Publications by authors named "Marco Gabbianelli"

21 Publications

  • Page 1 of 1

Phenotypical and functional abnormalities of circulating neutrophils in patients with β-thalassemia.

Ann Hematol 2020 Oct 15;99(10):2265-2277. Epub 2020 Aug 15.

Department of Cardiovascular and Endocrine-metabolic Diseases and Aging, Istituto Superiore di Sanità, Viale Regina Elena, 299, 00161, Rome, Italy.

β-Thalassemia is an inherited single gene disorder related to reduced synthesis of the β-globin chain of hemoglobin. Patients with β-thalassemia present variable clinical severity ranging from asymptomatic trait to severe transfusion-dependent anemia and multiple organs complications. Moreover, multiple immune abnormalities are a major concern in β-thalassemia patients. Aberrant neutrophil effector function plays a pivotal role in infection susceptibility in these patients. In severe and persistent inflammation, immature neutrophils are released from the bone marrow and are functionally different compared with mature ones. Despite some abnormalities reported for thalassemia patient's immune system, few data exist on the characterization of human neutrophils in β-thalassemia. The aim of this study was to investigate the phenotype and function of circulating neutrophil subsets in patients with β-thalassemia major and with β-thalassemia intermedia divided in transfusion-dependent and non-transfusion-dependent. By the use of immunochemical and cytofluorimetric analyses, we observed that patients' CD16+ neutrophils exhibit abnormalities in their phenotype and functions and the abnormalities vary according to the clinical form of the disease and to the neutrophil subset (CD16 and CD16). Abnormalities include altered surface expression of the innate immune receptor CD45, Toll-like receptor 4, and CD32, reduced ability to produce an oxidative burst, and elevated levels of membrane lipid peroxidation, especially in patients with a more severe form of the disease. Overall, our results indicating the occurrence of an immuno-senescent phenotype on circulating neutrophils from thalassemia patients suggest the usefulness of neutrophil feature assessment as a tool for better clinical management of β-thalassemia.
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http://dx.doi.org/10.1007/s00277-020-04213-0DOI Listing
October 2020

Platelet and megakaryocyte CD40L expression in β-Thalassemic patients.

Thromb Res 2020 05 2;189:108-111. Epub 2020 Mar 2.

Department of Cardiovascular, Endocrine-Metabolic Diseases and Ageing, Istituto Superiore di Sanità, Rome, Italy. Electronic address:

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http://dx.doi.org/10.1016/j.thromres.2020.02.026DOI Listing
May 2020

Neonatal Screening for Congenital Hypothyroidism: What Can We Learn From Discordant Twins?

J Clin Endocrinol Metab 2019 12;104(12):5765-5779

Department of Cardiovascular and Endocrine-Metabolic Diseases and Aging, National Institutes of Health, Rome, Italy.

Context: Newborn screening program for congenital hypothyroidism (CH) adopting rescreening in at-risk neonates.

Objectives: To estimate the concordance rate for CH in twin pairs discordant at the first screening; to verify whether long-term follow-up of healthy cotwins belonging to CH discordant pairs may be useful to diagnose thyroid hypofunction during development; to evaluate the importance of genetic and environmental influences on liability to permanent and transient CH.

Design And Patients: Forty-seven screening discordant twin pairs were investigated. Proband was defined as the twin in the pair with a positive test at the first screening and a confirmed diagnosis of CH.

Results: Seven screening discordant twin pairs became concordant for CH within the first month of life (pairwise concordance of 14.9%) because seven screening negative cotwins showed high TSH values when retested. During long-term follow-up (range, 3 to 21 years), hypothyroidism was diagnosed in two monozygotic screening negative cotwins at the age of 9 months and 12 years, respectively. Furthermore, the twin analysis showed that 95% of liability to transient CH was explained by genetic factors and 5% by environmental (unshared) factors, whereas 64% of phenotypic variance of permanent CH was explained by common environmental factors (shared during the fetal life) and 36% by unshared environmental factors.

Conclusions: This study showed that the introduction of rescreening permits the diagnosis of CH in a greater number of twins. It also showed the importance of long-term follow-up in both twins in the pair, and the role of nongenetic factors in the etiology of permanent CH.
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http://dx.doi.org/10.1210/jc.2019-00900DOI Listing
December 2019

SCF-mediated γ-globin gene expression in adult human erythroid cells is associated with KLF1, BCL11A and SOX6 down-regulation.

Blood Cells Mol Dis 2015 Jan 3;54(1):1-3. Epub 2014 Nov 3.

Dept. of Hematology, Oncology and Molecular Medicine, Istituto Superiore di Sanità, Rome, Italy. Electronic address:

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http://dx.doi.org/10.1016/j.bcmd.2014.10.004DOI Listing
January 2015

Aspirin influences megakaryocytic gene expression leading to up-regulation of multidrug resistance protein-4 in human platelets.

Br J Clin Pharmacol 2014 Dec;78(6):1343-53

Department of Experimental Medicine, Sapienza University of Rome, Rome.

Aim: The aim of the study was to investigate whether human megakaryocytic cells have an adaptive response to aspirin treatment, leading to an enhancement of multidrug resistance protein-4 (MRP4) expression in circulating platelets responsible for a reduced aspirin action. We recently found that platelet MRP4 overexpression has a role in reducing aspirin action in patients after by-pass surgery. Aspirin enhances MRP4-mRNA levels in rat liver and drug administration transcriptionally regulates MRP4 gene expression through peroxisome proliferator-activated receptor-α (PPARα).

Methods: The effects induced by aspirin or PPARα agonist (WY14643) on MRP4 modulation were evaluated in vitro in a human megakaryoblastic DAMI cell line, in megakaryocytes (MKs) and in platelets obtained from human haematopoietic progenitor cell (HPC) cultures, and in vivo platelets obtained from aspirin treated healthy volunteers (HV).

Results: In DAMI cells, aspirin and WY14643 treatment induced a significant increase in MRP4 and PPARα expression. In human MKs grown in the presence of either aspirin or WY14643, MRP4 and PPARα-mRNA were higher than in control cultures and derived platelets showed an enhancement in MRP4 protein expression. The ability of aspirin to modulate MRP4 expression in MKs and to transfer it to platelets was also confirmed in vivo. In fact, we found the highest MRP4 mRNA and protein expression in platelets obtained from HV after 15 days' aspirin treatment.

Conclusions: The present study provides evidence, for the first time, that aspirin treatment affects the platelet protein pattern through MK genomic modulation. This work represents an innovative and attractive approach, useful both to identify patients less sensitive to aspirin and to improve pharmacological treatment in cardiovascular high-risk patients.
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http://dx.doi.org/10.1111/bcp.12432DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4256623PMC
December 2014

MicroRNA-486-3p regulates γ-globin expression in human erythroid cells by directly modulating BCL11A.

PLoS One 2013 4;8(4):e60436. Epub 2013 Apr 4.

Department of Hematology, Oncology and Molecular Medicine, Istituto Superiore di Sanità, Rome, Italy.

MicroRNAs (miRNAs) play key roles in modulating a variety of cellular processes through repression of mRNAs target. The functional relevance of microRNAs has been proven in normal and malignant hematopoiesis. While analyzing miRNAs expression profile in unilineage serum-free liquid suspension unilineage cultures of peripheral blood CD34(+) hematopoietic progenitor cells (HPCs) through the erythroid, megakaryocytic, granulocytic and monocytic pathways, we identified miR-486-3p as mainly expressed within the erythroid lineage. We showed that miR-486-3p regulates BCL11A expression by binding to the extra-long isoform of BCL11A 3'UTR. Overexpression of miR-486-3p in erythroid cells resulted in reduced BCL11A protein levels, associated to increased expression of γ-globin gene, whereas inhibition of physiological miR-486-3p levels increased BCL11A and, consequently, reduced γ-globin expression. Thus, miR-486-3p regulating BCL11A expression might contributes to fetal hemoglobin (HbF) modulation and arise the question as to what extent this miRNA might contribute to different HbF levels observed among β-thalassemia patients. Erythroid cells, differentiated from PB CD34(+) cells of a small cohort of patients affected by major or intermedia β-thalassemia, showed miR-486-3p levels significantly higher than those observed in normal counterpart. Importantly, in these patients, miR-486-3p expression correlates with increased HbF synthesis. Thus, our data indicate that miR-486-3p might contribute to different HbF levels observed among thalassemic patients and, possibly, to the clinical severity of the disease.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0060436PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3617093PMC
November 2013

Autocrine role of angiopoietins during megakaryocytic differentiation.

PLoS One 2012 6;7(7):e39796. Epub 2012 Jul 6.

Department of Hematology, Oncology and Molecular Medicine, Istituto Superiore di Sanità, Rome, Italy.

The tyrosine kinase Tie-2 and its ligands Angiopoietins (Angs) transduce critical signals for angiogenesis in endothelial cells. This receptor and Ang-1 are coexpressed in hematopoietic stem cells and in a subset of megakaryocytes, though a possible role of angiopoietins in megakaryocytic differentiation/proliferation remains to be demonstrated. To investigate a possible effect of Ang-1/Ang-2 on megakaryocytic proliferation/differentiation we have used both normal CD34(+) cells induced to megakaryocytic differentiation and the UT7 cells engineered to express the thrombopoietin receptor (TPOR, also known as c-mpl, UT7/mpl). Our results indicate that Ang-1/Ang-2 may have a role in megakaryopoiesis. Particularly, Ang-2 is predominantly produced and released by immature normal megakaryocytic cells and by undifferentiated UT7/mpl cells and slightly stimulated TPO-induced cell proliferation. Ang-1 production is markedly induced during megakaryocytic differentiation/maturation and potentiated TPO-driven megakaryocytic differentiation. Blocking endogenously released angiopoietins partially inhibited megakaryocytic differentiation, particularly for that concerns the process of polyploidization. According to these data it is suggested that an autocrine angiopoietin/Tie-2 loop controls megakaryocytic proliferation and differentiation.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0039796PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3391299PMC
March 2013

Ferroportin and erythroid cells: an update.

Adv Hematol 2010 11;2010. Epub 2010 Aug 11.

Department of Hematology, Oncology and Molecular Medicine, Istituto Superiore di Sanità, Viale Regina Elena 299, 00161 Rome, Italy.

In recent years there have been major advances in our knowledge of the regulation of iron metabolism that have had implications for understanding the pathophysiology of some human disorders like beta-thalassemia and other iron overload diseases. However, little is known about the relationship among ineffective erythropoiesis, the role of iron-regulatory genes, and tissue iron distribution in beta-thalassemia. The principal aim of this paper is an update about the role of Ferroportin during human normal and pathological erythroid differentiation. Particular attention will be given to beta-thalassemia and other diseases with iron overload. Recent discoveries indicate that there is a potential for therapeutic intervention in beta-thalassemia by means of manipulating iron metabolism.
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http://dx.doi.org/10.1155/2010/404173DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2935194PMC
July 2011

Mechanism of human Hb switching: a possible role of the kit receptor/miR 221-222 complex.

Haematologica 2010 Aug 19;95(8):1253-60. Epub 2010 Mar 19.

Department of Hematology, Oncology and Molecular Medicine, Istituto Superiore di Sanità, 00161 Rome, Italy.

Background: The human hemoglobin switch (HbF-->HbA) takes place in the peri/post-natal period. In adult life, however, the residual HbF (<1%) may be partially reactivated by chemical inducers and/or cytokines such as the kit ligand (KL). MicroRNAs (miRs) play a pivotal role in normal hematopoiesis: downmodulation of miR-221/222 stimulates human erythropoietic proliferation through upmodulation of the kit receptor.

Design And Methods: We have explored the possible role of kit/KL in perinatal Hb switching by evaluating: i) the expression levels of both kit and kit ligand on CD34(+) cells and in plasma isolated from pre-, mid- and full-term cord blood samples; ii) the reactivation of HbF synthesis in KL-treated unilineage erythroid cell cultures; iii) the functional role of miR-221/222 in HbF production.

Results: In perinatal life, kit expression showed a gradual decline directly correlated to the decrease of HbF (from 80-90% to <30%). Moreover, in full-term cord blood erythroid cultures, kit ligand induced a marked increase of HbF (up to 80%) specifically abrogated by addition of the kit inhibitor imatinib, thus reversing the Hb switch. MiR-221/222 expression exhibited rising levels during peri/post-natal development. In functional studies, overexpression of these miRs in cord blood progenitors caused a remarkable decrease in kit expression, erythroblast proliferation and HbF content, whereas their suppression induced opposite effects.

Conclusions: Our studies indicate that human perinatal Hb switching is under control of the kit receptor/miR 221-222 complex. We do not exclude, however, that other mechanisms (i.e. glucocorticoids and the HbF inhibitor BCL11A) may also contribute to the peri/post-natal Hb switch.
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http://dx.doi.org/10.3324/haematol.2009.018259DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2913072PMC
August 2010

NFI-A directs the fate of hematopoietic progenitors to the erythroid or granulocytic lineage and controls beta-globin and G-CSF receptor expression.

Blood 2009 Aug 19;114(9):1753-63. Epub 2009 Jun 19.

Department of Histology and Medical Embryology, University La Sapienza, Rome, Italy.

It is generally conceded that selective combinations of transcription factors determine hematopoietic lineage commitment and differentiation. Here we show that in normal human hematopoiesis the transcription factor nuclear factor I-A (NFI-A) exhibits a marked lineage-specific expression pattern: it is upmodulated in the erythroid (E) lineage while fully suppressed in the granulopoietic (G) series. In unilineage E culture of hematopoietic progenitor cells (HPCs), NFI-A overexpression or knockdown accelerates or blocks erythropoiesis, respectively: notably, NFI-A overexpression restores E differentiation in the presence of low or minimal erythropoietin stimulus. Conversely, NFI-A ectopic expression in unilineage G culture induces a sharp inhibition of granulopoiesis. Finally, in bilineage E + G culture, NFI-A overexpression or suppression drives HPCs into the E or G differentiation pathways, respectively. These NFI-A actions are mediated, at least in part, by a dual and opposite transcriptional action: direct binding and activation or repression of the promoters of the beta-globin and G-CSF receptor gene, respectively. Altogether, these results indicate that, in early hematopoiesis, the NFI-A expression level acts as a novel factor channeling HPCs into either the E or G lineage.
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http://dx.doi.org/10.1182/blood-2008-12-196196DOI Listing
August 2009

Role of stem cell factor in the reactivation of human fetal hemoglobin.

Mediterr J Hematol Infect Dis 2009 Nov 13;1(1):e2009009. Epub 2009 Nov 13.

Department of Hematology, Oncology and Molecular Medicine, Istituto Superiore di Sanità, ROME, ITALY.

In humans the switch from fetal to adult hemoglobin (HbF → HbA) takes place in the perinatal and postnatal period, determining the progressive replacement of HbF with HbA synthesis (i.e., the relative HbF content in red blood cells decreases from 80-90% to <1%). In spite of more than twenty years of intensive investigations on this classic model, the molecular mechanisms regulating the Hb switching, as well as HbF synthesis in adults, has been only in part elucidated. In adult life, the residual HbF, restricted to F cell compartment, may be reactivated up to 10-20% of total Hb synthesis in various conditions associated with "stress erythropoiesis": this reactivation represented until now an interesting model of partial Hb switch reverse with important therapeutic implications in patients with hemoglobinopathies, and particularly in β-thalassemia. In vitro and in vivo models have led to the identification of several chemical compounds able to reactivate HbF synthesis in adult erythroid cells. Although the impact of these HbF inducers, including hypomethylating agents, histone deacetylase inhibitors and hydroxyurea, was clear on the natural history of sickle cell anemia, the benefit on the clinical course of β-thalassemia was only limited: particularly, the toxicity and the modest increase in γ-globin reactivation indicated the need for improved agents able to induce higher levels of HbF. In the present review we describe the biologic properties of Stem Cell Factor (SCF), a cytokine sustaining the survival and proliferation of erythroid cells, that at pharmacological doses acts as a potent stimulator of HbF synthesis in adult erythroid cells.
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http://dx.doi.org/10.4084/MJHID.2009.009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3033162PMC
November 2009

Effective erythropoiesis and HbF reactivation induced by kit ligand in beta-thalassemia.

Blood 2008 Jan 19;111(1):421-9. Epub 2007 Oct 19.

Department of Hematology, Oncology, and Molecular Medicine, Istituto Superiore di Sanità, Viale Regina Elena, 299-00161 Rome, Italy.

In human beta-thalassemia, the imbalance between alpha- and non-alpha-globin chains causes ineffective erythropoiesis, hemolysis, and anemia: this condition is effectively treated by an enhanced level of fetal hemoglobin (HbF). In spite of extensive studies on pharmacologic induction of HbF synthesis, clinical trials based on HbF reactivation in beta-thalassemia produced inconsistent results. Here, we investigated the in vitro response of beta-thalassemic erythroid progenitors to kit ligand (KL) in terms of HbF reactivation, stimulation of effective erythropoiesis, and inhibition of apoptosis. In unilineage erythroid cultures of 20 patients with intermedia or major beta-thalassemia, addition of KL, alone or combined with dexamethasone (Dex), remarkably stimulated cell proliferation (3-4 logs more than control cultures), while decreasing the percentage of apoptotic and dyserythropoietic cells (<5%). More important, in both thalassemic groups, addition of KL or KL plus Dex induced a marked increase of gamma-globin synthesis, thus reaching HbF levels 3-fold higher than in con-trol cultures (eg, from 27% to 75% or 81%, respectively, in beta-thalassemia major). These studies indicate that in beta-thalassemia, KL, alone or combined with Dex, induces an expansion of effective erythropoiesis and the reactivation of gamma-globin genes up to fetal levels and may hence be considered as a potential therapeutic agent for this disease.
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http://dx.doi.org/10.1182/blood-2007-06-097550DOI Listing
January 2008

Inhibition of TPO-induced MEK or mTOR activity induces opposite effects on the ploidy of human differentiating megakaryocytes.

J Cell Sci 2006 Feb 31;119(Pt 4):744-52. Epub 2006 Jan 31.

Department of Hematology, Oncology and Molecular Medicine, Istituto Superiore di Sanità, Viale Regina Elena, 299, 00161 Rome, Italy.

The megakaryocyte is a paradigm for mammalian polyploid cells. However, the mechanisms underlying megakaryocytic polyploidization have not been elucidated. In this study, we investigated the role of Shc-Ras-MAPK and PI3K-AKT-mTOR pathways in promoting megakaryocytic differentiation, maturation and polyploidization. CD34+ cells, purified from human peripheral blood, were induced in serum-free liquid suspension culture supplemented with thrombopoietin (TPO) to differentiate into a virtually pure megakaryocytic progeny (97-99% CD61+/CD41+ cells). The early and repeated addition to cell cultures of low concentrations of PD98059, an inhibitor of MEK1/2 activation, gave rise to a population of large megakaryocytes showing an increase in DNA content and polylobated nuclei (from 45% to 70% in control and treated cultures, respectively). Conversely, treatment with the mTOR inhibitor rapamycin strongly inhibited cell polyploidization, as compared with control cultures. Western blot analysis of PD98059-treated progenitor cells compared with the control showed a downmodulation of phospho-ERK 1 and phospho-ERK 2 and a minimal influence on p70S6K activation; by contrast, p70S6K activation was completely inhibited in rapamycin-treated cells. Interestingly, the cyclin D3 localization was nuclear in PD98059-induced polyploid megakaryocytes, whereas it was completely cytoplasmic in those treated with rapamycin. Altogether, our results are in line with a model in which binding of TPO to the TPO receptor (mpl) could activate the rapamycin-sensitive PI3K-AKT-mTOR-p70S6K pathway and its downstream targets in promoting megakaryocytic cell polyploidization.
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http://dx.doi.org/10.1242/jcs.02784DOI Listing
February 2006

Expression of alternative transcripts of ferroportin-1 during human erythroid differentiation.

Haematologica 2005 Dec;90(12):1595-606

Department of Hematology, Oncology and Molecular Medicine, Istituto Superiore di Sanità, Rome, Italy.

Background And Objectives: Ferroportin-1 (FPN1) is expressed in various types of cells that play critical roles in mammalian iron metabolism and appears to act as an iron exporter in these tissues. The aim of this study was to investigate whether erythroid cells possess specific mechanisms for iron export.

Design And Methods: The expression of FPN1 during human erythroid differentiation, the characterization of alternative transcripts, the modulation by iron and the subcellular localization of this protein were studied.

Results: FPN1 mRNA and protein are highly expressed during human erythroid differentiation. The iron-responsive element (IRE) in the 5'- untranslated region (UTR) of FPN1 mRNA is functional but, in spite of that, FPN1 protein expression, as well as mRNA level and half-life, seem not to be affected by iron. To explain these apparenthy discordant results we searched for alternative transcripts of FPN1 and found at least three different types of transcripts, displaying alternative 5' ends. Most of the FPN1 transcripts code for the canonical protein, but only half of them contain an IRE in the 5'-UTR and have the potential to be translationally regulated by iron. Expression analysis shows that alternative FPN1 transcripts are differentially expressed during erythroid differentiation. Finally, sustained expression of alternative FPN1 transcripts is apparently observed only in erythroid cells.

Interpretation And Conclusions: This is the first report describing the presence of FPN1 in erythroid cells at all stages of differentiation, providing evidence that erythroid cells possess specific mechanisms of iron export. The existence of multiple FPN1 transcripts indicates a complex regulation of the FPN1 gene in erythroid cells.
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December 2005

Transferrin receptor 2 protein is not expressed in normal erythroid cells.

Biochem J 2004 Aug;381(Pt 3):629-34

Department of Hematology, Oncology and Molecular Medicine, Istituto Superiore di Sanità, Viale Regina Elena 299, 00161 Rome, Italy.

Human TFR2 (transferrin receptor 2) is a membrane-bound protein homologous with TFR1. High levels of TFR2 mRNA were found mainly in the liver and, to a lesser extent, in erythroid precursors. However, although the presence of the TFR2 protein in hepatic cells has been confirmed in several studies, evidence is lacking about the presence of the TFR2 protein in normal erythroid cells. Using two anti-TFR2 monoclonal antibodies, G/14C2 and G/14E8, we have provided evidence that TFR2 protein is not expressed in normal erythroid cells at any stage of differentiation, from undifferentiated CD34+ cells to mature orthochromatic erythroblasts. In contrast, erythroleukaemic cells (K562 cells) exhibited a high level of expression of TFR2 at both the mRNA and the protein level. We can therefore conclude that an elevated expression of TFR2 protein is observed in leukaemic cells, but not in normal erythroblasts. The implications of this observation for the understanding of the phenotypic features of haemochromatosis due to mutation of the TFR2 gene are discussed.
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http://dx.doi.org/10.1042/BJ20040230DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1133871PMC
August 2004

HbF reactivation in sibling BFU-E colonies: synergistic interaction of kit ligand with low-dose dexamethasone.

Blood 2003 Apr 7;101(7):2826-32. Epub 2002 Nov 7.

Department of Hematology and Oncology, Istituto Superiore di Sanità, Rome, Italy.

Mechanisms underlying fetal hemoglobin (HbF) reactivation in stress erythropoiesis have not been fully elucidated. We suggested that a key role is played by kit ligand (KL). Because glucocorticoids (GCs) mediate stress erythropoiesis, we explored their capacity to potentiate the stimulatory effect of KL on HbF reactivation, as evaluated in unilineage erythropoietic culture of purified adult progenitors (erythroid burst-forming units [BFU-Es]). The GC derivative dexamethasone (Dex) was tested in minibulk cultures at graded dosages within the therapeutical range (10(-6) to 10(-9) M). Dex did not exert significant effects alone, but synergistically it potentiated the action of KL in a dose-dependent fashion. Specifically, Dex induced delayed erythroid maturation coupled with a 2-log increased number of generated erythroblasts and enhanced HbF synthesis up to 85% F cells and 55% gamma-globin content at terminal maturation (ie, in more than 80%-90% mature erythroblasts). Equivalent results were obtained in unicellular erythroid cultures of sibling BFU-Es treated with KL alone or combined with graded amounts of Dex. These results indicate that the stimulatory effect of KL + Dex is related to the modulation of gamma-globin expression rather than to recruitment of BFU-Es with elevated HbF synthetic potential. At the molecular level, Id2 expression is totally suppressed in control erythroid culture but is sustained in KL + Dex culture. Hypothetically, Id2 may mediate the expansion of early erythroid cells, which correlates with HbF reactivation. These studies indicate that GCs play an important role in HbF reactivation. Because Dex acts at dosages used in immunologic disease therapy, KL + Dex administration may be considered to develop preclinical models for beta-hemoglobinopathy treatment.
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http://dx.doi.org/10.1182/blood-2002-05-1477DOI Listing
April 2003

Autocrine-paracrine VEGF loops potentiate the maturation of megakaryocytic precursors through Flt1 receptor.

Blood 2003 Feb 24;101(4):1316-23. Epub 2002 Oct 24.

Department of Hematology-Oncology, Istituto Superiore di Sanità, Rome, Italy.

The expression/function of vascular endothelial growth factor (VEGF) receptors (VEGFR1/Flt1 and VEGFR2/KDR/Flk1) in hematopoiesis is under scrutiny. We have investigated the expression of Flt1 and kinase domain receptor (KDR) on hematopoietic precursors, as evaluated in liquid culture of CD34(+) hematopoietic progenitor cells (HPCs) induced to unilineage differentiation/maturation through the erythroid (E), megakaryocytic (Mk), granulocytic (G), or monocytic (Mo) lineage. KDR, expressed on 0.5% to 1.5% CD34(+) cells, is rapidly downmodulated on induction of differentiation. Similarly, Flt1 is present at very low levels in HPCs and is downmodulated in E and G lineages; however, Flt1 is induced in the precursors of both Mo and Mk series; ie, its level progressively increases during Mo maturation, and it peaks at the initial-intermediate culture stages in the Mk lineage. Functional experiments indicate that Mk and E, but not G and Mo, precursors release significant amounts of VEGF in the culture medium, particularly at low O(2) levels. The functional role of VEGF release on Mk maturation is indicated by 2 series of observations. (1) Molecules preventing the VEGF-Flt1 interaction on the precursor membrane (eg, soluble Flt1 receptors) significantly inhibit Mk polyploidization. (2) Addition of exogenous VEGF or placenta growth factor (PlGF) markedly potentiates Mk maturation. Conversely, VEGF does not modify Mo differentiation/maturation. Altogether, our results suggest that in the hematopoietic microenvironment an autocrine VEGF loop contributes to optimal Mk maturation through Flt1. A paracrine loop involving VEGF release by E precursors may also operate. Similarly, recent studies indicate that an autocrine loop involving VEGF and Flt1/Flk1 receptors mediates hematopoietic stem cell survival and differentiation.
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http://dx.doi.org/10.1182/blood-2002-07-2184DOI Listing
February 2003

Identification of the hemangioblast in postnatal life.

Blood 2002 Nov;100(9):3203-8

Kimmel Cancer Center, Room 609, Thomas Jefferson University, 233 S. 10th Street, Philadelphia, PA 19107-5541, USA.

Postnatal CD34(+) cells expressing vascular endothelial growth factor receptor 2 (KDR) generate hematopoietic or endothelial progeny in different in vitro and in vivo assays. Hypothetically, CD34(+)KDR(+) cells may comprise hemangioblasts bipotent for both lineages. This hypothesis is consistent with 2 series of experiments. In the first series, in clonogenic culture permissive for hematopoietic and endothelial cell growth, CD34(+)KDR(+) cells generate large hemato-endothelial (Hem-End) colonies (5% of seeded cells), whereas CD34(+)KDR(-) cells do not. Limiting-dilution analysis indicates that Hem-End colonies are clonally generated by single hemangioblasts. Sibling cells generated by a hemangioblast, replated in unicellular culture, produce either hematopoietic or Hem-End colonies, depending on the specific culture conditions. Identification of endothelial cells was based on the expression of VE-cadherin and endothelial markers and with lack of CD45 and hematopoietic molecules, as evaluated by immunofluorescence, immunocytochemistry, and reverse transcription-polymerase chain reaction. Furthermore, endothelial cells were functionally identified using low-density lipoprotein (LDL) uptake and tube-formation assays. In the second series, to evaluate the self-renewal capacity of hemangioblasts, single CD34(+)KDR(+) cells were grown in 3-month extended long-term culture (ELTC) through 3 serial culture rounds-that is, blast cells generated in unicellular ELTC were reseeded for a subsequent round of unicellular ELTC. After 9 months, 10% blasts from tertiary ELTC functioned as hemangioblasts and generated macroscopic Hem-End colonies in clonogenic culture. These studies identified postnatal hemangioblasts in a CD34(+)KDR(+) cell subset, endowed with long-term proliferative potential and bilineage differentiation capacity. Although exceedingly rare, hemangioblasts may represent the lifetime source/reservoir for primitive hematopoietic and endothelial progenitors.
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http://dx.doi.org/10.1182/blood-2002-05-1511DOI Listing
November 2002