberrant galactosylation, which in turn promote hepatic TPO synthesis regardless of platelet mass. Our findings provide a new paradigm for understanding aberrant megakaryopoiesis in MPNs and identify β4GalT1 as a potential actionable target for therapy.
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http://dx.doi.org/10.1182/blood.2020007265DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8057261PMC
April 2021

Therapeutic Potential of Endothelial Colony-Forming Cells in Ischemic Disease: Strategies to Improve their Regenerative Efficacy.

Int J Mol Sci 2020 Oct 7;21(19). Epub 2020 Oct 7.

Laboratory of General Physiology, Department of Biology and Biotechnology "L. Spallanzani", University of Pavia, 27100 Pavia, Italy.

Cardiovascular disease (CVD) comprises a range of major clinical cardiac and circulatory diseases, which produce immense health and economic burdens worldwide. Currently, vascular regenerative surgery represents the most employed therapeutic option to treat ischemic disorders, even though not all the patients are amenable to surgical revascularization. Therefore, more efficient therapeutic approaches are urgently required to promote neovascularization. Therapeutic angiogenesis represents an emerging strategy that aims at reconstructing the damaged vascular network by stimulating local angiogenesis and/or promoting de novo blood vessel formation according to a process known as vasculogenesis. In turn, circulating endothelial colony-forming cells (ECFCs) represent truly endothelial precursors, which display high clonogenic potential and have the documented ability to originate de novo blood vessels in vivo. Therefore, ECFCs are regarded as the most promising cellular candidate to promote therapeutic angiogenesis in patients suffering from CVD. The current briefly summarizes the available information about the origin and characterization of ECFCs and then widely illustrates the preclinical studies that assessed their regenerative efficacy in a variety of ischemic disorders, including acute myocardial infarction, peripheral artery disease, ischemic brain disease, and retinopathy. Then, we describe the most common pharmacological, genetic, and epigenetic strategies employed to enhance the vasoreparative potential of autologous ECFCs by manipulating crucial pro-angiogenic signaling pathways, e.g., extracellular-signal regulated kinase/Akt, phosphoinositide 3-kinase, and Ca signaling. We conclude by discussing the possibility of targeting circulating ECFCs to rescue their dysfunctional phenotype and promote neovascularization in the presence of CVD.
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http://dx.doi.org/10.3390/ijms21197406DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7582994PMC
October 2020

Cytogenetic study in primary myelofibrosis at diagnosis: Clinical and histological association and impact on survival according to WHO 2017 classification in an Italian multicenter series.

Hematol Oncol 2021 Feb 4;39(1):123-128. Epub 2020 Oct 4.

Division of Pathology, Department of Pathophysiology and Transplantation, University of Milan, Foundation IRCCS Ca' Granda Ospedale Maggiore Policlinico, Milan, Italy.

We analyzed cytogenetic data at diagnosis in 395 primary myelofibrosis (PMF) patients to evaluate any possible association between karyotype and WHO 2017 classification and its impact on prognosis. All the cases were diagnosed and followed at five Italian Hematological Centers between November 1983 and December 2016. An abnormal karyotype (AK) was found in 69 patients and clustered differently according to bone marrow fibrosis grade as it was found in 31 (27.0%) cases with overt fibrotic and 38 (13.6%) with pre-fibrotic PMF (p = 0.001). Sex, anemia, thrombocytopenia, circulating blasts ≥1%, higher lactate dehydrogenase, and International Prognostic Scoring System risk classes were all significantly associated with karyotype. At a median follow-up of >6 years, 101 deaths were recorded. Survival was different between AK and normal karyotype (NK) patients with an estimated median overall survival (OS) of 11.6 and 25.7 years, respectively (p = 0.0148). In conclusion, in our cohort around 20% of patients had an AK, more frequently in subjects with an advanced bone marrow fibrosis grade and clinical-laboratory features indicative of a more aggressive disease. This study shows that an AK confers a more severe clinical phenotype and impacts adversely on OS, thus representing an additional parameter to be considered in the evaluation of PMF prognosis.
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http://dx.doi.org/10.1002/hon.2808DOI Listing
February 2021

Nicotinic acid adenine dinucleotide phosphate activates two-pore channel TPC1 to mediate lysosomal Ca release in endothelial colony-forming cells.

J Cell Physiol 2021 01 24;236(1):688-705. Epub 2020 Jun 24.

Department of Medicine and Health Sciences "Vincenzo Tiberio", University of Molise, Campobasso, Italy.

Nicotinic acid adenine dinucleotide phosphate (NAADP) is the most recently discovered Ca -releasing messenger that increases the intracellular Ca concentration by mobilizing the lysosomal Ca store through two-pore channels 1 (TPC1) and 2 (TPC2). NAADP-induced lysosomal Ca release regulates multiple endothelial functions, including nitric oxide release and proliferation. A sizeable acidic Ca pool endowed with TPC1 is also present in human endothelial colony-forming cells (ECFCs), which represent the only known truly endothelial precursors. Herein, we sought to explore the role of the lysosomal Ca store and TPC1 in circulating ECFCs by harnessing Ca imaging and molecular biology techniques. The lysosomotropic agent, Gly-Phe β-naphthylamide, and nigericin, which dissipates the proton gradient which drives Ca sequestration by acidic organelles, caused endogenous Ca release in the presence of a replete inositol-1,4,5-trisphosphate (InsP )-sensitive endoplasmic reticulum (ER) Ca pool. Likewise, the amount of ER releasable Ca was reduced by disrupting lysosomal Ca content. Liposomal delivery of NAADP induced a transient Ca signal that was abolished by disrupting the lysosomal Ca store and by pharmacological and genetic blockade of TPC1. Pharmacological manipulation revealed that NAADP-induced Ca release also required ER-embedded InsP receptors. Finally, NAADP-induced lysosomal Ca release was found to trigger vascular endothelial growth factor-induced intracellular Ca oscillations and proliferation, while it did not contribute to adenosine-5'-trisphosphate-induced Ca signaling. These findings demonstrated that NAADP-induced TPC1-mediated Ca release can selectively be recruited to induce the Ca response to specific cues in circulating ECFCs.
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http://dx.doi.org/10.1002/jcp.29896DOI Listing
January 2021

Reduced CXCR4-expression on CD34-positive blood cells predicts outcomes of persons with primary myelofibrosis.

Leukemia 2021 02 14;35(2):468-475. Epub 2020 Jun 14.

Center for the Study of Myelofibrosis, Laboratory of Biochemistry, Biotechnology and Advanced Diagnostics, Istituto di Ricovero e Cura a Carattere Scientifico Policlinico S. Matteo Foundation, Pavia, Italy.

The expression of the CXCR4 chemokine receptor on CD34-positive blood cells is reduced in persons with primary myelofibrosis (PMF). We analyzed the relevance of cytofluorimetric assessment of the percentage of CD34-positive blood cells that had a positive CXCR4 surface expression (CD34/CXCR4-se) in a large cohort of subjects with myeloproliferative neoplasms. Mean CD34/CXCR4-se was lower in subjects with PMF compared with those with essential thrombocythemia (ET) or polycythemia vera (PV). A cutoff value of 39% was associated with a diagnosis of pre-fibrotic PMF vs. ET with a positive predictive value of 97%. In PMF male sex, older age, and MPL mutation were independent correlates of reduced CD34/CXCR4-se and associated with a briefer interval to development of severe anemia, large splenomegaly, thrombocytopenia, leukopenia, elevated CD34-positive blood cells, blast transformation and death. We constructed a prognostic model including age >65 years, hemoglobin < 100 g/L, CD34-positive blood cells > 50 × 10/L, and CD34/CXCR4-se <39% at diagnosis. The model identified three risk cohorts with greater accuracy compared with the International Prognostic Scoring System. In conclusion, CD34/CXCR4-se is a highly sensitive marker of disease activity and a new potential diagnostic and prognostic biomarker in PMF.
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http://dx.doi.org/10.1038/s41375-020-0926-6DOI Listing
February 2021

Systemic lupus erythematosus, endothelial progenitor cells and intracellular Ca signaling: A novel approach for an old disease.

J Autoimmun 2020 08 29;112:102486. Epub 2020 May 29.

Laboratory of General Physiology, Department of Biology and Biotechnology "L. Spallanzani", University of Pavia, via Forlanini 6, 27100, Pavia, Italy. Electronic address:

Systemic lupus erythematosus (SLE) is an autoimmune multisystem disease featured by an increased cardiovascular risk that may lead to premature patient's death. It has been demonstrated that SLE patients suffer from early onset endothelial dysfunction which is due to the impairment of endogenous vascular repair mechanisms. Vascular integrity and homeostasis are maintained by endothelial progenitor cells (EPCs), which are mobilized in response to endothelial injury to replace damaged endothelial cells. Two main EPCs subpopulations exist in peripheral blood: endothelial colony forming cells (ECFCs), which represent truly endothelial precursors and can physically engraft within neovessels, and myeloid angiogenic cells (MACs), which sustain angiogenesis in a paracrine manner. Emerging evidence indicates that ECFCs/MACs are down-regulated and display compromised angiogenic activity in SLE, thereby contributing to the pathogenesis of this disease. Intracellular calcium (Ca) signaling plays a crucial role in maintaining vascular integrity by stimulating migration, proliferation and tube formation in both ECFCs and MACs. Herein, we illustrate the evidences that support the role played by EPCs dysfunction in SLE. Subsequently, we discuss about the hypothesis that the Ca handling machinery is compromised in SLE-derived ECFCs and MACs, thereby resulting in their reduced pro-angiogenic activity. Finally, we speculate about the proposal to exploit intracellular Ca signaling to improve ECFCs' reparative phenotype and suggest this strategy as a new approach to treat SLE patients.
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http://dx.doi.org/10.1016/j.jaut.2020.102486DOI Listing
August 2020

Endothelial TRPV1 as an Emerging Molecular Target to Promote Therapeutic Angiogenesis.

Cells 2020 05 27;9(6). Epub 2020 May 27.

Laboratory of General Physiology, Department of Biology and Biotechnology "L. Spallanzani", University of Pavia, 27100 Pavia, Italy.

Therapeutic angiogenesis represents an emerging strategy to treat ischemic diseases by stimulating blood vessel growth to rescue local blood perfusion. Therefore, injured microvasculature may be repaired by stimulating resident endothelial cells or circulating endothelial colony forming cells (ECFCs) or by autologous cell-based therapy. Endothelial Ca signals represent a crucial player in angiogenesis and vasculogenesis; indeed, several angiogenic stimuli induce neovessel formation through an increase in intracellular Ca concentration. Several members of the Transient Receptor Potential (TRP) channel superfamily are expressed and mediate Ca-dependent functions in vascular endothelial cells and in ECFCs, the only known truly endothelial precursor. TRP Vanilloid 1 (TRPV1), a polymodal cation channel, is emerging as an important player in endothelial cell migration, proliferation, and tubulogenesis, through the integration of several chemical stimuli. Herein, we first summarize TRPV1 structure and gating mechanisms. Next, we illustrate the physiological roles of TRPV1 in vascular endothelium, focusing our attention on how endothelial TRPV1 promotes angiogenesis. In particular, we describe a recent strategy to stimulate TRPV1-mediated pro-angiogenic activity in ECFCs, in the presence of a photosensitive conjugated polymer. Taken together, these observations suggest that TRPV1 represents a useful target in the treatment of ischemic diseases.
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http://dx.doi.org/10.3390/cells9061341DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7349285PMC
May 2020

Plasma sIL-2Rα levels are associated with disease progression in myelofibrosis with JAK2 but not CALR mutation.

Leuk Res 2020 03 11;90:106319. Epub 2020 Feb 11.

Laboratory of Biochemistry-Biotechnology and Advanced Diagnosis, Fondazione Istituto di Ricovero e Cura a Carattere Scientifico (IRCCS) Policlinico S. Matteo, Pavia, Italy.

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http://dx.doi.org/10.1016/j.leukres.2020.106319DOI Listing
March 2020

Long-term efficacy and safety of ruxolitinib versus best available therapy in polycythaemia vera (RESPONSE): 5-year follow up of a phase 3 study.

Lancet Haematol 2020 Mar 23;7(3):e226-e237. Epub 2020 Jan 23.

The University of Texas MD Anderson Cancer Center, Houston, TX, USA.

Background: Polycythaemia vera is a myeloproliferative neoplasm characterised by excessive proliferation of erythroid, myeloid, and megakaryocytic components in the bone marrow due to mutations in the Janus kinase 2 (JAK2) gene. Ruxolitinib, a JAK 1 and JAK 2 inhibitor, showed superiority over best available therapy in a phase 2 study in patients with polycythaemia vera who were resistant to or intolerant of hydroxyurea. We aimed to compare the long-term safety and efficacy of ruxolitinib with best available therapy in patients with polycythaemia vera who were resistant to or intolerant of hydroxyurea.

Methods: We report the 5-year results for a randomised, open-label, phase 3 study (RESPONSE) that enrolled patients at 109 sites across North America, South America, Europe, and the Asia-Pacific region. Patients (18 years or older) with polycythaemia vera who were resistant to or intolerant of hydroxyurea were randomly assigned 1:1 to receive either ruxolitinib or best available therapy. Patients randomly assigned to the ruxolitinib group received the drug orally at a starting dose of 10 mg twice a day. Single-agent best available therapy comprised hydroxyurea, interferon or pegylated interferon, pipobroman, anagrelide, approved immunomodulators, or observation without pharmacological treatment. The primary endpoint, composite response (patients who achieved both haematocrit control without phlebotomy and 35% or more reduction from baseline in spleen volume) at 32 weeeks was previously reported. Patients receiving best available therapy could cross over to ruxolitinib after week 32. We assessed the durability of primary composite response, complete haematological remission, overall clinicohaematological response, overall survival, patient-reported outcomes, and safety after 5-years of follow-up. This study is registered with ClinicalTrials.gov, NCT01243944.

Findings: We enrolled patients between Oct 27, 2010, and Feb 13, 2013, and the study concluded on Feb 9, 2018. Of 342 individuals screened for eligibility, 222 patients were randomly assigned to receive ruxolitinib (n=110, 50%) or best available therapy (n=112, 50%). The median time since polycythaemia vera diagnosis was 8·2 years (IQR 3·9-12·3) in the ruxolitinib group and 9·3 years (4·9-13·8) in the best available therapy group. 98 (88%) of 112 patients initially randomly assigned to best available therapy crossed over to receive ruxolitinib and no patient remained on best available therapy after 80 weeks of study. Among 25 primary responders in the ruxolitinib group, six had progressed at the time of final analysis. At 5 years, the probability of maintaining primary composite response was 74% (95% CI 51-88). The probability of maintaining complete haematological remission was 55% (95% CI 32-73) and the probability of maintaining overall clinicohaematological responses was 67% (54-77). In the intention-to-treat analysis not accounting for crossover, the probability of survival at 5 years was 91·9% (84·4-95·9) with ruxolitinib therapy and 91·0% (82·8-95·4) with best available therapy. Anaemia was the most common adverse event in patients receiving ruxolitinib (rates per 100 patient-years of exposure were 8·9 for ruxolitinib and 8·8 for the crossover population), though most anaemia events were mild to moderate in severity (grade 1 or 2 anaemia rates per 100 patient-years of exposure were 8·0 for ruxolitinib and 8·2 for the crossover population). Non-haematological adverse events were generally lower with long-term ruxolitinib treatment than with best available therapy. Thromboembolic events were lower in the ruxolitinib group than the best available therapy group. There were two on-treatment deaths in the ruxolitinib group. One of these deaths was due to gastric adenocarcinoma, which was assessed by the investigator as related to ruxolitinib treatment.

Interpretation: We showed that ruxolitinib is a safe and effective long-term treatment option for patients with polycythaemia vera who are resistant to or intolerant of hydroxyurea. Taken together, ruxolitinib treatment offers the first widely approved therapeutic alternative for this post-hydroxyurea patient population.

Funding: Novartis Pharmaceuticals Corporation.
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http://dx.doi.org/10.1016/S2352-3026(19)30207-8DOI Listing
March 2020

Defective interaction of mutant calreticulin and SOCE in megakaryocytes from patients with myeloproliferative neoplasms.

Blood 2020 01;135(2):133-144

Department of Molecular Medicine, University of Pavia, Pavia, Italy.

Approximately one-fourth of patients with essential thrombocythemia or primary myelofibrosis carry a somatic mutation of the calreticulin gene (CALR), the gene encoding for calreticulin. A 52-bp deletion (type I mutation) and a 5-bp insertion (type II mutation) are the most frequent genetic lesions. The mechanism(s) by which a CALR mutation leads to a myeloproliferative phenotype has been clarified only in part. We studied the interaction between calreticulin and store-operated calcium (Ca2+) entry (SOCE) machinery in megakaryocytes (Mks) from healthy individuals and from patients with CALR-mutated myeloproliferative neoplasms (MPNs). In Mks from healthy subjects, binding of recombinant human thrombopoietin to c-Mpl induced the activation of signal transducer and activator of transcription 5, AKT, and extracellular signal-regulated kinase 1/2, determining inositol triphosphate-dependent Ca2+ release from the endoplasmic reticulum (ER). This resulted in the dissociation of the ER protein 57 (ERp57)-mediated complex between calreticulin and stromal interaction molecule 1 (STIM1), a protein of the SOCE machinery that leads to Ca2+ mobilization. In Mks from patients with CALR-mutated MPNs, defective interactions between mutant calreticulin, ERp57, and STIM1 activated SOCE and generated spontaneous cytosolic Ca2+ flows. In turn, this resulted in abnormal Mk proliferation that was reverted using a specific SOCE inhibitor. In summary, the abnormal SOCE regulation of Ca2+ flows in Mks contributes to the pathophysiology of CALR-mutated MPNs. In perspective, SOCE may represent a new therapeutic target to counteract Mk proliferation and its clinical consequences in MPNs.
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http://dx.doi.org/10.1182/blood.2019001103DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6952826PMC
January 2020

Shared and Tissue-Specific Expression Signatures between Bone Marrow from Primary Myelofibrosis and Essential Thrombocythemia.

Exp Hematol 2019 11 1;79:16-25.e3. Epub 2019 Nov 1.

Division of Hematology and Oncology, Tisch Cancer Institute, Icahn School of Medicine at Mount Sinai, New York, New York; Department of Biomedical and Neuromotorial Sciences, Alma Mater University, Bologna, Italy. Electronic address:

Megakaryocytes have been implicated in the micro-environmental abnormalities associated with fibrosis and hematopoietic failure in the bone marrow (BM) of primary myelofibrosis (PMF) patients, the Philadelphia-negative myeloproliferative neoplasm (MPN) associated with the poorest prognosis. To identify possible therapeutic targets for restoring BM functions in PMF, we compared the expression profiling of PMF BM with that of BM from essential thrombocytopenia (ET), a fibrosis-free MPN also associated with BM megakaryocyte hyperplasia. The signature of PMF BM was also compared with published signatures associated with liver and lung fibrosis. Gene set enrichment analysis (GSEA) identified distinctive differences between the expression profiles of PMF and ET. Notch, K-Ras, IL-8, and apoptosis pathways were altered the most in PMF as compared with controls. By contrast, cholesterol homeostasis, unfolded protein response, and hypoxia were the pathways found altered to the greatest degree in ET compared with control specimens. BM from PMF expressed a noncanonical transforming growth factor β (TGF-β) signature, which included activation of ID1, JUN, GADD45b, and genes with binding motifs for the JUN transcriptional complex AP1. By contrast, the expression of ID1 and GADD45b was not altered and there was a modest signal for JUN activation in ET. The similarities among PMF, liver fibrosis, and lung fibrosis were modest and included activation of integrin-α9 and tropomyosin-α1 between PMF and liver fibrosis, and of ectoderm-neural cortex protein 1 and FRAS1-related extracellular matrix protein 1 between PMF and lung fibrosis, but not TGF-β. These data identify TGF-β as a potential target for micro-environmental therapy in PMF.
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http://dx.doi.org/10.1016/j.exphem.2019.10.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6910948PMC
November 2019

A novel disorder involving dyshematopoiesis, inflammation, and HLH due to aberrant CDC42 function.

J Exp Med 2019 12 10;216(12):2778-2799. Epub 2019 Oct 10.

Baylor-Hopkins Center for Mendelian Genomics, Houston, TX.

Hemophagocytic lymphohistiocytosis (HLH) is characterized by immune dysregulation due to inadequate restraint of overactivated immune cells and is associated with a variable clinical spectrum having overlap with more common pathophysiologies. HLH is difficult to diagnose and can be part of inflammatory syndromes. Here, we identify a novel hematological/autoinflammatory condition (NOCARH syndrome) in four unrelated patients with superimposable features, including neonatal-onset cytopenia with dyshematopoiesis, autoinflammation, rash, and HLH. Patients shared the same de novo mutation (Chr1:22417990C>T, p.R186C) and altered hematopoietic compartment, immune dysregulation, and inflammation. mutations had been associated with syndromic neurodevelopmental disorders. In vitro and in vivo assays documented unique effects of p.R186C on CDC42 localization and function, correlating with the distinctiveness of the trait. Emapalumab was critical to the survival of one patient, who underwent successful bone marrow transplantation. Early recognition of the disorder and establishment of treatment followed by bone marrow transplant are important to survival.
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http://dx.doi.org/10.1084/jem.20190147DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6888978PMC
December 2019

Pegylated interferon alfa-2a for polycythemia vera or essential thrombocythemia resistant or intolerant to hydroxyurea.

Blood 2019 10;134(18):1498-1509

Department of Population Health and.

Prior studies have reported high response rates with recombinant interferon-α (rIFN-α) therapy in patients with essential thrombocythemia (ET) and polycythemia vera (PV). To further define the role of rIFN-α, we investigated the outcomes of pegylated-rIFN-α2a (PEG) therapy in ET and PV patients previously treated with hydroxyurea (HU). The Myeloproliferative Disorders Research Consortium (MPD-RC)-111 study was an investigator-initiated, international, multicenter, phase 2 trial evaluating the ability of PEG therapy to induce complete (CR) and partial (PR) hematologic responses in patients with high-risk ET or PV who were either refractory or intolerant to HU. The study included 65 patients with ET and 50 patients with PV. The overall response rates (ORRs; CR/PR) at 12 months were 69.2% (43.1% and 26.2%) in ET patients and 60% (22% and 38%) in PV patients. CR rates were higher in CALR-mutated ET patients (56.5% vs 28.0%; P = .01), compared with those in subjects lacking a CALR mutation. The median absolute reduction in JAK2V617F variant allele fraction was -6% (range, -84% to 47%) in patients achieving a CR vs +4% (range, -18% to 56%) in patients with PR or nonresponse (NR). Therapy was associated with a significant rate of adverse events (AEs); most were manageable, and PEG discontinuation related to AEs occurred in only 13.9% of subjects. We conclude that PEG is an effective therapy for patients with ET or PV who were previously refractory and/or intolerant of HU. This trial was registered at www.clinicaltrials.gov as #NCT01259856.
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http://dx.doi.org/10.1182/blood.2019000428DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6839950PMC
October 2019

Supporting data on in vitro cardioprotective and proliferative paracrine effects by the human amniotic fluid stem cell secretome.

Data Brief 2019 Aug 30;25:104324. Epub 2019 Jul 30.

Regenerative Medicine Laboratory, Department of Experimental Medicine, University of Genova, Genova, Italy.

The data and information presented here refer to the research article entitled: "Reactivating endogenous mechanisms of cardiac regeneration via paracrine boosting with the human amniotic fluid stem cell secretome" (Balbi et al., 2019, Apr 04). This dataset illustrates the in vitro paracrine effect exerted by the human amniotic fluid stem cell secretome on rodent neonatal cardiomyocytes, human endothelial progenitors and different subsets of cardiac progenitor cells. Cytokine/chemokine profiling of the human amniotic fluid stem cell secretome is provided as well. This data can provide useful insights in regenerative medicine as demonstrating the in vitro cardioprotective and proliferative secretory paracrine potential of human fetal stem cells.
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http://dx.doi.org/10.1016/j.dib.2019.104324DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6700494PMC
August 2019

Constitutive STAT5 phosphorylation in CD34+ cells of patients with primary myelofibrosis: Correlation with driver mutation status and disease severity.

PLoS One 2019 1;14(8):e0220189. Epub 2019 Aug 1.

Laboratory of Biochemistry, Biotechnology and Advanced Diagnosis, IRCCS Policlinico San Matteo Foundation, Pavia, Italy.

Primary Myelofibrosis (PMF) is a myeloproliferative disorder associated with JAK2V617F, Calreticulin (CALR) indels, and MPLW515L/K mutations activating the tyrosine kinase JAK2 and its downstream signaling pathway. The nature of signaling abnormalities in primary cells from PMF patients is poorly understood, since most of the work has been performed in cell lines or animal models. By flow cytometry we measured constitutive and cytokine induced phosphorylation of STAT5, STAT3, and ERK1/2 in circulating CD34+ cells from 57 patients with PMF (20 with prefibrotic-PMF) and 13 healthy controls (CTRLs). Levels of constitutive and TPO induced p-STAT5, and IL6 induced p-STAT3 were higher in patients than in CTRLs. Constitutive p-STAT5 values were lower in CALR than homozygous JAK2V617F mutated CD34+ cells from PMF patients. Moreover, constitutive p-STAT5 and IL6 induced p-STAT3 values correlated directly with circulating CD34+ cell number/L, and inversely with the frequency of circulating CD34+ cells expressing CXCR4. Constitutive p-STAT5 values of CD34+ cells were also inversely correlated with hemoglobin levels. When the patients were divided according with presence/absence of JAK2V617F mutation, all the correlations described characterized the JAK2V617F+ patients with prefibrotic-PMF (P-PMF). In conclusion, increased constitutive p-STAT5 and IL6 induced p-STAT3 values in circulating CD34+ cells characterize patients with PMF. Constitutive p-STAT5 and IL6 induced p-STAT3 values correlate with circulating CD34+ cell number/L, the frequency of circulating CD34+ cells expressing CXCR4 and hemoglobin levels within the prefibrotic JAK2V617F+ patient population. Our data point toward a complex activation of STAT5-dependent pathways in the stem/progenitor cell compartment, that characterize the phenotypic diversity of PMF.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0220189PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6675063PMC
March 2020

Generation of donor-derived Wilms tumor antigen 1-specific cytotoxic T lymphocytes with potent anti-leukemia activity for somatic cell therapy in children given haploidentical stem cell transplantation: a feasibility pre-clinical study.

Cytotherapy 2019 09 4;21(9):958-972. Epub 2019 Jul 4.

Laboratory of Immunology and Transplantation, Fondazione Istituto Ricovero e Cura a Carattere Scientifico (IRCCS) Policlinico San Matteo, Pavia, Italy; Cell Factory, Fondazione IRCCS Policlinico San Matteo, Pavia, Italy; Department of Clinic-Surgical, Diagnostic and Pediatric Sciences, University of Pavia, Italy. Electronic address:

Background: The Wilms tumor antigen 1 (WT1) is over-expressed in a vast majority of adult and childhood acute leukemia and myelodysplastic syndromes, being lowly or transiently expressed in normal tissues and hematopoietic stem cells (HSCs). A number of HLA-restricted WT1 epitopes are immunogenic, allowing the in vitro induction of WT1-specific cytotoxic T lymphocytes (CTLs) from patients and healthy donors.

Aim: The aim of the study was to investigate the feasibility of producing WT1-specific CTLs suitable for somatic cell therapy to prevent or treat relapse in children with acute myeloid or lymphoblastic leukemia given haploidentical HSC transplantation (haplo-HSCT).

Methods: For WT1-specific CTL production, donor-derived either peripheral blood mononuclear cells (PBMCs) or CD8+ lymphocytes were stimulated with WT1 peptide-loaded donor dendritic cells in the presence of interleukin (IL)-7 and IL-12. Effector cells were re-stimulated once with irradiated donor PBMCs pulsed with WT1-peptides, and then expanded in an antigen-independent way.

Results: WT1-specific CTLs, displaying high-level cytotoxicity against patients' leukemia blasts and negligible activity against patients' non-malignant cells, were obtained from both PBMCs and CD8+ lymphocytes. WT1-specific CTLs obtained from PBMCs showed a better expansion capacity and better anti-leukemia activity than those obtained from CD8+ lymphocytes, even though the difference was not statistically significant. In CTLs derived from PBMCs, both CD8+ and CD4+ subpopulations displayed strong anti-leukemia cytotoxic activity.

Discussion: Results of this pre-clinical study pave the way to a somatic cell therapy approach aimed at preventing or treating relapse in children given haplo-HSCT for WT1-positive leukemia.
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http://dx.doi.org/10.1016/j.jcyt.2019.06.007DOI Listing
September 2019

Reactivating endogenous mechanisms of cardiac regeneration via paracrine boosting using the human amniotic fluid stem cell secretome.

Int J Cardiol 2019 07 4;287:87-95. Epub 2019 Apr 4.

Regenerative Medicine Laboratory, Department of Experimental Medicine, University of Genova, Genova, Italy. Electronic address:

Background: The adult mammalian heart retains residual regenerative capability via endogenous cardiac progenitor cell (CPC) activation and cardiomyocyte proliferation. We previously reported the paracrine cardioprotective capacity of human amniotic fluid-derived stem cells (hAFS) following ischemia or cardiotoxicity. Here we analyse the potential of hAFS secretome fractions for cardiac regeneration and future clinical translation.

Methods: hAFS were isolated from amniotic fluid leftover samples from prenatal screening. hAFS conditioned medium (hAFS-CM) was obtained following hypoxic preconditioning. Anti-apoptotic, angiogenic and proliferative effects were evaluated on rodent neonatal cardiomyocytes (r/mNVCM), human endothelial colony forming cells (hECFC) and human CPC. Mice undergoing myocardial infarction (MI) were treated with hAFS-CM, hAFS-extracellular vesicles (hAFS-EV), or EV-depleted hAFS-CM (hAFS-DM) by single intra-myocardial administration and evaluated in the short and long term.

Results: hAFS-CM improved mNVCM survival under oxidative and hypoxic damage, induced Ca-dependent angiogenesis in hECFC and triggered hCPC and rNVCM proliferation. hAFS-CM treatment after MI counteracted scarring, supported cardiac function, angiogenesis and cardiomyocyte cell cycle progression in the long term. hAFS-DM had no effect. hAFS-CM and hAFS-EV equally induced epicardium WT1+ CPC reactivation. Although no CPC cardiovascular differentiation was observed, our data suggests contribution to local angiogenesis by paracrine modulation. hAFS-EV alone were able to recapitulate all the beneficial effects exerted by hAFS-CM, except for stimulation of vessel formation.

Conclusions: hAFS-CM and hAFS-EV can improve cardiac repair and trigger cardiac regeneration via paracrine modulation of endogenous mechanisms. While both formulations are effective in sustaining myocardial renewal, hAFS-CM retains higher pro-angiogenic potential, while hAFS-EV particularly enhances cardiac function.
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http://dx.doi.org/10.1016/j.ijcard.2019.04.011DOI Listing
July 2019

EDA fibronectin-TLR4 axis sustains megakaryocyte expansion and inflammation in bone marrow fibrosis.

J Exp Med 2019 03 7;216(3):587-604. Epub 2019 Feb 7.

Department of Molecular Medicine, University of Pavia, Pavia, Italy

The fibronectin EDA isoform (EDA FN) is instrumental in fibrogenesis but, to date, its expression and function in bone marrow (BM) fibrosis have not been explored. We found that mice constitutively expressing the EDA domain (EIIIA), but not EDA knockout mice, are more prone to develop BM fibrosis upon treatment with the thrombopoietin (TPO) mimetic romiplostim (TPO). Mechanistically, EDA FN binds to TLR4 and sustains progenitor cell proliferation and megakaryopoiesis in a TPO-independent fashion, inducing LPS-like responses, such as NF-κB activation and release of profibrotic IL-6. Pharmacological inhibition of TLR4 or TLR4 deletion in TPO mice abrogated Mk hyperplasia, BM fibrosis, IL-6 release, extramedullary hematopoiesis, and splenomegaly. Finally, developing a novel ELISA assay, we analyzed samples from patients affected by primary myelofibrosis (PMF), a well-known pathological situation caused by altered TPO signaling, and found that the EDA FN is increased in plasma and BM biopsies of PMF patients as compared with healthy controls, correlating with fibrotic phase.
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http://dx.doi.org/10.1084/jem.20181074DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6400533PMC
March 2019

Kinetic and Angiogenic Activity of Circulating Endothelial Colony Forming Cells in Patients with Infantile Haemangioma Receiving Propranolol.

Thromb Haemost 2019 Feb 4;119(2):274-284. Epub 2019 Jan 4.

Laboratory of Biochemistry, Biotechnology and Advanced Diagnosis, IRCCS Policlinico San Matteo Foundation, Pavia, Italy.

Endothelial progenitor cells (EPCs) have been suggested to contribute to the neovascularization of infantile haemangioma (IH). There is strong evidence of the efficacy of propranolol in the treatment of IH, possibly by inhibiting both vasculogenesis and angiogenesis in the tumour. We evaluate the frequency of circulating endothelial colony forming cells (ECFCs), as the best EPC surrogate, in patients with IH at diagnosis and while receiving propranolol by an ex vivo 12-month longitudinal study. Biological aspects of the ECFCs, such as their in vitro angiogenic potential, membrane CXCR4 expression and Ca signalling, were investigated. Circulating ECFCs were isolated by in vitro culture and expanded for 2 to 3 passages in 23 patients with IH (median age: 5.5 months, range: 5.5 weeks-11 months) before and 3, 6, 9 and 12 months after receiving propranolol. Twenty-four healthy subjects comparable for age were also assessed (CTRLs). Untreated patients with IH had a circulating ECFC frequency lower ( = 0.001) than CTRLs; nevertheless, in in vitro starving conditions, ECFCs showed enhanced capacity to form tube-like structures than those of CTRLs. Patients with IH following the therapy with propranolol had a significantly increased ( = 0.022) circulating ECFC frequency, that showed a diminished tube-like formation capacity in vitro, and an altered constitutive store-operated Ca entry. ECFCs play a role in IH pathogenesis; the response to propranolol therapy is associated with their increased frequency in the peripheral blood and a reduction of their vasculogenic activity.
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http://dx.doi.org/10.1055/s-0038-1676855DOI Listing
February 2019

The role of endothelial colony forming cells in kidney cancer's pathogenesis, and in resistance to anti-VEGFR agents and mTOR inhibitors: A speculative review.

Crit Rev Oncol Hematol 2018 Dec 18;132:89-99. Epub 2018 Sep 18.

Medical Oncology, Istituto di Ricovero e Cura a Carattere Scientifico (I.R.C.C.S.) Policlinico San Matteo Foundation, Piazzale Golgi 19, 27100, Pavia, Italy; present address: Department of Internal Medicine, University of Pavia, and Division of Translational Oncology, IRCCS Istituti Clinici Scientifici Maugeri, via S. Maugeri 10, 27100 Pavia, Italy. Electronic address:

Renal cell carcinoma (RCC) is highly dependent on angiogenesis, due to the overactivation of the VHL/HIF/VEGF/VEGFRs axis; this justifies the marked sensitivity of this neoplasm to antiangiogenic agents which, however, ultimately fail to control tumor growth. RCC also frequently shows alterations in the mTOR signaling pathway, and mTOR inhibitors have shown a similar pattern of initial activity/late failure as pure antiangiogenic agents. Understanding mechanisms of resistance to these agents would be key to improve the outcome of our patients. Circulating endothelial cells are a family of mainly bone marrow-derived progenitors, which have been postulated to be responsible of the reactivation of angiogenesis in different tumors. In this review, we shall discuss the complex nature and function of these cells, the evidence pro and contra their contribution to tumor vascularization, especially as far as RCC is concerned, and their possible role in determining resistance to presently available treatments.
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http://dx.doi.org/10.1016/j.critrevonc.2018.09.005DOI Listing
December 2018

Role of TGF-β1/miR-382-5p/SOD2 axis in the induction of oxidative stress in CD34+ cells from primary myelofibrosis.

Mol Oncol 2018 12 16;12(12):2102-2123. Epub 2018 Nov 16.

Department of Life Sciences, Centre for Regenerative Medicine, University of Modena and Reggio Emilia, Italy.

Primary myelofibrosis (PMF) is a myeloproliferative neoplasm characterized by an excessive production of pro-inflammatory cytokines resulting in chronic inflammation and genomic instability. Besides the driver mutations in JAK2, MPL, and CALR genes, the deregulation of miRNA expression may also contribute to the pathogenesis of PMF. To this end, we recently reported the upregulation of miR-382-5p in PMF CD34+ cells. In order to unveil the mechanistic details of the role of miR-382-5p in pathogenesis of PMF, we performed gene expression profiling of CD34+ cells overexpressing miR-382-5p. Among the downregulated genes, we identified superoxide dismutase 2 (SOD2), which is a predicted target of miR-382-5p. Subsequently, we confirmed miR-382-5p/SOD2 interaction by luciferase assay and we showed that miR-382-5p overexpression in CD34+ cells causes the decrease in SOD2 activity leading to reactive oxygen species (ROS) accumulation and oxidative DNA damage. In addition, our data indicate that inhibition of miR-382-5p in PMF CD34+ cells restores SOD2 function, induces ROS disposal, and reduces DNA oxidation. Since the pro-inflammatory cytokine transforming growth factor-β1 (TGF-β1) is a key player in PMF pathogenesis, we further investigated the effect of TGF-β1 on ROS and miR-382-5p levels. Our data showed that TGF-β1 treatment enhances miR-382-5p expression and reduces SOD2 activity leading to ROS accumulation. Finally, inhibition of TGF-β1 signaling in PMF CD34+ cells by galunisertib significantly reduced miR-382-5p expression and ROS accumulation and restored SOD2 activity. As a whole, this study reports that TGF-β1/miR-382-5p/SOD2 axis deregulation in PMF cells is linked to ROS overproduction that may contribute to enhanced oxidative stress and inflammation. Our results suggest that galunisertib may represent an effective drug reducing abnormal oxidative stress induced by TGF-β1 in PMF patients. DATABASE LINKING: GEO: https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE103464.
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http://dx.doi.org/10.1002/1878-0261.12387DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6275274PMC
December 2018
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