Publications by authors named "Raffaella Guerriero"

12 Publications

  • Page 1 of 1

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

Aspirin-Dependent Effects on Purinergic P2Y1 Receptor Expression.

Thromb Haemost 2019 May 13;119(5):726-734. Epub 2019 Feb 13.

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

Chronic treatment with aspirin in healthy volunteers (HVs) is associated with recovery of adenosine diphosphate (ADP)-induced platelet activation. The purinergic P2Y receptor exerts its effects via a G-protein, which is the same biochemical pathway activated by thromboxane-A2 receptor. We hypothesized that recovery of ADP-induced platelet activation could be attributed to increased P2Y expression induced by chronic aspirin exposure. We performed a multi-phase investigation which embraced both in vitro and in vivo experiments conducted in (1) human megakaryoblastic DAMI cells, (2) human megakaryocytic progenitor cell cultures, (3) platelets obtained from HVs treated with aspirin and (4) platelets obtained from aspirin-treated patients. DAMI cells treated with aspirin or WY14643 (PPARα agonist) had a significant up-regulation of P2Y mRNA, which was shown to be a PPARα-dependent process. In human megakaryocytic progenitors, in the presence of aspirin or WY14643, P2Y mRNA expression was higher than in mock culture. P2Y expression increased in platelets obtained from HVs treated with aspirin for 8 weeks. Platelets obtained from patients who were on aspirin for more than 2 months had increased P2Y expression and ADP-induced aggregation compared with patients on aspirin treatment for less than a month. Overall, our results suggest that aspirin induces genomic changes in megakaryocytes leading to P2Y up-regulation and that PPARα is the nuclear receptor involved in this regulation. Since P2Y is coupled to the same G-protein of thromboxane-A2 receptor, platelet adaptation in response to pharmacological inhibition seems not to be receptor specific, but may involve other receptors with the same biochemical pathway.
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http://dx.doi.org/10.1055/s-0039-1678707DOI Listing
May 2019

MiR-21 role in aspirin-dependent PPARα and multidrug resistance protein 4 upregulation.

Res Pract Thromb Haemost 2018 Jul 17;2(3):596-606. Epub 2018 May 17.

Department of Experimental Medicine Sapienza-University of Rome Rome Italy.

Background: A mechanism involved in high on-aspirin treatment residual platelet reactivity is platelet multidrug resistance protein 4 (MRP4) overexpression. Aspirin enhances platelet MRP4 expression with a PPARα-dependent mechanism and reduces miR-21 expression that, in turn, downregulates PPARα expression.

Objective: The aim of our study was to verify the relationship between miR-21 and MRP4-PPARα levels induced by aspirin treatment.

Methods: We evaluated the changes in MRP4-PPARα, mRNA, MRP4 protein, and miR-21 expression induced by aspirin in: (i) in vitro-treated megakaryoblastic cell line (DAMI), (ii) primary megakaryocytes cultures and derived platelets, (iii) healthy volunteers' platelets treated with aspirin, and (iv) aspirinated patients (aspirin-treated patients) and in a control population (control).

Results: We observed an aspirin-induced reverse relationship between the expression of miR-21 and PPARα-MRP4. In DAMI cells the miR-21 mimic transfection reduces PPARα and MRP4 expression, even if cells were treated with aspirin after transfection. MiR-21 inhibitor transfection induces PPARα and MRP4 expression that are not enhanced by aspirin treatment. In human megakaryocytes, aspirin treatment lead to a miR-21 downregulation and a MRP4 upregulation and this trend is confirmed in derived platelets. In aspirin-treated volunteers, an inverse relationship between miR-21 and MRP4 platelet expression was found after aspirin treatment. A similar negative relationship was found in aspirin-treated patients vs the control population.

Conclusion: The results reported in this study provide information that aspirin induces the modulation of platelet miR-21 expression levels and this modulation can be responsible for MRP4 enhancement in circulating platelets.
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http://dx.doi.org/10.1002/rth2.12104DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6046603PMC
July 2018

From Human Megakaryocytes to Platelets: Effects of Aspirin on High-Mobility Group Box 1/Receptor for Advanced Glycation End Products Axis.

Front Immunol 2017 12;8:1946. Epub 2018 Jan 12.

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

Platelets (PLTs) are the major source of high-mobility group box 1 (HMGB1), a protein that is involved in sterile inflammation of blood vessels and thrombosis. Megakaryocytes (MKs) synthesize HMGB1 and transfer both protein and mRNA into PLTs and PLT-derived microvesicles (MV). Free HMGB1 found in supernatants of differentiated MKs and in a megakaryoblastic cell line (DAMI cells). Aspirin "" and "" not only reduces HMGB1 and receptor for advanced glycation end products expression on MKs and PLTs but also drives the movement of HMGB1 from MKs into PLTs and PLT-derived MV. These findings suggest that consumption of low doses of aspirin reduces the risk of atherosclerosis complications as well as reducing PLT aggregation by the inhibition of COX-1.
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http://dx.doi.org/10.3389/fimmu.2017.01946DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5770369PMC
January 2018

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

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

Aspirin extrusion from human platelets through multidrug resistance protein-4-mediated transport: evidence of a reduced drug action in patients after coronary artery bypass grafting.

J Am Coll Cardiol 2011 Aug;58(7):752-61

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

Objectives: In this study we investigate: 1) the role of multidrug resistance protein-4 (MRP4), an organic anion unidirectional transporter, in modulating aspirin action on human platelet cyclooxygenase (COX)-1; and 2) whether the impairment of aspirin-COX-1 interaction, found in coronary artery bypass grafting (CABG) patients, could be dependent on MRP4-mediated transport.

Background: Platelets of CABG patients present a reduced sensitivity to aspirin despite in vivo and in vitro drug treatment. Aspirin is an organic anion and could be a substrate for MRP4.

Methods: Intracellular aspirin concentration and drug COX-1 activity, measured by thrombin-induced thromboxane B2 (TxB2) production, were evaluated in platelets obtained from healthy volunteers (HV) and hematopoietic-progenitor cell cultures reducing or not reducing MRP4-mediated transport. Platelet MRP4 expression was evaluated, in platelets from HV and CABG patients, by dot-blot or by immunogold-electromicrographs or immunofluorescence-microscopy analysis.

Results: Inhibition of MRP4-mediated transport by dipyridamole or Mk-571 increases aspirin entrapment and its in vitro effect on COX-1 activity (142.7 ± 34.6 pg/10(8) cells vs. 343.7 ± 169.3 pg/10⁸ cells TxB2-production). Platelets derived from megakaryocytes transfected with MRP4 small interfering ribonucleic acid have a higher aspirin entrapment and drug COX-1 activity. Platelets from CABG patients showed a high expression of MRP4 whose in vitro inhibition enhanced aspirin effect on COX-1 (349 ± 141 pg/10⁸ cells vs. 1,670 ± 646 pg/10⁸ cells TxB2-production).

Conclusions: Aspirin is a substrate for MRP4 and can be extruded from platelet through its transportation. Aspirin effect on COX-1 is little-related to MRP4-mediated aspirin transport in HV, but in CABG patients with MRP4 over-expression, its pharmacological inhibition enhances aspirin action in an efficient way.
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http://dx.doi.org/10.1016/j.jacc.2011.03.049DOI Listing
August 2011

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

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