The Experts below are selected from a list of 312 Experts worldwide ranked by ideXlab platform
Stefano Rivella - One of the best experts on this subject based on the ideXlab platform.
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minihepcidins improve Ineffective Erythropoiesis and splenomegaly in a new mouse model of adult β thalassemia major
Haematologica, 2020Co-Authors: Carla Casu, Yelena Ginzburg, Ritama Gupta, Roberta Chessa, Alison Liu, Hal Drakesmith, Robert E Fleming, Brian Macdonald, Stefano RivellaAbstract:Minihepcidins are hepcidin agonists that have been previously shown to reverse iron overload and improve Erythropoiesis in mice affected by non-transfusion-dependent thalassemia. Given the extreme anemia that occurred with the previous model of transfusion-dependent thalassemia, that model was inadequate for investigating whether minihepcidins can improve red blood cell quality, lifespan and Ineffective Erythropoiesis. To overcome this limitation, we generated a new murine model of transfusion-dependent thalassemia with severe anemia and splenomegaly, but sufficient red cells and hemoglobin production to test the effect of minihepcidins. Furthermore, this new model demonstrates cardiac iron overload for the first time. In the absence of transfusions, minihepcidins improved red blood cell morphology and lifespan as well as Ineffective Erythropoiesis. Administration of a minihepcidin in combination with chronic red blood cell transfusion further improved the Ineffective Erythropoiesis and splenomegaly and reversed cardiac iron overload. These studies indicate that drugs such as minihepcidins have therapeutic potential for patients with transfusion-dependent thalassemia.
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iron metabolism under conditions of Ineffective Erythropoiesis in β thalassemia
Blood, 2019Co-Authors: Stefano RivellaAbstract:β-Thalassemia (BT) is an inherited genetic disorder that is characterized by Ineffective Erythropoiesis (IE), leading to anemia and abnormal iron metabolism. IE is an abnormal expansion of the number of erythroid progenitor cells with unproductive synthesis of enucleated erythrocytes, leading to anemia and hypoxia. Anemic patients affected by BT suffer from iron overload, even in the absence of chronic blood transfusion, suggesting the presence of ≥1 erythroid factor with the ability to modulate iron metabolism and dietary iron absorption. Recent studies suggest that decreased erythroid cell differentiation and survival also contribute to IE, aggravating the anemia in BT. Furthermore, hypoxia can also affect and increase iron absorption. Understanding the relationship between iron metabolism and IE could provide important insights into the BT condition and help to develop novel treatments. In fact, genetic or pharmacological manipulations of iron metabolism or erythroid cell differentiation and survival have been shown to improve IE, iron overload, and anemia in animal models of BT. Based on those findings, new therapeutic approaches and drugs have been proposed; clinical trials are underway that have the potential to improve erythrocyte production, as well as to reduce the iron overload and organ toxicity in BT and in other disorders characterized by IE.
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Ineffective Erythropoiesis anemia and iron overload
Hematology-oncology Clinics of North America, 2017Co-Authors: Ritama Gupta, Khaled M Musallam, Ali T Taher, Stefano RivellaAbstract:Stress Erythropoiesis (SE) is characterized by an imbalance in erythroid proliferation and differentiation under increased demands of erythrocyte generation and tissue oxygenation. β-thalassemia represents a chronic state of SE, called Ineffective Erythropoiesis (IE), exhibiting an expansion of erythroid-progenitor pool and deposition of alpha chains on erythrocyte membranes, causing cell death and anemia. Concurrently, there is a decrease in hepcidin expression and a subsequent state of iron overload. There are substantial investigative efforts to target increased iron absorption under IE. There are also avenues for targeting cell contact and signaling within erythroblastic islands under SE, for therapeutic benefits.
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what can we learn from Ineffective Erythropoiesis in thalassemia
Blood Reviews, 2017Co-Authors: Paraskevi Rea Oikonomidou, Stefano RivellaAbstract:Erythropoiesis is a dynamic process regulated at multiple levels to balance proliferation, differentiation and survival of erythroid progenitors. Ineffective Erythropoiesis is a key feature of various diseases, including β-thalassemia. The pathogenic mechanisms leading to Ineffective Erythropoiesis are complex and still not fully understood. Altered survival and decreased differentiation of erythroid progenitors are both critical processes contributing to reduced production of mature red blood cells. Recent studies have identified novel important players and provided major advances in the development of targeted therapeutic approaches. In this review, β-thalassemia is used as a paradigmatic example to describe our current knowledge on the mechanisms leading to Ineffective Erythropoiesis and novel treatments that may have the potential to improve the clinical phenotype of associated diseases in the future.
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modified activin receptor iib ligand trap mitigates Ineffective Erythropoiesis and disease complications in murine β thalassemia
Blood, 2014Co-Authors: Rajasekhar Nvs Suragani, Mark J Alexander, Asya Grinberg, Sharon M Cawley, Robert Li, Samantha Wallner, Sara Gardenghi, Aaron W Mulivor, Stefano Rivella, Scott R PearsallAbstract:In β-thalassemia, unequal production of α- and β-globin chains in erythroid precursors causes apoptosis and inhibition of late-stage erythroid differentiation, leading to anemia, Ineffective Erythropoiesis (IE), and dysregulated iron homeostasis. Here we used a murine model of β-thalassemia intermedia (Hbbth1/th1 mice) to investigate effects of a modified activin receptor type IIB (ActRIIB) ligand trap (RAP-536) that inhibits Smad2/3 signaling. In Hbbth1/th1 mice, treatment with RAP-536 reduced overactivation of Smad2/3 in splenic erythroid precursors. In addition, treatment of Hbbth1/th1 mice with RAP-536 reduced α-globin aggregates in peripheral red cells, decreased the elevated reactive oxygen species present in erythroid precursors and peripheral red cells, and alleviated anemia by promoting differentiation of late-stage erythroid precursors and reducing hemolysis. Notably, RAP-536 treatment mitigated disease complications of IE, including iron overload, splenomegaly, and bone pathology, while reducing erythropoietin levels, improving erythrocyte morphology, and extending erythrocyte life span. These results implicate signaling by the transforming growth factor-β superfamily in late-stage Erythropoiesis and reveal potential of a modified ActRIIB ligand trap as a novel therapeutic agent for thalassemia syndrome and other red cell disorders characterized by IE.
Alan F List - One of the best experts on this subject based on the ideXlab platform.
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lenalidomide epoetin alfa versus lenalidomide monotherapy in myelodysplastic syndromes refractory to recombinant erythropoietin
Journal of Clinical Oncology, 2021Co-Authors: Alan F List, Amit Verma, Zhuoxin Sun, John M Bennett, Rami S Komrokji, Kathy L Mcgraw, Jaroslaw P Maciejewski, Jessica K Altman, Puneet Cheema, David F ClaxtonAbstract:PURPOSEImpaired response to erythropoietin underlies Ineffective Erythropoiesis and anemia in myelodysplastic syndromes (MDS). We investigated whether treatment with lenalidomide (LEN), which augme...
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age related inflammatory bone marrow microenvironment induces Ineffective Erythropoiesis mimicking del 5q mds
Leukemia, 2018Co-Authors: Yang Mei, Baobing Zhao, Ashley A Basiorka, Jing Yang, L Cao, J Zhang, Alan F ListAbstract:Anemia is characteristic of myelodysplastic syndromes (MDS). The mechanisms of anemia in MDS are unclear. Using a mouse genetic approach, here we show that dual deficiency of mDia1 and miR-146a, encoded on chromosome 5q and commonly deleted in MDS (del(5q) MDS), causes an age-related anemia and Ineffective Erythropoiesis mimicking human MDS. We demonstrate that the ageing bone marrow microenvironment is important for the development of Ineffective Erythropoiesis in these mice. Damage-associated molecular pattern molecules (DAMPs), whose levels increase in ageing bone marrow, induced TNFα and IL-6 upregulation in myeloid-derived suppressor cells (MDSCs) in mDia1/miR-146a double knockout mice. Mechanistically, we reveal that pathologic levels of TNFα and IL-6 inhibit erythroid colony formation and differentially affect terminal Erythropoiesis through reactive oxygen species-induced caspase-3 activation and apoptosis. Treatment of the mDia1/miR-146a double knockout mice with all-trans retinoic acid, which promoted the differentiation of MDSCs and ameliorated the inflammatory bone marrow microenvironment, significantly rescued anemia and Ineffective Erythropoiesis. Our study underscores the dual roles of the ageing microenvironment and genetic abnormalities in the pathogenesis of Ineffective Erythropoiesis in del(5q) MDS.
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dual deficiency of mdia1 and mir 146a in an age related inflammatory bone marrow microenvironment induces Ineffective Erythropoiesis in del 5q mds
Blood, 2016Co-Authors: Yang Mei, Baobing Zhao, Ashley A Basiorka, Jing Yang, Alan F ListAbstract:Abstract Myelodysplastic syndromes (MDS) are a group of age-related clonal hematologic diseases characterized by anemia, neutropenia, and thromobocytopenia. The mechanisms of anemia in MDS are unclear, which is partially due to the heterogeneity of MDS involving many cytogenetic and molecular abnormalities. Using a mouse genetic approach, here we show that dual deficiency of mDia1 and miR-146a, two genes located at chromosome 5q that is commonly deleted in MDS, causes an aged-related anemia and Ineffective Erythropoiesis that closely mimics human MDS. Bone marrow Erythropoiesis at various stages was dramatically affected in mDia1/miR-146a double knockout mice, which induced a massive splenomegaly with potent extramedullary Erythropoiesis. Old (> 1 year of age), but not young (2-4 months of age), wild type recipient mice that were transplanted with bone marrow cells from mDia1/miR-146a double knockout mice, exhibited severe anemia and rapid lethality, which indicates that the aged microenvironment is important for the development of Ineffective Erythropoiesis. Consistent with the roles of mDia1 and miR-146a in the innate immune response, the serum levels of TNFα and IL-6 were significantly elevated in mDia1/miR-146a double knockout mice. Pathogen-associated molecular pattern proteins (PAMPs), or damage-associated molecular pattern proteins (DAMPs), whose levels increase in aged microenvironment, both induced TNFα and IL-6 upregulation in mDia1/miR-146a double knockout granulocytes and T cells. Mechanistically, we demonstrated that the anemia and Ineffective Erythropoiesis was independent of hepcidin expression in mDia1/miR-146a double knockout mice. Instead, pathologic levels of TNFα and IL-6 inhibit erythroid colony formation and differentially affect terminal Erythropoiesis through reactive oxygen species-induced caspase-3 activation and cell apoptosis. Our study highlights the dual roles of age-related microenvironment and cytogenetic abnormalities in the pathogenesis of Ineffective Erythropoiesis in MDS. Disclosures No relevant conflicts of interest to declare.
Sara Gardenghi - One of the best experts on this subject based on the ideXlab platform.
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modified activin receptor iib ligand trap mitigates Ineffective Erythropoiesis and disease complications in murine β thalassemia
Blood, 2014Co-Authors: Rajasekhar Nvs Suragani, Mark J Alexander, Asya Grinberg, Sharon M Cawley, Robert Li, Samantha Wallner, Sara Gardenghi, Aaron W Mulivor, Stefano Rivella, Scott R PearsallAbstract:In β-thalassemia, unequal production of α- and β-globin chains in erythroid precursors causes apoptosis and inhibition of late-stage erythroid differentiation, leading to anemia, Ineffective Erythropoiesis (IE), and dysregulated iron homeostasis. Here we used a murine model of β-thalassemia intermedia (Hbbth1/th1 mice) to investigate effects of a modified activin receptor type IIB (ActRIIB) ligand trap (RAP-536) that inhibits Smad2/3 signaling. In Hbbth1/th1 mice, treatment with RAP-536 reduced overactivation of Smad2/3 in splenic erythroid precursors. In addition, treatment of Hbbth1/th1 mice with RAP-536 reduced α-globin aggregates in peripheral red cells, decreased the elevated reactive oxygen species present in erythroid precursors and peripheral red cells, and alleviated anemia by promoting differentiation of late-stage erythroid precursors and reducing hemolysis. Notably, RAP-536 treatment mitigated disease complications of IE, including iron overload, splenomegaly, and bone pathology, while reducing erythropoietin levels, improving erythrocyte morphology, and extending erythrocyte life span. These results implicate signaling by the transforming growth factor-β superfamily in late-stage Erythropoiesis and reveal potential of a modified ActRIIB ligand trap as a novel therapeutic agent for thalassemia syndrome and other red cell disorders characterized by IE.
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modified activin receptor iib ligand trap mitigates Ineffective Erythropoiesis and disease complications in murine β thalassemia
Blood, 2014Co-Authors: Rajasekhar Nvs Suragani, Mark J Alexander, Asya Grinberg, Sharon M Cawley, Robert Li, Samantha Wallner, Sara Gardenghi, Aaron W Mulivor, Stefano Rivella, Scott R PearsallAbstract:In β-thalassemia, unequal production of α- and β-globin chains in erythroid precursors causes apoptosis and inhibition of late-stage erythroid differentiation, leading to anemia, Ineffective Erythropoiesis (IE), and dysregulated iron homeostasis. Here we used a murine model of β-thalassemia intermedia (Hbbth1/th1 mice) to investigate effects of a modified activin receptor type IIB (ActRIIB) ligand trap (RAP-536) that inhibits Smad2/3 signaling. In Hbbth1/th1 mice, treatment with RAP-536 reduced overactivation of Smad2/3 in splenic erythroid precursors. In addition, treatment of Hbbth1/th1 mice with RAP-536 reduced α-globin aggregates in peripheral red cells, decreased the elevated reactive oxygen species present in erythroid precursors and peripheral red cells, and alleviated anemia by promoting differentiation of late-stage erythroid precursors and reducing hemolysis. Notably, RAP-536 treatment mitigated disease complications of IE, including iron overload, splenomegaly, and bone pathology, while reducing erythropoietin levels, improving erythrocyte morphology, and extending erythrocyte life span. These results implicate signaling by the transforming growth factor-β superfamily in late-stage Erythropoiesis and reveal potential of a modified ActRIIB ligand trap as a novel therapeutic agent for thalassemia syndrome and other red cell disorders characterized by IE.
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anemia Ineffective Erythropoiesis and hepcidin interacting factors in abnormal iron metabolism leading to iron overload in β thalassemia
Hematology-oncology Clinics of North America, 2010Co-Authors: Sara Gardenghi, Robert W Grady, Stefano RivellaAbstract:β-Thalassemia is a genetic disorder caused by mutations in the β-globin gene and characterized by chronic anemia caused by Ineffective Erythropoiesis, and accompanied by a variety of serious secondary complications such as extramedullary hematopoiesis, splenomegaly, and iron overload. In the past few years, numerous studies have shown that such secondary disease conditions have a genetic basis caused by the abnormal expression of genes with a role in controlling Erythropoiesis and iron metabolism. In this article, the most recent discoveries related to the mechanism(s) responsible for anemia/Ineffective Erythropoiesis and iron overload are discussed in detail. Particular attention is paid to the pathway(s) controlling the expression of hepcidin, which is the main regulator of iron metabolism, and the Epo/EpoR/Jak2/Stat5 signaling pathway, which regulates Erythropoiesis. Better understanding of how these pathways function and are altered in β-thalassemia has revealed several possibilities for development of new therapeutic approaches to treat of the complications of this disease.
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iron metabolism and Ineffective Erythropoiesis in β thalassemia mouse models
Annals of the New York Academy of Sciences, 2010Co-Authors: Pedro Ramos, Sara Gardenghi, Yelena Ginzburg, Luca Melchiori, Nico Vanroijen, Robert W Grady, Stefano RivellaAbstract:β-thalassemia is a disease associated with decreased β-globin production leading to anemia, Ineffective Erythropoiesis, and iron overload. New mechanisms associated with modulation of Erythropoiesis and iron metabolism have recently been discovered in thalassemic mice, improving our understanding of the pathophysiology of this disease. These discoveries have the potential to be translated into clinically-relevant therapeutic options to reduce Ineffective Erythropoiesis and iron overload. A new generation of therapies based on limiting Ineffective Erythropoiesis, iron absorption, and the correction of iron maldistribution could be on the way, possibly complementing and improving the current standard of patient care.
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thalassemia hijaking Ineffective Erythropoiesis and iron overload
Advances in Hematology, 2010Co-Authors: Luca Melchiori, Sara Gardenghi, Stefano RivellaAbstract:β-thalassemia encompasses a group of monogenic diseases that have in common defective synthesis of β-globin. The defects involved are extremely heterogeneous and give rise to a large phenotypic spectrum, with patients that are almost asymptomatic to cases in which regular blood transfusions are required to sustain life. As a result of the inefficient synthesis of β-globin, the patients suffer from chronic anemia due to a process called Ineffective Erythropoiesis (IE). The sequelae of IE lead to extramedullary hematopoiesis (EMH) with massive splenomegaly and dramatic iron overload, which in turn is responsible for many of the secondary pathologies observed in thalassemic patients. The processes are intimately linked such that an ideal therapeutic approach should address all of the complications. Although β-thalassemia is one of the first monogenic diseases to be described and represents a global health problem, only recently has the scientific community started to focus on the real molecular mechanisms that underlie this disease, opening new and exciting therapeutic perspectives for thalassemic patients worldwide.
Michael Dussiot - One of the best experts on this subject based on the ideXlab platform.
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fetal hemoglobin rescues Ineffective Erythropoiesis in sickle cell disease
Blood, 2020Co-Authors: Michael Dussiot, Hongxia Yan, Sara El Hoss, Sylvie Cochet, Auria Godard, Giacomo FratiAbstract:Sickle cell disease (SCD) is an autosomal hereditary recessive disorder caused by a point mutation in the β globin gene resulting in a Glu-to-Val substitution at the 6th position of the β globin protein. The resulting abnormal hemoglobin (HbS) polymerizes under hypoxic conditions driving red blood cell (RBC) sickling (Pauling et al., 1949). While pathobiology of circulating RBCs has been extensively analyzed in SCD, Erythropoiesis is surprisingly poorly documented. In β-thalassemia, Ineffective Erythropoiesis is characterized by high levels of apoptotic erythroblasts during the late stages of terminal differentiation, due to an accumulation of free β-globin chains (Arlet et al., 2016). Ineffective Erythropoiesis is the major cause of anemia in β-thalassemia patients. In contrast, a marked decrease in life span of circulating red cells, a feature of sickle red cells, is considered to be the major determinant of chronic anemia in SCD. It is generally surmised that Ineffective Erythropoiesis contributes little to anemia. The bone marrow environment has been well documented to be hypoxic (0.1 to 6% O2) (Mantel et al., 2015). As hypoxia induces HbS polymerization, we hypothesized that cell death may occur in vivo because of HbS polymer formation in the late stages of differentiation characterized by high intracellular hemoglobin concentration. In the present study, using both in vitro and in vivo derived human erythroblasts we assessed the extent of Ineffective Erythropoiesis in SCD. We explored the mechanistic basis of the Ineffective Erythropoiesis in SCD using biochemical, cellular and imaging techniques. In vitro erythroid differentiation using CD34+ cells isolated from SCD patients and from healthy donors was performed. A 2-phase erythroid differentiation protocol was used and cultures were performed at two different oxygen conditions, i.e. normoxia and partial hypoxia (5% O2). We found that hypoxia induces cell death of sickle erythroblasts starting at the polychromatic stage, positively selecting cells with high levels of fetal hemoglobin (HbF). This inference was supported by flow cytometry data showing higher percentages of dead cells within the non-F-cell population as compared to the F-cell population for SCD cells. Moreover, SCD dead cells showed higher levels of chaperon protein HSP70 in the cytoplasm than live cells, while no difference was detected between both subpopulations for control cells, suggesting that cell death of SCD erythroblasts was probably due to HSP70 cytoplasmic sequestration. This was supported by western-blot experiments showing less HSP70 in the nucleus of SCD erythroblasts under hypoxia, associated with decreased levels of GATA-1. At the molecular level, HSP70 was co-immunoprecipitated with HbS under hypoxia indicating that both proteins were in the same complex and suggesting interaction between HSP70 and HbS polymers in the cyotplasm. Importantly, we confirm these results in vivo by showing that in bone marrow of SCD patients (n = 5) cell loss occurs during terminal erythroid differentiation, with a significant drop in the cell count between the polychromatic and the orthochromatic stages (Figure 1). In order to specifically address the role of HbF in cell survival, we used a CRISPR-Cas9 approach to mimic the effect of hereditary persistence of fetal hemoglobin (HPFH). CD34+ cells were transfected either with a gRNA targeting the LRF binding site (-197) or a gRNA targeting an unrelated locus (AAVS1) (Weber, Frati, et al. 2020). As expected, the disruption of the LRF binding site resulted in HbF induction as shown by higher %F-cells compared to AAVS1 control. These higher levels of F-cells resulted in decreased apoptosis, under both normoxic and hypoxic conditions, clearly demonstrating the positive and selective effect of HbF on SCD cell survival (Figure 2). In summary, our study shows that HbF has a dual beneficial effect in SCD by conferring a preferential survival of F-cells in the circulation and by decreasing Ineffective Erythropoiesis. These findings thus bring new insights into the role of HbF in modulating clinical severity of anemia in SCD by both regulating red cell production and red cell destruction. Download : Download high-res image (734KB) Download : Download full-size image Disclosures No relevant conflicts of interest to declare.
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fetal hemoglobin rescues Ineffective Erythropoiesis in sickle cell disease
Haematologica, 2020Co-Authors: Sara El Hoss, Michael Dussiot, Hongxia Yan, Sylvie Cochet, Auria Godard, Giacomo Frati, Benedicte Boutonnatfaucher, Sandrine Laurance, Olivier Renaud, Laure JosephAbstract:While Ineffective Erythropoiesis has long been recognized as a key contributor to anemia in thalassemia, its role in anemia of sickle cell disease (SCD) has not been critically explored. Using in vitro and in vivo derived human erythroblasts we assessed the extent of Ineffective Erythropoiesis in SCD. Modeling the bone marrow hypoxic environment, we found that hypoxia induces death of sickle erythroblasts starting at the polychromatic stage, positively selecting cells with high levels of fetal hemoglobin (HbF). Cell death was associated with cytoplasmic sequestration of heat shock protein 70 and was rescued by induction of HbF synthesis. Importantly, we document that in bone marrow of SCD patients similar cell loss occurs during the final stages of terminal differentiation. Our study provides evidence for Ineffective Erythropoiesis in SCD and highlights an anti-apoptotic role for HbF during the terminal stages of erythroid differentiation. These findings imply that the beneficial effect on anemia of increased HbF levels is not only due to the increased life span of red cells but also a consequence of decreased Ineffective Erythropoiesis.
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XPO1 (Exportin-1) Is a Major Regulator of Human Erythroid Differentiation. Potential Clinical Applications to Decrease Ineffective Erythropoiesis of Beta-Thalassemia
Blood, 2015Co-Authors: Flavia Guillem, Jean-benoît Arlet, Michael Dussiot, Sebastien Causse, Guillaume Marcion, Emilie-fleur Gautier, Julien Rossignol, Mathilde Lamarque, Patrick Mayeux, Frédérique VerdierAbstract:Background We and others have shown that normal human erythroid cell maturation requires a transient activation of caspase-3 at late stages of maturation (Zermati et al, J Exp Med 2001). We further documented that, in human erythroblasts, the chaperone HSP70 is constitutively expressed and, at late stages of maturation, translocates into the nucleus and protects GATA-1, the master transcriptional factor critical for Erythropoiesis, from caspase-3 cleavage (Ribeil et al, Nature 2007). During the maturation of human β-TM erythroblasts, HSP70 is sequestrated by excess of α-globin chains in the cytoplasm and as a consequence, GATA-1 is no longer protected from caspase-3 cleavage resulting in end-stage maturation arrest and apoptosis (Arlet et al, Nature 2013). Understanding the molecular mechanisms that regulate the localization of HSP70 during erythroid differentiation may help to find new therapeutic targets to reduce Ineffective Erythropoiesis in beta-thalassemia. Methods CD34 positive cells from normal and thalassemic peripheral blood were cultured in IMDM/BIT media in the presence of SCF, IL3, IL6 for seven days and subsequently cultured for additional 7 to 9 days in media containing SCF, IL3 and Epo. Erythroblasts differentiation, HSP70 localization were analysed by FACS, AMNIS stream, confocal microscopy and western blot analysis. RNAseq and proteomic analysis of highly purified erythroid cells at all distinct stages of differentiation were used to assess expression levels of various exportins. Duolink and Octet analyses were used to assess protein proximity and affinity of interactions, respectively. Results During erythroid differentiation, Hikeshi, the cognate nuclear importin of HSP70, is constitutively expressed and enables HSP70 nucleus entry as assessed by siRNA experiments. However, its expression was not regulated during erythroid differentiation. In contrast, exportin expression analysis showed marked differences in expression levels of XPO1 and XPO7 during erythroid differentiation. XPO1 expression being reduced at the time of c-kit down-regulation and caspase 3 activation while there was a marked increase in XPO7 expression at the late stages of terminal erythroid differentiation. XPO1 interacted in vivo (Duolink analysis) and in vitro with HSP70 (Octet analysis). Likewise, the previously described HSP70 S400A mutant (in the Leucine-rich Nuclear Export Sequence), which is constitutively located in the nucleus interacted with XPO1 with lower affinity compared to HSP70 WT. Stem Cell Factor (SCF) starvation and Pi3k inhibition led to decreased in vivo HSP70/XPO1 interactions. However, neither phosphorylation of HSP70 nor XPO1 were detected by Nanopro and proteomic analysis, and XPO1 expression was not regulated by Pi3K pathway. Expression of RanGTP Activating Protein (RanGAP), a protein critical for XPO1/cargo interaction, was down-regulated at the moment of caspase 3 activation during erythroid maturation, which may explain the decrease in HSP70/XPO-1 interactions. Inhibitors of XPO1 (leptomycin B and KPT 251) were able to induce HSP70 nuclear localization at early stages of differentiation (proE). In erythroid progenitors from β-TM patients, treatment with the Selective Inhibitor of Nuclear Export compound KPT-251 rescued nuclear HSP70 localization and GATA1 expression, and resulted in improved of erythroid terminal differentiation, without cytotoxicity, of thalassemic erythroid progenitors. Conclusion XPO1 is a major regulator of Erythropoiesis through the regulation of HSP70 nuclear localization and is a potential new target to decrease Ineffective Erythropoiesis of thalassemia. Specific XPO-1 inhibitors currently in clinical development are being tested for potential therapy in thalassemic erythroid progenitors. Disclosures No relevant conflicts of interest to declare.
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exogenous apo transferrin increases monoferric transferrin decreasing cytosolic iron uptake and heme and globin synthesis in β thalassemic mice
Blood, 2014Co-Authors: Tenzin Choesang, Michael Dussiot, Laura Breda, Maria Feola, Huiyong Chen, Thiago Trovati Maciel, Weili Bao, Petra Pham, Daniel Garcia Santos, Antonia FollenziAbstract:β-thalassemia is an inherited blood disorder caused by reduced or absence of β-globin expression which results in imbalanced globin synthesis, Ineffective Erythropoiesis, and anemia. How the imbalance between α- and β-globin results in Ineffective Erythropoiesis, if apoptosis or dysfunctional differentiation of erythroid precursors results in Ineffective Erythropoiesis, and whether disrupted iron regulation and / or iron overload in β-thalassemia is directly involved in the pathophysiology of Ineffective Erythropoiesis is incompletely understood. Iron is critical for hemoglobin synthesis and Erythropoiesis is dependent on transferrin (Tf) bound iron. Tf functions as the main iron transporter in circulation, where it exists in three forms: as iron-free apo-transferrin (apoTf), monoferric Tf, or diferric Tf (holoTf). Typically, iron is bound to 30% of all Tf binding sites in circulation at which point monoferric Tf is found in the highest concentration relative to holoTf. . We have previously shown that exogenous apoTf ameliorates anemia in a mouse model of β-thalassemia intermedia (th1/th1), resulting in reduced splenomegaly, reticulocytosis, and α-globin precipitation on circulatory red blood cells (RBC). We also observe a decrease in mean corpuscular hemoglobin (MCH) and mean corpuscular volume (MCV), serum iron, Tf saturation, together suggesting that relative iron deficiency improves iron metabolism and Ineffective Erythropoiesis in apoTf-treated th1/th1 mice. We hypothesize that exogenous apoTf decreases cytosolic iron and heme as a consequence of increased monoferric Tf which results in less iron entering cells via Tf:TfR1 binding. Our current data reveals that in vitro incubation of purified apoTf and holoTf at 37C results in the formation of monoferric Tf, and injection of wild-type (WT) mice with a single intraperitoneal dose of apoTf (10mg) decreases holoTf (P=0.01) and increases monoferric Tf (P=0.02) in the serum 6 hours after injection. Using both calcium mobilization and anti-Tf antibodies in flow cytometry, we demonstrate that apoTf results in no TfR1 binding relative to holoTf in CHO cells (P Disclosures No relevant conflicts of interest to declare.
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hsp70 sequestration by free α globin promotes Ineffective Erythropoiesis in β thalassaemia
Nature, 2014Co-Authors: Jean-benoît Arlet, Olivier Negre, Jean-antoine Ribeil, Michael Dussiot, Ivan C. Moura, Flavia Guillem, Guillaume Marcion, Yves Beuzard, Adonis Hazoume, Samuel DemarestAbstract:β-Thalassaemia major (β-TM) is an inherited haemoglobinopathy caused by a quantitative defect in the synthesis of β-globin chains of haemoglobin, leading to the accumulation of free α-globin chains that form toxic aggregates. Despite extensive knowledge of the molecular defects causing β-TM, little is known of the mechanisms responsible for the Ineffective Erythropoiesis observed in the condition, which is characterized by accelerated erythroid differentiation, maturation arrest and apoptosis at the polychromatophilic stage. We have previously demonstrated that normal human erythroid maturation requires a transient activation of caspase-3 at the later stages of maturation. Although erythroid transcription factor GATA-1, the master transcriptional factor of Erythropoiesis, is a caspase-3 target, it is not cleaved during erythroid differentiation. We have shown that, in human erythroblasts, the chaperone heat shock protein70 (HSP70) is constitutively expressed and, at later stages of maturation, translocates into the nucleus and protects GATA-1 from caspase-3 cleavage. The primary role of this ubiquitous chaperone is to participate in the refolding of proteins denatured by cytoplasmic stress, thus preventing their aggregation. Here we show in vitro that during the maturation of human β-TM erythroblasts, HSP70 interacts directly with free α-globin chains. As a consequence, HSP70 is sequestrated in the cytoplasm and GATA-1 is no longer protected, resulting in end-stage maturation arrest and apoptosis. Transduction of a nuclear-targeted HSP70 mutant or a caspase-3-uncleavable GATA-1 mutant restores terminal maturation of β-TM erythroblasts, which may provide a rationale for new targeted therapies of β-TM
Yelena Ginzburg - One of the best experts on this subject based on the ideXlab platform.
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minihepcidins improve Ineffective Erythropoiesis and splenomegaly in a new mouse model of adult β thalassemia major
Haematologica, 2020Co-Authors: Carla Casu, Yelena Ginzburg, Ritama Gupta, Roberta Chessa, Alison Liu, Hal Drakesmith, Robert E Fleming, Brian Macdonald, Stefano RivellaAbstract:Minihepcidins are hepcidin agonists that have been previously shown to reverse iron overload and improve Erythropoiesis in mice affected by non-transfusion-dependent thalassemia. Given the extreme anemia that occurred with the previous model of transfusion-dependent thalassemia, that model was inadequate for investigating whether minihepcidins can improve red blood cell quality, lifespan and Ineffective Erythropoiesis. To overcome this limitation, we generated a new murine model of transfusion-dependent thalassemia with severe anemia and splenomegaly, but sufficient red cells and hemoglobin production to test the effect of minihepcidins. Furthermore, this new model demonstrates cardiac iron overload for the first time. In the absence of transfusions, minihepcidins improved red blood cell morphology and lifespan as well as Ineffective Erythropoiesis. Administration of a minihepcidin in combination with chronic red blood cell transfusion further improved the Ineffective Erythropoiesis and splenomegaly and reversed cardiac iron overload. These studies indicate that drugs such as minihepcidins have therapeutic potential for patients with transfusion-dependent thalassemia.
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additive effects of decreased tfr1 and ablated erfe improve both Ineffective Erythropoiesis and iron overload in β thalassemic mice
Blood, 2018Co-Authors: Marc Ruiz Martinez, Tomas Ganz, Maria Feola, Yelena GinzburgAbstract:Patients with β-thalassemias manifest anemia, Ineffective Erythropoiesis, extramedullary hematopoiesis, splenomegaly, and systemic iron overload. Even in non-transfusion dependent patients, iron overload in β-thalassemia develops because of increased intestinal iron absorption, leading to multiple organ dysfunction if untreated and accounts for most of the deaths in this disease. The main regulator of body iron content and distribution is hepcidin, inhibiting iron absorption in duodenal enterocytes and release of stored iron from macrophages and hepatocytes. Despite iron overload in patients and mice with β-thalassemia, hepcidin levels are insufficiently increased, as Ineffective Erythropoiesis dominates hepcidin regulation. Relatively low hepcidin causes iron overload in β-thalassemia. Recent evidence demonstrates that erythroferrone (ERFE), an erythroid regulator of hepcidin, is increased in bone marrow and serum from β-thalassemic patients and th3/+ mice [Kautz Nat Gen 2014] and its loss results in increased hepcidin, partially reversing iron overload in th3/+ mice [Kautz Blood 2015]. In addition, bone marrow ERFE expression normalizes in TfR1 haploinsufficient th3/+ mice [Li Blood 2017]. We hypothesize that the loss of ERFE and TfR1 influences Erythropoiesis and iron metabolism in complementary ways in th3/+ mice, and therefore aim to explore iron- and Erythropoiesis-related parameters in th3/+ TfR1+/- ERFE-/- (triple mutant (TM)) mice. All models are on a C57BL6 background and have been crossed to generate 4-6 mice for analysis at 6 weeks of age. We confirm our previous reports [Li Blood 2017] that th3/+TfR1+/- mice have increased RBC count and hemoglobin, decreased MCV and reticulocyte count (Table I), and reduce splenomegaly (Fig 1a and 1b) relative to th3/+ mice. We also confirm that th3/+ ERFE-/- mice do not reverse splenomegaly or improve peripheral blood circulating erythroid parameters compared to th3/+ mice [Kautz Blood 2015] (Table I) but exhibit further increase in TfR1 in late stage erythroid precursors (Fig 1c). Analysis of the bone marrow reveals that total erythroid mass is unaltered in triple mutants relative to th3/+, th3/+ ERFE-/-, and th3/+ TfR1+/- mice, but the number of late erythroblasts (poly-E and ortho-E stages) is normalized to WT levels (Fig 1d), strongly suggesting that, unlike in th3/+ Erythropoiesis, where the block in differentiation occurs at the poly-E stage, th3/+ TfR1+/- and especially triple mutant mice restore differentiation at this stage to generate a higher hemoglobin. No differences in erythroblast apoptosis or ROS concentration are evident in triple mutant relative to th3/+ ERFE-/- or th3/+ TfR1+/- mice. We also analyzed markers of Epo responsiveness and demonstrate that serum Epo and EpoR expression are increased in th3/+ relative to WT mice (Fig 1e and 1f), but while serum Epo is decreased, EpoR is further increased (Fig 1f). These findings suggest that Epo responsiveness is more optimized in triple mutant erythroblasts, enabling a smaller proportion of late stage erythroblasts to produce circulating RBCs with relatively less serum Epo. Remarkably, while neither th3/+ ERFE-/- and th3/+ TfR1+/- mice reverse iron overload or impact hepcidin expression at 6 weeks of age, triple mutant mice demonstrate fully normalized ratio of hepcidin expression relative to liver iron concentration (LIC) (Fig 1g). Taken together, these experiments provide evidence of the differential and additive effects of TfR1 and ERFE loss in th3/+ mice, with a predominantly erythropoietic benefit of TfR1 loss, a predominantly iron-homeostatic benefit of ERFE loss, and synergy of both in optimizing Epo responsiveness. Disclosures Ganz:Intrinsic LifeScience: Consultancy, Equity Ownership, Membership on an entity9s Board of Directors or advisory committees; Silarus Pharma: Consultancy, Equity Ownership; Keryx Pharma: Consultancy, Research Funding; Gilead: Consultancy; Ablynx: Consultancy; Vifor: Consultancy; Akebia: Consultancy, Research Funding; La Jolla Pharma: Consultancy, Patents & Royalties: Patent licensed to La Jolla Pharma by UCLA.
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role of activated pleckstrin 2 and down stream effects on Ineffective Erythropoiesis in β thalassemic mice
Blood, 2016Co-Authors: Maria Feola, Andrea Zamperone, Tenzin Choesang, Weili Bao, Antonia Follenzi, Shilpa M Hattangadi, Christopher E Mason, Yelena GinzburgAbstract:Erythropoiesis involves stem cell differentiation to mature red blood cells (RBCs). Erythropoietin (Epo) is essential for erythroipoiesis, and Epo binding to Epo receptor triggers a complicated and incompletely understood set of potentially related molecular signals influencing cell survival, differentiation, and enucleation. Although Epo is associated with increased survival of erythroid precursors, it induces reactive oxygen species (ROS), and high Epo concentration has an anti-enucleation effect in vitro.i Furthermore, diseases of Ineffective Erythropoiesis, e.g. β-thalassemia, are associated with increased Epo and ROS concentrations implicated in the expansion of and damage to erythroid precursors, respectively. Treating erythroblasts with low dose ROS scavenger promotes enucleation, but high dose ROS scavenger leads to cell death,i suggesting that an optimal ROS concentration is integral to effective Erythropoiesis. We and others have shown that ROS is increased in β-thalassemic erythroid precursors, but despite increased ROS, erythroid precursor apoptosis is not increased. We hypothesize that compensatory mechanisms prevent the ill-effects of increased ROS on erythroid precursors. Our prior experiments demonstrate disordered Erythropoiesis in β-thalassemic (th1/th1) mice, restored in transferrin-treated th1/th1 mice,ii despite which, ROS remained increased in erythroid precursor from transferrin-treated th1/th1 mice. To identify mechanisms responsible for transferrin's effect, we performed RNA seq analysis of erythroblasts from wild type (WT), th1/th1, and transferrin-treated th1/th1 mice. We identified increased pleckstrin-2 (plek2) in th1/th1 relative to WT mice, normalized in transferrin-treated th1/th1 mice. We hypothesize that plek2 activation counteracts the ill effects of ROS and promotes enucleation in β-thalassemia. Using confocal microscopy, we demonstrate that 1) plek2 co-localizes with actin on the cell membrane after the pro-erythroblast stage but is in the nucleus throughout terminal Erythropoiesis in WT mice; 2) membrane-associated plek2 is present earlier, in pro-erythroblasts, and remains membrane-associated until orthochromatophilic stage in th1/th1 mice; and 3) plek2 localization is normalized in transferrin-treated th1/th1 mice. Because plek2 activation leads to its association with the cell membrane and plek2 activation is increased in th1/th1 erythroblasts, we set out to explore the role of plek2 activation on Ineffective Erythropoiesis in transferrin-treated th1/th1 mice. Prior publications propose that plek2 interacts with and results in the phosphorylation of cofilin, preventing cofilin's translocation to the mitochondria as part of the apoptosis pathway in response to increased ROS.iii We demonstrate decreased in mitochondria cofilin localization and increased cellular p-cofilin in th1/th1 erythroblasts, normalized after transferrin treatment. These data suggest that despite an increase in ROS, plek2 and its induction of p-cofilin inhibit apoptosis in β-thalassemic erythroid precursors. Furthermore, in light of a prior report of an anti-enucleation effect of plek2 in vitroiii and the known regulation of enucleation by RacGTPasesiv, we hypothesize that plek2 activation triggers RacGTPase and prevents enucleation in th1/th1 mice. We demonstrate that in addition to changes in erythroblast RacGTPase concentration, membrane co-localization between plek2 and RacGTPase is enhanced and occurs earlier in th1/th1 erythroid differentiation relative to WT, normalized after transferrin treatment. Lastly, cleavage of Rho-associated kinase, Rock1, associated with enucleation,v is also decreased in th1/th1 erythroblasts, enhanced after transferrin treatment. Taken together, we speculate that plek2 haplo-insufficiency benefits β-thalassemic mice by enabling apoptosis of Ineffective erythroblasts, or result in worsening, possibly lethal, phenotype in light of the direct or indirect (through effects on RacGTPase or Rock1) role of plek2 in enucleation. Presently, we are testing this hypothesis by generating plek2+/- and plek-/- β-thalassemic mice. In conclusion, we demonstrate the important compensatory role of plek2 in β-thalassemic Erythropoiesis. i Zhao Exp Hematol 2016 ii Liu Blood 2013 iii Zhao haematol 2014 iv Ji Nat Cell Bio 2008 v Gabet Cell Death Diff 2011 Disclosures No relevant conflicts of interest to declare.
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β thalassemia a model for elucidating the dynamic regulation of Ineffective Erythropoiesis and iron metabolism
Blood, 2011Co-Authors: Yelena Ginzburg, Stefano RivellaAbstract:β-thalassemia is a disease characterized by anemia and is associated with Ineffective Erythropoiesis and iron dysregulation resulting in iron overload. The peptide hormone hepcidin regulates iron metabolism, and insufficient hepcidin synthesis is responsible for iron overload in minimally transfused patients with this disease. Understanding the crosstalk between Erythropoiesis and iron metabolism is an area of active investigation in which patients with and models of β-thalassemia have provided significant insight. The dependence of Erythropoiesis on iron presupposes that iron demand for hemoglobin synthesis is involved in the regulation of iron metabolism. Major advances have been made in understanding iron availability for Erythropoiesis and its dysregulation in β-thalassemia. In this review, we describe the clinical characteristics and current therapeutic standard in β-thalassemia, explore the definition of Ineffective Erythropoiesis, and discuss its role in hepcidin regulation. In preclinical experiments using interventions such as transferrin, hepcidin agonists, and JAK2 inhibitors, we provide evidence of potential new treatment alternatives that elucidate mechanisms by which expanded or Ineffective Erythropoiesis may regulate iron supply, distribution, and utilization in diseases such as β-thalassemia.
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apo transferrin injections reduce transferrin receptor 1 concentration on erythroid precursors and reverse Ineffective Erythropoiesis in splenectomized beta thalassemic mice
Blood, 2010Co-Authors: Jing Liu, William Breuer, Jianhua Zhang, Maya Shvartsman, Charles B Hall, Leni Vonbonsdorff, Zvi Ioav Cabantchik, Mohandas Narla, Yelena GinzburgAbstract:Abstract 4283 Beta-thalassemia results from a mutation in the beta-globin gene which leads to Ineffective Erythropoiesis (IE), shortened red blood cell (RBC) survival, and anemia requiring varying degrees of transfusion-dependence. Our previous data demonstrate that thalassemic (Hbb th1/th1 ) mice treated with apo-transferrin have more circulating RBCs, each with lower mean corpuscular hemoglobin (MCH), increased hemoglobin, and reversal of splenomegaly. These results suggest that anemia in beta-thalassemia is a consequence of excess intracellular heme in developing erythroblasts. Because iron in erythroid precursors is predominantly used for heme synthesis and iron uptake is limited to transferrin-bound iron through transferrin receptor 1 (TfR1), we hypothesized that exogenous apo-transferrin injections reduce MCH by decreasing surface TfR1 expression. To clarify the direct effect of splenic Erythropoiesis in this experimental system, we analyzed splenectomized thalassemic mice for the effect of apo-transferrin injections on erythroid precursor differentiation and expression of TfR1 in the bone marrow as well as all RBC parameters, serum erythropoietin levels, and degree of extramedullary Erythropoiesis (EMH) in the liver. We used intraperitoneal human apo-transferrin injection, 10 mg daily for 20 days. Apo-transferrin treated splenectomized thalassemic mice were compared with age and gender matched untreated splenectomized mice. A novel flow cytometry analysis using CD44 and TER119 in the bone marrow allowed us to measure the expression levels (using mean fluorescence intensity) of TfR1 at different stages of erythroid differentiation. Splenectomy in thalassemic mice resulted in decreased reticulocyte count ( P P P P =0.003, 0.01, 0.02, and 0.003 for proerythroblasts, basophilic, polychromatophilic, and orthochromatophilic erythroblasts, respectively) relative to age and gender matched non-splenectomized thalassemic mice. Despite this deterioration, apo-transferrin injections increased RBC counts ( P P P P P =0.0002) to levels similar to non-splenectomized apo-transferrin treated thalassemic mice. Additionally, serum erythropoietin concentration decreased ( P =0.009), RBC survival significantly improved (t½ = 33.5 vs. 10.6 days, P P =0.0007, 0.007, 0.01, and 0.004 for proerythroblasts, basophilic, polychromatophilic, and orthochromatophilic erythroblasts, respectively) compared with untreated splenectomized thalassemic mice. During a three month follow up after splenectomy, apo-transferrin injections decreased mortality in thalassemic mice (likely by reversal of severe anemia; hemoglobin of 11.3 vs. 7.5 g/dL in treated and untreated splenectomized thalassemic mice, respectively). In summary, exogenous apo-transferrin increases the efficiency of Erythropoiesis in splenectomized thalassemic mice as previously documented in non-splenectomized thalassemic mice, relieving their dependence on EMH in the liver and spleen. Decreased iron uptake in erythroid precursors of apo-transferrin treated splenectomized thalassemic mice is associated with a reduction in the amount of TfR1 on all stages of erythroid precursor development and likely explains the reduction of MCH in apo-transferrin treated mice. Our findings demonstrate a novel feedback inhibition of TfR1 expression by exogenous apo-transferrin injection. Taken together, these results support the use of apo-transferrin to 1) delay or prevent the need for surgical splenectomy and 2) ameliorate anemia, IE, and EMH in splenectomized patients with beta-thalassemia. Disclosures: Cabantchik: Aferrix Ltd: Consultancy, Membership on an entity9s Board of Directors or advisory committees.