review article ineffective erythropoiesis in...

Hindawi Publishing CorporationThe Scientific World JournalVolume 2013 Article ID 394295 11 pageshttpdxdoiorg1011552013394295

Review ArticleIneffective Erythropoiesis in 120573-Thalassemia

Jean-Antoine Ribeil12345 Jean-Benoit Arlet13456 Michael Dussiot13457

Ivan Cruz Moura3457 Geneviegraveve Courtois1345 and Olivier Hermine13458

1 Centre National de la Recherche Scientifique-Unite Mixte de Recherche 8147 Universite Paris V Rene DescartesHopital Necker Paris France

2 Departement de Biotherapie Faculte de Medecine Paris Descartes Sorbonne Paris-Cite et Assistance PubliquemdashHopitaux de ParisHopital Necker Paris France

3 Fondation Imagine Institut des Maladies Genetiques Faculte de Medecine Paris DescartesSorbonne Paris-Cite et Assistance PubliquemdashHopitaux de Paris Hopital Necker Paris France

4 Laboratoire drsquoExcellence des Globules Rouges (GR-ex) Paris France5 Fondation Imagine Universite Paris Descartes-Sorbonne Paris Cite Paris France6 Service de Medecine Interne Faculte de Medecine Paris DescartesSorbonne Paris-Cite et Assistance PubliquemdashHopitaux de Paris Hopital Europeen Georges Pompidou Paris France

7 INSERM U 699 Hopital Bichat Universite Paris Diderot Paris France8 Service drsquoHematologie Faculte de Medecine Paris DescartesSorbonne Paris-Cite et Assistance PubliquemdashHopitaux de Paris Hopital Necker Paris France

Correspondence should be addressed to Jean-Antoine Ribeil jean-antoineribeilnckaphpfr

Received 28 December 2012 Accepted 3 February 2013

Academic Editors Y Al-Tonbary M A Badr A El-Beshlawy and F Tricta

Copyright copy 2013 Jean-Antoine Ribeil et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

In humans 120573-thalassemia dyserythropoiesis is characterized by expansion of early erythroid precursors and erythroid progenitorsand then ineffective erythropoiesis This ineffective erythropoiesis is defined as a suboptimal production of mature erythrocytesoriginating from a proliferating pool of immature erythroblasts It is characterized by (1) accelerated erythroid differentiation(2) maturation blockade at the polychromatophilic stage and (3) death of erythroid precursors Despite extensive knowledge ofmolecular defects causing 120573-thalassemia less is known about the mechanisms responsible for ineffective erythropoiesis In thispaper we will focus on the underlying mechanisms leading to premature death of thalassemic erythroid precursors in the bonemarrow

1 Introduction

Normal human adult hemoglobin (Hb) A (HbA) consistsof two pairs of globin chains 120572

21205732 of which synthesis

is normally tightly coordinated to ensure equal produc-tion 120573-thalassemia one of the most common inheritedhemoglobinopathy in the world is due to autosomal muta-tions in the gene encoding 120573-globin which induce an absenceor low-level synthesis of this protein in erythropoietic cells[1] The consequence of these mutations is an imbalance of120572120573-globin chain synthesis mostly evident in the homozy-gous forms leading to the accumulation of free 120572-globinchains forming highly toxic aggregates [2] Thalassemic

patients suffer from anemia resulting from shortened redblood cell (RBC) survival by hemolysis and erythroidprecursors premature death in bone marrow (ineffectiveerythropoiesis)

The first description of thalassemia was reported by DrThomas Cooley in 1925 There are a multiplicity of differentgenetic mutations in 120573-thalassemia that give rise to a clin-ically heterogeneous spectrum ranging from asymptomaticexpression (thalassemia minor) and mild clinical anemia(thalassemia intermedia) to classical fatal Cooleyrsquos anemiaThe term ldquoCooleyrsquos anemiardquo now termed 1205730-thalassemiamajor (TM) has been used synonymously with clinicallysevere forms of 120573-thalassemia characterized by a very high

2 The Scientific World Journal

120572non-120572 chains ratio severe ineffective erythropoiesis anddependence on RBC transfusions to sustain live Regulartransfusions (average every month) expose these patients toiron overload and its live threatening systemic consequenceswhich require iron chelation [1]

It is well established that the 120572non-120572 ratio correlates withthe severity of disease [1 3] However genotypic variabilityimpairing globin chain synthesis at known loci is often insuf-ficient to explain the heterogeneity in clinical phenotypes ofindividual patients with the same genotype suggesting thatother genetic modulations might exist [1 4] The molecularmechanisms underlying the heterogeneity and occasionalseverity of the syndrome remain obscure and are not theobject of this paper

The pathophysiology of 120573-thalassemia has been the sub-ject of several extensive reviews particularly on its molecularand genetics basis [5] In this paper we will focus on themechanisms leading to end-stage maturation blockade andthalassemic erythroid precursors premature destruction inthe bone marrow Their understanding will probably be thekey for developing novel therapeutic approaches improvinganemia in 120573-thalassemia In order to describe mechanismsunderlying ineffective erythropoiesis we will first summarizethe current knowledge on normal hemoglobin synthesis andnormal erythropoiesis

2 Hemoglobin Synthesis

Two distinct globin chains 120572 and 120573 (each carrying an indi-vidual heme molecule) interact to form hemoglobin dimers120572120573 and two dimers combine to form a hemoglobin tetramer12057221205732 the functional form of hemoglobin carrying oxygenExcepted the very first weeks of embryogenesis in which

zeta chains are produced one of the globin chains is 120572 and thesecond chain is called ldquonon-120572rdquo The main (98) hemoglobintype in the normal human adult consists of two 120572 and two120573 chains (120572

21205732HbA) the minor type (2) consists of 2120572

and 2120575 chains (12057221205752HbA2) Usually globin chains synthesis

is relatively balanced even if some studies report a slightexcess of 120572 chains as a soluble pool [3 6 7] In contrast fetalerythrocytes contain another type of hemoglobin consistingof 2120572 and 2120574 chains (120572

21205742HbF) which can attract more

oxygen effectively from the maternal bloodGlobin chains originating from a common ancestral type

display a varying degree of homology Two clusters of globinsgenes are known the first one on chromosome 16 for 120572-likegenes (two 120572-globin genes 120572

1and 120572

2 and two zeta genes)

and the second one on chromosome 11 for 120573-like genes The51015840 to 31015840 order of 120573-like globin genes on chromosome 11 (120576- G120574-A120574-120575-120573 two 120574 genes) reflects their sequential activation andsilencing during the transition from embryonic to fetal andfrom fetal to adult ldquohemoglobinopoiesisrdquo called ldquohemoglobinontogenyrdquo or ldquohemoglobin chain switchrdquo

3 Erythropoiesis

Erythropoiesis is defined as the pathway producing matureRBC from hematopoietic stem cells This process includes

several steps restricting differentiation and proliferation ofcells which undergo this erythroid program depending onsequential and specific erythroid gene expression Erythro-poiesis is regulated by combined effects of microenviron-ment and growth factors that promote survival prolifer-ation andor differentiation of erythroid progenitors andnuclear factors that regulate transcription of genes involvedin survival and establishment of the erythroid phenotypeRBC production is orchestrated by a complex network oftranscription factors among which GATA-1 the master geneof erythropoiesis positively regulates specific erythroid genessuch as erythropoietin receptor (EpoR) glycophorin (GpA)and globin chains Moreover together with the transcriptionfactor STAT5 (activated through EpoR activation by erythro-poietin (Epo)) GATA-1 induces the expression of the anti-apoptotic protein Bcl-xL [8]

Committed erythroid progenitors differentiate into thefirst morphologically identified cell of the erythrocyte lin-eage the proerythroblast Next steps of erythroid differentia-tion are accompanied by temporally regulated changes in cellsurface protein expression reduction in cell size progressivehemoglobinization and nuclear condensation (successivelycalled basophilic erythroblast polychromatophilic erythrob-last and acidophilic erythroblast the last nucleated cell of themammalian erythrocyte lineage) which culminate in reticu-locytes cells by nucleus RNA andmitochondria extrusion Inaddition erythroidmaturation requires a transient activationof caspase-3 at the basophilic stage and translocation into thenucleus of the inducible heat shock protein 70 (Hsp70) toprotect GATA-1 from caspase-3 cleavage [9 10]

This process occurs within the erythroblastic island inwhich a macrophage is surrounded by erythroblasts at allstages of maturation [11 12] The production of erythrocytesis the largest quantitative output of the hematopoietic systemwith estimated production rates of 2 times 1011 erythrocytes perday The program of erythroid proliferation and differentia-tion must be positively and negatively regulated to ensure acontinuous but tightly controlled production of RBC

31 Positive Regulation of Erythropoiesis Erythropoiesis iscontrolled by the combined effect of two major cytokinesstem cell factor (SCF) and Epo SCF induces proliferationand survival and slows down differentiation of early ery-throid progenitors and precursors towards the basophilicerythroblast stage Epo is responsible of the finely tunedhomeostatic control of erythrocyte numbers by tissue oxy-genation Interaction of Epo with the EpoR induces throughJAK2 activation multiple signalling pathways involving PI3kinase Akt and STAT5 which prevent apoptosis supportingerythroid progenitors proliferation and allowing erythroidprogram to occur [13ndash15]

32 Negative Regulation of Erythropoiesis by Apoptosis Thenegative regulation of erythropoiesis ismainly due to apopto-sis a fundamental cellular mechanism allowing clearance ofunneeded or potentially dangerous cells Apoptotic programsrequire the action of a family of cysteine-dependent andaspartate-specific proteases called caspases Two classes of

The Scientific World Journal 3

caspases are described initiators (caspase-8 and -9) andeffectors (caspase-3 and -7) [16 17] Caspase-8 is activatedby the death receptor pathway after cell surface receptor-ligand interaction [18] In contrast caspase-9 is activatedby events causing intracellular damages and alterations inmitochondrial membrane potential (ie the mitochondrialpathway) [19 20] Activated caspase-8 and caspase-9 thenactivate effectors such as caspase-3 that cleaves GATA-1 Tal-1 [21 22] and proteins involved in cytoplasm nucleus andDNA integrity which allow the cell death program to occur

33 Role of Cell Death Receptors and Epo Death receptorsof the TNF receptor (TNF-R) superfamilies (Fas-L TNF-120572TRAIL) activate the extrinsic apoptotic pathway Fas and Fas-L are expressed in cultured erythroblasts but controversiesregarding the level and differentiation stage at which theyare expressed have been reported Some studies suggest theexistence of a negative regulatory feedback operating at lowEpo level in a paracrine pathway In this system Fas-Lexpressing mature erythroblasts displays cytotoxicity againstimmature erythroblasts expressing Fas [23 24] Epo is able topartially protect immature erythroid cells from Fas-mediatedapoptosis Fas and Fas-L are therefore major regulatorsof erythropoiesis In addition FasFas-L interaction resultsthrough caspase-8 activation in GATA-1 cleavage whichblocks erythroid differentiation and maturation [25]

The control of mature RBC production may be summa-rized as follow at low doses of Epo cells die by apoptosisat intermediary doses cells are arrested in their differentia-tion and maturation (through GATA-1 cleavage) or enter aprogram of apoptosis depending on the number of matureerythroblasts in the bone marrow at high dose erythroidprogenitors and precursors pursue theirmaturation indepen-dently of the number of mature erythroid precursors

34 Role of Caspases and Hsp70 in Differentiation andMaturation of Erythroid Cells The terminal differentiationof erythroid cells exhibits some similarities with apoptosissuch as reduction in cell size chromatin condensation anddegradation of nuclear components A transient activationof caspases by the mitochondrial pathway has been shownby our group and others to be required for erythroid cellsdifferentiation but to not induce neither GATA-1 cleavagenor apoptosis We have more recently reported that Hsp70an ubiquitous chaperone constitutively expressed duringerythroid differentiation protects GATA-1 in the nucleusfrom caspase-3-mediated proteolysis during caspase activa-tion These results strongly indicate that Hsp70 is anotherkey erythroid antiapoptotic protein protecting GATA-1 fromcaspase-3-mediated cleavage and consequently allowing Bcl-xL expression [9 10]

4 120573-Thalassemia Ineffective Erythropoiesis

41 Evidences for an Ineffective Erythropoiesis in 120573-Tha-lassemia Dyserythropoiesis in 120573-thalassemic patients wassuspected for a long time since it is largely recognized thatmany patients with an inadequate transfusional regimen

have a dramatic expansion of the hematopoietic marrow andextramedullary hematopoiesis which can lead to extensivebone deformity andor bonemarrowmass and splenomegalyErythrokinetic assays done in the 50

1015840

s showed that the rateof peripheral RBC destruction in 120573-thalassemia was insuf-ficient to explain severe anemia [26 27] Then ferrokineticstudies done in the 70

1015840

s studying incorporation of 59Fe intonewly formed RBC suggested that probably 60ndash80 oferythroid progenitors were arrested in proliferation andorunderwent death [28]

The bone marrow of patients suffering from 120573-tha-lassemia contains five to six times the number of ery-throid precursors observed in healthy controls [29] withincreased basophilic and polychromatophilic erythroblastsand decreased orthochromatic erythroblasts [29ndash32] More-over it has been shown that 120573-thalassemic bone marrowerythroblasts contain electron-dense alpha-globin inclusion(aggregates) beginning at early polychromatophilic stageswhich increase in size and frequency during subsequentmaturation [33]

Taken together these results resume the findings of120573-thalassemia dyserythropoiesis in human expansion ofvery early erythroid precursors (proerythroblasts and ear-lier stages) and then ineffective erythropoiesis Ineffectiveerythropoiesis defines the suboptimal production of matureerythrocytes from a proliferating pool of immature erythrob-lasts It is thus characterized by (1) accelerated erythroiddifferentiation (2) maturation blockade at the polychro-matophilic stage and (3) death of erythroid precursors [2930 32 34 35] (Figure 1)

Although early erythroid progenitors expansion isbelieved to be due to a dramatic increased in Epo level as aresult of the anemic state feedback [36] other mechanismsyet not known might be involved In addition precisepathophysiological mechanisms of accelerated erythroiddifferentiation and maturation arrest are still unknownHowever mechanisms underlying cell death were morestudied and we intend at that point to review the differentplayers of this death ldquogamerdquo

42 Enhanced Apoptosis Is a Key Feature of Ineffective Ery-thropoiesis in Human 120573-Thalassemia It was evidenced in the90s that 120573-TM erythroid precursors but neither lymphoidand nor myeloid precursors underwent increased apoptosis(among 3- to 4-fold increased compared to healthy controls)as detected in human and mice bone marrow by an increasein DNA laddering (a sign of enhanced nucleosomal DNAcleavage occurring specifically during apoptosis) and thenconfirmed by TUNEL labelling and exposure of phosphatidylserine assessed by Annexin V labelling by FACS analysis[29 30 32] In vitro findings corroborate the reduced cellexpansion in 120573-TM erythroid cultures and enhanced apop-tosis at the polychromatophilic stage of differentiation [32]

In spite of the markedly increased rate of apoptosisof 120573-thalassemic erythroid precursors BM smears of thesepatients do not show high increased number of dying ery-throblasts Indeed 15 to 20 of bone marrow erythroidprecursors (CD45minusCD71+) present apoptotic features in


Normal erythropoiesis

Pro-E

Term

inal

diff

eren

tiatio

nEa

rly er

ythr

opoi

esis

Bonemarrow

BloodRed blood

cellsEryptosishemolysis

Terminal maturation Apoptosismaturation arrest

Accelerated differentiation

Prog

enito

rs

expa

nsio

n

Thalassemiadyserythropoiesis

Ineff

ectiv

eer

ythr

opoi

esis

Figure 1 Difference between normal and 120573-thalassemia ineffective erythropoiesis Erythropoiesis is the pathway producing matureRBCs from hematopoietic stem cells including several proliferation and differentiation steps Erythroid differentiation is accompaniedby temporally regulated changes in cell surface protein expression reduction in cell size progressive hemoglobinization and nuclearcondensation and extrusion 120573-thalassemia dyserythropoiesis in human is characterized by expansion of very early erythroid precursors(proerythroblasts and earlier stages) and then ineffective erythropoiesis Ineffective erythropoiesis defines the suboptimal production ofmature erythrocytes from a proliferating pool of immature erythroblasts characterized by (1) accelerated erythroid differentiation (2)maturation blockade at the polychromatophilic stage and (3) death of erythroid precursors

aspirates [29 30 32] This paradox might be explained byincreased phagocytosis of abnormal precursors erythroblastsexpressing phosphatidyl serine by bonemarrowmacrophageswhose number and activation are enhanced respectively byabout 2-fold inTM[29 37 38] As a consequence the deliveryof thalassemic RBCs to the peripheral blood in the 120573-TMmajor patients is much reduced

43 Apoptotic Pathways Involved in 120573-Thalassemia IneffectiveErythropoiesis Studies of apoptotic death receptor pathwayshave shown that Fas and FasL are coexpressed early andat all stages of terminal differentiation Both proteins aredownregulated in bone marrow or spleen in proerythroblastand basophilic cells in 120573-thalassemic mice compared tocontrol mice in vivo No statistically difference was foundin more mature cells This down regulation in FasFasLexpressionmight be amarker of erythropoietic stress [39] andmight explain at least in part erythroid expansion

Regarding the intrinsic apoptotic pathway it wasexpected that it would have been also involved because itcould be induced by cellular oxidant injury Neverthelessthe involvement of this mitochondrial pathway has not beenevidenced to date in 120573-thalassemia [40]

44 Role of Oxidant Injury in 120573-Thalassemia IneffectiveErythropoiesis

441 Excess of Unmatched Globin Chains Generates ReactiveOxygen Species (ROS) Occurrence of increased death atthe polychromatophil stage of differentiation in TM [32]

coincides with the stage of intense hemoglobinization [4 2741 42]

It could partially be explained by accumulation andprecipitation of the unmatched 120572-globin chains at this stageforming aggregates [33] Indeed there is several evidence thatmembrane components oxidation might play an importantrole in 120573-thalassemia pathophysiology [5 43] Free 120572-globinwhich is highly unstable and bound to heme and ironcould generate reactive oxygen species (ROS) that damagecellular proteins lipids and nucleic acids [44 45] Datadescribing ROS production during erythroid differentiationin thalassemia are scarce [46] A significant increase inROS production both in early and late erythroid precursorscompared to normal erythroblasts was evidenced in 120573-thalassemia intermedia [35] or120573-thalassemiaHbE [34]Thusit was speculated that the excess of ROS by damagingcomponents of RBC might reduce lifespan of these cells andcause a premature RBC clearance by hemolysis (a passiveprocess) [45] However there is no robust evidence that ROSproduction is the direct cause of increased apoptosis in 120573-thalassemic erythroid cell precursors [5] The observationthat there are no differences [35] or a dramatic decrease [34]in ROS levels during thalassemic erythroid differentiationpleads against a direct mechanism leading to high ROS levelsand apoptosis since significant increased apoptosis was onlyobserved in late differentiation stage (polychromatophilicstage and after) [32] Actually the direct link between increaseof ROS and apoptosis has never been demonstrated in normalhuman erythroid cells However in other cell models ROSactivate apoptosis signal regulating kinase 1 (ASK1) and Jun-kinase which can induce apoptosis (extrinsic or intrinsic


signaling pathway) [47] ROS are also known to trigger theintrinsic apoptotic cascade via interactions with proteins ofthe mitochondrial permeability transition complex [48]

In conclusion even if increased ROS levels might bean actor of erythroid cell precursors death by inducingdamages of erythroid cell components the pathophysio-logical relationship between apoptosis and accumulation ofthe unmatched 120572-globin chain in erythroblasts needs to beclarified To date no data provide clear evidences that theapoptotic mechanism involves ROS On the other hand therole of ROS in accelerated cell differentiation another char-acteristic of ineffective erythropoiesis is questionable [34]since antioxidants inhibit the in vitro erythroid progenitorsdifferentiation from mice fetal liver [49]

442 Damaging Membrane Structures A Cause of IneffectiveErythropoiesis Unmatched 120572-globin chains and ROS pro-duction could induce alterations in membrane deformabilitystability and cellular hydration in addition to damages tocytoskeleton explaining peripheral hemolysis [50] Protein41 a major component of the skeleton controlling itscrosslinking is partially oxidized in 120573-thalassemiarsquos matureerythroblasts [51] 120572-globins accumulation can occur evenfrom the proerythroblast stage and such deposits were shownto colocalize with areas of defective assembly of the mem-brane skeletal proteins spectrin and protein 41 [31 52] Fur-thermore very early in erythropoiesis a defective assembly ofthe transmembrane band 3 protein was reported which wasnot spatially related to 120572-globin deposits This band 3 defectseems to disappear at the intermediate or late normoblaststage suggesting either that the defect was temporary orthat it severely affected band-3-deficient erythroid precursorswhich died and were removed Furthermore oxidant injuryled to clustering of band 3 which in turn produced aneoantigen that bound IgG and complement [5 53] Extra-membranous IgGcomplement complex provided signals formacrophages to remove such affected erythroid precursorsand RBC

All these studies showed that 120573-thalassemic RBC mem-branes exhibited abnormalities in membrane skeletal pro-teins Thus it could also be postulated that the accumulationof 120572 chains destabilizes the membrane and could participatein ineffective erythropoiesis but it cannot be the uniqueexplanation of the major increase of apoptosis [30 31]

443 The 120572-Hb-Stabilizing Protein (AHSP) Is an Erythroid-Specific Private Chaperone Protein That Specifically Binds 120572-Hb Recently an 120572-hemoglobin-stabilizing protein (AHSP)was identifiedThis protein specifically binds to and stabilizesfree 120572 chains AHSP is a 102 amino acid erythroid-specificprotein induced by the essential erythroid transcriptionfactor GATA-1 [54]AHSP is abundant in late-stage erythroidprecursors in which its expression kinetics match with 120572-globin ones [55] Stabilization of native folded 120572-globin byAHSP might be particularly important when heme quantityis limited for example during iron deficiency Indeed thepresence of functional iron response element (IRE) in the 31015840

untranslated region of human AHSP mRNA stabilizes thistranscript in low iron conditions [56 57]

A second AHSP function is to detoxify the excess of 120572-globin chains Protein interaction screening has shown thatAHSP binds free 120572 globin and 120572-hemoglobin (unmatched 120572-globin chains bound to heme) (120572-Hb) Subsequent investi-gations provide evidence that AHSP acts as a protein-specificmolecular chaperone to fold and stabilize 120572-globin for HbAsynthesis [58] and to protect erythroid cells against thedeleterious effects of excess free 120572-Hb [45] AHSP stabilizesthe structure of 120572-Hb and detoxifies it by inhibiting theability of heme iron to participate in chemical reactionsthat generate damaging ROS in RBC [45 54 59 60] In 120573-thalassemia mice model loss of AHSP deficiency exacerbatesanemia [45] 119860119867119878119875minusminus mice present mild hemolytic anemiaand hemoglobin precipitation in RBCs [45] 119860119867119878119875minusminus miceexhibited also an elevated proportion of immature erythroidprecursors a maturation arrest and excess of apoptosis [45]This effect could also reflect ineffective erythropoiesis as aconsequence of excess of free 120572 chains

The role of AHSP in human disease remains an openquestion Naturally occurring mutations that ablate AHSPexpression or alter the protein structure are rare [61 62]However in human studies it was found that quantitativevariation in AHSP expression between different individualsis extremely common [61] Causal relationships betweendecreased AHSP expression and severity of thalassemicsyndromes have not been established unequivocally [57]

45 Role of Heme and Heme Inhibitors in 120573-ThalassemiaIneffective Erythropoiesis Excess of 120572-globin chains are asso-ciated with reduced heme production in late erythroid pro-genitors [35] How the globin chain imbalance might affectthe rate of heme synthesis is still a matter of investigationThe reduction of heme biosynthesis in 120573-thalassemic ery-thropoiesis has nevertheless a positive action to prevent thecytotoxic effect of free heme excess Other cytoprotectivemechanisms in response to oxidative stress in 120573-thalassemicerythroid cells also probably involve PRDX2 protein PRDX2is abundantly expressed during 120573-thalassemic erythropoiesisand binds heme in erythroid precursors possibly playing anadditional role to protect maturing cells by free heme fromapoptosis [35]

Another protective factor in120573-thalassemic erythropoiesisinvolves the heme-regulated inhibitor of protein transla-tion which represses globin translation in heme-deficienterythroid precursors Heme-regulated inhibitor of proteintranslation plays a role inmurine120573-thalassemia since anemiais more severe in 120573-thalassemic mice genetically lackingthis protein [63 64] Roles of heme or heme inhibitors inineffective erythropoiesis are still not known

46 Role of Inflammatory Cytokines in Ineffective Erythro-poiesis in 120573-Thalassemia Patients Increased level of severalinflammatory cytokines has been reported in 120573-TM andmight contribute to ineffective erythropoiesis through thewell-knownmechanism of ldquoanemia associated with a chronicdiseaserdquo


Further studies have shown an increased of TNF-120572concentration in 120573-TM patients unrelated to splenectomy[65 66] or only in the splenectomised patients group [67ndash69]In these studies the authors suggested that themain cause forTNF-120572 rise was macrophage activation due to iron overloadand the antigenic stimulation induced by chronic transfusiontherapy

TNF-120572 inhibits erythropoiesis in vivo and in vitro [70ndash80] However the mechanism by which TNF-120572 inhibitserythroid progenitor cells remains unclear TNF-120572 induces anincrease of apoptosis within the compartment of immatureerythroblasts and a decrease in mature erythroblasts TNF-120572 inhibits directly the BFU-E colony growth [70 79 80]whereas inhibition of CFU-E colony growth by the TNF-120572is indirect via stimulation of 120573-interferon production fromaccessory cells [81 82] TNF-120572might act directly on the TNFreceptor expressed on immature erythroblasts or by inducingceramide synthesis lipids component of the cell membranewhich can act as a signaling molecule involved in TNF-induced apoptosis It was also reported that the inhibitoryeffect of TNF-120572 on erythroid maturation might be involvedin NF-120581B induction [70] TNF-120572 cannot only directly inhibiterythroid differentiation but also facilitate proliferation ofnonerythroid precursor cells (such as dendritics cells) inchronic disease with inflammatory syndrome [80]

Furthermore it was described that transforming growthfactor-120573 (TGF-120573) plasma level was higher in 120573-TM splenec-tomized patients as compared to control group as well asother cytokines from TGF-120573 superfamily [83] ThereforeTGF-120573 is a paradoxical inhibitor of normal erythropoiesisthat acts by blocking proliferation and accelerating differ-entiation of erythroid progenitors [84] Its potential role inpathophysiological mechanisms of 120573-thalassemia ineffectiveerythropoiesis has not been studied to date

47 Other Pathophysiological Pathways Involved in120573-Thalassemia Ineffective Erythropoiesis

471 Macrophages Number and Activation Are Enhanced Asdescribed abovemacrophages number and level of activationare enhanced in bone marrow of 120573-thalassemic patients [2937] It was suggested that macrophages activation was dueto iron overload and antigenic stimulation related to chronictransfusion therapy [83]Those activated macrophages selec-tively phagocyte apoptotic erythroblasts exhibiting ldquoeat mesignalrdquo [37 38 85] thereby contributing to the ineffectiveerythropoiesis

It has been demonstrated that thalassemic erythrocytesare phagocytosed by activated macrophages in vitro [85]and the mean number of 120573-thalassemic cells ingested bymonocytes was found to be approximatively 30 higher thanthat for normal monocytes [86] Mechanisms involved in therecognition of apoptotic erythroblasts by macrophages arenot fully understood but CD36 at the surface ofmacrophagesand phosphatidyl serine residues exposed on apoptotic ery-throblasts membrane appear to be involved [37] Further-more oxidant injury that produced a IgG-bound neoantigenband 3 associated to complement provided signals formacrophages to remove such affected erythroblasts [53]

472 Epo Response to Anemia in 120573-Thalassemia Major In120573-thalassemia Epo is dramatically increased in responseto anemia and hypoxia [87] Nevertheless Epo response isblunted as compared with Epo response in aplastic anemiaor iron deficiency anemia [88 89] Underlying mechanismsfor the blunted Epo response in patients with 120573-TM are notwell understood Three hypotheses are suggested

(i) increased capture and faster clearance of Epo byerythroid cells hyperplasia [90]

(ii) inhibition of Epo renal synthesis by inflammatorycytokines (IL-1120572 IL-1120573 TGF-120573 or TNF-120572) [91] asshowed in other anemias of chronic disease [92ndash96]or by impaired renal function [89 97ndash101] very highserum Epo levels were found in young patients with120573-TM and 120573-thalassemia intermedia comparable tothe levels found in patients with aplastic anemiawhich were different from the levels found in olderthalassemia patients with the same degree of anemiait is suggested therefore that a decrease in serumEpolevels could develop during the course of the disease[102 103]

(iii) The low oxygen-Hb affinity and subsequent rightshifting of oxygen-Hbdissociation curvewould facili-tate tissue oxygen availability decreasing the hypoxicburden to anemia Both low oxygen-Hb affinity andincreased 23-diphosphoglycerate levels [104] presentin 120573-TMmight induce an inadequate Epo response toa given degree of anemia

Nevertheless increased Epo level secondary to profoundanemia is believed to be the cause of early erythroid progen-itors and precursors expansion in 120573-TM [87] In 120573-TM micemodel it was shown that persistent activation of JAK2 as aconsequence of high levels of Epo drives erythroid expansionand extramedullary hematopoiesis thus might constitute atarget by using JAK2 inhibitors to treat this complication anddecrease transfusion burden [105]

473 Iron Overload in Thalassemia Tissue iron overload isthe most important complication of 120573-thalassemia and is amajor focus of therapeutic management [42 106]

Blood transfusion is a comprehensive source of iron load-ing for 120573-thalassemia patients Nevertheless iron overloadoccurs also in patients who have not received transfusionssuch as patients suffering from thalassemia intermedia [42107 108] Decreased levels of hepcidin in these patientsexplain this paradoxical feature Hepcidin is a key regulatorof iron homeostasis it blocks iron release from macrophagesand hepatocytes and inhibits intestinal iron absorptionIts liver expression increases in response to iron overloadand inflammatory stimuli [4 109] If hepcidin expressionwould be correctly regulated it should be increased in 120573-thalassemia patients in order to decrease intestinal ironabsorption However the opposite effect is observed [110111] Indeed two hepcidin erythroid regulators have beenreported the growth differentiation factor 15 (GDF15) andthe twisted gastrulation protein homolog 1 (TWSG1) [112113] High concentrations of both proteins members of the


TGF-120573 superfamily were evidenced in 120573-thalassemia serumcompared to normal human serum These proteins down-regulate hepcidin secretion by hepatocytes [112 114] GDF15expression is associated with cellular stress and apoptosisand is expressed at low level during normal erythropoiesisWhile 120573-thalassemic erythroid differentiation GDF15 hasbeen shown to be secreted by apoptotic erythroid cells atfinal stages In contrast the highest levels of TWSG1 weredetected at early stages of erythroblast differentiation beforehemoglobinization [113]

Red blood cell membranes from thalassemic patientscarry abnormal deposits of iron presumed to mediate avariety of oxidative induced membrane dysfunctions Thecombination of iron overload and increased outpouring ofcatabolic iron from the reticuloendothelial system over-whelms the iron-carrying reticuloendothelial system andthe iron capacity of transferrin the main transport proteinto bind and detoxify iron Non-transferrin-bound fractionof plasma iron might promote the generation of malonyl-dialdehyde [115] and free hydroxyl radicals propagators ofoxygen-related damage [42 116 117] In addition severalpathobiochemical consequences in thalassemic RBC mem-branes such as increased lipid peroxidation and protein thioloxidation have been linked to the deposition of genericiron on the cytosolic leaflet of plasma membrane Thismechanism could also contribute to erythroblast apoptosis[118]

It has been shown in mouse model of 120573-thalassemiaintermedia that decreasing iron availability of erythroid cellslimits the formation of toxic alpha-chainheme aggregatesand improves ineffective erythropoiesis and anemia [119]

Moreover it was hypothesized that oral iron chelatorswhich have an enhanced capacity to penetrate through cellmembrane might be useful in chelating these pathologiciron deposits responsible for ROS generation [118] Thissuggestion receives further support from in vitro and somein vivo studies using these treatments It was shown thatmembrane free-iron content decreased as did heme contentof RBC membranes from deferiprone-treated thalassemicpatients [118] It has been recently described that deferasiroxtherapy in 120573-TM patients is associated with higher levels ofcirculating erythroid burst-forming unit than controls andother iron chelators [120]

Furthermore 120573-TM patients with severe hemochromato-sis may develop severe endocrine complications due toiron overload Hypogonadism hypothyroidism and hypoad-renalism may also contribute to anemia Thus endocrinopa-thy must be monitored regularly and treated with hormonereplacement [121]

474 Masked Deficit of Folic Acid in Thalassemia Folic aciddeficiency has been reported in both thalassemia major andminor [122ndash124] as a consequence of increased folate usecaused by increased erythropoiesis It can lead to overesti-mation of RBC deficiency Daily folate supplementation iscurrently advised for patients with hemoglobinopathy [124]

5 Conclusion

The pathophysiological mechanisms of ineffective erythro-poiesis in 120573-thalassemia could be the conjunction of severalmechanisms of which the final consequence is the arrest ofmaturation and increased apoptosis of erythroblasts duringtheir terminal differentiation stage

Putative actors of ineffective erythropoiesis are suggestedto be (1) oxidative stress induced by the excess of 120572-globinsecondary to the 120572120573 globin imbalance (2) iron overloadand (3) endocrines and cytokine and environmental factorsKey questions still remain to be addressed how depositionof 120572-globin chains andor ROS production in erythroidprecursors induce apoptosis and if antiapoptotic processes(involving heat shock protein) in erythroid cells are deficientor overwhelm In the future it will be critical to decipher theprecise role and mechanisms of these components in orderto understand the ineffective erythropoiesis in 120573-thalassemiaand to develop new therapeutic strategies based on thesepotential targets

Authorsrsquo Contribution

J-A Ribeil J-B Arlet G Courtois and O Hermine con-tributed equally to this work

References

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[2] E Khandros C S Thom J DrsquoSouza and M J Weiss ldquoInte-grated protein quality-control pathways regulate free 120572-globinin murine 120573-thalassemiardquo Blood vol 119 no 22 pp 5265ndash52752012

[3] E Khandros and M J Weiss ldquoProtein quality controlduring erythropoiesis and hemoglobin synthesisrdquo Hematol-ogyOncology Clinics of North America vol 24 no 6 pp 1071ndash1088 2010

[4] D Rund and E Rachmilewitz ldquo120573-thalassemiardquo The New Eng-land Journal of Medicine vol 353 no 11 pp 1135ndash1146 2005

[5] S L Schrier ldquoPathophysiology of thalassemiardquoCurrent Opinionin Hematology vol 9 no 2 pp 123ndash126 2002

[6] F M Gill and E Schwartz ldquoFree 120572-globin pool in human bonemarrowrdquo Journal of Clinical Investigation vol 52 no 12 pp3057ndash3063 1973

[7] J R Shaeffer ldquoEvidence for soluble a chains as intermediatesin hemoglobin synthesis in the rabbit reticulocyterdquo Biochemicaland Biophysical Research Communications vol 28 no 4 pp647ndash652 1967

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[9] Y Zermati C Garrido S Amsellem et al ldquoCaspase activationis required for terminal erythroid differentiationrdquo Journal ofExperimental Medicine vol 193 no 2 pp 247ndash254 2001

[10] J A Ribeil Y Zermati J Vandekerckhove et al ldquoHsp70 regu-lates erythropoiesis by preventing caspase-3-mediated cleavageof GATA-1rdquo Nature vol 445 no 7123 pp 102ndash105 2007


[11] T D Allen and T M Dexter ldquoUltrastructural aspects of ery-thropoietic differentiation in long-term bone marrow culturerdquoDifferentiation vol 21 no 2 pp 86ndash94 1982

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[14] M Socolovsky M Murrell Y Liu R Pop E Porpiglia and ALevchenko ldquoNegative autoregulation by FAS mediates robustfetal erythropoiesisrdquo PLoS Biology vol 5 no 10 article e2522007

[15] M P Menon V Karur O Bogacheva O Bogachev B Cuetaraand D M Wojchowski ldquoSignals for stress erythropoiesis areintegrated via an erythropoietin receptor-phosphotyrosine-343-Stat5 axisrdquo Journal of Clinical Investigation vol 116 no 3pp 683ndash694 2006

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[19] S Orrenius ldquoMitochondrial regulation of apoptotic cell deathrdquoToxicology Letters vol 149 no 1ndash3 pp 19ndash23 2004

[20] D R Green and G Kroemer ldquoThe pathophysiology of mito-chondrial cell deathrdquo Science vol 305 no 5684 pp 626ndash6292004

[21] R de Maria A Zeuner A Eramo et al ldquoNegative regulationof erythropoiesis by caspase-mediated cleavage of GATA-1rdquoNature vol 401 no 6752 pp 489ndash493 1999

[22] A Zeuner A Eramo U Testa et al ldquoControl of erythroidcell production via caspase-mediated cleavage of transcriptionfactor SCLTal-1rdquo Cell Death and Differentiation vol 10 no 8pp 905ndash913 2003

[23] R deMaria U Testa L Luchetti et al ldquoApoptotic role of FasFasligand system in the regulation of erythropoiesisrdquo Blood vol 93no 3 pp 796ndash803 1999

[24] M Koulnis Y Liu K Hallstrom and M Socolovsky ldquoNegativeautoregulation by fas stabilizes adult erythropoiesis and accel-erates its stress responserdquo PLoS ONE vol 6 no 7 Article IDe21192 2011

[25] U Testa ldquoApoptotic mechanisms in the control of erythro-poiesisrdquo Leukemia vol 18 no 7 pp 1176ndash1199 2004

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[27] P Pootrakul P Sirankapracha S Hemsorach et al ldquoA correla-tion of erythrokinetics ineffective erythropoiesis and erythroidprecursor apoptosis in Thai patients with thalassemiardquo Bloodvol 96 no 7 pp 2606ndash2612 2000

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[30] J Yuan E Angelucci G Lucarelli et al ldquoAccelerated pro-grammed cell death (apoptosis) in erythroid precursors ofpatients with severe 120573-thalassemia (Cooleyrsquos anemia)rdquo Bloodvol 82 no 2 pp 374ndash377 1993

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[33] S N Wickramasinghe and V Bush ldquoObservations on theultrastructure of erythropoietic cells and reticulum cells in thebonemarrow of patients with homozygous 120573 thalassaemiardquoTheBritish Journal of Haematology vol 30 no 4 pp 395ndash399 1975

[34] A Leecharoenkiat T Wannatung P Lithanatudom et alldquoIncreased oxidative metabolism is associated with erythroidprecursor expansion in 1205730-thalassaemiaHb E diseaserdquo BloodCells Molecules and Diseases vol 47 no 3 pp 143ndash157 2011

[35] L de Franceschi M Bertoldi L de Falco et al ldquoOxidative stressmodulates heme synthesis and induces peroxiredoxin-2 as anovel cytoprotective response in 120573-thalassemic erythropoiesisrdquoHaematologica vol 96 no 11 pp 1595ndash1604 2011

[36] C Lacombe J L Da Silva P Bruneval et al ldquoPeritubular cellsare the site of erythropoietin synthesis in the murine hypoxickidneyrdquo Journal of Clinical Investigation vol 81 no 2 pp 620ndash623 1988

[37] E Angelucci H Bai F Centis et al ldquoEnhanced macrophagicattack on 120573-thalassemia major erythroid precursorsrdquoHaemato-logica vol 87 no 6 pp 578ndash583 2002

[38] F A Kuypers and K de Jong ldquoThe role of phosphatidylserinein recognition and removal of erythrocytesrdquo Cellular andMolecular Biology vol 50 no 2 pp 147ndash158 2004

[39] Y Liu R Pop C Sadegh C Brugnara V H Haase and MSocolovsky ldquoSuppression of Fas-FasL coexpression by erythro-poietinmediates erythroblast expansion during the erythropoi-etic stress response in vivordquo Blood vol 108 no 1 pp 123ndash1332006

[40] S L Schrier F Centis M Verneris L Ma and E AngeluccildquoThe role of oxidant injury in the pathophysiology of humanthalassemiasrdquo Redox Report vol 8 no 5 pp 241ndash245 2003

[41] D G Nathan and R B Gunn ldquoThalassemia the consequencesof unbalanced hemoglobin synthesisrdquoThe American Journal ofMedicine vol 41 no 5 pp 815ndash830 1966

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[43] D Tavazzi L Duca G Graziadei A Comino G Fiorelliand M D Cappellini ldquoMembrane-bound iron contributes tooxidative damage of 120573-thalassaemia intermedia erythrocytesrdquoThe British Journal of Haematology vol 112 no 1 pp 48ndash502001

[44] A Bank ldquoHemoglobin synthesis in 120573-thalassemia the proper-ties of the free alpha-chainsrdquo Journal of Clinical Investigationvol 47 no 4 pp 860ndash866 1968

[45] Y Kong S Zhou A J Kihm et al ldquoLoss of 120572-hemoglobin-stabilizing protein impairs erythropoiesis and exacerbates 120573-thalassemiardquo Journal of Clinical Investigation vol 114 no 10 pp1457ndash1466 2004

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[47] T Fujisawa K Takeda and H Ichijo ldquoASK family proteins instress response and diseaserdquo Molecular Biotechnology vol 37no 1 pp 13ndash18 2007


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[49] M Nagata N Arimitsu T Ito and K Sekimizu ldquoAntioxidantN-acetyl-l-cysteine inhibits erythropoietin-induced differenti-ation of erythroid progenitors derived from mouse fetal liverrdquoCell Biology International vol 31 no 3 pp 252ndash256 2007

[50] S L Schrier E Rachmilewitz and N Mohandas ldquoCellular andmembrane properties of 120572 and 120573 thalassemic erythrocytes aredifferent implication for differences in clinical manifestationsrdquoBlood vol 74 no 6 pp 2194ndash2202 1989

[51] R Advani S Sorenson E Shinar W Lande E Rachmilewitzand S L Schrier ldquoCharacterization and comparison of thered blood cell membrane damage in severe human 120572- and 120573-thalassemiardquo Blood vol 79 no 4 pp 1058ndash1063 1992

[52] M Aljurf L Ma E Angelucci et al ldquoAbnormal assembly ofmembrane proteins in erythroid progenitors of patients with 120573-thalassemia majorrdquo Blood vol 87 no 5 pp 2049ndash2056 1996

[53] J Yuan R Kannan E Shinar E A Rachmilewitz and P S LowldquoIsolation characterization and immunoprecipitation studiesof immune complexes from membranes of 120573-thalassemic ery-throcytesrdquo Blood vol 79 no 11 pp 3007ndash3013 1992

[54] A J Kihm Y Kong W Hong et al ldquoAn abundant erythroidprotein that stabilizes free 120572-haemoglobinrdquoNature vol 417 no6890 pp 758ndash763 2002

[55] C O Dos Santos A S S Duarte S T O Saad and F F CostaldquoExpression of 120572-hemoglobin stabilizing protein gene duringhuman erythropoiesisrdquo Experimental Hematology vol 32 no2 pp 157ndash162 2004

[56] C O Dos Santos L C Dore E Valentine et al ldquoAn ironresponsive element-like stem-loop regulates 120572-hemoglobin-stabilizing protein mRNArdquoThe Journal of Biological Chemistryvol 283 no 40 pp 26956ndash26964 2008

[57] M JWeiss andCODos Santos ldquoChaperoning erythropoiesisrdquoBlood vol 113 no 10 pp 2136ndash2144 2009

[58] X Yu Y Kong L C Dore et al ldquoAn erythroid chaperonethat facilitates folding of 120572-globin subunits for hemoglobinsynthesisrdquo Journal of Clinical Investigation vol 117 no 7 pp1856ndash1865 2007

[59] L Feng S Zhou L Gu et al ldquoStructure of oxidized 120572-haemoglobin bound to AHSP reveals a protective mechanismfor haemrdquo Nature vol 435 no 7042 pp 697ndash701 2005

[60] S Zhou J S Olson M Fabian M J Weiss and A J GowldquoBiochemical fates of 120572 hemoglobin bound to 120572 hemoglobin-stabilizing protein AHSPrdquo The Journal of Biological Chemistryvol 281 no 43 pp 32611ndash32618 2006

[61] C O Dos Santos S Zhou R Secolin et al ldquoPopulationanalysis of the alpha hemoglobin stabilizing protein (AHSP)gene identifies sequence variants that alter expression andfunctionrdquo American Journal of Hematology vol 83 no 2 pp103ndash108 2008

[62] V Viprakasit V S Tanphaichitr W Chinchang P SangklaM J Weiss and D R Higgs ldquoEvaluation of 120572 hemoglobinstabilizing protein (AHSP) as a geneticmodifier in patients with120573 thalassemiardquo Blood vol 103 no 9 pp 3296ndash3299 2004

[63] A PHanMD Fleming and J J Chen ldquoHeme-regulated eIF2120572kinase modifies the phenotypic severity of murine models oferythropoietic protoporphyria and 120573-thalassemiardquo Journal ofClinical Investigation vol 115 no 6 pp 1562ndash1570 2005

[64] J J Chen ldquoRegulation of protein synthesis by the heme-regulated eIF2120572 kinase relevance to anemiasrdquo Blood vol 109no 7 pp 2693ndash2699 2007

[65] G Lombardi R Matera M M Minervini et al ldquoSerum levelsof cytokines and soluble antigens in polytransfused patientswith 120573-thalassemia major relationship to immune statusrdquoHaematologica vol 79 no 5 pp 406ndash412 1994

[66] R Meliconi M Uguccioni E Lalli et al ldquoIncreased serumconcentrations of tumour necrosis factor in 120573 thalassaemiaeffect of bone marrow transplantationrdquo Journal of ClinicalPathology vol 45 no 1 pp 61ndash65 1992

[67] S Chuncharunee N Archararit P Hathirat U Udomsub-payakul and V Atichartakarn ldquoLevels of serum interleukin-6and tumor necrosis factor in postsplenectomized thalassemicpatientsrdquo Journal of the Medical Association ofThailand vol 80supplement 1 pp S86ndashS90 1997

[68] M Gharagozloo M Karimi and Z Amirghofran ldquoDouble-faced cell-mediated immunity in 120573-thalassemia major stimu-lated phenotype versus suppressed activityrdquo Annals of Hematol-ogy vol 88 no 1 pp 21ndash27 2009

[69] G C Del Vecchio F Schettini L Piacente A De SantisP Giordano and D de Mattia ldquoEffects of deferiprone onimmune status and cytokine pattern in thalassaemia majorrdquoActa Haematologica vol 108 no 3 pp 144ndash149 2002

[70] W Xiao K Koizumi M Nishio et al ldquoTumor necrosis factor-120572 inhibits generation of glycophorin A+ cells by CD34+ cellsrdquoExperimental Hematology vol 30 no 11 pp 1238ndash1247 2002

[71] G D Roodman A Bird D Hutzler and W MontgomeryldquoTumor necrosis factor-alpha and hematopoietic progenitorseffects of tumor necrosis factor on the growth of erythroidprogenitors CFU-E and BFU-E and the hematopoietic cell linesK562 HL60 and HEL cellsrdquo Experimental Hematology vol 15no 9 pp 928ndash935 1987

[72] R A Johnson T A Waddelow J Caro A Oliff and G DRoodman ldquoChronic exposure to tumor necrosis factor in vivopreferentially inhibits erythropoiesis in nude micerdquo Blood vol74 no 1 pp 130ndash138 1989

[73] H E Broxmeyer D E Williams L Lu et al ldquoThe suppressiveinfluences of human tumor necrosis factors on bone mar-row hematopoietic progenitor cells from normal donors andpatients with leukemia synergism of tumor necrosis factor andinterferon-120574rdquo Journal of Immunology vol 136 no 12 pp 4487ndash4495 1986

[74] T Murase T Hotta H Saito and R Ohno ldquoEffect of recom-binant human tumor necrosis factor on the colony growth ofhuman leukemia progenitor cells and normal hematopoieticprogenitor cellsrdquo Blood vol 69 no 2 pp 467ndash472 1987

[75] B Backx L Broeders F J Bot and B Lowenberg ldquoPositive andnegative effects of tumor necrosis factor on colony growth fromhighly purified normal marrow progenitorsrdquo Leukemia vol 5no 1 pp 66ndash70 1991

[76] L S Rusten and S E W Jacobsen ldquoTumor necrosis factor(TNF)-120572 directly inhibits human erythropoiesis in vitro role ofp55 and p75 TNF receptorsrdquo Blood vol 85 no 4 pp 989ndash9961995

[77] M Blick S A Sherwin M Rosenblum and J GuttermanldquoPhase I study of recombinant tumor necrosis factor in cancerpatientsrdquo Cancer Research vol 47 no 11 pp 2986ndash2989 1987

[78] H A Papadaki H D Kritikos V Valatas D T Boumpas andG D Eliopoulos ldquoAnemia of chronic disease in rheumatoidarthritis is associated with increased apoptosis of bone marrowerythroid cells improvement following anti-tumor necrosisfactor-120572 antibody therapyrdquo Blood vol 100 no 2 pp 474ndash4822002


[79] X F Li J Anderson D Hutzler and G D Roodman ldquoHemin-induced erythroid differentiation changes the sensitivity ofK562 cells to tumor necrosis factor-120572rdquo Experimental Hematol-ogy vol 17 no 11 pp 1059ndash1062 1989

[80] H FukayaWXiao K Inaba et al ldquoCodevelopment of dendriticcells along with erythroid differentiation from human CD34+cells by tumor necrosis factor-120572rdquo Experimental Hematology vol32 no 5 pp 450ndash460 2004

[81] R T Means Jr E N Dessypris and S B Krantz ldquoInhibition ofhuman colony-forming-unit erythroid by tumor necrosis factorrequires accessory cellsrdquo Journal of Clinical Investigation vol 86no 2 pp 538ndash541 1990

[82] R T Means Jr and S B Krantz ldquoInhibition of human erythroidcolony-forming units by tumor necrosis factor requires 120573interferonrdquo Journal of Clinical Investigation vol 91 no 2 pp416ndash419 1993

[83] G R Moshtaghi-Kashanian A Gholamhoseinian A Hosein-imoghadam and S Rajabalian ldquoSplenectomy changes thepattern of cytokine production in 120573-thalassemic patientsrdquoCytokine vol 35 no 5-6 pp 253ndash257 2006

[84] Y Zermati B Varet and O Hermine ldquoTGF-1205731 drives andaccelerates erythroid differentiation in the epo-dependent UT-7 cell line even in the absence of erythropoietinrdquo ExperimentalHematology vol 28 no 3 pp 256ndash266 2000

[85] A Knyszynski D Danon I Kahane and E A RachmilewitzldquoPhagocytosis of nucleated and mature 120573 thalassaemic redblood cells by mouse macrophages in vitrordquoThe British Journalof Haematology vol 43 no 2 pp 251ndash255 1979

[86] W Wanachiwanawin U Siripanyphinyo S Fucharoen et alldquoActivation of monocytes for the immune clearance of red cellsin 120573(o)-thalassaemiaHbErdquoThe British Journal of Haematologyvol 85 no 4 pp 773ndash777 1993

[87] Y Ginzburg and S Rivella ldquo120573-thalassemia a model for eluci-dating the dynamic regulation of ineffective erythropoiesis andiron metabolismrdquo Blood vol 118 no 16 pp 4321ndash4330 2011

[88] M Cazzola R Guarnone P Cerani E Centenara A Rovatiand Y Beguin ldquoRed blood cell precursor mass as an indepen-dent determinant of serum erythropoietin levelrdquo Blood vol 91no 6 pp 2139ndash2145 1998

[89] J S Chen K H Lin S T Wang C J Tsao and T F YehldquoBlunted serum erythropoietin response to anemia in patientspolytransfused for 120573-thalassemia majorrdquo Journal of PediatricHematologyOncology vol 20 no 2 pp 140ndash144 1998

[90] C Camaschella S Gonetta R Calabrese et al ldquoSerum ery-thropoietin and circulating transferrin receptor in thalassemiaintermedia patients with heterogeneous genotypesrdquo Haemato-logica vol 81 no 5 pp 397ndash403 1996

[91] W C Faquin T J Schneider and M A Goldberg ldquoEffectof inflammatory cytokines on hypoxia-induced erythropoietinproductionrdquo Blood vol 79 no 8 pp 1987ndash1994 1992

[92] M C Hochberg C M Arnold B B Hogans and J L SpivakldquoSerum immunoreactive erythropoietin in rheumatoid arthri-tis impaired response to anemiardquo Arthritis and Rheumatismvol 31 no 10 pp 1318ndash1321 1988

[93] C B Miller R J Jones S Piantadosi M D Abeloff and J LSpivak ldquoDecreased erythropoietin response in patients with theanemia of cancerrdquo The New England Journal of Medicine vol322 no 24 pp 1689ndash1692 1990

[94] A N Baer E N Dessypris E Goldwasser and S B KrantzldquoBlunted erythropoietin response to anaemia in rheumatoidarthritisrdquoThe British Journal of Haematology vol 66 no 4 pp559ndash564 1987

[95] H Johannsen W Jelkmann G Wiedemann M Otteand T Wagner ldquoErythropoietinhaemoglobin relationshipin leukaemia and ulcerative colitisrdquo European Journal ofHaematology vol 43 no 3 pp 201ndash206 1989

[96] J L Spivak D C Barnes E Fuchs and T C Quinn ldquoSerumimmunoreactive erythropoietin in HIV-infected patientsrdquo TheJournal of the American Medical Association vol 261 no 21 pp3104ndash3107 1989

[97] A Sumboonnanonda PMalasit V S Tanphaichitr et al ldquoRenaltubular function in120573-thalassemiardquoPediatric Nephrology vol 12no 4 pp 280ndash283 1998

[98] B Aldudak A K Bayazit A Noyan et al ldquoRenal function inpediatric patients with 120573-thalassemia majorrdquo Pediatric Nephrol-ogy vol 15 no 1-2 pp 109ndash112 2000

[99] P Cianciulli D Sollecito F Sorrentino et al ldquoEarly detection ofnephrotoxic effects in thalassemic patients receiving desferriox-amine therapyrdquoKidney International vol 46 no 2 pp 467ndash4701994

[100] B H Landing H C Gonick R L Nadorra et al ldquoRenal lesionsand clinical findings in thalassemia major and other chronicanemias with hemosiderosisrdquo Pediatric Pathology vol 9 no 5pp 479ndash500 1989

[101] V Smolkin R Halevy C Levin et al ldquoRenal function in chil-dren with 120573-thalassemia major and thalassemia intermediardquoPediatric Nephrology vol 23 no 10 pp 1847ndash1851 2008

[102] D Manor E Fibach A Goldfarb and E A RachmilewitzldquoErythropoietin activity in the serum of 120573 thalassemic patientsrdquoScandinavian Journal of Haematology vol 37 no 3 pp 221ndash2281986

[103] E A Rachmilewitz and M Aker ldquoThe role of recombinanthuman erythropoietin in the treatment of thalassemiardquo Annalsof the New York Academy of Sciences vol 850 pp 129ndash138 1998

[104] G Ricci G Castaldi G Zavagli G Lupi A Turati and T BezzildquoRed cell 23-diphosphoglycerate contents and oxygen affinityin heterozygous 120573-thalassaemiardquo Acta Haematologica vol 68no 1 pp 63ndash64 1982

[105] I V Libani E C Guy L Melchiori et al ldquoDecreased differenti-ation of erythroid cells exacerbates ineffective erythropoiesis in120573-thalassemiardquo Blood vol 112 no 3 pp 875ndash885 2008

[106] N F Olivieri and G M Brittenham ldquoIron-chelating therapyand the treatment of thalassemiardquo Blood vol 89 no 3 pp 739ndash761 1997

[107] M J Pippard S T Callender G TWarner andD JWeatherallldquoIron absorption and loading in 120573-thalassaemia intermediardquoThe Lancet vol 2 no 8147 pp 819ndash821 1979

[108] P Pootrakul K Kitcharoen P Yansukon et al ldquoThe effect oferythroid hyperplasia on iron balancerdquo Blood vol 71 no 4 pp1124ndash1129 1988

[109] T Ganz ldquoHepcidin a key regulator of iron metabolism andmediator of anemia of inflammationrdquo Blood vol 102 no 3 pp783ndash788 2003

[110] G Papanikolaou M Tzilianos J I Christakis et al ldquoHepcidinin iron overload disordersrdquoBlood vol 105 no 10 pp 4103ndash41052005

[111] A Kattamis I Papassotiriou D Palaiologou et al ldquoThe effectsof erythropoetic activity and iron burden on hepcidin expres-sion in patients with thalassemia majorrdquoHaematologica vol 91no 6 pp 809ndash812 2006

[112] T Tanno N V Bhanu P A Oneal et al ldquoHigh levels ofGDF15 in thalassemia suppress expression of the iron regulatoryprotein hepcidinrdquoNature Medicine vol 13 no 9 pp 1096ndash11012007


[113] T Tanno P Porayette O Sripichai et al ldquoIdentification ofTWSG1 as a second novel erythroid regulator of hepcidinexpression in murine and human cellsrdquo Blood vol 114 no 1pp 181ndash186 2009

[114] J Kanda C Mizumoto H Kawabata et al ldquoSerum hepcidinlevel and erythropoietic activity after hematopoietic stem celltransplantationrdquo Haematologica vol 93 no 10 pp 1550ndash15542008

[115] G Cighetti L Duca L Bortone et al ldquoOxidative status andmalondialdehyde in 120573-thalassaemia patientsrdquo European Journalof Clinical Investigation vol 32 supplement 1 pp 55ndash60 2002

[116] C Hershko and D J Weatherall ldquoIron-chelating therapyrdquoCritical Reviews in Clinical Laboratory Sciences vol 26 no 4pp 303ndash345 1988

[117] C Hershko A M Konijn and G Link ldquoIron chelators forthalassaemiardquo The British Journal of Haematology vol 101 no3 pp 399ndash406 1998

[118] O Shalev T Repka A Goldfarb et al ldquoDeferiprone (L1)chelates pathologic iron deposits from membranes of intactthalassemic and sickle red blood cells both in vitro and in vivordquoBlood vol 86 no 5 pp 2008ndash2013 1995

[119] H Li A C Rybicki S M Suzuka et al ldquoTransferrin therapyameliorates disease in beta-thalassemic micerdquoNature Medicinevol 16 no 2 pp 177ndash182 2010

[120] G L Forni M Podestarsquo M Musso et al ldquoDifferential effects ofthe type of iron chelator on the absolute number of hematopoi-etic peripheral progenitors in patients with 120573-thalassemiamajorrdquo Haematologica 2012

[121] D Tiosano and Z Hochberg ldquoEndocrine complications ofthalassemiardquo Journal of Endocrinological Investigation vol 24no 9 pp 716ndash723 2001

[122] P Danel R Girot and G Tchernia ldquoThalassemia major pre-senting asmegaloblastic anemiawith folate deficiencyrdquoArchivesFrancaises de Pediatrie vol 40 no 10 pp 799ndash801 1983

[123] F Ortuno A Remacha S Martin J Soler and E GimferrerldquoPrevalence of folate deficiency in 120573 and delta-beta heterozy-gous thalassemiardquo Haematologica vol 75 no 6 article 5851990

[124] A Mazzone M Vezzoli and E Ottini ldquoMasked deficit of B12and folic acid in thalassemiardquo American Journal of Hematologyvol 67 no 4 article 274 2001

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Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


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Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


120572non-120572 chains ratio severe ineffective erythropoiesis anddependence on RBC transfusions to sustain live Regulartransfusions (average every month) expose these patients toiron overload and its live threatening systemic consequenceswhich require iron chelation [1]

It is well established that the 120572non-120572 ratio correlates withthe severity of disease [1 3] However genotypic variabilityimpairing globin chain synthesis at known loci is often insuf-ficient to explain the heterogeneity in clinical phenotypes ofindividual patients with the same genotype suggesting thatother genetic modulations might exist [1 4] The molecularmechanisms underlying the heterogeneity and occasionalseverity of the syndrome remain obscure and are not theobject of this paper

The pathophysiology of 120573-thalassemia has been the sub-ject of several extensive reviews particularly on its molecularand genetics basis [5] In this paper we will focus on themechanisms leading to end-stage maturation blockade andthalassemic erythroid precursors premature destruction inthe bone marrow Their understanding will probably be thekey for developing novel therapeutic approaches improvinganemia in 120573-thalassemia In order to describe mechanismsunderlying ineffective erythropoiesis we will first summarizethe current knowledge on normal hemoglobin synthesis andnormal erythropoiesis

2 Hemoglobin Synthesis

Two distinct globin chains 120572 and 120573 (each carrying an indi-vidual heme molecule) interact to form hemoglobin dimers120572120573 and two dimers combine to form a hemoglobin tetramer12057221205732 the functional form of hemoglobin carrying oxygenExcepted the very first weeks of embryogenesis in which

zeta chains are produced one of the globin chains is 120572 and thesecond chain is called ldquonon-120572rdquo The main (98) hemoglobintype in the normal human adult consists of two 120572 and two120573 chains (120572

21205732HbA) the minor type (2) consists of 2120572

and 2120575 chains (12057221205752HbA2) Usually globin chains synthesis

is relatively balanced even if some studies report a slightexcess of 120572 chains as a soluble pool [3 6 7] In contrast fetalerythrocytes contain another type of hemoglobin consistingof 2120572 and 2120574 chains (120572

21205742HbF) which can attract more

oxygen effectively from the maternal bloodGlobin chains originating from a common ancestral type

display a varying degree of homology Two clusters of globinsgenes are known the first one on chromosome 16 for 120572-likegenes (two 120572-globin genes 120572

1and 120572

2 and two zeta genes)

and the second one on chromosome 11 for 120573-like genes The51015840 to 31015840 order of 120573-like globin genes on chromosome 11 (120576- G120574-A120574-120575-120573 two 120574 genes) reflects their sequential activation andsilencing during the transition from embryonic to fetal andfrom fetal to adult ldquohemoglobinopoiesisrdquo called ldquohemoglobinontogenyrdquo or ldquohemoglobin chain switchrdquo

3 Erythropoiesis

Erythropoiesis is defined as the pathway producing matureRBC from hematopoietic stem cells This process includes

several steps restricting differentiation and proliferation ofcells which undergo this erythroid program depending onsequential and specific erythroid gene expression Erythro-poiesis is regulated by combined effects of microenviron-ment and growth factors that promote survival prolifer-ation andor differentiation of erythroid progenitors andnuclear factors that regulate transcription of genes involvedin survival and establishment of the erythroid phenotypeRBC production is orchestrated by a complex network oftranscription factors among which GATA-1 the master geneof erythropoiesis positively regulates specific erythroid genessuch as erythropoietin receptor (EpoR) glycophorin (GpA)and globin chains Moreover together with the transcriptionfactor STAT5 (activated through EpoR activation by erythro-poietin (Epo)) GATA-1 induces the expression of the anti-apoptotic protein Bcl-xL [8]

Committed erythroid progenitors differentiate into thefirst morphologically identified cell of the erythrocyte lin-eage the proerythroblast Next steps of erythroid differentia-tion are accompanied by temporally regulated changes in cellsurface protein expression reduction in cell size progressivehemoglobinization and nuclear condensation (successivelycalled basophilic erythroblast polychromatophilic erythrob-last and acidophilic erythroblast the last nucleated cell of themammalian erythrocyte lineage) which culminate in reticu-locytes cells by nucleus RNA andmitochondria extrusion Inaddition erythroidmaturation requires a transient activationof caspase-3 at the basophilic stage and translocation into thenucleus of the inducible heat shock protein 70 (Hsp70) toprotect GATA-1 from caspase-3 cleavage [9 10]

This process occurs within the erythroblastic island inwhich a macrophage is surrounded by erythroblasts at allstages of maturation [11 12] The production of erythrocytesis the largest quantitative output of the hematopoietic systemwith estimated production rates of 2 times 1011 erythrocytes perday The program of erythroid proliferation and differentia-tion must be positively and negatively regulated to ensure acontinuous but tightly controlled production of RBC

31 Positive Regulation of Erythropoiesis Erythropoiesis iscontrolled by the combined effect of two major cytokinesstem cell factor (SCF) and Epo SCF induces proliferationand survival and slows down differentiation of early ery-throid progenitors and precursors towards the basophilicerythroblast stage Epo is responsible of the finely tunedhomeostatic control of erythrocyte numbers by tissue oxy-genation Interaction of Epo with the EpoR induces throughJAK2 activation multiple signalling pathways involving PI3kinase Akt and STAT5 which prevent apoptosis supportingerythroid progenitors proliferation and allowing erythroidprogram to occur [13ndash15]

32 Negative Regulation of Erythropoiesis by Apoptosis Thenegative regulation of erythropoiesis ismainly due to apopto-sis a fundamental cellular mechanism allowing clearance ofunneeded or potentially dangerous cells Apoptotic programsrequire the action of a family of cysteine-dependent andaspartate-specific proteases called caspases Two classes of









1015840


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Disease Markers



OncologyJournal of





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Journal of

ObesityJournal of
























1015840


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5 Conclusion





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Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















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Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















































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References






































































































































of






Disease Markers



OncologyJournal of





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Journal of

ObesityJournal of











































































































































of






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OncologyJournal of





PPAR Research



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