Pathophysiology of anemia and erythrocytosis

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Critical Reviews in Oncology/Hematology 64 (2007) 139–158

Pathophysiology of anemia and erythrocytosis

Vivien M. Hodges a,∗, Susan Rainey a, Terence R. Lappin a, A. Peter Maxwell b

a Haematology Research Group, Centre for Cancer Research and Cell Biology, Queen’s University, Belfast, United Kingdomb Regional Nephrology Unit, Belfast City Hospital and Queen’s University, Belfast, United Kingdom

Accepted 12 June 2007

ontents

. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1401.1. Erythropoiesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1401.2. Iron homeostasis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1411.3. Cytokine regulation of erythropoiesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1421.4. The erythropoietin receptor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1431.5. Control of EPO gene expression . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 144

. Anemia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1462.1. Anemias with high Epo levels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 146

2.1.1. Anemias due to bone marrow failure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1462.1.2. Anemias due to substrate deficiencies or protein/enzyme defects of the red blood cell . . . . . . . . . . . . . . . . . . . . . . . . . 147

2.2. Anemias with low Epo levels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1472.2.1. Anemia of chronic disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1472.2.2. Anemia of chronic renal failure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 148

2.3. ESAs for anemia treatment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 149. Erythrocytosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 149

3.1. Erythrocytosis with low Epo levels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1503.2. Erythrocytosis with high Epo levels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 151

3.2.1. Appropriately elevated Epo . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1513.2.2. Inappropriately elevated Epo . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 152

. Future directions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 152Reviewers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 153Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 153References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 153Biographies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 158

bstract

An increasing understanding of the process of erythropoiesis raises some interesting questions about the pathophysiology, diagnosis andreatment of anemia and erythrocytosis. The mechanisms underlying the development of many of the erythrocytoses, previously characterised
s idiopathic, have been elucidated leading to an increased understanding of oxygen homeostasis. Characterisation of anemia and erythrocytosisn relation to serum erythropoietin levels can be a useful addition to clinical diagnostic criteria and provide a rationale for treatment withrythropoiesis stimulating agents (ESAs). Recombinant human erythropoietin as well as other ESAs are now widely used to treat anemiasssociated with a range of conditions, including chronic kidney disease, chronic inflammatory disorders and cancer. There is also heightenedwareness of the potential abuse of ESAs to boost athletic performance in competitive sport. The discovery of erythropoietin receptors outsidef the erythropoietic compartment may herald future applications for ESAs in the management of neurological and cardiac diseases. The
∗ Corresponding author.E-mail address: [email protected] (V.M. Hodges).

040-8428/$ – see front matter © 2007 Elsevier Ireland Ltd. All rights reserved.oi:10.1016/j.critrevonc.2007.06.006

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40 V.M. Hodges et al. / Critical Reviews in Oncology/Hematology 64 (2007) 139–158

urrent controversy concerning optimal hemoglobin levels in chronic kidney disease patients treated with ESAs and the potential negativelinical outcomes of ESA treatment in cancer reinforces the need for cautious evaluation of the pleiotropic effects of ESAs in non-erythroidissues.

2007 Elsevier Ireland Ltd. All rights reserved.

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eywords: Anemia; Erythrocytosis; Erythropoietin; Chuvash polycythemia

. Introduction

In human adults the process of erythropoiesis involves theroduction of 2 × 1011 red blood cells each day, or 2.3 × 106

ells every second, and is regulated by a feedback mecha-ism primarily involving the hormone erythropoietin (Epo).ince red blood cells are the essential transporters of oxygen

hroughout the body, it is vital that this continual produc-ion is tightly regulated. Normally, Epo is present in thelasma at a concentration of 10−11 M, maintaining a hema-ocrit (HCT) of approximately 0.45 at sea level. Changesn circulating levels of Epo, its function, or its action onarget cells can lead to major changes in red blood num-ers and subsequently the oxygen-carrying capacity of thelood. Disorders with excessively high levels of erythrocytesre termed erythrocytoses, while states with abnormally lowevels of red blood cells are known as anemias. Here weeview the process of erythropoiesis, with an emphasis onecent advances in the understanding of Epo signaling andypoxic control of EPO expression. Anemia and erythrocyto-is have been examined, and mechanistically classified basedn the levels of Epo present in the circulation. Finally, in the
ase of anemia, the use of recombinant ESAs as treatment isiscussed.
ig. 1. The feedback loop mechanism of Epo production. The growth factor regulat

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omeostasis

.1. Erythropoiesis

Hematopoietic cell turnover in a human adult is estimatedo be close to 1 trillion cells per day including the forma-ion of 200 billion erythrocytes [1]. The circulating red cell

ass reflects a dynamic balance between Epo-regulated redell proliferation and the subsequent loss or destruction ofature erythrocytes. The production of Epo is controlled

y a classical feedback loop mechanism (see Fig. 1) and itsffects are mediated through the cell surface erythropoietineceptor (Epo-R). Factors which affect oxygen delivery tohe kidney, such as the inspired partial pressure of oxygenr defective cardio-pulmonary function perturbing normalenal perfusion, can increase Epo production and stimulaterythropoiesis. Conversely, increased oxygen supply to theidney reduces the stimulus to Epo synthesis. However, in ansolated perfused rat kidney model epo levels were found toncrease only moderately when renal blood flow was reducedo 10% of normal [2]. The optimal size of the red cell masss achieved when the rate of red cell production is closely

ed maturation of erythrocytes from HSC in the bone marrow is highlighted.

aintain a continuous population of maturing erythroid cells.his is achieved by the combined effects of growth factors

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V.M. Hodges et al. / Critical Reviews i

hat permit cellular proliferation and transcription factorshat activate lineage-specific gene expression. The renewalf hematopoietic cells, including those of the erythroid lin-age, is supported by a small population of self-renewingematopoietic stem cells (HSCs) (see Fig. 1). The earli-st identifiable erythroid progenitors, burst-forming unitsrythroid (BFU-E) and colony forming units erythroid (CFU-) are characterised by their in vitro colony formation inemisolid culture [3]. Early BFU-Es are not responsive topo and do not express the Epo-R. In vitro culture of theseells for 48–72 h in the presence of stem cell factor (SCF),nterleukin-3 (IL-3) or granulocyte-macrophage colony stim-lating factor (GM-CSF) results in the formation of matureFU-Es which express Epo-R and are weakly responsive

o Epo [4]. After 4–5 days in culture BFU-Es give rise toapidly dividing CFU-Es which are highly responsive to Epo3]. CFU-Es in turn give rise to morphologically recognisedrogenitors (proerythroblasts and subsequent progeny). Epoesponsiveness declines slowly as the cells undergo terminalaturation and proceed beyond the Epo-dependent stage of

evelopment, with concomitant reduction in the number ofpo-Rs per cell [5]. Mature erythrocytes are enucleate, havelifespan of ∼120 days and ultimately undergo a process of

enescence associated with distinct morphological changesnd removal from the circulation by macrophage-mediatedhagocytosis.

During the past six decades the biochemistry and physiol-gy of Epo have been studied extensively. The human EPOene is a single-copy gene in a 5.4 kb region of genomicNA [6,7] localized on chromosome 7 (7pter-q22) [8]. It

ontains four introns and five exons encoding a 193 aminocid peptide. A hydrophobic 27 amino acid leader sequence isleaved during secretion which leaves a peptide of 166 aminocids, MW 18,398 [7]. The carboxy-terminal arginine is lostn urinary and recombinant Epo, due to post-translationalrocessing by an intracellular carboxypeptidase [9]. Theolecule has a carbohydrate content of ∼40% with one O-

inked (Ser 126) and three N-linked (Asn 24, 38 and 83)lycosylation sites [10].

In adults, Epo is primarily produced in the cells of theenal cortex, accounting for ∼90% of plasma Epo levels.he liver is the main secondary site of Epo production but

urther extra-renal sites include the spleen, lung, testis, brainnd erythroid progenitor cells [11–14]. In recent years a wideange of other tissues has been reported to express Epo and itsognate receptor (reviewed in [13]). In general, plasma Epooncentrations show an inverse relationship with the oxygen-arrying capacity of the blood [15]. Normal Epo levels<5–25 U/L) [16] can increase exponentially by as much as000-fold in severe anemia. Conversely, inappropriate over-roduction of Epo can lead to erythrocytosis, increased bloodiscosity and decreased blood flow [17]. The increase in Epo
roduction is dependent on the de novo synthesis of Epoather than release of pre-formed stores from Epo producingells. The hypoxia-induced increase in EPO mRNA levelsesults from a combination of increased gene transcription
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logy/Hematology 64 (2007) 139–158 141

18] and stabilisation of EPO messenger RNA (EPO mRNA).he half-life of EPO mRNA during hypoxia is increased-fold due to decreased binding of the de-stabilising erythro-oietin mRNA-binding protein (ERBP) to the EPO mRNA′UTR [19]. The concentration of circulating Epo maintainsrythropoiesis by balancing the differentiation of erythroidrogenitor cells with the level of programmed cell death [20].argely quiescent early erythroid progenitors require Epo tonter the cell cycle while Epo acts as a survival factor forhe later progenitors, preventing their apoptosis and allowingerminal differentiation. In vivo physiological Epo levels areub-optimal, enabling only a fraction of progenitors to survivend proliferate; resulting in apoptosis of ∼20% of bone mar-ow erythroid cells [21]. The fine control of erythropoiesislso results from a graded response of erythroid progenitorso Epo, as cells with the same differentiation potential requireifferent concentrations of Epo to achieve terminal matura-ion [22]. Thus, the two key factors which determine the fatef individual cells are the concentration of Epo in the cellnvironment and the sensitivity of the cell to Epo (reviewedn [23]).

.2. Iron homeostasis

Iron is obligatory for erythropoiesis. In its ferrous (Fe2+)orm it is an essential component of heme in hemoglobin andany electron-transferring enzymes in the redox system of

he respiratory chain. The body balance of iron is regulatedolely by gastrointestinal absorption, which itself dependsn the bioavailability of iron in food. Dietary heme iron is anmportant nutritional source of iron and long recognised to be

ore readily absorbed than non-heme iron derived from veg-tables and grain. The protracted search for an intestinal hemeransporter recently identified a membrane protein, heme car-ier protein 1 (HCP 1), which is iron-regulated and localizedo the brush-border membrane of duodenal enterocytes intates of iron deficiency [24].

The principal regulator of systemic iron homeostasis isepcidin, a 25-amino acid peptide produced by hepato-ytes. Hepcidin controls the circulating iron concentration bynhibiting its efflux from duodenal enterocytes, macrophagesnd hepatocytes, by binding to the iron transporter, fer-eportin, on these cells. Whereas hepcidin production isecreased in anemia and hypoxia, it is increased in theresence of iron and inflammation. Consequently, in iron-eficiency anemia the hepcidin levels are low, but inron-loading anemias hepcidin levels reflect the divergentffects of the iron overload and ineffective erythropoiesis25,26]. Infection and inflammation induce the synthesisf hepcidin through a mechanism involving inflammatoryytokines. IL-6 alone causes rapid upregulation of hep-idin mRNA expression in primary hepatocytes in vitro and
ncreased urinary excretion of hepcidin in human volunteers27,28]. IL-1� and IL-1� are also capable of inducing hep-idin in vitro but in some cell models this effect is IL-6ependent [26]. Several proteins such as hemojuvelin, which

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s highly expressed in skeletal muscle, and bone morphogenicroteins and their receptors appear to play major roles inegulating hepcidin expression [29].

At the cellular level the acquisition and storage of iron areeciprocally regulated by post-transcriptional mechanismsn which the expression of both ferritin and the transferrineceptor are co-ordinately regulated by a cytosolic protein,he iron-responsive element binding protein (IRE-BP) [30].RE-BP has a high affinity for specific stem-loop structuresresent in ferritin and transferrin receptor mRNAs. It hashigh degree of sequence similarity to the mitochrondrial

nzyme aconitase and a labile Fe-S cluster involved in sens-ng intracellular iron levels. When intracellular iron levelsre low the IRE-BP binds to stem-loop structures in the 5′upstream) region of ferritin and the 3′ region of the transfer-in receptor mRNA thereby preventing translation of ferritinnd prolonging the half-life of transferrin receptor mRNA. Inontrast, when intracellular iron levels are high the affinity ofhe IRE-BP for the stem-loop structures is reduced, leadingo production of ferritin and reduced transferrin receptor pro-uction. Thus, changes in intracellular iron availability alterhe affinity of the IRE-BP for its cognate RNAs, leading toontrol of iron uptake and storage by the cell. The divalentetal transporter 1 (DMT1) and ferroportin 1 (FPN1) which

ontribute to the regulation of intestinal iron absorption alsoear iron-responsive elements [31].

Nutritional deficiencies of iron, vitamin B12 and folatean be limiting factors in the production of red blood cells.ron is required as a substrate for hemoglobin production;itamin B12 and folate are needed for DNA synthesis androliferation of progenitor cells. To maintain iron homeosta-is an adult requires the absorption of 1 mg iron per day. Andditional 1000 mg of iron is required in pregnancy and fol-owing blood donation or trauma 1 mg of iron is required toompensate for each mL of erythrocytes. Vitamin B12 referso a group of cobalt-containing compounds known as cobal-mines. Cyanocobaltamin, formed as the result of the cyanidesed in its purification, is the principal form of B12 utilisedn nutrient supplements and foods. Although the incidence ofitamin B12 deficiency in the general population is unknown,he risk of deficiency appears to increase with age [32]. Onetudy provided laboratory evidence of B12 deficiency in 15ercent of adults older than 65 years. Medication, such as pro-on pump inhibitors, that reduce gastric acidity can also leado decreased vitamin B12 levels [33]. The body stores nor-ally contain 3–5 years supply of B12 and several months’

upply of folate. Consequently, symptoms of deficiency mayake months to years to develop in adults but in infants andhildren with lower established reserves signs of deficiencyay become manifest more rapidly.

.3. Cytokine regulation of erythropoiesis

Erythropoiesis is a finely tuned balance between the pos-tive signals generated mainly by Epo and stem cell factorSCF)/c-kit ligand and the negative signals from death recep-

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logy/Hematology 64 (2007) 139–158

or ligands and inhibitory cytokines. SCF and its receptor c-kitre essential for normal erythropoiesis as SCF or c-kit nullice (the Sl and W, mutants, respectively) exhibit embryonic

ethality associated with severe macrocytic anemia [34]. C-it is expressed on high numbers of CD34+ cells and duringhe differentiation of BFU-Es through to CFU-Es but declinesuring terminal differentiation. Sl and W mutant mice showsevere reduction in CFU-E numbers, confirming the role

f c-kit in BFU-E to CFU-E transition. The combined actionf SCF and Epo results in the synergistic proliferation andxpansion of progenitors ([35]; reviewed in [36]) with acti-ation of the JAK-STAT, PI3K and MAPK pathways [37].hrombopoietin (Tpo) is the major regulator of megakary-poiesis but can synergise with early acting cytokines suchs SCF, IL-3, IL-6, IL-11 and G-CSF to support the prolif-ration of primitive hemopoietic progenitors [38]. Tpo alsocts in late stage erythropoiesis, acting in synergy with Epo toromote the proliferation and differentiation of progenitors39]. Insulin-like growth factor 1 (IGF-1) and insulin alsoave roles as positive erythropoietic regulators [40,41].

Chronic inflammation can inhibit erythropoiesis mediatedn part through the effects of IL-1, TNF�, TGF� and IFN�.hese pro-inflammatory cytokines are powerful inhibitors ofrythropoiesis in vivo and in vitro [42]. IL-1 and TNF� sup-ress Epo mRNA expression [43]. IL-1 inhibition may beediated in part by induction of NF-kB which can bind to

he Epo promoter [44] and negatively regulate Epo produc-ion [45]. TGF� reduces the proliferation and accelerates theifferentiation of late erythroid progenitors [42]. The actionf the pro-inflammatory cytokine IL-6 is less clear, with aroposed role in the onset of anemia of chronic disease butith positive effects also demonstrated in vitro [46] and in

ivo, as IL-6 deficient mice demonstrate severe anemia [47].L-6 may exert its effects on erythropoiesis though regula-ion of iron metabolism [48]. Erythroid differentiation canlso be negatively regulated by death receptor ligands, e.g.as, a classic pro-apoptotic activator of the caspase cascade,nd TRAIL (TNF-related apoptosis-inducing ligand). Withinrythroblastic islands of the bone marrow immature ery-hroid cells express death receptors which are activated byigands produced by mature erythroblasts leading to caspase

ediated apoptosis [49]. The action of death receptors, asith Epo-deprivation, is mediated in part by caspase-induced

leavage of the major erythroid transcription factor, GATA-150]. GATA-1 is a zinc finger protein which plays a cen-ral role in erythroid development. GATA-1 binding motifsWGATAR) have been identified in the promoters/enhancersf virtually all erythroid genes [51,52] including Epo andpo-R. Signaling through the Epo-R can increase the expres-ion and activation of GATA-1 which in turn upregulatespo-R resulting in increased receptor and GATA-1 expres-ion in late CFU-E/proerythroblast progenitors. GATA-1−/−
mbryonic stem (ES) cells undergo rapid apoptosis suggest-ng a key role for GATA-1 in erythroid survival [53]. Theate of proliferation and differentiation of progenitors is con-rolled by the balance between GATA-1 levels and caspase

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ctivity [50]. Another transcription factor Scl (TAL-1), likeATA-1, is rapidly cleaved after activation of the caspase

ascade but precedes GATA-1 downregulation [54]. Zeunert al. have demonstrated that caspase resistant cells maintainigh levels of bcl-XL which in turn exerts an anti-apoptoticffect by inhibiting amplification of the caspase cascade [54].FN� promotes apoptosis of erythroid progenitors via Fas55] with additional upregulation of multiple members of theNF family including TRAIL and TWEAK [56]. Maximal

nhibition by IFN� is seen at the BFU-E stage [57]. Interest-ngly, in this system SCF can overcome the effects mediatedy IFN� [57] and Nishio et al. [58] propose that SCF preventsas-mediated apoptosis by preventing activation of the cas-ase cascade. Rubiolo et al. [59] suggest that Fas, along withaf-1, acts as a key regulator of the balance between differen-

iation and proliferation of erythroid progenitors. TRAIL canlso negatively regulate erythropoiesis, reducing the numbersf erythroblasts [60] however, Epo can protect against theegative effects of TRAIL via induction of PKC epsilon [61].

.4. The erythropoietin receptor

Epo promotes the viability, proliferation and differenti-tion of mammalian erythroid progenitor cells via signals
ransduced by the specific cell surface Epo receptor (Epo-R).he Epo-R is a 508 amino acid protein and is a memberf the cytokine receptor superfamily. Bone marrow ery-hroid progenitors are the major Epo target cells. During
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ig. 2. Epo signaling pathways. The three main Epo signaling pathways are highlig401, STAT3 binding to Y431, activation of the MAPK pathway following assoc

ubunit to Y479. Negative regulation occurs via SHP-1 binding to Y429 and Y431OC3 can directly bind to JAK2 (signaling reviewed in [249]).

logy/Hematology 64 (2007) 139–158 143

rythroid differentiation cells become progressively moreensitive to Epo due to the appearance of Epo-Rs, with laterogenitors CFU-E and proerythroblasts having the high-st Epo-R expression (∼1000 receptors/cell) [4,62]. Miceith null mutations of the Epo-R die during embryogen-

sis at E12.5–13.5, a critical stage for increased erythroidell expansion [63]. Interestingly normal levels of progen-tors are still observed, indicating that Epo-R signaling isispensable at the earlier stages of erythropoiesis, includ-ng BFU-E formation [63]. Upon Epo binding, preformedeceptor dimers undergo a conformational change which acti-ates three major signal transduction pathways (JAK-STAT,I3K-Akt, Ras-MAPK) (see Fig. 2) [64].

Pre-associated JAK2 autophosphorylates and is respon-ible for phosphorylation of tyrosines in the cytoplasmicegion of the Epo-R [65]. Phosphorylated tyrosine residueshen act as docking sites for Src homology 2 (SH2) domainroteins including the signal transducers and activators ofranscription (STATs) 1, 3 and 5. STAT5a and STAT5become tyrosine phosphorylated, dimerize and the result-ng complex is translocated to the nucleus where it activatesene expression. The importance of STAT5 in both primitivend definitive erythropoiesis is highlighted as STAT5a−/−nd STAT5b−/− embryos are severely anemic with impaired
urvival of liver erythroid progenitors. Although only moder-tely anemic at birth the adult mice later exhibit a poor stressrythropoietic response. In stress erythropoiesis, for examplen acute anemia, splenic rather than bone marrow progenitors
hted. Positive signaling is associated with STAT5 binding to Mu Y343 andiation of the adaptor protein Grb2 withY464 and binding of the p85 PI3Kand CIS and SOCS3 competition for STAT5 binding at Y401. SOCS1 and

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re responsible for the erythropoietic response. One of theenes activated by Epo-induced STAT5 is bcl-XL, an anti-poptotic gene. Bcl-XL is activated through direct bindingf STAT5 to a consensus sequence in the bcl-XL promoternd studies using mice deficient in bcl-XL also indicate aritical role for this protein in terminal erythroid differenti-tion [66]. Bcl-XL is also induced by GATA-1 and is a keyrythroid progenitor survival factor, as bcl-XL

−/− mice showigh levels of apoptosis in fetal liver cells and bcl-XL

−/− ESells fail to generate mature erythroblasts [67,68]. Menon etl. [69] postulate that STAT5 can distinguish between home-static and stress-induced Epo-R signaling with activationf the cytokine oncostatin M, rather than bcl-XL, promotingrythroid survival. Roles for STAT1 and STAT3 in Epo-Rignaling have also been proposed [70]. The importance ofAK2 in Epo signaling is also highlighted as JAK2 knockoutice have an almost identical phenotype to Epo and Epo-R

ene disruptions with embryonic lethality [71,72].In addition to the JAK/STAT pathway the phosphatidyli-

ositol 3 (PI3)-kinase-Akt pathway is activated by bindingf the SH2 domain of the p85 subunit to Epo-R Tyr479,nd is associated with erythroblast survival signaling [73,74].kt phosphorylates the pro-apoptotic factor BAD, which dis-

ociates from the cell survival factor bcl-XL, resulting innhibition of apoptosis [75]. The mitogen activated proteininase (MAPK) pathway is required for expansion of pro-enitors in response to Epo and SCF [37]. Following receptorctivation Src-homology and collagen (SHC) is phospho-ylated and forms a complex with growth factor receptorinding protein (Grb2), son of sevenless (SOS) and the G-rotein Ras with the resulting activation of the Raf, MEK,APK pathway.Epo-induced signaling via tyrosine phosphorylation is

ransient and returns to basal levels within 30 min of stimula-ion suggesting rapid dephosphorylation [76]. Termination ofpo signaling occurs when hematopoietic cell phosphatase,HP-1 catalyses the dephosphorylation of JAK2 [77] by bind-

ng to Epo-R Tyr 429 via its SH2 domain. Signaling is alsonhibited by cytokine (including Epo) regulated SOCS pro-eins (suppressor of cytokine signaling), which operate via aegative feedback loop to suppress Epo-induced signal trans-uction. Negative Epo signaling is associated with SOCS-3hich associates with the Epo-R and JAK2 [78]; mice lack-

ng SOCS3 show embryonic lethality associated with massiverythrocytosis [79]. SOCS-1 inhibits JAK2 by binding to thectivation loop, recruiting ubiquitination machinery and tar-eting JAK2 for degradation [80]. CIS (Cytokine InducibleH2 containing protein) is also induced by Epo signalingia STAT5 and represses proliferation when recruited to thepo-R, negatively regulating STAT5 activation [81]. Lnk, andaptor protein implicated in cytokine receptor signaling,egatively regulates Epo-mediated erythropoiesis by atten-
ating JAK2 activation [82]. Epo signaling increases the ratef Epo-R internalisation and the receptor is degraded byysosomal and proteasomal targeting using JAK2 dependent
echanisms [83].

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Epo-R is expressed primarily in bone marrow erythroidrogenitors but is also found on myocytes, cortical neurons,rostatic cells and vascular, breast and ovarian epithelia [84].he presence of the Epo-R outside of the erythroid compart-ent remains controversial as the validity of the antibody

sed in many of the localisation studies has been called intouestion [85,86]. Epo exhibits neuroprotective effects and ispotent promoter of neuronal survival (reviewed in [87,88]).

nterestingly, transgenic mice with exclusive expression ofhe Epo-R in hematopoietic cells develop normally with noeurological abnormalities [89]. However, conditional neu-onal Epo-R knock down has demonstrated a role for Epo-Rn adult neurogenesis [90]. It has been reported that systemi-ally delivered recombinant human Epo (rHuEpo) can crosshe blood brain barrier, reducing tissue damage in animal

odels of ischemic stroke [91,92] and may have potentialherapeutic use for neuroprotection. CEPO, a carbamylatedpo derivative which is not erythropoietic, may also haveeuroprotective therapeutic potential [93]. Brines et al. [93]uggest that tissue protective signals in non-hematopoieticissues may be mediated by heterodimerization of the Epo-Rnd the common �-receptor (�cR), a subunit of the GM-CSF,L-3 and IL-5 receptors. Interestingly, �cR null mice exhibitormal development with only relatively minor haematopoi-tic alterations [94].

.5. Control of EPO gene expression

The EPO promoter, 117 bp 5′ to the transcription startite, acts synergistically with a 3′ enhancer to regulate thepo gene [18]. There are several important elements in theIS acting EPO enhancer, which lies 120 bp 3′ to the EPOolyadenylation signal (reviewed in [15]). The most impor-ant of these elements is a hypoxia inducible factor (HIF)inding site (HBS) with the sequence CACGTGCT. HIFcts as a global regulator of oxygen homeostasis (reviewedecently in [95]). Transcriptional regulation occurs throughinding of HIF heterodimers, consisting of the oxygen sensi-ive HIF-� subunit and the constitutively expressed HIF-1�ARNT), to hypoxia-responsive elements (a core pentanu-leotide sequence RCGTG) in target genes [96,97]. There arehree main isoforms of HIF-� (HIF-1�, -2� and -3�) whichre members of the PAS (Per/ARNT/Sim) family of basic-elix-loop-helix transcription factors. The isoforms can allimerise with HIF-1� in hypoxia but differ in their tissue-pecific expression pattern [98]. It is not yet clear whetherhe EPO gene is regulated primarily by HIF-1� or HIF-2�.IF-1� was the first isoform demonstrated to be involved

n EPO gene regulation in vitro [99]. Recently, Warnecke etl. have shown that the Epo response is HIF-2� dependentn hepatoma (Hep3B) and neuroblastoma (Kelly) cells [100].nterestingly, in both the kidney and liver it is the HIF-2� and
ot the HIF-1� isoform that is expressed in Epo-producingells [101,98]. All three HIF-� proteins contain a centralxygen-dependent degradation domain (ODD) as well as N-nd C-terminal transactivation domains (TADs) [102].

V.M. Hodges et al. / Critical Reviews in Oncology/Hematology 64 (2007) 139–158 145

Fig. 3. Hypoxic regulation of EPO expression In normoxia HIF-� is hydroxylated by PHDs in the presence of the co-substrates Fe2+ and 2-oxoglutarate leadingt by the pl nucleuE

oh(�rldOhahHpbsfa1sci8mtdtsp

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o association with a VHL-E3 ligase complex and ultimately degradationigases. In hypoxia, HIF-� remains unhydroxylated and is translocated to thePO.

Increases in Epo production are controlled by sensingf oxygen levels in the kidney by oxygen-dependent prolylydroxylases (PHDs) which regulate the stability of HIF-�see Fig. 3). There is continuous expression of HIF-� and

with mRNA levels remaining unchanged by hypoxia, andegulation of HIF activity occurring exclusively at the proteinevel. Under normoxic conditions, HIF-1� is not normallyetected since the protein has a half-life of <5 min at 21%2 [103]. In the presence of oxygen the HIF-1� protein isydroxylated at prolines 402 and 564 in the ODD [104,105],n oxygen-dependent process catalysed by PHDs. Prolyl-ydroxylated HIF-1� is then bound by a pVHL (the vonippel-Lindau tumor suppressor protein) multiprotein com-lex which functions as an E3 ubiquitin ligase [106]. HIF-�ecomes poly-ubiquitinated and is degraded by the protea-ome. In hypoxia, however, oxygen becomes rate-limitingor PHD mediated hydroxylation and HIF-� is stabilisednd translocated to the nucleus where it binds to HIF-� and activates transcription of hypoxia response genesuch as EPO. HIF-1� expression can be demonstrated inells exposed to hypoxia in as little as 30 min [103]. Dur-ng normoxia HIF-1� is also hydroxylated at asparagine03 by factor inhibiting HIF (FIH) in the c-TAD. Thisodification results in a decreased ability of the carboxy-

erminus of HIF-� to bind to its co-activator p300/CPBue to steric hindrance and prevents activation of the HIFranscriptional cascade [107,108]. Accumulating evidence
uggests that HIF-2� and HIF-3� are degraded by similarrocesses.
PHDs belong to a superfamily of iron and 2-oxoglutarateependent dioxygenases and in mammalian cells three iso-

kwme

roteasome. The levels of the PHDs are modulated in part by the Siah E3s, where it binds HIF-1� and stimulates expression of target genes including

orms of PHDs have been identified (PHD1, 2, and 3). Thesenzymes demonstrate an absolute requirement for dioxygens a co-substrate as well as ascorbate and Fe2+. The hydrox-lation reaction involves one oxygen atom inserted at theroline residues of HIF while the other is utilised in the con-ersion of 2-oxoglutarate (2-OG) to succinate and CO2. Fe2+

s crucial for activating oxygen and ascorbate functions in thee-activation process by reducing Fe3+ (for a recent reviewee [109]).

Oxygen sensing involves complex regulatory mechanismsith potentially 2–5% of the genome responsive to hypoxia

nd under HIF-1 regulation [95]. Unsurprisingly, the actionsf the PHDs are therefore modulated in a number of wayssing both HIF-dependent and independent mechanisms. TheHD enzymes only catalyse the hydroxylation of HIF and not

he reverse reaction so the actual protein levels determine thectivity at distinct oxygen tensions. The expression of PHD2nd PHD3 is induced by hypoxia in a HIF-dependent mannerromoting a negative feedback loop [110] with hypoxia-esponsive elements identified in the PHD2 and PHD3 generomoters [111,112]. The levels of the other hydroxylases,HD1 and FIH, are unaffected by hypoxia [113,114]. In addi-

ion, PHD1 and PHD3 are degraded by the proteasome, inHIF-independent mechanism, using the Siah1 and Siah23 ligases with Siah protein expression also induced duringypoxia [115]. The role of individual PHDs remains to beully elucidated. Berra et al. [116] report that PHD2 is the
ey oxygen sensor as siRNA silencing of this single proteinas sufficient to stabilise HIF-1� in normoxia. In addition, autation in PHD2 resulting in increased HIF stability, causes
rythrocytosis suggesting that PHDs 1 and 3 fail to compen-

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ate [117]. Appelhoff et al. [114] however suggested a role forll three PHDs to reflect the known variation in tissue-specificHD protein expression levels, with all three PHD proteinsontributing in a non-redundant manner to regulation of theIF system.An additional level of regulation of PHD activity has

lso been suggested at the levels of transcription and trans-ation. Two alternatively processed PHD3 transcripts haveeen identified [118] with the expression of one splice vari-nt restricted to primary cancer tissues. Tian et al. [119] haveemonstrated two PHD1 isoforms, resulting from differentranslation start sites, which exhibit similar hydoxylase activ-ty but different protein stabilities. Cellular localization maylso influence activity as PHD1 is almost exclusively foundn the nucleus; PHD2 is predominantly cytoplasmic whereasHD3 is found in both compartments [113]. The translocationf PHD2 to the nucleus was however demonstrated by Jok-lehto et al. [120] and along with increased PHD2 expressionas associated with tumor aggressiveness. The finely tuned

egulation of the hypoxic response allowing regulation overwide range of oxygen tensions may also be attributed to aifference in the oxygen dependence of PHD2 and FIH [121].he flexibility of regulation is demonstrated by differences

n preferences for the HIF isoforms by PHDs [114] and byariation in the specific activity for the HIF hydroxylationite [122]. The complex levels of regulation of hypoxia-nduced gene expression are only beginning to be fullynderstood.

. Anemia

Anemia is a deficiency of functional mature red bloodells, which leads to a decreased oxygen-carrying capac-ty of the blood, tissue hypoxia, and a variety of clinicalonsequences including weakness, fatigue, increased car-iac output, increased morbidity and mortality, and decreaseduality of life. Anemia is a very common disorder and prob-bly still quite under-recognised. Anemias may be classifiedccording to mechanisms such as levels of red blood cell pro-uction, destruction, or loss, and by parameters of red bloodell morphology, such as the size, color and shape, as well ashe RBC indicies, mean corpuscular volume (MCV), meanorpuscular hemoglobin concentration (MCHC) and meanorpuscular hemoglobin (MCH). Here, we consider anemiasased on levels of Epo present in the circulation, since Epos the primary hormone responsible for the maintenance ofhe erythroid lineage. Classifying anemias in relation to Epoevels is also useful in providing a rationale for treatment ofnemia by ESAs.

Epo concentrations will vary depending on both the degreend type of anemia. Availability of recombinant human
po (rHuEpo) has led to the replacement of in vitro bioas-ays, such as the ex-hypoxic mouse and mouse spleen cellssay, and the development of more sensitive and specificmmunoassays. Plasma/serum Epo levels are now measured
filaw


sing radioactive immunoassays (RIA) or more commonlymmunoassays using chemiluminescent (CLIA) or enzymaticetection methods (ELISA). Fully automated methods (e.g.PC Immunlite 2000) are also available. Samples are cali-rated against reference standards and concentrations of Epore expressed in International Units per liter (U/L) with aormal range of <5–25 U/L [16].

.1. Anemias with high Epo levels

.1.1. Anemias due to bone marrow failureFailure of the bone marrow to produce blood cells may

ccur at the level of the hemopoietic stem cell (HSC), ort later stages of hemopoietic cell development. The formereads to aplastic anemia, with a hypocellular bone marrow anddecrease in each of the blood cell subtypes. The main fea-

ures include anemia; leukopenia, which leads to an increasedisk of infection; and thrombocytopenia, which presents aleeding risk. Failure of blood cell production at later stagesill affect only distinct blood cell lineages. When only the

rythroid lineage is involved, it is termed pure red cell apla-ia (PRCA). These are usually acquired disorders, due to theutoimmune destruction of HSC or lineage-specific progeni-ors. This immune-mediated destruction may be due to priorxposure to toxins, viruses, or drugs, although in many caseshe cause is idiopathic. There has been an increase in theeported cases of red cell aplasia in patients treated withSAs, caused by the generation of autoantibodies againstoth the ESA and endogenous Epo [123–125]. Also, thereave been rare reports of aplastic anemia following therapyor chronic myelogenous leukemia (CML). In these cases,matinib, directed against bcr-abl positive CML cells, alsoills other blood cell types, presumably through less specificnteractions with other tyrosine kinase receptors [126]. Asith other forms of anemias, there is a varied clinical course,

rom patients with mild symptoms requiring little or no treat-ent to patients with severe life threatening pancytopenia.There are also rare congenital forms of bone marrow fail-

re. For example, Fanconi anemia (reviewed in [127]) isn autosomal recessive or X-linked form of aplastic anemiahat can also present with a variety of skeletal abnormalitiesncluding short stature and an increased risk of malignancies.

utations in at least eleven different genes are involved inhe pathogenesis of the disease. The treatment of choice isormally bone marrow transplantation. Diamond Blackfannemia (DBA) (reviewed in [128]) has an autosomal domi-ant inheritance pattern and is usually diagnosed in infancy.BA presents with a variety of congenital abnormalities,

ncluding facial, limb and urogenital malformations, and anncreased risk of developing leukemias. About 25% of theases are associated with a mutation in the ribosome protein19; others have been associated with an as yet unidenti-
ed gene on chromosome 8p, as well as at least one other
ocus. Recently, mutations in telomerase enzymes, leading tobnormally shortened telomeres, have also been associatedith marrow failure [129].

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.1.2. Anemias due to substrate deficiencies orrotein/enzyme defects of the red blood cell

Many proteins and substrates are required for normaled blood cell maturation and hemoglobin production (seeection 1.2), and a deficit in any one of these can lead tonemia. Iron deficiency is the most common cause of anemiaorldwide (reviewed in [130]). Substrate deficiencies also

nclude lack of vitamin B12 or folate (megaloblastic anemia)131,132] in the diet, but can also be due to problems affectingbsorption of these compounds. Malabsorption may be due toiver, pancreatic, or intestinal disease. Pernicious anemia is anutoimmune disorder where antibodies are directed againstntrinsic factor (a glycoprotein produced by the parietal cellsf the stomach which is needed for absorption of vitamin12 in the intestine) or against the parietal cells themselves,

eading to impaired vitamin B12 absorption [133].Anemia can be caused by a failure or insufficiency of

emoglobin production. This can be due to a heme synthesisefect (usually because of lack of iron, as described above),r a globin synthesis defect. One of the most common ofhese is thalassemia, a condition in which there is defectiveroduction of one of the globin chains of the hemoglobinrotein. This leads to an overall deficiency of hemoglobinnd toxicity caused by the unassembled globin chains whicheduces the red cell life span. This is usually inherited in anutosomal recessive manner but may also be acquired [134].nother type of hemoglobinopathy, sickle cell anemia, is also

nherited in an autosomal recessive manner, and has persistedt abnormally high levels because heterozygotes carrying theutant gene have significant resistance to malaria [135]. The

enetic defect leads to a single amino acid substitution andhe production of a hemoglobin molecule that can polymerizen the cell under conditions of low oxygen. These red bloodells undergo a change in shape and consistency which cancclude vessels (vaso-occulsive crisis), leading to ischemia,ain, and organ damage.

Other problems with red blood cell proteins includepherocytosis, which may be hereditary or non-hereditary136,137]. Here, defects in various red blood cell mem-rane proteins lead to abnormally shaped (round) cells, whichre rapidly removed from the circulation and destroyed byhe spleen. Sideroblastic anemias (characterised by largeeposits of iron in erythroid mitochondria) can also becquired or inherited [138], and may be seen in someyelodysplastic syndromes. Here, defects within HSCs lead

o impaired cellular differentiation and the production ofmmature blasts which are filled with iron.

Hemolytic anemias are characterised by shortened red cellurvival. Whereas senescent red cells normally undergo lysisfter approximately 120 days, in severe hemolytic disordershe red cell lifespan can be as a short as a few days. There arehree principal causes: (i) intracellular defects arising from

etabolic enzyme dysfunction and hemoglobinopathies; (ii)oss of structural integrity of the red cell membrane andytoskeleton as occurs in hereditary spherocytosis, paroxys-al nocturnal hemoglobinuria (PNH), autoimmune disorders

1ctm

logy/Hematology 64 (2007) 139–158 147

nd drug-associated antibody damage; (iii) extracellularamage arising from chemical toxins, mechanical trauma,hrombocytopenic purpura and microangiopathic conditions.n general in hemolytic conditions the plasma Epo levels areigher than normal, and treatment of these patients with ESAss seldom justified [139].

In all of these anemias Epo levels are high, as the defects lieownstream of Epo production. In the cases of bone marrowailure, the defect is in the inability of the immature erythro-ytes to appropriately respond to the Epo signal, because theyre destroyed or damaged. In nutritional deficiencies or redell protein disorders, there is either an inadequate supplyf substrates required for erythrocyte maturation, or therere functionally important mutations in essential enzymes ortructural proteins. In all of these cases, the lack of an ade-uate supply of red blood cells leads to a continued state ofypoxia, loss of negative feedback to the kidney, and thus aontinued stimulus to Epo production in an attempt to cor-ect the hypoxia. Therefore, treatment with ESAs in theseisorders is neither logical nor effective. Even a very highevel of Epo is unable to overcome the underlying defects inhe erythropoietic pathway. These anemias are largely treatedymptomatically with transfusion as indicated by clinicaleed. Nutritional supplementation can be highly effective forhe substrate deficiency anemias, and steroid therapy can beeneficial in immune-related anemias. For some disordersore radical therapy such as splenectomy and bone marrow

ransplants must be undertaken.

.2. Anemias with low Epo levels

.2.1. Anemia of chronic diseaseThe anemia of chronic disease (ACD) is the second most

revalent cause of anemia, after iron deficiency. In someases it can still be difficult to distinguish ACD from iron-eficiency anemia [140], except that ACD patients normallyxhibit normal or increased serum ferritin levels, rather thaneduced serum ferritin levels. Recently, is has also becomenown as the anemia of inflammation (AI), which includeshe rapid onset anemia of acute critical illness [141]. Theesult of an acute or chronic immune system activation, ACDay be seen in over 75% of patients with hematological can-

ers or solid tumors, viral infections (including HIV), andutoimmune diseases such as rheumatoid arthritis, systemicupus erythematosus and irritable bowel syndrome [142].nemia is also a common finding in diabetes, which cane associated with renal tubular failure, but also arises fromnknown causes [143]. ACD often co-exists with other typesf anemia, and is believed to be widely under-diagnosed andnder-treated. Infections, malignant cells, and autoimmuneysregulation all lead to activation of the immune systemnd the production of cytokines, most notably TNF�, IL-
, IL-6, IL-10, and IL-� (reviewed in [142]). Inflammatoryytokines can depress Epo production (Section 1.3) leadingo the development of anemia. Suppression of erythropoiesis
ay also be mediated in part by the generation of oxygen and

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Present evidence indicates that hepcidin is the key factorn the development of the hypoferremia associated with thenemia of inflammation. IL-6 can upregulate the productionf hepcidin in the liver [26]. Higher levels of hepcidin leado decreased iron absorption, and the iron that is absorbeds sequestered for storage in the reticuloendothelial system,o that there is reduced iron availability for hemoglobin pro-uction in maturing erythrocytes. Hepcidin may contributeo the anemia of chronic inflammation by decreasing theesponsiveness of bone marrow erythroid cells to low levelsf erythropoietin [145]. However, the relative contributionf hepcidin to the pathogenesis of this anemia comparedith other factors such as decreased Epo production, bluntedpo response and shortened red cell survival remains to bestablished.

In ACD, Epo levels are inappropriately low for the degreef anemia and there is a blunted response to any Epo thats produced. The total iron binding capacity of the blood issually normal, but the reticulocyte count is low. Ultimatelyhe best way to treat ACD is to identify and cure the under-ying cause of the chronic inflammatory disease. However,ince this may not be immediately possible, it is importanto alleviate the ACD. Hepcidin antagonists are being exam-ned as a possible treatment for ACD, but currently rHuEpos the usual treatment. This exogenous Epo is administeredo counteract the antiproliferative effects of the endogenousytokines being produced and rHuEpo reduces dependencen blood transfusions. However, the effects of long-term ane-ia treatment with ESAs on the underlying disease are less

lear with some clinical concerns raised over the safety ofHuEpo therapy for cancer-related anemias.

The microenvironments within tumors together with theirberrant blood supply create some areas within tumorshat are hypoxic. This phenomenon may be even moreevere in anemic patients. Tumor hypoxia can impair theffectiveness of radiation therapy and some chemotherapy146,147]. This has led to the hypothesis that strategieso diminish cancer-related anemia may not only allevi-te anemia-related symptoms but will also enhance tumoresponses to chemotherapy and radiotherapy and improveverall patient survival.

.2.2. Anemia of chronic renal failureAnemia associated with kidney failure may be present in

atients with acute or chronic renal failure. It is primarilyelated to an almost complete deficiency of Epo due to theoss of functional renal tissue. This may be compounded byn acute or chronic inflammatory state associated with theause of renal failure, e.g. glomerulonephritis. Many patientsith progressive kidney disease are treated with medication

hat inhibits the renin–angiotensin system such as angiotensinonverting enzyme inhibitors (ACEi) or angiotensin recep-or blockers (ARBs) that also blunts erythropoiesis. Theecrease in renal Epo production is inversely correlated with

bmep


he glomerular filtration rate [148,149]. In addition to abso-ute Epo deficiency there is also a contribution to the anemiaf chronic renal failure from uremic inhibitors of erythro-oiesis. Adequate dialysis, that reduces the levels of uremicoxins, can improve the therapeutic response to ESAs.

If a patient has progressed to end-stage renal diseaseESRD), the treatment of choice is renal transplantation150]. However, the new kidney may not function optimally,ailing to produce an adequate supply of Epo, leading to post-ransplantation anemia (PTA). Over 30% of patients exhibit

oderate to severe anemia at some point within 5 years post-ransplantation [151–153], the prevalence increasing withime after the transplant. In most cases there is an initialeak of Epo levels on the first day post-transplantation [154];fter that, there is a strong correlation between hemoglobinoncentrations and graft function, as measured by serum cre-tinine levels [150]. Although a successful graft can restoreoth the excretory and endocrine functions of the kidney,ts function may still be sub-optimal [155]. Most evidenceuggests that impaired Epo production by the graft is theain cause of the anemia [149,150], but other factors may

ontribute. The drugs mycophenolate mofetil (MMF) andzathioprine (immunosuppressants used to prevent organejection) may exacerbate anemia. MMF is a selective blockerf the de novo or ribose derived purine synthesis pathway,nd is expected to have a major effect on the proliferationf lymphocytes, but not erythrocytes, from the bone marrow150]. Thus, its association with poor erythrocyte produc-ion is unclear. Azathioprine leads to a general depression ofone marrow cellular proliferation [156] and also shows someelective erythroid toxicity, although the mechanism of thehenomenon is not well understood [157]. ARBs and ACEiused as anti-hypertensives after transplant to reduce the riskf cardiovascular events) are also associated with a higherikelihood of anemia [150,158]. Other risk factors for theevelopment of PTA include recent infections and increas-ng age of the donor [150]. It has also been observed that solidrgan transplant patients receiving immunosuppressive ther-py are susceptible to parvovirus B19 infections which canead to red cell aplasia. This anemia can usually be alleviatedy a reduction in dosage of immunosuppressant drugs [159]nd administration of immunoglobulin therapy [160] but thenemia is often recurrent [161].

Patients with both chronic renal failure associated ane-ia and PTA can benefit from treatment with exogenous

HuEpo. Although there are few data available concerninghe outcomes of treatment of PTA with Epo, there is no indi-ation that patients treated with rHuEpo have any greaterisk of deterioration in their graft function [155]. Neverthe-ess it is reported that fewer than 10% of transplant recipientseceive this treatment [150,152,158]. Some reports suggesthat PTA patients treated with Epo may develop hypertension,
ut overall, treatment of PTA may be beneficial, since ane-ia can contribute significantly to the risk of cardiovascular
vents, which are the main cause of death in renal trans-lant patients [162]. Treatment with Epo can improve left

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entricular structure and function as well as cardiac output,eading to a decrease in negative long-term outcomes as welln increased quality of life [163–165]. Recent studies suggesthat the presence of Epo receptors on normal kidney cellsllows Epo administration to have renal protective proper-ies, preventing ischemia-induced tissue damage in acute andhronic renal injuries [166,167].

.3. ESAs for anemia treatment

Treating anemia in patients can lead to decreased hos-italization times, quicker recoveries, improved clinicalutcomes, and better quality of life. Because Epo levels areow in patients with ACD and anemia of renal failure, ane-

ia can often be successfully treated by the administration ofxogenous Epo. One of the largest patient groups routinelyreated with ESAs are those with chronic kidney disease. Thearget hemoglobin values in these patients are 11–13 g/dLKDOQI guidelines [168]) or greater than 11 g/dL (Europeanest Practice Guidelines [169]), respectively. The optimumemoglobin in patients with renal anemia however remainsndetermined with recent reports demonstrating no benefitf rHuEpo therapy in maintaining higher target hemoglobinsn pre-dialysis or dialysis patients [168,170,171].

Originally, Epo was isolated and purified in small quanti-ies from the urine of patients with aplastic anemia [172], butn 1985, the human EPO gene was cloned [6,7] allowing thearge-scale production of the hormone by recombinant DNAechnology. Recombinant human Epo (rHuEpo), commonlynown as epoetin alpha, is highly effective at stimulatingrythropoiesis with a low immunogenicity. Only in a smallroportion of cases have patients developed neutralizing anti-odies that are also cross-reactive with native Epo, leading toevere pure red cell aplasia [173]. The increase in the num-er of PRCA cases has been attributed to leachates from theubber stoppers used in pre-filled syringe formulations of therug, which led to an increased immunogenicity [174,175].

For optimal efficacy and cost effectiveness epoetin isdministered 2–3 times per week by subcutaneous orntravenous injection. Novel Epo analogues with longer elim-nation half-lives, such as darbepoetin alfa (Aranesp) andontinuous erythropoietin receptor activator (CERA), haveeen developed which permit extended dosing schedules176–179]. A synthetic erythropoiesis protein (SEP), consist-ng of a 51 kDa protein–polymer, has also been developedhich stimulates activation of the erythropoietin receptor

180]. Biologically active erythropoietin dimers exhibitedrolonged half-life, but have not been clinically tested181]. Erythropoietin-mimetic peptides, which are struc-urally unrelated to Epo but are still able to activate the Epo-Rre also being investigated. These include Hematide, a pep-ide linked to polyethyleneglycol which increases solubility
nd circulation half-life, and is now entering phase II trials182,183]. Other strategies for alleviating anemia by stimu-ating erythropoiesis include inhibitors of hematopoietic cellhosphatases such as SHP-1, which negatively regulate the
aBae

logy/Hematology 64 (2007) 139–158 149

po-induced JAK-STAT pathway [77,184] and stabilizers ofIF-1� [179]. However, the existence of more than 70 known

argets for HIF-1� may severely limit this approach. Further-ore, naturally occurring mutations in VHL and PHD2 which

esult in HIF stabilisation can cause erythrocytosis [185,117].There has been some experimental interest in treating ane-

ia via EPO gene therapy but this approach is tempered byoncerns about safety in clinical practice. It is potentially fea-ible to achieve hypoxia-regulated EPO gene expression if thePO transgene is driven by a promoter containing a hypoxia

esponse element (HRE). This has been demonstrated in an inivo model where erythropoiesis was reconstituted in an Eponockout mouse after adenoviral vector delivery of an HREPO transgene [186]. There are persisting concerns that gene

herapy may cause additional unpredictable insertional muta-enic events which limit the applicability of this technique touman disease.

Recently some concerns have been raised about the long-erm safety of Epo treatment both for renal anemia andon-renal anemias. Large pharmacological Epo doses giveneriodically to patients do not replicate normal physiolog-cal kinetics [187]. Epo may have a protective effect, ineural [188,189] and cardiac tissues [190]. Conversely, Epoay be harmful to some groups of cancer patients. Henke

t al. found that although Epo treatment corrected the ane-ia in head and neck cancer patients it was associated with

ecreased progression-free survival [191]. The Breast Can-er Erythropoietin Survival Trial (BEST), in which overallurvival was the primary end-point, was terminated earlyecause of poorer survival in the rHuEpo arm [192]. Fur-hermore, a Cochrane review of 9353 cancer patients in 57rials established that recombinant Epo or Aranesp signifi-antly reduced the requirement for blood transfusion but alsoarried an increased risk of thrombo-embolic events (such astroke and myocardial infarction) with a relative risk ratio of.67 [193]. Recently, rHuEpo administration during primaryreatment of locally advanced cervical carcinoma was associ-ted with a poorer response to radiation therapy, including anncreased recurrence rate, increased risk of death due to dis-ase, and decreased disease-free overall survival time [194].aupel et al. point out that raising the Hb level above 14 g/dL

n patients with cancer-related anemia causes an increase inlood viscosity which may lead to adverse cardiovascularvents [195]. These studies indicate a need for caution inreating cancer patients with rHuEpo and have led to the pro-osal that they may enhance thrombosis, tumor growth andeovascularization [196].

. Erythrocytosis

The term erythrocytosis defines a group of disorders char-
cterised by an increase in circulating red blood cells (RBCs).oth erythrocytosis and polycythemia are used interchange-bly to describe a condition in which the hematocrit (HCT)xceeds the upper limit of normal. The term erythrocytosis is

150 V.M. Hodges et al. / Critical Reviews in Oncology/Hematology 64 (2007) 139–158

Table 1Classification of erythrocytoses

Classification Epo levels Cause

PrimaryCongenital Low Epo-R mutationAcquired Low PV

SecondaryCongenital Inappropriately normal or elevated Chuvash/VHL-dependent polycythemia

PHD2 mutationHb variants2,3BPGM mutation

Acquired Appropriately elevated High altitudeChronic pulmonary diseaseSleep apnoeaRight to left cardiac shunts

Inappropriately elevated TumorsExogenous Epo

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ore accurate when only the red cell lineage is involved whilehe term polycythemia can be confused with polycythemiaera (PV), a clonal stem cell disorder resulting in excessiveroduction of red cells, neutrophils and platelets. Erythrocy-osis is suspected in patients with an abnormally high HCT,emoglobin concentration or RBC count. All of these mea-urements are concentrations and are therefore dependent onlasma volume as well as circulating red cell mass (RCM).rythrocytosis can be further classified as relative, where the

ed cell mass is normal and the plasma volume is decreased orbsolute, where the red cell mass exceeds the normal upperimit. McMullin et al. [197] have provided guidelines foriagnosis, investigation and management of polycythemiand erythrocytosis.

Erythrocytosis may be classified as primary, secondary ordiopathic (see Table 1). In primary erythrocytosis there is anncrease in circulating RBC numbers caused by a molecu-ar defect in the erythroid lineage. Secondary erythrocytosisan arise due to appropriate changes such as adaptation toigh altitude, or inappropriate changes such as aberrantlyncreased circulating levels of Epo. The term idiopathic ery-hrocytosis denotes a heterogeneous group of rare disorders ofnknown etiology in which the cause may be either primaryr secondary. Similar to anemias, the treatment of patientsith erythrocytosis depends on the severity and cause of

he disorder. Some patients have a relatively benign diseasehilst others require venesection or cytoreductive therapy

197].Measurement of plasma Epo levels provides an important

nsight into the potential regulatory pathways which may beisrupted in the development of the erythrocytosis. Like thenemias, the erythrocytoses can also be classified using serum
po levels. Erythrocytosis with an inappropriately normal orlevated Epo level can be associated with abnormalities inhe oxygen sensing pathway or abnormal hemoglobin oxygenffinity while those with low Epo levels may be linked to dys-
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egulated Epo signaling and/or hypersensitivity of erythroidrecursors.

.1. Erythrocytosis with low Epo levels

Polycythemia vera (PV) is an acquired disorder lead-ng to clonal expansion of a single HSC containing ancquired mutation characterized by hyperplasia of threeone marrow lineages. It has an incidence of 2–3 per00,000 [198] and exhibits a risk of leukemic progres-ion. PV bone marrow progenitors can form colonies inhe absence of Epo in vitro, a criterion useful in distin-uishing PV from familial and congenital polycythemias.n 2005, an amino acid substitution V617F in JAK2 wasdentified in a high percentage of affected PV individuals199–202] and now forms a useful addition to PV diag-ostic criteria. This mutation is found in large numbers ofatients with myeloproliferative disorders, not only in PV butlso in essential thrombocytopenia and idiopathic myelofi-rosis. The mutation causes constitutive activation of JAK2nd downstream signaling pathways leading to erythrocyto-is. V617F is rare in patients with idiopathic erythrocytosis203].

Thirteen autosomal dominant mutations in the Epo-R haveeen described [204,205], which lead to erythrocytosis withow Epo levels and hypersensitivity of progenitors to Epo initro [206,207]. Mutation of the Epo-R was first described inlarge Finnish family (Trp439Ter) with thirty three individ-als over five generations demonstrating erythrocytosis withow Epo levels [208]. In total, ten Epo-R mutations cause aruncation of between 58 and 110 amino acids of the cyto-lasmic C-terminal of the receptor. This results in the loss of
ome of the Epo-R negative regulatory signaling pathways.he binding site for the phosphatase SHP-1 is lost in all of the
runcated receptors, resulting in prolonged JAK2 autophos-horylation and STAT5 activation leading to enhanced Epo

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timulation and a proliferative advantage [209,210]. Partialownregulation of Epo signaling may still occur in somef the receptor truncations as the binding site for the neg-tive regulators CIS and SOCS3 (Y401) is still retained (seeig. 2). In addition, SOCS1 and SOCS3 can still bind to thectivation loop of JAK2 leading to suppression of signaling.rcasoy and Karayal [211] reported that the tyrosines which

re retained in one truncated Epo-R (HuY285 and Y344),ncluding a tyrosine which binds STAT5, both contribute tohe sensitivity of the progenitors to Epo. Congenital erythro-ytosis was replicated in a mouse model where the murinepo-R was replaced by a truncated mutant human Epo-R212]. The other three reported Epo-R mutations result inither an amino acid substitution or a base change in intronand are not associated with the development of familial

rythrocytosis [204].

.2. Erythrocytosis with high Epo levels

Recent studies of patients with inherited erythrocytosisrom the Chuvash population in the Upper Volga region ofussia have identified an abnormality in the oxygen sensingathway. A genome-wide screen of this ethnically homo-eneous population localized a region on chromosome 3ssociated with the disease. This led to identification of a Co T transition at nucleotide 598 of the VHL gene leading ton arginine to tryptophan change at amino acid 200 (R200W)185]. Affected individuals are homozygous and carriers areeterozygous. R200W leads to a reduction in the interactionetween VHL and HIF-1�, decreasing the levels of ubiqui-inated HIF-1�, leading to more active HIF dimer and anncrease in the expression of hypoxia inducible downstreamenes, including Epo. Indeed, Gordeuk et al. [213] reportemoglobin adjusted Epo levels were ∼10-fold higher inomozygotes compared to controls with Epo-hypersensitiverythroid progenitors. Unlike classical VHL syndrome noumorigenesis is found in Chuvash polycythemia, withomozygosity instead associated with varicose veins, lowlood pressure and elevated basal expression of the HIF reg-lated genes VEGF and plasminogen activator inhibitor 1PAI-1) associated with thrombosis and vascular abnormali-ies [214,215].

Outside of Chuvashia, other families have been describedith this mutation including those of Caucasian, Africanmerican and Asian ancestries [198,216]. VHL-dependentolycythemia, with the Chuvash haplotype, has also beeneported as endemic on the island of Ischia with non-affectedeterozygotes also showing an increase in HIF-1 activity sug-esting a mutation maintenance advantage [217]. A founderffect has been established which arose in a single ancestor4,000–62,000 years ago [218], although Cario et al. [219]ecently reported a single case where the Chuvash mutation
rose on a separate genetic background. VHL mutations in allhree exons have now been described [217,219,220] includ-ng compound heterozygotes displaying Chuvash and otherHL mutations [221].
Enci

logy/Hematology 64 (2007) 139–158 151

However, a substantial number of patients with congenitalrythrocytosis with normal or elevated Epo do not have VHLutations but may have other defects in oxygen-dependent

ene regulation. Recently, Percy et al. [117] described autation in the hydroxylase PHD2 also leading to inap-

ropriate regulation of HIF and increased Epo expressionausing erythrocytosis. The C to G transition at base 950esults in a proline to arginine change in an evolutionar-ly conserved residue (P317R). This residue lies close tohe iron-chelating residue asparagine 315 in the active sitef the enzyme. The PHD2 mutation results in a markedoss of function by reducing binding to HIF and decreas-ng the prolyl-hydroxylase activity in vitro. Overexpressionf the mutant PHD was less effective at suppressing HIF-induced activation of a reporter gene. Unlike the recessivehuvash mutation this PHD mutation is autosomal dominant.hese studies of patients with erythrocytosis have provided

mportant insights into the regulatory mechanisms of oxygenomeostasis.

Hereditary erythrocytoses are extremely rare but alsonclude a wide range (>100) of hemoglobin variants ([222];ttp://globin.cse.psu.edu). The mutations are localized toegions of the hemoglobin molecule associated with oxy-en transport leading to an increased affinity for oxygen andcharacteristically abnormal “left-shifted” oxygen equilib-

ium curve. The red cell mass is increased to respond tohe resulting tissue hypoxia. In addition, deficiencies in theed cell enzyme bisphosphoglycerate mutase leading to lowevels of 2,3-bisphosphoglycerate (2,3 BPG) may cause ery-hrocytosis [223]. Reduced levels of 2,3 BPG, which bindso the central cavity of the hemoglobin molecule, lead to

failure to convert to a low oxygen affinity state. Thesenzyme deficiencies may be inherited in autosomal dominantr recessive traits depending on the severity of the deficiency224].

.2.1. Appropriately elevated EpoA number of relatively common clinical conditions are

ssociated with an elevated hematocrit secondary to anppropriately elevated serum Epo in the setting of chronicissue hypoxia. These include chronic respiratory diseasesssociated with hypoxia, right-to-left circulatory shunting,ost-renal transplant and residence at high altitude levels.

Chronic obstructive lung disease associated with loss ofunctional lung parenchyma and impaired pulmonary gasxchange results in chronic arterial hypoxia. This will trig-er increased renal Epo secretion to expand the red cell massnd enhance tissue oxygen delivery [225]. The rise in hema-ocrit may be compounded by smoking related reduction inlasma volume [226]. Alternatively, recurrent episodes ofypoventilation associated with sleep apnoea syndrome willlso result in decreased arterial oxygenation that will cause an
po-induced expansion in red cell mass, although the mag-itude of this response is disputed [227–229]. In congenitalyanotic heart disease a significant right-to-left cardiac shunts also associated with erythrocytosis [230] and in some cases
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ay be complicated by thrombotic events [231]. Serum Epoevels are high, driving an increase in red cell mass in affort to correct chronic tissue hypoxia caused by the shunt-ng of deoxygenated erythrocytes from the pulmonary arterialirculation directly into the systemic circulation.

Post-transplant erythrocytosis (PTE) occurs in up to 15%f renal transplant recipients [232]. Predisposing factorsnclude male gender, smoking, transplant renal artery steno-is, retention of native kidneys and a well-functioning graft233,234]. The source of Epo has been reported to be fromither the diseased native kidneys or the transplanted kid-ey itself [232,234,235]. Although Epo levels in most PTEatients are within the normal range the level is still inap-ropriate given the degree of erythrocytosis suggesting thathe normal physiological feedback regulation is disrupted.dditional stimuli to erythropoiesis may include the actionf factors such as androgens, insulin-like growth factor andngiotensin II [236,237]. This may explain the efficacy ofgents such as angiotensin converting enzyme inhibitors orngiotensin II type 1 receptor blockers that can reduce theaematocrit by inhibition of the renin–angiotensin system238,239].

A compensatory erythropoietin-induced increase in redell mass occurs secondary to hypoxia associated withesidence at high altitude. This contributes to the patho-hysiology of chronic mountain sickness. Some individualsxperience symptoms such as dizziness, headache, lethargy,nsomnia and paraesthesia described as burning hands andeet [240], and may develop cerebral or pulmonary edema.enetic and environmental adaptive factors are believed to

nfluence susceptibility to chronic mountain sickness since its more common in men than women, in post-menopausalhan premenopausal women and newcomers rather thanhronic mountain dwellers [241–243] although, Mejia et al.ould not show any genetic association between severe ery-hrocytosis and a number of candidate genes including EPO,PO-R, HIF-1α, VHL and PHDs [244].

.2.2. Inappropriately elevated EpoOne area of concern is the potential and actual misuse

f recombinant human erythropoietin to enhance athleticerformance. Epo doping is suspected in 3–7% of the top ath-etes involved in endurance sports [245] with an anticipatednhancement in performance by increasing the oxygen-arrying capacity of the blood. Numerous, often high-profile,ases have been detected in routine anti-doping controls buthe detection of recombinant proteins is challenging. Azzazyt al. [245] suggest that despite potential risks gene dopingay be in practice as soon as the Beijing 2008 Olympics. In

ddition, Lippi et al. [246] suggest that cobalt chloride abuseay become an alternative, currently undetectable, dopingethod as cobalt chloride administration can inhibit HIF-1�

egradation and thus lead to increased erythropoiesis.Erythrocytosis as a paraneoplastic syndrome has been

ssociated with a range of malignancies including renalell carcinoma, hepatoma, cerebellar hemangioblastoma,

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heochromocytomas, ovarian tumors and Wilm’s tumor197] and can be attributed to Epo production by the tumorr by normal parenchyma in response to hypoxia-inducedumor growth [247]. Erythrocytosis is reported in up to 5%f patients with renal cell carcinomas. The mechanism forncreased Epo production may be linked in the majority ofhese tumours to mutations in the VHL gene that disrupt theormal regulation of hypoxia-induced proteins resulting inbnormal sensing of the renal tissue oxygen tension [248].

Since most dialysis units treating renal disease will attempto achieve an audited standard of >80% of patients withaemoglobin values within the target range or above the targetevel it is inevitable that some ESA-treated patients inad-ertently end up with haemoglobin values above 13 g/dL. Itould however be extremely rare that a patient would be

ccidentally treated to a hemoglobin level that would qualifys erythrocytosis. Usually cessation of the ESA treatment isufficient to allow the haemoglobin concentration to fall backo the target level.

. Future directions

Identification of the mechanisms underpinning the nor-al physiology, cell and molecular biology of erythropoiesis

as been crucial to our understanding of the pathophysiologyf anemias and erythrocytoses. Development of recombinantNA-derived erythropoiesis stimulating agents represents a

pectacular example of translational research providing novelherapies for the anemias associated with low Epo levels.hese treatments have literally transformed the quality of

ife for millions of anemia sufferers, especially those withhronic kidney disease. Nevertheless there are some concernshat ESA therapy may have limits reflecting the differencesetween normal physiological erythropoiesis compared withharmacological doses of ESAs in maintaining red cell mass187]. The ideal target hemoglobin in patients with chronicidney disease remains controversial despite 20 years of clin-cal trials with ESAs. The effects of ESAs on cells outside ofhe erythroid lineage are also being explored with the promisef beneficial effects on injured cardiac and neural tissues.nterest in such novel indications is tempered by the possi-le adverse effects of ESA treatment on tumor biology andverall cardiovascular risk.

It is also interesting to contrast the fate of persons with dif-erent mechanisms resulting in erythrocytosis. The probandn the original Finnish study of erythrocytosis caused by

truncated Epo-R was an Olympic gold medal-winningross-country skier with a naturally advantageous increasedxygen-carrying capacity [208]. In contrast, the VHL muta-ion causing Chuvash polycythemia, whilst not associatedith the risk of tumor formation seen in VHL syndrome, is
onetheless linked with premature mortality in part due to anncreased risk of arterial thrombosis [213].
Future examination of the pathophysiology of anemiasnd erythrocytoses is likely to yield more surprising insights

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nto the complex inter-relationships of erythropoiesis andrythropoietin with overall human health.

eviewers

Josef Prchal, Professor of Medicine, Division of Hema-ology, University of Utah School of Medicine, RoomC416-SOM, 30N 1900E, Salt Lake City, UT 84132, Unitedtates.

Joachim Fandrey, Professor, Director, Institute for Phys-ology, University of Duisburg-Essen, Hufelandstrasse 55,-45122 Essen, Germany.Dr. Constance Tom Noguchi, PhD, Chief, Molecular Cell

iology Section, Molecular Medicine Branch, National Inst.iabetes & Digestive & Kidney Disease, National Institutesf Health, Building 10, Room 9N307, Bethesda, MD 20892-822, United States.

cknowledgements

The authors would like to thank Dr. Melanie Percy forritical reading of the manuscript and also to acknowledgeupport from RoFAR, the Northern Ireland Kidney Researchund, the Northern Ireland Leukaemia Research Fund and

he R&D Office, Northern Ireland.

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iographies

Vivien M. Hodges is Post-doctoral Fellow in Professorappin’s Laboratory in the Haematology Research Group,entre for Cancer Research and Cell Biology at Queen’sniversity, Belfast.

Susan Rainey is Post-doctoral Fellow in Professor Lap-in’s Laboratory in the Haematology Research Group, Centreor Cancer Research and Cell Biology at Queen’s University,elfast.

Terence R. Lappin is Professor of Haematology at Queen’sniversity, Belfast.

A. Peter Maxwell is Professor of Renal Medicine atueen’s University, Belfast.

Pathophysiology of anemia and erythrocytosis

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